PMID- 12369658 TI - The value of bone scintigraphy in the evaluation of osteoporotic patients with back pain. AB - We evaluated the role of bone scintigraphy in 60 osteoporotic patients with back pain. Thirty-four had scintigraphic evidence of vertebral fracture and were found to have a significantly lower bone density compared to those without fractures (p = 0.01). In only 14 patients was vertebral fracture considered to be the sole cause of pain with 38 having alternative abnormalities, the most common of which was facet joint disease (n = 30). Results of bone scintigraphy influenced a direct change in management in 18 patients and were able to exclude vertebral fracture as a cause of symptoms in 30. In symptomatic osteoporotic patients the bone scan may be helpful in elucidating the etiology of back pain and can impact on patient management. PMID- 12369659 TI - Intracardiac thrombosis and fever possibly triggered by ovulation induction in a patient with antiphospholipid antibodies. AB - We report on a 28-year old patient with polycystic ovary syndrome (PCOS) who presented with fever and laboratory markers of inflammation. Her medical history was relevant for multiple ovulation inductions (OI) and ovarian hyperstimulation syndrome (OHSS). She had two miscarriages and one preterm delivery. Intracardiac thrombosis was diagnosed in the presence of antiphospholipid antibodies. We suggest that primary antiphospholipid syndrome (APS) was possibly triggered by OI. PMID- 12369660 TI - Three-dimensional fetal sonography: use and misuse. PMID- 12369661 TI - Accuracy of real-time three-dimensional echocardiography for quantifying right ventricular volume: static and pulsatile flow studies in an anatomic in vitro model. AB - OBJECTIVE: The complex structural geometry of the right ventricle hinders accurate assessment of right ventricular volume and function on conventional two dimensional echocardiography. We sought to evaluate the accuracy of real-time three-dimensional echocardiography for quantifying the volume of the right ventricle in an in vitro experimental study. METHODS: We developed 39 anatomically accurate latex phantoms of human and porcine right ventricles (range, 24-108 mL) for 39 static and 10 pulsatile models. Real-time three dimensional scanning was performed with the models placed in a water bath and with a 3.5-MHz probe. In the dynamic models a pulsatile flow pump generated 2 different stroke volumes (29 and 64 mL/beat). Static chamber volumes and stroke volumes were verified by water displacement, which served as a reference standard. Three-dimensional echo right ventricle volumes were determined by tracing derived B- and C-scans, using the Simpson rule. RESULTS: Multiple regression analyses showed an excellent correlation between real-time three dimensional echocardiographic determinations and the static volumes (B-scan, r = 0.99; C-scan, r = 0.98; P < .001), as well as stroke volumes in the dynamic model (B-scan, r = 0.90; C-scan, r = 0.86; P < .001). However, the C-scans tended to underestimate cavity and stroke volumes more than the B-scans (mean difference for static volume: B-scan, 1.4% +/- 9.8%; C-scan, -7.4% +/- 8.0%; P < .001; mean difference for stroke volumes: B-scan, 3.0% +/- 19.1%; C-scan, -2.5% +/- 20.9%; P < .001). CONCLUSIONS: Real-time three-dimensional echocardiography can accurately quantify right ventricle cavity volumes and stroke volumes without geometric assumptions. PMID- 12369662 TI - Abdominal pain and preeclampsia: sonographic findings in the maternal liver. AB - OBJECTIVE: To describe sonographic findings in livers of pregnant women with severe preeclampsia and abdominal pain. METHODS: Over a 12-month period, we performed serial sonographic examinations on 32 pregnant women with severe preeclampsia and acute right upper quadrant and epigastric pain. On each sonogram we observed the liver size and texture, "periportal halo" sign, gallbladder wall, Glisson capsule thickness, painful compression of the liver and gallbladder, and ascites. The pancreas, spleen, kidneys, and uterus were also studied. Sonography was repeated after delivery. RESULTS: Initial sonograms showed liver abnormalities in 28 patients. Abnormalities consisted of liver hypertrophy (n = 24), hyperechoic thickening of the periportal area (periportal halo sign; n = 23), striated thickening of the gallbladder wall (n = 27), hyperechoic thickening of the Glisson capsule (n = 11), liver areas of increased echogenicity (n = 11), subcapsular hematoma (n = 1), and subcapsular calcification (n = 1). Probe compression of the liver enhanced abdominal pain (n = 13), whereas the gallbladder was painless in all cases. No gallbladder stones were detected. Ascites (n = 16) and pleural effusion (n = 11) were also present. In no case did we detect abnormalities of the pancreas, kidneys, or spleen. All patients eventually had hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome according to the American College of Obstetricians and Gynecologists classification. In 7 cases, HELLP syndrome developed postpartum. Three patients also had eclampsia. Follow-up sonograms highlighted quick regression of abnormalities after delivery. CONCLUSIONS: The livers of women with severe preeclampsia who had HELLP syndrome showed sonographic abnormalities before biological abnormalities. Serial sonographic examinations could therefore contribute to the obstetric care of these women. Preeclampsia and HELLP syndrome should be routinely checked for in all pregnant women with acute abdominal pain. PMID- 12369663 TI - The genetic sonogram: a method of risk assessment for Down syndrome in the second trimester. AB - OBJECTIVE: To determine the risk of Down syndrome in fetuses with sonographic markers using the Bayes theorem and likelihood ratios. METHODS: We prospectively evaluated the midtrimester sonographic features of fetuses with Down syndrome and compared them with euploid fetuses. Patients were referred for an increased risk of aneuploidy and evaluated for the presence of structural defects, a nuchal fold, short long bones, pyelectasis, an echogenic intracardiac focus, and hyperechoic bowel. All fetuses underwent amniocentesis at the time of sonographic assessment. The sensitivity, specificity, and likelihood ratios for markers were calculated both as nonisolated and isolated findings. RESULT: There were 164 fetuses with Down syndrome and 656 euploid fetuses. The presence of any marker resulted in sensitivity for the detection of Down syndrome of 80.5% with a false positive rate of 12.4%. The absence of any markers conferred a likelihood ratio of 0.2, decreasing the risk of Down syndrome by 80%. As an isolated marker, the nuchal fold had an "infinite" likelihood ratio for Down syndrome; a short humerus had a likelihood ratio of 5.8, whereas structural anomalies had a likelihood ratio of 3.3. Other isolated markers had low likelihood ratios because of the higher prevalence in the unaffected population. The likelihood ratios for the presence of 1, 2, and 3 of any of the markers were 1.9, 6.2, and 80, respectively. CONCLUSIONS: Although an isolated marker with a low likelihood ratio may not increase a patient's risk of Down syndrome, the presence of such a marker precludes reducing the risk of aneuploidy. Clusters of markers appear to confer a higher risk. PMID- 12369664 TI - The normal offset of the tricuspid septal leaflet in the fetus. AB - OBJECTIVE: To quantify the normal distance between the insertion of the medial leaflets of the mitral valve and tricuspid valve in the fetal heart. This mitral valve-tricuspid valve distance was compared with the distance from known cases of Ebstein anomaly. METHODS: An apical 4-chamber view was obtained at end diastole in fetuses between 18 and 41 weeks' gestation. Calipers were placed parallel to the ventricular septum, with 1 caliper on the medial insertion of the mitral valve and a second caliper on the medial insertion of the tricuspid valve. The distance recorded was plotted against gestational age. Statistical analysis was performed by descriptive and linear regression techniques. RESULTS: One hundred forty-five fetuses were studied. The mean +/- SD mitral valve-tricuspid valve distance in the second trimester was 2.8 +/- 0.9 mm with a range of 1.2 to 5.0 mm; in the third trimester it was 4.6 +/- 1.1 mm with a range of 2.2 to 6.9 mm. Regression analysis showed that with each 1-week increase in gestational age, there was an increase of 0.15 mm in separation between the medial leaflets of the mitral valve and tricuspid valve (beta = 0.15 +/- 0.011). CONCLUSIONS: A positive correlation between mitral valve-tricuspid valve distance and advancing gestational age was found. The reference range described allows for the identification of a fetal heart with normal variation in the mitral valve tricuspid valve distance. Further downward displacement of the medial tricuspid cusp suggests the possibility of Ebstein anomaly. PMID- 12369665 TI - Complex pelvic mass as a target of evaluation of vessel distribution by color Doppler sonography for the diagnosis of adnexal malignancies: results of a multicenter European study. AB - OBJECTIVE: To compare the diagnostic accuracy of gray scale sonography and color Doppler imaging in the differential diagnosis of adnexal malignancies from benign complex pelvic masses in a multicenter prospective study. METHODS: The study was performed as a collaborative work at 3 European university departments of obstetrics and gynecology. A total of 826 complex pelvic masses on which transvaginal sonography and evaluation of cancer antigen 125 plasma concentrations were performed before surgical exploration were included in the study. The scanning procedure was the same in the 3 institutions. An adnexal mass was first studied in gray scale sonography, and a probable histologic type was predicted. Second, solid excrescences or solid portions of the tumor were evaluated for vascular flow with color Doppler sonography (conventional or power). A mass was graded malignant if flow was shown within the excrescences or solid areas and benign if there was no flow. The overall agreement between the test result and the actual outcome was calculated by kappa statistics. RESULTS: Color Doppler evaluation was more accurate in the diagnosis of adnexal malignancies in comparison with gray scale sonography (kappa = 0.82 and 0.65, respectively) because of significantly higher specificity (0.94 versus 0.84; P < .001). The evaluation of the cancer antigen 125 plasma concentration did not seem to increase the accuracy of either method. CONCLUSIONS: The evaluation of vessel distribution by color Doppler sonography in complex adnexal cysts seems to increase the diagnostic accuracy of gray scale sonography in the detection of adnexal malignancies in a large study population. PMID- 12369666 TI - Correlation between sonographic and pathologic findings in muscle injury: experimental study in the rabbit. AB - OBJECTIVE: To evaluate the serial sonographic findings of experimental muscle injury and to correlate those findings with the pathologic findings at each period. METHODS: A muscle injury was artificially inflicted in 18 legs of 9 rabbits. Sonographic follow-up images were obtained 1, 3, and 7 days and 2, 3, 4, 6, and 8 weeks after infliction of muscle injury. Pathologic specimens were obtained for comparison with sonographic findings on each date. RESULTS: There was high echogenicity in the central portion after 3 days. It changed to low echogenicity after 7 days. There were linear echogenic lines in the central portion after 4 weeks, and these lines increased in number after 6 weeks. The peripheral portion exhibited high echogenicity up to 7 days. This high echogenicity was normalized after 2 weeks. Pathologic specimens showed hematomas, fibrin, and necrotic muscle fibers in the central portion up to 3 days after injury. Fibrin occupied most of the central portion after 2 weeks. Regenerating muscle fibers appeared within the fibrin clot after 4 weeks, and they became more prominent after 6 weeks. Necrotic muscle fibers, hemorrhage, and inflammatory cells of the peripheral portion disappeared after 2 weeks. CONCLUSIONS: Serial sonography of muscle injury was well correlated with the pathologic specimen up to 7 days after injury. After 4 weeks, regenerating muscle fibers showed a good correlation with the finding on sonography. Therefore, sonography can be helpful in diagnosis of muscle injury as well as in evaluation of the regenerating muscle fibers. PMID- 12369667 TI - Contrast-enhanced Doppler perfusion index: clinical and experimental evaluation. AB - OBJECTIVE: To assess the potential of the power Doppler signal intensity rate of enhancement due to contrast agent wash-in for assessment of hepatic hemodynamics. METHODS: With the use of standardized settings, power Doppler sonography was performed before and after administration of a contrast agent. Video-recorded examinations were digitized for offline analysis on a personal computer. The temporal changes of the power Doppler signal intensity were quantified to provide contrast agent wash-in curves. The contrast-enhanced Doppler perfusion index was defined by the ratio of the wash-in gradient of the hepatic artery and portal vein as contrast-enhanced Doppler perfusion index = hepatic artery gradient/(hepatic artery gradient + portal vein gradient). The contrast-enhanced Doppler perfusion index was evaluated at 4 contrast agent doses in each of 14 patients with liver metastases and 3 patients with hemangiomas. An in vitro flow model was used to determine the relationships between the power Doppler rate of enhancement and flow in vessels of 4, 8, and 12 mm in diameter. RESULTS: In vivo, there was a significantly higher (P < .0001) mean contrast enhanced Doppler perfusion index in patients with liver metastases (mean, 0.59; 95% confidence interval, 0.54-0.63), compared with patients with hemangiomas (mean, 0.33; 95% confidence interval, 0.24-0.41). The corresponding coefficients of variations were 25% for patients with liver metastases and 31% for patients with hemangiomas. In vitro, the power Doppler rate of enhancement was proportional to flow speed and independent of vessel diameter. CONCLUSIONS: Measurement of the contrast-enhanced Doppler perfusion index may have potential in assessment of hepatic hemodynamics and focal liver disease. PMID- 12369668 TI - Guidance of retrobulbar injection with real-time tomographic reflection. AB - OBJECTIVE: Retrobulbar and peribulbar injections are common ophthalmologic procedures used to deliver anesthetics and other medications for ophthalmic therapy and surgery. These injections, typically performed without any type of guidance, can lead to complications that are rare but visually devastating. The needle may penetrate the optic nerve, perforate the globe, or disperse toxic quantities of drugs intraocularly, causing major visual loss. Sonographic guidance may increase the accuracy of the needle placement, thereby decreasing the incidence of complications. However, difficulties arise in coordinating the relative location of the image, the needle, and the patient. Real-time tomographic reflection is a new method for in situ visualization of sonographic images, permitting direct hand-eye coordination to guide invasive instruments beneath the surface of the skin. METHOD: In this preliminary study, real-time tomographic reflection was used to visualize the eye and surrounding anatomic structures in a cadaver during a simulated retrobulbar injection. RESULT: The needle tip was easily followed as it was advanced into the retrobulbar space. CONCLUSIONS: The images presented in this preliminary study show the use of real time tomographic reflection to visualize insertion of an invasive instrument into the human body. PMID- 12369669 TI - Transvaginal and transabdominal extended field-of-view (EFOV) and power doppler EFOV sonography in gynecology: advantages and applications. AB - OBJECTIVE: To evaluate the possible advantages, applications, and usefulness of real-time transabdominal and transvaginal extended field-of-view sonography combined with power Doppler sonography in gynecology. METHODS: A series of 63 gynecologic patients, referred for preoperative sonographic examination and for whom the examiner thought that extended field-of-view sonography might be helpful in imaging pathologic findings, were selected. Patients were examined with conventional vaginal and abdominal B-mode sonography, extended field-of-view sonography, and power Doppler extended field-of-view sonography. A sonographic system with 3.5- to 7-MHz transducers was used to study and document pathologic findings. RESULTS: Extended field-of-view sonography provided a superior overview of pathologic findings and topography by creating a single image showing the relationship to reference structures. The combination of power Doppler extended field-of-view sonography provided additional information on the perfusion pattern in huge masses. In comparison with conventional sonographic images, the extended field-of-view sonographic images were easier to interpret by the referring or nonexamining physician. A list of proposed gynecologic applications for the use of extended field-of-view sonography was compiled. CONCLUSIONS: The extended field-of-view and power Doppler extended field-of-view sonographic technique provides documentation of surroundings, topographic orientation, and perfusion patterns in large pelvic masses and findings that exceed the limitations of the conventional sonographic sector. The clinical applications and advantages of extended field-of-view and power Doppler extended field-of-view imaging in gynecologic sonography are illustrated. PMID- 12369670 TI - The human fetal venous system: normal embryologic, anatomic, and physiologic characteristics and developmental abnormalities. AB - OBJECTIVE: The introduction of high-resolution ultrasonography combined with color-coded Doppler imaging offered a breakthrough in the evaluation of the human fetal venous system, considerably enhancing our understanding of fetal venous circulation in normal physiologic conditions, as well as providing us the ability to study circulatory changes in abnormal circumstances. The purpose of this study was to describe the normal anatomic development and complex of anomalies of the human fetal venous system and to review recently published series of these anomalies. METHODS: Normal embryologic and anatomic development is described. An English language literature search of recent MEDLINE listings was performed to glean data from recently published series reporting prenatal diagnosis of the various anomalies and their associated malformations. RESULTS: Anomalies of the human fetal venous system occur sporadically, often associated with cardiac or other malformations. The pathophysiologic mechanisms leading to abnormal in utero development of the human venous system remain largely undetermined. On the basis of the type of vein involved, embryologic precursor, and etiologic correlation (primary or secondary), classification into 4 major groups is described. CONCLUSIONS: Prenatal evaluation of fetuses found to have anomalies of the venous system should include a careful search for cardiac anomalies, including pulmonary venous drainage, and a detailed anatomic survey of the umbilical, portal, hepatic, and ductal systems to determine aberrant communication and, if possible, to discover clues to systemic diseases or thromboembolic phenomena. PMID- 12369671 TI - Ultrasonography of the accessory nerve: normal and pathologic findings in cadavers and patients with iatrogenic accessory nerve palsy. AB - OBJECTIVE: To determine feasibility of ultrasonography in detecting the normal accessory nerve as well as pathologic changes in cases of accessory nerve palsy. METHODS: Four patients with accessory nerve palsy were investigated by ultrasonography. Three cases of accessory nerve palsy after lymph node biopsy and neck dissection were primarily diagnosed on the basis of ultrasonography using a 5- to 12-MHz linear transducer. In addition, we performed ultrasonography in 3 cadaveric specimens to show the feasibility of detecting the accessory nerve. RESULT: Nerve transection (n = 2), scar tissue (n = 1), and atrophy of the trapezius muscle (n = 4) were confirmed by electroneurographic testing and surgical nerve inspection. In 1 case in which a patient had a whiplash injury with accessory nerve palsy, ultrasonography showed atrophy of the trapezius muscle with a normal nerve appearance. CONCLUSIONS: Ultrasonography allows visualization of the normal accessory nerve as well as changes after accessory nerve palsy. PMID- 12369672 TI - Sonographic findings of acute urinary retention secondary to an impacted pelvic mass. AB - OBJECTIVE: To describe the sonographic findings in a series of cases of acute urinary retention due to an impacted pelvic mass. METHODS: The anatomic changes of the lower urinary tract in 6 patients with impacted pelvic masses and acute urinary retention (3 cases of an impacted uterine leiomyoma and 3 cases of a retroverted gravid uterus) were evaluated with transabdominal and transvaginal sonography. RESULTS: When patients were in the supine position, the impacted pelvic masses displaced the cervix superiorly and anteriorly, compressing the lower bladder, leading to obstruction of the internal urethral orifice. During straining, there was no limitation of urethral mobility, but the increased abdominal pressure further compressed the lower bladder. When the subjects stood, the lower bladder filled with urine. There was descent of the bladder neck, and obstruction was relieved. CONCLUSIONS: Acute urinary retention in cases of an impacted pelvic mass is caused by a displaced cervix compressing the lower bladder, obstructing the internal urethral orifice. The urethra itself is not compressed or distorted. PMID- 12369673 TI - Preoperative sonographic diagnosis of primary fallopian tube carcinoma. PMID- 12369674 TI - Sonographic diagnosis of caudal regression in the first trimester of pregnancy. PMID- 12369675 TI - Prenatal sonographic findings of fetal megacalycosis. PMID- 12369676 TI - AIUM standard for the performance of an ultrasound examination of the abdomen or retroperitoneum. American Institute of Ultrasound in Medicine. PMID- 12369677 TI - AIUM standard for documentation of an ultrasound examination. American Institute of Ultrasound in Medicine. PMID- 12369678 TI - Breed variations in histopathologic features of chronic severe otitis externa in dogs: 80 cases (1995-2001). AB - OBJECTIVE: To compare pathologic changes of the horizontal ear canal associated with chronic severe otitis externa between Cocker Spaniels and dogs of other breeds. DESIGN: Retrospective study. ANIMALS: 80 dogs with severe otitis externa that required total ear canal ablation with lateral bulla osteotomy. PROCEDURE: Medical records were reviewed for breed, sex, and age at time of surgery. Histologic specimens from the horizontal ear canal were evaluated by a single examiner for overall tissue response pattern and scored for sebaceous gland hyperplasia, ceruminous gland hyperplasia, ceruminous gland ectasia, fibrosis, pigment-laden macrophages, and osseous metaplasia. RESULTS: 48 of 80 (60%) dogs were Cocker Spaniels. Thirty-five of 48 (72.9%) Cocker Spaniels had a predominately ceruminous tissue response pattern; only 9 of 32 (28.1 %) dogs of other breeds had the same pattern. Other breeds most commonly had a pattern dominated by fibrosis (n = 13 [40.6%]); fibrosis was the predominant pattern in only 4 of 48 (8.3%) Cocker Spaniels. Discriminant analysis and K-means clustering of 4 histopathologic criteria correctly classified 75% of the dogs as Cocker Spaniels or all other breeds. CONCLUSIONS AND CLINICAL RELEVANCE: Cocker Spaniels are at increased risk for chronic severe otitis externa requiring total ear canal ablation with lateral bulla osteotomy, indicating that earlier and more aggressive management of the primary otitis externa and secondary inflammation is warranted in this breed. Cocker Spaniels with chronic severe otitis externa have distinct differences in pathologic characteristics of the horizontal ear canal, compared with other breeds. PMID- 12369679 TI - Sensitivity and specificity of western blot testing of cerebrospinal fluid and serum for diagnosis of equine protozoal myeloencephalitis in horses with and without neurologic abnormalities. AB - OBJECTIVE: To determine sensitivity and specificity of western blot testing (WBT) of CSF and serum for diagnosis of equine protozoal myeloencephalitis (EPM) in horses with and without neurologic abnormalities. DESIGN: Prospective investigation. ANIMALS: 65 horses with and 169 horses without neurologic abnormalities. PROCEDURE: CSF and serum from horses submitted for necropsy were tested for Sarcocystis neurona-specific antibody with a WBT. Results of postmortem examination were used as the gold standard against which results of the WBT were compared. RESULTS: Sensitivity of WBT of CSF was 87% for horses with and 88% for horses without neurologic abnormalities. Specificity of WBT of CSF was 44% for horses with and 60% for horses without neurologic abnormalities. Regardless of whether horses did or did not have neurologic abnormalities, sensitivity and specificity of WBT of serum were not significantly different from values for WBT of CSF. Ninety-four horses without EPM had histologic evidence of slight CNS inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: The low specificity of WBT of CSF indicated that it is inappropriate to diagnose EPM on the basis of a positive test result alone because of the possibility of false-positive test results. The high sensitivity, however, means that a negative result is useful in ruling out EPM. There was no advantage in testing CSF versus serum in horses without neurologic abnormalities. Slight CNS inflammation was common in horses with and without S neurona-specific antibodies in the CSF and should not be considered an indication of CNS infection with S neurona. PMID- 12369680 TI - Use of a pool-raft system for recovery of horses from general anesthesia: 393 horses (1984-2000). AB - OBJECTIVE: To describe the pool-raft recovery system protocol and to evaluate the clinical outcome in horses that underwent recovery from general anesthesia using this system. DESIGN: Retrospective study. ANIMALS: 393 horses that underwent recovery from general anesthesia in the pool-raft system. PROCEDURE: Anesthetic records were examined from horses recovered from anesthesia in the pool-raft system between January 1984 and December 2000. Complete medical records of horses were examined when available. Information regarding the anesthetic and recovery period was recorded. Horses first recovered from general anesthesia in the pool raft and, once awake, were transported to a recovery stall and lowered to the floor in a standing position. RESULTS: 351 horses underwent 1 pool-raft recovery, and 42 horses underwent multiple pool-raft recoveries. Most horses were recovered from general anesthesia within the pool-raft system to safeguard repair of a major orthopedic injury. During 471 pool-raft recoveries, 34 (7%) horses had complications within the recovery pool and 62 (13%) had complications within the recovery stall. Deaths resulted from complete failure of internal fixation, pulmonary dysfunction, or a combination of pulmonary dysfunction and fixation failure in 2% (10/471) of horses that underwent pool-raft recoveries. CONCLUSIONS AND CLINICAL RELEVANCE: The pool-raft system is a good option for recovery from general anesthesia. Although not a fail-safe system, it appears to decrease the complications of recovering horses in a high-risk category. Potential disadvantages of this system are added expense and manpower necessary in building, maintenance, and usage, as well as size limitations of the raft itself. PMID- 12369681 TI - Medetomidine, ketamine, and sevoflurane for anesthesia of injured loggerhead sea turtles: 13 cases (1996-2000). AB - OBJECTIVE: To determine safety and efficacy of an anesthetic protocol incorporating medetomidine, ketamine, and sevoflurane for anesthesia of injured loggerhead sea turtles. DESIGN: Retrospective study. ANIMALS: 13 loggerhead sea turtles. PROCEDURE: Anesthesia was induced with medetomidine (50 microg/kg [22.7 microg/lb], IV) and ketamine (5 mg/kg (2.3 mg/lb], IV) and maintained with sevoflurane (0.5 to 2.5%) in oxygen. Sevoflurane was delivered with a pressure limited intermittent-flow ventilator. Heart rate and rhythm, end-tidal partial pressure of CO2, and cloacal temperature were monitored continuously; venous blood gas analyses were performed intermittently. Administration of sevoflurane was discontinued 30 to 60 minutes prior to the end of the surgical procedure. Atipamezole (0.25 mg/kg [0.11 mg/lb], IV) was administered at the end of surgery. RESULTS: Median induction time was 11 minutes (range, 2 to 40 minutes; n = 11). Median delivered sevoflurane concentrations 15, 30, 60, and 120 minutes after intubation were 2.5 (n = 12), 1.5 (12), 1.25 (12), and 0.5% (8), respectively. Heart rate decreased during surgery to a median value of 15 beats/min (n = 11). End-tidal partial pressure of CO2 ranged from 2 to 16 mm Hg (n = 8); median blood gas values were within reference limits. Median time from atipamezole administration to extubation was 14 minutes (range, 2 to 84 minutes; n = 7). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a combination of medetomidine and ketamine for induction and sevoflurane for maintenance provides safe, effective, controllable anesthesia in injured loggerhead sea turtles. PMID- 12369682 TI - Campaign to outlaw sow housing in Florida advances. PMID- 12369683 TI - New companion animal tumor registry in the works. PMID- 12369684 TI - Food safety, FMD, and animal welfare. PMID- 12369685 TI - Strongly protests avian influenza virus response. PMID- 12369686 TI - Chlorpyrifos poisoning over-looked as potential diagnosis. PMID- 12369687 TI - Readers disturbed about letter on free-roaming cats. PMID- 12369688 TI - Readers disturbed about letter on free-roaming cats. PMID- 12369689 TI - Readers disturbed about letter on free-roaming cats. PMID- 12369690 TI - Comments on dosing lufenuron. PMID- 12369691 TI - JAVMA cover receives complaints. PMID- 12369692 TI - JAVMA cover receives complaints. PMID- 12369693 TI - Client confidence in veterinarians: how can it be sustained? PMID- 12369694 TI - What is your diagnosis? Several smoothly marginated, mineralized lesions cranial, lateral and caudal to the distal portion of the left femur. PMID- 12369695 TI - ECG of the month. Atrial fibrillation. PMID- 12369696 TI - Mechanisms of action and potential uses of hyaluronan in dogs with osteoarthritis. PMID- 12369697 TI - Healthy People 2010--new opportunities for veterinary medicine in the 21st century. PMID- 12369698 TI - Individual animal medicine and animal production skills expected of entry-level veterinarians in bovine practice. PMID- 12369699 TI - Surgery, anesthesia, and restraint skills expected of entry-level veterinarians in bovine practice. PMID- 12369700 TI - Use of vagal nerve stimulation as a treatment for refractory epilepsy in dogs. AB - OBJECTIVE: To evaluate safety and efficacy of vagal nerve stimulation in dogs with refractory epilepsy. DESIGN: Placebo-controlled, double-masked, crossover study. ANIMALS: 10 dogs with poorly controlled seizures. PROCEDURE: A programmable pacemaker-like device designed to deliver intermittent stimulation to the left cervical trunk of the vagus was surgically implanted in each dog. Dogs were assigned randomly to two 13-week test periods, 1 with nerve stimulation and 1 without nerve stimulation. Owners recorded data on seizure frequency, duration, and intensity, as well as adverse effects. RESULTS: No significant difference in seizure frequency, duration, or severity was detected between overall 13-week treatment and control periods. During the final 4 weeks of the treatment period, a significant decrease in mean seizure frequency (34.4%) was detected, compared with the control period. Complications included transient bradycardia, asystole, and apnea during intraoperative device testing, and seroma formation, subcutaneous migration of the generator, and transient Horner's syndrome during the 14-day period between surgery and suture removal. No adverse effects of stimulation were detected, and most owners were satisfied with the treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Vagal nerve stimulation is a potentially safe approach to seizure control that appears to be efficacious in certain dogs and should be considered a possible treatment option when antiepileptic medications are ineffective. PMID- 12369701 TI - Retroperitoneal fibrosis in four cats following renal transplantation. AB - Four cats developed fibrosis within the retroperitoneal space following renal transplantation. In human transplant patients, retroperitoneal fibrosis is an uncommon complication following surgery and may be secondary to operative trauma, infection, deposition of foreign material in the operative field, urinary extravasation, and perirenal hemorrhage caused by trauma to the allograft. Possible causes of fibrosis in the cats of this report include abdominal inflammation associated with allograft rejection, pyelonephritis, and septic peritoneal effusion. All of the cats of this report were readmitted to the veterinary teaching hospital following renal transplantation because of recurrence of azotemia 1 to 5 months after transplantation. Abdominal ultrasonography revealed a 2- to 4-mm-thick capsule surrounding the allograft in 2 of 4 cats, hydronephrosis in 4 cats, and hydroureter proximally in 2 cats. An exploratory laparotomy was performed in all cats to remove the fibrotic tissue causing the ureteral obstruction. Normal renal function was restored in all cats following surgery. Histologic evaluation of biopsy specimens revealed smooth muscle (3 cats) and fibrous connective tissue (4). All 4 cats, regardless of the cause, responded well to surgical resection of the scar tissue that was causing a ureteral obstruction. None of the cats had recurrence of obstruction following surgery. PMID- 12369702 TI - Use of thoracoscopy to determine the etiology of pleural effusion in dogs and cats: 18 cases (1998-2001). AB - OBJECTIVE: To assess use of thoracoscopy to determine causes of pleural effusion in dogs and cats. DESIGN: Retrospective study. ANIMALS: 15 dogs and 3 cats with pleural effusion. PROCEDURE: Medical records were reviewed from 1998 to 2001 for dogs and cats that had exploratory thoracoscopy, biopsy, and histologic analysis to determine the etiology of pleural effusion. Intraoperative and postoperative complications were recorded. Surgical biopsy specimens were evaluated for quantity and quality for providing a histologic diagnosis. RESULTS: Biopsy specimens were deemed adequate in quantity and quality to render a histologic diagnosis in all animals. Etiology of the effusion was neoplasia in 8 animals and non-neoplastic pleuritis in 10 animals. Median survival time of animals with neoplasia was 15 days, whereas those with inflammatory diseases had median survival time of > 785 days. Postoperative pneumothorax was encountered in 2 animals subsequent to pulmonary biopsy. No other major complications were recorded. CONCLUSIONS AND CLINICAL RELEVANCE: Thoracoscopy is a diagnostic option that provides excellent viewing of intrathoracic structures and adequate biopsy specimens with minimal complications. This technique provides a less invasive alternative to thoracotomy for evaluating the etiology of pleural effusion. PMID- 12369703 TI - Influence of vestibulovaginal stenosis, pelvic bladder, and recessed vulva on response to treatment for clinical signs of lower urinary tract disease in dogs: 38 cases (1990-1999). AB - OBJECTIVE: To determine influence of vestibulovaginal stenosis, pelvic bladder, and recessed vulva on response to treatment for clinical signs of lower urinary tract disease in dogs. DESIGN: Retrospective study. ANIMALS: 38 spayed female dogs. PROCEDURE: Medical records and client follow-up were reviewed for dogs evaluated via excretory urography because of clinical signs of lower urinary tract disease. Clinical signs, results of radiography, and response to surgical or medical treatment were analyzed. RESULTS: Clinical signs included urinary tract infection (n = 24), urinary incontinence (20), vaginitis (11), pollakiuria or stranguria (10), and perivulvar dermatitis (4). Vaginocystourethrographic findings included vestibulovaginal stenosis (n = 28), pelvic bladder (17), and ureteritis or pyelonephritis (4). Ten dogs had a vestibulovaginal ratio of < 0.20 (severe stenosis), 9 dogs had a ratio of 0.20 to 0.25 (moderate stenosis), 9 dogs had a ratio of 0.26 to 0.35 (mild stenosis), and 10 dogs had a ratio of > 0.35 (anatomically normal). Lower urinary tract infection, incontinence, and pelvic bladder were not associated with response to treatment for recessed vulva. Vestibulovaginal stenosis with a ratio < 0.20 was significantly associated negatively with response to treatment. Dogs without severe vestibulovaginal stenosis that received vulvoplasty for a recessed vulva responded well to treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Vestibulovaginal stenosis is likely an important factor in dogs with vestibulovaginal ratio < 0.20. Vaginectomy or resection and anastomosis should be considered in dogs with severe vestibulovaginal stenosis and signs of lower urinary tract disease. PMID- 12369704 TI - Ebb and tide of glucokinase. PMID- 12369705 TI - Substrate-induced nuclear export and peripheral compartmentalization of hepatic glucokinase correlates with glycogen deposition. AB - Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopy and quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60 min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface. PMID- 12369706 TI - Human C-peptide dose dependently prevents early neuropathy in the BB/Wor-rat. AB - In order to explore the neuroprotective and cross-species activities of C-peptide on type 1 diabetic neuropathy, spontaneously diabetic BB/W-rats were given increasing doses of human recombinant C-peptide (hrC-peptide). Diabetic rats received 10, 100, 500, or 1000 microg of hrC-peptide/kg body weight/day from onset of diabetes. After 2 months of hrC-peptide administration, 100 microg and greater doses completely prevented the nerve conduction defect, which was associated with a significant but incomplete prevention of neural Na+/K+-ATPase activity in diabetic rats with 500 microg or greater C-peptide replacement. Increasing doses of hrC-peptide showed increasing prevention of early structural abnormalities such as paranodal swelling and axonal degeneration and an increasing frequency of regenerating sural nerve fibers. We conclude that hrC peptide exerts a dose dependent protection on type 1 diabetic neuropathy in rats and that this effect is probably mediated by the partially conserved sequence of the active C-terminal pentapeptide. PMID- 12369707 TI - The diabetic nephropathy and the development of hypertension in rats. AB - The present study was designed to examine the development of hypertension in diabetic rats treated with streptozotocin (STZ, 1 mg/g bw). The rats were studied at 3, 6, 9, 12 and 15 weeks. From the third week the rats were divided in diabetic rats according their glycemias and controls, along 15 weeks. After the third week a group of rats showed increased urinary protein excretion (93, 134, 155 and 191%) compared to controls. In this group of rats the urinary kallikrein excretion was lower than control and the systolic blood pressure became significantly elevated between 3 and 6 weeks and persisted up to 15 weeks. On the other hand a group of diabetic rats were normotensive with urinary protein excretion similar to controls and urinary kallikrein lower compared to control but significantly higher compared diabetic hypertensive rats. These data suggest that the association of progressive diabetic nephropathy with abnormal endothelium-dependent vasodilation may produce a high prevalence of hypertensive diabetes. PMID- 12369708 TI - Brain-derived neurotrophic factor regulates energy expenditure through the central nervous system in obese diabetic mice. AB - It has been previously demonstrated that brain-derived neurotrophic factor (BDNF) regulates glucose metabolism and energy expenditure in rodent diabetic models such as C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice. Central administration of BDNF has been found to reduce blood glucose in db/db mice, suggesting that BDNF acts through the central nervous system. In the present study we have expanded these investigations to explore the effect of central administration of BDNF on energy metabolism. Intracerebroventricular administration of BDNF lowered blood glucose and increased pancreatic insulin content of db/db mice compared with vehicle treated pellet pair-fed db/db mice. While body temperatures of the pellet pair fed db/db mice given vehicle were reduced because of restricted food supply in this pair-feeding condition, BDNF treatment remarkably alleviated the reduction of body temperature suggesting the enhancement of thermogenesis. BDNF enhanced norepinephrine turnover and increased uncoupling protein-1 mRNA expression in the interscapular brown adipose tissue. Our evidence indicates that BDNF activates the sympathetic nervous system via the central nervous system and regulates energy expenditure in obese diabetic animals. PMID- 12369709 TI - Characterization of oxidative stress in various tissues of diabetic and galactose fed rats. AB - Rats fed a galactose-rich diet have been used for several years as a model for diabetes to study, particularly in the eye, the effects of excess blood hexoses. This study sought to determine the utility of galactosemia as a model for oxidative stress in extraocular tissues by examining biomarkers of oxidative stress in galactose-fed rats and experimentally-induced diabetic rats. Sprague Dawley rats were divided into four groups: experimental control; streptozotocin induced diabetic; insulin-treated diabetic; and galactose-fed. The rats were maintained on these regimens for 30 days, at which point the activities of catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase, as well as levels of lipid peroxidation and reduced and oxidized glutathione were determined in heart, liver, and kidney. This study indicates that while there are some similarities between galactosemic and diabetic rats in these measured indices of oxidative stress (hepatic catalase activity levels and hepatic and renal levels of oxidized glutathione in both diabetic and galactosemic rats were significantly decreased when compared to normal), overall the galactosemic rat model is not closely parallel to the diabetic rat model in extra-ocular tissues. In addition, several effects of diabetes (increased hepatic glutathione peroxidase activity, increased superoxide dismutase activity in kidney and heart, decreased renal and increased cardiac catalase activity) were not mimicked in galactosemic rats, and glutathione concentration in both liver and heart was affected in opposite ways in diabetic rats and galactose-fed rats. Insulin treatment reversed/prevented the activity changes in renal and cardiac superoxide dismutase, renal and cardiac catalase, and hepatic glutathione peroxidase as well as the hepatic changes in lipid peroxidation and reduced and oxidized glutathione, and the increase in cardiac glutathione. Thus, prudence should be exercised in the use of experimentally galactosemic rats as a model for diabetes until the correspondence of the models has been more fully characterized. PMID- 12369710 TI - Leptin and its relation to obesity and insulin in the SHR/N-corpulent rat, a model of type II diabetes mellitus. AB - The spontaneously hypertensive/NIH-corpulent (SHR/N-cp) rat is a genetic animal model that exhibits obesity, metabolic features of hyperinsulinemia, hyperglycemia, and hyperlipidemia, which are characteristic of type II diabetes and mild hypertension. To determine the role of leptin, the protein product of the ob gene, in the development of obesity and diabetes in this model, we measured steady-state circulating levels of leptin in obese and lean SHR/N-cp rats and examined the relation between plasma leptin levels and metabolic variables at the stage of established obesity in these animals. Mean fasting plasma leptin concentration was 8-fold higher in obese than in lean rats (p < 0.01). This was associated with a 6-fold elevation in plasma insulin in the obese group. Fasting levels of plasma glucose, cholesterol, and triglyceride were all significantly higher in obese rats than in lean controls. Spearman correlation analysis showed a significant positive correlation between plasma leptin concentration and body weight among the animals (r = 0.73, p < 0.01). Similarly, plasma insulin concentration was significantly correlated with BW in all animals (r = 0.54, p < 0.05). There was also a significant positive correlation between plasma leptin and plasma insulin in the entire group (r = 0.70, p < 0.01). However, this relationship was significant only for lean rats but not for obese rats (r = 10.59, p < 0.05 for lean rats, and r = 0.23, p = NS, for obese rats). Plasma leptin also correlated positively with fasting plasma glucose (r = 0.75, p < 0.05), total cholesterol (r = 0.63, p < 0.05), and triglyceride (r = 0.67, p < 0.05). The marked elevation of plasma leptin in obese SHR/N-cp rats suggests that obesity in this animal model is related to up-regulation of the ob gene. Circulating leptin appears to be one of the best biological markers of obesity and that hyperleptinemia is closely associated with several metabolic risk factors related to insulin resistance in the diabesity syndrome. PMID- 12369711 TI - Effects of aminoguanidine on glomerular basement membrane thickness and anionic charge in a diabetic rat model. AB - We investigated the effect of aminoguanidine (AG) administration on GBM thickness, glomerular heparan sulfate (HS) content, and urinary albumin and HS excretion in diabetic rats. After induction of diabetes, female Wistar rats were divided into 2 groups: Group AGDM (n = 11) received 1 g/L aminoguanidine bicarbonate in drinking water, group DC (n = 12) was given only tap water. Control rats received AG (group AGH, n = 8) or tap water (group HC, n = 8). At the end of a period of 8 weeks, urinary albumin and glycosaminoglycan (GAG) excretion was detected. GBM heparan sulfate distribution and count was determined under the electron microscope. The AGDM group had lower urinary albumin and GAG excretion than diabetic controls. GBM thickness was increased in diabetic rats compared to groups of AGDM and HC. In AGDM group alcian blue stained particle distribution and count in the GBM was similar to healthy controls. In conclusion AG prevents the decrease of anionic charged molecules in the GBM and GBM thickening. This can be one of the mechanisms by which AG decreases albuminuria in diabetic rats. PMID- 12369712 TI - Role of protein kinase C, PI3-kinase and tyrosine kinase in activation of MAP kinase by glucose and agonists of G-protein coupled receptors in INS-1 cells. AB - MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase and cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [32P]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 microM PD 098059 (IC50 = 5 microM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton ("downregulation") of PKC by a long term (22 h) pretreatment with 1 microM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 microM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 microM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [3H]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but not for its insulin secretory response with respect to major initiators and modulators of insulin release. The data indicate that MAP kinase is active and under the control of MAP kinase. PKC is upstream of a genistein-sensitive tyrosine kinase and probably downstream of a PI3-kinase in INS-1 cells. PMID- 12369713 TI - Insulin and glucagon impairments in relation with islet cells morphological modifications following long term pancreatic duct ligation in the rabbit--a model of non-insulin-dependent diabetes. AB - Plasma levels of glucose, insulin and glucagon were measured at various time intervals after pancreatic duct ligation (PDL) in rabbits. Two hyperglycemic periods were observed: one between 15-90 days (peak at 30 days of 15.1 +/- 1.2 mmol/l, p < 0.01), and the other at 450 days (11.2 +/- 0.5 mmol/l, p < 0.02). The first hyperglycemic episode was significantly correlated with both hypoinsulinemia (41.8 +/- 8 pmol/l, r = -0.94, p < 0.01) and hyperglucagonemia (232 +/- 21 ng/l, r = 0.95, p < 0.01). However, the late hyperglycemic phase (450 days), which was not accompanied by hypoinsulinemia, was observed after the hyperglucagonemia (390 days) produced by abundant immunostained A-cells giving rise to a 3-fold increase in pancreatic glucagon stores. The insulin and glucagon responses to glucose loading at 180, 270 and 450 days reflected the insensitivity of B- and A-cells to glucose. The PDL rabbit model with chronic and severe glycemic disorders due to the predominant role of glucagon mimicked key features of the NIDDM syndrome secondary to exocrine disease. PMID- 12369714 TI - Placental transfer of lactate, glucose and 2-deoxyglucose in control and diabetic Wistar rats. AB - Placental transfer of lactate, glucose and 2-deoxyglucose was examined employing the in situ perfused placenta. Control and streptozotocin induced diabetic Wistar rats were infused with [U-14C]-glucose and [3H]-2-deoxyglucose (2DG). The fetal side of the placenta was perfused with a cell free medium and glucose uptake was calculated in the adjacent fetuses. Despite the 5-fold higher maternal plasma glucose concentration in the diabetic dams the calculated fetal glucose metabolic index was not significantly different between the 2 groups. Placental blood flow was reduced in the diabetic animals compared with controls but reduction of transfer of [U-14C]-glucose and [3H]-2-deoxyglucose and endogenously derived [14C]-Lactate to the fetal compartment, could not be accounted for by reduced placental blood flow alone. There was no significant net production or uptake of lactate into the perfusion medium that had perfused the fetal side of the placenta in either group. The plasma lactate levels in the fetuses adjacent to the perfused placenta were found to be higher than in the maternal plasma and significantly higher in the fetuses of the diabetic group compared with control group. In this model the in-situ perfused placenta does not secrete significant quantities of lactate into the fetal compartment in either the control or diabetic group. PMID- 12369715 TI - Leptin in children with newly diagnosed type 1 diabetes: effect of insulin therapy. AB - Leptin, the gene product of adipose tissue that signals caloric plentitude via central nervous system receptors, may also have diverse peripheral metabolic actions. Of paramount interest has been the potential interaction(s) between leptin and insulin. Insofar as insulin alters leptin secretion/action (or vice versa), dysregulation of this system could contribute to disease states such as diabetes. The purpose of this study was to examine the effect of exogenous insulin on serum leptin in children with newly-diagnosed Type 1 diabetes. Since these patients are hypoinsulinemic (insulin-depleted) at diagnosis, they present an ideal opportunity to examine the effect of insulin repletion on serum leptin. Seventeen patients were enrolled. At baseline (prior to insulin therapy), leptin levels were 4.3 +/- 1.1 ng/ml; they were not statistically related to the baseline serum insulin or illness severity. There was no significant change in serum leptin before, shortly (1-6 days) or several weeks (3-26 weeks) after insulin treatment even when the data was corrected for changes in BMI, hemoglobin A1C, and daily insulin dose. Since repletion of the insulin deficiency that is present in non-acidotic, ambulatory patients with new onset Type 1 diabetes did not alter serum leptin, these results argue against an effect of insulin on serum leptin in the absence of the acute diabetic ketoacidosis. Because as the recuperative months following the diagnosis of new onset Type 1 diabetes are marked by weight gain, the absence of a rise in serum leptin might also indicate either an adaptive (weight permissive) or pathologic (impaired secretory) deficit. PMID- 12369716 TI - Prevention of diabetes in the NOD mouse by intra-muscular injection of recombinant adeno-associated virus containing the preproinsulin II gene. AB - Using the Adeno-associated virus (AAV) as a gene delivery vehicle, we have constructed a recombinant vector containing the full length rat preproinsulin gene (vLP-1). Utilizing the well described non-obese diabetic (NOD) mouse model, an experimental group (n = 10) of animals were intramuscularly (i.m.) injected with 10(7) rAAV virions containing the insulin gene and compared to a mock injected control group (n = 10). Blood glucose (glc) was then measured weekly for 16 weeks. Data showed that the experimental group contained 70% euglycemic animals (defined as glc<200 mg/dL) versus 10% of the control animals (P < .05) at 14 weeks. Mean weight in the treated group was greater than the untreated group. Insulin mRNA was detected at the injection site of all of the treated animals, but not controls. Complete destruction of islets was confirmed by histology ruling out the possibility of spontaneous reversal of insulinitis. We conclude that i.m. delivery of the insulin gene in the NOD mouse was able to prevent clinical DM up to 14 weeks in a majority of treated animals. Our experimental data suggests that gene therapy may be an alternative treatment for IDDM in the future. PMID- 12369717 TI - Diabetes in old male offspring of rat dams fed a reduced protein diet. AB - Restricted fetal growth is associated with increased risk for the future development of Type 2 diabetes in humans. The study aim was to assess the glucose tolerance of old (seventeen months) male rats, which were growth restricted in early life due to maternal protein restriction during gestation and lactation. Rat mothers were fed diets containing either 20% or 8% protein and all offspring weaned onto a standard rat diet. In old-age fasting plasma glucose concentrations were significantly higher in the low protein offspring: 8.4 (1.3) mmol/l v. 5.3 (1.3) mmol/l (p = 0.005). Areas under the curves were increased by 67% for glucose (p = 0.01) and 81% for insulin (p = 0.01) in these rats in intravenous glucose tolerance tests, suggesting (a degree of) insulin resistance. These results show that early growth retardation due to maternal protein restriction leads to the development of diabetes in old male rat offspring. The diabetes is predominantly associated with insulin resistance. PMID- 12369718 TI - Proinsulin C-peptide--a consensus statement. AB - In recent years the physiological role of the proinsulin C-peptide has received increasing attention, focusing on the potential therapeutic value of C-peptide replacement in preventing and ameliorating type 1 diabetic complications. In order to consolidate these new data and to identify the immediate directions of C peptide research and its clinical usefulness, an International Symposium was held in Detroit, Michigan, on October 20-21, 2000, under the auspices of the Wayne State University/Morris Hood Jr. Comprehensive Diabetes Center. In this communication, we review the cellular, physiological and clinical effects of C peptide replacement in animal models and in patients with type 1 diabetes. Finally, recommendations are presented as to the most urgent studies that should be pursued to further establish the biological action of C-peptide and its therapeutic value. PMID- 12369719 TI - Has C-peptide come of age? PMID- 12369720 TI - Islet cell antibodies represent autoimmune response against several antigens. AB - To study the antigens involved in the islet cell antibody (ICA) reaction we selected 30 patient serum samples (ten in each group) positive for ICA and one other additional autoantibody, such as glutamic acid decarboxylase antibodies (GADA), thyrosine phosphatase antibodies (IA-2A) or insulin autoantibodies (IAA). The serum samples were incubated with the specific antigen (GAD65, IA-2 or insulin) and the ICA analysis and the corresponding immunoprecipitation assay were performed before and after the absorption. We could then demonstrate that specific autoantibodies against GAD65 and IA-2 could be absorbed with the corresponding antigen, since ten GADA positive and six IA-2A samples turned completely negative. However, the ICA reaction after absorption with GADA, IA-2A and insulin was still present, although at significantly lower levels. The results strongly indicate that the ICA reaction represents simultaneous autoimmunity against several other antigens beside GAD65, IA-2 and insulin. PMID- 12369721 TI - In vitro effect of fenugreek extracts on intestinal sodium-dependent glucose uptake and hepatic glycogen phosphorylase A. AB - Fenugreek (Trigonella foenum-graecum L. seed) is a food with traditional medicinal use in diabetes. Beneficial effects have been demonstrated in diabetic animals and both insulin-dependent and non-insulin-dependent diabetic subjects. Effects of a lipid extract A, crude ethanolic extract B, further sub-fractions of B (saponin-free C, saponin D and sapogenin E) and a gum fibre fraction F on intestinal sodium-dependent glucose uptake were investigated in vitro using rabbit intestinal brush border membrane vesicles. All fractions except A inhibited glucose-uptake at 0.33 and/or 3.3 mg/mL (p < 0.001). Greatest inhibition was observed with fractions D and E. Diosgenin and trigonelline (compounds reported in fenugreek) also inhibited glucose-uptake (IC50 values approximately 3 mg/ml, equivalent to 8 mM and 19 mM respectively) but did not account for the activity of the crude extracts. Fenugreek extracts had no effect on basal levels of glycogen phosphorylase a (HGPa) activity in rat hepatocyte suspensions. However fractions C and E caused a marginal but statistically significant inhibition (18.9 and 15.1% respectively, p < 0.05) of glucagon induction of this enzyme suggesting a glucagon-antagonist effect. Diosgenin (1.65 mg/ml; 4 mM) inhibited glucagon-induced HGPa activity by 20% (p < 0.05), and was more effective than trigonelline (non significant inhibition of 9.4% at 1.65 mg/ml, 10 mM). PMID- 12369722 TI - Differential acute and long term actions of succinic acid monomethyl ester exposure on insulin-secreting BRIN-BD11 cells. AB - Esters of succinic acid are potent insulin secretagogues, and have been proposed as novel antidiabetic agents for type 2 diabetes. This study examines the effects of acute and chronic exposure to succinic acid monomethyl ester (SAM) on insulin secretion, glucose metabolism and pancreatic beta cell function using the BRIN BD11 cell line. SAM stimulated insulin release in a dose-dependent manner at both non-stimulatory (1.1 mM) and stimulatory (16.7 mM) glucose. The depolarizing actions of arginine also stimulated a significant increase in SAM-induced insulin release but 2-ketoisocaproic acid (KIC) inhibited SAM induced insulin secretion indicating a possible competition between the preferential oxidative metabolism of these two agents. Prolonged (18 hour) exposure to SAM revealed decreases in the insulin-secretory responses to glucose, KIC, glyceraldehyde and alanine. Furthermore, SAM diminished the effects of non-metabolized secretagogues arginine and 3-isobutyl-1-methylxanthine (IBMX). While the ability of BRIN-BD11 cells to oxidise glucose was unaffected by SAM culture, glucose utilization was substantially reduced. Collectively, these data suggest that while SAM may enhance the secretory potential of non-metabolized secretagogues, it may also serve as a preferential metabolic fuel in preference to other important physiological nutrients and compromise pancreatic beta cell function following prolonged exposure. PMID- 12369723 TI - Functional enhancement of electrofusion-derived BRIN-BD11 insulin-secreting cells after implantation into diabetic mice. AB - Electrofusion-derived BRIN-BD11 cells are glucose-sensitive insulin-secreting cells which provide an archetypal bioengineered surrogate beta-cell for insulin replacement therapy in diabetes mellitus. 5x10(6) BRIN-BD11 cells were implanted intraperitoneally into severely hyperglycaemic (>24 mmol/l) streptozotocin induced insulin-treated diabetic athymic nude (nu/nu) mice. The implants reduced hyperglycaemia such that insulin injections were discontinued by 5-16 days (<17 mmol/l) and normoglycaemia (<9 mmol/l) was achieved by 7-20 days. Implanted cells were removed after 28 days and re-established in culture. After re-culture for 20 days, glucose-stimulated (16.7 mmol/l) insulin release was enhanced by 121% (p<0.001) compared to non-implanted cells. Insulin responses to glucagon-like peptide-1 (10(-9) mol/l), cholecystokinin-8 (10(-8) mol/l) and L-alanine (10 mmol/l) were increased by 32%, 31% and 68% respectively (p<0.05-0.01). Insulin content of the cells was 148% greater at 20 days after re-culture than before implantation (p<0.001), but basal insulin release (at 5.6 mmol/l glucose) was not changed. After re-culture for 40 days, insulin content declined to 68% of the content before implantation (p<0.01), although basal insulin release was unchanged. However, the insulin secretory responses to glucose, glucagon-like peptide-1, cholecystokinin-8 and L-alanine were decreased after 40 days of re culture to 65%, 72%, 73% and 42% respectively of the values before implantation (p<0.05-0.01). The functional enhancement of electrofusion-derived surrogate beta cells that were re-cultured for 20 days after implantation and restoration of normoglycaemia indicates that the in vivo environment could greatly assist beta cell engineering approaches to therapy for diabetes. PMID- 12369724 TI - Approaches to type 1 diabetes prevention by intervention in cytokine immunoregulatory circuits. AB - Type 1 (insulin-dependent) diabetes mellitus, like other organ specific autoimmune diseases, results from a disorder of immunoregulation. T cells specific for pancreatic islet beta cell constituents (autoantigens) exist normally but are restrained by regulatory mechanisms (self-tolerant state). When regulation fails, beta cell-specific autoreactive T cells become activated and expand clonally. Current evidence indicates that islet beta cell-specific autoreactive T cells belong to a T helper 1 (Th1) subset, and these Th1 cells and their characteristic cytokine products, IFNgamma and IL-2, are believed to cause islet inflammation (insulitis) and beta cell destruction. Immune-mediated destruction of beta cells precedes hyperglycemia and clinical symptoms by many years because these become apparent only when most of the insulin-secreting beta cells have been destroyed. Therefore, several approaches are being tested or are under consideration for clinical trials to prevent or arrest complete autoimmune destruction of islet beta cells and insulin-dependent diabetes. Approaches that attempt to correct underlying immunoregulatory defects in autoimmune diabetes include interventions aimed at i) deleting beta cell autoreactive Th1 cells and cytokines (IFNgamma and IL-2) and/or ii) increasing regulatory Th2 cells and/or Th3 cells and their cytokine products (IL-4, IL-10 and TGFbeta1). PMID- 12369725 TI - Non insulin dependent diabetes in sand rat (Psammomys obesus) and production of collagen in cultured aortic smooth muscle cells. influence of insulin. AB - In this report, we have shown that the standard laboratory diet administered to Psammomys obesus (sand rat) from Beni Abbes in Algeria, induced a non-insulin dependent diabetes, characterised by increase of body weight (p<0.001) as well as hyperinsulinemia, hyperglycemia and hypercholesterolemia. In cultured aortic smooth muscle cells (SMC) of sand rats, type I and type III collagen biosynthesis and insulin effects, at low dose, on these parameters were investigated. In all experimental conditions of cultured SMC study, The alpha chains of type I collagen were analysed by immunoblotting in media and cells. Metabolic radiolabelling and Immunochemical procedures revealed that, in diabetic state, synthetic SMC (SMCs) actively produce type I and III collagen which are synthesised in the cells and secreted in the medium; type I collagen was predominant as compared with type III collagen. Diabetes enhanced the collagen synthesis. Low dose of Insulin added to the medium, during 48 h of incubation, induced a marked reduction in the synthesis of collagen types, especially type I collagen. PMID- 12369726 TI - High insulin requirements and poor metabolic control do not modify the expression, regulation and PKC mediated activation of the p21ras pathway in PBMC from type II diabetic patients. AB - AIMS: To asses whether clinically severe insulin resistance and poor metabolic control in patients with type 11 diabetes are associated with aberrant expression or function of the p21ras pathway. METHODS: We examined the expression and function of the p21ras pathway in resting and activated PBMC from 10 insulin treated patients with type II diabetes characterized by high insulin requirements and poor metabolic control (IR group) and 10 age and sex matched well controlled patients treated by diet alone or oral hypoglycemic medications (WC group). RESULTS: Levels of p21ras and its regulatory elements: p21rasGAP and hSOS1, were comparable in the two groups. The induced activities of p21ras and its associated down-stream regulatory enzyme MAP-kinase following TPA stimulation were also comparable in the IR and WC patients. CONCLUSIONS: Taken together, these data indicate that clinically significant severe insulin resistance does not modify the expression, regulation and activation of p21ras pathway in PBMC of patients with type II diabetes. PMID- 12369727 TI - Streptozocin diabetes elevates all isoforms of TGF-beta in the rat kidney. AB - Transforming growth factor beta (TGF-beta) is a major promoter of diabetic nephropathy. While TGF-beta1 is the most abundant renal isoform, types 2 and 3 are present as well and have identical in vitro effects. Whole kidney extracts were studied 2 weeks after induction of streptozocin diabetes and in control rats. Mean glomerular area was 25% greater in the diabetic animals. TGF-beta1 showed a 2-fold increase in message with a 3-fold increase in protein. TGF-beta2 mRNA increased approximately 6% while its protein doubled. TGF-beta-message increased by 25%, producing a 35% increase in its protein. TGF-beta-inducible gene H3 mRNA was increased 35% in the diabetic animals, consistent with increased activity of this growth factor. All isoforms of TGF-beta are increased in the diabetic rat kidney. Future studies need to address the specific role that each isoform plays in diabetic nephropathy as well as the impact of therapies on each isoform. PMID- 12369728 TI - Glucose-dependent and glucose-sensitizing insulinotropic effect of nateglinide: comparison to sulfonylureas and repaglinide. AB - Nateglinide, a novel D-phenylalanine derivative, stimulates insulin release via closure of K(ATP) channels in pancreatic beta-cell, a primary mechanism of action it shares with sulfonylureas (SUs) and repaglinide. This study investigated (1) the influence of ambient glucose levels on the insulinotropic effects of nateglinide, glyburide and repaglinide, and (2) the influence of the antidiabetic agents on glucose-stimulated insulin secretion (GSIS) in vitro from isolated rat islets. The EC50 of nateglinide to stimulate insulin secretion was 14 microM in the presence of 3 mM glucose and was reduced by 6-fold in 8 mM glucose and by 16 fold in 16 mM glucose, indicating a glucose-dependent insulinotropic effect. The actions of glyburide and repaglinide failed to demonstrate such a glucose concentration-dependent sensitization. When tested at fixed and equipotent concentrations (approximately 2x EC50 in the presence of 8 mM glucose) nateglinide and repaglinide shifted the EC50s for GSIS to the left by 1.7 mM suggesting an enhancement of islet glucose sensitivity, while glimepiride and glyburide caused, respectively, no change and a right shift of the EC50. These data demonstrate that despite a common basic mechanism of action, the insulinotropic effects of different agents can be influenced differentially by ambient glucose and can differentially influence the islet responsiveness to glucose. Further, the present findings suggest that nateglinide may exert a more physiologic effect on insulin secretion than comparator agents and thereby have less propensity to elicit hypoglycemia in vivo. PMID- 12369729 TI - Effectiveness of nateglinide on in vitro insulin secretion from rat pancreatic islets desensitized to sulfonylureas. AB - Chronic exposure of pancreatic islets to sulfonylureas (SUs) is known to impair the ability of islets to respond to subsequent acute stimulation by SUs or glucose. Nateglinide (NAT) is a novel insulinotropic agent with a primarily site of action at beta-cell K(ATP) channels, which is common to the structurally diverse drugs like repaglinide (REP) and the SUs. Earlier studies on the kinetics, glucose-dependence and sensitivity to metabolic inhibitors of the interaction between NAT and K(ATP) channels suggested a distinct signaling pathways with NAT compared to REP, glyburide (GLY) or glimepiride (GLI). To obtain further evidence for this concept, the present study compared the insulin secretion in vitro from rat islets stimulated acutely by NAT, GLY, GLI or REP at equipotent concentrations during 1-hr static incubation following overnight treatment with GLY or tolbutamide (TOL). The islets fully retained the responsiveness to NAT stimulation after prolonged pretreatment with both SUs, while their acute response to REP, GLY, and GLI was markedly attenuated, confirming the desensitization of islets. The insulinotropic efficacy of NAT in islets desensitized to SUs may result from a distinct receptor/effector mechanism, which contributes to the unique pharmacological profile of NAT. PMID- 12369730 TI - Function of nociceptin and opioid OP4 receptors in the regulation of the cardiovascular system. AB - Nociceptin is the endogenous ligand of the opioid OP4 or ORL1 (opioid receptor like1) receptor. It decreases blood pressure and heart rate in anesthetized and conscious rats and mice after its intravenous and intracerebroventricular injection in a manner sensitive to OP4 but not to OP1-3 (or delta, kappa and mu opioid) receptor antagonists. OP4 receptors involved in the cardiovascular effects of nociceptin were identified on sensory afferent fibres, in brain areas including the nucleus tractus solitarii and the rostral ventrolateral medulla, on preganglionic and/or postganglionic sympathetic and parasympathetic nerve fibres innervating blood vessels and heart or directly on these target organs. These receptors do not seem to be tonically activated but may play a role in the pathophysiology of inflammation, arterial hypertension and cardiac or brain circulatory ischemia. PMID- 12369731 TI - Nitric oxide interferes with salivary mucin synthesis: involvement of ERK and p38 mitogen-activated protein kinase. AB - BACKGROUND: Nitric oxide (NO), a pluripotent molecule, is an important biological messenger that plays a role in the regulation of tissue homeostasis and pathophysiological processes. METHODS: Using sublingual salivary gland acinar cells in culture, we investigated the effect of NO on mucus glycoprotein synthesis, apoptotic processes, and the involvement of extracellular signal regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK). RESULTS: Exposure of the acinar cells to NO donor led to a dose-dependent decrease (up to 42.8%) in mucus glycoprotein synthesis, and this effect of NO was accompanied by a marked increase in caspase-3 activity and apoptosis. Inhibition of ERK with PD98059 accelerated (up to 35.4%) the NO-induced decrease in the glycoprotein synthesis, and cause further enhancement in caspase-3 (up to 27.2%) activity and apoptosis (64.9%). On the other hand, blockade of p38 kinase with SB203580 produced a dose-dependent reversal (up to 42%) in the NO-induced reduction in the glycoprotein synthesis, and substantially countered the NO-induced increases in caspase-3 activity (by 62.8%) and apoptosis (by 57.6%). Moreover, caspase-3 inhibitor, Ac-DEVD-CHO, not only blocked the NO-induced increase in caspase-3 activity but also produced an increase in the glycoprotein synthesis. CONCLUSIONS: Together, our data indicate that the modulatory influence of NO on salivary mucin synthesis is closely linked to ERK and p38 protein kinase activation, in conjunction with caspase-3 activation and apoptosis. PMID- 12369732 TI - Reduction of rise in blood pressure and cortisol release during stress by Ginkgo biloba extract (EGb 761) in healthy volunteers. AB - The standardized extract of Ginkgo biloba (EGb 761) was found not only to improve memory and aging associated cognitive deficits but also to exert beneficial effects on mood. An antistress action of the extract has been suggested but not directly proven. The present study was aimed to evaluate the effects of EGb 761 on salivary cortisol and blood pressure responses during stress in healthy young volunteers (n = 70) in a double blind placebo controlled design. A stress model involving a combination of static exercise (handgrip) and mental stimuli was used. Single treatment with EGb 761 (120 mg) reduced stress-induced rise in blood pressure without affecting the heart rate. Salivary cortisol responses showed differences with respect to the gender and the time of day of the stress exposure, with the activation only in male subjects in the afternoon. This activation was absent if they were treated with EGb 761. The performance in a short memory test with higher scores achieved by women remained unaffected by EGb 761 treatment. Thus, this study provides evidence that EGb 761 has an inhibitory action on blood pressure and it may influence cortisol release in response to some stress stimuli. PMID- 12369733 TI - Vasopressin V1a, V1b and V2 receptors mRNA in the kidney and heart of the renin transgenic TGR(mRen2)27 and Sprague Dawley rats. AB - Vasopressin plays significant role in regulation of blood pressure by means of V1 and V2 receptors, however regulation of synthesis of these receptors in hypertension is only poorly recognized. The purpose of the present study was to compare expression of V1a, V1b and V2 vasopressin (R) mRNA in the renal cortex, renal medulla and the heart of hypertensive renin transgenic TGR(mRen2)27 rats (TGR) and of their parent normotensive Sprague Dawley (SD) strain. The study was performed on 12 weeks old TGR and SD rats. Competitive PCR method was used for quantitative analysis of V1a, V1b and V2 receptors mRNA in fragments of renal cortex, renal medulla and apex of the left ventricle of the heart. In both strains expression of V1aR and V2R mRNA was significantly greater in the renal medulla than in the renal cortex. In the renal medulla but not in the cortex expression of V1aR mRNA was significantly greater in TGR than in SD rats. V2R mRNA expression was similar in the renal cortex and renal medulla of both strains. V1aR mRNA was well expressed in the heart of SD and TGR rats, however there was no significant difference between these two strains. V2R mRNA was not present in the heart. V1bR mRNa could not be detected either in the kidney or in the heart. The results provide evidence for specific increase of expression of V1a receptors mRNA in the renal medulla of TGR rats. PMID- 12369734 TI - Influence of the stimulation of carotid body chemoreceptors on the gastric mucosal blood flow in artificially ventilated and spontaneously breathing rats. AB - The cardiovascular effects of the stimulation of arterial chemoreceptors are different in spontaneously breathing and artificially ventilated animals. Respiratory failure and long term sojourn at high altitude coincide frequently with the occurrence of gastric ulceration. In both these situations a profound stimulation of arterial chemoreceptors is present. The purpose of the paper was to investigate the reflex effect of stimulation carotid chemoreceptors on gastric mucosal blood flow in the rat. Arterial chemoreceptors were stimulated by two methods (I) substitution gas mixture of 10% oxygen in nitrogen for room air and (II) direct injection of acid saline ( 0.05 ml, pH = 6.8) into the distal part of left common carotid artery. In artificially ventilated rats stimulation of arterial chemoreceptors caused significant increase in gastric mucosal vascular resistance, accompanied by marked decline in blood flow. This effect was mediated by adrenergic mechanism. On the contrary to artificially ventilated rats, decline of gastric mucosal vascular resistance with concomitant increase in blood flow was found in spontaneously breathing animals. This effect was not abolished either by phentolamine or atropine. As vasodilatatory effect of arterial chemoreceptors stimulation was abolished by bilateral vagotomy, we postulate that non adrenergic and non cholinergic vagal fibers mediate observed vascular changes in gastric mucosa in spontaneously breathing rats. We hypothesize that in artificially ventilated patients with respiratory failure stimulation of arterial chemoreceptors by hypoxemia and or acidosis may contribute to the development of gastric mucosal lesions. PMID- 12369735 TI - Accumulation of specific ceramides in ischemic/reperfused rat heart; effect of ischemic preconditioning. AB - Ceramide signalling has been implicated in the mechanism of myocardial ischemia/reperfusion injury (IR). This study tested the hypothesis that ceramides containing a specific amino-linked acyl residue mediate the injury, and that ischemic preconditioning (IPC) affords myocardial protection because it prevents increased ceramide accumulation in IR myocardium. Perfused rat hearts were subjected either to the sham perfusion or to 30 min global ischemia, 30 min ischemia/30 min reperfusion (IR) or were preconditioned prior to the standard IR. The ventricles were harvested for biochemical assay that involved transmethylation of ceramide amino-linked acyl residues, and gas liquid chromatography measurement of acyl methyl esters. Fourteen ceramides containing myrystic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, arachidonic, eicosapentaenoic, behenic, docosapentaenoic, docosahexaenoic or nervonic acid were identified in the myocardium of rats. The total basal ceramide concentration in the myocardium was 135 nmol/g tissue, and it was increased by 14.1% and 48.4% in the ischemia and IR group, respectively. However, in fact, IR increased the accumulation of only 7 out of 14 ceramides identified in the heart (i.e., those containing palmitic, stearic, oleic, linoleic, and arachidonic acid), and the relative magnitude of these increases varied between the particular ceramides and was independent from their basal tissue concentration. IPC improved postischemic hemodynamic recovery and partially prevented the reperfusion-induced increases in these 7 ceramides, while the other ceramides were unaffected by IPC. These results support the role of the specific ceramide signalling in the mechanism of myocardial IR injury. We speculate that by preventing tissue accumulation of certain ceramides, IPC attenuates this signalling, that adds to the mechanism of myocardial protection afforded by IPC. PMID- 12369736 TI - Effects of 5-HT1B receptor ligands microinjected into the accumbal shell or core on the cocaine-induced locomotor hyperactivity in rats. AB - The present study was designed to examine the effect of 5-HT1B receptor ligands microinjected into the subregions of the nucleus accumbens (the shell and the core) on the locomotor hyperactivity induced by cocaine in rats. Male Wistar rats were implanted bilaterally with cannulae into the accumbens shell or core, and then were locally injected with GR 55562 (an antagonist of 5-HT1B receptors) or CP 93129 (an agonist of 5-HT1B receptors). Given alone to any accumbal subregion, GR 55562 (0.1-10 microg/side) or CP 93129 (0.1-10 microg/side) did not change basal locomotor activity. Systemic cocaine (10 mg/kg) significantly increased the locomotor activity of rats. GR 55562 (0.1-10 microg/side), administered intra accumbens shell prior to cocaine, dose-dependently attenuated the psychostimulant induced locomotor hyperactivity. Such attenuation was not found in animals which had been injected with GR 55562 into the accumbens core. When injected into the accumbens shell (but not the core) before cocaine, CP 93129 (0.1-10 microg/side) enhanced the locomotor response to cocaine; the maximum effect being observed after 10 microg/side of the agonist. The later enhancement was attenuated after intra-accumbens shell treatment with GR 55562 (1 microg/side). Our findings indicate that cocaine induced hyperlocomotion is modified by 5-HT1B receptor ligands microinjected into the accumbens shell, but not core, this modification consisting in inhibitory and facilitatory effects of the 5-HT1B receptor antagonist (GR 55562) and agonist (CP 93129), respectively. In other words, the present results suggest that the accumbal shell 5-HT1B receptors play a permissive role in the behavioural response to the psychostimulant. PMID- 12369737 TI - DOI, an agonist of 5-HT2A/2C serotonin receptor, alters the expression of cyclooxygenase-2 in the rat parietal cortex. AB - The hallucinogenic effect of DOI, serotonin 5-HT2A/2C receptor agonist, is known to be associated with the activation of cortical 5-HT2 receptors. However, the effect of DOI on excitability of cortical neurons and their subsequent function is still not quite understood. Previous immunohistochemical studies using Fos proteins expression as a marker of neuronal activity showed the involvement of arachidonic acid cascade, particularly cyclooxygenase metabolic pathway, in DOI induced Fos proteins expression in the rat parietal cortex. DOI increases arachidonic acid release which is transformed itself via acceleration of cyclooxygenase metabolic pathway to biologically active metabolites, such as prostaglandins and thromboxanes. Since cyclooxygenase-2 (COX-2) expression correlates with neuronal activity, it was of interest to investigate whether DOI is capable of influencing the level of COX-2 protein and mRNA expression in the rat parietal cortex. It was observed that neurons which were positive for 5-HT2A receptors showed constitutive COX-2 immunoreactivity. It was found further, that COX-2 protein level was increased at 1 h, and returned to the control level at 3 and 6 h after DOI (5 mg/kg) administration. In contrast, DOI decreased the COX-2 mRNA expression at all tested time points (1 h, 3h and 6h after DOI treatment). The obtained results further support the suggestion that COX-2 activation and possibly arachidonic acid metabolites generated by COX-2 may be considered as important mediators of functional responses generated by activation of cortical 5 HT2A/2C receptors. PMID- 12369738 TI - Effect of different muscle shortening velocities during prolonged incremental cycling exercise on the plasma growth hormone, insulin, glucose, glucagon, cortisol, leptin and lactate concentrations. AB - BACKGROUND: Strenuous exercise was reported to involve the alteration in the release of some "stress" hormones such as growth hormone (GH), cortisol, catecholamines and appropriate adjustment of energy metabolism but the relative contribution of these hormones to metabolic response, to cycling exercise performed at different muscle shortening velocities, has not been clarified. AIMS: The purpose of this experiment was to assess the effect of applying different pedalling rates during a prolonged incremental cycling exercise test on the changes in the plasma levels of growth hormone, cortisol, insulin, glucagon and leptin in humans. MATERIAL AND METHODS: Fifteen healthy non-smoking men (means +/- SD: age 22.9 +/- 2.4 years; body mass 71.9 +/- 8.2 kg; height 178 +/- 6 cm; with VO2max of 3.896 +/- 0.544 1 x min(-1), assessed in laboratory tests, were subjects in this study. The subjects performed in two different days a prolonged incremental exercise tests at two different pedalling rates, one of them at 60 and another at 120 rev x min(-1). During this tests the power output has increased by 30 W every 6 minutes. The tests were stopped when the subject reached about 70 % of the VO2max. RESULTS AND CONCLUSIONS: We have found that choosing slow or fast pedalling rates (60 or 120 rev min(-1)), while generating the same external mechanical power output, had no effect on the pattern of changes in plasma cortisol, insulin, glucagon, glucose and leptin concentrations. But, generation of the same external mechanical power output at 120 rev min(-1) causes more stepper increase (p < 0.01) in the plasma growth hormone concentration [GH]pl and plasma lactate concentrations [La]pl when compared to that observed during cycling at 60 rev x min(-1). We have also found that the onset of a significant increase in [GH]pl during cycling at 60 rev x min(-1) was not accompanied by significant increase in [La]pl. While during cycling at 120 rev x min(-1) the onset of a significant increase in [La]pl occurred without increase in [GH]pl, but with continuation of exercise when plasma [La]pl increased, there was also a parallel rise in plasma [GH]pl, as reported before. This results indicates that the increase in [GH]pl during exercise is not closely related to the increase in [La]pl. PMID- 12369739 TI - Vasopressin and oxytocin release as influenced by thyrotropin-releasing hormone in euhydrated and dehydrated rats. AB - Since the thyrotropin-releasing hormone (TRH) can modulate the processes of vasopressin (AVP) and oxytocin (OT) biosynthesis and release mainly at the hypothalamo-neurohypophysial level, the present experiments were undertaken to estimate whether TRH, administered intravenously in different doses, modifies these mechanisms under conditions of osmotic stimulation, brought about by dehydration. AVP and OT contents in the hypothalamus and neurohypophysis as well as plasma levels of AVP, OT, free thyroxine (FT4) and free triiodothyronine (FT3) were studied after intravenously TRH treatment in euhydrated and dehydrated for two days male rats. Under conditions of equilibrated water metabolism TRH diminished significantly the hypothalamic and neurohypophysial AVP and OT content but was without the effect on plasma oxytocin level; however, TRH in a dose of 100 ng/100 g b.w. raised plasma AVP level. TRH, injected i.v. to dehydrated animals, resulted in a diminution of AVP content in the hypothalamus but did not affect the hypothalamic OT stores. After osmotic stimulation, neurohypophysial AVP and OT release was significantly restricted in TRH-treated rats. Under the same conditions, injections of TRH were followed by a significant decrease of plasma OT level. I.v. injected TRH enhanced somewhat FT3 concentration in blood plasma of euhydrated animals but diminished FT4 plasma level during dehydration. Data from the present study suggest that TRH displays different character of action on vasopressin and oxytocin secretion in relation to the actual state of water metabolism. PMID- 12369740 TI - The influences of GnRH, oxytocin and vasoactive intestinal peptide on LH and PRL secretion by porcine pituitary cells in vitro. AB - The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n = 8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n = 8) and Group 3 (n = 8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30 36 h and 60-66 h before slaughter; and in Group 4 (n = 9) with progesterone (P4) at the dose 120 mg/ 100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (10(6)/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37 degrees C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37 degrees C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p < 0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p < 0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p < 0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals. PMID- 12369741 TI - Effect of constitutive- and inducible-cyclooxygenase in the carbachol-induced pituitary-adrenocortical response during social stress. AB - Acetylcholine potently stimulates the hypothalamic-pituitary-adrenal (HPA) axis. Cholinergic receptor agonist carbachol, given intraperitoneally (i.p.) or into the lateral cerebral ventricle (i.c.v.) to non-anesthetized rats acts via multiple pathways to stimulate the HPA axis. The present study sought to determine 1) the functional selectivity of carbachol for cholinergic muscarinic and/or nicotinic receptors involved in the stimulation of HPA axis; 2) the involvement of prostaglandins (PGs) generated by constitutive and inducible cyclooxygenase (COX-1 and COX-2) in the carbachol-induced ACTH and corticosterone secretion in non-stressed rats and animals exposed to social crowding stress for 7 days (24 per a cage for 6). Carbachol was given i.c.v. or i.p. and cholinergic receptor antagonists or cyclooxygenase isoenzyme antagonists were given by the same routes 15 min earlier. One hour after the last injection trunk blood was taken for ACTH and corticosterone determinations. Atropine (0.1 microg i.c.v.), a cholinergic receptor antagonist, totally abolished the carbachol (2 microg i.c.v.)-induced ACTH and corticosterone secretion and mecamylamine (20 microg i.c.v.), a selective nicotinic receptor antagonist, did not affect this secretion. This finding indicates that carbachol functions as a selective central cholinergic muscarinic receptor agonist for the HPA axis stimulation. Crowding stress significantly diminished the carbachol (0.2 mg/kg i.p.)-induced plasma ACTH and corticosterone levels measured 1 hr after administration. Pretreatment with indomethacin (2 mg/kg i.p.), a non-selective cyclooxygenase inhibitor, significantly diminished the ACTH and corticosterone responses to carbachol (0.2 mg/kg i.p.) in control rats and moderately decreased these responses in stressed rats. Piroxicam (0.2 and 2.0 mg/kg i.p.), a COX-1 inhibitor, considerably impaired the carbachol-induced ACTH and corticosterone responses in control rats and markedly diminished these responses in stressed rats. A selective COX-2 blocker, compound NS-398 (0.2 and 2.0 mg/kg i.p.), substantially decreased the carbachol-induced hormones secretion in control rats but did not markedly alter this secretion in stressed rats. These results indicate that in the carbachol induced HPA axis activation PGs generated by COX-1 are considerably and to a much greater extent involved than PGs generated by COX-2. Social stress markedly diminishes the mediation of PGs generated by COX-1 but PGs synthesized by COX-2 do not substantially participate in the carbachol-induced HPA response. PMID- 12369742 TI - The effect of peroxisome proliferator-activated receptors alpha (PPARalpha) agonist, fenofibrate, on lipid peroxidation, total antioxidant capacity, and plasma paraoxonase 1 (PON 1) activity. AB - The aim of this study was to investigate the effect of peroxisome proliferator activated receptors alpha agonist, fenofibrate, on the level of oxidative stress, total antioxidant capacity, and plasma paraoxonase 1 (PON 1) activity in the rat. The adult male Wistar rats received fenofibrate for 7 days. The drug was added to food at concentrations 0.005%, 0.05% and 0.5%, which corresponded to doses of 3, 30 and 300 mg/kg/day, respectively. Fenofibrate treatment dose-dependently reduced plasma concentration of malonyldialdehyde and 4-hydroxydialkenals. The level of these lipid peroxidation products in animals treated with 0.005%, 0.05% and 0.5% fenofibrate was lower than in control group by 52.8%, 62.7% and 87.1%, respectively. Lipid hydroperoxides in plasma decreased by 29.7%, 23.4% and 27.5% in these groups, respectively. The drug had no significant effect on total antioxidant capacity measured as ferric reducing ability of plasma (FRAP). Paraoxon-hydrolyzing activity (PON) of plasma paraoxonase was 81.5% lower in animals receiving 0.05% fenofibrate and 69.2% lower in rats treated with 0.5% fenofibrate than in control. Phenyl acetate hydrolyzing activity (arylesterase, AE) was reduced by 15.2%, 49.6% and 55.8% in rats receiving 0.005%, 0.05% and 0.5% fenofibrate, respectively. PON/AE ratio decreased following 0.05% and 0.5% fenofibrate by 64.9% and 30.4%, respectively. The drug had no significant effect on total plasma triglycerides and cholesterol concentrations. The results indicate that fenofibrate treatment favourably modulates oxidant-antioxidant balance and unfavourably affects plasma PON 1 activity in normolipidemic rats. These effects can contribute to the influence of PPARalpha agonists on pathological processes involved in atherogenesis. PMID- 12369743 TI - Poly(ADP-ribose) polymerase activity in the cat carotid body in hypoxia and hyperoxia. AB - Reactive oxygen species (ROS) induce DNA damage with the ensuing activation of the chromosomal repair enzyme poly(ADP-ribose) polymerase (PARP). ROS also interact with the function of carotid body chemoreceptor cells. The possibility arises that PARP is part of the carotid chemosensing process. This study seeks to determine the presence of PARP and its changes in response to contrasting chemical stimuli, hypoxia and hyperoxia, both capable of generating ROS, in cat carotid bodies. The organs were dissected from anesthetized cats exposed in vivo to acute normoxic (PaO2 approximately 90 mmHg), hypoxic (PaO2 approximately 25 mmHg), and hyperoxic (PaO2 > 400 mmHg) conditions. Carotid body homogenate was the source of PARP and [adenine 14C] NAD was the substrate in the assay. Specimens of the superior cervical ganglion and brainstem were used as reference tissues. We found that PARP activity amounted to 27 pmol/mg protein/min in the normoxic carotid body. The activity level more than doubled in both hypoxic and hyperoxic carotid bodies. Changes of PARP in the reference tissues were qualitatively similar. We conclude that PARP is present in the carotid body but the augmentation of the enzyme activity in both hypoxia and hyperoxia reflects DNA damage, induced likely by ROS and being universal for neural tissues, rather than a specific involvement of PARP in the chemosensing process. PMID- 12369744 TI - An evidence-based approach to improving care of patients with heart failure across the continuum. AB - A coordinated initiative for patients with heart failure was planned and implemented across this healthcare system to: (1) incorporate best evidence-based practice to rapidly stabilize the patient, and (2) establish early, coordinated patient education to promote self-care at home with the support of appropriate resources. Length of stay, readmission frequency, ACE inhibitor and beta blocker prescribing patterns at discharge were the outcomes selected for ongoing study and cross-site efforts toward improvement. These outcomes did improve following cross-site implementation with the collaboration of all appropriate disciplines and the coordination of new and existing services to serve this population. PMID- 12369745 TI - Road to excellence in pain management: research, outcomes and direction (ROAD). AB - Effective pain management is a critical patient care goal mandated by numerous health care organizations. There remains great opportunity to improve pain management across all sites of care. This article describes an interdisciplinary model for process improvement within an integrated health care system. An outcome based approach to pain management resulted in the development of four key clinical indicators that are measured across sites, including acute care, long term, ambulatory, and home care. Early outcome data are presented. Strategies for improving pain management focus on visibility, staff accountability, patient rights, and education. PMID- 12369746 TI - Enhancing the accuracy of hemodynamic monitoring. AB - Assuring that critical care nurses have the skill and knowledge necessary to accurately utilize the many diagnostic monitoring functions available within critical care is essential for optimizing patient outcomes. This performance improvement project focused on the hemodynamic monitoring skills and knowledge level of the critical care nurses in both a cardiovascular and medical/surgical intensive care unit. A four-step progressive intervention strategy was initiated based on our pre-implementation assessment. Post-implementation measures demonstrated a substantial increase in the skill and knowledge level of the nurses. PMID- 12369747 TI - A model to enhance staff response in cardiopulmonary arrest. AB - Dissatisfaction with doing a yearly written test to evaluate competency in the management of cardiopulmonary arrest led to our exploration of ways to change our processes at St. Joseph Regional Medical Center. Guided by our Mission, Vision, and Values, we strove to create a process that would deliver exceptional health care to our patients, while contributing to the personal and professional growth of our staff. Cardiopulmonary arrest is a low volume, high-risk occurrence, which is anxiety provoking for staff. Therefore, it is difficult for staff to feel comfortable and maintain competence in these situations. A process was implemented incorporating a hands-on approach to manage a simulated cardiopulmonary arrest on an annual basis for registered nurses (RNs) and assistive staff. The opportunity for hands-on practice and skill development has enhanced the confidence of RNs and assistive staff, affording them the ability to handle real emergencies. PMID- 12369748 TI - Putting outcomes into practice in physician offices. AB - Implementing outcomes in physician offices is a challenging area. Unlike hospitals, clinics typically have much fewer support staff and resources, electronic clinical data is difficult to access, and physician resistance may be significant. Yet, accountability for outcomes is coming to physician offices. In all outcome efforts, the key steps for guideline implementation are awareness, agreement, decision to adopt, and commitment to adherence. This article describes outcome management efforts in a 15-clinic medical group in the areas of diabetes, asthma, preventive health, pneumonia, heart failure, and patient satisfaction. Implementation strategies, barriers, impact, and outcome data results are described. PMID- 12369749 TI - The value of mentoring: a strategic approach to retention and recruitment. AB - The issues of recruitment, training, and retention of experienced nursing staff remains an ongoing business strategy of nursing service in many health care facilities. The implementation of a structured mentoring program recognizes the need to develop and maintain relationships between the new and the experienced nurses. The terms of mentor and mentee are defined within a structured orientation program, highlighting specific roles and responsibilities of each. The use of other staff as preceptors and resources is discussed as a mechanism to enhance diversity in skill and knowledge development. The value of clinical tracking forms, planning calendars, and feedback mechanisms are stressed to ensure success in monitoring this program in a longitudinal way. Problems associated with the assignment of mentors are addressed as an area for future investigation in different care settings. PMID- 12369750 TI - Scared to go to work: a home care performance improvement initiative. AB - The incident report read. "His arm was tightly locked around my neck. His free arm was touching me while he said terrible, obscene things he intended to do. My heart was pounding. My mind was racing, unable to think rationally. I needed to focus on an escape plan. I was too terrified to think. I just aimlessly struggled. It seemed like an eternity, then, somehow I broke loose." Reading the words on the incident report was horrible. But, nothing compared to seeing the young nurse's eyes, as she stood, barely able to speak, in the office doorway of her manager. There was no doubt that immediate intervention was needed to improve safety at our home health agency. PMID- 12369751 TI - Treatment of the patient with acute myocardial infarction: reducing time delays. AB - There is a critical relationship of time to treatment and myocardial salvage in the patient with acute myocardial infarction (AMI). The challenge lies in developing a process that minimizes delays in assessment and initiation of reperfusion therapy. Three target areas were identified-time to EKG. thrombolytic therapy, and primary PTCA. A multidisciplinary team reviewed the existing standard of care and identified critical areas that were causing delays. An emergency department algorithm was developed to minimize delays, while data analysis tracked our progress. A collaborative multidisciplinary effort can reduce delays in the treatment for the patient with AMI. PMID- 12369752 TI - Metabolism of drugs by leukocytes. AB - Neutrophils and monocytes can metabolize drugs to reactive metabolites, especially those drugs that have nitrogen or sulfur in a low oxidation state. The major system involved in this oxidation is the combination of NADPH oxidase and myeloperoxidase which generates HOCl. Although this system is unlikely to be quantitatively important, i.e. it is unlikely to have a significant effect on the pharmacokinetics of a drug, the reactive metabolites produced appear to have significant biological effects. Reactive metabolites, by their very nature, have short half-lives, and most of their effects will be exerted on the cells that formed them. Therefore, they are likely to be important for adverse reactions that involve leukocytes, such as agranulocytosis and immune-mediated reactions. However, the mechanism of these reactions is unknown and evidence for the association of leukocyte-derived reactive metabolites with such reactions is circumstantial at present. There is also circumstantial evidence to link the formation of such reactive metabolites to the antiinflammatory effects of some drugs. Possible mechanisms include the scavenging of other reactive species or inhibition of cells, especially neutrophils and macrophages, involved in inflammation. The oxidation of drugs by leukocytes requires activation of the cells; therefore, infection or other inflammatory conditions that activate leukocytes may represent one of the risk factors for idiosyncratic drug reactions. PMID- 12369753 TI - Effect of environmental pollutants on hepatocellular function in rats: 3 methylcholanthrene and Aroclor-1254. AB - Environmental pollutants, Aroclor-1254 (PCB) and 3-methylcholanthrene (MC), were employed in this study to investigate some aspects of the induction of hepatic drug metabolism in rats. PCB and MC treatments increased 7-ethoxyresorufin and 7 ethoxycoumarin O-deethylase activities related to cytochrome P-448. Cytochrome P 450 reductase activity was increased by PCB while no effect was observed by MC treatment. Pretreatment with PCB resulted in approximately 50% increase in the phospholipid content of the microsomes whereas MC caused no change. Liver microsomal cholesterol content was decreased while triglycerides were increased by PCB. The ratio between saturated and unsaturated fatty acids (saturation index) decreased in the total microsomes and phospholipids with PCB treatment, whereas MC did not alter the ratio, except that the major effect of MC was observed in the acyl derivatives of microsomal phosphatidylethanolamine. It is proposed that the uniaxial rotation and mobility of hemoproteins may be restricted by an increase in the saturation index of the membrane, while a decreased index may facilitate contact with reductases for electron transfer by enhanced membrane fluidity. The decreased saturation index after treatment with MC may play a role in carcinogenicity by triggering induction of free radicals. PMID- 12369754 TI - Modulation of bile acids induced by paraquat in rabbits. AB - Rabbit bile was examined for changes in composition induced by paraquat. Paraquat was administered intraperitoneally and changes in bile components were monitored by high performance liquid chromatography. Alterations in the ratios of total glycine/taurine conjugated bile acids (TGC/TTC), cholic acid/deoxycholic acid (CA/DC), cholic acid/chenodeoxycholic acid (CA/CDC) and cholic acid/cholesterol (CA/CH) were measured as an index of paraquat toxicity. A statistically significant increase in the ratio of TGC/TTC was observed, while CA/DC, CA/CDC and CA/CH showed a decrease. Phospholipids, protein, sugar, bilirubin, beta carotene, vitamin A and vitamin E in the bile and serum of the experimental animals were also monitored. In bile, the levels of cholesterol, phospholipids, protein, sugar, and total bile acids increased while the levels of the antioxidants beta-carotene, vitamin A and vitamin E decreased. A decrease in the bilirubin content of the bile was also observed. These modifications may be useful clinically for assessment of paraquat toxicity. PMID- 12369755 TI - Circadian rhythms of paracetamol metabolism in healthy subjects; a preliminary report. AB - Paracetamol was used as a "probe" drug to study the circadian rhythms of metabolite ratios in man. Paracetamol was orally administered to six volunteers at different times of day, 0-8 h and 8-24 h urine samples being measured for sulphate and glucuronide formation. Results showed a wide interindividual variation in paracetamol metabolite excretion among the six subjects. However, when a 500 mg dose was administered, free paracetamol excretion was minimal when the dose was given at 12.00 h and maximal when given at 20.00 h for the 0-8 h collection period. Sulphate excretion rose slightly at night and decreased gradually during the day. Glucuronide excretion was greatest with drug administration at 16.00 h and least if paracetamol was ingested at 08.00 h. The 8 24 h profiles were roughly similar. At a higher dose (1500 mg), free paracetamol excretion showed a minimum from dosing at 20.00 h and a maximum from dosing at 24.00 h in both 0-8 h and 8-24 h collections, while the sulphate conjugate peaked for doses at 20.00 h and 8.00 h with collections at 0-8 h and 8-24 h respectively. The glucuronide conjugate was maximal for paracetamol administration at 16.00 h for both 0-8 h and 8-24 h collections. There appears to be a 12 hour phase variation in excretion; this may result from circadian rhythms in absorption and enzyme activities. These parameters may also affect metabolism at higher dose levels, so that the hepatotoxicity of paracetamol could vary with the time of dose. PMID- 12369756 TI - Absolute bioavailability of glimepiride (Amaryl) after oral administration. AB - Twelve healthy fasting male volunteers received a single 1.0 mg dose of glimepiride either as an intravenous injection over one minute or as a tablet. Blood and urine samples were taken before drug administration and afterwards for up to 24 hours (blood) and 48 hours (urine) to determine serum and urinary concentrations of glimepiride and its hydroxy- and carboxy-metabolites (M1 and M2). There were no statistically significant differences between mean serum pharmacokinetic parameters for the oral and intravenous formulations either with glimepiride or M1. Mean urinary recovery of M1 plus M2 was 50% of the dose for the glimepiride tablet and 51% for the intravenous injection. The absolute bioavailability of the tablet formulation was 107% (AUDC(glimepiride)), 109% (AUDC(M1)) and 97% (urinary recovery). The tablet formulation of glimepiride is completely bioavailable and was safe and well tolerated in healthy volunteers. PMID- 12369757 TI - Dose linearity assessment of glimepiride (Amaryl) tablets in healthy volunteers. AB - Twelve healthy fasting male volunteers received glimepiride in 1, 2, 4 or 8 mg single oral doses. On the days when glimepiride was taken, the subjects were given a standardised carbohydrate diet (18 bread exchange units) and drank 125 ml of water hourly. Blood and urine samples were taken before drug administration and afterwards for up to 36 hours (blood) and 48 hours (urine) to determine serum and urinary concentrations of glimepiride and its hydroxy- and carboxy metabolites (M1 and M2). The areas under the curve for glimepiride after oral doses of 1 to 8 mg and the urinary recovery of its metabolites M1 and M2 were dose linear. All confidence intervals were well contained within the bioequivalence range of 80-125%. There was a statistically significant difference for Cmax values of glimepiride between doses after dose normalisation. A dose dependent increase for Cmax was nevertheless clearly observed with a correlation coefficient of r=0.90. The pharmacokinetics of glimepiride are dose linear in the dose range 1 to 8 mg, and glimepiride was safe and well tolerated in healthy volunteers. PMID- 12369758 TI - Leaching of trifluralin, metolachlor, and metribuzin in a clay loam soil of Louisiana. AB - Trifluralin[2,6-dinitro-N,N-dipropyl-4-(trifluormethyl)benzenamine], metolachlor[2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1 methylethyl)acetamide], and metribuzin[4-amino-6-(1,1-dimethylethyl)-3 (methylthio)-1,2,4-triazin-5(4H)one] were applied in field plots located on a Commerce clay loam soil near Baton Rouge, Louisiana at the rate of 1683 g/ha, 2759 g/ha and 609 g/ha, respectively. The half-lives of trifluralin, metolachlor, and metribuzin in the top 0-15 cm soil depth were found to be 54.7 days, 35.8 days and 29.8 days, respectively. The proportion of trifluralin, metolachlor, and metribuzin in the top 0-15 cm soil depth was 94.7%, 86.6%, and 75.4%, respectively of that found in the top 0-60 cm soil depth 30 days after application. Trifluralin concentrations were within a range of 0.026 ng/mL to 0.058 ng/mL in 1 m deep well water, and between 0.007 ng/mL and 0.039 ng/mL in 2 m deep well water over a 62 day period after application. Metolachlor concentrations in the 1 m and 2 m wells ranged from 3.62 ng/mL to 82.32 ng/mL and 8.44 ng/mL to 15.53 ng/mL, respectively. Whereas metribuzin concentrations in the 1 m and 2 m wells ranged from 0.70 ng/mL to 27.75 ng/mL and 1.71 ng/mL to 3.83 ng/mL, respectively. Accordingly, trifluralin was found to be strongly adsorbed on the soil and showed negligible leaching. Although metolachlor and metribuzin were also both readily adsorbed on the soil, their leaching potential was high. As a result, in the clay loam soil studied, metribuzin concentration in groundwater with shallow aquifers is likely to exceed the 10 mg/L US Environmental Protection Agency (EPA) advisory level for drinking water early in the application season, whereas trifluralin and metolachlor concentrations are expected to remain substantially lower than their respective 2 ng/mL and 175 ng/mL EPA advisory levels. PMID- 12369759 TI - Runoff of trifluralin, metolachlor, and metribuzin from a clay loam soil of Louisiana. AB - Trifluralin[2,6-dinitro-N,N-dipropyl-4-(trifluormethyl)benzenamine], metolachlor[2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] and metribuzin[4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4 triazin-5(4H)one] were applied as pre-emergent herbicides to soybean plots in Louisiana (LA) at the rate of 1683 g/ha, 2759 g/ha and 609 g/ha, respectively. The concentrations of trifluralin in the runoff water ranged between 0.09 ng/mL and 0.02 ng/mL, which is lower than the 2 ng/mL US Environmental Protection Agency (EPA) advisory level for trifuralin in drinking water. Metolachlor concentrations in the runoff water ranged from 9.0 ng/mL to 221.5 ng/mL, which is both lower and higher than the 175 ng/mL EPA advisory level for metolachlor. Similarly, metribuzin concentrations in the runoff water ranged between 1.5 ng/mL and 56.2 ng/mL, which is also lower and higher than the 10 ng/mL EPA advisory level for metribuzin. Accordingly, from the field plots located on a Commerce clay loam soil in LA, although the concentration of trifluralin in runoff water were substantially lower than the EPA advisory level, metolachlor and metribuzin concentrations are likely to exceed the EPA advisory levels early on in the application season with a subsequent rapid decrease to safe levels. The total loss of trifluralin in runoff water was 0.005% of the applied amount over an 89 day period after application. The total loss of metolachlor and metribuzin in the runoff water was 4.67% and 5.36% of the applied amount, respectively, over a 22 day period after application. As such, there was almost no movement of trifluralin in the runoff water, whereas metolachlor and metribuzin were much more easily moved. PMID- 12369760 TI - Impact of four pesticides on the growth and metabolic activities of two photosynthetic algae. AB - The acute toxicity was determined for soil algae Chlorella kesslerei and Anabaena inaequalis, exposed to pesticides lindane, pentachlorophenol (PCP), isoproturon (IPU), and methyl parathion (MP). Toxicity markers included growth inhibition, chlorophyll biosynthesis, and total carbohydrate content, as a function of dose and time. Concentration response functions (EC50) were estimated by probit data transformation and weighted linear regression analyses. Lindane's toxicity to Chlorella increased sharply with time (EC50 = 7490, 10.3, 0.09 mg L(-1); 24, 48, 72 h), but remained nearly constant through 72 h with Anabaena (8.7-6.7 mg L(-1); 24-72 h). PCP at low concentrations stimulated algal growth and chlorophyll a production, an effect reversed at higher doses. Anabaena was less tolerant of PCP and MP than was Chlorella. The 96-h static EC50 values for Chlorella were: 0.003, 34, 0.05, and 291 mg L(-1) for lindane, PCP, isoproturon, and MP, respectively; for Anabaena, these were 4.2, 0.13, 0.21, and 19 mg L(-1). Carbohydrate production responses were similar to those of cell density (growth) and chlorophyll biosynthesis, with MP having the lowest adverse impact. The overall relative toxicity among the four tested pesticides was: for Chlorella, lindane > IPU >> PCP >> MP; and for Anabaena, PCP > IPU > lindane > MP. The results confirm that toxicants such as these pesticides may affect individual (though related) species to significantly different degrees. PMID- 12369761 TI - Abiotic degradation of imazethapyr in aqueous solution. AB - The photodegradation of imazethapyr [2-(4,5-dihydro-4-methyl-4-(1-methylethyl)-5 oxo-1H-imidazol-2-yl)-5-ethyl-3-pyridinecarboxylic acid] in aqueous solution in the presence of titranium dioxide (TiO2) and humic acids (HA) at different ratios of herbicide/TiO2 and herbicide/humic acids was studied at pH 7.0. Irradiation was carried out with polychromatic light using Heraeus apparatus equipped with xenon lamp to simulate sunlight having a spectral energy distribution similar to solar irradiation (>290 nm). The concentration of remaining herbicide was followed using a High Pressure Liquid Chromatograph (HPLC) equipped with UV detector at 230 nm. In pure aqueous solution imazethapyr degrades slowly and the photodegradation leads to the formation of two metabolites labelled A and B. The presence of TiO2 caused enhancement of the degradation rate. The presence of HA induced an increase of the photodegradation of the pesticide with respect to pure aqueous solution. PMID- 12369762 TI - Adsorption of paraquat on the physically activated bleaching earth waste from soybean oil processing plant. AB - A series of regeneration experiments with physical activation were carried out on bleaching earth waste from the soybean refining process in a rotary reactor. The influence of activation parameters on the spent clay by varying the holding time of 1 to approximately 4 hours and temperature of 700 to approximately 900 degrees C was determined. The variations of pore properties as well as the change of chemical characteristics in the resulting solids were also studied. Results showed that the resulting samples were type IV with hysteresis loops corresponding to type H3 from nitrogen adsorption-desorption isotherms, indicating slit-shaped mesoporous characteristics. However, the regenerated clays had smaller surface areas (70 to approximately 117 m2/g) than that (245 m2/g) of fresh bleaching earth. Under the physical activation conditions investigated, the holding time of 1 hour and temperature of 700 degrees C were found to be optimal conditions for producing mesoporous clay with physical activation. The adsorption of paraquat on regenerated sample was also evaluated. The isotherm showed that the regenerated sample still had a high affinity for this herbicide. Thus, the regeneration of this agro-industrial waste is one option for utilizing the clay resource, and it may be used for water treatment applications to remove organic contaminants. PMID- 12369763 TI - Pesticide residues in thermal mineral water in Greece. AB - Eight different hot springs (SPA) in Greece were monitored over a one-year survey for priority pesticide residues. A specific and effective procedure including solid phase extraction in combination with HPLC and GC analytical methods were applied. Samples that were sensitive to nitrogen-phosphorus (NPD) and/or electron capture (ECD) detectors were analysed by capillary gas chromatography. From the twenty-six water samples, pesticide residues were detected in fourteen of them (54%) but no one exceeding the European Union Maximum Acceptable Concentration (MAC). Lindane (gamma-BHC) was the most frequently detected pesticide. It was found in nine samples (35%) in concentrations from < 0.005 to 0.01 microg/L. Other pesticides detected were phorate (in five samples), propachlor (in two samples) and chlorpyriphos ethyl (in three samples) but in concentrations far below the permissible levels. PMID- 12369764 TI - Alterations of lipid profile in plasma and liver of diabetic rats: effect of hypoglycemic herbs. AB - The effect of three species of hypoglycemic herbs (Termis, Halfa barr, or Kammun Quaramany) on the lipid profile was investigated in plasma and liver tissues of diabetic and herbs-treated diabetic rats. This profile includes total lipids (TL), triglycerides (TG), cholesterol, high-density lipoprotein (HDL) and low density lipoprotein (LDL). A dose of 1.5 ml of aqueous suspension of each herb/100 g body weight (equivalent to 75 mg/100 g body weight) was orally administered daily to alloxan-diabetic rats for four weeks. The present study showed 2-fold increase (p<0.05) in the plasma glucose level of diabetic rats, which received alloxan as a single dose of 120 mg/kg body weight, relative to the mean value of control group. This elevated glucose level was restored to its normal level after treatment with any one of the three herbs. Furthermore, the levels of TL, TG, cholesterol, LDL and VLDL were significantly (p<0.05) increased in the plasma and the liver tissues of diabetic rats compared to the control group, whereas HDL level was significantly (p<0.05) decreased. The plasma levels of all above parameters were normalized after treatment of the diabetic rats with Kammun Quaramany. Treatment of diabetic rats with Tennis normalized TG, cholesterol, LDL and VLDL levels, but Halfa barr restored the induced levels of plasma cholesterol, LDL and HDL to their normal levels. On the other hand, treatment with any of the three herbal suspensions could not restore the concentrations of the all tested parameters in the liver. These data demonstrated that the glycemic control of any of the three herbal suspensions was associated with their hypocholesterolemic effects on the hypercholesterolemia of the alloxan induced diabetic rats. Moreover, the Kammun Quaramany showed the most potent effect. PMID- 12369765 TI - Methionine auxotroph Escherichia coli growth assay kinetics in antibiotic and antifungal amended selective media. AB - The objective of this work was to determine if Escherichia coli methionine bioassay characteristics were influenced by selective media amended with antibiotics and the antifungal compound cycloheximide. Bacterial cells were grown in minimal media with increasing concentrations of methionine and were incubated at 37 degrees C with vigorous agitation for 6 hours. Addition of antistatic agents to the media did not change the growth kinetic response (P>0.05) to methionine concentration (3.4 to 26.8 microM). This supports the utility of this strain as a methionine bioassay organism for feed and other environmental sources. PMID- 12369766 TI - Cool temperature performance of a wheat straw biofilter for treating dairy wastewater. AB - A wheat straw biofilter was evaluated for attenuating pollutants in dairy (milkhouse and milking parlor) wastewater. During the 14-day study, the biofilter was operated in a sequential aerobic-anaerobic mode in a temperature range of 8 14 degrees C. While the biofilter was very effective (89% removal) in attenuating total suspended solids and moderately effective (76% removal) in attenuating oil and grease, its effectiveness in attenuating chemical oxygen demand was low (37% removal). The biofilter was ineffective in attenuating nitrate, while its effectiveness in attenuating ammonium (20% removal) and total Kjeldahl nitrogen (15% removal) was low. The biofilter was not effective in attenuating ortho phosphate, total phosphorus, and fecal coliform. Though microbial degradation accounted for some pollutant removal, filtration seemed to be the primary mechanism. Lower temperature of operation and high oil and grease concentration (that reduced nutrient transfer to the biofilm) decreased microbial activity, reducing pollutant attenuation. Biofilter performance could be enhanced by using residual heat in the wastewater to raise the operating temperature of the biofilter and by removing oil and grease prior to applying the wastewater to the biofilter. PMID- 12369767 TI - Interfacial transfer velocities of ozone dry deposition over agricultural soils: experimental and theoretical analysis. AB - A mathematical dry deposition model was developed and an experiment performed to verify that the interfacial transfer velocity (V(S)) of ozone dry deposition includes the interfacial reactive velocity (V(Sr)) and interfacial kinetic velocity (V(Sk)), as determined by measuring the ozone depletion over agricultural field soils in a dry deposition chamber. Experimental results indicate that the chemical reaction (O3 + NO --> NO2 + O2) produces the reactive velocity. Observed interfacial transfer velocities depend on nitrogen oxide emission (NO) and soil temperature (T(S)). Additionally, observed kinetic velocities of conditioned field soils increased linearly with soil temperature. Moreover, observed reactive velocities of field soils increased exponentially with soil temperature, and depend on the emission rate of nitrogen oxide. Results in this study demonstrate that interfacial transfer velocities are variable velocities for long-term transportation, that influenced factors are chemical kinetics, thermodynamics and biochemical mechanisms. PMID- 12369768 TI - Cross-cultural assessment of childhood temperament. A confirmatory factor analysis of the French Emotionality Activity and Sociability (EAS) questionnaire. AB - The Emotionality Activity Sociability (EAS) questionnaire focuses on heritable individual differences in reactivity and behavior which are often referred to in developmental temperament research. Psychometric properties of the French version of EAS were examined in a sample of 197 school-children aged six to 12 years. Parents, teachers and children aged nine years and more completed parallel forms of the EAS questionnaire. Confirmatory factor analysis was used to examine the fit between the original factors and the data. Internal consistency of each subscale, inter-rater and external validity were also examined. Children-rated EAS showed the best indices of fit between the four hypothesized factors and the data, but internal consistency was generally lower than in adult-rated questionnaires. Shyness and sociability showed significant overlap in both parent and teacher-rated EAS. The low concordance between child and adult-ratings indicates that temperament evaluation and interpretation of items may be influenced by subjective and/or developmental factors. Results are discussed in the perspective of validity versus cross-cultural comparability of temperament measurement. The theoretical four-factor structure was not completely replicable in our sample. PMID- 12369769 TI - Childhood disintegrative disorder. Re-examination of the current concept. AB - Childhood disintegrative disorder (CDD), which is classified as a sub-type of pervasive developmental disorder (PDD), has been recognised for many years. Research data on CDD, however, is sparse and it primarily describes the clinical parameters. In this research report clinical data on 12 cases of CDD and 21 cases of typical autism, seen during a specified period, are compared and critically evaluated in reference to the diagnostic criteria in ICD-10 for these disorders. While the findings support the clinical validity of CDD, these also highlight the limitations of the current criteria (ICD-10) particularly the age of onset in CDD and the conceptual confusion in labelling it as a 'PDD'. Need for more research in the areas of the biology, course and outcome of CDD is emphasised. PMID- 12369770 TI - Recurrent abdominal pain and headache--psychopathology, life events and family functioning. AB - We assessed the psychopathology of children and adolescents with Recurrent Abdominal Pain (RAP) and tension-type headaches (TTH), the psychopathology and Expressed Emotion (EE) of their mothers and family functioning. Additionally, we assessed the relationship of negative Life Events (LE) to RAP and headaches. Sixty-nine children and adolescents with either RAP or headaches, and their mothers were examined and compared to controls. Of the children with RAP or headache, 81.6% and 83.9% respectively carried a psychiatric diagnosis, primarily anxiety or depressive disorder, in contrast to 15% of controls. Mothers of patients with RAP showed more symptoms of anger and hostility than controls. Index mothers had higher EE than control mothers. More problems were reported in the families of patients with RAP. Families of patients with headache were similar to those with RAP but differed from controls in terms of behaviour control and general functioning. More negative LE were experienced by both index groups. If psychological intervention is decided for certain children with RAP or TTH, it should address their depression, anxiety, the impact of negative LE and family functioning. PMID- 12369771 TI - Hysteria. Pretending to be sick and its consequences. AB - Hysteria, as it involves the medical profession, is a form of sickness that is defined as being without disease or illness. This lack of a biomedical explanation has limited progress in its understanding. In this essay we propose that hysteria might be better thought of as a form of pretending, elaborated in transaction with the medical system. In medicine, to pretend usually means to deceive. From the perspective of play, however, pretend is a state more akin to acting, magic, belief, and hypnosis. We provide a number of reasons why sickness is an attractive focus for pretending. We show how enactments of sickness can be scripted by a group of involved persons, each contributing from their own perspective, as occurs in the parlour game of 'Consequences', except in hysteria the consequences are often dire. PMID- 12369772 TI - Triglyceride, cholesterol and weight changes among risperidone-treated youths. A retrospective study. AB - The purpose of this retrospective chart review was to assess triglyceride, cholesterol and weight changes among risperidone-treated youths. The charts of 22 child and adolescent inpatients were abstracted. The sample's mean (+/- SD) age was 12.8 (+/- 2.6) years, daily risperidone dose 2.7 (+/- 2.2) mg, and average length of exposure 4.9 (+/- 1.0) months. Repeated measures analysis of variance revealed statistically and clinically significant weight gain averaging 7.0 (+/- 4.7) kg (95% confidence interval [CI] for the mean = 4.9,9.1; F = 49.421, df = 1,21, p < 0.001). No significant changes in serum triglyceride or cholesterol levels were seen in the group as a whole. Triglyceride levels and weight were strongly correlated with each other: almost 25% of the variance in triglyceride level changes could be explained by weight gain alone (R2 = 0.22, F = 5.526, p = 0.029), although such association weakened when excluding subjects (N=5) concurrently treated with lithium or divalproex (R2 = 0.06, p > 0.05). On the basis of this preliminary report it seems prudent to be clinically vigilant and conservative, recommending regular laboratory monitoring until a clearer picture emerges regarding lipid dysregulation associated with risperidone and other atypical antipsychotic use in children and adolescents. PMID- 12369773 TI - Cerebral palsy and juvenile-onset bipolar disorder. A preliminary report. AB - Cerebral palsy refers to a heterogeneous group of congenital and early acquired brain disorders. Children with cerebral palsy and other brain disorders have an increased rate of psychiatric disorder. The pattern of disorder is not particularly distinctive and no specific association has been found. We report two cases of spastic diplegia of prematurity comorbid with juvenile onset bipolar disorder, which highlight some of the diagnostic difficulties in these cases. An interesting association between the periventricular leucomalacia as an aetiological factor in cerebral palsy and the white matter lesions seen on magnetic resonance imaging in cases of bipolar disorder is noted. PMID- 12369774 TI - A clinical case series of six extremely aggressive youths treated with olanzapine. AB - Olanzapine is an atypical neuroleptic drug with mood-stabilising properties and few of the side effects commonly associated with conventional neuroleptic treatment. We used olanzapine, 5-20 mg/day, to treat severe aggression in six non psychotic teenage boys with neuropsychiatric disorders. All but one started to respond within one week. The therapeutic effect in four of the patients clearly outweighed the side effects (weight gain and sedation). The subjects described a markedly increased sense of well being during the olanzapine treatment. PMID- 12369775 TI - Autistic symptoms following herpes encephalitis. AB - Autism is a childhood onset neurodevelopmental disorder characterized by reciprocal social deficits, communication impairment, and rigid ritualistic interests, with the onset almost always before three years of age. Although the etiology of the disorder is strongly influenced by genes, environmental factors are also important. In this context, several reports have described its association with known medical conditions, including infections affecting the central nervous system. In this report, we describe an 11-year-old Asian youngster who developed the symptoms of autism following an episode of herpes encephalitis. In contrast to previous similar reports, imaging studies suggested a predominant involvement of the frontal lobes. At follow-up after three years, he continued to show the core deficits of autism. This case further supports the role of environmental factors, such as infections, in the etiology of autism, and suggests that in a minority of cases, autistic symptoms can develop in later childhood. PMID- 12369776 TI - Parathyroid hormone and periosteal bone expansion. PMID- 12369777 TI - Klotho gene polymorphisms associated with bone density of aged postmenopausal women. AB - Because mice deficient in klotho gene expression exhibit multiple aging phenotypes including osteopenia, we explored the possibility that the klotho gene may contribute to age-related bone loss in humans by examining the association between klotho gene polymorphisms and bone density in two genetically distinct racial populations: the white and the Japanese. Screening of single-nucleotide polymorphisms (SNPs) in the human klotho gene identified 11 polymorphisms, and three of them were common in both populations. Associations of the common SNPs with bone density were investigated in populations of 1187 white women and of 215 Japanese postmenopausal women. In the white population, one in the promoter region (G-395A, p = 0.001) and one in exon 4 (C1818T, p = 0.010) and their haplotypes (p < 0.0001) were significantly associated with bone density in aged postmenopausal women (> or = 65 years), but not in premenopausal or younger postmenopausal women. These associations were also seen in Japanese postmenopausal women. An electrophoretic mobility shift analysis revealed that the G-A substitution in the promoter region affected DNA-protein interaction in cultured human kidney 293 cells. These results indicate that the klotho gene may be involved in the pathophysiology of bone loss with aging in humans. PMID- 12369778 TI - Mapping quantitative trait loci that influence femoral cross-sectional area in mice. AB - Size and shape are critical determinants of the mechanical properties of skeletal elements and can be anticipated to be highly heritable. Moreover, the genes responsible may be independent of those that regulate bone mineral density (BMD). To begin to identify the heritable determinants of skeletal geometry, we have examined femoral cross-sectional area (FCSA) in male and female mice from two inbred strains of mice with divergent FCSA (C57BL/6 [B6] and DBA/2 [D2]), a large genetically heterogeneous population (n = 964) of B6D2F2 mice and 18 BXD recombinant inbred (RI) strains derived from their F2 cross. Femora were harvested from 16-week-old mice and FCSA (bone and marrow space enclosed within the periosteum) was measured at the midshaft by digital image analysis. In all mouse populations examined, FCSA was positively correlated with body weight and weight-corrected FCSA (WC-FCSA) values were normally distributed in the BXD-RI and F2 populations, suggesting polygenic control of this trait. Genome-wide quantitative trait locus (QTL) analysis of the B6D2F2 population revealed regions on four different chromosomes that were very strongly linked to WC-FCSA (chromosomes 6, 8, 10, and X) in both genders. Evidence of gender-specific genetic influences on femoral geometry was also identified at three other chromosomal sites (chromosomes 2, 7, and 12). Supporting evidence for the WC-FCSA QTLs on chromosomes 2, 7, 8, 10, and 12 also was present in the RI strains. Interestingly, none of these WC-FCSA QTLs were identified in our previous QTL analysis of whole body BMD in the same B6D2F2 population. Thus, the genetic determinants of bone size appear to be largely, if not entirely, distinct from those that regulate BMD attainment. The identification of the genes responsible for geometric differences in bone development should reveal fundamentally important processes in the control of skeletal integrity. PMID- 12369779 TI - Localization of the mutation responsible for osteopetrosis in the op rat to a 1.5 cM genetic interval on rat chromosome 10: identification of positional candidate genes by radiation hybrid mapping. AB - Osteopetrosis is caused by a heterogenous group of bone diseases that result in an increase in skeletal mass because of inadequate osteoclastic bone resorption. In the op osteopetrotic rat, the disease has been linked to a single genetic locus located at the proximal end of rat chromosome 10. In this study, we identified a 1.5-cM genetic interval that contains the mutation. We then generated an improved radiation hybrid (RH) map of this region to identify potential functional and positional candidates for the op gene. Using the rat genome radiation hybrid panel, we mapped 57 markers including 24 genes (14 that have not yet been mapped in the rat) and 10 expressed sequence tag markers. Included in the mapped genes are several candidate genes that might significantly influence the biochemical pathways involved in osteopetrosis. These include genes involved in osteoclast differentiation, apoptosis, and the functional capabilities of mature osteoclasts to resorb bone. Further analysis of the genes and expressed transcripts mapped to this region may yield important insights into the multifactorial control of osteoclast function and the mechanisms of failed bone homeostasis in diseases such as osteopetrosis, osteoporosis, and rheumatoid arthritis in which failed bone homeostasis is an instigating or exacerbating circumstance of the disease process. PMID- 12369780 TI - The effects of vitamin D receptor polymorphism on secondary hyperparathyroidism and bone density after renal transplantation. AB - Immunosuppresive treatment and secondary hyperparathyroidism (SHPT) are considered among the most important pathogenetic factors for postrenal transplant bone disease. The aim of this study was to investigate the relationships among vitamin D receptor (VDR) gene polymorphism, parathyroid hormone (PTH) levels, and bone density in renal transplant recipients. We enrolled 69 patients (47 men and 22 women; mean age, 47 +/- 11 years) who had undergone kidney transplantation 51 +/- 5 months before. All patients underwent an evaluation of the main biochemical parameters of bone metabolism as well as bone densitometry. VDR alleles were typed by a polymerase chain reaction (PCR) assay based on a polymorphic BsmI restriction site. When the patients were categorized according to the VDR genotype (BB, Bb, and bb), serum creatinine, and the cumulative doses of immunosuppressive drugs were similar across the groups. PTH levels higher than 80 pg/ml were found in 53.6% of the patients, with the highest values being detected in the bb VDR genotype (p < 0.05). PTH was significantly correlated to urinary type I collagen cross-linked N-telopeptide (NTx) values. Bone density was low in the whole population; however, spinal bone density was lower in the bb subgroup (p < 0.02). In the whole population, only PTH (p < 0.05) and body mass index (BMI; p < 0.01) were independent predictors of spinal bone density. When grouping the patients by the VDR gene polymorphism, only PTH continued to be an independent predictor of spinal bone density in the bb allele subgroup (R2 adj. = 0.17). We can conclude that the VDR genotype polymorphism affects bone density of renal transplant recipients via its effects on the severity of SHPT. PMID- 12369781 TI - Involvement of Rho and p38 MAPK in endothelin-1-induced expression of PGHS-2 mRNA in osteoblast-like cells. AB - Prostaglandins (PGs) play an important role in bone remodeling because eicosanoids are local mediators of bone metabolism, which can induce physiological and pathological responses of bone tissue. Biosynthesis of PGs is catalyzed by constitutively expressed PG endoperoxide G/H synthase (PGHS) 1 and by the inducible isoform PGHS-2. In MC3T3-E1 osteoblast-like cells, expression of PGHS-2 was shown by mechanical forces, cytokines, growth factors, and hormones. Recently, endothelin (ET) 1-stimulated PGHS-2 mRNA expression was described, leading to a burst in prostaglandin E2 (PGE2) production. In this study, we investigated ET-1-induced signal transduction pathway(s) involved in the PGHS-2 mRNA production. Time course of PGHS-2 mRNA expression reaching the maximum within 45 minutes is in good agreement with the concept of an immediate early gene product. Inhibition of phospholipase C (PLC), phospholipase D (PLD), phosphatidylinositol-3 kinase (PI-3-kinase), and protein kinase C (PKC) had no influence on PGHS-2 synthesis. Using specific blockers of tyrosine kinases indicated involvement of p38 MAPK but not p42/44 MAPK. By preloading cells with exoenzyme C3, we were able to show requirement of the Rho family of G proteins for p38 MAPK phosphorylation and PGHS-2 mRNA synthesis, whereas pertussis toxin (PTX) and cholera toxin (CTX) had no remarkable effect. PMID- 12369782 TI - Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix. AB - We screened the small molecule compounds that stimulate osteogenesis by themselves or promote bone morphogenetic protein (BMP)-induced bone formation. We found that a specific inhibitor for MAPK/extracellular signal-regulated kinase kinase (MEK)-1, promoted the early osteoblastic differentiation and mineralization of extracellular matrix (ECM) in C2Cl2 pluripotent mesenchymal cells treated with recombinant human BMP-2 (rhBMP-2) and MC3T3-E1 preosteoblastic cells. ALP activity was synergistically increased by the treatment with a specific MEK-1 inhibitor PD98059 and rhBMP-2 in both cell lines. Twenty-five micromolar PD98059 promoted mineralization of ECM in rhBMP-2-treated C2Cl2 cells and MC3T3-E1 cells. In contrast, PD98059 reduced osteocalcin (OCN) secretion and its transcriptional level in rhBMP-2-treated C2Cl2 cells but increased its secretion and mRNA level in MC3T3-E1 cells. Stable expression of a dominant negative MEK-1 mutant in C2Cl2 cells represented high ALP activity and low osteocalcin production in the presence of rhBMP-2, while a constitutively active mutant of MEK-1 attenuated both of them. Together, our results indicated that BMP 2-induced mineralization of ECM of pluripotent mesenchymal stem cells and preosteoblastic cells could be controlled by a fine tuning of the MAPK signaling pathway. Further, MEK-1 inhibitors would be useful for the promotion of bone formation, for instance, the treatments for delayed fracture healing or advance of localized osteoporotic change after fracture healing. PMID- 12369783 TI - L-type calcium channels mediate mechanically induced bone formation in vivo. AB - Cell and tissue culture studies suggest that the long-lasting (L-type) voltage sensitive calcium channels (VSCC) play a role in the signaling cascade in bone cells after mechanical loading. We investigated whether the L-type VSCC mediates mechanically induced bone formation in vivo using two L-type VSCC antagonists verapamil and nifedipine. Female Sprague-Dawley rats were divided into five groups: control group (Veh), two verapamil-treated groups (20 mg/kg, Vera-L; 100 mg/kg, Vera-H), and two nifedipine-treated groups (20 mg/kg, Nife-L; 100 mg/kg, Nife-H). One bout of mechanical loading was applied to the right tibia 90 minutes after oral administration of verapamil or 30 minutes after oral administration of nifedipine. Mechanical loading increased mineralizing surface (MS/bone surface [BS]), mineral apposition rate (MAR), and bone formation rate (BFR/BS) on the endocortical surface in loaded tibias of control animals compared with nonloaded (left) tibias. Verapamil and nifedipine suppressed the load-induced increase in BFR/BS observed in vehicle-treated controls by 56-61% (p < 0.01) and 56-74% (p < 0.01), respectively. Yet, significant differences in MS/BS and BFR/BS between right and left limbs were found in verapamil- and nifedipine-treated animals, indicating that the treatments did not completely abolish load-induced bone formation. This study shows that blocking the L-type calcium channel in vivo substantially suppresses the mechanically induced increase in bone formation that normally would occur and suggests that the L-type calcium channel mediates mechanically induced bone adaptation in vivo. PMID- 12369784 TI - Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization. AB - It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular P, and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5-7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary. PMID- 12369785 TI - Strong static magnetic field stimulates bone formation to a definite orientation in vitro and in vivo. AB - The induction of bone formation to an intentional orientation is a potentially viable clinical treatment for bone disorders. Among the many chemical and physical factors, a static magnetic field (SMF) of tesla order can regulate the shapes of blood cells and matrix fibers. This study investigated the effects of a strong SMF (8 T) on bone formation in both in vivo and in vitro systems. After 60 h of exposure to the SMF, cultured mouse osteoblastic MC3T3-E1 cells were transformed to rodlike shapes and were orientated in the direction parallel to the magnetic field. Although this strong SMF exposure did not affect cell proliferation, it up-regulated cell differentiation and matrix synthesis as determined by ALP and alizarin red stainings, respectively. The SMF also stimulated ectopic bone formation in and around subcutaneously implanted bone morphogenetic protein (BMP) 2-containing pellets in mice, in which the orientation of bone formation was parallel to the magnetic field. It is concluded that a strong SMF has the potency not only to stimulate bone formation, but also to regulate its orientation in both in vitro and in vivo models. This is the first study to show the regulation of the orientation of adherent cells by a magnetic field. We propose that the combination of a strong SMF and a potent osteogenic agent such as BMP possibly may lead to an effective treatment of bone fractures and defects. PMID- 12369786 TI - Dentin matrix protein 1, a target molecule for Cbfa1 in bone, is a unique bone marker gene. AB - Dentin matrix protein 1 (Dmp1), a phosphoprotein highly linked to dentin formation, has also been reported to be expressed in the skeleton. However, the role of Dmp1 in skeletal tissues remains unclear. To clarify the role of Dmp1 in bone formation, we characterized the expression profile of Dmp1 in bone and cartilage and examined whether Dmp1 expression was regulated by core-binding factor a1 (Cbfa1). Studies of fetal rat calvarial (FRC) cell cultures showed that the expression of Dmp1 was associated closely with "bone nodule" formation and mineralization in vitro. In situ hybridization studies were performed to examine the spatial and temporal expression patterns of Dmp1 during development in mouse embryos from 12.5 day postcoitus (dpc) to 8 weeks postnatal; these studies showed that Dmp1 first appeared in hypertrophic cartilage cells, followed by osteoblasts, and later was expressed strongly in osteocytes. The expression profiles of Cbfa1 and Dmp1 overlapped in both cartilage and bone during development, with Cbfa1 preceding Dmp1. Examination of Dmp1 expression in Cbfa1-/ mice revealed that Dmp1 was absent in the developing bones of Cbfa1-null mice, whereas there was essentially no change in Dmp1 expression in the arrested tooth bud. Transient transfection studies showed forced expression of Dmp1 under the control of Cbfa1 and gel shift data indicated the presence of a functional osteocalcin-specific element (OSE)-2 response element in the Dmp1 proximal promoter region. However, in vitro promoter studies suggested that regulation of Dmp1 by Cbfa1 was not mediated by direct binding of Cbfa1 to this site and may be through indirect mechanisms. These studies highlight Dmp1 as a unique marker gene for osteoblastic differentiation. The close association of Dmp1 and Cbfa1 in the developing skeleton suggests that Dmp1 may play an important role in bone formation. PMID- 12369787 TI - Establishment of a novel chondrocytic cell line N1511 derived from p53-null mice. AB - We established a clonal chondrocytic cell line N1511 derived from rib cartilage of a p53-null mouse. N1511 cells proliferated in polygonal shape and elicited differentiation at confluence when treated with combination of bone morphogenetic protein (BMP) 2 and insulin or parathyroid hormone (PTH) and dexamethasone. BMP 2/insulin-treated cells became refractile without forming cartilaginous nodules and reached terminal differentiation, became positive for alizarin red staining, and developed considerable ALP activity. In contrast, PTH/dexamethasone-treated cells formed Alcian blue-positive nodules but remained negative for alizarin red staining and ALP activity. Northern blot analysis revealed that BMP-2/insulin treated cells sequentially expressed type II, IX, and X collagens, whereas PTH/dexamethasone-treated cells slowly expressed type II collagen and then type IX, and they did not exhibit type X collagen expression. These results show that BMP-2/insulin treatment induces full differentiation toward hypertrophy, whereas treatment with PTH/dexamethasone slows and limits differentiation. Recovery of p53 expression in N1511 cells by transient transfection inhibited cell proliferation, suggesting that cell proliferation could be regulated with p53 in this cell line. These results indicate that N1511 is the only cell line with known genetic mutation, which undergoes multiple steps of chondrocyte differentiation toward hypertrophy, and because proliferation could be regulated by expression of p53, N1511 could be an excellent model for studies of chondrogenesis, the function of p53, and genetic engineering of cartilage tissue. PMID- 12369788 TI - Accelerated chondrogenesis of the rabbit cranial base growth plate by oscillatory mechanical stimuli. AB - How mechanical stimuli modulate chondral growth is not well understood. To test a hypothesis that chondral growth is accelerated by oscillatory mechanical stimuli rather than the peak magnitude of mechanical force, we delivered 2-N tensile forces with static (frequency = 0 Hz) and cyclic (f = 1 Hz) profiles noninvasively to the maxillae of growing New Zealand white rabbits for 20 minutes/day over 12 days. Computerized histomorphometry revealed significantly greater maximum height of the cranial base growth plate (GP) treated with cyclic forces (870 +/- 130 microm) than static forces (654 +/- 29 microm) and sham controls (566 +/- 47 microm). In addition, the average total GP area treated with cyclic forces (2.63 +/- 0.17 mm2) was significantly greater than static forces (2.12 +/- 0.99 mm2) and sham controls (1.65 +/- 0.13 mm2). The proliferating zone of GPs treated with cyclic forces (158 +/- 38.5 microm) was significantly longer than the corresponding zones of static forces (117 +/- 8.6 microm) and sham controls (54 +/- 14.9 microm). The average number of chondrocytes in the proliferating zone treated with cyclic forces (1045 +/- 127) was significantly greater than static forces (632 +/- 85) and sham controls (632 +/- 60) in standardized grids. Like natural GPs, the cartilage matrix treated with cyclic and static tensile forces consisted of abundant aggrecan-like proteoglycans. These findings indicate that oscillatory components of mechanical force rather than its peak magnitude are potent anabolic stimulus for chondral growth. A cascade of oscillatory mechanical stimuli is likely capable of engineering chondral growth beyond naturally occurring chondrogenesis. PMID- 12369789 TI - Annexin VIII is differentially expressed by chondrocytes in the mammalian growth plate during endochondral ossification and in osteoarthritic cartilage. AB - Endochondral ossification is the developmental process that leads to the formation and coordinated longitudinal growth of the majority of the vertebrate skeleton. Central to this process is chondrocyte differentiation occurring in the growth plate that lies at the junction between the epiphyseal cartilage and the bone. To identify novel factors involved in this differentiation process, suppression subtractive hybridization was performed to amplify preferentially cDNAs uniquely expressed in fetal bovine growth plate chondrocytes as opposed to epiphyseal chondrocytes. The subtracted product was used to screen a fetal bovine chondrocyte cDNA library. One of the cDNA clones identified encoded the bovine orthologue of annexin VIII, a protein not previously described in the growth plate. Northern and Western blotting confirmed that annexin VIII was expressed by growth plate chondrocytes and not by epiphyseal chondrocytes. Immunohistochemistry of the fetal bovine growth plate identified a gradient of increasing annexin VIII protein from the proliferative to the hypertrophic zone. Immunofluorescence localized annexin VIII largely to the chondrocyte cell membrane. In a preliminary study, we examined the distribution of annexin VIII in normal and osteoarthritic (OA) articular cartilage. In OA cartilage, the protein was located in a subset of mid- to deep zone chondrocytes and in the matrix surrounding these cells; no annexin VIII was detected in normal articular cartilage. Thus annexin VIII is a marker for chondrocyte differentiation during normal endochondral ossification and may act as a marker for cells undergoing inappropriate differentiation in OA. PMID- 12369790 TI - Basic fibroblast growth factor stimulates osteoclast recruitment, development, and bone pit resorption in association with angiogenesis in vivo on the chick chorioallantoic membrane and activates isolated avian osteoclast resorption in vitro. AB - Increased local osteoclast (OC)-mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor-associated osteolysis and inflammatory related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre-OC to emigrate from the circulation and develop into bone-resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo. Angiogenesis elicited by bFGF on the CAM was accompanied by increased OC formation and bone pit resorption (both overall and on a per OC basis) on the bone implants in vivo. In complementary in vitro assays, bFGF did not directly stimulate avian OC development from bone marrow mononuclear cell precursors, consistent with their low mRNA expression of the four avian signaling FGF receptors (FGFR)-1, FGFR-2, FGFR-3, and FGFR-like embryonic kinase (FREK). In contrast, bFGF activated isolated avian OC bone pit resorption via mechanisms inhibited by a selective cyclo-oxygenase (COX)-2 prostaglandin inhibitor (NS-398) or p42/p44 MAPK activation inhibitor (PD98059), consistent with a relatively high expression of FGFR-1 by differentiated avian OCs. Thus, bFGF may sensitively regulate local bone resorption and remodeling through direct and indirect mechanisms that promote angiogenesis and OC recruitment, formation, differentiation, and activated bone pit resorption. The potential for bFGF to coinduce angiogenesis and OC bone remodeling may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis. Alternatively, inhibiting such bFGF-dependent processes may aid in the treatment of inflammatory related or metastatic bone loss. PMID- 12369791 TI - Involvement of cyclic adenosine monophosphate-dependent protein kinase A and pertussis toxin-sensitive G proteins in the migratory response of human CD14+ mononuclear cells to katacalcin. AB - Katacalcin (KC) belongs to a small family of polypeptides that are encoded by the calc-1 gene and also include calcitonin (CT) and procalcitonin NH2-terminal cleavage peptide (N-ProCT). Biological roles of KC or N-ProCT are unknown. To determine whether these polypeptides affect leukocyte function, forearm venous blood polymorphonuclear neutrophils and CD14+ peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donors. Cell migration was assessed in a blindwell chemotaxis chamber using nitrocellulose micropore filters. Cellular levels of cyclic adenosine monophosphate (cAMP) were measured by HPLC; activation of protein kinase A was studied by Western blot. Fluorochrome-labeled peptide binding to cells was studied by fluorescence-activated cell sorting (FACS) and intracellular calcium transients were studied by confocal microscopy with FLUO-3. KC elicited concentration-dependent migration of CD14+ PBMC at concentrations from the atomolar to the micromolar range and deactivated attractant-induced chemotaxis. CT N-terminal flanking peptide had no such effect. Neutrophils did not migrate toward any of those peptides and their oxygen-free radical release was not affected as measured fluorometrically. Functional responses of CD14+ PBMC to KC correlated to forskolin-sensitive cAMP accumulation in cells and were inhibited by protein kinase A inhibitor (PKI) and Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate. Treatment of CD14+ PBMC with KC activated protein kinase A(C alpha). Intracellular calcium was decreased with CT, KC, and procalcitonin (PCT). Binding studies showed that KC might share the binding site with CT and PCT. Data indicate that KC regulates human CD14+ PBMC migration via signaling events involving protein kinase A-dependent cAMP pathways. PMID- 12369792 TI - Three-dimensional-line skeleton graph analysis of high-resolution magnetic resonance images: a validation study from 34-microm-resolution microcomputed tomography. AB - The resolution achievable in vivo by magnetic resonance imaging (MRI) techniques is not sufficient to depict precisely individual trabeculae and, thus, does not permit the quantification of the "true" trabecular bone morphology and topology. Nevertheless, the characterization of the "apparent" trabecular bone network derived from high-resolution MR images (MRIs) and their potential to provide information in addition to bone mineral density (BMD) alone has been established in studies of osteoporosis. The aim of this work was to show the ability of the three-dimensional-line skeleton graph analysis (3D-LSGA) to characterize high resolution MRIs of trabecular bone structure. Fifteen trabecular bone samples of the distal radius were imaged using the high-resolution MRI (156 x 156 x 300 microm3) and microcomputed tomography (microCT; 34 x 34 x 34 microm3). After thresholding, the 3D skeleton graph of each binary image was obtained. To remove the assimilated-noise branches of the skeleton graph and smooth this skeleton graph before it was analyzed, we defined a smoothing length criterion (l(c)), such that all "termini" branches having a length lower than l(c) were removed. Local topological and morphological LSGA measurements were performed from MRIs and microCT images of the same samples. The correlations between these two sets of measurements were dependent on the smoothing criterion l(c), reaching R2 = 0.85 for topological measurements and R2 = 0.57-0.64 for morphological measurements. 3D-LSGA technique could be applied to in vivo high-resolution MRIs of trabecular bone structure, giving an indirect characterization of the microtrabecular bone network. PMID- 12369793 TI - Relationship of serum leptin concentration with bone mineral density in the United States population. AB - Overweight is associated with both higher bone mineral density (BMD) and higher serum leptin concentrations. In humans, little is known about the relationship of leptin concentration and bone density. We studied this relationship in a large, national population-based sample. Participants included 5815 adults in the Third U.S. National Health and Nutrition Examination Survey (NHANES III; 1988-1994) who underwent DXA of the proximal femur and measurement of fasting serum leptin. Mean +/- SE BMD (gm/cm2) of the total hip was 1.01 +/- 0.005 in men, 0.94 +/- 0.004 in premenopausal women, and 0.78 +/- 0.007 in postmenopausal women. Bone density increased with increasing leptin concentration in men (p = 0.003), premenopausal women (p < 0.001), and postmenopausal women (p < 0.001). However, after adjusting for body mass index (BMI) and other bone density-related factors, an inverse association emerged in men (p < 0.001), being most evident among men < 60 years old. There was no association of leptin and BMD in premenopausal women (p = 0.66) or postmenopausal women (p = 0.69) in multivariate analysis. Controlling for leptin had no effect on the strong positive association of BMI and BMD in either men or women. Serum leptin concentration did not appear to affect directly BMD. If present, the association appeared to be limited to younger men who are at lower risk of osteoporosis. PMID- 12369794 TI - Effects of genistein and hormone-replacement therapy on bone loss in early postmenopausal women: a randomized double-blind placebo-controlled study. AB - The natural isoflavone phytoestrogen genistein has been shown to stimulate osteoblastic bone formation, inhibit osteoclastic bone resorption, and prevent bone loss in ovariectomized rats. However, no controlled clinical trial has been performed so far to evaluate the effects of the phytoestrogen on bone loss in postmenopausal women. We performed a randomized double-blind placebo-controlled study to evaluate and compare with hormone-replacement therapy (HRT) the effect of the phytoestrogen genistein on bone metabolism and bone mineral density (BMD) in postmenopausal women. Participants were 90 healthy ambulatory women who were 47-57 years of age, with a BMD at the femoral neck of <0.795 g/cm2. After a 4 week stabilization on a standard fat-reduced diet, participants of the study were randomly assigned to receive continuous HRT for 1 year (n = 30; 1 mg of 17beta estradiol [E2] combined with 0.5 mg of norethisterone acetate), the phytoestrogen genistein (n = 30; 54 mg/day), or placebo (n = 30). Urinary excretion of pyridinoline (PYR) and deoxypyridinoline (DPYR) was not significantly modified by placebo administration either at 6 months or at 12 months. Genistein treatment significantly reduced the excretion of pyridinium cross-links at 6 months (PYR = 54 +/- 10%; DPYR = -55 +/- 13%; p < 0.001) and 12 months (PYR = -42 +/- 12%; DPYR = -44 +/- 16%; p < 0.001). A similar and not statistically different decrease in excretion of pyridinium cross-links was also observed in the postmenopausal women randomized to receive HRT. Placebo administration did not change the serum levels of the bone-specific ALP (B-ALP) and osteocalcin (bone Gla protein [BGP]). In contrast, administration of genistein markedly increased serum B-ALP and BGP either at 6 months (B-ALP = 23 +/- 4%; BGP = 29 +/- 11%; p < 0.005) or at 12 months (B-ALP = 25 +/- 7%; BGP = 37 +/- 16%; p < 0.05). Postmenopausal women treated with HRT had, in contrast, decreased serum B-ALP and BGP levels either at 6 months (B-ALP = -17 +/- 6%; BGP = -20 +/- 9%; p < 0.001) or 12 months (B-ALP = 20 +/- 5%; BGP = -22 +/- 10%; p < 0.001). Furthermore, at the end of the experimental period, genistein and HRT significantly increased BMD in the femur (femoral neck: genistein = 3.6 +/- 3%, HRT = 2.4 +/- 2%, placebo = -0.65 +/- 0.1%, and p < 0.001) and lumbar spine (genistein = 3 +/- 2%, HRT = 3.8 +/- 2.7%, placebo = -1.6 +/- 0.3%, and p < 0.001). This study confirms the genistein positive effects on bone loss already observed in the experimental models of osteoporosis and indicates that the phytoestrogen reduces bone resorption and increases bone formation in postmenopausal women. PMID- 12369795 TI - Agreement or disagreement. PMID- 12369796 TI - Parathyroid hormone type 1 receptor and human osteoclasts. PMID- 12369797 TI - Behavioral analysis of the saccharin deprivation effect in rats. AB - The deprivation effect (DE)--an increase in the level of free-choice consumption of alcohol after a period of forced abstinence--may reflect relapselike drinking and be relevant for modeling alcohol abuse. However, the behavioral mechanisms of the DE are unclear. In these experiments, rats had unlimited free-choice access to water and saccharin-containing solutions and underwent repeated episodes of saccharin deprivation. It was found that DE magnitude correlates positively with the deprivation phase duration, expression of the DE is highly context dependent, and the DE can be prevented by extinguishing response to the saccharin-associated stimuli. Thus, DE procedures may be useful for studying the effects of continued exposure to stimuli associated with various primary reinforcers such as drugs of abuse. PMID- 12369798 TI - Brief access to sucrose engages food-entrainable rhythms in food-deprived rats. AB - Two studies examined whether brief access to sucrose solutions engages food entrainable rhythms when pitted against a light-dark cycle. In Experiment 1, 32% sucrose was given once daily for 5-6 min at Zeitgeber Time 4 (ZT 4) for 17-18 days to 8 food-deprived and 8 ad-lib-fed rats. Chow was provided at ZT 16. Temperature, wheel running, activity, and corticosterone (B) anticipated sucrose only in food-deprived rats. Ad-lib rats were similar to controls. In Experiment 2, 32% sucrose was presented at ZT 4 for 5 or 10 min for 17 days to food-deprived rats. Chow was given at ZT 12. Exogenous cues were minimized. Both groups showed anticipation of sucrose, whereas room controls did not. After a shift to 4% sucrose for 8 days, the 10-min group showed signs of damping of these rhythms, whereas the 5-min group did not. Data indicated that brief access to sucrose engages food-entrainable rhythms in food-deprived rats. PMID- 12369799 TI - Flavor avoidance induced by LiCl and dexfenfluramine in rats and mice using nondeprivation protocols. AB - Using dexfenfluramine as unconditional stimulus (US), the authors confirmed that sham-operated and area postrema (AP)-lesioned rats form comparable conditioned flavor avoidances. When lithium chloride (LiCI) was used as the US, AP-lesioned rats did not learn to avoid a drug-paired flavor conditional stimulus (CS+). Sham operated, but not AP-lesioned, rats had low intakes of the placebo-paired flavor (CS-), which suggests that the lesions disrupted generalization of avoidance. Generalized avoidance in intact rats was similar when either sweetened milk or Polycose was used as the caloric vehicle for the CSs. When flavored gels of Polycose were used as CSs, C57BL/6J mice developed flavor avoidance with either LiCl or dexfenfluramine as US. Compared with rats, mice required higher doses of these agents, avoidance was not complete after many pairings, and there was no generalization to the CS-. PMID- 12369800 TI - Ventral tegmental area dopamine neurons mediate the shock sensitization of acoustic startle: a potential site of action for benzodiazepine anxiolytics. AB - Dopamine (DA)-containing neurons in the ventral tegmental area (VTA) are thought to play an important role in fear motivation. The primary objective of the present study was to determine the connection between DA D2, gamma aminobutyric acid (GABA)A, and benzodiazepine receptors in the VTA and footshock-associated emotionality. Microinfusion of the DA D2 receptor agonist quinpirole. the GABA(A) receptor agonist muscimol, and the benzodiazepine receptor agonist flurazepam into the VTA was observed to suppress the shock enhancement of acoustic startle amplitudes. None of the drugs depressed baseline startle responding or footshock reactivity. The results indicate the involvement of VTA DA neurons in the fear arousing properties of footshock and implicate the VTA as a possible neural site for the anxiolytic actions of benzodiazepines. PMID- 12369801 TI - The effects of electrical stimulation of the nucleus basalis on the electroencephalogram, heart rate, and respiration. AB - The nucleus basalis (NB) mediates cortical electroencephalograph (EEG) activation; NB stimulation also modulates cortical responses to sensory stimuli and can induce learning-related receptive field plasticity. However, little is known about the behavioral effects of NB stimulation. This study concerns the effects of NB stimulation on cardiac and respiratory behavior and quantifies its EEG effects in freely moving rats. The EEG exhibited stimulation-induced decreases in theta and alpha power and increases in gamma power. NB stimulation elicited biphasic heart rate changes and disrupted ongoing respiration patterns. Neither EEG nor behavioral effects exhibited habituation or facilitation. These results indicate that the NB may serve not only as a cortical, but also as a behavioral, activation system that is normally engaged during learning. PMID- 12369802 TI - Appetitive conditioning-induced plasticity is expressed during paradoxical sleep in the medial geniculate, but not in the lateral amygdala. AB - This study examined whether neurons in the medial division of the medial geniculate (MGm) and the dorsal part of the lateral amygdala (LAd) express learning-induced plasticity in paradoxical sleep (PS) after appetitive conditioning, as they do in PS after fear conditioning. Rats received tone-food pairings in 3 sessions. After each session, the tone was presented at a nonawakening intensity during PS. Multiunit activity was simultaneously recorded in MGm and LAd. During waking, increases in tone-evoked discharges developed in MGm and LAd; however, as training continued, they lessened in LAd, but not in MGm. During PS, conditioned tone responses were expressed in MGm, but not in LAd. Thus, these results demonstrate dissociation of MGm and LAd plasticity. Moreover, compared with fear conditioning results, they suggest that expression of amygdalar plasticity in PS depends on the emotional salience of the stimulus. PMID- 12369803 TI - Novel factors contributing to the expression of latent inhibition. AB - Behavioral and neural correlates of latent inhibition (LI) during eyeblink conditioning were studied in 2 experiments. In Experiment 1, rabbits (Oryctolagus cuniculus) were conditioned after 8 days of tone conditioned stimulus (CS) presentations or 8 days of context-alone experience. LI was seen in the CS preexposed rabbits when a relatively intense (5 psi) airpuff unconditioned stimulus was paired with the CS. In Experiment 2, rabbits were given 0, 4, or 8 days of CS preexposures or context-alone experience. Hippocampal activity was monitored from the 8-day CS- or context-exposure rabbits. The LI effect was seen only in rabbits given 4 days of CS preexposure, thus suggesting that LI depended largely on the rate of acquisition in the context-preexposed control group. The neural recordings showed that the hippocampus was sensitive to the relative novelty of the stimuli and the overall context, regardless of whether exposure to stimuli and context promoted LI. PMID- 12369804 TI - Coordination of grip and load forces during vertical point-to-point movements with a grasped object in Parkinson's disease. AB - Thirteen patients with Parkinson's disease and 13 age-matched control subjects performed vertical point-to-point arm movements with an instrumented object, starting and ending with the object being held stationary. All Parkinsonian patients were tested on medication. Parkinsonian patients retained all aspects of predictive grip force control. Compared with healthy controls, they generated similar static grip forces during stationary holding and similar force ratios between maximum grip and load force, reflecting effective grip force adjustments in relation to movement-induced inertial loads. Grip and load force maximums coincided very closely, indicating that temporal aspects of predictive grip force regulation were also unaffected. However, Parkinsonian subjects showed additional oscillations in acceleration and grip force due to tremor and produced significantly slower arm accelerations due to bradykinesia. The results suggest that Parkinson's disease does not significantly impair the anticipation of movement-induced load fluctuations during voluntary arm movements with a grasped object performed on medication. PMID- 12369805 TI - Effects of age on virtual environment place navigation and allocentric cognitive mapping. AB - This study assessed age differences in navigational behavior in a virtual Morris water maze (vMWM) and examined the ability of older adults to develop cognitive maps after vMWM experience. Compared with younger participants, older volunteers traversed a longer linear distance to locate the hidden platform. On the probe trial, younger volunteers spent a greater proportion of their total distance traveled in proximity to the platform and had more platform intersections. Analysis of map reproductions demonstrated that older participants used proximal objects to locate the goal but did not use room-geometry cues to aid navigation. These findings demonstrate age-related deficits on a laboratory measure of place learning and suggest that deficiencies in allocentric mapping may contribute to these deficits. PMID- 12369806 TI - The effects of NMDA lesions centered on the postrhinal cortex on spatial memory tasks in the rat. AB - Rats with bilateral N-methyl-D-aspartate lesions centered on the postrhinal cortex (POR) and sham lesions were tested in a series of spatial memory tasks. The POR-lesioned rats were significantly impaired compared with sham rats in the reference memory version of both the water maze and radial arm maze tasks and in the standard radial arm maze working memory task. The POR-lesioned rats displayed a delay-independent impairment in the working memory versions of the water maze and in a delayed nonmatching-to-place (DNMP) version of the radial arm maze task. The POR-lesioned rats were also impaired in a DNMP procedure conducted in the T maze. These findings indicate that the POR has a delay-independent role in the processing of spatial information. PMID- 12369807 TI - Two forms of spatial memory: a double dissociation between the parietal cortex and the hippocampus in the rat. AB - Two variants of a continuous recognition training procedure were designed in order to query 2 forms of spatial memory. A continuous reinforcement condition (reflecting perceptual memory) and a differential reinforcement condition (reflecting episodic-like memory) were used to test rats on a 12-arm radial maze. After total hippocampal lesions, rats demonstrated intact performance on the continuous reinforcement condition, but impaired performance on the differential reinforcement condition. After parietal lesions, rats demonstrated the reverse pattern of performance: impaired performance on the continuous reinforcement condition and intact performance on the differential reinforcement condition. Thus, a double dissociation appears to exist between parietal cortex and hippocampus for the continuous reinforcement condition (reflecting perceptual memory) versus the differential reinforcement condition (reflecting episodic memory) for spatial location information. PMID- 12369808 TI - Double dissociation of function within the hippocampus: spatial memory and hyponeophagia. AB - Complete and dorsal hippocampal lesions impaired spatial performance on 2 working memory tasks: rewarded alternation on the T maze and matching to position in the water maze. In contrast, ventral hippocampal lesions had no effect on these tasks, even when task difficulty was increased by the introduction of delays. Ventral lesions did resemble complete lesions in reducing anxiety in 3 commonly used tests of anxiety (social interaction, plus-maze, and hyponeophagia). Dorsal lesions also appeared to be anxiolytic in the social interaction and plus-maze tests, but they did not affect hyponeophagia. Complete- and dorsal-lesioned rats displayed hyperactivity, whereas ventral-lesioned rats did not. These results show a double dissociation between dorsal and ventral hippocampal lesions (hyponeophagia vs. spatial memory), suggesting differentiation of function along the septotemporal axis of this structure. PMID- 12369809 TI - Long-term estrogen therapy worsens the behavioral and neuropathological consequences of chronic brain inflammation. AB - Alzheimer's disease (AD) is accompanied by chronic neuroinflammation and occurs with greater incidence in postmenopausal women. The increased incidence may be delayed by estrogen replacement therapy (ERT). The authors investigated the interaction of chronic ERT and lipopolysaccharide (LPS)-induced neuroinflammation in the female rat. Ovariectomy did not impair water maze performance; however, addition of chronic ERT or neuroinflammation resulted in an impairment that became exacerbated by the simultaneous occurrence of both conditions. Chronic LPS activated microglia, which was not reduced by ERT. Intact females receiving LPS infusion were not impaired in the water maze and had significantly fewer activated microglia. Results suggest that chronic ERT in postmenopausal women may exacerbate the memory impairment induced by the chronic neuroinflammation associated with AD. PMID- 12369810 TI - The union of the state: myoclonic twitching is coupled with nuchal muscle atonia in infant rats. AB - Active sleep (AS), as measured by the occurrence of myoclonic twitching (MT), is the most prevalent behavioral state in newborn rats. Historically, AS has been considered a developmental precursor of REM sleep, but recently this idea has been questioned. In the present study, the authors assess, in 2-, 5-, and 8-day old rats, the relationship between MT and nuchal muscle atonia, a widely recognized component of REM sleep. At all ages, muscle atonia preceded MT and persisted until awake behaviors occurred. In addition, muscle tone decreased gradually during transitions from awake behavior to twitching. Thus, MT during infancy occurs against a backdrop of muscle atonia, a result that is consistent with the view that AS is a developmental precursor of REM sleep. PMID- 12369811 TI - Fourth ventricular CART reduces food and water intake and produces a conditioned taste aversion in rats. AB - Cocaine- and amphetamine-regulated transcript peptide (CART) reduces rats' intake of liquid diet if the peptide reaches the 4th ventricle (4V). To test for specificity of 4V CART effects on feeding, the authors compared its ability to reduce intakes of liquid diet and water and tested for conditioned taste aversion (CTA). CART reduced 30-min intakes of both water and Ensure at a threshold of 1 microg. Lithium chloride (0.15 M, 20 ml/kg i.p.) and 4V CART (1 microg) paired with novel saccharin solution reduced saccharin preferences similarly in subsequent 2-bottle tests, compared with saline. Thus, CART can produce CTA. These data demonstrate that 4V CART's actions in ingestive behavior are not specific to nutrients and suggest that aspects of 4V CART's actions in reducing intake may be secondary to the production of an aversive state. PMID- 12369812 TI - Posttraining intra-basolateral amygdala scopolamine impairs food- and amphetamine induced conditioned place preferences. AB - The present study investigated the role of cholinergic muscarinic receptor function within the basolateral amygdala memory in the consolidation of conditioned place preference (CPP) memory. Adult male Long-Evans rats were confined to treatment- or nontreatment-paired compartments for 30 min on 4 alternating days. After training, rats received intrabasolateral amygdala infusions of scopolamine (2.5 microg or 5.0 microg/0.5 microl) or saline. The rats were then given a 20-min test session, and the time spent in each of the compartments was recorded. Immediate posttraining (but not delayed 2 hr) scopolamine (5.0 microg) blocked acquisition of food- and amphetamine-induced CPPs. The findings indicate a time-dependent role for basolateral amygdala muscarinic receptors in memory consolidation underlying CPPs for natural and drug rewards. PMID- 12369813 TI - Low levels of estradiol facilitate, whereas high levels of estradiol impair, working memory performance on the radial arm maze. AB - Previous investigations of estradiol's effects on learning and memory yielded equivocal results. This study was designed to determine whether these inconsistencies were due to dose-dependent effects of estradiol on different memory processes. Ovariectomized female rats were injected daily with estradiol benzoate (EB; 0.32, 1.00, or 5.00 microg) or vehicle. Approximately 3 hr after injection, rats were run on a hippocampus-dependent working/reference memory version of the radial arm maze. Total number of working (WME), reference, and combined working/reference memory errors were scored. Compared with vehicle, 1.00 or 5.00 microg EB (high physiological) impaired performance by increasing the number of WME, whereas 0.32 microg EB (low physiological) facilitated performance by decreasing the number of WME. Taken together, these data demonstrate a dose dependent effect of EB on working memory. PMID- 12369814 TI - The GAAA tetraloop-receptor interaction contributes differentially to folding thermodynamics and kinetics for the P4-P6 RNA domain. AB - Tetraloops with the generic sequence GNRA are commonly found in RNA secondary structure, and interactions of such tetraloops with "receptors" elsewhere in RNA play important roles in RNA structure and folding. However, the contributions of tetraloop-receptor interactions specifically to the kinetics of RNA tertiary folding, rather than the thermodynamics of maintaining tertiary structure once folded, have not been reported. Here we investigate the role of the key GAAA tetraloop-receptor motif in folding of the P4-P6 domain of the Tetrahymena group I intron RNA. Insertions of one or more nucleotides into the tetraloop significantly disrupt the thermodynamics of tertiary folding; single-nucleotide insertions shift the folding free energy by 2-4 kcal/mol (DeltaDeltaG(o)'). The folding kinetics of several modified P4-P6 domains were determined by stopped flow fluorescence spectroscopy, using an internally incorporated pyrene residue as the chromophore. In contrast to the thermodynamic results, the kinetics of Mg(2+)-induced folding were barely affected by the tetraloop modifications, with a DeltaDeltaG(++) of 0.2-0.4 kcal/mol and a Phi value (ratio of the kinetic and thermodynamic contributions) of <0.1. These data indicate an early transition state for folding of P4-P6 with respect to forming the tetraloop-receptor contact, consistent with previous results for modifications elsewhere in P4-P6. We conclude that the GAAA tetraloop-receptor motif contributes little to the stabilization of the transition state for Mg(2+)-induced P4-P6 folding. Rather, the tetraloop-receptor motif acts to clamp the RNA once folding has occurred. This is the first report to correlate the kinetic and thermodynamic contributions of an important RNA tertiary motif, the GNRA tetraloop-receptor. The results are related to possible models for the Mg(2+)-induced folding of the P4-P6 RNA, including a model invoking rapid nonspecific electrostatic collapse. PMID- 12369815 TI - Locally disordered conformer of the hamster prion protein: a crucial intermediate to PrPSc? AB - A crucial step for transformation of the normal cellular isoform of the prion protein (PrP(C)) to the infectious prion protein (PrP(Sc)) is thought to entail a previously uncharacterized intermediate conformer, PrP*, which interacts with a template PrP(Sc) molecule in the conversion process. By carrying out (15)N-(1)H two-dimensional NMR measurements under variable pressure on Syrian hamster prion protein rPrP(90-231), we found a metastable conformer of PrP(C) coexisting at a population of approximately 1% at pH 5.2 and 30 degrees C, in which helices B and C are preferentially disordered. While the identity is still unproven, this observed metastable conformer is most logically PrP* or a closely related precursor. The structural characteristics of this metastable conformer are consistent with available immunological and pathological information about the prion protein. PMID- 12369816 TI - NMR solution structure of ATTp, an Arabidopsis thaliana trypsin inhibitor. AB - The three-dimensional structure of the precursor form of the Arabidopsis thaliana trypsin inhibitor (ATT(p), GenBank entry Z46816), a 68-residue (approximately 7.5 kDa) rapeseed class proteinase inhibitor, has been determined in solution at pH 5.0 and 25 degrees C by multinuclear magnetic resonance spectroscopy. The protein contains one alpha-helix and two strands of antiparallel beta-sheet, with a type IV beta-turn connecting the two strands. The alpha-helix and the inhibitory loop are connected to the beta-sheet through three disulfide bridges; a fourth disulfide bridge connects the N- and C-termini. The overall structural topology of ATT(p) is similar to those of the sweet tasting protein brazzein (rmsd of 3.0 A) and the antifungal protein Rs-Afp1 [a knottin protein from radish (Raphanus sativus), rmsd of 2.7 A]. The precursor segment in ATT(p) is disordered, as visualized by the final 20-conformer ensemble and as confirmed by (15)N heteronuclear NOE analysis. The overall fold of ATT(p) is distinct from those of other classes of serine proteinase inhibitors except in the inhibitor loop; therefore, it represents a new inhibitor fold. PMID- 12369817 TI - Biochemical characterization and structural analysis of a highly proficient cocaine esterase. AB - The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site. PMID- 12369818 TI - Populating partially unfolded forms by hydrogen exchange-directed protein engineering. AB - The native-state hydrogen exchange of a redesigned apocytochrome b(562) suggests that at least two partially unfolded forms (PUFs) exist for this four-helix bundle protein under native conditions. The more stable PUF has the N-terminal helix unfolded. To verify the conclusion further and obtain more detailed structural information about this PUF, five hydrophobic core residues in the N terminal helix were mutated to Gly and Asp to destabilize the native state selectively and populate the PUF for structural studies. The secondary structure and the backbone dynamics of this mutant were characterized using multidimensional NMR. Consistent with the prediction, the N-terminal region of the mutant was found to be unfolded while other parts of the proteins remained folded. These results suggest that native-state hydrogen exchange-directed protein engineering can be a useful approach to populating partially unfolded forms for detailed structural studies. PMID- 12369819 TI - Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding. AB - (S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Active site structures of three homologous enzymes, including MDH, show the presence of two conserved arginine residues in close juxtaposition (Arg165 and Arg277 in MDH). Arg277 has an important catalytic role; it stabilizes both the ground and transition states through its positive charge as well as a hydrogen bond [Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065]. In this study, we examined the role of Arg165 and the overall importance of the Arg165/Arg277 pair. Single mutants at Arg165 as well as double mutants at Arg165 and Arg277 were characterized. Our results show that Arg165 has a role similar to, but less critical than, that of Arg277. It stabilizes the transition state through its positive charge and the ground state through a charge-independent interaction, most likely, a hydrogen bond. Though the k(cat)s for the charge-conserved mutants, R165K and R277K, were only 3-5-fold lower than those of wild-type MDH (wtMDH), the k(cat) for R165K/R277K was approximately 350-fold lower. Thus, at least one arginine residue is required for the optimal substrate orientation and catalysis. Stopped-flow studies show that the FMN reduction step is completely rate-limiting for both wtMDH and the arginine mutants, with the possible exception of R165E. Substrate isotope effects indicate that the carbon-hydrogen bond-breaking step is only partially rate-limiting for wtMDH but fully rate-limiting for the mutants. pH profiles of R165M conclusively show that the pK(a) of 9.3 in free wtMDH does not belong to Arg165. PMID- 12369820 TI - Aminophosphonate inhibitors of dialkylglycine decarboxylase: structural basis for slow binding inhibition. AB - The kinetics of inhibition of dialkylglycine decarboxylase by five aminophosphonate inhibitors are presented. Two of these [(R)-1-amino-1 methylpropanephosphonate and (S)-1-aminoethanephosphonate] are slow binding inhibitors. The inhibitors follow a mechanism in which a weak complex is rapidly formed, followed by slow isomerization to the tight complex. Here, the tight complexes are bound 10-fold more tightly than the weak, initial complexes. The slow onset inhibition occurs with t(1/2) values of 1.3 and 0.55 min at saturating inhibitor concentrations for the AMPP and S-AEP inhibitors, respectively, while dissociation of these inhibitor complexes occurs with t(1/2) values of 13 and 4.6 min, respectively. The X-ray structures of four of the inhibitors in complex with dialkylglycine decarboxylase have been determined to resolutions ranging from 2.6 to 2.0 A, and refined to R-factors of 14.5-19.5%. These structures show variation in the active site structure with inhibitor side chain size and slow binding character. It is proposed that the slow binding behavior originates in an isomerization from an initial complex in which the PLP pyridine nitrogen-D243 OD2 distance is approximately 2.9 A to one in which it is approximately 2.7 A. The angles that the C-P bonds make with the p orbitals of the aldimine pi system are correlated with the reactivities of the analogous amino acid substrates, suggesting a role for stereoelectronic effects in Schiff base reactivity. PMID- 12369821 TI - Mutations in the extracellular domain and in the membrane-spanning domains interfere with nicotinic acetylcholine receptor maturation. AB - The deg-3(u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects of this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3(u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutations in the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while others are likely to disrupt trafficking. PMID- 12369822 TI - The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal Cl-/HCO3- exchange to Na+/H+ exchange. AB - Intestinal electroneutral NaCl absorption is mediated by parallel operation of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange in the enterocyte apical membrane. The ion transporters involved are Na(+)/H(+) exchanger 3 (NHE3) and the down regulated in adenoma (dra) gene product. cAMP-mediated inhibition of NHE3 requires the transporter to bind to the second PDZ (PSD95, disk large, ZO1) domain of the adapter protein NHE3 kinase A regulatory protein (E3KARP). Because the C-terminal four amino acids of dra are ETKF (glutamate-threonine-lysine phenylalanine), resembling a PDZ interaction motif, we hypothesized that dra may also bind to one of the PDZ domains of E3KARP. In vitro the ETKF motif of dra binds to the second PDZ domain of E3KARP, the affinity being comparable to that of the known ligand CFTR. The C-terminal phenylalanine, which is an unconventional residue in PDZ interaction motifs, can only be substituted by the classical residue leucine, but not by other hydrophobic residues (valine, isoleucine). Immunofluorescence colocalizes dra, NHE3, and E3KARP in the apical compartment of human proximal colon. We suggest a model in which both NHE3 and dra bind to the second PDZ domain of E3KARP and that linking of the transporters occurs through dimerization of E3KARP. In such a model, the first PDZ domain would remain available for instance for signal transduction proteins. PMID- 12369823 TI - Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands. AB - A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic, and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of nonequilibrium UV-melting and annealing curves in the range of pH 5.1-6.7 in a 10 mM citrate/phosphate buffer containing 0.1 M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both the association and dissociation process in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability, as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications. PMID- 12369824 TI - Antiapoptotic Cdc42 mutants are potent activators of cellular transformation. AB - Cdc42 is a small GTP-binding protein which has been implicated in a number of cellular activities, including cell morphology, motility, cell-cycle progression, and malignant transformation. While GTPase-defective forms of Cdc42 inhibit cell growth, a mutation [Cdc42(F28L)] that allows the constitutive exchange of GDP for GTP and is GTPase-competent induces cellular transformation. These results suggest that Cdc42 must cycle between its GTP- and GDP-bound states to stimulate cell growth. In attempting to design Cdc42 molecules with more potent transforming activity, we set out to generate other types of Cdc42 mutants capable of constitutive GDP-GTP exchange. Here, we describe one such mutant, generated by changing a conserved aspartic acid residue at position 118 to an asparagine. The Cdc42(D118N) protein exchanges GDP for GTP more rapidly than wild type Cdc42, but significantly more slowly than the Cdc42(F28L) mutant. Despite its slower rate of activation, the Cdc42(D118N) mutant is more potent at inducing cellular transformation than the Cdc42(F28L) protein, and causes a significant loss in actin stress fibers, reminiscent of what is observed with fibroblasts transformed by oncogenic Ras mutants. Effector-loop mutations made within the D118N background inhibit Cdc42-induced transformation and Cdc42-mediated antiapoptotic (survival) activity to similar extents. In addition, mutating aspartic acid 121 (to asparagine), which forms part of a caspase cleavage site (DLRD, residues 118-121 of Cdc42), in combination with the F28L mutation generates a Cdc42 molecule [Cdc42(F28L/D121N)] with transforming activity significantly stronger than that of Cdc42(F28L). Thus, mutations that combine some capacity for cycling between the GTP- and GDP-bound states with increased survival against apoptotic signals yield Cdc42 molecules with the maximum capability for inducing cellular transformation. PMID- 12369825 TI - Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives. AB - Tachyplesin I is a 17-residue peptide isolated from the horseshoe crab, Tachypleus tridentatus. It has high antimicrobial activity and adopts a beta hairpin conformation in solution stabilized by two cross-strand disulfide bonds. We report an NMR structural investigation of wild-type tachyplesin I and three linear derivatives (denoted TPY4, TPF4, and TPA4 in which the bridging cysteine residues are uniformly replaced with tyrosine, phenylalanine, and alanine, respectively). The three-dimensional aqueous solution structures of the wild type and the active variant TPY4 reveal very similar beta-hairpin conformations. In contrast, the inactive variant TPA4 is unstructured in solution. The arrangement of the tyrosine side chains in the TPY4 structure suggests that the beta-hairpin is stabilized by aromatic ring stacking interactions. This is supported by experiments in which the beta-hairpin structure of TPF4 is disrupted by the addition of phenol, but not by the addition of an equimolar amount of cyclohexanol. We have also determined the structures of wild-type tachyplesin I and TPY4 in the presence of dodecylphosphocholine micelles. Both peptides undergo significant conformational rearrangement upon micelle association. Analysis of the micelle-associated peptide structures shows an increased level of exposure of specific hydrophobic side chains and an increased hydrophobic integy moment. Comparison of the structures in micelle and aqueous solution for both wild-type tachyplesin I and TPY4 reveals two requirements for high antimicrobial activity: a beta-hairpin fold in solution and the ability to rearrange critical side chain residues upon membrane association. PMID- 12369826 TI - Specificity of the basic side chains of Lys114, Lys125, and Arg129 of antithrombin in heparin binding. AB - The anticoagulant polysaccharide heparin binds and activates the plasma proteinase inhibitor antithrombin through a pentasaccharide sequence. Lys114, Lys125, and Arg129 are the three most important residues of the inhibitor for pentasaccharide binding. To elucidate to what extent another positively charged side chain can fulfill the role of each of these residues, we have mutated Lys114 and Lys125 to Arg and Arg129 to Lys. Lys114 could be reasonably well replaced with Arg with only an approximately 15-fold decrease in pentasaccharide affinity, in contrast to an approximately 10(5)-fold decrease caused by substitution with an noncharged amino acid of comparable size. However, a loss of approximately one ionic interaction on mutation to Arg indicates that the optimal configuration of the network of basic residues of antithrombin that together interact with the pentasaccharide requires a Lys in position 114. Replacement of Lys125 with Arg caused an even smaller, approximately 3-fold, decrease in pentasaccharide affinity, compared with that of approximately 400-fold caused by mutation to a neutral amino acid. An Arg in position 125 is thus essentially equivalent to the wild-type Lys in pentasaccharide binding. Substitution of Arg129 with Lys decreased the pentasaccharide affinity an appreciable approximately 100-fold, a loss approaching that of approximately 400-fold caused by substitution with a neutral amino acid. Arg is thus specifically required in position 129 for high affinity pentasaccharide binding. This requirement is most likely due to the ability of Arg to interact with other residues of antithrombin, primarily, Glu414 and Thr44, in a manner that appropriately positions the Arg side chain for keeping the pentasaccharide anchored to the activated state of the inhibitor. PMID- 12369827 TI - The Mg2+ requirements for rho transcription termination factor: catalysis and bicyclomycin inhibition. AB - Kinetic studies document that the essential Escherichia coli transcription termination factor rho utilizes Mg(2+) and ATP as a substrate and requires a second Mg(2+) ion for maximum poly(C)-dependent ATP hydrolysis activity. The velocity curves show a classic nonessential Mg(2+) activation pattern in which Mg(2+) augments hydrolysis by 39% and gives a K(1)' for MgATP of 9.5 microM in the presence of excess Mg(2+) and a K(1) for MgATP of 21.2 microM under limiting Mg(2+) concentrations. Bicyclomycin (1), a commercial antibiotic that inhibits rho, weakened Mg(2+) binding at the nonessential site and disrupted the nonessential Mg(2+) activation pathway for poly(C)-dependent ATP hydrolysis. The K(i) values for 1 were 23 microM and 35 microM under excess and limiting Mg(2+) conditions, respectively, while the K(Mg(app)) for nonessential Mg(2+) increased with increasing 1 concentrations. These findings, when combined with reported mechanistic studies, provide an emerging picture of key catalytic and substrate binding sites that are necessary for rho function and that are proximal to the 1 binding site. PMID- 12369828 TI - Essential features of the catalytic core of peptidyl-alpha-hydroxyglycine alpha amidating lyase. AB - Bioactive peptides frequently terminate with an essential alpha-amide that is generated from a COOH-terminal Gly in a two-step enzymatic process occurring within the lumen of the secretory pathway. The first enzyme, peptidylglycine alpha-hydroxylating monooxygenase, is a member of the copper- and ascorbate dependent monooxygenase family. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL, EC 4.3.2.5), has no known homologues. Examination of the catalytic core of PAL (PALcc) using trypsin, BNPS skatole, and COOH terminally truncated proteins failed to identify stable subdomains. Treatment of PALcc with divalent metal ion chelators inactivated the enzyme and increased its protease and thermal sensitivity, suggesting a structural role for bound metal. Purified PALcc contained 0.7 +/- 0.4 mol of zinc/mol of enzyme. Since the four Cys residues in PALcc form two disulfide bonds, potential Zn ligands include conserved Asp, Glu, and His residues. The secretion and activity of PALcc bearing mutations in each conserved Asp, Glu, and His residue were evaluated. Mutation of three conserved Asp residues and two conserved His residues yielded a protein that could not be secreted, suggesting that these residues play a structural role. Analysis of mutants that were efficiently secreted identified three His residues along with single Asp residue that may play a role in catalysis. These essential residues occur in a pattern unique to PAL. PMID- 12369829 TI - Substrate specificity of the Plasmodium falciparum glycosylphosphatidylinositol biosynthetic pathway and inhibition by species-specific suicide substrates. AB - The substrate specificities of the early glycosylphosphatidylinositol biosynthetic enzymes of Plasmodium were determined using substrate analogues of D GlcN(alpha)1-6-D-myo-inositol-1-HPO(4)-sn-1,2-dipalmitoylglycerol (GlcN-PI). Similarities between the Plasmodium and mammalian (HeLa) enzymes were observed. These are as follows: (i) The presence and orientation of the 2'-acetamido/amino and 3'-OH groups are essential for substrate recognition for the de-N-acetylase, inositol acyltransferase, and first mannosyltransferase enzymes. (ii) The 6'-OH group of the GlcN is dispensable for the de-N-acetylase, inositol acyltransferase, all four of the mannosyltransferases, and the ethanolamine phosphate transferase. (iii) The 4'-OH group of GlcNAc is not required for recognition, but substitution interferes with binding to the de-N-acetylase. The 4'-OH group of GlcN is essential for the inositol acyltransferase and first mannosyltransferase. (iv) The carbonyl group of the natural 2-O-hexadecanyl ester of GlcN-(acyl)PI is essential for substrate recognition by the first mannosyltransferase. However, several differences were also discovered: (i) Plasmodium-specific inhibition of the inositol acyltransferase was detected with GlcN-[L]-PI, while GlcN-(2-O-alkyl)PI weakly inhibited the first mannosyltransferase in a competitive manner. (ii) The Plasmodium de-N-acetylase can act on analogues containing N-benzoyl, GalNAc, or betaGlcNAc whereas the human enzyme cannot. Using the parasite specificity of the later two analogues with the known nonspecific de-N-acetylase suicide inhibitor [Smith, T. K., et al. (2001) EMBO J. 20, 3322-3332], GalNCONH(2)-PI and GlcNCONH(2)-beta-PI were designed and found to be potent (IC(50) approximately 0.2 microM), Plasmodium specific suicide substrate inhibitors. These inhibitors could be potential lead compounds for the development of antimalaria drugs. PMID- 12369830 TI - Membrane insertion and dissociation processes of a model transmembrane helix. AB - An important subject for elucidating membrane protein (MP) folding is how transmembrane helices (TMHs) insert into and dissociate from membranes. We investigated helix dissociation kinetics and insertion topology by means of intervesicular transfer of the fluorophore-labeled completely hydrophobic model transmembrane helix NBD-(LALAAAA)(3)-NH(2) (NBD = 7-nitro-2-1,3-benzoxadiazol-4 yl). The peptide forms a topologically stable transmembrane helix, which is in a monomer-antiparallel dimer equilibrium [Yano, Y., Takemoto, T., Kobayashi, S., Yasui, H., Sakurai, H., Ohashi, W., Niwa, M., Futaki, S., Sugiura, Y., and Matsuzaki, K. (2002) Biochemistry 41, 3073-3080]. The helix transfer kinetics, representing the helix dissociation process, was monitored by fluorescence recovery of the quenched peptide in donor vesicles containing a quencher upon its transfer to acceptor vesicles without the quencher. The transfer kinetics and vesicle concentration dependence demonstrated that the transfer was mediated by monomer in the aqueous phase. Furthermore, the activation enthalpy was estimated to be +17.7 +/- 1.3 kcal mol(-1). Helix insertion topology, detected by chemical quenching of the NBD group in the outer leaflet by dithionite ions, was found to be controlled by transmembrane electric potential-helix macro dipole interaction. On the basis of these observations, a model for the helix insertion/dissociation processes was discussed. PMID- 12369831 TI - A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step. AB - The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme. PMID- 12369832 TI - A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity. AB - An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg 116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin. PMID- 12369833 TI - The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c, regulates microtubule binding and stability. AB - Phosphorylation of microtubule-associated protein 2 (MAP2) has a profound effect on microtubule stability and organization. In this work a consensus protein kinase A (PKA) phosphorylation site, T(220), of juvenile MAP2c is characterized. As confirmed by mass spectrometry, this site can be phosphorylated by PKA but shows less than average reactivity among the 3.5 +/- 0.5 phosphate residues incorporated into the protein. In contrast, T(220) is uniquely sensitive to dephosphorylation: three major Ser/Thr protein phosphatases, in the order of efficiency PP2B > PP2A(c) > PP1(c), remove this phosphate group first. MAP2c specifically dephosphorylated at this site binds and stabilizes microtubules stronger than either fully phosphorylated or nonphosphorylated MAP2c. Phosphorylation of this site also affects proteolytic sensitivity of MAP2c, which might represent a further level of control in this system. Thus, the phosphorylation state of T(220) may be a primary determinant of microtubule function. PMID- 12369834 TI - Location and properties of the taxol binding center in microtubules: a picosecond laser study with fluorescent taxoids. AB - The interaction of two bioactive, fluorescent analogues of the anticancer drug Taxol, Flutax1 [7-O-[N-(fluorescein-4'-carbonyl)-L-alanyl]taxol] and Flutax2 [7-O [N-(2,7-difluorofluorescein-4'-carbonyl)-L-alanyl]taxol], with microtubules in solution has been studied with picosecond laser methods. As shown here, although a mixture of the fluorescein mono- and dianion species of Flutax1 is present in solution, the bound taxoid contains only the dianion form of the dye. This indicates strong electrostatic interactions at the microtubule lattice with the appending dye, most likely with charged residues of the M-loop of the beta tubulin subunit. Moreover, analysis of the dynamic depolarization of microtubule bound Flutax at low binding site occupancy was consistent with a protein active center with significant conformational flexibility. On the other hand, for microtubules fully saturated with the taxoid, a new, additional depolarizing process was observed, with relaxation times of 14 ns (Flutax1) and 8 ns (Flutax2), which is due to Forster resonance energy homotransfer (FREHT) between neighboring dye molecules. Application of a detailed analysis of FREHT-induced depolarization in a circular array of dye molecules presented here yielded a separation between nearest-neighbor Flutax moieties of 40 +/- 5 A, for microtubules made up of between 12 and 14 protofilaments, a value that is only compatible with the Taxol binding site being located at the inner wall of the microtubule. The internal position of the drug molecular target as measured here is also consistent with other spectroscopic observations and confirms existing predictions based on microtubule structures modeled from high-resolution, electron density maps of alphabeta-tubulin. PMID- 12369835 TI - Mechanism of regulation of Acanthamoeba myosin-IC by heavy-chain phosphorylation. AB - The ATPase activity of myosin-Is from lower eukaryotes is activated by phosphorylation by the p21-activated kinase family at the TEDS site on an actin binding surface-loop. This actin-binding loop is the site of a cardiac myosin-II mutation responsible for some forms of familial hypertrophic cardiomyopathy. To determine the mechanism of myosin-I regulation by heavy-chain phosphorylation (HCP) and to better understand the importance of this loop in the function of all myosin isoforms, we performed a kinetic investigation of the regulatory mechanism of the Acanthamoeba myosin-IC motor domain (MIC(IQ)). Phosphorylated and dephosphorylated MIC(IQ) show actin-activated ATPase activity; however, HCP increases the ATPase activity >20-fold. HCP does not greatly affect the rate of phosphate release from MIC in the absence of actin, as determined by single turnover experiments. Additionally, HCP does not significantly affect the affinity of myosin for actin in the absence or presence of ATP, the rate of ATP induced dissociation of actoMIC(IQ), the affinity of ADP, or the rate of ADP release. Sequential-mix single-turnover experiments show that HCP regulates the rate of phosphate release from actin-bound MIC(IQ). We propose that the TEDS containing actin-binding loop plays a direct role in regulating phosphate release and the force-generating (A-to-R) transition of myosin-IC. PMID- 12369836 TI - Mutations in the putative pore-forming segment favor short-lived wild-type Kir2.1 pore conformations. AB - We have characterized single and double mutations in the M1-M2 segment of an inwardly rectifying K(+) channel, Kir2.1, using the cell-attached configuration of the patch-clamp technique. These mutations generated novel N-glycosylation sites at positions 128, 140, 143, and 147. Previously, we showed that these mutants were glycosylated, functional, and at the cell surface, which indicated that the putative pore-forming segment, including the invariant G(Y/F)G sequence of K(+) channels, was extracellular [Schwalbe, R. A., Rudin, A., Xia, S.-L., and Wingo, C. S. (2002) J. Biol. Chem. 277, 24382-24389]. In this study, three conductance states, corresponding to the main open state and two subconductance states, were identified in WT Kir2.1 channels expressed in infected Sf9 cells. Kir2.1 channels with mutations in the M1-M2 linker had at least one distinguishable conductance state of WT channels. In addition, these mutations altered the transitions, duration, and frequency of the defined populations of permeating and nonpermeating states. Of note, S128N had permeation rates similar to those of WT Kir2.1, but the total duration of the lower subconductance state was 3-5 times longer. Mutations in the signature sequence, I143N/Y145T, produced channels with permeation rates similar to those of the main open state and lower subconductance state of WT Kir2.1; however, the frequencies of these states were substantially different. These results demonstrate a novel functional role of the M1-M2 segment in regulating the transitions of the Kir2.1 channel and therefore suggest that this segment is a critical structural determinant in adjustments of pore conformations. Additionally, our results indicate that these mutants are correctly folded and thus further substantiate that the M1-M2 segment, including the G(Y/F)G sequence, is topologically extracellular. PMID- 12369837 TI - Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A tracts. AB - We have investigated by NMR the effects of NH(4)(+) on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](2), the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH(4)(+) binding in the minor groove. The cross peak intensities of the NOESY spectra collected at low and high NH(4)(+) concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH(4)(+) concentration is kept larger than 1 M, the A.T imino proton exchange times of A tract sequences exhibit a linear dependence versus the inverse of the NH(3) proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (Warmlander, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615). PMID- 12369838 TI - Thermodynamic contributions for the incorporation of GTA triplets within canonical TAT/TAT and C+GC/C+GC base-triplet stacks of DNA triplexes. AB - Nucleic acid triple helices may be used in the control of gene expression. One limitation of using triplex-forming oligonucleotides as therapeutic agents is that their target sequences are limited to homopurine tracts. To increase the repertoire of sequences that can be targeted, it has been postulated that a guanine can target a thymidine forming a stable GTA mismatch triplet. In this work, we have used a combination of optical and calorimetric techniques to determine thermodynamic unfolding profiles of two triplexes containing a single GTA triplet, d(A(3)TA(3)C(5)T(3)AT(3)C(5)T(3)GT(3)) (ATA) and d(AGTGAC(5)TCACTC(5)TCGCT) (GTG), and their control triplexes, d(A(7)C(5)T(7)C(5)T(7)) (TAT7) and d(AGAGAC(5)TCTCTC(5)TCTCT) (AG5T). In general, the presence of a GTA mismatch in DNA triplexes is destabilizing; however, this destabilization is greater when placed in a C(+)GC/C(+)GC base-triplet stack than between a TAT/TAT stack. These destabilizations are accompanied by a reduced unfolding enthalpy of approximately 10 kcal/mol, suggesting a decrease in the base stacking contributions surrounding the mismatch. Relative to their corresponding control triplexes, the folding of ATA is accompanied by a lower counterion uptake and a similar proton uptake, while GTG folding is accompanied by an increase in the counterion and proton uptakes. These effects are consistent with the observed decrease in stacking interactions. The overall results indicate that the main difficulty of targeting pyrimidine interruptions is that the decrease in stacking contributions, due to the incorporation of a GTA mismatch, affects the stability of the neighboring base triplets. This suggests that nucleotide analogues that increase the strength of these base-triplet stacks will result in a more effective targeting of pyrimidine interruptions. PMID- 12369839 TI - Stereodefined phosphorothioate analogues of DNA: relative thermodynamic stability of the model PS-DNA/DNA and PS-DNA/RNA complexes. AB - Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively. PMID- 12369840 TI - The three-dimensional structure of the gallium complex of azoverdin, a siderophore of Azomonas macrocytogenes ATCC 12334, determined by NMR using residual dipolar coupling constants. AB - In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer membrane receptor. This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked. This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins. We report the three-dimensional structure of azoverdin-Ga(III) in solution. The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta. The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion. This conformation leaves a large open space permitting access to the gallium ion. The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three dimensional structures of other pyoverdins. PMID- 12369841 TI - A solution NMR molecular model for the aspartate-ligated, cubane cluster containing ferredoxin from the hyperthermophilic archeaon Pyrococcus furiosus. AB - A solution molecular model for the conformationally dynamically heterogeneous Pyrococcus furiosus ferredoxin with an intact disulfide bond has been constructed on the basis of reported (1)H NMR spectral parameters using distance geometry and simulated annealing protocols. Conventional long-mixing time NOESY and H-bonding constraints have been augmented by previously reported short-mixing time NOESY, steady-state NOE, and cluster paramagnetism-induced relaxation. The family of 15 structures with inconsequential violations exhibited low rms deviations for backbone atoms for the overwhelming majority of the residues, including the cluster ligating loop with the unprecedented ligated Asp14. Larger rms deviations were observed across the disulfide bond, but closer inspection revealed that the 15 structures can be factored into 10 substructures exhibiting an "S" or right handed disulfide orientation and 5 exhibiting an "R" or left-handed disulfide orientation. The remainder of the structure is indistinguishable for the two disulfide orientations but confirms stabilizing extensions of secondary structural elements in the lengthening of the long helix and both the lengthening and incorporation of a third strand into the beta-sheet involving the termini, with these extensions interacting strongly in a modular fashion through the rings of Tyr46 and Trp2. These extensions of stabilizing interactions in Pyrococcus furiosus Fd, however, lead to strong destabilization of the disulfide bond and destabilization of the highly conserved first and last beta-turns in the sequence. It is concluded that the structural alternations in Pyrococcus Fd relative to other hyperthermostable Fds are not to increase thermostability but to place "stress" on the disulfide bond and render it more reducible. The possible physiological implications of this unique reducible disulfide bond are discussed. PMID- 12369842 TI - Controlling membrane cholesterol content. A role for polyunsaturated (docosahexaenoate) phospholipids. AB - The molecular organization of cholesterol in 1,2 didocosahexaenoylphosphatidylcholine (22:6-22:6PC) and 1-stearoyl-2 docosahexaenoylphosphatidylcholine (18:0-22:6PC) bilayers was investigated. Using low- and wide-angle X-ray diffraction (XRD), we determined that the solubility of the sterol at 20 degrees C was 11 +/- 3 mol % in 22:6-22:6PC vs 55 +/- 3 mol % in 18:0-22:6PC bilayers. Solubility in the dipolyunsaturated membrane rose to 17 +/- 3 mol % at 40 degrees C, while in the saturated-polyunsaturated membrane there was no change within experimental uncertainty. We compared the molecular orientation of [3alpha-(2)H(1)]cholesterol incorporated into 22:6-22:6PC bilayers to its solubility limit and into 18:0-22:6PC bilayers to a comparable concentration (10 mol %) in solid-state (2)H NMR experiments. The sterol possessed a tilt angle alpha(0) = 24 degrees +/- 1 degrees in 22:6-22:6PC that was independent of temperature over a range from 20 to 40 degrees C. In contrast, the value was alpha(0) = 21 degrees +/- 1 degrees in 18:0-22:6 bilayers at 20 degrees C and increased to alpha(0) = 24 degrees +/- 1 degrees at 40 degrees C. We attribute the low solubility of cholesterol in 22:6-22:6PC membranes to steric incompatibility between the rigid steroid moiety and the highly disordered docosahexaenoic acid (DHA) chain, which has the potential to promote lateral heterogeneity within DHA-rich membranes. Considering 22:6-22:6PC to be the most unsaturated phospholipid found in vivo, this model membrane study provides a point of reference for elucidating the role of sterol-lipid interactions in controlling local compositional organization. Our results form the basis for a model that is consistent with cholesterol's ability to modulate the activity of certain neural transmembrane proteins. PMID- 12369843 TI - GTPase activation of elongation factors Tu and G on the ribosome. AB - The GTPase activity of elongation factors Tu and G is stimulated by the ribosome. The factor binding site is located on the 50S ribosomal subunit and comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the sarcin ricin loop of 23S rRNA. The role of these ribosomal elements in factor binding, GTPase activation, or functions in tRNA binding and translocation, and their relative contributions, is not known. By comparing ribosomes depleted of L7/12 and reconstituted ribosomes, we show that, for both factors, interactions with L7/12 and with other ribosomal residues contribute about equally and additively to GTPase activation, resulting in an overall 10(7)-fold stimulation. Removal of L7/12 has little effect on factor binding to the ribosome. Effects on other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and translocation, are fully explained by the inhibition of GTP hydrolysis. Based on these results, we propose that L7/12 stimulates the GTPase activity of both factors by inducing the catalytically active conformation of the G domain. This effect appears to be augmented by interactions of other structural elements of the large ribosomal subunit with the switch regions of the factors. PMID- 12369844 TI - Critical role of chromium (Cr)-DNA interactions in the formation of Cr-induced polymerase arresting lesions. AB - The genotoxicity associated with the metabolic reduction of hexavalent chromium [Cr(VI)] is complex and can impede DNA polymerase-mediated replication in vitro. The exact biochemical nature of Cr-induced polymerase arresting lesions (PALs) is not understood, but is believed to involve the formation of Cr-DNA interstrand cross-links (ICLs). The aim of this investigation was to determine the dependence of direct Cr-DNA interactions on the development of PALs in DNA treated with trivalent Cr [Cr(III)] or with Cr(VI) in the presence of ascorbic acid (Asc), a major intracellular reductant, using an in vitro, acellular system. The formation of Cr-DNA adducts, ICLs, and PALs was maximal at Asc:Cr(VI) molar ratios of 0.5 2, but gradually decreased at higher ratios. EDTA, a Cr(III) chelator, significantly decreased Cr-DNA binding and ICL and PAL formation. Co-treatment of DNA with Cr(VI)/Asc and mannitol, a Cr(V) chelator, selectively inhibited the formation of mono/bifunctional DNA adducts and PALs produced by Cr(VI) reduction, but had no effect on Cr(III)-DNA binding or Cr(III)-induced polymerase arrest. Blocking Cr-DNA phosphate interaction by preincubation of DNA with MgCl(2) abrogated DNA binding and ICL and PAL production. DNA strand breaks and abasic sites may lead to the in vitro arrest of DNA polymerases; however, we failed to detect significant increases in the frequency of these lesions following Cr(VI)/Asc treatment. These data indicate that the bifunctional adduction of Cr to DNA phosphates (ICLs) constitutes a major PAL. Furthermore, the generation of DNA strand breaks and abasic sites by Cr(VI) reduction is insufficient to explain PALs observed in vitro. PMID- 12369845 TI - Evidence that phospholipids play a key role in pre-beta apoA-I formation and high density lipoprotein remodeling. AB - The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2 oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2 docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation. PMID- 12369846 TI - Conformational prerequisites for alpha-lactalbumin fibrillation. AB - Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein, formed amyloid fibrils at low pH, where it adopted the classical molten globule-like conformation. Fibrillation was accompanied by a dramatic increase in the beta structure content and a characteristic increase in the thioflavin T fluorescence intensity. S-(Carboxymethyl)-alpha-lactalbumin, a disordered form of the protein with three out of four disulfide bridges reduced, was even more susceptible to fibrillation. Other partially folded conformations induced in the intact protein at neutral pH, either by the removal of Ca(2+) or by the binding of Zn(2+) to the Ca(2+)-protein complex, did not fibrillate, although Zn(2+)-loaded alpha lactalbumin precipitated out of solution as amorphous aggregates. Our data suggest that the transformation of a protein into an essentially unfolded (thus, highly flexible) conformation is required for successful fibril formation, whereas more rigid (but still flexible) species may form amorphous aggregates. PMID- 12369847 TI - A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay. AB - The bacterial cell wall is a polymer consisting of alternating N acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, cross-linked via peptides appended to MurNAc. The final steps in the formation of cell wall, also referred to as murein, are catalyzed by high-molecular-weight, class A penicillin-binding proteins (PBPs). These bifunctional enzymes catalyze both glycosyltransfer, to form the carbohydrate backbone of murein, and transpeptidation, to form the interstrand peptide linkages. Using PBP1b from Eschericia coli, an in vitro kinetic characterization of the glycosyltransfer reaction was carried out. Initial studies with unlabeled substrate (Lipid II) revealed that activity is strongly influenced by DMSO, as well as metal and detergent. In addition, a continuous fluoresence assay was developed and used to determine the effect of pH on the reaction. A single basic residue was titrated, with a pK(a) of 7.0. Taken together, these data suggest a mechanism for PBP1b where the glycosyltransfer reaction is catalyzed by the concerted effect of an active site base to deprotonate the glycosyl acceptor and a divalent metal to assist departure of the leaving group of the glycosyl donor. PMID- 12369848 TI - Protein kinase C activation induces phosphatidylserine exposure on red blood cells. AB - We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+) when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin E(2), that are released during clot formation. Since calcium loading of RBCs can lead to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we decided to investigate the possible linkage between PKC activation and membrane PS scrambling using phorbol 12-myristate-13 acetate (PMA), a commonly used activator of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate PS exposure in an RBC subpopulation. The size of the subpopulation did not change upon further incubation, indicating that not all RBCs are equally susceptible to this treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated intracellular calcium levels. In the absence of extracellular calcium, no PS exposure was found. However, we did find cells with high levels of calcium that did not expose PS, and a variable percentage of PS-exposing cells that did not show elevated calcium concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding site, or chelerythrine chloride, an inhibitor of the active site, diminished the level of formation of PS-exposing cells. However, the inhibitors had different effects on calcium internalization, indicating that a high calcium concentration alone was not responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC inhibition could prevent PS exposure induced by calcium and ionophore treatment of RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid scrambling. PMID- 12369850 TI - Rationale for Ras and raf-kinase as a target for cancer therapeutics. AB - Improvements in our understanding of the intrinsic aberrancies in cancer cells have enabled the design and development of novel therapeutics that specifically target these changes. Among the many complex cellular pathways and mechanisms which have been unveiled by new molecular techniques, RAS-mediated signal transduction is one met with tremendous research interests. Activation of RAS initiates several signaling cascades, of which the RAS-RAF-MEK-ERK pathway is among the better delineated, and is the main focus of this review. Other cellular consequences of RAS activation including interactions with the RHO-family proteins, the PI3-kinase pathway, and other mitogen activated protein kinase cascades, will be discussed. The intricate balance and coordination of multiple RAS-mediated signals lead to ultimate effects on cell growth, differentiation, cycling and survival. Pharmacological strategies such as analog development, synthesis of small molecule inhibitors, antisense technology, and vaccine therapy have been utilized to intervene with key RAS-signaling proteins, in an attempt to provide rational therapeutic solutions in malignant diseases. PMID- 12369851 TI - Inhibition of raf kinase in the treatment of acute myeloid leukemia. AB - Adult acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) remain a formidable therapeutic challenge. Although 30-40% of young adults with AML may have prolonged remissions, following either intensive chemotherapy or bone marrow transplantation, the remainder relapse and ultimately die of their disease. Older adults, or those with leukemia following an antecedent hematologic disorder such as MDS, experience only brief remissions with modern chemotherapy, and in this population the rate of long-term disease-free survival is less than 10%. Treatment of MDS consists mainly of supportive care with antibiotics and transfusion therapy, and only a small minority of patients are cured with allogeneic stem cell transplantation. As the biology of the acute leukemias and MDS has unfolded, new potential targets for arresting malignant cell growth and restoring normal hematopoiesis have been identified. This article will discuss the relevance of the ras-raf signaling pathway in the pathogenesis of myeloid leukemia, and describes some early results in the use of novel small molecule inhibitors of farnysltransferase and raf protein kinase in leukemia therapy. PMID- 12369852 TI - BAY 43-9006: early clinical data in patients with advanced solid malignancies. AB - Various signalling pathways can confer the malignant phenotype to a cell. Ras signalling proteins have been found to play an important role in controlling cellular growth. Raf-1 is a protein kinase that exerts its effects downstream of Ras in the mitogen-activated protein kinase pathway and is thus likely to be crucial in the development of the malignant phenotype. BAY 43-9006 is an orally administered selective inhibitor of Raf-1 and the first compound of its class to enter clinical trials. This article describes the early clinical data of BAY 43 9006 in patients with advanced, refractory solid tumours. To date, over 60 patients have been treated as part of four Phase I clinical trials. Dose levels have ranged from 50mg once weekly to 200mg twice-daily in continuous administration. The drug has been generally well tolerated with no dose limiting toxicity yet encountered. The more common toxicities have involved the gastrointestinal tract (diarrhea, nausea, abdominal cramping) and the skin (pruritus, rash, cheilitis). Pharmacokinetic evaluations have found BAY 43-9006 to have considerable interpatient variability. However, there seems to be an increase in C(max) and AUC values with increasing dose. There is no clear effect of food on bioavailability. Splitting the dose to twice-daily administration has shown increases in C(max) and AUC values but is also accompanied by considerable interpatient variability. PMID- 12369853 TI - BAY 43-9006: preclinical data. AB - The drug design and discovery efforts described in the previous section led to the development of a novel, small molecule Raf-1 kinase inhibitor, BAY 43-9006, which belongs to a class that can be broadly described as bis-aryl ureas (Figure 1). BAY 43-9006 was identified during a large medicinal chemistry optimization program, and this compound was selected for further pharmacological characterization based on its potent inhibition of Raf-1 (IC(50) 12 nM) and its favorable kinase selectivity profile. In vitro and in vivo experiments were designed to demonstrate effective blockade of the Raf/MEK/ERK signaling pathway in tumor cells and for anti tumor efficacy in human xenograft models. PMID- 12369854 TI - Agents targeting ras signaling pathway. AB - Ras genes encode proteins that activate in an intracellular signaling network controlling differentiation, proliferation and cell survival. Mutated Ras oncogenes encoding proteins that are constitutively active can induce malignancies in a variety of laboratory models. In human malignancies, Ras mutations are common, having been identified in approximately 30% of cancers. Given the importance of Ras and downstream targets Raf and MEK in the development of malignancies and their frequent expression in human cancers, it is not surprising that a variety of agents disrupting signaling through Ras and downstream proteins are under development. These agents can be broadly classified structurally as small molecules and anti-sense oligonucleotides. They can be characterized functionally as those inhibiting Ras protein expression such as the oligodeoxynucleotide ISIS 2503, those inhibiting Ras processing, in particular the farnesyl transferase inhibitors R115777, SCH 66336 and BMS 214662, and those inhibiting downstream effectors Raf, such as ISIS 5132 and MEK, which is inhibited by CI-1040. The purpose of this review is to highlight recent advances in the development of these agents. PMID- 12369855 TI - Design and discovery of small molecules targeting raf-1 kinase. AB - Raf kinase, an enzyme which acts downstream in the Ras signaling pathway, is involved in cancerous cell proliferation. Thus, small molecule inhibitors of Raf kinase activity may be important agents for the treatment of cancer. A novel class of Raf-1 inhibitors was discovered, using a combination of medicinal and combinatorial chemistry approaches. This effort culminated in the identification of the clinical candidate BAY 43-9006, currently undergoing Phase I clinical trials. The present review summarizes the medicinal chemistry development of ureas as highly potent inhibitors of Raf-1 kinase. PMID- 12369856 TI - The design of clinical trials for new molecularly targeted compounds: progress and new initiatives. AB - Investigators involved in the development of cancer therapeutics are testing new trial designs and endpoints in order to accommodate the perceived challenges in defining appropriate doses and schedules for further testing. Many new agents with specific molecular targets have entered clinical development or are being considered for development. While some of the agents have both toxicity and antitumour efficacy apparent at clinically achievable doses, thus the use of traditional algorithms is appropriate, others have significant clinical activity at doses considerably lower than the maximum tolerated dose. New initiatives in clinical trial design, both phase I and phase II may allow the development of appropriate plans for the development of these new molecularly targeted agents. Measures of target effect (tissue or imaging) are now commonly included in early trials of new targeted compounds, in an attempt to demonstrate proof of principle as well as guide dose selection. Phase II trial designs including novel correlative, imaging and clinical endpoints are being tested. Alternate endpoints such as progression or time to progression are being increasingly considered, and novel designs such as randomized discontinuation designs, multinomial designs and growth modulation indices are being prospectively tested. Progress in this area of early trial design are reviewed. PMID- 12369857 TI - The shape of the messenger: using protein structure information to design novel cytokine-based therapeutics. AB - The cloning and mass production of recombinant cytokine proteins opened a new world of treatment possibilities. While some cytokines, including several haematopoietic factors and interferons, are now used routinely in the clinic, there are still many problems with side effects. These are due to the many different activities of cytokines on different cell populations. In some cases, activities responsible for side effects have been attributed to discreet areas of the proteins and "structure driven design" can be used to generate novel proteins with better clinical profile. In other cases, structural alterations can enhance activity by increasing serum half-life. This review summarizes the structures of cytokines and their receptor complexes deposited in the Protein Data Base (PDB) and introduces the other articles in this issue on structure-driven design of cytokines for therapy. Cytokines fall into only a few structural classifications. Most of the growth regulatory cytokines including serum factors, growth hormone, haematopoietic growth factors, colony stimulating factors, erythropoietin, IL-3, IL-2 and interferons, are four or five helix bundles. Factors which primarily induce inflammatory responses, including TNF, lymphotoxin and IL-1, form beta barrel structures that resemble the FGF family. Chemokines and factors that regulate multicellular responses, such as macrophage migration, neutrophil invasion and chemotaxis, have similar structures, classified as alpha + beta. One biological paradox is that many cytokines that vary greatly in function have a similar structure and share receptors. However, homologous cytokines may differ considerably in their mode of interaction with a shared receptor. A few structures for the extracellular regions of cytokine receptors are known, in several cases complexed with their biological target. These structures, coupled with structural alignment of families, indicate areas that control binding to receptors, as opposed to specific areas responsible for the specific activities of this diverse group of proteins. Methods to use cytokine structure to derive better therapeutics are summarized. PMID- 12369858 TI - Interferon-alpha/beta-receptor interactions: a complex story unfolding. AB - The Type I interferons (IFN-alpha/beta) exhibit pleiotropic biological activities. Notably, the different IFN subtypes activate the same cell surface receptor complex to mediate variable responses. Accumulating evidence suggests that distinct differences in critical amino acid residues among the different IFN alphas and IFN-beta determine the nature of the ligand-receptor interaction and the subsequent responses. This review focuses on IFN-receptor interactions, the key residues involved in this interaction and the potential for targeted modifications of the ligand to enhance bioactivity. PMID- 12369859 TI - Structure, biology, and therapeutic implications of pegylated interferon alpha 2b. AB - Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients. PMID- 12369860 TI - Designing decoys for chemokine-chemokine receptor interaction. AB - Aberrant expression of chemokines and their receptors play causative roles in the pathophysiology of numerous autoimmune and inflammatory disease processes. Moreover, an integral step in HIV infection involves binding to chemokine receptors, and hence chemokines are intimately linked to HIV-related diseases. Therefore, chemokines and their receptors are excellent targets for developing drugs that are more specific and may be of benefit in the management of disease. Knowledge of the chemokine and chemokine receptor structures, and an understanding of the structural basis of their function are essential for structure-aided design of receptor decoys. Chemokine ligands bind their receptors with nanomolar (nM) affinity, and successful design of a small molecule antagonist should bind the receptor with similar high affinity and specificity. Chemokines bind receptors that belong to the 7-transmembrane class on leukocytes, and highly negatively charged proteoglycans that are present on the cell surface. Structure-function studies have identified regions in both the ligand and the receptor that mediate binding and activation. Structures of numerous chemokines have been solved though very little is known regarding receptor structures. This review will summarize the current knowledge on the structures, structure function, and the efficacy of chemokine derivatives and functional domain peptides as antagonists, and discuss strategies for exploiting this information for designing decoys for inflammatory, autoimmune, and HIV-related diseases. PMID- 12369861 TI - Therapeutic enhancement of IL-2 through molecular design. AB - A recombinant human IL-2 analog (rIL-2, Proleukin) is currently being evaluated for clinical benefit in HIV infected patients. It is approved for therapy of patients with metastatic melanoma and renal cell carcinoma. Treatment of cancer patients with rIL-2 results in durable responses but is associated with life threatening toxicity, which limits its use to patients in relatively good health. Antitumor efficacy associated with rIL-2 therapy are hypothesized to be mediated by distinct types of cells that express structurally different forms of the IL-2 receptor. This hypothesis suggests that it might be possible to engineer an IL-2 variant addressing the risks associated with the therapeutic use of IL-2. In this article, we review the clinical experience with IL-2 and its analogs, the evidence that different IL-2 receptors may dissociate efficacy and toxicity, and describe the generation of a novel IL-2 variant with the potential for a superior therapeutic index. PMID- 12369862 TI - Structure-based design of mimetics for granulocyte-macrophage colony stimulating factor (GM-CSF). AB - Granulocyte-macrophage colony stimulating factor (GM-CSF) activity has been linked to pro-inflammatory effects in autoimmune syndromes, such as rheumatoid arthritis. Thus GM-CSF mimetics with antagonist activity might play a therapeutic role in these diseases. The human GM-CSF core structure consists of a four alpha helix bundle, and GM-CSF activity is controlled by its binding to a two-subunit receptor. A number of residues located on the B and C helices of GM-CSF are postulated to interact with the alpha chain of the GM-CSF receptor (GM-CSFR). Several approaches have been successfully utilized to develop peptide mimetics of this site, including peptides from the native sequence, a peptide derived from a recombinant antibody (rAb) light chain which mimicked GM-CSF receptor binding activity, and structurally guided de novo design. Analysis of the rAb light chain had suggested mimicry of GM-CSF with residues mostly contributed by the CDR I region. Key residues involved in CDR I peptide/GM-CSFR binding were identified by truncation and alteration of individual residues, while the structural elements required to antagonize the biological action of GM-CSF were separately tested in binding and inhibitory activity assays of multiple cyclic analogues. A peptide designed to retain the loop conformation of the CDR I region of the rAb light chain competed with GM-CSF for both antibody and receptor binding, but the role of specific residues in antibody versus receptor binding differed markedly. These studies suggest that structural analysis of peptide mimetics can reveal differences in receptor and antibody binding, perhaps including key interactions that impact binding kinetics. Peptide mimetics of other four-helix bundle cytokines are reviewed, including helical and reverse turn mimetics of helical structures. Use of peptide mimetics coupled with structural and kinetic analysis provides a powerful approach to identifying important receptor-ligand interactions, which implications for rational design of novel therapeutics. PMID- 12369863 TI - Small molecule peptidomimetic ligands of neurotrophin receptors, identifying binding sites, activation sites and regulatory sites. AB - Neurotrophins (NTFs) are a family of polypeptide growth factors that control the apoptotic death or survival, growth, and differentiation of neurons. NTFs also regulate several other cell populations such as lymphoid, epithelial, oligoglia, and mast cells. Disregulation of the NTFs or their receptors plays a key role (etiological or upstream) in certain human pathologies. Hyperactivity may lead to inflammatory pain, or some forms of cancer by autocrine/paracrine growth. Loss of activity may lead to neurodegeneration, neuropathic pain, or some forms of cancer by absence of differentiation. Consequently the NTFs and their receptors are important therapeutic targets, and pharmacological modulation may have applications ranging from treatment of chronic or acute neurodegeneration, some forms of cancer, and chronic pain (with agonists); and some forms of cancer or acute pain (with antagonists). PMID- 12369864 TI - Rational design and development of RDP58. AB - We used a novel rational design approach in the development of novel immunomodulatory peptides, in particular RDP58, with at least two primary biological activities, inhibition of TNF production and upregulation of heme oxygenase-1 (HO-1) activity. The design strategy used a variety of topological and shape descriptors in combination with an analysis of molecular dynamics trajectories to identify potential drug candidates. The process was initiated using a panel of 19 lead molecules derived from a functionally-important region of HLA I, on the premise that the peptides might modulate immune responses by blocking HLA/T lymphocyte interactions. Each of these 19 peptides was tested in vivo for potential cardiac allograft protection in a mouse model. Outcome of graft survival was the primary data available to describe nine of the peptides as active and ten as inactive. A virtual combinatorial library of 279,936 peptides was then generated, based on physical properties associated with efficacy in the original learning set of 19 peptides--in particular, the distribution of lipophilic residues. The library was screened using descriptors that fell into 2 categories: static (physico-chemical) and dynamic (conformational). The screenings identified 5 peptides with theoretic potential efficacy. These were synthesized and tested for activity in vitro and in vivo. Besides prolonging graft survival in rodents, RDP58 was found to inhibit TNF and increase HO-1 activity; these biological activities were tested exhaustively in various in vivo disease models. RDP58 demonstrated convincing potential to alleviate disease symptoms in many of the experimental diseases. PMID- 12369865 TI - Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. AB - As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested. PMID- 12369866 TI - Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins. AB - The protein serine/threonine phosphatases constitute a unique class of enzymes that are critical for cell regulation, as they must counteract the activities of thousands of protein kinases in human cells. Uncontrolled inhibition of phosphatase activity by toxic inhibitors can lead to widespread catastrophic effects. Over the past decade, a number of natural product toxins have been identified that specifically and potently inhibit protein phosphatase-1 and 2A. Amongst these are the cyanobacteria-derived cyclic heptapeptide microcystin-LR and the polyether fatty acid okadaic acid from dinoflagellate sources. The molecular mechanism underlying potent inhibition of protein phosphatase-1 by these toxins is becoming clear through insights gathered from diverse sources. These include: 1. Comparison of structure-activity relationships amongst the different classes of toxins. 2. Delineation of the structural differences between protein phosphatase-1 and 2A that account for their differing sensitivity to toxins, particularly okadaic acid and microcystin-LR. 3. Determination of the crystal structure of protein phosphatase-1 with microcystin-LR, okadaic acid and calyculin bound. 4. Site-specific mutagenesis and biochemical analysis of protein phosphatase-1 mutants. Taken together, these data point to a common binding site on protein phosphatase-1 for okadaic acid, microcystin-LR and the calyculins. However, careful analysis of these data suggest that each toxin binds to the common binding site in a subtly different way, relying on distinct structural interactions such as hydrophobic binding, hydrogen bonding and electrostatic interactions to different degrees. The insights derived from studying the molecular enzymology of protein phosphatase-1 may help explain the different sensitivities of other structurally conserved protein serine/theonine phosphatases to toxin inhibition. Furthermore, studies on the binding of structurally diverse toxins at the active site of protein phosphatase-1 are leading to a clearer understanding of potential enzyme-substrate interactions in this important class of cell regulatory proteins. PMID- 12369867 TI - The microcystins and nodularins: cyclic polypeptide inhibitors of PP1 and PP2A. AB - The serine/threonine phosphatases are inhibited by a variety of natural toxins, including the microcystins and nodularins. Progress in understanding the details of the biosynthetic origin and the binding of these compounds is discussed, as is the progress made in synthesizing the members of these families. Additionally, the work by several groups to either synthesize simplified analogues that are still potent, or introduce selectivity for PP1 over PP2A are discussed. Finally, the properties of a series of five new truncated analogues are examined. PMID- 12369868 TI - Fostriecin: chemistry and biology. AB - A review of the current status of the chemistry and biology of fostriecin (CI 920) is provided. Fostriecin is a structurally unique, naturally-occurring phosphate monoester that exhibits potent and efficacious antitumor activity. Initially it was suggested that its activity could be attributed to a direct, albeit weak, inhibition of the enzyme topoisomerase II. However, recent studies have shown that fostriecin inhibits the mitotic entry checkpoint through the much more potent and selective inhibition of protein phosphatase 2A (PP2A) and protein phosphatase 4 (PP4). In fact, it is the most selective small molecule inhibitor of a protein phosphatase disclosed to date. The contribution, if any, that topoisomerase II versus PP2A/PP4 inhibition makes to fostriecin's antitumor activity has not yet been fully defined. Initial phase I clinical trials with fostriecin never reached dose-limiting toxicity or therapeutic dose levels and were halted due to its storage instability and unpredictable chemical purity. Hence, the total synthesis of fostriecin has been pursued in order to confirm its structure and stereochemistry, to provide access to quantities of the pure natural product, and to access key partial structures or simplified/stable analogs. Several additional natural products have been isolated which contain similar structural features (phospholine, phoslactomycins, phosphazomycin, leustroducsins, sultriecin, and cytostatin), and some exhibit comparable biological properties. PMID- 12369869 TI - Synthesis of specific protein phosphatase inhibitors, tautomycin and tautomycetin toward structure-activity relationship study. AB - The recent progress in synthetic and SAR studies of the specific protein phosphatase inhibitors tautomycin and tautomycetin is reviewed. This article covers the total synthesis of tautomycin and synthetic studies of the spiroketal and the anhydride segments and tautomycetin, and SAR studies on PP inhibition and apoptosis-inducing activity of tautomycin. PMID- 12369870 TI - Regulators of serine/threonine protein phosphatases at the dawn of a clinical era? AB - Reversible phosphorylation is a key mechanism for regulating the biological activity of many human proteins that affect a diverse array of cellular processes, including protein-protein interactions, gene transcription, cell-cycle progression and apoptosis. Once viewed as simple house keeping enzymes, recent studies have made it eminently clear that, like their kinase counterparts, protein phosphatases are dynamic and highly regulated enzymes. Therefore, the development of compounds that alter the activity of specific phosphatases is rapidly emerging as an important area in drug discovery. Because >98% of protein phosphorylation occurs on serine and threonine residues, the identification of agents that alter the activity of specific serine/threonine phosphatases seems especially promising for drug development in the future. This review is focused on the enzymes encoded by the PPP-gene family, which includes PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7. The structure/functions of human phosphatases will be addressed briefly, as will the natural product inhibitors of PP1-PP6 (e.g. okadaic acid, microcystins, nodularin, cantharidin, calyculin A, tautomycin, and fostriecin). The development of chimeric antisense oligonucleotides that support RNAase H mediated degradation of the targeted mRNA has resulted in compounds capable of specifically suppressing the expression of PP5 (ISIS 15534) and PP1gamma 1 (ISIS 14435) in human cells. Such compounds have already proven useful for the validation of drug targets, and if difficulties associated with systemic delivery of antisense oligonucleotides can be overcome, antisense is poised to have a major impact on the clinical management of many human disorders. PMID- 12369871 TI - Selective cyclooxygenase-2 inhibitors and non-small cell lung cancer. AB - Lung cancer is the leading cause of death from cancer in most developed nations. The most common type of lung cancer is of non-small cell histology, representing approximately 80% of the total. Despite aggressive treatments in early stages and improvement of polychemotherapy outcomes in advanced disease, the five years survival rate for lung cancer remains under 15%. Fortunately, our improved knowledge of tumor biology and mechanisms of oncogenesis suggests several new potential targets for clinical research in cancer therapy. A substantial body of evidence indicates that cyclooxigenase (COX)-2 and prostaglandins (PGs) play an important role in tumorigenesis. Mechanisms involved in COX-2 participation in tumorigenesis and tumor growth include xenobiotic metabolism, angiogenesis stimulation, inhibition of immune surveillance and inhibition of apoptosis. COX-2 is frequently overexpressed in bronchial premalignancy, lung adenocarcinoma and squamous cell carcinoma and COX-2 overexpression is a marker of poor prognosis in surgically resected stage I non-small cell lung cancer. Treatment with COX-2 inhibitors reduces the growth of NSCLC cells in vitro and in xenograft studies. Recent studies have defined some of the mechanisms involved in COX-2 participation in NSCLC development and diffusion. These evidences support the hypothesis that selective COX-2 inhibitors (coxibs) may prove beneficial in the prevention and treatment of NSCLC. PMID- 12369872 TI - BM 212 and its derivatives as a new class of antimycobacterial active agents. AB - During our investigation in pyrrole antibacterial area we have identified a subclass with a good potent in vitro activity against mycobacteria and fungi. We have individuated the salient structural feature and BM 212 as lead for the class. SAR studies allowed us to synthesize several analogue derivatives. Some of them revealed more active than BM 212 against mycobacteria, but they lost antifungal activity. In particular the Protection Index (PI) was very interesting for some derivatives, comparable to that of reference compounds, Isoniazid (INH), Streptomycin (SM) and Rifampin (RF). Many of the synthesized compounds revealed active against intracellular mycobacteria and they showed to be inhibitory to drug-resistant mycobacteria of clinical origin. On the base of microbiological results we have hypothesized a pharmacophore model that was also optimized. The rational design, and the evaluation of the in vitro activity against mycobacteria are described. PMID- 12369873 TI - Drug evolution: p-aminobenzoic acid as a building block. AB - The core or the building block is an important component in drug development. In this article, we propose and review p-aminobenzoic acid (PABA) as a building block used in the design of drugs or drug candidates. PABA is frequently found as a structure moiety in drugs. For example, in a database of 12,111 commercial drugs, 1.5% (184 drugs) were found to contain the PABA moiety. These drugs have a wide range of therapeutic uses, such as: sun-screening, antibacterial, antineoplastic, local anesthetic, anticonvulsant, anti-arrhythmic, anti-emetic, gastrokinetic, antipsychotic, neuroleptic, and migraine prophylactic. This article reviews the molecular targets and the mechanisms of these activities. Drugs containing PABA also show a wide range of structural diversity. Of the 184 PABA containing drugs identified, 95 different substitutions were found at the carboxylic group and 61 were found at the amino group of the building block. Substitution on the aromatic ring was also diverse. 13, 3, and 13 different side chains were found to modify positions 2, 3 and 5 of the aromatic ring respectively. In some drugs, the amino group is further substituted to form tertiary amine (4 different side chains). Substitutions at the carboxyl and amino groups of PABA are particularly suitable for the generation of combinatorial libraries. Just by reshuffling the identified side chains of the 184 PABA containing drugs, 4.5 million compounds can be generated. Consequently, PABA fits well as a building block for a general chemical library of "drug-like" molecules with a wide range of functional and structural diversity. PMID- 12369874 TI - The role of monocytes and macrophages in the pathogenesis of HIV-1 infection. AB - Cells of the macrophage lineage play an important role in initial infection with HIV-1 and contribute to the pathogenesis of the disease throughout the course of infection. Both blood monocytes and tissue macrophages can be infected with HIV-1 in vivo and in vitro, although the latter are more susceptible to infection. They express the CD4 receptor and chemokine co-receptors for HIV-1 entry, and hence are targets for HIV-1 infection. Cells of the macrophage lineage can be infected predominantly with macrophage (M)-tropic strains, although infection with some T cell line (T)-tropic strains or dual-tropic isolates of HIV-1 (exhibiting features of both M-tropic and T-tropic isolates) has also been reported. Following infection with HIV-1, monocyte/macrophages are resistant to cytopathic effects and persist throughout the course of infection as long-term stable reservoirs for HIV-1 capable of disseminating the virus to tissues. Infectious virus can be recovered from blood monocytes obtained from patients receiving highly active antiretroviral therapy with no detectable HIV-1 in blood. Cells of the macrophage lineage play an important role in the neuropathogenesis of HIV-1 infection and contribute to HIV-induced dementia via production of proinflammatory cytokines and neurotoxins. Following HIV-1 infection, effector functions carried out by monocyte/macrophages are also impaired, including phagocytosis, intracellular killing, chemotaxis and cytokine production. Such defects contribute to the pathogenesis of AIDS by allowing reactivation and development of opportunistic infections. This review focuses on the overall role of monocytes and macrophages in the pathogenesis of HIV-1 infection and considers the mechanisms underlying defective monocyte/macrophage function. PMID- 12369875 TI - HIV protease inhibitors: peptidomimetic drugs and future perspectives. AB - A literature review on the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). This review includes its life cycle, HIV protease structure, function, and substrates, as well as the mechanism and the design of inhibitors including the clinically approved drugs. Moreover the review mentioned the problems that hindered the development of peptidomimetic drug candidates as HIV protease inhibitors and the different approaches used by medicinal chemists to overcome these problems. A special attention was made to the design rationale as well as the lead optimization processes that provided inhibitors that possess high potency, reduced molecular weight and lower lipophilicity of the allophenylnorstatine (Apns) containing HIV protease inhibitors. PMID- 12369876 TI - Adenosine A(1) receptor: analysis of the potential therapeutic effects obtained by its activation in the central nervous system. AB - Adenosine modulates several physiological functions in the CNS and in peripheral tissues via membrane receptors which have been classified into four adenosine subtypes. A(1) activation produces neuronal depression: this inhibition allows A(1) agonists to produce ischemic tolerance and protection in neuronal tissue. In order to selectively reproduce these effects, several A(1) selective ligands have been synthesised and evaluated to understand how they interact with the adenosine A(1) receptor. The investigation methods include SAR studies using native and chemically modified A(1) receptors, molecular cloning of native and mutant adenosine A(1) receptors, molecular modeling and thermodynamic analysis of drug receptor interaction. Despite the great quantity of information available on the adenosine A(1) receptor, no A(1) agonist has so far entered in clinical use against brain diseases in view of the side effects; moreover selective A(1) agonists appear to be poorly adsorbed into the brain and can be quickly degraded in vivo or in the whole blood. In an attempt to overcome these problems studies have been undertaken dealing with the use of partial agonists to inhibit side effects and the employment of prodrugs to increase stability and diffusion through lipid barriers of A(1) ligands. Other attempts involve either the use of A(1) receptor enhancers as modulators able to locally enhance the action of endogenously produced adenosine, or the encapsulation of A(1)agonists in drug delivery systems targeted to the brain. In this review, these approaches will be described together with the effects of adenosine A(1) receptor ligands and their binding mechanisms on the central nervous system. PMID- 12369877 TI - The role of CD8+ T cell soluble factors in human immunodeficiency virus infection. AB - Cell-mediated immune responses are important for the control of HIV replication in vivo. Cytotoxic CD8+ T cells (CTL) recognize and kill HIV-infected cells which display MHC class-I proteins. In addition to the recognition and killing of infected cells, CD8+T cells can interfere with stages of the HIV life-cycle. Chemokines produced by CD8+ T cells bind to their seven-transmembrane G protein coupled receptors resulting in a block in the entry of HIV into macrophages and T cells. In addition, activated CD8+ T cells produce factors which strongly modulate HIV at the level of transcription. This review will focus primarily on the current knowledge of the multifactorial functions of CD8+ T cells in HIV infection. An understanding of the mechanisms involved in the CD8-mediated control of transcription may identify other factors with potential value in the treatment of HIV infection. PMID- 12369878 TI - The cyanobacterial origin of potent anticancer agents originally isolated from sea hares. AB - It is increasingly evident that the true biological origin of many metabolites originally isolated from certain marine macroorganisms is cyanobacterial. For example, several dolastatins, potent cytotoxic compounds originally derived from the sea hare Dolabella auricularia, have now been isolated from marine cyanobacteria of the genera Lyngbya and Symploca. This review discusses the isolation of dolastatins and close structural analogues from cyanobacteria. Biosynthetic signatures of metabolites isolated from sea hares, but which are most probably cyanobacterial in origin, are also presented. Finally, some more complex ecology involving movement of cyanobacterial metabolites through the marine food web is presented. PMID- 12369879 TI - A review of chemical agents in the pharmacotherapy of addiction. AB - Chemical substance abuse has tormented mankind throughout history. A number of chemical approaches have been employed in an attempt to treat chemical addiction. Unfortunately, most of these have proven unsuccessful though several chemical entities have been shown to be moderately effective. The naturally occurring alkaloid ibogaine has been reported to interrupt the cravings for alcohol, cocaine and opiates. Other alkaloids from Tabernanthe iboga, such as ibogamine and tabernanthine, provide insight into the structure activity relationship at the different receptors believed to be involved in addiction. The synthetic iboga alkaloid congener, 18-MC, also shows potential as an anti-addictive agent without the hallucinogenic effects of ibogaine. Additionally, acamprosate, BP 897, GBR12909, lofexidine and memantine have shown promising results in the treatment of addiction. All of these leads provide a start for the medicinal chemist to design anti-addictive agents, since currently no drugs are approved in the U.S. for the treatment of addictions to cocaine, methamphetamine, other stimulants or PCP. PMID- 12369880 TI - Reliability of logP predictions based on calculated molecular descriptors: a critical review. AB - Correct QSAR analysis requires reliable measured or calculated logP values, being logP the most frequently utilized and most important physico-chemical parameter in such studies. Since the publication of theoretical fundamentals of logP prediction, many commercial software solutions are available. These programs are all based on experimental data of huge databases therefore the predicted logP values are mostly acceptable - especially for known structures and their derivatives. In this study we critically reviewed the published methods and compared the predictive power of commercial softwares (CLOGP, KOWWIN, SciLogP/ULTRA) to each other and to our recently developed automatic QS(P)AR program. We have selected a very diverse set of 625 known drugs (98%) and drug like molecules with experimentally validated logP values. We have collected 78 reported "outliers" as well, which could not be predicted by the "traditional" methods. We used these data in the model building and validation. Finally, we used an external validation set of compounds missing from public databases. We emphasized the importance of data quality, descriptor calculation and selection, and presented a general, reliable descriptor selection and validation technique for such kind of studies. Our method is based on the strictest mathematical and statistical rules, fully automatic and after the initial settings there is no option for user intervention. Three approaches were applied: multiple linear regression, partial least squares analysis and artificial neural network. LogP predictions with a multiple linear regression model showed acceptable accuracy for new compounds therefore it can be used for "in-silico-screening" and/or planning virtual/combinatorial libraries. PMID- 12369881 TI - Clinical significance of pleiotropic effects of statins: lipid reduction and beyond. AB - Statins significantly reduce cardiovascular-related morbidity and mortality in patients with and without coronary artery disease. The potential of this drug class has yet to be fully explored. Accumulating evidence from basic research and clinical trials indicates that statins have pleiotropic effects that may largely account for the clinical benefits observed. Potential beneficial effects of these agents include enhancement of nitric oxide production in vasculature and the kidney. Statins have been shown to stabilize unstable plaques, improve vascular relaxation, and promote new vessel formation. Clinical trials and animal studies have shown that these agents reduce cardiovascular disease (CVD) risks and events, progression of nephropathy, development of diabetes, and fracture rates; these are benefits that go beyond lipid lowering alone. Potential beneficial effects are due to the positive impact on vascular and glomerular nitric oxide (NO) production and attenuation of vascular inflammation. Effects on bone, including fracture reduction, are thought to be mediated by direct action on bone formation. Moreover, potential reduction in the development of diabetes as observed in the West of Scotland Coronary Prevention Study (WOSCOPS) may relate to the improvement in insulin sensitivity. These actions are mediated, in part, by the effects on small G-proteins, modulation of signaling cascades, transcription, and gene expression. In particular, the inhibition of small GTP binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the direct cellular effects of statins on the vascular wall. The clinical relevance of these effects is beginning to be recognized, and ongoing studies will be able to answer these many questions in the near future. Actions of statins on vascular, glomerular, bone, and insulin-sensitive tissue as well as effects of statins on cognitive function and oncoprotection will be discussed in this review. PMID- 12369882 TI - Conformational changes preceding amyloid-fibril formation of amyloid-beta and stefin B; parallels in pH dependence. AB - Amyloid beta (A beta) protein is the key component of amyloid plaques in Alzheimer's disease brain whereas stefin B is an intracellular cysteine proteinase inhibitor, broadly distributed in different tissue and recently reported to form amyloid fibrils in vitro. By reducing the pH to 4.6, the native conformation of both polypeptides are changed into less ordered metastable intermediates that are stabilized by formation of the more stable fibrils. In A beta, the Glu at position 11 was found to be responsible for the conformational change at pH 4.6. Metal ions, including copper and zinc, could also induce conformational changes of A beta at neutral pH. The acid modified A beta conformer exhibited protease K resistance, preferential internalization and accumulation in the human glial cells. In stefin B, reducing the pH to pH 3.3 results in another intermediate of the molten-globule type which also leads to amyloid fibril formation. Multiple sequence alignment revealed distinct similarities of A beta (1-42) peptide, stefin B (13 to 61 residues) and prion fragment (90 to 144 residues). PMID- 12369883 TI - Mechanistic studies of the process of amyloid fibrils formation by the use of peptide fragments and analogues: implications for the design of fibrillization inhibitors. AB - The process of amyloid fibrils formation is a common mechanism of a large number of unrelated infectious, genetic and spontaneous diseases. A partial list includes the bovine spongiform encephalopathy (BSE), Alzheimer's diseases, Type II diabetes, Creutzfeldt-Jakob disease, and various unrelated amyloidosis diseases. In spite of its significant clinical importance, the mechanism of fibrillization is not fully understood. This review discusses the recent advancements in the mechanistic studies of amyloid formation by the use peptide fragments and analogues of amyloid-forming proteins and polypeptides. The use of short peptide shed much light of the mechanism of amyloid fibrillization. Recent studies clearly prove that very short peptide fragments (as short as pentapeptides) can form well-ordered amyloidal structures. Therefore, the molecular recognition and self-assembly process that lead to the formation of order structures is being mediated by small structural elements. Analysis of short amyloid-related fragment by the use of an alanine-scan and sequence analysis of a variety of unrelated peptide and protein fragments suggest that aromatic interaction may play a central role in the process of amyloid formation. Inhibitors that are based on the short aromatic elements already demonstrated clear potency in arresting the process of amyloid fibrils formation. Taken together, the recent advancement in the mechanistic understanding of the process of amyloid fibrils formation has a major importance in the development of inhibitors of fibrillization that may serve as future therapeutic means to treat amyloid diseases. PMID- 12369884 TI - Anti-aggregating antibodies, a new approach towards treatment of conformational diseases. AB - More and more evidence shows that Alzheimer's and prion-related diseases belong to the family of conformational diseases characterized by protein self association and tissue deposition as amyloid fibrils. Regardless of the nature of the protein constituent, all forms of amyloid are stable assemblies based on noncovalent interactions between subunits of crossed beta-sheet structure. Understanding the mechanism and molecular details of the pathological conformational conversion of amyloidogenic proteins may be of importance to the development of approaches towards prevention and treatment of such diseases. We previously found that monoclonal antibodies (mAbs) interact at strategic sites where protein unfolding is initiated, thereby stabilizing the protein and preventing further precipitation. Indeed, site-directed mAbs raised against the N terminal region of Alzheimer's beta-peptide (A beta P) disaggregate A beta P fibrils, restore peptide solubility and prevent its neurotoxic effects. Similarly, selected mAbs raised against the human prion peptide 106-126 modulate conformational changes occurring in the prion peptide exposed to aggregating conditions, preventing its aggregation and related neurotoxicity on cultivated neural-like cells. All these data and related procedures bring more attention to the immunological concept in the treatment of conformational diseases, and the recent performance of such antibodies in transgenic mice, as a model for human diseases, suggests the development of vaccination approaches against such diseases. PMID- 12369885 TI - Protein conformational misfolding and amyloid formation: characteristics of a new class of disorders that include Alzheimer's and Prion diseases. AB - The accumulation of proteinaceous deposits has been recognised to occur in several neurodegenerative conditions including Prion diseases, Alzheimer's disease, Parkinson's disease, and Huntington's disease. Over the last two decades interest in these conditions has increased markedly, fueled partially by an increasing prevalence of these diseases in the Western world. Evidence indicates that anomalous protein misfolding and aggregation, with an accompanying "toxic gain of function" is central to the neuropathogenesis of these diseases. An increased understanding of the similarities and differences in the production, aggregation and accumulation of the respective proteins involved in these diseases, and the associated mechanisms of neurodegeneration, should aid in the development of new therapeutic agents to treat this group of related disorders. PMID- 12369886 TI - Pathological peptide folding in Alzheimer's disease and other conformational disorders. AB - Main neuropathological hallmarks of Alzheimer's disease (AD) and other neurodegenerative disorders are the deposition of neurofibrillary tangles consisting of abnormally phosphorylated protein tau and of senile plaques largely containing insoluble beta-amyloid peptides (A beta), containing up to 43 amino acid residues derived from the beta-amyloid precursor protein. Such A beta-sheets become visible by using suitable histochemical methods. Molecular simulation showed that the central, alpha-helical, lipophilic, antigenic folding domain of the A beta-peptide loop is a promising molecular target of beta-sheet breakers that thus prevent the polymerization of A beta into aggregates. It seems that di- and tetramers of A beta-peptides have a beta-barrel- like structure. In the present review, an optimized neural network analysis was applied to recognize possible structure-activity relationships of peptidomimetic beta-sheet breakers. The anti-aggregatory potency of beta-sheet breakers largely depends upon their total, electrostatic, and hydration energy as derived from their geometry optimized conformations using the hybrid Gasteiger-molecular mechanics approach. Moreover, we also summarize peptide misfolding in several disorders with distinct clinical symptoms, including prion diseases and a broad variety of systemic amyloidoses, as the common pathogenic step driving these disorders. In particular, conversion of nontoxic alpha-helix/random-coils to beta-sheet conformation was recognized as being critical in producing highly pathogenic peptide assemblies. Whereas conventional pharmacotherapy of AD is mainly focused on restoring cholinergic activity and diminishing inflammatory responses as a consequence of amyloid accumulation, we here survey potential approaches aimed at preventing or reserving the transition of neurotoxic peptide species from alpha helical/random coil to beta-sheet conformation and thus abrogating their effects in a broad variety of disorders. PMID- 12369887 TI - The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. AB - Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s. PMID- 12369888 TI - Modulation of phase II drug metabolizing enzyme activities by N-heterocycles. AB - Drugs and other chemicals that contain one or more heterocyclic nitrogen atoms are most widely known for their ability to both inhibit and induce cytochrome p450s. Their ability to affect a wide range of Phase II drug metabolizing enzyme activities has received much less attention and exposure. For some N heterocycles, induction of Phase II metabolism occurs in the absence of induction effects on cytochrome p450, a property with potential utility in reducing drug and chemical toxicity (chemoprotection). The mechanism of this induction, and an accounting of the Phase II selectivity, has not been rigorously investigated. This review gathers and documents existing information on the simultaneous inductive effects of N-heterocycles on glucuronidation, sulfation (sulfonation) and glutathione conjugation reactions, as well epoxide hydrolase and quinone oxidoreductase activities. Basic information on cytochrome p450 induction is provided for comparative purposes. The review will serve as a base for any future interest in the phenomenon. The existing information on enzyme changes derives largely from laboratory animal studies. Chemicals investigated range from both simple and complex pyridines and imidazoles to benzoquinolines and phenanthrolines. Isomeric differences can have a major impact on induction characteristics and Phase II selectivity. Promising indications for the effectiveness of N-heterocycle elicited enzyme changes in chemoprotection in vivo awaits investigation. PMID- 12369889 TI - Cytochrome P450-based gene therapy for cancer treatment: from concept to the clinic. AB - In the last 16 years, more than a dozen gene-directed enzyme prodrug therapies for cancer treatment have been evaluated in preclinical studies. However, only few of them have evolved to the stage of clinical trial. This review assesses current knowledge in the area of cancer gene therapy, emphasizing cytochrome p450 (CYP)-based prodrug activation systems. This approach is intuitively highly suitable for the treatment of cancers, since several major anticancer drugs are activated by liver CYP enzymes. Important features of this strategy include: 1) use of human CYP genes to avoid immune complications that may hamper expression of therapeutic genes of non-human origin and thereby inhibit prodrug activation, 2). use of well established and clinically effective anticancer prodrugs, 3). strong bystander cytotoxic effect seen with all liver-activated CYP prodrugs, 4). the potential to inhibit liver CYP activity and expression to increase the bioavailability of prodrugs for CYP-transduced tumors, 5). possible extension to many CYP enzymes and their potential anticancer prodrug substrates, and 6). it can be used to arm therapeutic conditionally replicating viruses. Historically, this strategy utilized CYP 2B1 to activate oxazaphosphorines. It is now becoming clear that the repertoire of prodrugs is expandable and that CYP gene candidates are not limited to naturally occurring CYP genes, but may also encompass engineered CYP enzymes, improved by site directed mutagenesis or other approaches. Encouraging results from a recent phase I/II clinical trial that have implemented this strategy, as well as emerging problems related to gene delivery are discussed in this review. PMID- 12369890 TI - Complexities of glucuronidation affecting in vitro in vivo extrapolation. AB - Glucuronidation is responsible for the clearance of a diverse range of drug and chemicals whose topology confers properties that complicate in vitro-in vivo clearance correlations as compared to those possible for oxidative metabolism. The active site of the UGTs faces the inside of the luminal space of the endoplasmic reticulum, thus presenting diffusional barriers for substrates, the cosubstrate, UDPGA, and resultant glucuronide products. Transport processes for the cosubstrate UDPGA and glucuronidated products likely contribute to the well known latency phenomena in which exogenous detergents or alamethicin are required for maximal UGT activity in microsomes. This complicates the extrapolation of results of in vitro clearance studies to the in vivo situation. Even with activation, the microsomal-based clearance values still underestimate the actual in vivo UGT-mediated clearance; therefore latency is not the only explanation for the poor correlation. Recent data indicate that hepatocytes are a promising in vitro system that can be used for the early evaluation of human clearance behavior of drug candidates. Both induction and inhibition of UGT-mediated clearance are a source of clinical drug-drug interactions. Emerging evidence indicates that the same mechanisms identified in the regulation of CYP enzymes also are involved in regulation of the UGTs, i.e., CAR, AH and probably PXR mediate regulation of UGT1A1, 1A6 and UGT2B7, respectively. In contrast to CYP mediated interactions, with a few exceptions, the magnitude of UGT-mediated interactions are less than 2-fold because of the relatively high UGT Km values and substrate overlap among the multiple isozymes. PMID- 12369891 TI - Assessment of blood-brain barrier penetration: in silico, in vitro and in vivo. AB - The amount of drug achieved and maintained in the brain after systemic administration is determined by the agent's permeability at blood-brain barrier (BBB), potential involvement of transport systems, and the distribution, metabolism and elimination properties. Passive diffusion permeability may be predicted by an in silico method based on a molecule's structure property. In vitro cell culture is another useful tool for the assessment of passive permeability and BBB transports (e.g. PGP, MRP). In situ or in vivo techniques like carotid artery single injection or perfusion, brain microdialysis, autoradiography, and others are used at various stages of drug discovery and development to estimate CNS penetration and PK/PD correlation. Each technique has its own application with specific advantages and limitations. PMID- 12369892 TI - Whole-body autoradiography in drug discovery. AB - In the drug discovery process the pharmacokinetic screening, drug stability studies, evaluation of metabolites, CYP involvement, enzyme induction and inhibition, and excretion studies play a major role. The use of more sensitive and novel detection systems have made the discovery process less cumbersome than in previous years. In particular, the use of whole-body autoradiography (WBA) for tissue distribution, which was once considered an impractical tool, owing to the long turn around time (4-10 weeks), is coming to the forefront for rapidly resolving issues encountered in discovery. In today's research environment early lead compounds can be radio-labeled and whole-body sections imaged quickly (3-5 days) using new techniques, which has made (14)C- and (3)H-WBA a viable tool. The technique has been used in vivo in species from mice to monkeys, and ex vivo and/or in vitro in larger animals and humans. WBA has considerable merit in identifying "pharmacodeficient" compounds and providing insight on mechanistic questions. WBA data can provide information related to tissue pharmacokinetics, routes of elimination, CYP or Pgp mediated drug-drug interactions, tissue distribution, site specific drug localization and retention, metabolism, clearance, compound solubility issues, routes of administration, penetration into specific targets (e.g., tumors), tissue binding (e.g., melanin), and interspecies kinetics. Thus, WBA is quickly becoming part of the battery of studies conducted during the lead optimization process to select optimal drug candidates. Examples of the use of the WBA tool in early discovery are reviewed. PMID- 12369893 TI - Analysis of anticancer drugs and their metabolites by mass spectrometry. AB - There is an increasing awareness that the metabolites of anticancer drugs can contribute to the pharmacodynamic effects that are observed, which has stimulated a much greater emphasis on metabolic and pharmacokinetic issues. This has coincided with the development of electrospray and related atmospheric pressure ionization mass spectrometry techniques such as ionspray (nebulizer assisted electrospray), turboionspray (heated nebulizer assisted electrospray) and atmospheric pressure chemical ionization (nebulization coupled with corona discharge). The combination of collision induced dissociation and tandem mass spectrometry coupled with a soft ionization process that produces abundant molecular species provides very powerful methodology for the trace analysis of drugs and their metabolites. The present review has emphasized the more rigorous quantitative applications that have appeared in the literature over the last five years. It is evident that modern techniques of liquid chromatography tandem mass spectrometry coupled with stable isotope dilution methodology have had a profound effect on our ability to analyze anticancer drugs and their metabolites. As new drugs emerge into the clinic, this methodology will clearly be the method of choice, particularly when many samples have to be analyzed over a short time. This approach was beautifully demonstrated in the study of the novel signal transduction inhibitor, Gleevec where thousands of clinical samples were analyzed for drug and metabolites over a relatively short period of time. The need to analyze anticancer drugs and their metabolites with such prompt turn around times has stimulated even more rapid approaches to analysis using robotic-based purification methodology and short LC chromatographic run times. PMID- 12369894 TI - Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. AB - Drug or xenobiotics metabolizing enzymes (DMEs or XMEs) play central roles in the biotransformation, metabolism and/or detoxification of xenobiotics or foreign compounds, that are introduced to the human body. In general, DMEs protect or defend the body against the potential harmful insults from the environment. Once in the body, many xenobiotics may induce signal transduction events either specifically or non-specifically leading to various cellular, physiological and pharmacological responses including homeostasis, proliferation, differentiation, apoptosis, or necrosis. For the body to minimize the insults caused by these xenobiotics, various tissues/organs are well equipped with diverse DMEs including various Phase I and Phase II enzymes, which are present in abundance either at the basal level and/or increased/induced after exposure. To better understand the pharmacogenomic/gene expression profile of DMEs and the underlying molecular mechanisms after exposure to xenobiotics or drugs, we will review our current knowledge on DNA microarray technology in gene expression profiling and the signal transduction events elicited by various xenobiotics mediated by either specific receptors or non-specific signal transduction pathways. Pharmacogenomics is the study of genes and the gene products (proteins) essential for pharmacological or toxicological responses to pharmaceutical agents. In order to assess the battery of genes that are induced or repressed by xenobiotics and pharmaceutical agents, cDNA microarray or oligonucleotide-based DNA chip technology can be a powerful tool to analyze, simultaneously, the gene expression profiles that are induced or repressed by xenobiotics. The regulation of gene expression of the various phase I DMEs such as the cytochrome P450 (CYP) as well as phase II DMEs generally depends on the interaction of the xenobiotics with the receptors. For instance, the expression of CYP1 genes can be induced via the aryl hydrocarbon receptor (AhR) which dimerizes with the AhR nuclear translocator (ARNT), in response to many polycyclic aromatic hydrocarbon (PAHs). Similarly, the steroid family of orphan receptors, the constitutive androstane receptor (CAR) and pregnane X receptors (PXR), heterodimerize with the retinoid X receptor (RXR), transcriptionally activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as phenobarbital-like compounds (CAR) and dexamethasone and rifampin-type of agents (PXR). The peroxisome proliferator activated receptor (PPAR) which is one of the first characterized members of the nuclear hormone receptor, also dimerizes with RXR and it has been shown to be activated by lipid lowering agent fibrate-type of compounds leading to transcriptional activation of the promoters on the CYP4A genes. The transcriptional activation of these promoters generally leads to the induction of their mRNA. The physiological and the pharmacological implications of common partner of RXR for CAR, PXR, and PPAR receptors largely remain unknown and are under intense investigations. For the phase II DMEs, phase II gene inducers such as phenolic compounds butylated hydroxyanisol (BHA), tert-butylhydroquinone (tBHQ), green tea polyphenol (GTP), (-)-epicatechin-3-gallate (EGCG) and the isothiocyanates (PEITC, sulforaphane) generally appear to be electrophiles. They can activate the mitogen-activated protein kinase (MAPK) pathway via electrophilic-mediated stress response, resulting in the activation of bZIP transcription factors Nrf2 which dimerizes with Mafs and binds to the antioxidant/electrophile response element (ARE/EpRE) enhancers which are found in many phase II DMEs as well as many cellular defensive enzymes such as thioredoxins, gammaGCS and HO-1, with the subsequent induction of gene expression of these genes. It appears that in general, exposure to phase I or phase II gene inducers or xenobiotics may trigger a cellular "stress" response leading to the increase in the gene expression of these DMEs, which ultimately enhance the elimination and clearance of the xenobiotics e xenobiotics and/or the "cellular stresses" including harmful reactive intermediates such as reactive oxygen species (ROS), so that the body will remove the "stress" expeditiously. Consequently, this homeostatic response of the body plays a central role in the protection of the organism against environmental insults such as xenobiotics. Advances in DNA microarray technologies and mammalian genome sequencing will soon allow quantitative assessment of expression profiles of all genes in the selected tissues. The ability to predict phenotypic outcomes from gene expression profiles is currently in its infancy, however, and will require additional bioinformatic tools. Such tools will facilitate information gathering from literature and gene databases as well as integration of expression data with animal physiology studies. The study of pharmacogenomic/gene expression profile and the understanding of the regulation and the signal transduction mechanisms elicited by pharmaceutical agents can be of potential importance during drug discovery and the drug development. PMID- 12369895 TI - Biological activities, mechanisms of action and biomedical prospect of the antitumor ether phospholipid ET-18-OCH(3) (edelfosine), a proapoptotic agent in tumor cells. AB - The antitumor ether lipid ET-18-OCH(3) (edelfosine) is the prototype of a new class of antineoplastic agents, synthetic analogues of lysophosphatidylcholine, that shows a high metabolic stability, does not interact with DNA and shows a selective apoptotic response in tumor cells, sparing normal cells. Unlike currently used antitumor drugs, ET-18-OCH(3) does not act directly on the formation and function of the replication machinery, and thereby its effects are independent of the proliferative state of target cells. Because of its capacity to modulate cellular regulatory and signaling events, including those failing in cancer cells, like defective apoptosis, ET-18-OCH(3), beyond its putative clinical importance, is an interesting model compound for the development of more selective drugs for cancer therapy. Although ET-18-OCH(3) enhances host defense mechanisms against tumors, its major antitumor action lies in a direct effect on cancer cells, inhibiting phosphatidylcholine biosynthesis and inducing apoptosis in tumor cells. Recent progress has allowed unraveling the molecular mechanism underlying the apoptotic action of ET-18-OCH(3), leading to the notion that ET-18 OCH(3) is selectively incorporated into tumor cells and induces cell death by intracellular activation of the cell death receptor Fas/CD95. This intracellular Fas/CD95 activation is a novel mechanism of action for an antitumor drug and represents a new way to target tumor cells in cancer chemotherapy that can be of interest as a new framework in designing novel antitumor drugs. ET-18-OCH(3) and some analogues are pleiotropic agents that affect additional biomedical important diseases, including parasitic and autoimmune diseases, suggesting new therapeutic indications for these compounds. PMID- 12369896 TI - The influence of DMPK as an integrated partner in modern drug discovery. AB - In response to the challenge laid down by advances in other drug discovery functions, DMPK has now established an array of automated, miniaturised in vitro screens, rapid bioanalytical methodologies and in silico tools with which to optimise or predict passive absorption, metabolic clearance and minimise drug drug interaction potential. The awareness of the pivotal role that physicochemical properties play in the control of many of these processes has been key. This review highlights some of these structure-activity relationships with emphasis on drug absorption, clearance, protein binding and distribution. However, some fundamental processes remain to be elucidated fully, including the in vivo impact of non-specific or futile binding in in vitro screens and the functional significance of intestinal and hepatobiliary transporter proteins. Transgenic animals should soon add value to our understanding of the contribution of transporter proteins to drug bioavailability (intestinal and hepatic drug uptake/efflux) and drug interactions and in validating projections for Man. Future studies should also focus on the evaluation of the various in vitro human CYP induction screens available, with particular emphasis on their predictive value for the clinical scenario. PMID- 12369897 TI - Permeability characteristics of endocrine-disrupting chemicals using an in vitro cell culture model, Caco-2 cells. AB - The purpose of this study was to evaluate the permeability characteristics of endocrine disrupting chemicals utilizing epithelial monolayers of Caco-2 cells. The drugs tested in this study were bisphenol A (BPA), tert-octylphenol (tOP), tert-butylphenol (tBP), di(2-ethylhexyl)phthalate (DOP), dibutylphthalate (DBP), and butylbenzylphthalate (BBP), all of which are used in plastic materials. The Caco-2 cell line was grown on cell culture inserts with polyethylene terephthalate membranes, and Hank's balanced salt solution (HBSS, pH 7.4) was used for the transport experiments. The barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a volt ohmmeter, and transport of these endocrine disrupting chemicals was examined in both directions. The permeated amounts of these chemicals within 180 min in the apical to basolateral (A-to-B) and the basolateral to apical (B-to-A) directions without verapamil, a P-glycoprotein (P-gp) inhibitor, were in the rank order of tBP > tOP > BPA > DOP > DBP > BBP and BPA >> tBP > tOP > DOP > DBP > BBP, respectively. In the presence of 100 microM verapamil, the permeated amounts of BPA, tOP and tBP within 180 min in the B-to-A direction decreased by 12-, 2.6- and 3.1-fold, respectively. In the case of phthalate esters, the permeated amount of DOP within 180 min in the B-to-A direction decreased by 1.6-fold, while that of DBP and BBP showed no significant changes. The ratios of apparent permeability coefficient of B-to-A against A-to-B, P(app) ratios, for BPA, tOP and tBP were markedly decreased in the presence of 100 microM verapamil. These findings indicated that both BPA and alkyl phenols are substrates of the P-gp located in the apical side of Caco-2 cells, and suggested that the P-gp in the small intestine may act as an organic barrier against BPA and alkyl phenols. PMID- 12369898 TI - Structural biology of the cell adhesion protein CD2: alternatively folded states and structure-function relation. AB - Cluster of differentiation 2 (CD2) is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells and plays an important role in mediating cell adhesion in both T-lymphocytes and in signal transduction. The understanding of the biochemical basis of molecular recognition by the cell adhesion molecule CD2 has been advanced greatly through the determination of structures and the dynamic properties of the complexes and their individual components and through site-directed mutagenesis. A number of general principles can be derived from the structural and functional studies of the extracellular domains of CD2 and CD58 and their complex. Significant electrostatic interactions within the protein-protein interfaces contribute directly to the formation of macromolecular complexes of CD2 and CD58. Also, residues located on the protein protein interface demonstrate a certain degree of conformational change upon the formation of a complex. Structural analysis of CD2 has revealed that this adhesion molecule exhibits strong conformational flexibility with a partial non native helical conformation at high temperatures and in the presence of an organic solvent. In addition, it can be converted into a domain swapped dimer, or trimer and tetramer through hinge deletion. Thus, the conformational status of the adhesive proteins contributes to the regulation of cell adhesion and the folding of CD2. PMID- 12369899 TI - Protein-X, Pancreatic Stone-, Pancreatic thread-, reg-protein, P19, lithostathine, and now what? Characterization, structural analysis and putative function(s) of the major non-enzymatic protein of pancreatic secretions. AB - Reg protein was first found in pancreatic stones. It was named Pancreatic Stone Protein and later renamed lithostathine, as it was assumed to prevent stone formation. The 144 amino acid protein is O-glycosylated on Thr-5. The glycan chain is variable in length and in charge. Lithostathine 3-D organization is of the C-lectin type, even though it is unlikely to have any functional calcium binding site. The Arg11-Ile12 bond is readily cleaved by trypsin; the resulting C terminal polypeptide precipitates at physiological pH and tends to form fibrils. The protein was more recently found in the regenerating endocrine pancreas and it was named Reg (for regenerating) protein. Numerous proteins related to Reg have been identified successively in several mammalian species. They constitute the Reg superfamily. Reg genes show the same organization and are located in the same chromosome region. These genes are therefore likely to derive from a common ancestor gene by duplication. In the course of evolution, they may have diverged in tissue-related expression and function. In the endocrine pancreas, Reg protein stimulates islet beta-cell growth and reduces experimental diabetes via the activation of a high affinity receptor. The role of the protein produced by the exocrine pancreas, however, is controversial. Not only is Reg/lithostathine unlikely to be a physiologically relevant pancreatic stone inhibitor, but it may contribute to stone formation. We suggest that it might help prevent the harmful activation of protease precursors in the pancreatic juice. The protein provides a useful model for examining the conformational changes associated with globular to fibril transformation. PMID- 12369900 TI - Structure and function of complement activating enzyme complexes: C1 and MBL MASPs. AB - The complement system is a major effector arm of the immune defense contributing to the destruction of invading pathogens. There are three possible routes of complement cascade activation: the classical, the alternative and the lectin pathways. The activation of the classical and lectin pathways is initiated by supramolecular complexes, which resemble each other. Each complex has a recognition subunit (C1q in the classical and mannose-binding lectin (MBL) in the lectin pathway), which associates with serine protease zymogens (C1q with C1r and C1s, and MBL with MBL-associated serine proteases: MASP-1, MASP-2) to form the C1 and MBL-MASPs complexes, respectively. As the recognition subunits bind to activator structures, subsequent activation of the serine protease zymogens occurs. The precise structure of the complexes and the exact mechanism of their activation have not been solved, yet. In this review we summarize the recent advances about the structure and function of the individual subcomponents of both complexes achieved by genetic engineering, molecular modeling, physico-chemical and functional studies. Special emphasis will be laid on the serine proteases: the role of the individual domains in the assembly of the C1s-C1r-C1r-C1s tetramer and in the control of the protease activity will be discussed. We will then focus on recent functional models of the supramolecular complexes. The question of how a non-enzymatic signal (the binding of C1q or MBL to activators) can be converted into enzymatic events (activation of serine protease zymogens) will be addressed. The similarities and differences between C1 and MBL-MASPs will also be discussed. PMID- 12369901 TI - Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase and caspase-3 by nitric oxide. AB - The regulation of enzyme activity function is a major factor in the cellular response to a changing environment. One mechanism of enzyme activity regulation includes post-translational protein thiol modification by nitric oxide (NO) or its redox species. Major routs used by NO to modify cysteine residues of proteins include S-nitrosation, oxidation, mixed disulfide formation with glutathione, and the covalent attachment of nucleotide cofactors, i.e NAD(+)/NADH. Critical thiol centers serve as recognition sites for NO, thus channeling the NO signal through post-translational modifications and oxidation into cellular functions. Here, we summarize current knowledge on active site thiol modification of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and caspase-3 by nitric oxide. Although very different in their cellular function, both enzymes contain highly reactive cysteines which represent sensitive targets for NO. Our studies are supportive of a potential role of S-nitrosation and mixed disulfide formation as a general signaling mechanism that allows sensing of nitrosative stress. At the same time, modification of GAPDH and caspase-3 by NO show the diversity of mechanisms (S nitrosation versus oxidations) that we are confronted with as a result of NO delivery, especially comparing in vitro studies with cellular systems. In the future it will be challenging to dissect how nitrosative and oxidative signaling mechanisms overlap and how intracellular communication systems allow their activation in a selective way. PMID- 12369902 TI - Concepts and misconcepts in the analysis of simple kinetics of protein folding. AB - Unusually simple two-state kinetics characterizes the folding of a number of small proteins possessing a variety secondary structures. This limits dramatically the number of experimentally resolvable parameters that may characterize this process and also suggests the possibility to describe it based on simple theories borrowed from the field of ordinary chemical reactions. An attempt is made to critically evaluate the basic concepts, which are in the background of this approach. We demonstrate their limitations, which may cast doubt on the interpretation of experimental data. It is shown also that, in contrast to provisions of transition state theory, the simple kinetics of protein folding does not correlate with folded state stability or with the size of the folding unit. Moreover, the folding kinetics exhibits anomalous dependence on temperature and pressure and surprisingly strong dependence on solvent viscosity. The possible role in folding of fluctuations, relaxations and gradient dynamics is discussed. Being overlooked or underestimated, these mechanisms may determine the rate and specificity of the process. PMID- 12369903 TI - PPAR alpha-mediated responses in the rodent liver: a holistic biochemical view. AB - Carcinogenesis through the direct action of genotoxic, DNA damaging chemicals is an established and well-studied paradigm. As yet there are no short term tests available for non-genotoxic rodent carcinogens that do not damage DNA but cause liver tumours in long term rodent bioassays. A key aim is to develop short term in vitro screens for the detection of nongenotoxic carcinogens, and this requires knowledge of the mode or mechanism of action of this class of chemicals. The largest and most chemically diverse family of non-genotoxic hepatocarcinogens is the peroxisome proliferators (PPs) such as hypolipidaemic fibrate drugs, plasticizers used in clingwrap/medical tubing and certain pesticides and solvents. PPs mediate their biological responses via activation of the transcription factor PPAR alpha (peroxisome proliferator activated receptor alpha), a member of the nuclear hormone receptor superfamily. PPAR alpha activation is responsible for the pleiotropic effects of PPs in rodent liver such as the induction of enzymes of the b-oxidation pathway, hepatocyte DNA synthesis, liver enlargement and tumourigenesis. Although much is known, we are far from defining the key cell cycle regulating targets of PPs, due perhaps to past limitations of technology. The technology of proteomics allows quantitative measurement of the expression levels of potentially thousands of individual genes at the protein level on exposure to toxic insult. This is predicted to revolutionise the way many biological systems are investigated. Here we review the current knowledge of proteins involved in the response to peroxisome proliferators and describe the impact of proteomics in this field. PMID- 12369904 TI - Proteins that convert from alpha helix to beta sheet: implications for folding and disease. AB - The sequence of a protein normally determines which amino acid residues will form alpha helices, and which one beta sheets, to an extent that allows secondary structure prediction to be made with a reasonable reliability. Nevertheless, non native helical structures are observed during in vitro folding of several model proteins and may even occur during protein biosynthesis within the ribosomal exit tunnel. Moreover, non-native beta sheet structures are common in amyloid fibrils formed by a variety of pathogenic and even non-pathogenic proteins and peptides. In all of these cases, the formation of alpha helix precedes the appearance of beta sheet, which suggests that conversion from the simpler, more local helix structure to the often more convoluted sheet architecture during folding and pathogenic misfolding processes could be a unifying principle of general importance. A better understanding of this switching process, and the ability to design molecular systems which can be induced to switch between these conformations will have a significant impact on fields ranging from fundamental biochemistry through to applied technology and medicine. PMID- 12369905 TI - The use of circular dichroism in the investigation of protein structure and function. AB - Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as alpha-helix and beta-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising molten globule intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications. PMID- 12369906 TI - Aldosterone- and progesterone-membrane-binding proteins: new concepts of nongenomic steroid action. AB - n the classical theory of steroid action steroids penetrate into cells and bind to intracellular receptors resulting in modulation of nuclear transcription and protein synthesis within hours. In addition, rapid actions of steroids have been identified, which are incompatible with the classic model of steroid action. Specific binding sites for aldosterone and progesterone have been reported in membrane preparations of liver, vascular smooth muscle cells and kidney. These sites are discussed to be involved in rapid nongenomic steroid actions, such as the rapid activation of the Na(+)/H(+) exchanger and elevation of [Ca(2+)]i in vascular smooth muscle cells by aldosterone. In addition, rapid progesterone induced increases of [Ca(2+)]i have been reported in spermatozoa. A high affinity progesterone-membrane binding protein from porcine liver has been identified and cloned. The derived amino acid sequence showed no significant identity with any functional protein suggesting a binding site completely different to classic progesterone receptors. These binding sites are possibly involved in rapidly induced meiotic maturation of amphibian oocytes and the spermatozoan acrosome reactions as evidenced by recent studies, where the progesterone induced acrosome reactions and calcium signaling was blocked by a specific antibody raised against the membrane binding site for progesterone. In addition to data on specific steroid binding and rapid steroid signaling in vitro, results of nongenomic steroid effects in vivo are presented and their physiological relevance are discussed in the review. PMID- 12369907 TI - Engineering novel bioactive mini-proteins from small size natural and de novo designed scaffolds. AB - Mini-proteins, polypeptides containing less than 100 amino acids, such as (animal toxins, protease inhibitors, knottins, zinc fingers, etc.) represent successful structural solutions to the need to express a specific binding activity in different biological contexts. Artificial mini-proteins have also been designed de novo, representing simplified versions of natural folds and containing natural or artificial connectivities. Both systems have been used as structural scaffolds in the engineering of novel binding activities, according to three main approaches: i) incorporation of functional protein epitopes into structurally compatible regions of mini-protein scaffolds; ii) random mutagenesis and functional selection of particular structural regions of mini-protein scaffolds; iii) minimization of protein domains by the use of sequence randomization and functional selection, combined with structural information, in an iterative process. These newly engineered mini-proteins, with specific and high binding affinities within a small size and well-defined three-dimensional structure, represent novel tools in biology, biotechnology and medical sciences. In addition, some of them can also be directly used in therapy or present high potential to serve as drugs. In all cases, they represent precious structural intermediates useful to identify frameworks for peptidomimetic design or directly lead to new small organic structures, representing novel drug candidates. The engineering of novel functional mini-proteins has the potential to become a fundamental step towards the conversion of a protein functional epitope or a flexible peptide lead into a classical pharmaceutical. PMID- 12369908 TI - Mechanism of action of D-xylose isomerase. AB - The present knowledge on the stereochemical mechanism of action of glucose (or xylose) isomerase, one of the highest tonnage industrial enzymes, is summarized. First we deal shortly with experimental methods applied to study the structure and function of this enzyme: enzyme kinetics, protein engineering, X-ray crystallography, nuclear magnetic and electron paramagnetic resonance spectroscopy. Computational methods like homology modeling, molecular orbital, molecular dynamics and continuum electrostatic methods are also shortly treated. We discuss mostly those results and their contribution to the elucidation of the mechanism of action that have been published in the last decade. Structural characteristics of free xylose isomerase as well as its complexes with various ligands are depicted. This information provides a tool for the study of structural details of the enzyme mechanism. We present a general mechanism where the first step is ring opening, which is followed by the extension of the substrate to an open-chain conformation, a proton shuttle with the participation of a structural water molecule and the rate-determining hydride shift. The role of metal ions in the catalytic process is discussed in detail. Finally we present main trends in efforts of engineering the enzyme and delineate the prospective future lines. The review is completed by an extended bibliography with over 100 citations. PMID- 12369909 TI - Automated protein design and sequence optimisation: scoring functions and the search problem. AB - Advances in molecular biology may mean that almost any protein sequence can be synthesised, but perhaps this has served to highlight the inadequacy of theoretical work. For a given protein fold, it is probably not possible to reliably predict an "ideal" sequence. We identify and survey several aspects of the problem. Firstly, it is not clear what is the best way to score a sequence structure pair. Secondly, there is no consensus as to what the score function should represent (free energy or some abstract measure of sequence-structure compatibility). Finally, the number of possible sequences is astronomical and searching this space poses a daunting optimisation problem. These problems are discussed in the light of recent experimental successes. PMID- 12369910 TI - Computational methods for protein secondary structure prediction using multiple sequence alignments. AB - Efforts to use computers in predicting the secondary structure of proteins based only on primary structure information started over a quarter century ago [1-3]. Although the results were encouraging initially, the accuracy of the pioneering methods generally did not attain the level required for using predictions of secondary structures reliably in modelling the three-dimensional topology of proteins. During the last decade, however, the introduction of new computational techniques as well as the use of multiple sequence information has lead to a dramatic increase in the success rate of prediction methods, such that successful 3D modelling based on predicted secondary structure has become feasible [e.g., Ref 4]. This review is aimed at presenting an overview of the scale of the secondary structure prediction problem and associated pitfalls, as well as the history of the development of computational prediction methods. As recent successful strategies for secondary structure prediction all rely on multiple sequence information, some methods for accurate protein multiple sequence alignments will also be described. While the main focus is on prediction methods for globular proteins, also the prediction of trans-membrane segments within membrane proteins will be briefly summarised. Finally, an integrated iterative approach tying secondary structure prediction and multiple alignment will be introduced [5]. PMID- 12369911 TI - Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies. AB - Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins. PMID- 12369912 TI - Group II chaperonins as mediators of cytosolic protein folding. AB - Protein folding and assembly in the cell requires the assistance of molecular chaperones. These components prevent off-pathway folding reactions that lead to aggregation. They are also critical factors in organismal stress physiology, protecting cells against heat shock and providing thermotolerance. Among this important protein family are chaperonins. They form large cylindrical double ring complexes with a central cavity where protein binding and folding takes place in an ATP-dependent manner. Recently, components functionally related to the eubacterial and organellar chaperonins have been found in the cytosol of archaebacteria and of eukaryotic cells. Based on their sequences and structural features, they have been classified as group II chaperonins, to distinguish them from the group I chaperonins occurring in bacteria. Of particular interest in the group II family is the eukaryotic CCT complex, whose function in protein folding and assembly has been demonstrated mainly for the cytoskeletal proteins tubulin and actin. Together with the Hsp70 chaperone system, it can be considered as an essential helper factor to facilitate the folding of native proteins in the eukaryotic cytosol. Recent structural data have opened the path to a molecular understanding of group II chaperonins and have helped to define their role in cellular protein folding. PMID- 12369913 TI - Different approaches to potentiate the immune response induced by a 12-mer synthetic peptide. AB - Interest in synthetic immunogenic peptides is increasingly growing due to the continuous achievements in the understanding of the molecular basis of the immune response. Moreover, the use of peptide fragments to generate antibodies capable of recognizing and neutralizing viral particles could be a valuable alternative to conventional vaccines: they are safe, they can be designed to induce defined immune responses and they can be synthesised in large quantities in high purity. However, their relatively low immunogenicity requires the development of effective adjuvants and/or their incorporation into controlled release formulations. Several different strategies have been used for the induction and analysis of protective immune responses induced by short peptides against infectious diseases. In the present article we summarise the different approaches used to potentiate the immune response induced by a continuous epitope of hepatitis A virus: co-linear link of T-cell epitopes in different orientations, incorporation into liposomes, and preparation of homogeneous and heterogeneous Multiple Antigenic Peptides. PMID- 12369914 TI - DNA replication in the third domain (of life). AB - DNA replication is the process underlying evolution and the propagation of living organisms. Since the discovery of DNA-dependent DNA polymerases more than 40 years ago, the mechanisms governing DNA replication have been extensively studied in bacteria and eukarya. During the last several years, these studies have been extended to the third domain of life, the archaea. Although archaea are prokaryotes, their replication machinery and the proteins participating in the initiation of DNA replication are more similar to those found in eukarya than bacteria. It appears, however, that replication in archaea is a simpler version of the eukaryotic one as fewer polypeptides participate in each phase of the replication process. The archaeal replication apparatus also has several unique features not found in eukaryotic organisms. Furthermore, like bacteria, members of this domain thrive under a broad range of environmental conditions including extreme temperature, high salt, pH, etc. Thus, the replication machinery had to adapt to these extreme conditions. This article summarizes our current understanding of the mechanisms governing DNA replication in archaea and highlights similarities and differences between archaeal replication and that of bacteria and eukarya. PMID- 12369915 TI - Phage display of antibody fragments. AB - In recent years, phage display of peptides and proteins has become a very popular method in oncology, immunology, protein engineering and ligand-receptor studies among others. Antibody fragments, as Fabs or single chain Fv, have been among the first proteins to be displayed on the surface of a filamentous bacteriophage with a procedure initially described in 1990 by McCafferty et al. (Nature, 348, 552 554). From that time, molecular biology techniques have allowed the creation of large repertoires of antibody fragments from antibody V genes, bypassing hybrydoma technology and even immunisation. A large number of phage antibody libraries, from which molecules of the desired functional properties can be rapidly selected, have been built and distributed in many laboratories world wide. Antibody fragments recovered from phage libraries generally show moderate binding strength; with different systems of biopanning binders can be obtained with dissociation constant ranged between 10-(5) to 10-(8) M. Nevertheless, antibody fragments can be furtherly modified to improve affinity or avidity, respectively by mutating crucial residues of complementarity determining regions or by increasing the number of binding sites making dimeric, trimeric or multimeric molecules. Here, we summarise the latest progress in this field, with particular reference to applications of scFv in the diagnosis and therapy of solid tumours and in the molecular mimicry of viral antigens and membrane receptors. In fact, the production of artificial protein epitopes by phage antibodies is becoming a valid system to overcome problems caused by difficult cloning and low expression of particular recombinant proteins. PMID- 12369916 TI - Prediction of protein structural classes and subcellular locations. AB - The structural class and subcellular location are the two important features of proteins that are closely related to their biological functions. With the rapid increase in new protein sequences entering into data banks, it is highly desirable to develop a fast and accurate method for predicting the attributes of these features for them. This can expedite the functionality determination of new proteins and the process of prioritizing genes and proteins identified by genomics efforts as potential molecular targets for drug design. Various prediction methods have been developed during the last two decades. This review is devoted to presenting a systematic introduction and comparison of the existing methods in respect to the prediction algorithm and classification scheme. The attention is focused on the state-of-the-art, which is featured by the covarient discriminant algorithm developed very recently, as well as some new classification schemes for protein structural classes and subcellular locations. Particularly, addressed are also the physical chemistry foundation of the existing prediction methods, and the essence why the covariant-discriminant algorithm is so powerful. PMID- 12369917 TI - Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms. AB - The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined. Relationships between active site anatomy, topology and conformational rearrangements in the protein molecule is discussed in the context of enzyme mechanism of action. PMID- 12369918 TI - Computational tools for protein modeling. AB - Protein modeling is playing a more and more important role in protein and peptide sciences due to improvements in modeling methods, advances in computer technology, and the huge amount of biological data becoming available. Modeling tools can often predict the structure and shed some light on the function and its underlying mechanism. They can also provide insight to design experiments and suggest possible leads for drug design. This review attempts to provide a comprehensive introduction to major computer programs, especially on-line servers, for protein modeling. The review covers the following aspects: (1) protein sequence comparison, including sequence alignment/search, sequence-based protein family classification, domain parsing, and phylogenetic classification; (2) sequence annotation, including annotation/prediction of hydrophobic profiles, transmembrane regions, active sites, signaling sites, and secondary structures; (3) protein structure analysis, including visualization, geometry analysis, structure comparison/classification, dynamics, and electrostatics; (4) three dimensional structure prediction, including homology modeling, fold recognition using threading, ab initio prediction, and docking. We will address what a user can expect from the computer tools in terms of their strengths and limitations. We will also discuss the major challenges and the future trends in the field. A collection of the links of tools can be found at http://compbio.ornl.gov/structure/resource/. PMID- 12369919 TI - Recent progress in the solid-phase synthesis of glycopeptide. AB - The recent understanding of the biological role of glycoproteins has brought about a demand for the highly homogeneous glycopeptides as the functional model for glycoproteins. Thus, much efforts have been made to establish easy and efficient method for glycopeptide synthesis. In this paper, we briefly review the recent advances in the synthesis of O- and N-linked glycopeptide based on the solid-phase method. In O-glycopeptide section, the preparation of glycosylated amino acid units with mucin type and other O-linked carbohydrate chains and their use for solid-phase synthesis are summarized. Other approaches, such as the glycosylation of resin bound peptide are also overviewed. In N-glycopeptide section, the synthesis using glycosylated amino acid units as well as other methods are described. PMID- 12369920 TI - Construction of macromolecular assemblages in eukaryotic processes and their role in human disease: linking RINGs together. AB - Members of the Really Interesting New Gene (RING) family of proteins are found throughout the cells of eukaryotes and function in processes as diverse as development, oncogenesis, viral replication and apoptosis. There are over 200 members of the RING family where membership is based on the presence of a consensus sequence of zinc binding residues. Outside of these residues there is little sequence homology; however, there are conserved structural features. Current evidence strongly suggests that RINGs are protein interaction domains. We examine the features of RING binding motifs in terms of individual cases and the potential for finding a universal consensus sequence for RING binding domains (FRODOs). This review examines known and potential functions of RINGs, and attempts to develop a framework within which their seemingly multivalent cellular roles can be consistently understood in their structural and biochemical context. Interestingly, some RINGs can self-associate as well as bind other RINGs. The ability to self-associate is typically translated into the annoying propensity of these domains to aggregate during biochemical characterization. The RINGs of PML, BRCA1, RAG1, KAP1/TIF1beta, Polycomb proteins, TRAFs and the viral protein Z have been well characterized in terms of both biochemical studies and functional data and so will serve as focal points for discussion. We suggest physiological functions for the oligomeric properties of these domains, such as their role in formation of macromolecular assemblages which function in an intricate interplay of coupled metal binding, folding and aggregation, and participate in diverse functions: epigenetic regulation of gene expression, RNA transport, cell cycle control, ubiquitination, signal transduction and organelle assembly. PMID- 12369921 TI - Serratia type pore forming toxins. AB - The Serratia marcescens hemolysin represents a new type of hemolysin and has been studied in great molecular detail with regard to structure, activation and secretion. It has nothing in common with the pore forming toxins of E. coli type (RTX toxins), the Staphylococcus aureus alpha-toxin or the thiol activated toxin of group A beta-hemolytic streptococci (Streptolysin O). Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins. The S. marcescens hemolysin proteins ShlB and ShlA exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Edwardsiella tarda and Erwinia chrysantemi. Furthermore, sequence motifs present in ShlA and Shlb have been shown to be important for activity and secretion of the S. marcescens hemolysin. Thus, the S. marcescens hemolysin forms the prototype of a new class of hemolysins and of a new secretory mechanism. The uniqueness of this new mechanism is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor. New data implicate a conformational change in ShlA during activation. In addition, ShlA not only forms pores in erythrocytes but also in fibroblasts and epithelial cells. The cytotoxic action of ShlA is mainly determined by lysis of infected cells in vitro. In sublytic doses, as will normally be the situation in vivo, ShlA exerts additionally effects which are currently under investigation. The knowledge of the structure, activation, secretion and mode of action of S. marcescens hemolysin has implications for proteins, related in sequence or in mode of secretion and activation. PMID- 12369922 TI - The C-terminal domain of pancreatic lipase: functional and structural analogies with c2 domains. AB - The 3D structure of pancreatic lipase (PL) consists of two functional domains. The N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site, which involves a catalytic triad analogous to that present in serine proteases. The beta-sandwich C-terminal domain of PL plays an important part in the binding process between the lipase and colipase, the specific PL cofactor. Recent structure-function studies have suggested that the PL C-terminal domain may have an extra role apart from that of binding colipase. This domain contains an exposed hydrophobic loop (beta5') which was found to be located on the same side as the hydrophobic loops surrounding the active site, and it may be involved in the lipid binding process. Indirect evidence for this new function of the PL C terminal domain has been provided by studies with monoclonal antibodies directed against the beta5' loop. The catalytic activity of the PL-antibody complexes on water insoluble substrates decreased drastically, whereas their esterase activity on a soluble substrate remained unchanged. During the last few years, a number of protein structures (15-lipoxygenase, alpha-toxin from Clostridium perfringens) have been determined that contain domains with close structural homologies with the beta-sandwich C-terminal domain of PL. Generally speaking, these domains show structural homologies with the C2 domains occurring in a wide range of proteins involved in signal transduction (e.g. phosphoinositide-specific phospholipase C, protein kinase C, cytosolic phospholipase A2), membrane traffic (e.g. synaptotagmin I, rabphilin) and membrane disruption (e.g. perforin). Here it is proposed to review the structure and function of the C2 domains, based on the recent 3D structures and improved sequence alignments. PMID- 12369923 TI - Chitinolytic enzymes: catalysis, substrate binding, and their application. AB - After the epoch-making report on X-ray crystal structure of a lysozyme-N acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 +) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate assisted mechanism. Hen egg white lysozyme has the binding cleft represented by ( 4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)( 1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application. PMID- 12369925 TI - Introduction. dUTPases. PMID- 12369926 TI - Homotrimeric dUTPases; structural solutions for specific recognition and hydrolysis of dUTP. AB - Prevention of incorporation of dUTP into DNA is essential for maintenance of the genetic information. Prompt and specific removal of dUTP from the nucleotide pool, as expedited by the ubiquitous enzyme dUTPase, is therefore required for full viability in most biological systems. Conserved structural features perpetuate specificity in choice of substrate, which is crucial as hydrolysis of the structurally closely related nucleotides dTTP, dCTP and UTP would debilitate DNA and RNA synthesis. The most common family of dUTPases is the homotrimeric variety where X-ray structures are available for one bacterial, one mammalian and two retroviral dUTPases. These four enzymes have similar overall structural layouts, but the interactions that stabilise the trimer vary markedly, ranging from exclusively hydrophobic to water-mediated interactions. Trimeric dUTPases contain five conserved sequence motifs, positioned at the subunit interfaces where they contribute to the formation of the active sites. Each of the three identical active sites per trimer is built of residues contributed by all three subunits. One subunit provides residues involved in base and sugar recognition, where a beta-hairpin acts to maintain exquisite selectivity, while a second subunit contributes residues for phosphate interactions. The third subunit supplies a glycine-rich consensus motif located in the flexible C-terminal part of the subunit, known from crystallographic studies to cover the active site in the presence of substrate and certain substrate analogues. All dUTPases studied require the presence of a divalent metal ion, preferably Mg(2+), for optimal activity. The putative position of the essential metal ion has been identified in the structure of one retroviral dUTPase. Structure-function studies are essential if the properties of dUTPases are to be understood fully in relation to their biological role. In this review the structural arrangement of the homotrimeric dUTPases is discussed in the context of active site geometry, achievement of specificity and subunit interactions. PMID- 12369927 TI - Glycine rich P-loop motif in deoxyuridine pyrophosphatase. AB - Deoxyuridine pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester bond of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for dTTP synthesis. Similar to other nucleotide binding proteins, dUTPase also consists of a sequence motif rich in glycine residues known as P-loop motif. The P-loop motif of the nucleotide binding proteins are involved in substrate binding, catalysis, recognition and regulation of activity. In dUTPase the function of the P-loop motif is not well understood. One of the main reasons for this limited information is the lack of the three dimensional structure of a dUTPase enzyme with an ordered Gly-rich P-loop motif with a bound substrate and Mg(2+) ion. This review presents an insight into the role of Gly-rich P-loop motif in the function of dUTPase as revealed from the crystal structure. The analysis reveals the Gly-rich P-loop motif of dUTPase to be the longest in terms of its amino-acid composition as compared to other nucleotide binding proteins and exhibit a high-degree of sequence conservation among spectrum of species. The enzyme utilizes adaptive recognition to bind to the phosphate groups of the nucleotide. In particular, the alpha-beta phosphodiester bond adopts an unfavorable eclipsed conformation in the presence of the Gly-rich P-loop motif. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage. PMID- 12369928 TI - Evolution of the DUT gene: horizontal transfer between host and pathogen in all three domains of life. AB - The ubiquity of the dut gene in Eukarya, Eubacteria, and Archaea implies its existence in the last common ancestor of the three domains of life. The dut gene exists as single, tandemly duplicated, and tandemly triplicated copies. The dUTPase is encoded as an auxiliary gene in the genomes of several DNA viruses and two distinct lineages of retroviruses. A comprehensive analysis of dUTPase amino acid sequence relationships explores the evolutionary dynamics of dut genes in viruses and their hosts. The data set was comprised of representative sequences from available Eukaryotes, Archaea, Eubacteria cells and viruses. A multiple alignment of these protein sequences was generated using a hidden Markov model (HMM) approach developed to align divergent data. Phylogenetic analysis revealed that horizontal transfer from hosts to virus genomes has occurred in all three domains of life. The evidence for horizontal transfers is particularly interesting in Eukaryotes as these dut genes have introns, while DNA virus dut genes do not. This implies an intermediary Retroid Agent facilitated the horizontal transfer process, via reverse transcription, between host mRNA and DNA viruses. The horizontal transfer of the dut gene from Eukaryotic, Eubacterial, and Archaeal organisms to both DNA and RNA viruses is the first documented case of host to pathogen transfer that has occurred in all three domains of life. PMID- 12369929 TI - Evolution of the dUTPase gene of mammalian and avian herpesviruses. AB - Sequences of dUTPases encoded by Alpha- and Gammaherpesviruses resemble other dUTPases in their possession of five conserved motifs, but differ in having greater chain lengths (about twice as long) and in the location of Motif 3 at an N terminal location relative to the other motifs. It was proposed that the herpesvirus gene arose by intragenic duplication of a standard dUTPase coding sequence and subsequent loss of one copy of each motif from the double length chain, and that the resulting enzyme was active as a monomer. With knowledge of the trimeric 3D structure of standard dUTPases, it is possible to suggest transformations that occurred in evolutionary development of the herpesvirus dUTPase. The distinct location of Motif 3 can indeed be seen to be consistent with it contributing to a single intramolecular active site with the other motifs. Separately, the occurrence in herpesvirus dUTPases of around 20 to 40 additional residues between Motifs 4 and 5 allows the C-terminal Motif 5 to reach the active site intramolecularly. The driving force behind these evolutionary changes remains obscure. We speculate that they may have allowed acquisition of a novel, presently unknown function by the protein. Consistent with this idea is the observation that in Alpha- and Gammaherpesvirus dUTPases the original locus of Motif 3 is occupied by a distinct conserved sequence (Motif 6); perhaps this element constitutes part of a separate functional capability. Notably, the apparently orthologous protein in Betaherpesviruses lacks the standard motifs while Motif 6 is still present. PMID- 12369930 TI - The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity. AB - The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons. Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5prie; exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles. There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N. This review will summarize the current information on isoform characteristics of both dUTPase and uracil-DNA glycosylase. It will also focus on evidence for phosphorylation and speculate as to the purpose of these post-translational events. PMID- 12369931 TI - dUTPase in human neoplastic cells as a potential target for therapeutic intervention. PMID- 12369932 TI - Infective proteins: the prion puzzle. AB - According to the Koch postulates an infectious organism is the one that can be isolated from an host suffering from a disorder, can be propagated in laboratory, can cause the same disease when introduced in another host, and finally, can be re-isolated from the host itself. If we change the word "organism" with the word "protein" we have a quite exact description of prions. Prion related disorders are a very unique category of infectious diseases. The ethiology of the so-called prionoses is related to the conversion of a normal protein (PrP(C), the cellular isoform of the prion protein) into a pathological form (the scrapie isoform of the prion protein, PrP(Sc)) which is able to propagate. The striking difference between the two forms seems to consist in a conformational modification of a mainly alpha-helix structured PrP(C) into a mainly beta-sheet PrP(Sc). The latter forms amyloid-like fibrils which precipitate into insoluble aggregates leading to the neurodegenerative changes specific of Spongiform Encephalopathies. This review will focus on the structure of the prion proteins and on PrP(C) cellular cycle, and it will discuss some hypothesis about the protein biochemical function. Finally, the various molecular mechanisms proposed for the development of conformational modifications will be reviewed, i.e. how a protein can become infectious by simply changing its structure. PMID- 12369933 TI - Chaperone-like activity of alpha-crystallin and other small heat shock proteins. AB - Small heat shock proteins (sHsps) are a large family of proteins with monomeric molecular weight of 12-43 kDa, present within the prokaryotic and eukariotic cell as large oligomeric complexes, ranging in size from 200-800 kDa. Unlike the high molecular weight Hsps, which are involved in protein folding in vivo, under normal conditions, sHsps play an important role in protecting organism from stress. SHsps share an evolutionarily conserved sequence of 80-100 amino acids, located in the C-terminal region, and called "alpha-crystallin domain"; its role in subunits interactions has been recently underlined by site-directed spin labeling studies and by fluorescence resonance energy transfer data. The N terminal region, preceding the alpha-crystallin domain, is variable in length and amino acid sequence, contributing to structural diversity between different sHsps and having a role in multimerization. The alpha-Crystallin domain is followed by C-terminal extension, a polar structure, involved in protein solubility, which share no sequence homology. Expression of sHsps is induced in response to various kinds of stress including heat shock, oxidative stress, osmostress, or ischemia, but some sHsps are expressed constitutively under physiological conditions. In vitro, sHsps selectively bind and stabilize proteins and prevent their aggregation at elevated temperatures in an ATP-independent way and protect enzymes against heat-induced inactivation. Our own studies focused on the chaperone-like activity of alpha-crystallin, the major protein component of vertebrate lens, using another system than heat-induced aggregation. Our data demonstrated that alpha-crystallin specifically protects enzymes against inactivation by different posttranslational modifications such as glycation, carbamylation and aldehyde binding, and also reactivates GuHCl-denatured enzymes. Complex formation between alpha-crystallin and the denatured enzymes, was suggested as a mechanism of protection. PMID- 12369934 TI - Chaperone-assisted protein folding in the cell cytoplasm. AB - Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones. Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions. There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins. Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation. Chaperones can also promote the disaggregation of preformed protein aggregates. Many of the identified chaperones are also heat shock proteins. The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides. The four main chaperone systems in the Escherichia coli cytoplasm are as follows. (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains. (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE. This system recognizes polypeptide chains in an extended conformation. (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces. (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins. These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation. Several advances have recently been made in characterizing the structure and function of all of these chaperone systems. These advances have provided us with a better understanding of the protein folding process in the cell. PMID- 12369935 TI - Life and death in the placenta: new peptides and genes regulating human syncytiotrophoblast and extravillous cytotrophoblast lineage formation and renewal. AB - Differential techniques have revealed several novel genes and peptides involved in trophoblast development including PL74/gdf15/MIC-1, a TGFbeta family cytokine that controls apoptosis and differentiation, PL48, a new serine-threonine protein kinase, serum and glucocorticoid-induced kinase, PBK-1, a tunicamycin-responsive gene, a cathepsin D-like gene (DAP-1) and hypoxia- regulated genes HRF-1,2,6,8 and HIF-1alpha, HIF-1beta, and hEPAS-1. Syncytin, a cell fusion- inducing gene, has been cloned from placenta where it regulates cell fusion. ERV-3 has also been demonstrated to promote cell fusion. These two genes represent the first demonstrated functions of endogenous retroviral sequences in human tissues. Endoglin, PlGF, TGFbeta3, IGF-II, IGFBP-1, and a placental IGFBP protease have found new roles in regulating cytotrophoblast proliferation and invasiveness. A specific placental p105 rasGAP protein has been identified. The homeobox genes DLX4, HB24, MSX2 and MOX2 also likely play a role in development at the epithelial-mesenchymal boundary. Transcription factors such as TEF-5, Hand1, HEB, HASH-2 and two genes represented by ESTs may have regulatory roles in placental development. Evidence suggests that the placenta has an unusual two-cell system for apoptosis regulation in which the cytotrophoblast may direct later apoptotic events in the syncytium, and with syncytialization possibly triggered by the "phosphatidylserine flip". Thus, the placenta is both a rich source of new growth regulatory substances, and a model system for originating new paradigms of developmental biology. PMID- 12369936 TI - Development of neurotrophic molecules for treatment of neurodegeneration. AB - Over the past several years, neurotrophic factors have made considerable progress from the laboratory into the clinic. Evidence from preclinical and clinical studies indicates that it may be possible to use neurotrophic factors to prevent, slow the progression of, or even reverse the effects of a number of neurodegenerative diseases and other types of insults in both the central and peripheral nervous system. Their potential importance in the development of therapeutic agents against neurodegenerative disorders and nerve injury has led to a flurry of activity towards understanding their structure, function and signalling mechanisms. Approaches to develop pharmacological agents that target neurotrophic factors, their receptors or neurotrophic factors signalling pathways have been attempted. This review focuses on some of the major themes and lines of mechanistic and therapeutic advances in this fast-moving field of neuroscience. PMID- 12369937 TI - Amyloid forming proteases: therapeutic targets for Alzheimer's disease. AB - Alzheimer's disease is an age-related neurodegenerative disease which causes global loss of cognitive function. Drug treatment for Alzheimer's disease has been limited to agents that ameliorate behavioral symptoms but these agents are without effect on disease progression. Rational drug design for the treatment of Alzheimer's disease now seems possible. As a result of major advances in medical research in this area, knowledge of the etiology of Alzheimer's disease is rapidly being understood. This information has uncovered relevant and novel targets for treatment. At the center of the etiological progression of this disease is beta-amyloid. A substantial body of evidence strongly suggests that this small protein is critical to the development of Alzheimer's disease. The beta-amyloid protein is generated by two different proteolytic cleavages. Recently, the proteases responsible for producing the beta-amyloid protein have been identified. The proteases represent viable targets for therapeutic intervention against Alzheimer's disease. In this review, we describe the biological characteristics of the beta-amyloid-forming proteases in the context of pharmaceutical development. PMID- 12369938 TI - Peptidic inhibitors of the hepatitis C virus serine protease within non structural protein 3. AB - New treatments for HCV (Hepatitis C virus) infections are likely to arise from inhibition of the essential, virally-encoded enzymes. These targets include the serine protease required for processing of the HCV polyprotein. The protease constitutes one functional domain of the bifunctional HCV NS3 (non-structural protein 3). Here, insights regarding the NS3 structure and recently synthesized NS3 inhibitors are reviewed. Interestingly, many NS3 protease inhibitors have taken advantage of an unusual product inhibition by N-terminal products of cleavage at the polyprotein processing sites. PMID- 12369939 TI - Inhibitors of the protease domain of urokinase-type plasminogen activator. AB - Human urokinase-type plasminogen activator (uPA or uPA) has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Since Urokinase plays a role in tissue remodeling, it may be responsible, in part, for the disease progression of cancer. Inhibitors of urokinase may then be useful in the treatment of cancer by retarding tumor growth and metastasis. Urokinase is a multidomain protein, two regions of the protein are most responsible for the observed proteolytic activity in cancer disease and progression. The N-terminal domain or ATF binds to a Urokinase receptor (uPAR) on the cell surface and the C-terminal serine protease domain, then, activates plasminogen to plasmin, beginning a cascade of events leading to the progression of cancer. Investigations of urokinase inhibition has been an area of ongoing research for the past 3 decades. It began with the discovery of small natural and unnatural amino acid derivatives or peptide analogs which exhibited weak inhibition of uPA. The last decade has seen the generation of several classes of potent and selective Urokinase inhibitor directed to the serine protease domain of the protein which have shown potential anti-cancer effects. The availability of structural information of enzyme-inhibitor complexes either by nuclear magnetic spectroscopy (NMR) or crystallography has allowed a detailed analysis of inhibitor protein interactions that contribute to observed inhibitor potency. Structural studies of specific inhibitor-uPA complexes will be discussed as well as the contributions of specific inhibitor protein interactions that are important for overall inhibitor potency. These data were used to discover a class of urokinase inhibitor based on the 2-Naphthamidine template that exhibits potent urokinase inhibition and excellent selectivity for urokinase over similar trypsin family serine proteases. PMID- 12369940 TI - The determination and use of optimized protease substrates in drug discovery and development. AB - There is an increasing need to rapidly determine the specificity of proteases that potentially play a role in human and animal disease. Substrates for novel proteases can be discovered by testing standard protease substrates such as oxidized insulin B-chain, by screening commercially available substrates for other proteases, or by preparing derivatives of known biological targets. The relative importance of each substrate residue can be determined through alanine scanning, or by preparing incremental changes at one or more positions within the known substrate. More efficient methods such as coupled liquid chromatography - mass spectrometry (LC-MS) or C-terminal/N-terminal sequencing of reaction products allow the selection of improved substrates from mixtures of peptides. In other cases mixtures of substrates can be spatially segregated prior to protease treatment during chemical synthesis on beads or membranes. Positional scanning libraries can be used to find substrates for proteases with interdependent subsites, while minimizing required synthetic and screening effort. As proteases catalyze both hydrolysis and amide bond formation, acyl transfer from protease substrate intermediates to mixtures of peptide nucleophiles provide substrate sequence information. Genetic methods including substrate phage display, retroviral display, bacterial display, and yeast alpha-halo assays combine selection with the ability to propagate selected sequences and directly deconvolute the cleaved peptide via sequencing of substrate-coding DNA. This review describes various methods for optimizing protease substrates for biological activity and the use of optimized substrates in pharmaceutical discovery. PMID- 12369941 TI - Designed enediyne antitumor agents. AB - The enediynes remain among the most potent antitumoral agents to have been discovered in the past decade. Following prodrug activation, the enediynes undergo cycloaromatization reactions resulting in formation of highly reactive diradical intermediates. The diradical species engage in atom-transfer chemistry to produce neutral arene products, in the process inducing damage to key macromolecules. Several of the naturally occurring members of the enediyne family of antibiotics have entered clinical trials, and this has prompted the design of synthetic enediynes, where the enediyne lquo;warheadrquo; is conjugated to a targeted delivery vehicle. This review will describe ecent efforts using chemical synthesis to identify and improve the target specificity of designed enediynes, and to establish efficient methods to achieve prodrug activation. Finally, new horizons will be examined, including the use of post-cycloaromatized enediyne templates as recognition elements for unique DNA and RNA microenvironments. PMID- 12369942 TI - Cationic lipids in gene delivery: principles, vector design and therapeutical applications. AB - Gene therapy will change medicine by treating the diseases at their core levels revolutionizing the way to deliver functional proteins. The development of this technology relies in designing optimal systems for DNA transfer and expression (transfection), cationic lipids being a promising alternative. Being safer than viral vectors, they also allow the delivery of larger plasmids and can be easily GMP-manufactured and stored. The main problem associated with the use of these vectors is their transfection efficiency, which is still inferior to viral methods. In this paper we present an overview of the correlations between the chemical structure and biological activity for the principal classes of cationic lipids. Key issues in the design of this class of transfection agents are presented, as well as the future trends. PMID- 12369943 TI - Design of telomerase inhibitors for the treatment of cancer. AB - Telomerase is a cellular ribonucleoprotein reverse transcriptase responsible for the maintenance of telomeres, the tandemly repeating guanine-rich nucleic acid sequences at the 3'-ends of eukaryotic chromosomes that serve to protect chromosomal stability and maintain integrity. Telomerase enzyme activity is essential for the sustained proliferation of most immortal cells, including cancer cells, and is currently an important recognised target for the development of novel and potentially tumour-specific anticancer chemotherapeutics. Herein, we review recent advances in the design and development of telomerase inhibitors for the treatment of cancer. To date, these have included antisense strategies, reverse transcriptase inhibitors, and agents capable of interacting with high order telomeric DNA tetraplex (or "G-quadruplex") structures in such a way as to prevent enzyme access to its required linear telomeric DNA substrate. Critical appraisal of each distinct approach is provided together with highlighted areas for continued development necessary to further refine the present disparate classes of telomerase inhibitors for use in clinically viable therapies. PMID- 12369944 TI - Current status and perspectives in the development of camptothecins. AB - Camptothecins are cytotoxic agents with a wide spectrum of antitumor activity. The unique mechanism of action, the impressive preclinical efficacy and the clinical success of irinotecan and topotecan have stimulated intensive efforts to identify novel analogues. The development of novel camptothecins was recently rationalized on the basis of the detailed knowledge of mechanism of drug-target interaction and was aimed to overcome the major limitations of these drugs (i.e. lactone ring instability and reversibility of topoisomerase I-DNA cleavage complexes). The development of novel series of analogues (7-substituted camptothecins, silatecans and homocamptothecins) resulted in identification of promising compounds, which are currently in clinical development. Considering the lack of precise correlations between preclinical activity and clinical efficacy of camptothecins, the potential advantages of novel analogs in clinical therapy remains to be documented. However, a rational basis for drug selection and development is now provided by the recognition of major limitations of these agents and by a detailed knowledge of multiple interactions between drug, cellular target and serum albumin. Inhibition of the nuclear enzyme DNA topoisomerase I has proven to be a promising strategy in the design of antitumor agents, in spite of a limited cellular basis of selectivity in cytotoxic action of camptothecins (i.e., overexpression of the target enzyme in tumor cells, and increased sensitivity of proliferating cells). The interest in topoisomerase I as a therapeutic target promoted various efforts to identify other chemotypes effective as topoisomerase inhibitors and chemical/modelling efforts to rationally design specific analogs among known inhibitors. Additional approaches, including drug delivery/formulation, optimization of dose/schedule and route of administration, are expected to improve the therapy with camptothecins and other inhibitors. PMID- 12369945 TI - The aryl hydrocarbon receptor in anticancer drug discovery: friend or foe? AB - Binding of ligands such as polycyclic aromatic hydrocarbons to the Aryl hydrocarbon Receptor (AhR) and the sequence of events leading to induction of xenobiotic-metabolising enzymes such as the cytochrome P450 isoform 1A1 and subsequent generation of DNA adducts is historically associated with the process of chemical carcinogenesis. Cancer chemopreventative agents, on the other hand, often exert their biological effect at least in part through antagonism of AhR induced carcinogenesis. A third scenario associated with AhR binding could occur if the induction of xenobiotic enzymes and subsequent DNA damage causes apoptosis. If this occurs selectively in tumour cells whilst sparing normal tissue, the AhR ligand would have a therapeutic cytotoxic effect. In this review we survey for the first time the major classes of reported AhR ligands and discuss the biological consequences of AhR binding in each case. The use of AhR ligands as cancer chemotherapeutic agents, as illustrated by the case of the 2-(4 aminophenyl)benzothiazole prodrug Phortress, is discussed as a therapeutic strategy. PMID- 12369946 TI - Purine nucleosides bearing 1-alkynyl chains as adenosine receptor agonists. AB - The synthesis and the pharmacological activity of alkynyl derivatives of adenosine (Ado) and N-ethylcarboxamidoadenosine (NECA), that have been tested on adenosine receptors from different sources, have been reviewed. Most of compounds have been characterized in the last ten years by using radioligand binding assays on rat brain membranes and functional studies on different animal models. More recently, the four human adenosine receptor subtypes have been stably transfected into Chinese hamster ovary (CHO) cells allowing for comparative studies in a similar cellular background, utilizing radioligand binding studies (A(1), A(2A), A(3)) or adenylate cyclase activity assays (A(2B)). From the whole pattern of studies the following structure-activity relationships have been drown: The activities of 2-alkynylAdos resulted slightly higher at A(1) and lower at A(3) and A(2B) subtypes than the corresponding NECA derivatives, whereas the affinities at A(2A) subtype are similar for the two series of nucleosides. The presence of a methyl group on N(6) of the 2-alkynyladenosines, inducing a contemporary increase in affinity at the human A(3) receptor and a decrease at the other subtypes, resulted in a relevant increase in A(3) selectivity. In particular, 2-phenylethynyl-N(6)-methylAdo showed an A(3) affinity in the low nanomolar range (K(i) A(3) = 3.4 nM), and about 500 fold A(1)/A(3) and about 2500 fold A(2A)/A(3) selectivity. The presence of a hydroxyl group in some alkynyl side chains led to potent inhibitors of platelet aggegation induced by ADP. Introduction of particular substituents, such as the racemic 2 phenylhydroxypropynyl group, both in adenosine and in NECA analogues, led to highly potent, non selective agonists at all the four subtypes. For the potency at A(2B) receptor it seems to be very important the type of alkynyl chain in 2 position and the presence of the carboxyamido group on the sugar; in fact, the (S)-2-phenylhydroxypropynylNECA [(S)-PHPNECA, EC(50) A(2B) = 220 nM] proved to be one of the most potent A(2B) agonist reported so far. The introduction of alkynyl chain in 8-position of adenosine led to very selective ligands for the A(3) receptor subtype. These nucleosides behave as adenosine antagonists, since they do not stimulate basal [(35)S]GTPgammaS binding, but inhibit NECA-stimulated binding. PMID- 12369947 TI - Pyrazolo-triazolo-pyrimidine derivatives as adenosine receptor antagonists: a possible template for adenosine receptor subtypes? AB - Adenosine, a widely distributed modulator, regulates many physiological functions through specific cell membrane G-protein-coupled receptors classified as A(1), A(2A), A(2B) and A(3). An intense medicinal chemistry effort made over the last 20 years has led to a variety of selective adenosine receptor agonists and antagonists. In particular, the pyrazolo-triazolo-pyrimidine nucleus has been strongly investigated in the last years by our group. All the modifications performed and a tentative of structure-activity-relationship is reported. In fact, the combination of different substitutions at the N(7), N(8) and N(5) positions afford compounds which showed good affinity and selectivity for the different adenosine receptor subtypes. The data herein summarized, permit to speculate on the use of this nucleus as possible template for the adenosine receptor subtypes. PMID- 12369948 TI - Intrinsic activity at adenosine A1 receptors: partial and inverse agonism. AB - Novel phenomena, such as constitutive activity and inverse agonism, have led to a ligand (re)classification along an agonists-neutral antagonist-inverse agonist continuum. This review focuses on adenosine A(1) receptor ligands and their intrinsic activity (alpha). The intrinsic activity of a ligand depends both on the chemical characteristics of the compound itself and on the experimental conditions in which it is assayed. Consequently, due to this tissue-dependency determination of a ligand s intrinsic activity is not always easily performed. Meanwhile, this feature may also be used in a profitable manner, namely to separate desired and unwanted effects of the drug. Briefly, this review provides possible screening methods to distinguish the different classes of ligands. It also deals with the structural elements and functional groups in the adenosine A(1) receptor ligands that determine their intrinsic activity. PMID- 12369949 TI - Applications of adenosine receptor ligands in medical imaging by positron emission tomography. AB - In the last decade the field of purinergic pharmacology has continued to grow as the complexity of the receptor families and the various enzymes involved in purine metabolism have been defined in molecular terms. Adenosine receptors (ARs) are currently divided into the four subclasses A(1)-, A(2A)-, A(2B)- and A(3)AR. The most intensively studied subtypes are the high-affinity A(1) and A(2A) receptors, which are activated by adenosine in nano- to submicromolar concentrations. The clinical importance of the A(1) adenosine receptor (A(1)AR) and the A(2A)adenosine receptor (A(2A)AR) makes them attractive targets for radionuclide in vivo imaging. Positron Emission Tomography (PET) is an imaging modality which can determine biochemical and physiological processes in vivo in a quantitative way by using radiopharmaceuticals labeled with positron emitting radionuclides as (11)C, (13)N, (15)O and (18)F and by measuring the annihilation radiation using a coincidence technique. This includes also measurement of the pharmacokinetics of labeled drugs and the assessment of the effects of drugs on metabolism. In the present article we review the radioligands which are currently available for visualisation and quantification of ARs using PET with a special focus on the A(1)AR and A(2A)AR. PMID- 12369950 TI - P2-pyrimidinergic receptors and their ligands. AB - Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y(2), P2Y(4), P2Y(6) and P2Y(14). Pharmacological experiments indicate that further subtypes may exist. P2Y(2) and P2Y(4) receptors are activated by UTP (the P2Y(2) and the rat, but not the human P2Y(4) receptor are also activated by ATP), the P2Y(6) receptor is activated by UDP, and the P2Y(14) receptor by UDP-glucose. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. P2Y(2) agonists are currently in clinical development for cystic fibrosis and dry eye syndrome. Selective antagonists for pyrimidinergic P2Y receptors are still lacking. PMID- 12369951 TI - Structure-activity relationships of suramin and pyridoxal-5'-phosphate derivatives as P2 receptor antagonists. AB - Extracellular adenine and uracil 5'-nucleotides are important signalling molecules that exert a great variety of effects in numerous tissues and cell types through the activation of P2 receptors. In the past eight years, an extended series of P2 receptors (P2X(17), ionotropic subunits; P2Y(1,2,4,6,11,12), metabotropic receptors) has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2 receptor subtypes to the diverse physiological responses mediated by the pharmacological phenotypes of native P2 receptors. Unfortunately, subtype-selective P2 ligands, especially potent and selective antagonists, have been only slowly forthcoming, and this acts as a considerable impediment to progress. However, a number of new P2 receptor antagonists have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2 receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2 receptors and their ligands. It then focuses on structure-activity relationships of PPADS and suramin analogues and will finish with a brief discussion of some related therapeutic possibilities. PMID- 12369952 TI - Molecular modeling as a tool to investigate molecular recognition in P2Y receptors. AB - Nucleotides are emerging as an ubiquitous family of extracellular signaling molecules. These effects are mediated through a specific class of plasma membrane receptors called P2 receptors that, according to the molecular structure, are further subdivided into two subfamilies: P2Y and P2X. Specifically, P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors. In this review, we focus our attention to GPCRs molecular architecture, with the special emphasis on our work on the human P2Y(1) receptor. In fact, despite an enormous amount of research on the structure and function of these receptors, fundamental understanding of the molecular details of ligand/GPCR interactions remains very rudimentary. How agonist binding transforms a resting GPCR into its active form and the microscopic basis of binding site blockade by an antagonist are generally still unclear. In the absence of high-resolution structural knowledge of GPCRs, such questions only can be addressed by building models, which are tested through pharmacological and biochemical studies. In this review, we underline how different molecular modeling approaches can help the investigation of both receptor architecture and ligand/receptor molecular recognition. PMID- 12369953 TI - Alzheimer's disease: targets for drug development. AB - The numerous advances in the understanding of the neurobiology of Alzheimer's disease in the past 15 years have suggested many new potential targets for therapeutic intervention. This article gives a broad overview of the spectrum of targets for AD treatment, with particular emphasis on amyloid beta-peptides and tau protein. PMID- 12369954 TI - Cholinergic drugs in pharmacotherapy of Alzheimer's disease. AB - The cholinergic hypothesis of Alzheimer's disease has spurred the development of numerous structural classes of compounds with different pharmacological profiles aimed at increasing central cholinergic neurotransmission, thus providing a symptomatic treatment for this disease. Indeed, the only drugs currently approved for the treatment of Alzheimer's disease are cholinomimetics with the pharmacological profile of acetylcholinesterase inhibitors. Recent evidence of a potential disease modifying role of acetylcholinesterase inhibitors and M(1) muscarinic agonists have led to a revival of this approach, which might be considered as more than a symptomatic treatment. PMID- 12369955 TI - Rational design of reversible acetylcholinesterase inhibitors. AB - A large amount of structural information on AChE and AChE-inhibitor complexes is currently available. Based on that, molecular modeling studies can be intensively used to gain insight into the mechanism of action of the enzyme and the molecular determinants that modulate the potency of inhibitors. In turn, this knowledge can be exploited to design new compounds leading to more effective cholinergic strategies. This manuscript reviews recent developments in the design of reversible acetylcholinesterase inhibitors. PMID- 12369956 TI - Non-cholinergic pharmacotherapy approaches to the future treatment of Alzheimer's disease. AB - Research on the molecular basis of Alzheimer's disease has elucidated pathogenic pathways from which a range of rational pharmacological interventions has emerged. The most promising strategies involve approaches to retarding, halting or preventing the formation or accumulation of beta amyloid plaques and neurofibrillary tangles. Other therapeutic approaches include those acting via excitatory amino acid receptors, limiting the oxidative stress and inflammatory response associated with dementia, molecules with nerve growth factor like activity. In the present article these and the other recent advances in the neurobiology and pharmacotherapy of AD will be reviewed. PMID- 12369957 TI - Transgenic mouse models with tau pathology to test therapeutic agents for Alzheimer's disease. AB - The deposit of two proteins in the brain characterizes Alzheimer's disease: deposits of beta-amyloid protein to form senile plaques and tau protein in neurofibrillary tangles. This review discusses transgenic animals overexpressing normal or mutated tau protein as well as kinases involved in tau hyperphosphorylation. These animals hold a great potential as tools to test the effects of forthcoming therapeutical agents for Alzheimer's disease. PMID- 12369958 TI - Pharmacogenomics in Alzheimer's disease. AB - Alzheimer's disease (AD) is a complex disorder associated with multiple genetic defects either mutational or of susceptibility. Information available on AD genetics does not explain in full the etiopathogenesis of AD, suggesting that environmental factors and/or epigenetic phenomena may also contribute to AD pathology and phenotypic expression of dementia. The genomics of AD is still in its infancy, but is helping to understand novel aspects of the disease including genetic epidemiology, multifactorial risk factors, pathogenic mechanisms associated with genetic networks and genetically-regulated metabolic cascades. AD genomics is also helping to develop new strategies in pharmacogenomic research and prevention. Functional genomics, proteomics, pharmacogenomics, high throughput methods, combinatorial chemistry and modern bioinformatics will greatly contribute to accelerate drug development for AD and other complex disorders. Main genes involved in AD include mutational loci (APP, PS1, PS2, TAU) and multiple susceptibility loci (APOE, A2M, AACT, LRP1, IL1A, TNF, ACE, BACE, BCHE, CST3, MTHFR, GSK3B, NOS) distributed across the human genome. Genomic associations integrate bigenic, trigenic, tetragenic or polygenic matrix models to investigate the genomic organization of AD in comparison to the control population. Similar genetic models are used in pharmacogenomics to elucidate genotype-specific responses of AD patients to a particular drug or combination of drugs. Using APOE-related monogenic models it has been demonstrated that the therapeutic response to drugs in AD is genotype-specific. A multifactorial therapy combining 3 different drugs yielded positive results during the 6-12 months in approximately 60% of the patients. With this therapeutic strategy, APOE 4/4 carriers were the worst responders, and patients with the APOE-3/4 genotype were the best responders. In bigenic and trigenic models it was possible to differentiate the influencial effect of PS1 and PS2 polymorphic variants on mental performance in response to multifactorial therapy. The application of functional genomics to AD can be a suitable strategy for harmonization in molecular diagnosis and drug clinical trials. Furthermore, the pharmacogenomics of AD may contribute in the future to optimise drug development and therapeutics, increasing efficacy and safety, and reducing side-effects and unnecessary costs. PMID- 12369959 TI - Towards Alzheimer's disease vaccination. AB - Active and passive immunization against fibrillar beta-amyloid of various mice models of Alzheimer's disease leads to the disaggregation and inhibition of plaque formation. Preliminary results showing improved behaviour and memory function obtained after administration of anti-beta-amyloid vaccines to transgenic mice encourage these and related approaches for testing in the treatment and prevention of Alzheimer's disease. PMID- 12369960 TI - Recent advances in the medicinal chemistry of polyamine toxins. AB - This review describes the recent developments in the field of polyamine toxins, with focus on structure activity relationship investigations, including studies of importance of the polyamine moiety for biological activity, photolabeling studies using polyamine toxins as templates, as well as use of solid phase methods for the synthesis of polyamine toxins. The review is mainly concerned with effects of polyamine toxins on nicotinic acetylcholine receptors and ionotropic glutamate receptors. PMID- 12369961 TI - Fullerenes in medicinal chemistry and their biological applications. AB - The isolation and preparation ten years ago of the fullerenes in bulk quantities, sparked off a truly remarkable interdisciplinary research activity, encompassing diverse fields of chemistry, physics, materials science and medicinal chemistry. Pharmaceutical and biological studies have revealed that fullerene-based compounds exhibit various types of biological activity either in vitro or in vivo and are discussed in this review. PMID- 12369962 TI - Carbohydrates and derivatives as potential drug candidates with emphasis on the selectin and linear-B area. AB - Enzymatic carbohydrate synthesis using glycosyltransferases is highly regio- and stereospecific and does not require extensive protecting group designs. Naturally occurring carbohydrates have been prepared by this biomimetic pathway successfully. As more and more transferases are isolated and get cloned and overexpressed, non-natural substrates were probed with these biocatalysts. Key polar groups and non-essential residues of the substrates have been determined. Consequently, this technique was employed to generate natural and non-natural carbohydrate libraries for pharmaceutical purposes. The synthesis of sialyl Lewis(a)- and sialyl-Lewis(x) libraries and non-natural Linear-B derivatives applying glycosyltransferases is presented in this article. The respective transferases investigated are alpha(1-3)galactosyltransferase, beta(1 3)galactosyltransferase, beta(1-4)galactosyltransferase, alpha(2 3)sialyltransferase, alpha(1- 3)fucosyltransferase III and alpha(1 3)fucosyltransferase VI. With respect to the natural acceptors, the aglycon part and the N-acetyl group of the glucosamide have been varied. All enzymes tolerate an unexpected wide range of non-natural acceptors, which is not yet exploited in its full scale. In addition, fucosyltransferase III and VI can be employed to convert also non-natural donors with non-natural acceptors at the same time. Thus sialyl- Lewis(a)- and sialyl-Lewis(x)-libraries which differ in three positions compared to the natural tetrasaccharides are generated very efficiently. Also a library of linear-B trisaccharides, a reactive xenoantigen, has been prepared enzymatically. The aglycon part and the natural N-acetyl group of the glucosamine which is part of the acceptor substrate have been altered widely. This convenient methodology is compared with the evolving solid-phase carbohydrate synthesis using conventional chemistry. The potential use of transferases in solid-phase carbohydrate chemistry is discussed together with the possibility to use these biocatalysts to synthesize carbohydrate mimetics. The presented findings may also be useful to design potential glycosyltransferase inhibitors. PMID- 12369963 TI - Non peptidic ligands at the opioid receptor like-1 (ORL-1). AB - Opioid receptor like-1 (ORL-1) has recently been indicated as a potentially useful target for the treatment of a number of central disorders and several other diseases. This review deals with non peptidic ligands at the ORL-1 receptor, focusing on their structural and binding properties. Agonism or antagonism evidenced from functional experiments is also commented. For some compounds, possible therapeutic applications are considered. PMID- 12369964 TI - FKBP ligands as novel therapeutics for neurological disorders. AB - Given their clinical importance for the treatment of acute and chronic neurodegenerative diseases in humans including nerve injuries (e.g. Alzheimer's disease, Parkinson's disease, diabetic neuropathy) a number of different approaches were pursued to obtain selectively acting FK506-binding protein (FKBP) ligands: computational methods and target-oriented screening of natural compound and synthetic product libraries. The resulting monofunctional ligands, which inhibit the peptidyl prolyl cis/trans isomerase activity of FKBPs, highlight the role of these enzymes in neuronal signaling. The exploration of the mechanisms of neuroregenerative and neuroprotective action of some of these compounds is the main focus of ongoing neuropharmaceutical research. PMID- 12369965 TI - Antibody directed enzyme prodrug therapy (ADEPT) and related approaches for anticancer therapy. AB - In antibody directed enzyme prodrug therapy (ADEPT) an antibody bound enzyme is targeted to tumor cells. This allows for selective activation of a nontoxic prodrug to a cytotoxic agent at the tumor site for cancer therapy. Site-selective prodrug activation results in reduced side effects in remote tissue. This article reviews ADEPT with emphasis on the chemical viewpoint and recent developments. PMID- 12369966 TI - Peptides neutralizing lipopolysaccharide - structure and function. AB - Lipopolysaccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. In the last decade peptidic antagonists have become increasingly important in providing leads for treatment of LPS-mediated diseases. In this review an overview of the sources, functions and structures of antiseptic and antibacterial peptides that interact with LPS is presented. PMID- 12369967 TI - A(3) adenosine receptor antagonists. AB - During the past years a number of potent and selective antagonists for the human A(3) adenosine receptor (AR) have been developed, including tricyclic compounds, such as triazoloquinazoline, pyrazolo-triazolopyridine, imidazopurinone, triazoloquinoxaline and pyrazoloquinoline derivatives. Bicyclic compounds include isoquinoline and related quinazoline derivatives. Monocyclic dihydropyridine and pyridine also proved to be potent selective A(3) AR antagonists. So far, no potent, selective antagonist is available for rodent A(3) ARs. Most of the A(3) AR antagonists are highly lipophilic and exhibit very poor water-solubility. Potential therapeutic applications for A(3) AR antagonists include inflammatory diseases, asthma, stroke, and glaucoma. PMID- 12369968 TI - Technological advances in antigen delivery and synthetic peptide vaccine developmental strategies. AB - Significant advances have been made in the field of peptide chemistry, especially in the design of synthetic peptide immunogens which has led to new concepts and strategies for human vaccine development. This article reviews some of these technological advances and approaches to vaccine design including the multiple antigenic peptide system, the lipid polylysine core peptide system, the polymerization of peptides into multivalent immunogens and self adjuvanting synthetic peptide-based immunogens. PMID- 12369969 TI - N-oxides as hypoxia selective cytotoxins. AB - N-oxide-containing compounds have been developed as prodrugs that are selectively bioactivated in the hypoxic cells in tumors. This selectivity is based on the net reduction of the N-oxide moiety in the absence of oxygen, in a one or two electron process, by reductive enzymes. A wide range of N-oxides have been studied and some of them are currently in clinical use. This review covers the principal families of compounds under study and in clinical trials. PMID- 12369970 TI - Recent advances in the design of iron chelators against oxidative damage. AB - Iron imbalance plays a pivotal role in oxidative damages associated with a wide range of pathological conditions. However, owing to the essential role of iron in biological processes, the beneficial effects of iron chelation therapy against oxidative damage have to be balanced against potential toxicity. The present review briefly introduces iron redox biochemistry and oxidative-stress associated pathologies, surveys recent advances in iron chelating strategies and summarizes some of our recent findings in this field, with a special emphasis on the chemical design constraints one must satisfy in order to synthesize iron chelators which could be beneficial against oxidative stress without inducing iron depletion of the body. The concept of oxidative stress activatable iron chelators is presented as a new paradigm for safe and efficient treatment of oxidative-stress associated conditions. PMID- 12369971 TI - Synthesis and structure-activity relationships of 2,3-benzodiazepines as AMPA receptor antagonists. AB - There is increasing evidence of the potential therapeutic utility of glutamate receptor antagonists in the treatment of several neurodegenerative disorders, including stroke and epilepsy. In the last few years noncompetitive AMPA receptor antagonists have received considerable attention due to their therapeutic potentiality. The discovery of GYKI 52466, the prototype of noncompetitive AMPA receptor antagonists endowed with anticonvulsant and neuroprotective properties, induced growing interest on 2,3-benzodiazepine derivatives. This review covers the chemistry and pharmacology of this important class of AMPA receptor antagonists. PMID- 12369972 TI - Towards improved acetylcholinesterase inhibitors: a structural and computational approach. AB - During the last years, solving the X-ray crystallographic structure of both the unliganded acetylcholinesterase (AChE) and AChE complexes with various inhibitors has provided valuable knowledge of the interactions that mediate inhibitor binding. This structural information allows us to rationalize differences in binding affinities for related analogues, and more importantly opens new strategies to design compounds with improved pharmacological properties. This is illustrated in the case of the recently reported huprines, which are a new class of very potent and selective acetylcholinesterase inhibitors. PMID- 12369973 TI - Peripheral and dual binding site acetylcholinesterase inhibitors: implications in treatment of Alzheimer's disease. AB - Recently advances in understanding the molecular basis of Alzheimer's disease have led to the consideration of the relationship between cholinergic inhibitors and amyloid deposition as a new hypothesis for the future rational design of effective anti-Alzheimer drugs. In the present review, the non-cholinergic functions of acetylcholinesterase (AChE) and the therapeutic potential of peripheral and dual binding site AChE inhibitors in delaying the neurodegenerative process will be discussed. PMID- 12369974 TI - Designing peptide mimetics for the treatment of multiple sclerosis. AB - Multiple sclerosis is a chronic inflammatory disease of the central nervous system. While the molecular basis of the disease is still unknown, research effort is currently under progress to prevent or ameliorate its effects. There are two major approaches currently in the pursuing of improved therapeutics for the treatment of multiple sclerosis. The first approach focuses on peptide or mimetic therapy and the second on immunotherapy by preventing or controlling disease through the release of appropriate cytokines. PMID- 12369975 TI - Viral protein functions study by affinity modification. AB - The knowledge of virus reproduction is necessary to design new safe drugs for inhibition of infections. Ultra-violet irradiation of virus proteins with labeled virus genome fragments permits to identify specific nucleic acid binding proteins. Affinity modification of enzymes with nucleotide derivatives could help to determine NTP-binding proteins and those involved in viral genome replication. Photoreactive analogues of nucleic acids are among the tools used to detect elongation subunits of replicative complexes. Affinity modification approach has already resulted in successful treatment of virus diseases. PMID- 12369976 TI - Aerobic nitroreduction by flavoproteins: enzyme structure, mechanisms and role in cancer chemotherapy. AB - NQO1 (DT-diaphorase) and its truncated isoenzyme, the metalloenzyme NQO2, can reduce quinone substrates by two-electron transfer. While NQO1 is a known detoxification enzyme, the function of NQO2 is less well understood. Both rat NQO1 and human NQO2 reductively bioactivate the dinitroarene CB 1954 to a cytotoxic product that behaves as a difunctional DNA-crosslinking species with potent anti-tumour activity, although human NQO1 is much less effective. A FMN dependent nitroreductase from E. coli B also reduces quinones and reductively bioactivates CB 1954. However, this enzyme reduces CB 1954 to the 2- and 4 hydroxylamines in equivalent yield, whereas NQO1 and NQO2 generate only the 4 isomer. The reduction profile is a key factor in the development of anti-tumour prodrugs, where distinct delivery strategies are being evaluated: prodrug therapy, antibody-, macromolecule and gene-directed enzyme prodrug therapy (ADEPT, MDEPT or GDEPT). The flavoprotein enzymes are explored in terms of structure and bioreduction mechanism, particularly for use in the design of novel prodrugs with potential application as chemotherapeutic agents. PMID- 12369977 TI - Targeting "hydrolytic" activity of the S-adenosyl-L-homocysteine hydrolase. AB - Substrates that are specific for the "hydrolytic" activities of AdoHcy hydrolase have been recently identified. Upon interaction with the AdoHcy hydrolase such substrates generate the "active" electrophiles which then react with the enzyme nucleophiles to produce covalent inhibition. Dihalohomovinyl and haloacetylene analogues derived from adenosine as well 5'-S-allenyl-5'--thioadenosine derivative have been characterized as the first type II mechanism-based inhibitors of AdoHcy hydrolase that rely only on the "hydrolytic" activity. Design and synthesis of the novel adenine nucleosides as well their interaction with AdoHcy hydrolase are discussed in this review. PMID- 12369978 TI - The therapeutic potential for catalytic antibodies: from a concept to a promise. AB - More than ten years have now elapsed since the first reports confirmed that antibodies not only label antigenic targets but can also perform catalytic functions. Much of the initial research in this area focussed on exploring the scope and utility of these biocatalysts both as enzyme mimics and as programmable protein catalysts. However, their potential in the biomedical field has also been probed. This review details the present perspective of catalytic antibodies as new tools for immunotherapy and specifically focuses on their application to prodrug activation and drug inactivation. PMID- 12369979 TI - Leucine aminopeptidase as a target for inhibitor design. AB - In this review we focus on the most effective and the most promising inhibitors of leucine aminopeptidase. Their binding modes to the enzyme, the attempt to explain the origin of the inhibitory activity, as well as the structure-activity relationship for some of these compounds are discussed. Besides, the structural and electronic requirements of the enzyme active site and the binding pockets, together with the specificity towards the ligands, based on the structural and kinetic data, are presented. PMID- 12369980 TI - Origin of chiral pharmacology: stereochemistry in metalloprotease inhibition. AB - The stereospecificity shown by a wide variety of inhibitors that are effective for carboxypeptidase A (CPA), a representative zinc protease is analyzed on the basis of inhibitor type. In cases of ground state analog inhibitors and transition state analog inhibitors, the stereoisomers having the stereochemistry that corresponds to stereochemistry of substrate are more potent, but in the case of irreversible inhibitors including mechanism-based inactivators the preferred inhibitory stereochemistry cannot be predicted simply from the substrate stereospecificity. The Ogston's three point fit concept may be of great value in understanding the inhibitory stereochemistry of reversible competitive inhibitors. On the other hand, the stereochemistry of irreversible inhibitors may possibly be predicted on the ground of the transition state structure that plays a critical role in the inactivation pathway. PMID- 12369981 TI - Tacrine-huperzine A hybrids (huprines): a new class of highly potent and selective acetylcholinesterase inhibitors of interest for the treatment of Alzheimer's disease. AB - Tacrine-huperzine A hybrids (huprines) are a new class of very potent and selective acetylcholinesterase (AChE) inhibitors. Huprines were designed from tacrine and (-)-huperzine A through a conjunctive approach. They combine the 4 aminoquinoline substructure of tacrine with the carbobicyclic substructure of (-) huperzine A. Structural variations on several parts of a lead structure have allowed to complete a structure-activity relationship exploration of this new structural family and have led to several huprines more active than other known AChE inhibitors. PMID- 12369982 TI - Beta-amyloid aggregation inhibitors for the treatment of Alzheimer's disease: dream or reality? AB - Amyloid (Abeta) deposition remains a hallmark in the pathology of Alzheimer's disease (AD). Important drug discovery efforts dedicated to the inhibition of the polymerization process leading to amyloid neurotoxicity are pursued by academic groups and the pharmaceutical industry as a potential preventive treatment for AD. The aim of this review is to up-date current knowledge on the amyloid aggregation process and the various available peptidic and non-peptidic Abeta aggregation inhibitors. PMID- 12369983 TI - Quantitative structure-activity relationship (QSAR) paradigm--Hansch era to new millennium. AB - The analysis of structure-activity relationships started probably more than hundred years ago but the concept of quantitatively correlating physicochemical properties of molecules with their biological activities, termed as quantitative structure-activity relationship (QSAR), was initiated by Corwin Hansch and his groups in early 1960. Many new methods have emerged since then. The concept evolved from 2D QSAR to 3D QSAR and lately another dimension (4D QSAR) has been added. This evolution is briefly reviewed here. PMID- 12369984 TI - Pharmaceutical target identification by gene expression analysis. AB - The majority of newly-identified genes in the human genome show no significant sequence similarity to genes whose function is known, so they are not easily recognized as potential drug targets. Expression analysis is an alternative method to suggest possible functions of genes. We review statistical methods for gene expression analysis to identify potential pharmaceutical targets. Specifically, we illustrate the analysis of differential gene expression (using discriminant analysis, t-tests, and analysis of variance) and co-expression (using correlation, clustering, and chi-square). We present an example of the use of expression analysis to identify co-expressed cardiomyopathy-associated genes. PMID- 12369985 TI - Antihypertensive drugs that act on Renin-Angiotensin System with emphasis in AT(1) antagonists. AB - Angiotensin II, the primary active hormone in the Renin-Angiotensin System is a major vasoconstrictor implicated in the cause of hypertension. Research efforts have focused in the treatment of disease by blocking its release and more recently by competing its action on AT(1) receptors. This approach generated in the pharmaceutical market, losartan, and other derivatives. To better understand the stereoelectronic requirements that lead to the molecular basis of hypertension the stereochemical features of angiotensin II and its antagonists are studied. PMID- 12369986 TI - Molecular moments for computer-aided drug discovery. AB - Certain of the fundamental concepts underlying the utilization of comparative molecular moment (CoMMA) descriptors as measures of three-dimensional molecular similarity are reviewed. The results of a principal component regression (PCR) analyses of the five data sets previously examined by partial least squares (PLS) calculations are provided. The results further substantiate the utility of the CoMMA descriptors in predicting chemical and biological activity. PMID- 12369987 TI - Modifying TNFalpha for therapeutic use: a perspective on the TNF receptor system. AB - TNFalpha is an inflammatory mediator that is relevant to several autoimmune diseases. Macromolecular inhibitors of TNFalpha have proven therapeutically useful in some preliminary studies. We have developed small molecule TNFalpha antagonist based on the crystal structure of TNF receptor complex. The TNFalpha inhibitor is specific and mediates biological function similar to the inhibitory soluble TNF receptor. This review focuses on development of small molecule anti TNFalpha mimetics by us and current status of other agents. PMID- 12369988 TI - Recent advances in bioreductive drug targeting. AB - Advances in the chemistry of bioreductive drug activation have led to the design of hypoxia-selective drug delivery systems. These prodrugs, comprising a bioreductive "trigger", "linker" and "effector" were first explored with nitrobenzyl quaternary ammonium mustards. Alternative nitroheterocycles were subsequently developed, together with new avenues of prodrug activation in ADEPT and GDEPT. Major advances have also been made in utilising indolequinone reductive chemistry based upon an appreciation of the kinetics of oxygen sensitive reductive elimination. PMID- 12369989 TI - DNA tetraplex-binding drugs: structure-selective targeting is critical for antitumour telomerase inhibition. AB - Four-stranded tetraplex ("G-quadruplex") DNA represents a new paradigm for the design of DNA-interactive antitumour drugs, as the formed DNA-drug complexes have been suggested to interfere with critical telomerase function. The unique structural features presented by tetraplex over duplex DNA have stimulated the design of small ligand molecules able to selectively promote the formation and/or stabilisation of such higher-order DNA structures. Current developments in tetraplex-targeted telomerase inhibitors, and importantly their DNA structural selectivity, are explored. PMID- 12369990 TI - Small molecule inhibitors of serine/threonine protein phosphatases. AB - Serine/threonine protein phosphatases have long been ignored as potential therapeutic targets for two reasons, one the biochemical significance of these proteins has not been appreciated and two, many natural protein phosphatase inhibitors are potent toxins and are considered unsuitable for clinical use. This review outlines the biochemical role of this protein family in cancer, cystic fibrosis, immunosuppression and, cardiac and neurological disorders. Particular emphasis is also given to the synthesis of selective small molecule inhibitors and their clinical exploitation. PMID- 12369991 TI - Nitric oxide-releasing nonsteroidal anti-inflammatory drugs: novel gastrointestinal-sparing drugs. AB - Nonsteroidal anti-inflammatory drugs (NSAIDs) have unacceptable morbidity and mortality due to their gastrointestinal toxicity. Attempts so far to improve the safety profile of NSAIDs have met with limited clinical acceptance. Nitric oxide (NO) functions as an endogenous mediator of gastric mucosal health and defense. Recent medicinal chemistry approaches attempt to exploit the tissue-protective function of NO against NSAID-induced gastric injury. Both nitroxybutyl-ester and nitrosothiol NSAID derivatives have been synthesized. Profiling of these NO donating NSAIDs in both the laboratory and the clinic suggests that they might offer a unique solution to the problem of NSAID-induced gastropathy without sacrificing the well-accepted pharmacological activity of these agents in the management of pain and inflammation. PMID- 12369992 TI - Enterohepatic recirculation: a powerful incentive for drug discovery in the inosine monophosphate dehydrogenase field. AB - With the exception of organ transplant immunosuppression, the treatment of various IMPDH-dependent hyperproliferative diseases by MPA has failed due to the drug's EHC-induced GIT adverse effects. To influence its therapeutic index, novel formulations such as gastro-resistant MPA-Na (ERL080) or MPA/cholestyramine combinations have been developed. Structurally novel IMPDH inhibitors have been discovered based on high throughput screening (pyridazoles) and rational design (methoxyphenyloxazoles). The clinical data on methoxyphenyloxazole derivatives such as VX-497 that is not expected to undergo EHC, will bring improved understanding of the relationship between IMPDH blockade and GIT toxicity. PMID- 12369993 TI - A comparison of methods for pharmacophore generation with the catalyst software and their use for 3D-QSAR: application to a set of 4-aminopyridine thrombin inhibitors. AB - The method of structure-based pharmacophores for use in 3D-QSAR as implemented by Gillner and Greenidge is further examined. Conformational models are generated using both Catalyst and Macromodel. K(i) estimates obtained with the pharmacophore models are compared with observed values for a set of 4 aminopyridine thrombin inhibitors. PMID- 12369994 TI - Recent advances in the development of dopamine D(3) receptor agonists and antagonists. AB - Advances in molecular cloning techniques have allowed the characterization of five subtypes (D(1)-D(5)) of dopamine (DA) receptors. The limbic location of the D(3) receptor has led to speculation about its possible role in schizophrenia and drug abuse. Since the D(3) receptor is localized in the limbic region rather than the striatum, antipsychotics with D(3) receptor selectivity could be devoid of extrapyramidal side effects commonly seen with D(2) receptor antagonists. Recent work in our laboratory revealed that the benz[e] indole cis-(+/-)-44b demonstrated high selectivity for the D(3) receptor. This compound exhibits a typical antipsychotic profile without the motor effects found in commonly used antipsychotic agents. This mini-review will give a brief introduction on D(3) receptors and a detailed description of selectively-acting D(3) agonists and antagonists which have recently appeared in the literature. PMID- 12369995 TI - Structure-metabolism relationships: steric effects and the enzymatic hydrolysis of carboxylic esters. AB - After a brief review of a number of issues related to the enzymatic hydrolysis of carboxylic esters, scuh as interspecies variability, mechanism, stereospecificity, and activation energy, and after and overview of relevant aspects related to the quantitative modeling of steric effects, the results of a recently developed quantitative structure-metabolism relationship model are discussed. They were obtained for in vitro human blood enzymatic hydrolysis of noncongener esters by introduction of the inaccessible solid angle as a novel measure of steric hindrance. PMID- 12369996 TI - Structure-function relationships in chloroquine and related 4-aminoquinoline antimalarials. AB - Recent publication have provided strong evidence that activity and cellular uptake of 4-aminoquinoline antimalarials depends on vacuolar haemoglobin degradation and that haematin is the drug target. Studies on haematin-quinoline interactions have provided insight into the structural requirements for these interactions and indications are that 4-aminoquinolines may act by inhibiting haemozoin formation. Structural requirements for this activity have also been reported recently and have led to construction of an empirical structure-function relationship for 4-aminoquinolines. PMID- 12369997 TI - Molecular mechanism of P-glycoprotein assembly into cellular membranes. AB - In the past decade major advances have been made towards understanding the mechanisms by which polytopic membrane proteins fold and assemble in cellular membranes. In eukaryotes, this process is mediated by a complex set of machinery in the endoplasmic reticulum (ER) that facilitates translocation of peptide loops across and integration of hydrophobic helices into the lipid bilayer. Studies evaluating the biogenesis of P-glycoprotein (P-gp) have been at the forefront of this rapidly expanding field. They have revealed a fascinating although sometimes confusing picture that has challenged our notions about general mechanisms of polytopic protein assembly and questioned specific predictions about the details and uniqueness of P-gp transmembrane topology. This review will attempt to summarize and consolidate our current knowledge of the sequence of events that gives rise to P-gp topology in the ER compartment and the implications of these events for polytopic protein biogenesis and function. PMID- 12369999 TI - Monoclonal antibodies as a tool for structure-function studies of the MDR1-P glycoprotein. AB - P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon. PMID- 12369998 TI - Multidrug resistance and cancer: the role of the human ABC transporter ABCG2. AB - A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional chemotherapy, a phenomenon called multidrug resistance. This broad-based resistance results in large part, but not solely, from overexpression of members of the ATP-binding cassette (ABC) superfamily of membrane transporters, including P-glycoprotein, various members of the multidrug resistance associated proteins (MRPs), and ABCG2, also known as MXR1, BCRP, and ABCP. When overexpressed in cell lines, ABCG2 has the ability to confer high levels of resistance to anthracyclines, mitoxantrone, bisantrene, and the camptothecins topotecan and SN-38. This review focuses on the discovery, the biochemistry and the normal physiology of human ABCG2, a novel ABC half transporter expressed abundantly in placenta, as well as in liver, intestine and stem cells. ABCG2 may serve a protective function by preventing toxins from entering cells as well as potentially playing a role in regulating stem cell differentiation. We also discuss the involvement of ABCG2 in multidrug resistance in cancer, especially with regard to acute myeloid leukemia. The mechanism by which substrates are recognized by ABCG2 and how the energy of ATP hydrolysis is transduced into transport remain elusive. A complete understanding of the mechanism and biological function of ABCG2 will be important for understanding its normal physiology as well as potentially for the development of novel chemotherapeutic treatment strategies. PMID- 12370000 TI - Mechanistic parallels in bacterial and human multidrug efflux transporters. AB - Bacteria carry a battery of multidrug transporters, which belong to six families of transporters. Members of at least three families the ATP-Binding Cassette superfamily, the Major Facilitator Superfamily and the Multidrug Endosomal Transporter family have been shown to contribute to multidrug resistance phenotype in eukaryotic cells. This review is focused on comparison of bacterial and eukaryotic transporters that do not have a common evolutionary trait and use different sources of energy to perform the transport. Yet they demonstrate an impressive resemblance. All multidrug transporters are capable of recognizing a broad spectrum of structurally diverse compounds. The accumulated data suggest that structural and mechanistic determinants of such ability are similar among unrelated proteins. Despite the apparent similarity, many features are still unique for different classes of transporters. Intriguingly, some cells appear to simultaneously express transporters belonging to different classes. Depending on mechanistic particularities of transporters such concurrent expression can result in synergistic or non-synergistic effects. PMID- 12370001 TI - Phylogenetic and functional classification of ATP-binding cassette (ABC) systems. AB - ATP binding cassette (ABC) systems constitute one of the most abundant superfamilies of proteins. They are involved in the transport of a wide variety of substances, but also in many cellular processes and in their regulation. In this paper, we made a comparative analysis of the properties of ABC systems and we provide a phylogenetic and functional classification. This analysis will be helpful to accurately annotate ABC systems discovered during the sequencing of the genome of living organisms and to identify the partners of the ABC ATPases. PMID- 12370002 TI - Probing the phosphopeptide specificities of protein tyrosine phosphatases, SH2 and PTB domains with combinatorial library methods. AB - Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains. This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed. PMID- 12370003 TI - Anthrax fusion protein therapy of cancer. AB - Most patients with cancer are treated with chemotherapy but die from progressive disease or toxicities of therapy. Current chemotherapy regimens primarily use cytotoxic drugs which damage cell DNA or impair cell proliferation in both malignant and normal tissues. After several treatment courses, the patients' tumor cells often overexpress multi-drug resistance genes which prevent further tumor cytoreduction. Novel agents which can kill such resistant tumor cells are needed. One such class of agents are targeted peptide toxins. Targeted peptide toxins consist of peptide toxins covalently linked to tumor selective peptide ligands. These molecules bind tumor cell surface receptors, internalize, and facilitate transfer of the toxin catalytic domains to the cytosol. Once in the cytosol, the enzyme activity leads to cell death. A number of plant, bacterial and fungal toxins have been used, and clinical trials with several of these have produced complete remissions in chemoresistant neoplasms. Nevertheless, there is a continuing need for novel targeted toxins. Many patients have pre-existing antibodies against the currently clinically used toxins and many toxins are inactive when used for myeloid malignancies where internalized proteins are rapidly routed and degraded in lysosomes. Anthrax toxins are the cytotoxic components of Bacillus anthracis. While the bacteria has been the source of serious illness, deaths and global anxieties related to past or future bioterrorism, the isolated toxins do not pose public health hazards. In fact, toxin treated patients will likely develop protective antibodies. Anthrax toxin is an excellent choice for tumor cell surface targeting. Other than U.S. military personnel immunized during the Gulf War, most people lack pre-existing antibodies. This may change in the future due to threats of additional terrorist acts, but for the present few patients will have antibodies to anthrax proteins. The separate subunits for binding, translocation and cell killing facilitate genetic engineering to yield tumor-specific cell killing. The toxins are more potent than most of the other peptide toxins and may yield highly efficacious targeted molecules. This essay will review anthrax toxin structure-function, preliminary experiments with re-targeted anthrax toxin and potential designs for new ligand-anthrax therapeutics. PMID- 12370004 TI - Matrix metalloproteinases in lung diseases. AB - Matrix metalloproteinases (MMPs) are a pivotal family of zinc enzymes responsible for degradation of the extracellular matrix (ECM) components including basement membrane collagen, interstitial collagen, fibronectin, and various proteoglycans, during normal remodeling and repair processes. The potent proteolytic activities of MMPs is mainly regulated by the balance with specific tissue inhibitors of Matrix metalloproteinases (TIMP). Excessive or inappropriate expression of MMP may contribute to the pathogenesis of tissue destructive processes in a wide variety of diseases including lung diseases. Although the precise mechanisms are still unknown, the contribution of individual MMPs are worth investigating in seeking the pathogenesis of various lung diseases such as lung cancer, bronchial asthma, chronic obstructive pulmonary disease, acute lung injury, pulmonary hypertension and interstitial lung diseases. In particular, the close association of each lung disease with the destructive effects of gelatinase A and B (also called MMP-2 and MMP-9) on the basement membrane in early alveolar remodeling, and that of collagenase-1 (MMP-1) on the major interstitial structural protein of ECM have received considerable attention. The interaction of MMPs with chemical mediators and inflammatory cytokines has also been reported in some recent studies. Several promising therapeutic approaches to inhibit MMPs have just started in the field of oncology, while more specific MMP inhibitors may be required for further investigation in other fields of lung diseases. In this review, the main focus is on the recent clinical and experimental findings and the contributions of MMPs and/or TIMPs in the lung diseases. PMID- 12370005 TI - PACAP and its receptors exert pleiotropic effects in the nervous system by activating multiple signaling pathways. AB - Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from the ovine brain in 1989 as a novel hypothalamic hormone that potently activates adenylate cyclase to produce cyclic AMP in pituitary cells. This neuropeptide belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) superfamily, and exists in two amidated forms as PACAP38 (38-amino acid residues) and PACAP27 derived from the same precursor. The primary structure of PACAP has been remarkably conserved throughout evolution among tunicata, ichthyopsida, amphibia and mammalia, and a PACAP-like neuropeptide has also been determined in Drosophila. Both PACAP and its receptors are mainly distributed in the nervous and endocrine systems showing pleiotropic functions with high potency. There are three types of receptors with high PACAP-binding affinity and with different tissue-distribution patterns. All of them belong to G-protein coupled receptor superfamily with seven transmembrane domains. PAC(1) is the PACAP-specific receptor and exists in at least eight splice variants which couple to different intracellular signal transduction pathways. VPAC(1) and VPAC(2) are the common receptors for both PACAP and VIP, which are coupled to adenylate cyclase. This review article presents and discusses an update on PACAP research and its pleiotropic physiological functions based on multiple receptor-mediated signaling mechanisms in both the central and peripheral nervous system, including the regulation of hypothalamic neurosecretion, homeostatic control of circadian clock and behavioral actions, involvement in learning and memory processes, neuroprotective effects such as anti-apoptosis and response to injury and inflammation, and neural ontogenetic functions on proliferation/differentiation processes from early stages. PMID- 12370006 TI - Recent progress in protein 3D structure comparison. AB - Quantitation of protein 3-D structure similarity is crucial in such fields as evolutionary studies, structural modeling and prediction of biological function. There are various approaches, many of which are tailored to specific problems. This review summarizes the recent developments in this field with particular interest in two main areas: i) improvements to and statistical interpretation of the root-man-square distance between equivalent atoms, rmsd; and ii) methods of protein structural classification based on geometrical features. Special attention is given to fast methods capable of analyzing large structural databases. PMID- 12370007 TI - Structures and interactions of proteins involved in the coupling function of the protonmotive F(o)F(1)-ATP synthase. AB - The mitochondrial F(1)F(o) ATP synthase complex has a key role in cellular energy metabolism. The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F(1), protruding out of the inner side of the membrane, a membrane integral proton translocating moiety F(o), and a stalk connecting F(1) to F(o). The X-ray crystallographic analysis of the structure of the bovine mitochondrial F(1) ATPase has provided a structural basis for the binding-change rotary mechanism of the catalytic process in F(1), in which the gamma subunit rotates in the central cavity of the F(1) alpha3/beta3 hexamer. Rotation of gamma and eta subunits in the E. coli enzyme and of, gamma and delta subunits in the mitochondrial enzyme, is driven, during ATP synthesis, by proton motive rotation of an oligomer of c subunits (10-12 copies) within the F(o) base piece. Average analysis of electron microscopy images and cross-linking results have revealed that, in addition to a central stalk, contributed by gamma and delta/eta subunits, there is a second lateral one connecting the peripheries of F(o) and F(1). To gain deeper insight into the mechanism of coupling between proton translocation and catalytic activity (ATP synthesis and hydrolysis), studies have been undertaken on the role of F(1) and F(o) subunits which contribute to the structural and functional connection between the catalytic sector F(1) and the proton translocating moiety F(o). These studies, which employed limited proteolysis, chemical cross-linking and functional analysis of the native and reconstituted F(1)F(o) complex, as well as isolated F(1), have shown that the N-terminus of alpha subunits, located at the top of the F(1) hexamer is essential for energy coupling in the F(1)F(o) complex. The alpha N terminus domain appears to be connected to F(o) by OSCP (F(o) subunit conferring sensitivity of the complex to oligomycin). In turn, OSCP contacts F(o)I-PVP(b) and d subunits, with which it constitutes a structure surrounding the central gamma and delta rotary shaft. Cross-linking of F(o)I-PVP(b) and gamma subunits causes a dramatic enhancement of downhill proton translocation decoupled from ATP synthesis but is without effect on ATP driven uphill proton transport. This would indicate the existence of different rate-limiting steps in the two directions of proton translocation through F(o). In mitochondria, futile ATP hydrolysis by the F(1)F(o) complex is inhibited by the ATPase inhibitor protein (IF(1)), which reversibly binds at one side of the F(1)F(o) connection. The trans-membrane deltapH component of the respiratory deltap displaces IF(1) from the complex; in particular the matrix pH is the critical factor for IF(1)association and its related inhibitory activity. The 42L-58K segment of the IF(1) has been shown to be the most active segment of the protein; it interacts with the surface of one alpha/beta pairs of F(1), thus inhibiting, with the same pH dependence as the natural IF(1), the conformational interconversions of the catalytic sites involved in ATP hydrolysis. IF(1) has a relevant physiopathological role for the conservation of the cellular ATP pool in ischemic tissues. Under these conditions IF(1), which appears to be over expressed, prevents dissipation of the glycolytic ATP. PMID- 12370008 TI - Ordered heme binding ensures the assembly of fully functional hemoglobin: a hypothesis. AB - The exact mechanism by which four Fe-Protoporphyrin-IX (heme) moieties and four nascent globin chains combine to form human hemoglobin (alpha(2)beta(2)) remains a mystery. Recent Soret spectral static and kinetic studies of the incorporation of CN-Hemin derivatives into an array of human globin species have provided in vitro evidence of an ordered assembly pathway, through an alphaheme-betaglobin intermediate, that ensures correct formation of active hemoglobin tetramers. PMID- 12370009 TI - Protein regulators of eicosanoid synthesis: role in inflammation. AB - A variety of factors contribute to the complex course of inflammation. Microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. In the early phase of inflammation, excessive amounts of cytokines and inflammatory mediators are released. These factors activate, in addition to other signaling pathways, the lipid synthesis pathways, which play a crucial role in the pathogenesis of organ dysfunction. Arachidonic acid (AA), the precursor of pro-inflammatory eicosanoids, is released from membrane phospholipids by the action of phospholipase A(2) (PLA(2)), and is metabolized to prostaglandins (PGs) and leukotrienes (LTs) by the action of cyclooxygenase (COX) and lipoxygenase (LO) enzymes, respectively. Disordered activation of PLA(2), LO and COX enzymes have been implicated in many inflammatory diseases. PLA(2) is activated by phospholipase-A(2)-activating protein (PLAP) and LO by 5-lipoxygenase-activating protein (FLAP). The inducible form of COX-2 enzyme, which is usually not present under basal conditions, is induced in inflammation. In this article the function of these enzymes in eicosanoid synthesis, their regulation, and their implication in inflammatory disorders will be reviewed. The properties, function and regulation of the protein activators PLAP and FLAP will also be discussed. PMID- 12370010 TI - Protein synthesis at atomic resolution: mechanistics of translation in the light of highly resolved structures for the ribosome. AB - Our understanding of the process of translation has progressed rapidly since the availability of highly resolved structures for the ribosome. A wealth of information has emerged in terms of both RNA and protein structure and the interplay between them. This has prompted us to revisit the astonishing "treasure trove" of functional data regarding the ribosome that has accumulated over the past decades. Here we try a systematic synopsis of these ribosomal functions in light of the cryo-electron microscopic structures (resolution >7 A) and the atomic x-ray structures (>2.4 A) of the ribosome. PMID- 12370011 TI - Initiation and inhibition of protein biosynthesis studies at high resolution. AB - Analysis of the high resolution structure of the small subunit from Thermus thermophilus shed light on its inherent conformational variability and indicated an interconnected network of features allowing concerted movements during translocation. It also showed that conformational rearrangements may be involved in subunit association and that a latch-like movement guarantees processivity and ensures maximized fidelity. Conformational mobility is associated with the binding and the anti association function of initiation factor 3, and antibiotics interfering with prevent the initiation of the biosynthetic process. Proteins stabilize the structure mainly by their long basic extensions that penetrate into the ribosomal RNA. When pointing into the solution, these extensions may have functional roles in binding of non-ribosomal factors participating in the process of protein biosynthesis. In addition, although the decoding center is formed of RNA, proteins seem to serve ancillary functions such as stabilizing ist required conformation and assisting the directionality of the translocation. PMID- 12370012 TI - High-resolution structures of large ribosomal subunits from mesophilic eubacteria and halophilic archaea at various functional States. AB - Structural analysis of the recently determined high resolution structures of the small and the large ribosomal subunits from three bacterial sources, assisted by the medium resolution structure of a complex of the entire ribosome with three tRNAs, led to a quantum jump in our understanding of the process of the translation of the genetic code into proteins. Results of these studies highlighted dynamic aspects of protein biosynthesis; illuminated the modes of action of several antibiotics; indicated strategies adopted by ribosomes for maximizing their functional activity and revealed a wealth of architectural elements, including long tails of proteins penetrating the particle s cores and stabilizing the intricate folds of the RNA chains. Binding of substrate analogues showed that the decoding and the peptide-bond formation are accomplished mainly by RNA. However, several proteins may be functionally relevant in directing the mRNA and in mediating the proper orientation of the tRNA molecules within the ribosomal rRNA frame. Elements involved in intersubunit contacts or in substrate binding are inherently flexible, but maintain well-ordered characteristic conformations in unbound particles. The ribosomes utilize this conformational variability for optimizing their efficiency and minimizing non-productive interactions, hence disorder of functionally relevant features may be linked to less active conformations or to far from physiological conditions. Clinically relevant antibiotics bind almost exclusively to rRNA. In the small subunit they affect the decoding accuracy or limit conformational mobility and in the large subunit they either interfere with substrate binding, by interacting with components of the peptidyl transferase cavity, or hinder the progression of the growing peptide chain. PMID- 12370013 TI - Three-dimensional electron cryomicroscopy of ribosomes. AB - Single particle electron cryomicroscopy is nowadays routinely used to generate three-dimensional structural information of ribosomal complexes without the need of crystallization. A large number of structures of functional important ribosomal complexes have thus been determined using this technique. In E. coli 70S ribosomes all three tRNA binding sites could be localized. The ternary complex of EF-TutRNAGTP that delivers the tRNA to the ribosome was directly visualized in a ribosomal complex blocked by the antibiotic kirromycin. Three different functional states of translocation have been studied and the respective EF-G binding sites have been mapped. The level of resolution achievable with electron cryomicroscopy allows conformational changes in the domain structures of elongation factors to be modelled in terms of rigid body movements. Structural information on eukaryotic ribosomes is also available for yeast and mammalian 80S ribosomes. The structural differences between rabbit 80S and E. coli 70S ribosomes could be interpreted in terms of ribosomal RNA expansion segments in the 18S and 23S RNA. The EF-G homologue EF2 was mapped analysing the structure of an 80SEF2sodarin complex and most recently the binding of a hepatitis C virus IRES element to a yeast 40S subunit has been studied. The first electron cryomicroscopical 3D reconstructions have further been used to overcome the initial phasing problems in X-ray crystallographic studies of the ribosome facilitating structure determination of the recent atomic resolution structures of the 30S and 50S ribosomal subunits. In turn, the knowledge of the atomic structure of the ribosome makes detailed interpretations of cryo-EM maps possible at approximately 20 A resolution. PMID- 12370014 TI - Structure and function of the acidic ribosomal stalk proteins. AB - The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified. GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a "landing platform" for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown. While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 EF-G and P-protein EF-2 couples. The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an alpha-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The physiological role of the other dimerization mode is unclear and is in apparent contradiction to distances measured by fluorescence resonance energy transfer. The discrepancies between the crystal structures and results from other physico chemical methods may partly be a consequence of the dynamic functions of the proteins, necessitating a high flexibility. PMID- 12370015 TI - Structure and function of bacterial initiation factors. AB - Bacteria require three initiation factors, IF1, IF2 and IF3, to start protein synthesis. In the last few years the elucidation of both structural and mechanistic aspects pertaining to these proteins has made substantial progress. In this article we outline the translation initiation process in bacteria and review these recent developments giving a summary of the main features of the structure and function of the initiation factors. PMID- 12370016 TI - Inhibitory mechanisms of antibiotics targeting elongation factor Tu. AB - Since the pioneering discovery of the inhibitory effects of kirromycin on bacterial elongation factor Tu (EF-Tu) more than 25 years ago [1], a great wealth of biological data has accumulated concerning protein biosynthesis inhibitors specific for EF-Tu. With the subsequent discovery of over two dozen naturally occurring EF-Tu inhibitors belonging to four different subclasses, EF-Tu has blossomed into an appealing antimicrobial target for rational drug discovery efforts. Very recently, independent crystal structure determinations of EF-Tu in complex with two potent antibiotics, aurodox and GE2270A, have provided structural explanations for the mode of action of these two compounds, and have set the foundation for the design of inhibitors with higher bioavailability, broader spectra, and greater efficacy. PMID- 12370017 TI - Is tRNA binding or tRNA mimicry mandatory for translation factors? AB - tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both. PMID- 12370018 TI - Protein factors mediating selenoprotein synthesis. AB - The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis. PMID- 12370019 TI - Fusion proteins from artificial and natural structural modules. AB - The purpose of preparing fusion proteins from designed and natural sequences is mainly twofold; it aims at the stabilization of structure and at the modification of biological activity. Fusion with beta-galactosidase, for example, can increase the intracellular stability and DDT-degrading activity of an artificial DDT binding peptide, and fusions with a leucine zipper produce mono- and bifunctional single-chain variable domain antibody fragments or homodimeric and heterodimeric DNA-binding proteins like an artificial homodimeric HIV-1 enhancer-binding protein with increased binding specificity and repressor activity. Of importance are also short leader sequences that mediate the translocation of proteins across the cytoplasmic and the nuclear membrane. An interesting by-product of the leucine zipper-mediated dimerization of an HIV-1 enhancer-binding protein was the synthesis and the structural as well as functional characterization of a retro leucine zipper. PMID- 12370020 TI - Revisiting proteus: do minor changes in lectin structure matter in biological activity? Lessons from and potential biotechnological uses of the Diocleinae subtribe lectins. AB - Significant differences in function have been observed among lectins structurally similar to concanavalin A, but their high homology with this widely used lectin has kept them in obscurity. The observation of large differences in the potency of many of these Diocleinae lectins as stimulators of Interferon-g production by human peripheral blood mononuclear cells has lead to a major effort to unravel their chemical structure and biological activity. Modeling studies of some of these lectins reveal conformational changes in side chains of some residues involved in the carbohydrate-binding site, with possible effects on the ability of these proteins to recognize specific carbohydrate structures. Additionally, all them constitute in fact a mixture of isolectins, which in different proportions could lead to diverse effects. The present review of the biological actions of Diocleinae lectins includes several in vitro and in vivo immunological findings, as well as their effects on insect growth and reproduction. In these systems Diocleinae lectins proved to be quite diverse in their potency. Such diversity in the biological activity of highly related proteins recalls the origin of the name protein: like Proteus, the capability of assuming various forms is the essential feature of this class of molecules. PMID- 12370021 TI - Inhibition of cysteine protease activity by NO-donors. AB - Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed. PMID- 12370022 TI - How the sequestration of a protein interferes with its mechanism of action: example of a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain involved in gene expression regulation. AB - We describe here a new family of proteins characterized by a particular cysteine rich carboxy-terminal domain and involved in gene expression regulation. This family presently includes three members: I-mfa (inhibitor of MyoD family), HIC p40 and HIC p32 (human I-mfa domain-containing protein). I-mfa, by interacting with MyoD family members, represses both transcriptional activation and myogenesis mediated by these factors. HIC two isoforms, HIC p40 and HIC p32, are involved in the positive regulation of Tax-mediated HTLV-I (human T-cell leukemia virus type 1) promoter activation and in the negative regulation of Tat-mediated HIV-1 (human immunodeficiency virus type 1) promoter transcription. The common carboxy-terminal region of HIC p40 and HIC p32, which is clearly involved in these regulations, shares 77% homology with the carboxy-terminal domain of I-mfa. This suggests that I-mfa, HIC p40 and HIC p32 are part of a new family of proteins involved in gene expression regulation and characterized by a specific cysteine-rich carboxy-terminal domain. Moreover, the three proteins present different subcellular localizations: I-mfa and HIC p32 are mainly cytoplasmic while HIC p40 is mainly nucleolar. The specific localization of each member of this new family will be discussed, possibly explaining how they work. Effectively, a mechanism of protein sequestration in a particular compartment, cytoplasm or nucleolus, could be involved in their function, as it is the case for many other proteins. This relationship between sequestration and function regulation will be exemplified for several cellular factors. PMID- 12370023 TI - Mif1: a missing link between the unfolded protein response pathway and ER associated protein degradation? AB - Eukaryotic cells have three different mechanisms to deal with the accumulation of unfolded proteins in the endoplasmic reticulum: (1) In cells in which unfolded polypeptides accumulate, translation initiation is inhibited to prevent further accumulation of unfolded proteins. (2) Expression of proteins involved in polypeptide folding is strongly enhanced by a process called the Unfolded Protein Response (UPR). (3) Proteins missing the proper tertiary structure are degraded by the ER-Associated protein Degradation (ERAD) mechanism. Recent studies in S. cerevisiae have shown that the processes of UPR and ERAD are functionally linked to each other. Cells lacking a functional ERAD show a constitutive activation of UPR. In addition, many of the components of ERAD are under the direct transcriptional control of UPR. Finally, while neither UPR nor ERAD are essential for cell viability, deletion of both pathways results in severe growth impairment. UPR and ERAD are conserved between yeast and mammalian cells. One of the components of mammalian UPR is the protease presenilin-1. Mutations in the gene for presenilin-1 cause early-onset familial Alzheimer disease. Interestingly, inhibition of proteolysis by the ubiquitin-26S proteasome system has also been described for Alzheimer s disease. This suggests a link between UPR and ERAD in mammalian cells. The recently identified gene Mif1 is a possible candidate to form a direct link between UPR and ERAD in mammalian cells. The Mif1 gene is under the direct control of UPR. Mif1 is a trans-ER-membrane protein, with both the N- and the C-termini facing the cytoplasmic side of the ER membrane. It contains an N-terminal ubiquitin-like domain. It is anticipated that Mif1 may associate through its ubiquitin-like domain with the 26S proteasome, in this way connecting the protein degradation machinery to the ER membrane and resulting in an efficient ERAD. PMID- 12370024 TI - Protein structure to function via dynamics. AB - Protein folding, binding, catalytic activity and molecular recognition all involve molecular movements, with varying extents. The molecular movements are brought upon via flexible regions. Stemming from sequence, a fine tuning of electrostatic and hydrophobic properties of the protein fold determine flexible and rigid regions. Studies show flexible regions usually lack electrostatic interactions, such as salt-bridges and hydrogen-bonds, while the rigid regions often have larger number of such electrostatic interactions. Protein flexible regions are not simply an outcome of looser packing or instability, rather they are evolutionally selected. In this review article we highlight the significance of protein flexibilities in folding, binding and function, and their structural and thermodynamic determinants. Our electrostatic calculations and molecular dynamic simulations on an antibody-antigen complex further illustrate the importance of protein flexibilities in binding and function. PMID- 12370025 TI - The use of DODT as a non-malodorous scavenger in Fmoc-based peptide synthesis. AB - We have synthesized a random group of peptides and performed cleavages using various cleavage cocktails including 3,6-dioxa-1,8-octanedithiol (DODT). Purity of the peptides was compared to that obtained with standard protocols for cleavage using RP-HPLC and Maldi-Tof mass spectrometry. We show that stinking thiols can be replaced by the almost odourless (DODT) without negatively affecting the purity of the end product. PMID- 12370026 TI - Kinetic analysis of the cleavage of human protease-activated receptor-1 / 2 / 3 and 4 using quenched-fluorescent peptide substrates. AB - Protease-activated receptors [PARs] are a family of G-protein-coupled seven transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten residue peptide spanning the receptor activation cleavage site and using progress curve kinetics, k(cat) / K(m) values were determined. PMID- 12370027 TI - Antibiotic activity of reversed peptides of alpha-helical antimicrobial peptide, P18. AB - P18 (KWKLFKKIPKFLHLAKKF-NH(2)), an a-helical antimicrobial peptide designed from cecropin Amagainin 2 hybrid, was known to have potent antimicrobial activity against bacteria as well as fungi without hemolytic activity. To find the peptides comparable or superior to the antimicrobial activity of P18, the two reversed peptides (Rev-1 and Rev-2) of P18 were designed and synthesized. These peptides were found to have similar antimicrobial activity against bacterial and fungal cells without hemolytic activity as compared with P18. Furthermore, a reversed peptide, Rev-2 was shown to have a two-fold higher activity in killing some bacterial cells than P18. Therefore, these results suggested that Rev-2 peptide seems to be an excellent candidate for developing novel peptide antibiotics. PMID- 12370028 TI - Conformational analysis of sea cucumber (Caudina arenicola) C globin 94-97 fragment, Pro-Glu-Leu-Leu. AB - The tetrapeptide Pro-Glu-Leu-Leu forms the 94-97 fragment of C globin in sea cucumber. 2% Butanediol dimethacrylate-cross linked polystyrene (2% BDDMA-PS), which had been optimized, was used for the synthesis of the tetrapeptide Pro-Glu Leu-Leu. The peptide was synthesized by using Boc-amino acid strategy. The peptide purity was checked by RP-HPLC and the peptide was characterized by (1)H NMR spectroscopy and amino acid analysis. Conformation of the peptide was studied by 1D- and 2D- homonuclear (1)H NMR, in DMSO-d6 at 300K. The conformation of the synthetic tetrapeptide (extended backbone conformation) is not in agreement with that in C globin. PMID- 12370029 TI - A synthetic strategy for ON-resin amino acid specific multiple fatty acid acylation of peptides. AB - Covalent modification with fatty acids is observed in several proteins that play crucial roles in cellular physiology. In this paper, a convenient method for the generation of multiple fatty acylated synthetic peptides is described. Peptides were synthesized using solid phase procedures with fluorenylmethoxycarbonyl a amino protected amino acids. Acetamidomethyl protected cysteines were employed. The thiol protecting group was selectively deprotected and acylation was carried out on the resin-bound peptides. The strategy described in this report is applicable to any peptide sequence. PMID- 12370030 TI - Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells. AB - BmK ITa1 cDNA was cloned and highly expressed in E. coli and insect cell. SDS PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively. The expression product purified by a Ni(2+)-IDA-sepharose 6B column was toxic for insect, which indicated that it was biologically activity. Furthermore, Quantitative estimation show that the biological activity of recombinant BmK ITa1 from Tn cells was more powerful than from E. coli. PMID- 12370032 TI - Effects of stereochemistry of sugars on protein stabilities. AB - We investigated thermal stabilities of four proteins in the presence of four kinds of sugars to analyze the mechanism of stabilization of proteins by additives. These proteins were stabilized by the addition of sugars, and the degree of stabilization correlated to the partial molar isentropic compressibility of the sugar. PMID- 12370031 TI - Functionally important residues for the anticoagulant activity of a basic phospholipase A(2) from the Agkistrodon halys pallas. AB - To identify the anticoagulant region of the phospholipase A(2) (PLA(2)) from the Agkistrodon halys Pallas (class II), four mutants E53G, W70M, T56K, and D67K were produced according to the prediction from the crystal structure and the sequence comparison of the strong, weak and non-anticoagulant PLA2s. A test of blood clotting revealed that E53G and W70M had lost their effects on the blood clotting, while T56K and D67K had enhanced activity. The four residues are located on the same face in the tertiary structure of this enzyme. The result supported the prediction that there exists an anticoagulant region that is composed of some residues that are close to each other in tertiary structure to form a functional face. PMID- 12370034 TI - Binding potency of peptide fragments of type 1 collagen cross-linked N telopeptide measured by an enzyme-linked immunosorbant assay. AB - Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans. PMID- 12370033 TI - Purification, crystallization and initial structural solution of a new alpha-like toxin with cardiac toxicity from scorpion Buthus martensii Karsch. AB - An alpha-like toxin named BmK M7 active on both mammals and insects has been purified from the venom of scorpion Buthus martensii Karsch (BmK) recently. The electrophysiological experiments showed that M7 can bind to human cardiac Na+ channel and modify its normal properties, hence can be considered as a cardiotoxin. Single crystals of M7 have been obtained by hanging-drop vapor diffusion method using ammonium sulfate as precipitant in Tris-HCl buffer at pH 8.5. A data set to 1.40 A resolution was collected using synchrotron radiation and CCD detector in Photon Factory in Japan. Data analysis showed that the crystals belonged to space group P3(1)21/P3(1)21, with cell dimensions a=b=32.76 A, c=176.82 A. Assuming two molecules per asymmetric unit, the Vm value is 1.92 A3/Da. The initial structural analysis was carried out by molecular replacement, which showed the correct space group (P3(1)21), and the orientations and positions of the two molecules in the asymmetric unit. PMID- 12370035 TI - Crystallization and preliminary X-ray diffraction analysis of FKBP12 complexed with a novel neurotrophic ligand. AB - A novel neurotrophic ligand, (3R)-4-(p-Toluenesulfonyl)-1,4-thiazane-3-carboxylic acid-L-Leucine ethyl ester, has been complexed with FKBP12 and crystallized using the hanging-drop vapor-diffusion method. Crystals belong to P2(1) space group, with unit cell parameters a=41.2, b=29.6, c=41.5 A, beta=114.0 degrees. The crystals diffract to 1.8 A resolution limit. PMID- 12370036 TI - Nitric oxide in atherosclerosis. AB - *NO is produced endogenously from L-arginine by NOSs. Among its multiple activities, the homeostatic control of the vascular endothelium is crucial for atherosclerosis, a pathogenic condition connected with elevated levels of LDL, the main plasma cholesterol carrier. Oxidised LDL is proatheromatic, and toxic peroxidation products contribute to further endothelial damage. *NO controls vascular tone, inhibits LDL oxidation and has hypocholesterolaemic activity. This review is referred to the chemistry, biology and role of *NO on atherosclerosis and its treatment by *NO donors or modulators. PMID- 12370037 TI - Tubulin as an antiprotozoal drug target. AB - Since tubulin is a known anticancer and anthelmintic drug target, the investigation of protozoal tubulin could lead to the development of new antiparasitic drugs. This review outlines the current state of knowledge concerning drug-mammalian tubulin interactions, the effects of antimicrotubule agents on parasites and parasite tubulin, and our current hypotheses regarding the development of selective ligands for protozoal tubulin as antiparasitic drug candidates. PMID- 12370038 TI - Vitronectin receptor alpha(V)beta(3) integrin antagonists: chemical and structural requirements for activity and selectivity. AB - Alpha(V)beta(3) integrin, a cell surface protein, has been targeted by a variety of natural and synthetic antagonists in the search for potential cancer and osteoporosis drug candidates. This review discusses chemical and structural requirements for activity and selectivity deduced from SAR studies and draws a tentative picture of the pharmacophore. PMID- 12370039 TI - Structural development of biological response modifiers based on retinoids and thalidomide. AB - The full-scale commercial appearance of antibiotics in the 1950's caused a shift of the nature of our lethal diseases from infectious/acute to non infectious/chronic. In this situation, biological response modifiers (BRM's), which are not based on selective toxicity, are expected to be useful. There exist several types of BRM's, including retinoids which act directly on cells at the gene expression level, and thalidomide (and related molecules) which modulate internal circumstances of our body. We have been engaged in medicinal chemical/structural development studies based on these bio-active compounds. Retinoids include all-trans-retinoic acid (ATRA), a major active form of vitamin A (retinol), and its bio-isosters, which elicit their biological effects by binding to their nuclear receptors, RAR's. ATRA has been used in differentiation therapy [typically for the treatment of acute promyelocytic leukemia (APL)] and the treatment of dermatological diseases. Our structural development studies of retinoids, including computer-assisted molecular design has yielded class/subtype selective agonists, synergists and antagonists of RAR's and their partner nuclear receptors, RXR's. Thalidomide elicits a wide range of pharmacological effects, including anti-cachexia, anti-angiogenic and anti-metastatic activities. We have found that thalidomide is a multi-target drug. Hypothetical target events/molecules of thalidomide include TNF-alpha production, nuclear androgen receptor, aminopeptidases, and alpha-glucosidase. Specific and potent compounds for each of these target phenomena/molecules have been prepared by appropriate modification of the thalidomide structure, and are expected to be superior lead compounds for novel immunomodulators, anti-angiogenic agents, and anti-tumor promoting agents. PMID- 12370040 TI - Polyamine metabolism as chemotherapeutic target in protozoan parasites. AB - Polyamines are essential cell constituents for all organisms. The present review highlights important differences in the synthesis, degradation, and interconversion of polyamines between the protozoan parasites (Trypanosoma brucei, Trypanosoma cruzi, Cryptosporidium parvum and Trichomonas vaginalis) and their mammalian hosts. Approaches include development of mono- and di-substituted polyamine analogs targeting polyamine interconversion, as well as more traditional targeting of synthetic enzymes and related pathways. PMID- 12370041 TI - Structure-activity relationship, conformation and pharmacology studies of morphiceptin analogues--selective mu-opioid receptor ligands. AB - Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) is one of the most selective agonists for the mu-opioid receptor. In this review structure-activity relationships of morphiceptin analogues and studies resulting in defining low energy conformations are discussed. Finally, new developments in the control of tumour growth and cell proliferation by morphiceptin analogues are surveyed, which open future perspectives in the diagnosis and treatment of various cancers. PMID- 12370042 TI - Malaria: new chemotherapeutic peroxide drugs. AB - Chemical insights into artemisinin's biological mechanism of action have allowed rational design of some new trioxane and endoperoxide antimalarial drug candidates that are efficacious and safe. This review summarizes recent achievements in this area of peroxide drug development for malaria chemotherapy. PMID- 12370043 TI - Protection against cancer by plant phenylpropenoids: induction of mammalian anticarcinogenic enzymes. AB - Chemoprotection has established itself as a "major arm" in the "war against cancer" and induction of phase 2 detoxification enzymes as an effective strategy. Prominent among inducers are Michael reaction acceptors. Such functionalities are intrinsic to many phenylpropanoids present in edible plants, where they play roles in plant defense. This minireview focuses on the ability of such plant metabolites to elevate phase 2 enzymes in various cell culture and animal models and ultimately to protect against carcinogenesis. PMID- 12370044 TI - Recent advances in topoisomerase I-targeting agents, camptothecin analogues. AB - The present review concentrates on camptothecin (CPT) analogues, the most extensively studied topoisomerase I (topo I) inhibitors, and provides concise information on the structural features of human topo I enzyme, mechanisms of interaction of CPT with topo I, structure-activity relationship study of CPT analogues including the influence of lactone stability on antitumor activity, and recent updates of valuable CPT analogues. PMID- 12370045 TI - Mimetics of the peptide beta-strand. AB - Bioactive structures of peptides represent important clues for drug discovery and development although peptides themselves have substantial limitations as drugs. One promising approach to overcoming the limitations of peptides is to progressively replace amide bonds in peptides with non-peptidic constraints that bring drug-like properties like stability and bioavailability to the molecules. These constraints can also be used to mould molecules into shapes which mimic key elements of protein secondary structure that confer bioactivity to protein surfaces. Preorganizing a molecule into the shape recognized by a receptor results in high affinity binding though a considerable entropy saving and is an effective approach to engineering highly bioactive drug leads. One peptide structure, the extended beta strand, has only recently been identified as a fundamental recognition element in physiological processes. Relatively few molecules have been described as constrained mimics of extended peptide conformations. We now summarize some approaches to mimicking peptide beta strands, and illustrate these with examples of bioactive, stable and bioavailable molecules that are conformationally biased to mimic the extended peptide beta strand. PMID- 12370046 TI - Design, synthesis, and application of peptide secondary structure mimetics. AB - The secondary structure peptidomimetic approach is a rational way to develop novel nonpeptide pharmaceutical agents based upon biologically significant proteinaceous leads. A part of this approach elaborated in this laboratory over the past ten years is reviewed along with the recent developments in this field. PMID- 12370047 TI - Peptidomimetics and peptide backbone modifications. AB - The replacement of the amide bond in a peptide backbone is a widely used form of peptide mimicry. Several of the most common amide bond surrogates, including peptidomimetic work done in this laboratory, and their biological applications are presented in this review. PMID- 12370048 TI - SRC homology-2 inhibitors: peptidomimetic and nonpeptide. AB - The structural and functional characterization of Src homology-2 (SH2) domains and their relationship to catalytic proteins (e.g., kinases, phosphatases, and lipases) or non-catalytic proteins (e.g., upstream adapters, and downstream transcription factors) has significantly impacted our understanding of signal transduction pathways and the identification of promising therapeutic targets for drug discovery. Such SH2-containing proteins are known to be intimately involved in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, and gene transcription. Molecular recognition and biochemical selectivity exists for various SH2 domains based on their binding to phosphotyrosine (pTyr) and contiguous C-terminal amino acids of cognate protein 'partners' in a sequence-dependent manner (i.e., -pTyr-AA(1)-AA(2)-AA(3) ) which result in the formation of signal transduction protein complexes in cells. In recent years, drug discovery efforts have advanced peptidomimetic and nonpeptide inhibitors of such protein-protein interactions based on mimicking pTyr-containing peptide ligands as well as SH2 structure-based de novo design of nonpeptide templates that can capture key binding sites on the target protein. Noteworthy are peptidomimetic and nonpeptide inhibitors of Src, Lck, Grb2, PI-3K, and Zap70 from pioneering efforts that led to the first examples of cellularly and in vivo active SH2 inhibitors. This mini-review highlights key achievements in SH2 inhibitor drug discovery with an emphasis on peptidomimetic and nonpeptide lead compounds in terms of structure-based design, key chemical and biological properties, and proof-of-concept studies relative to further defining the role(s) of SH2 domains in signal transduction processes, cellular functions, and in vivo disease models. PMID- 12370049 TI - Peptidomimetics and angiogenesis. AB - Angiogenesis is the sprouting of new blood capillaries from surrounding preexisting blood vessels. This process is fundamental for embryonic development, wound healing and inflammation. In healthy adults angiogenesis is of minor importance. However, aberrant angiogenesis is essentially involved in disorders as diabetic retinopathy, rheumatoid arthritis and tumor growth, and blocking angiogenesis has emerged as a promising target for antagonizing these diseases. Therefore the development of new anti-angiogenic drugs is of great interest in academic and industrial research. This review focuses on the employment of peptidomimetics in inhibiting pathologic angiogenesis. It will survey the individual aspects of angiogenesis where the usage of peptidomimetics is favored and will consider the current progresses on this field. PMID- 12370050 TI - Recent advances in the development of nonpeptide somatostatin receptor ligands. AB - Somatostatin (SRIF) is a cyclic peptide that occurs in two biologically active forms, SRIF-14 and SRIF-28. These peptides inhibit the secretion of many other peptides, including insulin and glucagon, function as neurotransmitters or neuromodulators, and exhibit potent antiproliferative activity. Recent research has led to the development of nonpeptide SRIF ligands with high affinity and selectivity at all SRIF receptor subtypes. Additionally, the newly discovered sst(2)and sst(3) antagonists will greatly facilitate our understanding of these receptors. These novel nonpeptide SRIF agonists and antagonists may have therapeutic potential in a variety of disease states. PMID- 12370051 TI - Biomembrane permeability of peptides: strategies to improve their mucosal uptake. AB - In order to gain a therapeutic response after mucosal administration peptide drugs have to permeate the absorption membrane based on the mucus layer (I) and the epithelial tissue (II) in significant quantities. The peptide drug transport across the membrane can be improved by the use of mucolytic agents and the permeation enhancers. The generation of novel, more potent permeation enhancers, based on an improved knowledge of the absorption membrane in combination with the appropriate delivery systems will strongly improve the bioavailability of mucosally applied peptide drugs. PMID- 12370052 TI - Cell membranes as barriers for the use of antisense therapeutic agents. AB - Antisense oligonucleotides are promising therapeutical agents for numerous diseases resulting from overexpression of genes, expression of mutant genes and viral infections. As most oligonucleotides are polyanions they can not readily pass cellular membranes in adequate amounts to show activity. Therefore, different types of carrier systems and modifications have been developed to enhance absorption and distribution at the level of tissues and cells. The current state of delivery systems will be reviewed with a major part devoted to the commonly used cationic lipids. PMID- 12370053 TI - Liposomes for intravenous drug targeting: design and applications. AB - Drug targeting with liposomes has been studied for over 25 years and has demonstrated its value in clinical practice. This mini review offers an overview of the design and application of liposomes for i.v. drug targeting. Two approaches are outlined: passive and active targeting. The former approach is based on liposomes with prolonged circulation and selective target localization properties, while in the latter approach specific targeting ligands are coupled to the liposome surface in order to achieve enhanced interaction with target cell membranes. PMID- 12370054 TI - How do channel- and pore-forming helical peptides interact with lipid membranes and how does this account for their antimicrobial activity? AB - Animals and plants defend themselves against pathogenic micro-organisms by the rapid mobilization of polycationic helical amphipathic peptides. Interactions with membranes induce optimal orientation and mutual structural changes, allowing for example to form transbilayer ion channels or pores whose properties are compared in this review. Physicochemical studies of peptide-lipid interactions provide attractive approaches for drug design. PMID- 12370056 TI - Cytosolic calcium oscillations in signal transduction pathways. AB - The oscillatory nature of the intracellular calcium signal has been recognized as soon as the methodological developments allowed us to record calcium fluctuations at the single cell level. While the principal mechanisms responsible for the generation of these oscillations have been partially resolved, more attention has been recently focused on signal decoding and more particularly on the role of cell structure organization in transducing this signal to the molecular targets of the calcium messenger. PMID- 12370055 TI - The biochemical and physiological characteristics of surface receptors of gram negative bacteria. AB - This review focuses on the properties of ferric iron surface receptors of Gram negative bacteria. We discuss the different strategies to acquire iron, and the fundamental role of these receptors in pathogenicity. The structure of some of these receptors, iron transport and regulation mechanisms are presented here. PMID- 12370057 TI - Heterotrimeric G proteins control diverse pathways of transmembrane signaling, a base for drug discovery. AB - Heptahelical receptors are coupled to heterotrimeric GTP-binding proteins (G proteins) which transduce most signals through their alpha and betagamma subunits to effectors including adenylylcyclases, ion channels, phospholipases Cbeta, and phosphoinositide 3-kinases. The diversity of G proteins, their effectors and regulators (RGS proteins), supports the interest of these protein families as potential drug targets. PMID- 12370058 TI - Structure, function and modulation of chemokine receptors: members of the g protein-coupled receptor superfamily. AB - Chemokine receptors are membrane proteins that play an important role in inflammation and the cellular entry of human immunodeficiency virus type I (HIV 1). Understanding the structure-function relationship of chemokine receptor ligand interactions and developing novel strategies to control these interactions have important implications for therapeutic intervention of human diseases such as HIV-1 infection. This article reviews the work carried out in our laboratory in molecular modeling and site-directed mutagenesis of chemokine receptor-ligand interactions and chemical synthesis of chemokine-derived peptide agonists and antagonists. These studies demonstrate a paradigm for exploring and controlling membrane protein-protein interactions. PMID- 12370059 TI - Molecular and physicochemical aspects of local anesthetics acting on nicotinic acetylcholine receptor-containing membranes. AB - Local anesthetics inhibit the ion channel activity of nicotinic acetylcholine receptors in a noncompetitive fashion. This inhibitory action is ascribed to two possible inhibitory mechanisms: an open-channel-blocking mechanism and/or an allosteric process where the drug binds either to the closed channel or to other nonluminal sites, respectively. PMID- 12370060 TI - Non-genomic effects of steroid hormones on membrane channels. AB - Steroid hormones may possess two distinct actions, a delayed genomic influence and the rapid nongenomic effects, which may act in concert. Nongenomic effect may be mediated by putative membrane receptors or due to allosteric interactions of steroids with membrane proteins (e.g. ionic channels), inducing rapid changes in protein/receptor/channel activation or inhibition. PMID- 12370061 TI - Nerve membrane ion channels as the target site of insecticides. AB - Most insecticides are potent neurotoxicants that act on various neuroreceptors and ion channels. However, the major target receptors are limited to sodium channels, GABA receptors, and nicotinic acetylcholine receptors. DDT and pyrethroids act similarly on sodium channels to keep them open leading to hyperexcitation. Indoxacarb inhibits sodium channels and certain subtypes of nicotinic receptors. Dieldrin, lindane and fipronil block GABA receptors. Imidacloprid modulates nicotinic receptors in a complex manner. Spinosad's major target site appears to be nicotinic receptors. PMID- 12370062 TI - N(7)-substituted-5-aryl-pyrrolo[2,3-d]pyrimidines represent a versatile class of potent inhibitors of the tyrosine kinase c-Src. AB - 5-Aryl-pyrrolo[2,3-d]pyrimidines incorporating different N(7)-substituents have been prepared and evaluated for their inhibitory potency towards the tyrosine kinase c-Src. Optimization of these compounds resulted in highly potent c-Src inhibitors, some (e.g. 4g, 6g, 7h, 8l) with excellent specificity towards other receptor and nonreceptor tyrosine kinases. In addition compounds 4g, 5b and 5c are characterized by a good pharmacokinetic profile. PMID- 12370063 TI - Strategies for access to enantiomerically pure ecadotril, dexecadotril and fasidotril: a review. AB - Ecadotril and dexecadotril are powerful and selective inhibitors of neprilysin (NEP, EC 3.4.24.11) and are being developed as therapeutic agents, since they behave as prodrugs of the enantiomers of thiorphan. They exhibit different pharmaceutical profiles (intestinal antisecretatory action for the (R) enantiomer, i.e. dexecadotril, and cardiovascular activity for the (S) enantiomer, i.e. ecadotril). Fasidotril is a related compound which has special interest as an equipotent dual inhibitor of NEP and ACE (EC 3.4.15.1). This behavior confers on fasidotril powerful pharmaceutical properties in the cardiovascular field. This review deals with various synthetic approaches, either published or patented, for access to the enantiomerically pure or highly enriched forms of these drugs. Thus, different methods have been studied, which are taken from different methodologies of resolution procedures and asymmetric synthesis, namely : i- Synthesis from a chiron from the chiral pool ii- Chemical resolution of racemic precursors iii- Enzymatic resolution and desymmetrization of meso starting materials iv- Asymmetric synthesis, including enantioselective catalytic hydrogenation, alkaloid catalyzed asymmetric Michael additions, and diastereoselective alkylation of a chiral derivative. Some of these methods are used in industrial processes leading to the indicated compounds. PMID- 12370064 TI - cycloSal-pronucleotides design of chemical trojan horses. AB - Pronucleotides represent a promising alternative to improve the biological activity of nucleoside analogues against different viral diseases. The basic idea is to achieve nucleotide delivery into cells bypassing limitations encountered during the intracellular formation of nucleotides. The cycloSal-concept is one of several pronucleotide systems reported so far. For some nucleoside analogues, the cycloSal-approach improved antiviral potency thus broadening the applicability of nucleosides. The initial design, chemistry, the proof-of-principle and different applications of the cycloSal-strategy will be discussed in this review. PMID- 12370065 TI - Dimeric 4-Aryl-1,4-dihydropyridines: development of a third class of nonpeptidic HIV-1 protease inhibitors. AB - Cross-resistance development against most peptidic HIV-1 protease inhibitors (PI) forces the development of nonpeptidic alternatives. The classes of nonpeptidic protease inhibitors was limited so far to cyclic ureas and 4-hydroxy-2-pyrones with problems of limited bioavailability by extensive metabolism and protein binding. Cage dimeric 4-aryl-1,4-dihydropyridines have been developed as third class of nonpeptidic PIs. In the following synthesis, molecular modeling and biological activities of a first series of the novel PIs are reviewed. Bioavailability of the dimers will not be limited by protein binding and metabolism as far as evaluated. PMID- 12370066 TI - Recent advances in the identification and development of 20S proteasome inhibitors. AB - The involvement of the 20S proteasome in the degradation of critical intracellular regulatory proteins has suggested the potential use of proteasome inhibitors as novel anti-inflammatory agents and for the treatment of cancer and auto-immune diseases. Early inhibitors of the 20S proteasome were relatively non specific compounds and used for in vitro studies of the ubiquitin/proteasome dependent degradation pathway. The inherent drawbacks of these inhibitors (e.g., non-target specific, too reactive or unstable) has prompted medicinal chemists to search for alternative subunit-specific proteasome inhibitors. This manuscript summarises recent salient medicinal chemistry achievements in this area of research. PMID- 12370067 TI - Cytochrome p450 retinoic acid 4-hydroxylase inhibitors: potential agents for cancer therapy. AB - Retinoids play a crucial role in cellular differentiation and proliferation of epithelial tissue and their utility in oncology and dermatology is well documented. This mini review focuses on the role of all-trans-retinoic acid (ATRA or RA), the principal endogenous retinoid and its metabolism in cancer therapy. ATRA has been used successfully in differentiating therapy of acute promyelecytic leukemia and other types of cancers. However, its usefulness is limited by the rapid emergence of ATRA resistance due (in part) to ATRA - induced acceleration of ATRA metabolism. A novel strategy to subjugate the limitation associated with exogenous ATRA therapy has been to modulate and/or increase the levels of endogenous ATRA by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme(s) responsible for ATRA metabolism. These inhibitors are also referred to as retinoic acid metabolism blocking agents (RAMBAs). This review highlights development in the design, synthesis and evaluation of RAMBAs since 1987. Major emphasis is given to liarozole, the most studied and only RAMBA to undergo clinical investigation and also the recently developed novel and highly potent 4 azoly retinoids. The potential role of a new family of cytochrome P450 enzymes, CYP26, with specificity towards ATRA is also discussed. PMID- 12370068 TI - New approaches to raising the HDL cholesterol level. AB - Not only a high level of low-density lipoprotein (LDL) cholesterol, but also a low level of high-density lipoprotein (HDL) cholesterol, is a critical risk factor for atherosclerosis and coronary heart disease. Although fibrates and niacin can be used to improve low HDL cholesterol levels, their effect is not wholly satisfactory, so better drugs for the elevation of HDL cholesterol are desired. Among the many methods that may be used to raise HDL cholesterol levels, this review focuses on inhibitors of cholesteryl ester transfer protein (CETP) and on nuclear orphan receptor agonists that mediate the expression of ATP binding cassette transporter 1 (ABC1). PMID- 12370069 TI - Refining retinoids with heteroatoms. AB - Retinoids are a group of synthetic compounds designed to refine the numerous biological activities of retinoic acid into pharmaceuticals for several diseases, including cancer. Designs that conformationally-restricted the rotation of the structures resulted in arotinoids that were biologically active, but with increased toxicity. Incorporation of a heteroatom in one cyclic ring of the arotinoid structures drastically reduced the toxicity, while retaining biological activity. Clinical trials of a heteroarotinoid, Tazarotene, confirmed the improved chemotherapeutic ratio (efficacy/toxicity). PMID- 12370070 TI - Phytoecdysteroids effects on mammalians, isolation and analysis. AB - Ecdysteroids are known insect moulting hormones, regulating the insects' metamorphosis. At the same time, ecdysteroids reveal beneficial effects on humans and animals alike. Medicinal plants have been subjected to an intensive research, addressing the presence of ecdysteroids. The possible utilization of medicinal plant deals with their use as raw materials for health preparations and also for the isolation of new phytoecdysteroids. Research on the plant ecdysteroids involves two basic lines. Isolation of major compounds and scout their physiological effects; and isolation of minor ecdysteroids to find new compounds. This review summarizes the recent efforts in the ecdysteroid research including their indication as health improvement preparations, chromatography of ecdysteroids and certain methods for identification of new ecdysteroids. PMID- 12370071 TI - Molecular pharmacophore determination of lipid lowering drugs with the receptor mapping method. AB - Hypolipidemic pharmacophoric moieties of statins, fibrates, ACAT inhibitors and beta-sitosterol analog series were identified by computational modeling, and compared with the computed structure of new potential glycyrrhetinic acid derivatives lipid-lowering drugs. Their electronic and geometric domains, similar to those of fibrates, suggest a fibrate -like mechanism matching biochemical data. PMID- 12370072 TI - Biomolecular targets for platinum antitumor drugs. AB - Cis-diamminedichloroplatinum(II) (cisplatin) is widely used for the treatment of testicular, ovarian, and other forms of cancer. Several second generation platinum centered antitumor drugs have been approved or undergoing phase-3 clinical trial. Cisplatin arrests the cell cycle at the G2 phase by a mechanism commonly known as apoptosis. At the molecular level, it is generally believed that the anticancer properties of these compounds are due to the covalent binding to DNA. In addition to DNA binding, the platinum drugs bind and interact with proteins and enzymes. The toxic effects of the drugs have been usually attributed to protein binding. However, a growing body of work points to much more complex anticancer mechanisms involving direct and indirect interactions of platinum compounds with proteins and enzymes. In this review, a discussion on the strength and weaknesses of DNA binding mechanism followed by enzymes and protein interactions with the drugs are presented for the comprehensive understanding of apoptosis. The purpose of this review is to encourage researchers to explore metallobiochemistry of platinum drugs focusing attention to cellular and molecular events beyond DNA binding. PMID- 12370073 TI - Synthesis and antimalarial activity of 1,2,4,5-tetraoxanes. AB - Methods for formation of 1,2,4,5-tetraoxanes are summarized and antimalarial activities of 1,2,4,5-tetraoxanes are discussed. PMID- 12370074 TI - Medicinal chemistry of nicotinamide in the treatment of ischemia and reperfusion. AB - Nicotinamide can facilitate DNA repair by inhibiting poly(ADP-ribose) polymerase, increasing NAD levels and adjusting other related enzyme activities. This review will summarize recent work on the design of poly(ADP-ribose) polymerase inhibitors, poly(ADP-ribose) glycohydrolase inhibitors and will discuss the possible use of drugs that interact with NAD synthetic enzymes. PMID- 12370075 TI - Gas chromatographic determination of prostaglandins. AB - Progress in separation and detection of prostaglandins and the other metabolites of arachidonic acid by means of GC-ECD, GC-MS, and GC-MS-MS in the course of the past fifteen years was reviewed. One discussed the problems of sample preparation, selection of proper chromatographic conditions, and detection modes available. Finally, applications of the methods developed to detection and quantification of prostanoids in biological material was presented. PMID- 12370076 TI - QRAR models for central nervous system drugs using biopartitioning micellar chromatography. AB - The capability of biopartitioning Micellar Chromatography, BMC, to describe and estimate pharmacokinetic and pharmacodynamic parameters of central nervous system drugs is reviewed in this article. BMC is a mode of micellar liquid chromatography, MLC, that uses micellar mobile phases of Brij35 (polyoxyethilene(23) lauryl ether) prepared in physiological conditions (pH, ionic strength). The retention of a drug in this system depends on its hydrophobic, electronic and steric properties, which also determine its biological activity. The results of BMC studies suggest that this in vitro approach is an attractive useful tool to be implemented into the lead optimization step of drug development scheme. PMID- 12370077 TI - Highlights in the development of new antiviral agents. AB - The potential of a large variety of new compounds and new strategies for the treatment of virtually all major virus infections has been addressed. This includes, for the treatment of HIV infections, virus adsorption inhibitors (cosalane derivatives, cyanovirin-N), co-receptor antagonists (TAK-779, AMD3100), viral fusion inhibitors (pentafuside T-20, betulinic acid derivatives), viral uncoating inhibitors (azodicarbonamide), nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs: emtricitabine, amdoxovir, dOTC, d4TMP prodrugs, tenofovir disoproxil fumarate), non-nucleoside reverse transcriptase inhibitors (NNRTIs: thiocarboxanilide UC-781, capravirine, SJ-3366, DPC 083, TMC 125/R165335), integrase inhibitors (diketo acids), transcription inhibitors (temacrazine, flavopiridol), protease inhibitors (atazanavir, mozenavir, tipranavir); for the treatment of RSV and paramyxovirus infections, viral fusion inhibitors (R170591, VP-14637, NMS03); for the treatment of picornavirus infections, viral uncoating inhibitors (pleconaril); for the treatment of pesti- (hepaci-, flavi-) virus infections, RNA replicase inhibitors (VP-32947); for the treatment of herpesvirus (HSV, VZV, CMV) infections, DNA polymerase inhibitors (A 5021, L- and D-cyclohexenylguanine); for the treatment of VZV infections, bicyclic furopyrimidine analogues; for the treatment of CMV infections, fomivirsen; for the treatment of DNA virus infections at large (papilloma-, polyoma-, herpes-, adeno- and poxvirus infections), cidofovir; for the treatment of influenza, neuraminidase inhibitors (zanamivir, oseltamivir, RWJ-270201); for the treatment of HBV infections, adefovir dipivoxil; for the treatment of HBV and HCV infections, N-glycosylation inhibitors (N-nonyl-deoxynojirimycin); and, finally, IMP dehydrogenase inhibitors and S-adenosylhomocysteine hydrolase inhibitors, for the treatment of various virus infections, including hemorrhagic fever virus infections. PMID- 12370078 TI - Selective agonists and antagonists for kainate receptors. AB - Kainate receptors have only recently been characterized both from the pharmacological and biological point of view. Due to the limited number of truly kainate selective ligands, most of the known agonists and antagonists are generally classified as AMPA/kainate receptors ligands. The increasing interest in the search for selective kainate ligands aims at understanding the physiological role played by these receptors and finding out potential therapeutic approaches for the treatment of a number of neurological pathologies, i.e. schizophrenia, as well as acute and chronic neurodegenerative diseases, i.e. epilepsy, cerebral ischaemia, Parkinson's and Alzheimer's diseases. This review will focus on the recently discovered ligands for kainate receptors, with a particular attention given to those molecules displaying a selectivity for the different subunits of the kainate receptors and, on the other hand, to the role played by these receptor subtypes in the pathophysiology of the central nervous system. PMID- 12370079 TI - Daphnane-type diterpene orthoesters and their biological activities. AB - Daphnane orthoesters are the active ingredients of plant remedies from the Western, Chinese and African traditional medicine, and have provided important tools to investigate medicinally relevant processes like tumour promotion, apoptosis, neurotrophism, and VR1 activation. The occurrence, biological activity, and molecular pharmacology of these compounds will be reviewed. PMID- 12370080 TI - Inhibition of phosphatidylcholine synthesis induces expression of the endoplasmic reticulum stress and apoptosis-related protein CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153). AB - Inhibition of de novo synthesis of phosphatidylcholine (PC) by some anti-cancer drugs such as hexadecylphosphocholine leads to apoptosis in various cell lines. Likewise, in MT58, a mutant Chinese hamster ovary (CHO) cell line containing a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), an important regulatory enzyme in the CDP-choline pathway, inhibition of PC synthesis causes PC depletion. Cellular perturbations like metabolic insults and unfolded proteins can be registered by the endoplasmic reticulum (ER) and result in ER stress responses, which can lead eventually to apoptosis. In this study we investigated the effect of PC depletion on the ER stress response and ER-related proteins. Shifting MT58 cells to the non-permissive temperature of 40 degrees C resulted in PC depletion via an inhibition of CT within 24 h. Early apoptotic features appeared in several cells around 30 h, and most cells were apoptotic within 48 h. The temperature shift in MT58 led to an increase of pro-apoptotic CCAAT/enhancer-binding protein-homologous protein (CHOP; also known as GADD153) after 16 h, to a maximum at 24 h. Incubation of wild-type CHO-K1 or CT-expressing MT58 cells at 40 degrees C did not induce differences in CHOP protein levels in time. In contrast, expression of the ER chaperone BiP/GRP78, induced by an increase in misfolded/unfolded proteins, and caspase 12, a protease specifically involved in apoptosis that results from stress in the ER, did not differ between MT58 and CHO-K1 cells in time when cultured at 40 degrees C. Furthermore, heat shock protein 70, a protein that is stimulated by accumulation of abnormal proteins and heat stress, displayed similar expression patterns in MT58 and K1 cells. These results suggest that PC depletion in MT58 induces the ER-stress related protein CHOP, without raising a general ER stress response. PMID- 12370081 TI - Characterization of the Aalpha and Abeta subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution. AB - Protein phosphatase 2A (PP2A) is very versatile owing to a large number of regulatory subunits and its ability to interact with numerous other proteins. The regulatory A subunit exists as two closely related isoforms designated Aalpha and Abeta. Mutations have been found in both isoforms in a variety of human cancers. Although Aalpha has been intensely studied, little is known about Abeta. We generated Abeta-specific antibodies and determined the cell cycle expression, subcellular distribution, and metabolic stability of Abeta in comparison with Aalpha. Both forms were expressed at constant levels throughout the cell cycle, but Aalpha was expressed at a much higher level than Abeta. Both forms were found predominantly in the cytoplasm, and both had a half-life of approx. 10 h. However, Aalpha and Abeta differed substantially in their expression patterns in normal tissues and in tumour cell lines. Whereas Aalpha was expressed at similarly high levels in all tissues and cell lines, Abeta expression varied greatly. In addition, in vivo studies with epitope-tagged Aalpha and Abeta subunits demonstrated that Abeta is a markedly weaker binder of regulatory B and catalytic C subunits than Aalpha. Construction of phylogenetic trees revealed that the conservation of Aalpha during the evolution of mammals is extraordinarily high in comparison with both Abeta and cytochrome c, suggesting that Aalpha is involved in more protein-protein interactions than Abeta. We also measured the binding of polyoma virus middle tumour antigen and simian virus 40 (SV40) small tumour antigen to Aalpha and Abeta. Whereas both isoforms bound polyoma virus middle tumour antigen equally well, only Aalpha bound SV40 small tumour antigen. PMID- 12370082 TI - The 'four principles of bioethics' as found in 13th century Muslim scholar Mawlana's teachings. AB - BACKGROUND: There have been different ethical approaches to the issues in the history of philosophy. Two American philosophers Beachump and Childress formulated some ethical principles namely 'respect to autonomy', 'justice', 'beneficence' and 'non-maleficence'. These 'four principles' were presented by the authors as universal and applicable to any culture and society. Mawlana, a great figure in Sufi tradition, had written many books which not only guide people how to worship God to be close to Him, but also advise people how to lead a good life to enrich their personality, as well as to create a harmonious society and a peaceful world. METHODS: In this study we examined the major works of Mawlana to find out which of these 'four principles of bioethics' exist in Mawlana's ethical understanding. RESULTS: We have found in our study that all these principles exist in Mawlana's writings and philosophy in one form or another. CONCLUSIONS: We have concluded that, further to Beachump and Childress' claim that these principles are universal and applicable to any culture and society, these principles have always existed in different moral traditions in different ways, of which Mawlana's teaching might be presented as a good example. PMID- 12370083 TI - Is age an independent determinant of mortality in cardiac surgery as suggested by the EuroSCORE? AB - BACKGROUND: The proportion of older patients in cardiac surgery is continuously increasing. 37% of patients undergoing heart surgery in Germany in the year 2000 were 70 years of age and older. We have studied the role of age as a determinant of mortality in cardiac surgery in our institutional patient population. METHODS: We have calculated the EuroSCORE and the corresponding age-adjusted EuroSCORE in 8769 patients who underwent heart surgery between January 1996 and January 2002 and collected the information on the occurrence of postoperative complications and 30-days mortality. RESULTS: The multimorbidity increased with ascending age. Both the EuroSCORE and the age-adjusted EuroSCORE values increased significantly with age in the whole group of patients as well as in the group of patients who were alive 30 days after heart surgery. The incidence of postoperative complications and 30-days mortality increased significantly with age. In patients who died within 30 days after surgery, the EuroSCORE increased significantly with age, whereas the age-adjusted EuroSCORE did not. The occurrence of diabetes mellitus, arterial hypertension and atrial fibrillation, i.e., the risk factors not considered by the EuroSCORE, exhibited a significant age dependence in our patients. The univariate analysis identified the significant dependence of 30 days mortality on diabetes and atrial fibrillation. The stepwise logistic regression analysis showed the dependence of mortality on diabetes. CONCLUSIONS: On the background of the well-known age-dependent structural and functional changes of different body organs, our data show that age is a significant risk indicator in cardiac surgery, strongly correlating with morbidity and mortality. Consequently, special preventive and therapeutic measures are required in clinical environment in the case of elderly patients undergoing cardiac surgery. PMID- 12370084 TI - Appendicular bone mass and knee and hand osteoarthritis in Japanese women: a cross-sectional study. AB - BACKGROUND: It has been reported that there is an inverse association between osteoarthritis (OA) and osteoporosis. However, the relationship of bone mass to OA in a Japanese population whose rates of OA are different from Caucasians remains uncertain. METHODS: We studied the association of appendicular bone mineral density (second metacarpal; mBMD) and quantitative bone ultrasound (calcaneus; stiffness index) with knee and hand OA among 567 Japanese community dwelling women. Knee and hand radiographs were scored for OA using Kellgren Lawrence (K/L) scales. In addition, we evaluated the presence of osteophytes and of joint space narrowing. The hand joints were examined at the distal and proximal interphalangeal (DIP, PIP) and first metacarpophalangeal/carpometacarpal (MCP/CMC) joints. RESULTS: After adjusting for age and body mass index (BMI), stiffness index was significantly higher in women with K/L scale, grade 3 at CMC/MCP joint compared with those with no OA. Adjusted means of stiffness index and mBMD were significantly higher in women with definite osteophytes at the CMC/MCP joint compared to those without osteophytes, whereas there were no significant differences for knee, DIP and PIP joints. Stiffness index, but not mBMD, was higher in women with definite joint space narrowing at the CMC/MCP joint compared with those with no joint space narrowing. CONCLUSIONS: Appendicular bone mass was increased with OA at the CMC/MCP joint, especially among women with osteophytes. Our findings suggest that the association of peripheral bone mass with OA for knee, DIP or PIP may be less clearcut in Japanese women than in other populations. PMID- 12370085 TI - HLA-A and -B alleles and haplotypes in hemochromatosis probands with HFE C282Y homozygosity in central Alabama. AB - BACKGROUND: We wanted to quantify HLA-A and -B allele and haplotype frequencies in Alabama hemochromatosis probands with HFE C282Y homozygosity and controls, and to compare results to those in other populations. METHODS: Alleles were detected using DNA-based typing (probands) and microlymphocytotoxicity (controls). RESULTS: Alleles were determined in 139 probands (1,321 controls) and haplotypes in 118 probands (605 controls). In probands, A*03 positivity was 0.7482 (0.2739 controls; p = or < 0.0001; odds ratio (OR) 7.9); positivity for B*07, B*14, and B*56 was also increased. In probands, haplotypes A*03-B*07 and A*03-B*14 were more frequent (p < 0.0001, respectively; OR = 12.3 and 11.1, respectively). The haplotypes A*01-B*60, A*02-B*39, A*02-B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03 B*35, A*03-B*44, A*03-B*47, and A*03-B*57 were also significantly more frequent in probands. 37.3% of probands were HLA-haploidentical with other proband(s). CONCLUSIONS: A*03 and A*03-B*07 frequencies are increased in Alabama probands, as in other hemochromatosis cohorts. Increased absolute frequencies of A*03-B*35 have been reported only in the present Alabama probands and in hemochromatosis patients in Italy. Increased absolute frequencies of A*01-B*60, A*02-B*39, A*02 B*62, A*03-B*13, A*03-B*15, A*03-B*27, A*03-B*44, A*03-B*47, and A*03-B*57 in hemochromatosis cohorts have not been reported previously. PMID- 12370086 TI - A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies. AB - BACKGROUND: Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases affecting both humans and animals. They are associated with post-translational conversion of the normal cellular prion protein (PrPC) into a heat- and protease-resistant abnormal isoform (PrPSc). Detection of PrPSc in individuals is widely utilized for the diagnosis of prion diseases. METHODS: TSE brain tissue samples have been processed in order to quantitatively isolate PrPSc. The protocol includes an initial homogenization, digestion with proteinase K and salt precipitation. RESULTS: Here we show that over 97 percent of the PrPSc present can be precipitated from infected brain material using this simple salting-out procedure for proteins. No chemically harsh conditions are used during the process in order to conserve the native quality of the isolated protein. CONCLUSION: The resulting PrPSc-enriched preparation should provide a suitable substrate for analyzing the structure of the prion agent and for scavenging for other molecules with which it may associate. In comparison with most methods that exist today, the one described in this study is rapid, cost effective and does not demand expensive laboratory equipment. PMID- 12370087 TI - Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation. AB - BACKGROUND: The 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear. RESULTS: MEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126. CONCLUSIONS: These results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2. PMID- 12370088 TI - MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function. AB - BACKGROUND: Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN containing proteins as it is highly conserved and important for proteasome function. RESULTS: We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. CONCLUSIONS: The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1 removing activity. PMID- 12370089 TI - Ischemic preconditioning of myocardium. AB - Preconditioning of the myocardium with short episodes of sublethal ischemia will delay the onset of necrosis during a subsequent lethal ischemic insult. Ischemic preconditioning seems to involve a variety of stress signals which include activation of membrane receptors and signaling molecules such as protein kinase C, mitogen-activated protein kinases, opening of ATP-sensitive potassium channel, and expression of many protective proteins. The purpose of this review is to assess the current position in this field and to facilitate future research. PMID- 12370090 TI - Neuroprotective effect of ONO-1078, a leukotriene receptor antagonist, on focal cerebral ischemia in rats. AB - AIM: To determine whether ONO-1078 (pranlukast), a potent leukotriene receptor antagonist, has neuroprotective effect on focal cerebral ischemia in the rat. METHODS: Focal cerebral ischemia was induced by 30 min of middle cerebral artery (MCA) occlusion and followed by 24 h reperfusion. ONO-1078 (0.003-1.0 mg/kg) or vehicle (saline 1 mL/kg) was ip injected 30 min before MCA occlusion and 2 h after reperfusion. The neurological score, infarct volume, neuron density (in cortex, hippocampus, and striatum), brain edema, and albumin exudation around the vessels were determined 24 h after reperfusion. RESULTS: ONO-1078 slightly improved the neurological deficiency, and dramatically decreased infarct volume and neuron loss which showed a bell shaped dose response effect with highest effect at doses of 0.01-0.3 mg/kg. Enlargement of the ischemic hemisphere and albumin exudation were inhibited at doses of 0.01-1.0 mg/kg. CONCLUSION: ONO-1078 has the protective effect on focal cerebral ischemia in rats, which is partially attributed to the inhibition of brain edema. This may represent a novel approach to the treatment of acute cerebral ischemia with cysteinyl leukotriene receptor antagonists. PMID- 12370091 TI - Beta-endorphin suppresses release of thyrotropin-releasing hormone in rat hypothalamus during acute hypoxia exposure. AB - AIM: To study the influences of beta-endorphin (beta-EP) on the responses of thyrotropin-releasing hormone (TRH) in median eminence (ME) and paraventricular nucleus (PVN) of hypothalamus to acute hypoxia in conscious rats. METHODS: Brain TRH, serum T3 and T4 were measured by radioimmunoassay. The male Wistar rats were exposed in a simulated hypobaric chamber at 7000 m altitude (8.2 % O2) for 2 h. beta-EP was given by intraventricular injection (icv) before hypoxia. RESULTS: beta-EP (0.1 or 1 micromol/L, icv) elevated TRH levels of ME by 12 % (P <0.05) and 15 % (P < 0.05) in treated groups comparing with saline control group (4.8+/ 0.3) microg/g protein, and enhanced TRH of PVN by 24 % (P <0.05) and 44 % (P < 0.01) in treated groups comparing with control group (180+/-21) ng/g protein during hypoxia. Meanwhile, serum T3 and T4 were significantly decreased (P < 0.05 or P < 0.01). Naloxone 10 micromol/L abolished the effects of beta-EP (0.1 micromol/L) on TRH in ME (P <0.01) and PVN (P < 0.01) as well as T3 and T4. Naloxone (10 micromol/L, icv) alone reduced contents of TRH in ME and PVN (P <0.05 or P <0.01), but increased the levels of serum T3 and T4 (P <0.01). CONCLUSION: beta-Endorphin was involved in the modulation of hypothalamic TRH release of rats during hypoxia, through an inhibitory mechanism of TRH release in ME and PVN of hypothalamus. PMID- 12370092 TI - Melatonin reduces colon immunological injury in rats by regulating activity of macrophages. AB - AIM: To investigate the effects of melatonin on the colon immunological injury of rats and the role of macrophages in this process. METHODS: The rats colitis was established by intrarectal injection with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and ethanol. The animals were randomized into 6 groups: normal group, model group, 5-aminosalicylic acid group (100 mg/kg), and melatonin group (2.5, 5.0, and 10.0 mg/kg), treated intrarectally with saline, saline, 5-aminosalicylic acid, and melatonin, respectively (once a day, from d 7 after colitis established to d 28). At the end of the experiment, the colon mucosa damage index (CMDI), the score of histology (HS), the level of myeloperoxidase(MPO), and the sore of occult blood test (OBT) were evaluated. Meanwhile, the activity of interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha, and nitric oxide (NO) were also detected. RESULTS: After treated with TNBS and ethanol, the extents of CMDI, HS, OBT, and the level of MPO in model group were more higher than that in normal group. Melatonin could alleviate the colon injury, and reduce the level of MPO and the degree of OBT. The activity of IL-1, TNF-alpha, and NO which released mainly from macrophages was elevated remarkably. Melatonin could depress all this parameters. CONCLUSION: Melatonin could reduce the colon damage in the colitis rats by regulating macrophage activity. PMID- 12370093 TI - Growth hormone increases lung microvascular injury in lipopolysaccharide peritonitis rats: possible involvement of NF-kappaB activation in circulating neutrophils. AB - AIM: To investigate the effects of growth hormone (GH) on NF-kappaB activity in neutrophils and neutrophils-mediated organ injury induced by lipopolysaccharide (LPS) in rats. METHODS: Male Wistar rats challenged with or without LPS (5 mg/kg) were treated with varied doses of GH (0.5, 1.0, and 2.0 mg/kg) for 2 or 4 h. NF kappaB activities in circulating neutrophils were measured with electrophoretic mobility shift assays (EMSA), and I-kappaB levels in circulating neutrophils were detected by Western blot. Lung neutrophils sequestration and lung microvascular permeability were measured at 4 h after LPS challenge. RESULTS: Circulating neutrophils in LPS challenged rats had increased NF-kappaB activity and decreased I-kappaB level as compared with controls. GH dramatically increased NF-kappaB activity and I-kappaB degradation induced by LPS challenge in neutrophils. Also, subsequently, GH treatment increased lung neutrophils sequestration and lung microvascular injury induced by LPS. CONCLUSION: These results suggest that treatment of GH is harmful, instead of beneficial, to LPS-induced organ injury. Increased neutrophils' NF-kappaB activity and lung neutrophils sequestration are critical in vivo mechanisms mediating GH action on LPS-induced organ injury. PMID- 12370094 TI - Low dose of resveratrol enhanced immune response of mice. AB - AIM: To study the immune modulating effect of low dose of resveratrol. METHODS: Concanavalin A (ConA) and Staphylococcus aureus Cowan (Sac) were used to induce the activation of T lymphocyte and antigen presenting cell and cytokine production. [3H]-Thymidine incorporation was used to evaluate the proliferation of lymphocyte. Cytokine production was detected by ELISA method. Dinitrofluorobenzene (DNFB) was used to induce mice delayed type hypersensitivity (DTH, delayed hypersensitivity) response and ear swelling was used as an evaluating indicator. Changes of lymphocyte subtypes were detected by flow cytometry. RESULTS: Resveratrol (0.75-6 micromol/L) concentration-dependently promoted lymphocyte proliferation and IL-2 production induced by ConA. Sac induced IL-12 and IFN-gamma (interferon type II) production were also concentration-dependently enhanced by resveratrol, while IL-10 production was inhibited. Resveratrol (4 mg/kg, ig) promoted DTH response of mouse, which was suppressed by ethanol (16 %, w/v) consumption. Resveratrol treatment had no significant influence on lymphocyte subtypes in mice, however it could reverse the suppressive effect of ethanol both on macrophage percentage and on macrophage MHC-II molecule expression. CONCLUSION: Low dose resveratrol enhanced cell mediate immune response. Promoting Th1 cytokine production and influencing on macrophage function might be its mechanisms. PMID- 12370095 TI - Effects of astragaloside IV on myocardial calcium transport and cardiac function in ischemic rats. AB - AIM: To explore the effects of astragaloside IV (XGA) on myocardial calcium transport and cardiac function in ischemic rats. METHODS: Eighty-four Wistar rats were divided into three groups: control group (n=12); ischemic group (n=12) was given isoprenaline injection sc at a dose of 30 mg/kg; and XGA group (n=60) was given XGA after isoprenaline administration. The changes of the parameters of hemodynamics, cardiac function, and intra- and extracellular calcium concentration of the myocardial cells were determined. The dose- and time-effect relationship of XGA on myocardial calcium transport and cardiac function were observed. RESULTS: After XGA administration, there was significant improvement in cardiac function and hemodynamics in ischemic rats. The cardiac output, heart rate, stroke volume, mean aortic pressure, systolic aortic pressure, the stroke work of left ventricule, the right and left ventricle systolic pressure, and +dp/dt of the right ventricle of ischemic rats gradually recovered to the level of the control group with increasing the dose of XGA and prolongation of the action of XGA. The ionized calcium of the myocardium and the total amount of calcium of the myocardial tissue decreased markedly compared to those in ischemic group, and the activity of calcium pump of erythrocyte membrane increased significantly in comparison to that of ischemic group, but their changes had no trend of increase with increasing dose of the XGA. However, there was a gradual decrease of the ionized calcium of the myocardium with the prolongation of acting time of XGA. CONCLUSION: XGA improves the cardiac function in ischemic rats, and the reduction of excessive accumulation of intracellular calcium within myocardial cells plays an important role. PMID- 12370097 TI - Effects of anthopleurin-Q on myocardial hypertrophy in rats and physiologic properties of isolated atria in guinea pigs. AB - AIM: To study effects of anthopleurin-Q (AP-Q) on myocardial hypertrophy in rats and isolated atria in guinea pigs. METHODS: Two myocardial hypertrophy models in rats were established, one introduced by levothyroxine, the other by stenosis of abdominal aorta. Cardiac myocytes morphometry and functional experiments were employed to investigate effects of AP-Q. RESULTS: Low dose of AP-Q (1 microg/kg/d, ip) reduced morphologic changes of myocardial hypertrophy in both rat models. While high dose of AP-Q (10 microg/kg/d, ip) did not, and caused mild hydropic degeneration in cardiomyocytes. High concentration of AP-Q (30 nmol/L) enhanced the contractility, raised automaticity, and prolonged the functional refractory period (FRP) in isolated left atria of guinea pigs; higher concentration (100 nmol/L) triggered arrhythmia in right atria; low concentration of AP-Q (1 nmol/L)did not affect any myocardial properties above. CONCLUSION: Low dose of AP-Q without inotropic effect can hinder the experimental myocardial hypertrophy in rats; high dose with positive inotropic effect may be responsible for its toxic reaction. PMID- 12370096 TI - Inhibitory effects of Ginkgo biloba extract on vascular endothelial growth factor in rat aortic endothelial cells. AB - AIM: To study the protective effects of Ginkgo biloba extract (GbE) against rat aortic endothelial cells (RAEC) damage induced by lysophosphatidylcholine (LPC). METHODS: Cell injury were determined by MTT assay and LDH release. Vascular endothelial growth factor (VEGF) protein production from RAEC was determined by enzyme-linked immunosorbent assay (ELISA). VEGF mRNA expression was examined by in situ hybridization and dot blot. RESULTS: GbE 0.01-1 microg/L prevented LPC induced injury in cultured RAEC in a concentration-dependent manner. Cultured RAEC could express VEGF protein and VEGF mRNA was induced by LPC 5 mg/L. GbE could inhibit the expression of VEGF protein and VEGF mRNA in co-cultured RAEC with LPC. CONCLUSION: LPC could induce a strong expression of VEGF in RAEC. GbE could protect RAEC against the LPC-induced damage and downregulate VEGF protein and VEGF mRNA expression in cultured RAEC. PMID- 12370098 TI - Effect of magnesium lithospermate B on calcium and nitric oxide in endothelial cells upon hypoxia/reoxygenation. AB - AIM: To investigate the effect of magnesium lithospermate B (MLB) on hypoxia/ reoxygenation (H/R)-induced elevation of intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) release in endothelial cells. METHODS: The cultured human umbilical vein endothelial cells (ECV304) were exposed to hypoxia for 30 min under 95 % N2 and 5 % CO2, then reoxygenation for 30 min under air and 5 % CO2. Cell injury was evaluated by dye exclusion test, superoxide dismutase (SOD) assay, and molondialdehyde (MDA) assay. [Ca2+]i was determined by Fura 2 AM. NO content was examined by a NO assay kit. Endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) mRNA expressions were measured by semi-quantitative RT-PCR. RESULTS: Cell viability was decreased from (93.1+/ 1.2) % in normoxia to (88+/-3) % in H/R (P < 0.01), and SOD activity was also decreased from (0.24+/-0.07) kNU/L to (0.18+/-0.03) kNU/L in H/R (P >0.05), but MDA production was increased from (1.12+/-0.06) mmol/L in normoxia to (3.78+/ 0.03) mmol/L in H/R (P < 0.01) in ECV304 cultured under calcium conditions. MLB 2.5, 5, and 10 mg/L increased cell viability and SOD activity, and inhibited MDA formation in ECV304. H/R increased [Ca2+]i (F340/F380 from 1.65+/-0.16 to 1.89+/ 0.28), NO release [from (7.5+/-1.3) micromol/L to (16+/-5) micromol/L], and eNOS mRNA expression, but decreased iNOS mRNA expression in ECV304 (P <0.05). However, it did not affect them under calcium-free conditions. MLB inhibited H/R-induced increases in [Ca2+]i and eNOS mRNA expression, stimulated NO release and iNOS mRNA expression (P <0.05). CONCLUSION: MLB attenuates H/R-induced cell injury and increases NO release in ECV304. This increase of NO production is possibly associated with preventing cell injury induced by H/R in MLB-treated ECV304. PMID- 12370099 TI - Influence of nimodipine on elimination of soman in rabbit blood and distribution of [3H]soman in mice. AB - AIM: To investigate the effect of nimodipine on the elimination of soman in rabbit blood and distribution of [3H]soman in mice. METHODS: Chirasil capillary gas chromatographic analysis method with large volume injections was used to determine the concentration of C(+/-)P(-)soman in rabbit blood. [3H]soman trace method was used to study the effect of nimodipine on soman distribution in mice. RESULTS: Nimodipine (10 mg/kg, ip, 1 h pre-treated) could significantly reduce the concentration of C(+/-)P(-)soman in rabbit blood from (54+/-13) to (19+/-12) microg/L blood at 15 s after soman injection (43.2 microg/kg, iv). Nimodipine could increase clearance rate [CL(S)] from (20.8+/-1.5) to (31+/-11) mL/kg/s and reduce AUC of C(+/-)P(-)soman from (2.08+/-0.15) to (1.6+/-0.4) mg/s. Nimodipine (10 mg/kg, ip, 1 h pre-treated) treatment could significantly reduce the distribution amount of bound [3H]soman in plasma, brain, lung, and liver, moreover increased the distribution amount of bound [3H]soman in small intestine during 0-120 min after mice received [3H]soman (0.544 GBq*119 microg/kg, sc) compared to soman control group. CONCLUSION: Nimodipine might alter the distribution of soman and reduce the initial concentration of soman in rabbit blood, then accelerated the metabolic detoxication of soman. PMID- 12370100 TI - Effects of bicyclol on aflatoxin B1 metabolism and hepatotoxicity in rats. AB - AIM: To study the effect of new antihepatitis drug, bicyclol, on the metabolism and hepatotoxicity of aflatoxin B1 (AFB1) in rats. METHODS: Rats were given bicyclol 300 mg/kg/d ig for 3 d and then injected ip with AFB1 1.5 mg/kg. Liver damages were examined 16 h after ip AFB1. The in vitro metabolism of AFB1 by bicyclol-pretreated liver microsomes was investigated by HPLC assay. RESULTS: Bicyclol (300 mg/kg/d for 3 d) pretreatment provided protection against AFB1 hepatotoxicity as evidenced by the decrease of AFB1-elevated serum aminotransferase and hepatic malondialdehyde in rats. Bicyclol pretreatment slightly increased the production of the less toxic metabolite aflatoxin Q1. Bicyclol increased liver cytochrome P450 content, CYP 2B1-mediated 7 pentoxyresorufin O-dealkylase (PROD) activity, cytosolic glutathione (GSH) level, and GSH S-transferase (GST) activities. Moreover, bicyclol increased CYP 3A mediated erythromycin-demethylase and CYP 1A-mediated 7-ethoxyresorufin O deethylase (EROD) activities. CONCLUSION: Bicyclol protected rats against AFB1 hepatotoxicity by increasing the detoxifying metabolism of AFB1 in the liver. PMID- 12370101 TI - Comparison of antitumor effect of recombinant L-asparaginase with wild type one in vitro and in vivo. AB - AIM: To investigate the antitumor effect of recombinant L-asparaginase on the growth of several tumors such as P388, L1210, hepatocellular carcinoma (Heps), K562, P815, and sarcoma 180 (S180). METHODS: Tumor cells (K562, L1210, and P815) were cultured in vitro and the morphology of those cells was observed with inverse microscope and transmission electron microscope. MTT assay was performed to measure the cell proliferation and inhibition rate. DNA content was assayed by flow cytometry. According to protocols of transplant tumor research, mice were transplanted with tumor cells L1210, P388, Heps, and S180. The survival rate and weight of tumor were observed after the treatment of test drugs. RESULTS: The antitumor effects of L-asparaginase were observed in vitro with tumor cells K562, L1210, and P815 (P <0.01). In vivo experiments showed that ip administration of L asparaginase significantly increased the survival rate and life span of mice with P388 or L1210 tumor cells (P <0.01). Tumor growth induced with Heps was also significantly suppressed. Furthermore, significant suppression of tumor growth was observed in mice induced with Heps and S180 by iv administration of L asparaginase. CONCLUSION: Recombinant L-asparaginase markedly inhibited tumors tested in this study and the results strongly suggest that recombinant L asparaginase has great potential for clinical treatment of these tumors. PMID- 12370102 TI - Serine 331 is major site of phosphorylation and desensitization induced by protein kinase C in thromboxane receptor alpha. AB - AIM: To identify the specific serine/threonine residues in the C-terminal tail of thromboxane receptor alpha (TPalpha) being phosphorylated and desensitized, and various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. METHODS: To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKC, and then the cDNA of phosphorylated protein was mutagenized to localize the major site of receptor phosphorylation induced by protein kinase C. Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged wild type or mutant TPalpha were used to study the phosphorylation and desensitization. RESULTS: Only the C-terminal tail can be used as a substrate for the purified PKC. Ser-331 (mP4) was demonstrated to be heavily phosphorylated, Ser-324 (mP1) was shown to be slightly phosphorylated, Ser-329 was illustrated to be faintly phosphorylated, and other Ser/Thr residues were not found to be phosphorylated. Phorbol-12-myristate-13-acetate (PMA) induced receptor phosphorylation in HEK 293 cells expressing the wild type TPalpha. However, PMA did not significantly trigger receptor phosphorylation in HEK 293 cells expressing the S331A mutant receptor. Pretreatment of the cells expressing the wild type with PMA inhibited I-BOP induced Ca2+ release, however, pretreatment of the cells expressing the S331A mutant receptor with PMA did not abolish I-BOP induced Ca2+ release. CONCLUSION: Ser-331 is the major and crucial site of receptor phosphorylation and desensitization. PMID- 12370103 TI - Insights into obesity and insulin resistance from the study of extreme human phenotypes. AB - The detailed study of rare, extreme human phenotypes has a long and distinguished history in endocrinology. Such individuals have often acted as 'experiments of nature' providing important novel information regarding endocrine physiology and mechanistic insights relevant to the study of more common endocrine disorders. This review presents a personal experience of the study of two such extreme phenotypes, obesity and severe insulin resistance. PMID- 12370104 TI - Thyroid dysfunction during pregnancy and in the first postpartum year in women with diabetes mellitus type 1. AB - BACKGROUND: The prevalence of thyroid dysfunction in pregnancy and in the first postpartum year (postpartum thyroid dysfunction (PPTD)) in women with diabetes mellitus type 1 (DM1) is known to be higher than in the general population. To assess prevalence, incidence and risk factors in The Netherlands we performed a prospective cohort study. DESIGN: From 1994 to 1998, 126 women with DM1 from eight Dutch clinics were included. TSH, free thyroxine, free tri-iodothyronine and anti-thyroid peroxidase antibodies (TPO-ab) were measured pre-pregnancy, in the first and last trimester of pregnancy and at 1.5, 3, 6, 9 and 12 months after delivery. RESULTS: Eighty-two women completed the study. Thyroid dysfunction during pregnancy was observed in 22.5% (first trimester) and 18.4% (third trimester), and mostly consisted of subclinical hypothyroidism. Baseline characteristics of women with thyroid dysfunction in pregnancy did not differ from those without thyroid dysfunction. Overt PPTD was seen in 15.9%. Incidence of PPTD was 10%. Patients with PPTD were slightly older than those without PPTD and the prevalence of TPO-ab was higher in these women. CONCLUSION: In women with DM1 the prevalence of thyroid dysfunction during pregnancy and overt PPTD is 3 fold higher than in the general Dutch population. Risk factors are age and TPO ab. Given the possible impact on psychomotor development of the offspring and on well-being of the mother these data suggest there is a case for screening (pre )pregnant women with DM1 for TSH and TPO-ab. PMID- 12370105 TI - Increased intima-media thickness of the carotid artery wall, normal blood pressure profile and normal left ventricular mass in subjects with primary hyperparathyroidism. AB - OBJECTIVE: Despite the increasing evidence that primary hyperparathyroidism (PHPT) contributes to greater risk of cardiovascular morbidity and mortality, its exact role in the development of cardiovascular changes and its clinical significance are still controversial. Given the multiple influence of PHPT on the cardiovascular system, this study aimed to assess the effects of PHPT on blood pressure profile, and on features of the heart and arterial vessels in normotensive symptomless patients. DESIGN: Twenty patients (8 males and 12 females) with a median age of 51.5 years (range 44 to 65 years) were evaluated and the results were compared with those of 20 controls matched for age, gender and body mass index. Patients' parathyroid hormone levels ranged from 172 to 454 pg/ml and Ca levels ranged from 11.4 to 13.5 mg/dl. Fasting levels of glucose, insulin, total and high density lipoprotein cholesterol and triglycerides were within the normal range in all subjects recruited. METHODS: Twenty-four-hour blood pressure profile, left ventricle (LV) dimension and carotid artery anatomy were investigated, the latter two by ultrasonography. RESULTS: No difference was found between the patients and controls in blood pressure profile, when the following parameters were considered: supine systolic/diastolic pressure, average 24-h systolic, diastolic and mean arterial pressure, day-time mean arterial pressure and fall in nocturnal blood pressure (-17% and -18% respectively). Heart rate and all parameters of LV mass were similar in patients and controls. The only alteration found in patients was in significantly greater carotid intimal medial thickness (IMT) (P<0.001). Atherosclerotic plaques were more frequent in patients than in controls, with a difference reaching a trend (40% vs 10%, chi(2)=4.8; P=0.091). Considering that the carotid IMT is considered to be a marker of systemic atherosclerosis, our finding suggests early atherosclerotic changes in PHPT. No correlation was found between the severity and cardiovascular manifestation of PHPT. CONCLUSIONS: Vascular changes may occur due to a combination of structural and functional impairments in PHPT patients, likely as a result of altered calcium metabolism and impaired equilibrium of other factors regulating vascular function. Both extent and duration of PHPT can play a relative role in the development of cardiovascular complications. Considering that PHPT is now recognized as a quite common and often symptomless endocrine disorder, the evidence of cardiovascular manifestation in normotensive patients, found by this morphological study, suggests a possible implication for the management of such patients. In this light, screening for abnormalities in cardiovascular system function should be recommended in all PHPT subjects. PMID- 12370106 TI - Effects of long-lasting raloxifene treatment on serum prolactin and gonadotropin levels in postmenopausal women. AB - OBJECTIVE: To evaluate the effects of a 6 month administration of raloxifene hydrochloride, a selective estrogen receptor modulator which was recently approved for the prevention of osteoporosis, on serum gonadotropin and prolactin (PRL) levels and on TRH-stimulated PRL responsiveness in postmenopausal women who have not undergone estrogen replacement therapy. DESIGN AND METHODS: Sixteen healthy postmenopausal women were divided into two groups on the basis of their bone status, evaluated by dual energy X-ray absorptiometry at the lumbar level. Eight women (chronological age 52.4+/-4.1 (s.d.) years, menopausal age 42.4+/-3.9 years), in whom T-score L2-L4 was less than -2.5 s.d., were treated with raloxifene (60 mg p.o.) administered daily for 6 months (group 1), while the other eight women (chronological age 52.6+/-2.5 years, menopausal age 42.1+/-3.6 years), in whom the T-score L2-L4 ranged between -1 and -2.5 s.d., were used as a control group (group 2). Serum PRL, FSH, LH and 17beta-estradiol (E2) levels were evaluated at baseline and after 3 and 6 months of treatment. In all subjects, PRL responsiveness to TRH (200 microg i.v.) administration was evaluated at baseline and at the end of the study. RESULTS: At baseline, mean PRL, LH and FSH levels were not significantly different in the two groups (PRL 133.6+/-21.7 vs 136.7+/ 28.1 mIU/l (NS), LH 25.1+/-6.8 vs 24.4+/-6.7 mIU/ml (NS), FSH 74.4+/-25.0 vs 71.1+/-24.1 mIU/ml (NS), in group 1 and group 2 respectively). No significant variations in serum FSH and LH values, in either group, or in serum PRL levels in group 2, were observed at the 3 and 6 month examinations. On the contrary, serum PRL values decreased significantly in group 1 after 3 months (100.1+/-47.7 mIU/l, P<0.05) and 6 months (81.5+/-30.2 mIU/l, P<0.001). At baseline, no significant differences were observed in the TRH-stimulated serum PRL peak between the groups (1015.4+/-30.5 vs 1030.2+/-25.7 mIU/l in group 1 and in group 2 respectively), while it decreased significantly at the 6 month examination in group 1 (770.5+/ 47.4 mIU/l, P<0.001) and it was significantly lower than in group 2 (1068.1+/ 301.8 mIU/l, P=0.02). Serum E2 was not detected at baseline and at each examination, in all patients. CONCLUSIONS: The decrease of PRL values induced by long-term raloxifene administration in postmenopausal women could be explained by a direct antiestrogenic effect of raloxifene on lactotrope cells or by the recently suggested increase of opiatergic tone on the hypothalamic-pituitary region. PMID- 12370107 TI - Comparison of high-dose finasteride (5 mg/day) versus low-dose finasteride (2.5 mg/day) in the treatment of hirsutism. AB - OBJECTIVE: To compare the clinical efficacy and safety of high-dose (5 mg/day) and low-dose (2.5 mg/day) finasteride in the treatment of hirsutism in women. DESIGN: A prospective, randomized and controlled clinical trial. METHODS: Fifty six hirsute women with moderate to severe hirsutism were prospectively evaluated to see the effects of low-dose (2.5 mg/day) and high-dose (5 mg/day) finasteride. Patients were randomly divided into two treatment groups. Group I (n=29) received 2.5 mg finasteride/day and group II (n=27) received 5 mg finasteride/day orally for 1 year. Hirsutism score, body mass index and hormonal parameters (FSH, LH, estradiol, androstenedione, testosterone, free testosterone, 17alpha hydroxyprogesterone, dehydroepiandrosterone sulfate and sex hormone-binding globulin) were measured in all the patients before treatment and repeated at six monthly intervals. RESULTS: The hirsutism scores decreased significantly at months 6 and 12 from a mean+/-s.d. of 18.4+/-4.6 to 13.3+/-5.2 (P<0.001) and 18.4+/-4.6 to 8.6+/-4.2 (P<0.001) in group I and from 18.7+/-5.2 to 13.9+/-5.3 (P<0.001) and 18.7+/-5.2 to 10.3+/-5.0 (P<0.001) in group II respectively. No significant changes in the blood chemistry and hormonal parameters except estradiol levels were observed. No serious side-effects were seen in the two groups. In group II, estradiol levels increased significantly at 6 and 12 months. CONCLUSIONS: In this study, hirsutism scores decreased significantly at 6 and 12 months in both groups I and II. Low-dose (2.5 mg/day) finasteride is safe and cost effective in the treatment of hirsutism and may be used instead of high-dose finasteride (5 mg/day) therapy. PMID- 12370108 TI - A comparison between the effects of low dose (1 microg) and standard dose (250 microg) ACTH stimulation tests on adrenal P450c17alpha enzyme activity in women with polycystic ovary syndrome. AB - OBJECTIVE: Numerous studies have found elevated androgen production by the adrenal glands in patients with polycystic ovary syndrome (PCOS). However, the role and the mechanisms responsible for the adrenal androgen excess in women with PCOS are not well understood. DESIGN: Our aim was to compare 17 hydroxyprogesterone (17-OHP), androstenedione, dehydroepiandrosterone sulfate (DHEAS) and cortisol responses to a low dose (1 microg) ACTH stimulation test (LDT) with the responses to a standard dose (250 microg) ACTH stimulation test (SDT) in patients with PCOS. METHODS: Fifty women with PCOS (mean age 25.4+/-0.7 years) and 20 healthy women (mean age 27.3+/-2.2 years) were included in the study. The patients and controls underwent ACTH stimulation tests with 1 microg and 250 microg synthetic ACTH in the follicular phase of their cycles. Venous blood was drawn at 0, 30 and 60 min for determination of serum cortisol, 17-OHP, androstenedione and DHEAS levels. RESULTS: In PCOS subjects, peak and area under the curve (AUC) 17-OHP (9.3+/-0.3 nmol/l, 378.4+/-61 nmol/lx60 min), androstenedione (15.6+/-0.6 nmol/l, 806.4+/-52 nmol/lx60 min) and DHEAS (7.5+/ 0.4 micromol/l, 385.6+/-25.5 micromol/lx60 min) responses to SDT were significantly higher than the levels in healthy women (respectively 5.7+/-0.3 nmol/l and 249.4+/-52.2 nmol/lx60 min for 17-OHP; 9.1+/-0.3 nmol/l and 413.7+/ 31.6 nmol/lx60 min for androstenedione; 4.3+/-0.4 micromol/l and 224.9+/-24.5 micromol/lx60 min for DHEAS) (P<0.05). Peak and AUC cortisol responses to SDT were similar in PCOS and control subjects. Peak and AUC cortisol and 17-OHP responses to LDT in women with PCOS were similar to the values obtained in healthy women. Peak androstenedione (12.5+/-0.6 nmol/l) and peak (6.5+/-0.5 nmol/l) and AUC (336.3+/-22.4 micromol/lx60 min) DHEAS responses to LDT were significantly higher in women with PCOS. CONCLUSIONS: These results show that LDT is capable of revealing the adrenal hyperactivity in women with PCOS. Adrenal P450c17alpha enzyme dysregulation in PCOS is revealed by ACTH stimulation at a pharmacological dose (250 microg) but not by a physiological dose (1 microg). LDT is able to demonstrate adrenal hyperactivity characterized by an increase in DHEAS levels. PMID- 12370109 TI - Impact of constitutional genetic variation in androgen/oestrogen-regulating genes on age-related changes in human prostate. AB - OBJECTIVE: Benign prostatic hyperplasia (BPH) is the most common benign tumour in ageing men. While the etiopathology remains unsolved, a disruption in the endocrine/autocrine-paracrine prostatic homeostasis, involving steroid hormones, contributes to the pathogenesis of BPH. DNA polymorphisms in genes involved in hormone synthesis, signalling and metabolism may, therefore, be responsible for these changes. We have evaluated the correlation between specific genotypes in androgen- and oestrogen-regulating genes (AR, SRD5A2, CYP17 and CYP19), and age related prostatic changes. METHODS: We have tested genetic susceptibility to morphological and pathological criteria in 195 French Caucasians, using allelic variants for candidate genes involved in androgen/oestrogen prostatic activity: androgen receptor (CAG repeats), 5alpha-reductase type 2 (TA repeats, V89L and A49T mutations), A2 variant of the 17alpha-hydroxylase (CYP17) and the simple tandem repeat polymorphism (STRP) aromatase (CYP19) polymorphisms. RESULTS: The A2 variant of 17alpha-hydroxylase (CYP17) and allele 191 of STRP aromatase (CYP19) showed an opposite effect on age-related prostate hyperplasia: CYP17 being associated with increased risk of prostate enlargement and CYP19 with reduced risk. The 5alpha-reductase type II variants studied did not show links with prostate hyperplasia. The androgen receptor gene CAG repeat length showed a low correlation with the increase of prostate weight, suggesting some effect on age-related prostate growth. CONCLUSION: These results suggested that common variants of the CYP17 gene are associated with prostate enlargement and therefore may increase the risk of development of BPH in this population, while infrequent variants of the aromatase gene (CYP19) could be of a protective nature. PMID- 12370110 TI - Molecular defects of the CYP21 gene in Spanish girls with isolated precocious pubarche. AB - OBJECTIVE: To determine the frequency of mutant alleles in the CYP21 gene in Spanish girls presenting with precocious pubarche (PP) and to assess the relationships between genotype and endocrine-metabolic variables. DESIGN: Fifty three unrelated girls with a history of PP (14 prepubertal, 8 pubertal and 31 postmenarcheal) and 35 controls were studied. METHODS: Genomic DNA was extracted from peripheral blood leukocytes. After selection against the pseudogen, an allele-specific PCR was used to identify 14 known mutations in the CYP21 gene. The mutations studied were Pro30Leu, splice intron 2, Ilel72Asn, Cluster E(6), Glyl92Ser, Ins T, GT-CT, Gln318-stop, Arg357Trp, Trp406-stop, Pro453Ser, Arg483Pro, Arg483 frameshift and Val281Leu. A standard 2-h oral glucose tolerance test was performed in all PP girls. Ovarian 17-hydroxyprogesterone (17-OHP) responses to gonadotrophin-releasing hormone-agonist stimulation was assessed in postmenarcheal PP girls. RESULTS: Thirteen PP girls and eight control girls were heterozygous for one of the mutations studied. The frequency of the carrier status was 25% and 23% in the PP and control groups respectively. Severe mutations were found in 33% of the carrier girls. Serum 17-OHP responses to ACTH stimulation were similar in carriers and non-carriers (351+/-65 vs 334+/-22 ng/dl). The presence of ovarian hyperandrogenism and/or hyperinsulinism was also not related to the carrier status. CONCLUSION: The incidence of molecular defects in the CYP21 gene in the present study was comparable in the PP and control groups. We found no relationship between the presence of carrier status and endocrine-metabolic abnormalities. Prospective studies of larger cohorts of PP girls are needed to ascertain the long-term clinical relevance of CYP21 heterozygosity. PMID- 12370111 TI - Long-term follow-up study of patients with adrenal incidentalomas. AB - BACKGROUND: The incidence of adrenal incidentalomas has sharply increased in recent decades and concurrent subtle endocrine abnormalities, or even subclinical conditions, have been identified. Nonetheless, data concerning possible changes in adrenal size and/or hormonal pattern during follow-up are still inadequate. OBJECTIVE: To evaluate long-term morphological and functional evolution of adrenal incidentalomas after initial diagnosis and to identify possible risk factors for hormonal hyperactivity and mass enlargement. PATIENTS: Sixty-four patients (34-79 years) were followed-up for 12-120 months (median 25.5 months). Initial computerized tomography scan showed a unilateral mass in 51 patients and bilateral lesions in 13 patients. Average mass diameter at diagnosis was 2.5+/ 0.1 cm (range 1.0-4.0). Twelve patients had subclinical Cushing's syndrome, 41 had mild hormonal alterations, and 11 had normal adrenal function at baseline. All patients were investigated by morphological and functional evaluation 6 and 12 months after diagnosis, and then at 1-year intervals. RESULTS: During follow up, a mass size increase >/=1 cm was observed in 13 patients, and 18 developed further subtle endocrine alterations. Cumulative risk of developing endocrine abnormalities was 17% at 1 year, 29% at 2 years, and 47% at 5 years. The risk was higher in the first 2 years of follow-up if the initial tumor diameter was >or=3 cm. Overall, cumulative risk of mass enlargement was 6% at 1 year, 14% at 2 years, and 29% at 5 years, and it was greater in patients with normal adrenal function than in those with subtle hormonal abnormalities (P<0.05). One female subject showed a mass enlargement after 6 months of follow-up and was eventually diagnosed with non-Hodgkin's lymphoma. CONCLUSIONS: Patients with an adrenal incidentaloma are at risk for tumor growth and development of hormonal alterations. The risk of adrenal malignancy, although not elevated, also indicates the need for long-term follow-up. PMID- 12370112 TI - Effect of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor gamma-2 gene on adiposity, insulin sensitivity and lipid profile in the Spanish population. AB - OBJECTIVE: To investigate the role of the Pro12Ala peroxisome proliferator activated receptor (PPAR) gamma-2 polymorphism in the susceptibility to the insulin resistance syndrome and its metabolic complications in a population-based nationwide multicenter study in Spain. DESIGN: 464 unrelated adults (45.3% men and 54.7% women) aged between 35 and 64 years were randomly chosen from a nationwide population-based survey of obesity and related conditions including insulin resistance and cardiovascular risk factors. METHODS: Anthropometric determinations included: body mass index (BMI), waist-to-hip ratio, sagittal abdominal diameter; biochemical determinations included: fasting plasma glucose concentration and concentration 2 h after an oral glucose tolerance test (OGTT), total cholesterol, high and low density lipoprotein-cholesterol, triglycerides, leptin and insulin. Systolic and diastolic blood pressure were also measured. Genotyping of the PPARgamma-2 Pro12Ala polymorphism was determined by polymerase chain reaction and single strand conformation polymorphism analysis. RESULTS: The Ala12 allele frequency was higher in obese men than in lean men (0.15 vs 0.08, P=0.03). Men carriers of the Ala12 allele had a higher BMI than non-carriers (38.9% vs 21.3%; adjusted odds ratio 2.36, 95% confidence interval 1.10-5.05, P=0.03). However, despite higher BMI obese men carriers of the Ala12 allele had lower sagittal abdominal diameter than Pro12 homozygotes (24.1+/-3.2 vs 26.3+/ 2.5 cm, P=0.01). The Ala12 allele was associated with lower total triglycerides levels in the overall population and it was also associated with lower fasting insulin levels and a higher insulin sensitivity by homeostasis model assessment (HOMA) in women. CONCLUSIONS: Our results suggest that the Pro12Ala polymorphism of the PPARgamma-2 gene promotes peripheral deposition of adipose tissue and increased insulin sensitivity for a given BMI. The results in women might be due to their different adipose tissue distribution. PMID- 12370113 TI - Apolipoprotein E gene determines serum testosterone and dehydroepiandrosterone levels in postmenopausal women. AB - OBJECTIVE: Apolipoprotein E (ApoE) is believed to play an important role in lipid metabolism and has been found to be related to diseases associated with ageing, the important characteristic of which is decline in circulating sex steroids, including androgen. DESIGN: To find the relationships of levels of serum testosterone and its precursor, dehydroepiandrosterone (DHEA), to ApoE polymorphism in 113 postmenopausal Caucasian women. METHODS: The ApoE genotype was assessed by polymerase chain reaction and CfoI endonuclease digestion. ApoE genotype distribution was as follows: E2/3, 15%; E3/3, 71.7%; E2/4, 1.8%; E3/4, 10.6; and E4/4, 0.89%. The differences in serum androgen levels between genotypes were evaluated by ANCOVA and least significant difference (LSD) multiple comparisons test after adjustment for body mass index, age and/or years since menopause. RESULTS: Significant intergroup differences between the most frequent allele combination (2/3, 3/3 and 3/4) in serum DHEA levels were found (P<0.05, ANCOVA). DHEA levels were higher in women with the E3/4 allele combination than in the E3/3 genotype (P<0.01, LSD multiple comparisons). In serum testosterone levels, borderline intergroup differences were found (P<0.07, ANCOVA). Higher testosterone levels were found in the E3/4 allele combination as compared with E3/3 (P<0.05, LSD multiple comparisons). Dose effect of E4 allele analysis indicated higher serum DHEA and testosterone levels in women with the E4 allele present than in women with the E4 allele absent (P<0.003 for DHEA, P<0.007 for testosterone, ANCOVA). CONCLUSIONS: Circulating testosterone and DHEA are associated with the ApoE genotype, which may render women carrying the allele E4 more susceptible to the development of some diseases associated with ageing and menopause [corrected]. PMID- 12370114 TI - Chromosome 22q in pancreatic endocrine tumors: identification of a homozygous deletion and potential prognostic associations of allelic deletions. AB - OBJECTIVE: A variety of human tumors frequently show allelic deletions of chromosome 22q, suggesting that inactivation of one or more tumor suppressor genes in this region is important for their tumorigenesis. METHODS: In this study, 23 patients with pancreatic endocrine tumors (PETs), including gastrinomas, VIPomas and non-functioning islet cell carcinomas, were analyzed for loss of heterozygosity (LOH) on chromosome 22q with 12 microsatellite and 7 sequence tagged site markers. RESULTS: LOH on chromosome 22q was identified in 22 of 23 (96%) PETs. Markers in the chromosomal region 22q12.1 revealed LOH rates up to 85%. Notably, one tumor revealed a homozygous deletion in a second region at 22q12.3. LOH at this locus occurred more frequently in tumors with distant metastases (10 of 11) compared with tumors without distant metastases (3 of 12; P=0.0057) and, overall, allelic loss of 22q is positively correlated with distant metastases (r=0.78; P<0.0001). CONCLUSIONS: These findings are suggestive for novel tumor suppressor gene loci at chromosome 22q that might contribute to the pathogenesis of PETs, especially to the development of distant metastases. PMID- 12370115 TI - Role of galectin-3 immunodetection in the cytological diagnosis of thyroid cystic papillary carcinoma. AB - OBJECTIVE: Cystic thyroid lesions can harbour an occult papillary carcinoma, which fine needle aspiration (FNA) biopsy may fail to detect. Recently, new markers such as galectin-3 lectin have been proposed to distinguish benign from malignant thyroid lesions of follicular origin. The aim of this study was to assess the role of galectin-3 immunodetection in a series of FNA cytological samples of benign and malignant thyroid cystic nodules. METHODS: We retrospectively analysed galectin-3 expression by immunoperoxidase staining on 32 cytological paraffin-embedded samples of cystic papillary carcinoma and on 12 samples of benign cysts, both obtained by FNA biopsy. Specificity, sensitivity, positive/negative predictive values, and accuracy of standard cytological examination and galectin-3 immunodetection were assessed. RESULTS: Among cystic papillary carcinomas, 29 of 32 samples were galectin-3 positive, whereas standard FNA cytology made a correct diagnosis in only 25 of 32 samples. All the benign cysts were negative for galectin-3. In comparing the sensitivity and specificity of the two methods, it appeared that both had a 100% specificity, whereas the sensitivity of cytological examination alone was 75% versus 89.3% obtained by galectin-3 immunohistochemistry. CONCLUSIONS: Galectin-3 immunostaining represents a valid pre-operative adjunct to pick up malignant cells in those cases where a very poor number of epithelial cells may lead to a cytological misdiagnosis. Therefore, we suggest that in poorly cellular FNA biopsies of simple or complex thyroid cysts, galectin-3 expression by epithelial cells is consistent with a cystic carcinoma and supports surgical treatment indication. PMID- 12370116 TI - GH and IGF-I regulate the expression of endothelial nitric oxide synthase (eNOS) in cardiovascular tissues of hypophysectomized female rats. AB - OBJECTIVE: This study explored whether short-term replacement therapy with growth hormone (GH) affects blood pressure (BP), heart rate (HR) and endothelial nitric oxide synthase (eNOS) expression in cardiovascular tissues in hypophysectomized (Hx) female rats. DESIGN AND METHODS: BP, HR and the expression of eNOS in the aorta, caval vein and heart were studied in Hx female rats and in Hx female rats that underwent 7 days treatment with GH and thyroxine+glucocorticoids ([T(4)+GC]). Insulin-like growth factor-I (IGF-I) was included in a second experimental protocol to explore the indirect effect of GH. The expression and localisation of eNOS was analysed by immunoblotting and immunohistochemistry. RESULTS: Decreased BP (Hx 98+/-1, Intact 129+/-3 mmHg, P<0.05), HR (Hx 297+/-14, Intact 399+/-31 beats/min, P<0.05) and unchanged eNOS expression was demonstrated in Hx compared with intact rats. None of the hormones affected BP, but both GH and IGF-I increased HR compared with Hx rats (GH 358+/-10, IGF-I 337+/-7, Hx 306+/-11 beats/min, P<0.05). Replacement of GH, GH+[T(4)+GC] and IGF-I resulted in an increased aortic eNOS expression (GH 161+/-24, GH+[T(4)+GC] 177+/-25, IGF-I 153+/-21, Hx 109+/-7%, P<0.05), whereas in caval vein only GH+[T(4)+GC] affected eNOS expression. None of the hormones changed the level of eNOS in the heart. eNOS was localised in the intima layer of the aorta, whereas in the caval vein eNOS was localised in all cell layers. CONCLUSIONS: These findings support the suggested positive role of GH in the regulation of the cardiovascular homeostasis. The observed up-regulation of eNOS, and presumably an increased NO bioavailability, may result in improved endothelial and cardiovascular function. PMID- 12370117 TI - Growth hormone protects against radiotherapy-induced cell death. AB - BACKGROUND: In vivo treatment with growth hormone reduces radiation-associated mortality. The molecular mechanisms underlying this effect are unknown. It has been described that increased sensitivity to ionising radiation can be due to defects in machinery involved in detection and/or repair of DNA double-strand breaks. OBJECTIVE: To study the mechanisms involved in growth hormone action on the increased survival in irradiated cells. MATERIALS AND METHODS: CHO-4 cells stably expressing the growth hormone receptor were used. A cell viability assay was carried out to analyse the increase in survival induced by growth hormone in irradiated cells. To investigate whether the DNA repair mechanism could be implicated in this effect we performed DNA reactivation assays using pHIV-LUC and pCMV-betagal plasmids as control. Identical studies were also conducted using the radiomimetic drug, bleomycin. RESULTS: Growth hormone protects CHO-4 cells from bleomycin- and radiation-induced cell death. In pHIV-LUC transfected cells, a time-dependent decrease in luciferase activity was observed after irradiation in the absence of growth hormone. However, cells pretreated with this hormone maintained reporter activity. When cells were transfected with irradiated pHIV LUC plasmid, only the hormone-treated cells recovered the transcriptional activity. CONCLUSIONS: Growth hormone exerts a radioprotective effect in CHO-4 cells stably transfected with the complementary DNA for the rat growth hormone receptor. The radioprotection is triggered directly by the hormone and it is also observed with bleomycin. The increased survival in response to radiation and bleomycin treatment induced by growth hormone correlates with an enhanced ability of the cells to repair damaged DNA. PMID- 12370118 TI - Efficacy of 1400 W, a novel inhibitor of inducible nitric oxide synthase, in preventing interleukin-1beta-induced suppression of pancreatic islet function in vitro and multiple low-dose streptozotocin-induced diabetes in vivo. AB - OBJECTIVE: Nitric oxide (NO), generated by inducible nitric oxide synthase (iNOS), has been implicated in beta-cell destruction in type 1 diabetes. In the present study, we tested a highly selective iNOS inhibitor, 1400 W, against interleukin-1beta (IL-1beta) induced suppression of rat pancreatic islets, and investigated whether 1400 W could prevent multiple low-dose streptozotocin (MLDS) induced diabetes in mice. Furthermore, we studied if 1400 W affected lipopolysaccharide (LPS) induced increase in plasma nitrite+nitrate (NO(x)) in mice. DESIGN AND METHODS: Precultured rat pancreatic islets were exposed for 48 h to 0, 1, 10 or 50 micromol/l 1400 W in the presence or absence of 25 U/ml IL 1beta, whereupon islet functions were analyzed. MLDS-treated mice were given 5.9 mg/kg body weight of 1400 W intraperitoneally daily or 14 mg/kg body weight twice a day. Blood glucose was monitored and degree of pancreatic mononuclear infiltration was determined. Mice previously injected intraperitoneally with LPS (500 microg) were given 1400 W (14 mg/kg body weight) intraperitoneally and plasma NO(x) was determined after 3, 6 and 10 h. RESULTS: The inhibitor alone did not affect islet functions. 1400 W (50 micromol/l) fully counteracted both the suppression of glucose oxidation rate, (pro)insulin biosynthesis and nitrite accumulation caused by IL-1beta. Cytokine-induced decrease in medium insulin accumulation and glucose-stimulated insulin release was partly counteracted by 1400 W, suggesting that inhibition of insulin release was partially NO independent. LPS-induced increase in plasma NO(x) was markedly inhibited for up to 10 h after 1400 W administration. Irrespective of 1400 W treatment, animals treated with MLDS developed hyperglycemia and pancreatic insulitis. CONCLUSIONS: 1400 W counteracted IL-1beta-induced suppression of rat islets in vitro and LPS induction of NO(x) in vivo, however, it failed to protect against MLDS diabetes in vivo. The latter might be due to a failure by 1400 W in vivo to inhibit NO formation at the level of the pancreatic islet. PMID- 12370119 TI - Negative regulation of adipose-expressed galectin-12 by isoproterenol, tumor necrosis factor alpha, insulin and dexamethasone. AB - OBJECTIVE: Galectin-12 has recently been shown to be a predominantly adipocyte expressed protein which is stimulated by insulin-sensitizing thiazolidinediones and possesses apoptosis-inducing activity. METHODS: To further clarify galectin 12 regulation and its potential involvement in the development of insulin resistance, 3T3-L1 adipocytes were chronically treated with various hormones known to impair insulin sensitivity, and galectin-12 mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Treatment of 3T3-L1 cells for 16 h with 10 micromol/l isoproterenol, 100 nmol/l insulin, 0.6 nmol/l tumor necrosis factor alpha (TNFalpha), and 100 nmol/l dexamethasone reduced galectin-12 gene expression between 47% and 85%. These negative effects were dose-dependent with significant inhibition detectable at concentrations as low as 10 nmol/l isoproterenol, 0.06 nmol/l TNFalpha, and 1 nmol/l dexamethasone. Furthermore, the inhibitory effect of isoproterenol could be almost completely reversed by pretreatment with the beta-adrenergic antagonist propranolol and mimicked by stimulation of G(S)-proteins with cholera toxin or by activation of adenylyl cyclase with forskolin and dibutyryl-cAMP. CONCLUSIONS: Our results suggest that galectin-12 is an adipocyte-expressed protein which is downregulated by various insulin resistance-inducing hormones. These findings imply a role for galectin-12 in the pathogenesis of insulin resistance. PMID- 12370120 TI - Development of cDNA microarray for expression profiling of estrogen-responsive genes. AB - Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed. PMID- 12370121 TI - The murine gene encoding parathyroid hormone: genomic organization, nucleotide sequence and transcriptional regulation. AB - The type 1 parathyroid hormone receptor (PTHR1) binds, with equal affinity, two ligands with distinct biological functions: PTH, the major peptide hormone controlling calcium homeostasis, and the paracrine factor, PTH-related peptide (PTHrP), a local regulator of cellular proliferation and differentiation. To clarify the complexity of possible interactions between two distinct ligands, PTH and PTHrP, and their common receptor in the intact organism, and to identify as yet unrecognized roles for PTH in normal physiology, we have cloned and characterized the structural organization, nucleotide sequence and transcriptional regulation of the murine gene encoding PTH. One recombinant clone isolated from a mouse genomic library contained 14 kb of DNA, encompassing the entire Pth gene. The transcriptional unit spans 3.2 kb of genomic DNA and, analogous to the human PTH gene, it is interrupted by two introns. The deduced mRNA encodes the 115-amino acid precursor, preproPTH. Comparison of the murine preproPTH sequence with other mammalian forms of the protein shows it to be highly conserved and to share limited structural similarity to PTHrP at the amino terminal region, a domain critical for binding and activation of their common receptor. Putative binding motifs for the transcription factors sex-determining region Y gene product, transcriptional repressor CDP, hepatic nuclear factor 3beta, GATA-binding factor 1, glucocorticoid receptor, SRY-related high mobility group box protein 5 and cAMP response element binding protein were identified in the 5' flanking region of the Pth gene. When placed upstream of a reporter gene, these sequences failed to confer transcriptional regulation in response to 1,25(OH)(2) vitamin D(3), but responded positively to the addition of isoproterenol and forskolin. Mutational analysis identified a cAMP-response element in the Pth promoter. PMID- 12370122 TI - Identification and characterization of a human cDNA and gene encoding a ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein. AB - Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a human cDNA and gene encoding a ubiquitously expressed G6Pase catalytic subunit related protein (UGRP). The ORF of this UGRP cDNA encodes a protein (346 amino acids (aa); M(r) 38 709) which shares 36% overall identity to the human G6Pase catalytic subunit (357 aa; M(r) 40 487). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis. UGRP mRNA was detected by RNA blot analysis in every tissue examined with the highest expression in muscle. Database analysis showed that the human UGRP gene is composed of six exons, spans approximately 5.4 kbp of genomic DNA and is located on chromosome 17q21 with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined. PMID- 12370123 TI - A COUP-TF/Svp homolog is highly expressed during vitellogenesis in the mosquito Aedes aegypti. AB - In the mosquito Aedes aegypti, vitellogenesis is activated via an ecdysteroid hormonal cascade initiated by a blood meal. The functional ecdysone receptor is a heterodimer composed of the ecdysone receptor (EcR) and ultraspiracle, the homolog of the retinoid X receptor. The precise tuning of this hormonal response requires participation of both positive and negative transcriptional regulators. In Drosophila, Svp, a homolog of chicken ovalbumin upstream promoter transcription factor (COUP-TF), inhibits ecdysone receptor complex-mediated transactivation in vitro and in vivo. Here we report the cloning and characterization of the Svp homolog in mosquito Aedes aegypti, AaSvp. It possesses a high degree of amino acid sequence similarity to the members of the COUP-TF/Svp subfamily. AaSvp transcripts and protein are present in the fat body at high levels from the state of arrest to about 60 h post blood meal. AaSvp binds strongly to a variety of direct repeats of the sequence AGGTCA, but weakly to inverted repeats such as hsp27 EcRE. Transient transfection assays in Drosophila S2 cells showed that AaSvp was able to repress 20-hydroxyecdysone (20E)-dependent transactivation mediated by the mosquito ecdysteroid receptor complex. These data suggest that AaSvp negatively regulates the 20E signaling in the fat body during mosquito vitellogenesis. PMID- 12370124 TI - Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens. AB - Phytohormones and chemical compounds revealing estrogenic effects are of increasing interest for their possible influence on the physiology of the reproductive tract. The gap junction connexin (Cx) genes Cx26 and Cx43, the plasma glycoprotein clusterin gene and the complement C3 gene are highly regulated by estrogen in rat endometrium. To test the value of these genes as markers for estrogenic responsiveness we analyzed the effects of estradiol, diethylstilbestrol, the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen, the phytoestrogens genistein and daidzein, and the industrial compounds DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane) and polychlorinated biphenyl (PCB) on the transcription of these genes in rat endometrium in vivo. Enhancement of Cx26 and decrease of clusterin transcripts expression by estradiol was observed at 0.03 micro g/250 g body weight (BW), and induction of C3 expression was observed at 0.05 micro g/250 g BW. A comparable effect was obtained by a tenfold higher concentration of diethylstilbestrol. Tamoxifen had a regulatory effect on this set of genes at about a 300-fold higher concentration, while raloxifen revealed much weaker estrogenic activity. No effect on Cx43 transcripts was observed with any of the compounds at the concentrations used. An effect of genistein was observed only on Cx26 expression, while PCB decreased clusterin transcripts. These results show that Cx26, C3 and clusterin reveal a comparable sensitivity to estrogens and SERMs. With respect to the phytoestrogen genistein, however, Cx26 seems to be the most sensitive gene. The analysis of clusters of estrogen-sensitive endometrial genes could help to identify estrogenic substances, assess their potency, and elucidate their mechanism of action. PMID- 12370125 TI - Human alpha 2A-adrenergic receptor gene expressed in transgenic mouse adipose tissue under the control of its regulatory elements. AB - Catecholamines regulate white adipose tissue function and development by acting through beta- and alpha2-adrenergic receptors (ARs). Human adipocytes express mainly alpha 2A- but few or no beta 3-ARs while the reverse is true for rodent adipocytes. Our aim was to generate a mouse model with a human-like alpha2/beta adrenergic balance in adipose tissue by creating transgenic mice harbouring the human alpha 2A-AR gene under the control of its own regulatory elements in a combined mouse beta 3-AR-/- and human beta 3-AR+/+ background. Transgenic mice exhibit functional human alpha 2A-ARs only in white fat cells. Interestingly, as in humans, subcutaneous adipocytes expressed higher levels of alpha2-AR than perigonadal fat cells, which are associated with a better antilipolytic response to epinephrine. High-fat-diet-induced obesity was observed in transgenic mice in the absence of fat cell size modifications. In addition, analysis of gene expression related to lipid metabolism in isolated adipocytes suggested reduced lipid mobilization and no changes in lipid storage capacity of transgenic mice fed a high-fat diet. Finally, the development of adipose tissue in these mice was not associated with significant modifications of glucose and insulin blood levels. Thus, these transgenic mice constitute an original model of diet-induced obesity for in vivo physiological and pharmacological studies with respect to the alpha2/beta-AR balance in adipose tissue. PMID- 12370126 TI - Cowden disease or multiple hamartoma syndrome--cutaneous clue to internal malignancy. AB - Cowden disease (CD) #158350, also known as multiple hamartoma syndrome, is a multisystemic cancer predisposition disorder, inherited in an autosomal dominant pattern. Mucocutaneous lesions are the most constant features: facial papules, acral keratoses and oral papillomatosis. The most common associated cancers are breast, thyroid and endometrial carcinomas. The CD gene locus has been mapped to chromosome 10q22-23. Subsequently the tumor suppressor gene PTEN was located to this chromosomal region and soon after germline mutations in the PTEN gene were demonstrated in CD patients. Somatic PTEN mutations have been found in a variety of sporadic cancers. So CD is an important clinical and genetic model for carcinogenesis. We recently observed four cases of CD and reviewed the literature on CD over the last 40 years, in particular the clinical and histopathological features, genetics, and diagnostic criteria. Based on these data we propose a possible management of CD patients. With increased knowledge and awareness of the typical mucocutaneous lesions an earlier diagnosis and an appropriate cancer surveillance of these patients might be possible. PMID- 12370127 TI - Neuropeptides and sebaceous glands. AB - This review provides a new insight into the participation of neuropeptides, notably substance P (SP), in the pathophysiology of acne. We show morphological alterations of sebaceous glands elicited by SP and differences in expression of various neurogenic factors in association with sebaceous glands in acne-prone versus normal facial skin. In vitro studies reveal that SP promotes both the proliferation and the differentiation of sebaceous glands. SP induces the expression of neutral endopeptidase, a potent neuropeptide-degrading enzyme, in sebaceous germinative cells and of E-selectin by perisebaceous venules. Facial skin from acne patients is characterized by rich innervation, by increased numbers of SP-containing nerves and mast cells, and by strong expression of neutral endopeptidase in sebaceous glands and E-selectin in venules around sebaceous glands, compared with normal skin. Mast cell-derived IL-6 and TNF alpha, followed by SP-stimulated degranulation, have the potential to induce nerve growth factor expression by sebaceous cells which results in the promotion of innervation and in the expression of E-selectin, respectively. SP enhances mast cell proliferation through up-regulation of stem cell factor expression in fibroblasts. These findings suggest the involvement of neurogenic factors, such as neuropeptides, in the disease process of acne and explain the possible mechanism of the exacerbation of acne from a neurological point of view. PMID- 12370128 TI - Acrogeric Ehlers-Danlos syndrome type IV: report of a new patient with additional findings. AB - We report a 21 year-old Turkish male with acrogeric Ehlers-Danlos syndrome type IV, a rare autosomal dominant disorder. In addition to the usual findings, the patient also had glaucoma and some unusual skeletal features including impressio digitalis in the skull, prognathism of the lower jaw and maxiller hypoplasia, not previously described as part of acrogeric Ehlers-Danlos syndrome type IV. These features expand the phenotypic spectrum of acrogeric Ehlers-Danlos syndrome type IV. PMID- 12370130 TI - Proposal for a new UVA protection factor: use of an in vitro model of immediate pigment darkening. AB - In order to define a new method for measuring UVA photoprotection, we built an in vitro immediate pigment darkening model (IPD). IPD is a photochemical reversible reaction induced by UVA on the skin. Our model consists of aqueous solutions of melanocytic compounds (dihydroxyphenylalanine/pheomelanins). Irradiation of these solutions with UVA induces an increase in their absorbance. Oxygen deprivation inhibits the solution darkening and light turn-off induces a decrease in the absorbance as observed in vivo. A UVA photoprotection parameter (PUVA) was defined using the ability of a sunscreen to inhibit the model reaction. A calibration of the reaction inhibition is realised using neutral beam attenuators. PUVA is defined as the percentage absorbance of a beam attenuator which would have the same inhibitory effect as the sunscreen tested. A correlation between PUVA and Diffey-Robson parameter is presented. The method developed here could be use as a indicative tool before human experiments. PMID- 12370129 TI - Interleukin-4 induces apoptosis in cultured human follicular keratinocytes, but not in dermal papilla cells. AB - The importance of apoptosis in hair follicle cycling is still not clearly understood, however, its regulation in follicular keratinocytes (FK) during bulb regression (catagen) may be essential for hair regrowth. So far, the control of FK apoptosis remains unknown. In this study, the anti-inflammatory cytokine IL-4 was found to induce apoptosis dose and time dependently in cultured human FK, in contrast to other agents known to inhibit hair growth such as IL-1alpha, IL 1beta, TNFalpha and TGFbeta, as shown by DNA fragmentation. On the other hand, cytokines reported to be involved in hair follicle cycling including IL-4 were not able to induce apoptosis in dermal papilla cells (DPC), in contrast to staurosporine. This PKC inhibitor revealed dose-dependent apoptotic signals not only for DPC but also for FK in vitro. In further experiments the expression of apoptosis regulating proteins, possibly involved in catagen formation, was analyzed in FK and DPC. However, no striking difference in RNA expression was seen in either cell population under culture conditions and after incubation with IL-4. We conclude, therefore, that IL-4 mediated apoptosis may participate in regulating catagen formation in the hair follicle, acting selectively on cultured FK and being independent of bcl-2 and bax expression. PMID- 12370131 TI - Effects of dexamethasone and sex hormones on cytokine-induced cellular adhesion molecule expression in human endothelial cells. AB - The cause or mechanism of the female predisposition in systemic lupus erythematosus and progressive systemic sclerosis is largely unknown. Accumulating evidence shows that dysfunction or activation of endothelial cells plays an important role in these conditions. In this study, we investigated the influence of various steroid hormones on the IL-1beta (50 U/mL)/TNF-alpha (50 U/mL) stimulated human dermal microvascular endothelial cell line (HMEC-1) and human umbilical vein endothelial cells (HUVEC). Dexamethasone showed significant inhibition of cytokine-induced ICAM-1 expression in HMEC-1, and E-selectin, VCAM 1 and ICAM-1 expressions in HUVEC. Androgens, especially dihydrotestosterone had a small, but statistically significant suppressive effect in HMEC-1 only. Estrogen exhibited no regulatory function in either cell line. No obvious expression of estrogen and androgen receptors could be demonstrated in either cell by immunostaining. Our study provided some pharmacological evidence that the superior anti-inflammatory effect of glucocorticoids on vasculitis was partly due to their inhibition of the CAM expression in endothelial cells. More studies are needed to determine if androgens could have a protective effect in vasculitis or vasculopathy associated with connective tissue diseases. PMID- 12370132 TI - Evidence of in situ cytotoxicity in American cutaneous leishmaniasis. AB - The role of cytotoxicity in the defense mechanisms or pathogenesis of human cutaneous leishmaniasis is not yet well known. In the present work we assessed the presence of NK, CD8+ and CD45RO+ T cells, as well as the expression of a molecule associated with cytotoxic properties (TIA-1) in the lesions of cutaneous leishmaniasis. CD8+ T cells, NK and activated T cells were found within the dermal cell infiltrate. We found a heterogeneous but usually strong expression of TIA-1, a marker of cytotoxic granules of T and NK cells, in human cutaneous leishmaniasis lesions. These data suggest that cytotoxic activity occurs in situ in American cutaneous leishmaniasis and that both NK cells and activated CD8+ T cells are involved in this reaction. PMID- 12370133 TI - Skin changes in geriatric nurses prior to training heralding a particular risk of hand dermatitis. AB - Irritant skin changes are a well known problem in nursing services. Especially geriatric nurses often complain of hand dermatitis, most likely induced by frequent washing and hand disinfections. In this cohort study, demographic data and skin changes from 521 nurse trainees were recorded. The data of geriatric nurse trainees (n = 149) were compared to that of other nurse trainees (n = 372), mostly of surgery, internal medicine, pediatric and obstetrics. Geriatric nurse trainees were significantly older and had noticeably severer irritant skin changes at the start of the training. Geriatric nurse trainees were more often undergoing retraining, because they had other jobs before. Interestingly, some nurses performed the retraining because they had problems with hand dermatitis in their previous job. More education concerning the risk of irritant dermatitis in health care occupations is desirable, not only for the starting nurse but also for the employment offices. PMID- 12370134 TI - Chronic actinic dermatitis treated with cyclosporine-A. AB - Chronic actinic dermatitis (CAD) groups together all chronic photodermatosis with light photosensitivity. We report the case of a 69-year-old man who, for over one year, had presented a reddish-brown erythema and shedding, with thickened and hypo-elastic skin on the face, scalp, neck and on the back of the hands and forearms. Patch tests were positive to isoeugenolo 1% and photo-patch tests showed a positivity to phenotiazine 2%. After a short and ineffective treatment with beta-carotene and photo-protectors, the clinical picture was resolved with the administration of oral steroids but with a relapse of the dermatitis once the dosage was lowered. We therefore started treatment with cyclosporine-A (4.5 mg/kg/die), which resulted in a rapid improvement of the clinical picture, but with a reappearance of the manifestations when the dosage was lowered. The treatment was resumed and we observed that the dose of 1.5/mg/kg/die resulted morbus-static. The patient is still being treated with this drug at this dosage. The result is that the disease is under control and no side effects are present. As we consider CAD an invalidating disease it seems to us that Cy-A could be taken into consideration as an alternative to traditional treatments. PMID- 12370135 TI - The cytoprotective effect of amifostine in acute radiation dermatitis: a retrospective analysis. AB - To study the impact of amifostine as a cytoprotective agent against acute radiation dermatitis, we reviewed 220 patient records. One hundred cancer patients, with tumors localised in the pelvis (bladder, rectum, prostatic carcinomas, or gynecological cancer), who received radiotherapy and cytoprotective treatment with intravenous infusion of amifostine (group A) were included in this study. Retrospectively, we randomly selected from a database in our hospital 120 historical controls, who received only radiotherapy without cytoprotection (group B). Mean gross dermatitis score (MGDS) was the mean value of recorded acute radiation dermatitis according to common toxicity criteria. In group A versus B patients, a significantly reduced severity of dermatitis (P < 0.001, Fisher's exact test) and significant reduction of MGDS as well as mean interruption treatment time (P < 0.001, Mann-Whitney U test) was observed. The relative risk of the outcome of the two study groups was 0.23 (95% CI: 0.15 to 0.34). The significant dermato-cytoprotective effect of amifostine noticed in our retrospective analysis warrants further investigation with randomised trials. PMID- 12370136 TI - Long-term safety and efficacy of high-concentration (20 microg/g) tacalcitol ointment in psoriasis vulgaris. AB - A multi-center open prospective research was conducted in order to assess the safety and efficacy of tacalcitol 20 microg/g ointment once daily (maximum 10 g/day) in the long-term treatment of psoriasis vulgaris. For the 74 subjects included in the 54-week efficacy analysis, the mean PASI score at the beginning of the study was 22.49 10.20 (mean SD), which was 5.73 6.04 after 54 weeks. A significant decrease (p < 0.001) in the mean PASI score was seen after 1 week of application, and the score remained almost constant after 18 weeks through 54 weeks. Twenty-five local adverse drug reactions were noticed in 16 of the 154 subjects included in the safety analysis. No increase in the incidence of severe adverse drug reactions was seen in the long-term administration of tacalcitol 20 microg/g ointment. Although a significant decrease in the intact parathyroid hormone (PTH) and 1alpha,25-(OH)2D3 was observed, the homeostasis of the corrected serum calcium was maintained. Tacalcitol 20 microg/g ointment, applied once daily at doses of up to 10 g/day (200 microg tacalcitol), is safe and effective, even in long-term administration, in the treatment of patients with psoriasis vulgaris. Serum calcium should be monitored in patients with decreased renal function and other suspected impairment of calcium metabolism, before and during the treatment with tacalcitol 20 microg/g ointment. PMID- 12370137 TI - A famous Turkish dermatologist, Dr. Hulusi Behcet. AB - Dr. Hulusi Behcet (1889-1948) is a famous Turkish dermatologist. He was born in Istanbul on February 20, 1889. His father was Ahmet Behcet and his mother Ayqse Behcet was also Ahmet's cousin. After the Turkish Republic was established and the "Family Name Law" was accepted, his father Ahmet Behcet, who was among the friends of Mustafa Kemal Ataturk, the founder of Turkish Republic, received private permission to use his father's name Behcet. Dr. Hulusi Behcet pursued his education at Gulhane Military Medical Academy. After he had become a medical doctor, he specialized in dermatology and venereal disease at Gulhane Military Medical Academy and he completed his specialization in 1914. His first observations on Behcet's Disease started with a patient he met between 1924-1925. Dr. Behcet followed the symptoms of three patients whom he had had for years, then he decided that they were the symptoms of a new disease (1936). He published these cases in the Archives of Dermatology and Veneral Disease. He died from a sudden heart attack on March 8, 1948. Today, this disease is universally called Behcet's Disease in medical literature. PMID- 12370138 TI - Benediktos Adamantiades and his forgotten contributions to medicine. AB - Benediktos Adamantiades was born in Prousa, Asia Minor in 1875, studied at the Medical School of the University of Athens and specialised in ophthalmology in Paris. After being director of the Ophthalmology Department of Hippocration Hospital in Athens he developed significant scientific activities. During the annual meeting of the Medical Society of Athens on November 15, 1930, Adamantiades presented "A case of relapsing iritis with hypopyon" identifying the three major signs of the so called Adamantiades-Behcet's disease and insisting on a single clinical entity. In the same year, his lecture was published in the Proceedings of the Medical Society of Athens, and in 1931 in the French journal Annales d'Oculistique. In the year 1946, Adamantiades defined thrombophlebitis as the 4th major sign of the disease. Later, he presented the first classification of the disease by describing the ocular, mucocutaneous and systemic forms in a review work. He pointed out that the disease can occur for years as a monosymptomatic or oligosymptomatic disorder and that eye involvement and severe prognosis are more common in men than in women. In this work he also proposed the first diagnostic criteria. In addition to Adamantiades-Behcet's disease, Adamantiades described the interstitial keratitis in trachomatic patients to be a bacterial infection and classified the epidemic idiopathic hemeralopia. Further pioneer works were those on the marginal corneal degeneration, the posterior vitreous detachment, the measurement of the optic fundi and of the ocular pressure as well as investigations on trachoma and the pathogenesis of glaucoma. Adamantiades compiled over 150 scientific papers many of which marked a new stage in his field. He died in 1962 in Athens. PMID- 12370139 TI - Acute generalized exanthematous pustulosis with erythema multiforme-like lesions. AB - Acute generalized exanthematous pustulosis (AGEP) resembles generalized pustular psoriasis, but may manifest targetoid lesions, purpura, and blisters in addition to pustules. We describe a case of AGEP with erythema multiforme (EM)-like features in a 35-year-old woman who presented with acute onset of high fever and a strikingly polymorphic eruption consisting of numerous tiny pustules on erythematous bases, marked facial edema, oral and genital erosions, targetoid vesicular and purpuric lesions, pustules in string-of-pearl configuration and ring-like vesicles. The histology revealed, in addition to subcorneal pustules, vacuolar interface dermatitis with involvement of eccrine glands, and microabscesses in pilosebaceous structures. Systemic corticorsteroid and antibiotics were initiated, resulting in rapid resolution without recurrence. Recognition of EM-like lesions on a background of generalized pustular eruption could facilitate the diagnosis of AGEP and the institution of appropriate treatment. PMID- 12370140 TI - Granulomatous mycosis fungoides responsive to gemcitabine. AB - We report a 61-year-old woman with a 1-year-history of widespread erythematous scaly patches and plaques as well as red/purplish to brownish confluent plaques. Ulcerated lesions with a purulent, hemorrhagic exudate and sharp elevated borders were located on the lower extremities. Diagnosis of granulomatous mycosis fungoides was supported by histopathologic findings showing an inflammatory reaction with epithelioid and large giant cells associated with features characteristic of mycosis fungoides. Immunohistochemical studies showed a T helper phenotype of neoplastic cells (CD3+, CD4+, CD45RO+) with expression of the cytotoxic protein TIA-1. Molecular analysis of TCRgamma gene demonstrated a monoclonal rearrangement in the lesional skin. After failure of conventional therapies, 6 cycles of gemcitabine treatment produced partial remission of cutaneous lesions and stable disease throughout a 12-month follow-up period, suggesting that gemcitabine is a promising chemotherapeutic agent for refractory mycosis fungoides. PMID- 12370141 TI - Multiple, bilateral and painful ear nodules of the anthelices: a variant of chondrodermatitis nodularis? AB - A case of a distinctive clinicopathologic condition of the ear cartilage is presented, characterized by multiple, bilateral and painful nodules of the anthelices without epidermal involvement. Histologically, there was a peri chondrial lymphohistiocytic infiltrate and a small focus of degenerate, basophilic cartilage as well as cystic chondromalacia containing an amorphous mass. This condition is both clinically and histopathologically distinct from other causes of ear nodules, although the lesions seen in our patient exhibit features of chondrodermatitis nodularis helices and therefore could well be a variant of the latter. PMID- 12370142 TI - Pyoderma gangrenosum in a patient with bullous systemic lupus erythematosus. AB - We report a 55-year-old woman with bullous systemic lupus erythematosus, who later developed pyoderma gangrenosum (PG). Dapsone was effective for the eruption of bullous bullous systemic lupus erythematosus but not for pyoderma gangrenosum. Cyclosporine was effective for the skin lesions of pyoderma gangrenosum. This is the first reported case of PG associated with bullous systemic lupus erythematosus. PMID- 12370143 TI - Dimorphic exanthema manifested as reticular maculopapular exanthema and erythema multiforme major associated with pyrazolon derivatives. AB - The non-steroidal anti-inflammatory drugs, especially various pyrazole or pyrazolon derivatives, were one of the classes of drugs commonly implicated in erythema multiforme or Stevens-Johnson syndrome/toxic epidermal necrolysis. Reticular exanthem was a rare morphological pattern of maculopapular drug eruptions. Here we report a case of dimorphic exanthema presenting with partly reticular maculopapular exanthema and erythema multiforme-like lesions, associated with pyrazolon derivatives. Patch testing showed negative reaction to the suspected drugs. The possible mechanism underlying the association of dimorphic exanthema with NSAIDs is discussed. PMID- 12370144 TI - Aggressive digital papillary adenocarcinoma arising on the right great toe. AB - A 20-year-old Japanese man with aggressive digital papillary adenocarcinoma on the right great toe was presented. The tumor was comprised of multiple lobules of epithelial cells exhibiting solid areas, lacking cystic spaces. A pattern of fused back-to-back glands lined by cuboidal to low columnar epithelial cells with little evidence of papillary formations was observed. Apocrine-like decapitation secretion was not observed, and continuity of the tumor to the epidermis was evident. The neoplastic cells were immunohistochemically positive for S-100 protein, but negative for epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and gross cystic disease fluid protein (GCDFP). Intraluminal contents, however, showed positive reactivity with CEA and EMA. On the basis of their histologic and immunohistochemical features, the tumor was diagnosed as aggressive digital papillary adenocarcinoma. PMID- 12370145 TI - Pronounced and early acne in Apert's syndrome: a case successfully treated with oral isotretinoin. AB - An unusual case of widespread acne unresponsive to treatment with early onset in a child with Apert's syndrome is presented. The patient eventually responded to oral isotretinoin therapy. The morphological profile of the sebaceous glands and the expression of proliferative markers and androgen receptors were evaluated in seboblasts and sebocytes using morphological, ultrastructural and immunohistochemical techniques. There were no significant differences in staining for proliferative markers and nuclear expression of androgen receptors in the glands from the patient and four healthy controls. Our results support the view that acne in Apert's syndrome is not sustained by abnormalities of the sebaceous glands demonstrable with conventional morphological techniques, and that it does not depend on an increased expression of androgen receptors. PMID- 12370146 TI - Porokeratosis of the lower lip. AB - The porokeratoses are defined as specific disorders of keratinization that are histologically characterised by the presence of a dense, parakeratotic column, well-circumscribed from the rest of the corneocytes, known as the cornoid lamella. In this article, we report two cases of porokeratosis of the lower lip, drawing attention to the unusual site. We also emphasize the clinical appearance of these lesions, which may easily be misdiagnosed if not examined correctly. As a consequence of this misdiagnosis, the prevalence of these lesions is probably greater than at present estimated. Moreover, a good response to cryotherapy is shown. PMID- 12370147 TI - Anticonvulsant hypersensitivity syndrome with fatal outcome. AB - Anticonvulsant hypersensitivity syndrome is a disease characterized by multisystemic involvement, fever, lymphadenopathy, mucocutaneous rash, hypertransaminasemia and peripheral eosinophilia. This rare syndrome seems to be related to arene oxide metabolites of aromatic anticonvulsants (phenytoin, phenobarbital and carbamazepine). Anticonvulsant hypersensitivity seems to be much more aggressive in patients undergoing concomitant radiotherapy. We report a case of anticonvulsant hypersensitivity syndrome developing toxic epidermal necrolysis with fatal outcome in a patient receiving cranial irradiation and aromatic anticonvulsants for seizure prophylaxis. This report attempts to emphasize the importance of an early diagnosis of this syndrome, the knowledge of the common cross-reactivity among the major anticonvulsants and the need for an appropriate measurement of the true benefits of seizure prophylaxis in patients with brain tumors. PMID- 12370148 TI - Epicutaneous patch testing. AB - Epicutaneous patch testing is still regarded as the best method of diagnosing allergic contact dermatitis. The present patch test technique is the result of a continuous process of development and improvement since its first application in the late 19th century. During the last decades of the 20th century a lot of effort was put into standardization of materials and methods used in patch testing. Patch tests can be used to confirm a suspected allergic contact dermatitis and either to recommend avoidance of particular products or to recommend alternative products in a particular patient. The true rate of clinically relevant hypersensitivity in positive patch test reactions remains to a great extent unknown. The ideal patch test should cause as few adverse reactions as possible, but a lot of adverse reactions have been described. How ever, it has to be noted that the overall risk-benefit equation of patch testing is in favor of the benefit, if performed correctly and with the proper indications. A careful history taking and attention to the clinical picture are key actions to facilitate the interpretation of the clinical relevance of the epicutaneous patch test results. PMID- 12370149 TI - Make resistance training part of your fitness plan. PMID- 12370150 TI - Bone health. Nutrients beyond calcium. PMID- 12370151 TI - Got directions? Women's and men's navigational differences. PMID- 12370152 TI - Treating hot flashes with drugs: an update. PMID- 12370153 TI - By the way, doctor. What does an abnormal Pap mean? I recently had an abnormal Pap test and my doctor told me to come back in three months to be tested again. Isn't there some way for me to find out what's wrong now? PMID- 12370154 TI - By the way, doctor. Should I test my cholesterol at home? I recently saw an ad for a cholesterol test kit for home use. It seems like a good way to keep track of my cholesterol. Is there any reason not to use something like this? PMID- 12370155 TI - By the way, doctor. Is MSM as good as it sounds? Can you tell me anything about the dietary supplement MSM? I've heard it's supposed to relieve arthritis pain. PMID- 12370156 TI - Herpes simplex virus and risk of cervical cancer: a longitudinal, nested case control study in the nordic countries. AB - Human papillomaviruses (HPVs) play the major role in cervical carcinogenesis. The authors reevaluated the role of herpes simplex virus type 2 (HSV-2) in this multistage process by conducting a longitudinal, nested case-control study using 1974-1993 data and comparing the results with those from a meta-analysis of studies. A Nordic cohort of 550,000 women was followed up for an average of 5 years, after which 178 cervical carcinoma cases and 527 controls were identified. HSV-2; HPV-16, HPV-18, and HPV-33; and Chlamydia trachomatis antibodies were determined at baseline by HSV-2 glycoprotein gG-2 and HPV virus-like-particle enzyme immunoassays and by using the microimmunofluorescence method. The relative risk of cervical carcinoma was calculated by conditional logistic regression. Longitudinal studies on HSV-2 and cervical neoplasia were identified through MEDLINE (National Library of Medicine, Bethesda, Maryland), and weighted mean relative risks were calculated. Smoking (relative risk = 1.6, 95% confidence interval (CI): 1.1, 2.3) and HPV-16/HPV-18/HPV-33 (relative risk = 2.9, 95% CI: 1.9, 4.3) were both associated with cervical carcinoma. The smoking- and HPV 16/HPV-18/HPV-33-adjusted relative risks for HSV-2 were 1.0 (95% CI: 0.6, 1.7) and 0.7 (95% CI: 0.3, 1.6), respectively, for HPV seropositives. In the meta analysis, the relative risk for HSV-2 was 0.9 (95% CI: 0.6, 1.3). In both sets of data, HSV-2 did not play a role in cervical carcinogenesis. PMID- 12370157 TI - Association of galactose-1-phosphate uridyltransferase activity and N314D genotype with the risk of ovarian cancer. AB - Deficiency in the galactose-1-phosphate uridyltransferase (GALT) enzyme results in accumulation of galactose and its metabolites in the ovary (Am J Epidemiol 1989;130:904-10). Galactose may raise gonadotropin levels, resulting in proliferation of ovarian epithelium. In 1993-1999, the authors conducted a population-based case-control study of ovarian cancer in Hawaii and Los Angeles, California, to examine the hypothesis that reduced GALT activity is associated with an increased risk of ovarian cancer. A total of 239 ovarian cancer cases and 244 population controls were interviewed. A blood sample was collected to measure levels of GALT and to assay for the N314D (A940G) polymorphism of the GALT gene. Covariate-adjusted mean GALT activity was similar between cases (23.8 micro mol per hour/g hemoglobin (Hb)) and controls (23.7 micro mol per hour/g Hb) (p = 0.83). No evidence was found for a dose-response relation between the odds ratios for ovarian cancer and GALT activity or the ratio of lactose intake to GALT activity. The risk associated with the presence of at least one variant Asp314 allele was 0.77 (95% confidence interval: 0.42, 1.41). This study did not support the hypothesis that reduced galactose metabolism is a risk factor for ovarian cancer, although increased GALT activity attenuated the inverse association of oral contraceptive pill use with risk. PMID- 12370158 TI - Familial aggregation of diabetes and hypertension in a case-control study of colorectal neoplasia. AB - Familial aggregation of diseases potentially associated with metabolic syndrome (diabetes mellitus, hypertension, and cardiovascular diseases) was assessed in a colonoscopy-based case-control study of colorectal neoplasia in Toronto and Ottawa, Canada, in 1993-1996. Each familial disease was analyzed by logistic regression using generalized estimating equations. Case probands had incident adenomatous polyps (n = 172) or incident (n = 25) or prevalent (n = 132) colorectal cancer (CRC), while control probands (n = 282) had a negative colonoscopy and no history of CRC or polyps. Significant effect modification was evident in the data, with the strongest positive associations between familial diabetes and colorectal neoplasia among older probands with symptoms (parents: odds ratio (OR) = 2.4, 95% confidence interval (CI): 1.2, 4.8; siblings: OR = 5.8, 95% CI: 2.6, 13.3). Familial hypertension was also associated with colorectal neoplasia among probands with symptoms (OR = 1.7, 95% CI: 1.1, 2.6). In stratified analyses, familial diabetes, hypertension, and stroke were positively associated with adenomatous polyps in subgroups of probands who were older and/or had symptoms, while only familial diabetes was possibly associated with CRC. Associations in other proband groups may have been obscured by high cumulative incidence of parental CRC. Family studies are needed to understand the contribution of specific environmental and genetic factors in accounting for the disease aggregations. PMID- 12370159 TI - Age and the burden of death attributable to diabetes in the United States. AB - Diabetes is a well-established cause of cardiovascular disease (CVD) and all cause mortality. The burden of death attributable to diabetes in the United States is not well quantified, particularly with regard to age. The authors analyzed data from the Second National Health and Nutrition Examination Survey (NHANES II) (1976-1980) and the NHANES II Mortality Study, in which a nationally representative cohort of 9,250 adults aged 30-75 years was followed for 12-16 years, to determine all-cause and cause-specific mortality. Overall, between 1976 and 1980, the prevalence of diagnosed diabetes was 4.3%. By 1992, the relative hazard of all-cause mortality was 1.9 (95% confidence interval: 1.5, 2.3), and the population attributable risk (PAR) was 3.6%. The relative hazard of CVD mortality was 2.3 (95% confidence interval: 1.8, 2.8), and the PAR was 5.2%. Including participants with undiagnosed diabetes in the estimates increased the PAR for all-cause mortality to 5.1% and that for CVD mortality to 6.8%. Women had a higher prevalence of diagnosed diabetes than men and a greater relative hazard of death than nondiabetic women, leading to a higher PAR for women (3.8% for all causes and 7.3% for CVD) versus men (3.3% for all causes and 3.8% for CVD). These data suggest that diabetes accounts for at least 3.6% of all deaths and 5.2% of CVD deaths in US adults. Improvements in diabetes prevention and treatment should produce noticeable effects on US life expectancy. PMID- 12370160 TI - Longitudinal analysis of changes in indices of obesity from age 8 years to age 18 years. Project HeartBeat! AB - To compare growth patterns of obesity indices derived from body composition and anthropometric measures, the authors analyzed data from Project HeartBeat!, a longitudinal study of cardiovascular disease risk factors in childhood and adolescence. A total of 678 children initially aged 8, 11, and 14 years in The Woodlands and Conroe, Texas, were enrolled and followed with 4-monthly examinations between October 1991 and August 1995. Trajectories of change from age 8 years to age 18 years were estimated for body mass index, percent body fat, abdominal circumference, the sum of two skinfolds, and the sum of six skinfolds. All indices varied importantly with age. Percent body fat, sum of two skinfolds, and sum of six skinfolds shared similar growth patterns, with strong divergence between males and females. Males' body fat decreased with age and females' increased or remained nearly constant with age. In contrast, both body mass index and abdominal circumference increased monotonically with age in both sexes, exhibiting little sex difference as children reached late adolescence. Sex differences were more striking among Blacks than among non-Blacks. The authors conclude that growth patterns of adiposity differ according to the measure chosen. Furthermore, changes in different obesity indices may not relate in the same way to changes in blood pressure or blood lipid concentrations. PMID- 12370161 TI - Chronic disease mortality in a cohort of smokeless tobacco users. AB - The purpose of this study was to characterize the relation between smokeless tobacco use and the risk of all-cause and disease-specific mortality. Using data from the First National Health and Nutrition Examination Survey Epidemiologic Followup Study, the authors assessed the 20-year mortality experience of smokeless tobacco users. Subjects aged 45 years or more at baseline (1971-1975) were categorized as either smokeless tobacco users (n = 1,068) or non-smokeless tobacco users (n = 5,737). Subjects were further stratified by smoking status and gender. Proportional hazard ratios were used to assess associations. After adjustment for confounders, no association between smokeless tobacco use and all cause (hazard ratio = 1.1, 95% confidence interval (CI): 0.9, 1.3), all cancer (hazard ratio = 1.1, 95% CI: 0.6, 1.9), or all cardiovascular (hazard ratio = 1.1, 95% CI: 0.8, 1.5) mortality was found. There was an increase in all cancer mortality of borderline significance among female smokeless tobacco users (hazard ratio = 1.7, 95% CI: 1.0, 2.8). The lung cancer mortality rate among combined users (smokeless tobacco and cigarettes), based on the rates for exclusive smokeless tobacco users and exclusive smokers, was higher than expected, possibly because of heavier smoking among these subjects. The mortality experience of smokeless tobacco users was not significantly greater than that of non-tobacco users and was appreciably less than that of cigarette smokers. Furthermore, combined use of smokeless tobacco and cigarettes did not increase overall mortality beyond that expected from use of the individual products. PMID- 12370162 TI - Association between chronic obstructive pulmonary disease and employment by industry and occupation in the US population: a study of data from the Third National Health and Nutrition Examination Survey. AB - Data from the US population-based Third National Health and Nutrition Examination Survey, conducted from 1988 to 1994, were used to estimate the population prevalence, prevalence odds ratios, and attributable fractions for the association of chronic obstructive pulmonary disease (COPD) with employment by industry and occupation. The aim was to identify industries and occupations at increased risk of COPD. COPD was defined as forced expiratory volume in 1 second (FEV(1))/forced vital capacity <70% and FEV(1 )<80% predicted. The authors used SUDAAN software (Research Triangle Institute, Research Triangle Park, North Carolina) to estimate the weighted population prevalence and odds ratios using 9,823 subjects aged 30-75 years who underwent lung function tests. Odds ratios for COPD, adjusted for age, smoking status, pack-years of smoking, body mass index, education, and socioeconomic status, were increased for the following industries: rubber, plastics, and leather manufacturing; utilities; office building services; textile mill products manufacturing; the armed forces; food products manufacturing; repair services and gas stations; agriculture; sales; construction; transportation and trucking; personal services; and health care. Occupations associated with increased odds ratios for COPD were freight, stock, and material handlers; records processing and distribution clerks; sales; transportation-related occupations; machine operators; construction trades; and waitresses. The fraction of COPD attributable to work was estimated as 19.2% overall and 31.1% among never smokers. PMID- 12370163 TI - Cognitive function after 11.5 years of alcohol use: relation to alcohol use. AB - The authors investigated the relation between alcohol use and cognitive decline after 11.5 years in a community sample. Findings were based on a study of 1,488 participants in the Baltimore arm of the Epidemiologic Catchment Area study, who completed the Mini-Mental State Examination (MMSE) at three time points in 1981, 1982, and 1993-1996. The participants were divided into five groups based on the amount and frequency of alcohol intake and the Diagnostic and Statistical Manual of Mental Disorders: DSM IIIR diagnosis of alcohol use disorders. The relation between level of alcohol use and MMSE score change between waves 2 and 3 of the study was examined using analysis of variance, which was then adjusted for the effects of age, race, and education. Alcohol use was associated with significantly less cognitive decline in alcohol drinkers when compared with nondrinkers for both sexes. When adjusted, a trend toward significantly less cognitive decline was seen in women drinkers, but not in men. Among female users, there was a trend toward less cognitive decline in women who used alcohol habitually as compared with those who were nonusers or heavy users. The authors conclude that, over long time periods, alcohol use is not associated with cognitive decline and, in women, may be associated with less decline. PMID- 12370164 TI - Modeling treatment effects on binary outcomes with grouped-treatment variables and individual covariates. AB - During evaluation of treatment effects in observational studies, confounding is a constant threat because it is always possible that patients with a better prognosis, not adequately characterized by measured covariates, are chosen for a specific therapy. Ecologic analyses may avoid confounding that would be present in analysis at the individual level because variations in regional or hospital practice may be unrelated to prognosis. The authors used simulated data with an excluded confounder to evaluate the reliability and limitations of the grouped treatment approach, a method of incorporating an ecologic measure of treatment assignment into an individual-level multivariable model, similar to the instrumental variable approach. Estimates based on the grouped-treatment approach were closer to the true value than those of standard individual-level multivariable analysis in every simulation. Furthermore, confidence intervals based on the grouped-treatment approach achieved approximately their nominal coverage, whereas those based on individual-level analyses did not. The grouped treatment approach appears to be more reliable than standard individual-level analysis in situations where the grouped-treatment variable is unassociated with the outcome except via the actual treatment assignment and measured covariates. PMID- 12370165 TI - Empirically calibrated model of hepatitis C virus infection in the United States. AB - This study presents a comprehensive epidemiologic model of hepatitis C in the United States. Through empirical calibration of model parameter values, the objectives were to gain insights into uncertain aspects of the natural history of hepatitis C and to improve the basis for projecting the future course of the epidemic. A systematic review of the published literature was conducted to define plausible ranges around model parameters, and multiple simulations of the model were undertaken using sampled values from these ranges. Model predictions produced by each set of sampled values were compared with available epidemiologic data on infection prevalence and mortality from liver cancer, and various goodness-of-fit criteria were used to identify the range of parameter values that were consistent with these data. The results of the study indicate that rates of progression to advanced liver disease may be lower than previously assumed. The authors also found that a wide range of plausible assumptions about heterogeneity in these rates, beyond that explained by age and sex, is consistent with observed epidemiologic trends. These findings have important implications both for individual clinical decisions and for broader public health policy. PMID- 12370166 TI - Use of multiple imputation to correct for nonresponse bias in a survey of urologic symptoms among African-American men. AB - The Flint Men's Health Study is an ongoing population-based study of African American men designed to address questions related to prostate cancer and urologic symptoms. The initial phase of the study was conducted in 1996-1997 in two stages: an interviewer-administered survey followed by a clinical examination. The response rate in the clinical examination phase was 52%. Thus, some data were missing for clinical examination variables, diminishing the generalizability of the results to the general population. This paper is a case study demonstrating the application of multiple imputation to address important questions related to prostate cancer and urologic symptoms in a data set with missing values. On the basis of the observed clinical examination data, the American Urological Association Symptoms Score showed a surprising reduction in symptoms in the oldest age group, but after multiple imputation there was a monotonically increasing trend with age. It appeared that multiple imputation corrected for nonresponse bias associated with the observed data. For other outcome measures-namely, the age-adjusted 95th percentile of prostate-specific antigen level and the association between urologic symptoms and prostate volume results from the observed data and the multiply imputed data were similar. PMID- 12370169 TI - Efficiency of correct nucleotide insertion governs DNA polymerase fidelity. AB - DNA polymerase fidelity or specificity expresses the ability of a polymerase to select a correct nucleoside triphosphate (dNTP) from a pool of structurally similar molecules. Fidelity is quantified from the ratio of specificity constants (catalytic efficiencies) for alternate substrates (i.e. correct and incorrect dNTPs). An analysis of the efficiency of dNTP (correct and incorrect) insertion for a low fidelity mutant of DNA polymerase beta (R283A) and exonuclease deficient DNA polymerases from five families derived from a variety of biological sources reveals that a strong correlation exists between the ability to synthesize DNA and the probability that the polymerase will make a mistake (i.e. base substitution error). Unexpectedly, this analysis indicates that the difference between low and high fidelity DNA polymerases is related to the efficiency of correct, but not incorrect, nucleotide insertion. In contrast to the loss of fidelity observed with the catalytically inefficient R283A mutant, the fidelity of another inefficient mutant of DNA polymerase beta (G274P) is not altered. Thus, although all natural low fidelity DNA polymerases are inefficient, not every inefficient DNA polymerase has low fidelity. Low fidelity polymerases appear to be an evolutionary solution to how to replicate damaged DNA or DNA repair intermediates without burdening the genome with excessive polymerase initiated errors. PMID- 12370170 TI - Nudix hydrolases that degrade dinucleoside and diphosphoinositol polyphosphates also have 5-phosphoribosyl 1-pyrophosphate (PRPP) pyrophosphatase activity that generates the glycolytic activator ribose 1,5-bisphosphate. AB - A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5 phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and P(i). Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non nucleotide diphosphoinositol polyphosphates. K(m) and k(cat) values for PRPP hydrolysis for the Deinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s(-1), 0.13 mm and 0.057 s(-1), and 0.38 mm and 1.0 s(-1), respectively. Active site mutants of the Caenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for the D. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes. PMID- 12370171 TI - Kainate-binding proteins are rendered functional ion channels upon transplantation of two short pore-flanking domains from a kainate receptor. AB - Kainate-binding proteins belong to an elusive class of putative ionotropic glutamate receptors that to date have not been shown to form functional ion channels in heterologous expression systems, despite binding glutamatergic agonists with high affinity. To test the hypothesis that inefficient or interrupted signal transduction from the ligand-binding site via linker domains to the ion pore (gating) might be responsible for this apparent lack of function, we transplanted the short homologous linker sequences from the fully functional rat kainate receptor GluR6 into frog kainate-binding protein. We were able to generate chimeric receptors that are functional in the Xenopus oocyte expression system and in human embryonic kidney 293 cells. The linker domains A and B in particular appear to be crucial for gating, because a functional kainate-binding protein was observed when at least parts of both linkers were derived from GluR6. We speculate that to enable signal transduction from the ligand-binding site to the ion pore of the frog kainate-binding protein, the linker structure of the protein has to undergo an essential conformational alteration, possibly mediated by an as yet unknown subunit or modulatory protein. PMID- 12370172 TI - Crystal structure of venus, a yellow fluorescent protein with improved maturation and reduced environmental sensitivity. AB - Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 A resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37 degrees C by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the Calpha-Cbeta bond of Tyr(66) during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site. PMID- 12370173 TI - Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo. AB - Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites. PMID- 12370174 TI - Novel target sequences for Pax-6 in the brain-specific activating regions of the rat aldolase C gene. AB - Upstream activating sequences of the rat aldolase C gene are shown here to confer brain-specific expression in transgenic mice. In addition to binding sites described previously for the brain-expressed POU proteins Brn-1 and Brn-2 (Skala, H., Porteu, A., Thomas, M., Szajnert, M. F., Okazawa, H., Kahn, A., and Phan-Dinh Tuy, F. (1998) J. Biol. Chem. 273, 31806-31814), we have identified two novel DNA elements critical for an interaction with a brain-specific, high affinity DNA binding protein. Characterization of this binding protein showed it to be sensitive to thiol oxidation and stable to heat at 100 degrees C. This protein was purified on the basis of its thermostability and its selective adsorption to streptavidin magnetic particles via a biotinylated multimer of its target DNA binding site. Liquid chromatography coupled to tandem mass spectrometry analysis, binding competition with consensus oligonucleotides, and antibody supershift assays led to its identification as the homeodomain paired protein Pax-6. This result suggests that the brain-specific aldolase C gene could constitute a new target for the transcription factor Pax-6, which is implicated increasingly in neurogenesis. PMID- 12370175 TI - Characterization of the two coactivator-interacting surfaces of the androgen receptor and their relative role in transcriptional control. AB - The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer. PMID- 12370176 TI - Cell surface targeting of pregnancy-associated plasma protein A proteolytic activity. Reversible adhesion is mediated by two neighboring short consensus repeats. AB - The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547 residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP. PMID- 12370177 TI - Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid. AB - 4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5 diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry. PMID- 12370178 TI - Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation. AB - Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin 1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin 1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation. PMID- 12370179 TI - Analysis of O-acetyl-ADP-ribose as a target for Nudix ADP-ribose hydrolases. AB - The Sir2 family of NAD(+)-dependent histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life span extension, and chromosomal stability. Recent evidence has indicated that these proteins produce a novel metabolite O-acetyl-ADP-ribose (OAADPr) during deacetylation. Cellular studies have demonstrated that this metabolite exhibits biological effects when microinjected in living cells. However, the molecular targets of OAADPr remain to be identified. Here we have analyzed the ADP-ribose-specific Nudix family of hydrolases as potential in vivo metabolizing enzymes of OAADPr. In vitro, we found that the ADP-ribose hydrolases (yeast YSA1, mouse NudT5, and human NUDT9) cleaved OAADPr to the products AMP and acetylated ribose 5'-phosphate. Steady-state kinetic analyses revealed that YSA1 and NudT5 hydrolyzed OAADPr with similar kinetic constants to those obtained with ADP ribose as substrate. In dramatic contrast, human NUDT9 was 500-fold less efficient (k(cat)/K(m) values) at hydrolyzing OAADPr compared with ADP-ribose. The inability of OAADPr to inhibit the reaction of NUDT9 with ADP-ribose suggests that NUDT9 binds OAADPr with low affinity, likely due to steric considerations of the additional acetylated-ribose moiety. We next explored whether Nudix hydrolytic activities against OAADPr could be observed in cell extracts from yeast and human. Using a detailed analysis of the products generated during the consumption of OAADPr in extracts, we identified two robust enzymatic activities that were not consistent with the known Nudix hydrolases. Instead, we identified cytoplasmic esterase activities that hydrolyze OAADPr to acetate and ADP-ribose, whereas a distinct activity residing in the nucleus is consistent with an OAADPr specific acetyltransferase. These findings establish for the first time that select members of the ADP-ribose hydrolases are potential targets of OAADPr metabolism. However, the predominate endogenous activities observed from diverse cell extracts represent novel enzymes. PMID- 12370180 TI - The position of cysteine relative to the transmembrane domain is critical for palmitoylation of H1, the major subunit of the human asialoglycoprotein receptor. AB - The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation. PMID- 12370181 TI - Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate. AB - The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex. PMID- 12370182 TI - Characterization of a unique glycosylated anchor endopeptidase that cleaves the LPXTG sequence motif of cell surface proteins of Gram-positive bacteria. AB - The precursors of most surface proteins on Gram-positive bacteria have a C terminal hydrophobic domain and charged tail, preceded by a conserved LPXTG motif that signals the anchoring process. This motif is the substrate for an enzyme, termed sortase, which has transpeptidation activity resulting in the cleavage of the LPXTG sequence and ultimate attachment of the protein to the peptidoglycan. While screening a group A streptococcal membrane extract for cleavage activity of the LPXTG motif, we identified an enzyme (which we term "LPXTGase") that differs significantly from sortase but also cleaves this motif. The enzyme is heavily glycosylated, which is required for its activity. Amino acid composition and sequence analysis revealed that LPXTGase differs from other enzymes, in that the molecule, which is about 14 kDa in size, has no aromatic amino acids, is rich in alanine, and is 30% composed of uncommon amino acids, suggesting a nonribosomal construction. A similar enzyme found in the membrane extract of Staphylococcus aureus, indicates that this unusual molecule may be common among Gram-positive bacteria. Whereas peptide antibiotics have been reported from bacillus species that also contain unusual amino acids and are synthesized non-ribosomally on amino acid-activating polyenzyme templates, this would be the first reported enzyme that may be similarly synthesized. PMID- 12370183 TI - Strand transfer occurs in retroviruses by a pause-initiated two-step mechanism. AB - Recombination promotes retrovirus evolution. It involves transferring a growing DNA primer from one genomic RNA template in the virus to the other. Strand transfer results in vitro suggested that pausing of the reverse transcriptase during synthesis allows enhanced RNase H cleavage of the initial, or donor, RNA template that facilitates primer interaction with the acceptor template. Hairpins are common structures in retrovirus RNAs that induce pausing. Analyzing primer transfers in hairpins by base substitution markers showed transfer sites well beyond the site of pausing. We developed methods to distinguish the initial site of primer-acceptor template interaction from the site of primer terminus transfer. The strand transfer mechanism was confirmed to involve two steps. In the first, the acceptor template invades the primer-donor complex. However, the primer terminus continues elongation on the donor RNA. The interacting primer and acceptor strands then propagate by branch migration to catch the advancing primer terminus. Some distance downstream of the invasion site the primer terminus transfers, marking the genetic shift from donor to acceptor. Nucleocapsid protein (NC) is known to influence primer elongation and strand exchange. The presence of NC increased the efficiency of transfers but did not appear to alter the fundamental transfer mechanism. PMID- 12370184 TI - Structure-based design of p18INK4c proteins with increased thermodynamic stability and cell cycle inhibitory activity. AB - p18(INK4c) is a member of the INK4 family of proteins that regulate the G(1) to S cell cycle transition by binding to and inhibiting the pRb kinase activity of cyclin-dependent kinases 4 and 6. The p16(INK4a) member of the INK4 protein family is altered in a variety of cancers and structure-function studies of the INK4 proteins reveal that the vast majority of missense tumor-derived p16(INK4a) mutations reduce protein thermodynamic stability. Based on this observation, we used p18(INK4c) as a model to test the proposal that INK4 proteins with increased stability might have enhanced cell cycle inhibitory activity. Structure-based mutagenesis was used to prepare p18(INK4c) mutant proteins with a predicted increase in stability. Using this approach, we report the generation of three mutant p18(INK4C) proteins, F71N, F82Q, and F92N, with increased stability toward thermal denaturation of which the F71N mutant also showed an increased stability to chemical denaturation. The x-ray crystal structures of the F71N, F82Q, and F92N p18INK4C mutant proteins were determined to reveal the structural basis for their increased stability properties. Significantly, the F71N mutant also showed enhanced CDK6 interaction and cell cycle inhibitory activity in vivo, as measured using co-immunoprecipitation and transient transfection assays, respectively. These studies show that a structure-based approach to increase the thermodynamic stability of INK4 proteins can be exploited to prepare more biologically active molecules with potential applications for the development of molecules to treat p16(INK4a)-mediated cancers. PMID- 12370185 TI - Closely related CC- and A-adding enzymes collaborate to construct and repair the 3'-terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans. AB - The 3'-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CC-adding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334-1336). Here we show that in Synechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC- and A adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time. PMID- 12370186 TI - Liver protection from apoptosis requires both blockage of initiator caspase activities and inhibition of ASK1/JNK pathway via glutathione S-transferase regulation. AB - Hepatoprotection mediated by free radical scavenging molecules such as dimethyl sulfoxide (Me(2)SO) arose the question as to whether this effect involved one or several anti-apoptotic signals. Here, using primary cultures of rat hepatocytes and in vivo thioacetamide-induced liver failure, we showed that Me(2)SO failed to prevent any cleavage of initiator caspase-8 and -9 but constantly inhibited procaspase-3 maturation and apoptosis execution, pointing to an efficient inhibition of cleaved initiator caspase activities. Evidence was recently provided that apoptosis might require both caspase and ASK1/JNK-p38 activities. We demonstrated that this kinase pathway was strongly inhibited in the presence of Me(2)SO whereas overexpression of ASK1 was able to restore caspase-3 activity and apoptosis. Interestingly, we also found that GST M1/2 and GST Alpha1/2 dropped under apoptotic conditions; furthermore transfection of GST M1, A1, or P1 to cells overexpressing ASK1, abolished caspase-3 activity and restored viability. This role of GSTs was further assessed by showing that their high expression level was tightly associated with inhibition of ASK1 activity in Me(2)SO-protected hepatocytes. Together, these results demonstrate that Me(2)SO mediated hepatoprotection involves a dual inhibition of cleaved initiator caspase and ASK1/JNK-p38 activities. Furthermore, in highlighting the control of apoptosis by GSTs, these data provide new insights for analyzing the complex mechanisms of hepatoprotection. PMID- 12370187 TI - Beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. AB - Chemotaxis mediated by chemokine receptors such as CXCR4 plays a key role in lymphocyte homing and hematopoiesis as well as in breast cancer metastasis. We have demonstrated previously that beta-arrestin2 functions to attenuate CXCR4 mediated G protein activation and to enhance CXCR4 internalization. Here we show further that the expression of beta-arrestin2 in both HeLa and human embryonic kidney 293 cells significantly enhances the chemotactic efficacy of stromal cell derived factor 1alpha, the specific agonist of CXCR4, whereas the suppression of beta-arrestin2 endogenous expression by antisense or RNA-mediated interference technology considerably attenuates stromal cell-derived factor 1alpha-induced cell migration. Expression of beta-arrestin2 also augmented chemokine receptor CCR5-mediated but not epidermal growth factor receptor-mediated chemotaxis, indicating the specific effect of beta-arrestin2. Further analysis reveals that expression of beta-arrestin2 strengthened CXCR4-mediated activation of both p38 MAPK and ERK, and the suppression of beta-arrestin2 expression blocked the activation of two kinases. Interestingly, inhibition of p38 MAPK activation (but not ERK activation) by its inhibitors or by expression of a dominant-negative mutant of p38 MAPK effectively blocked the chemotactic effect of beta-arrestin2. Expression of a dominant-negative mutant of ASK1 also exerted the similar blocking effect. The results of our study suggest that beta-arrestin2 can function not only as a regulator of CXCR4 signaling but also as a mediator of stromal cell-derived factor 1alpha-induced chemotaxis and that this activity probably occurs via the ASK1/p38 MAPK pathway. PMID- 12370188 TI - Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3. AB - Cytoplasmic tails of LIMPII and the invariant chain contain similar leucine-based sorting signals, but the invariant chain interacts only with AP1 and AP2, whereas LIMPII interacts strongly with AP3. In a series of in vitro experiments, we investigated the effect of residues upstream of the leucine pairs and demonstrated that these residues determine adapter binding, and certain residues favor interactions with AP3. Furthermore, constructs that interacted stronger with AP3 interacted weakly with AP1 and vice versa. Exchanging residues upstream of the leucine-based signal in LIMPII with those of the invariant chain reduced LIMPII binding to AP3 in vitro, and in vivo the corresponding LIMPII mutant was rerouted via the plasma membrane like the invariant chain. These preferential interactions of different leucine signals with different AP complexes may thus be the determining step sorting proteins from the trans-Golgi network to their final destinations. Proteins that interact with AP3 are sorted directly to endosomes/lysosomes, whereas proteins that interact with AP1 are sorted via a different route. At the same time, constructs that exhibited specificity for either AP1 or AP3 might still interact with AP2, suggesting that AP2 may recognize a wider variety of leucine signals. This is consistent with the suggested role of AP2 in internalization of proteins containing general leucine based signals, including proteins that have been missorted to the plasma membrane. PMID- 12370189 TI - Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate. AB - In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE. During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity. PMID- 12370191 TI - Cell killing by HIV-1 protease. AB - The human immunodeficiency virus protease (HIV-1 PR) was expressed both in the yeast Saccharomyces cerevisiae and in mammalian cells. Inducible expression of HIV-1 PR arrested yeast growth, which was followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Given that neither poliovirus 2A protease nor 2BC protein, both being highly toxic for S. cerevisiae, were able to produce similar effects, it seems that this lytic phenotype is specific of HIV-1 PR. Drastic alterations in membrane permeability preceded the lysis in yeast expressing HIV-1 PR. Cell killing and lysis provoked by HIV-1 PR were also observed in mammalian cells. Thus, COS7 cells expressing the protease showed increased plasma membrane permeability and underwent lysis by necrosis with no signs of apoptosis. Strikingly, the morphological alterations induced by HIV-1 PR in yeast and mammalian cells were similar in many aspects. To our knowledge, this is the first report of a viral protein with such an activity. These findings contribute to the present knowledge on HIV-1-induced cytopathogenesis. PMID- 12370190 TI - On the specificity of interaction between the Saccharomyces cerevisiae clamp loader replication factor C and primed DNA templates during DNA replication. AB - Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K(d) approximately 10 nm). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k(off) > 0.2 s(-1) versus 0.025 s(-1), respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly. PMID- 12370192 TI - In vivo evidence for interferon-gamma-mediated homeostatic mechanisms in small intestine of the NHE3 Na+/H+ exchanger knockout model of congenital diarrhea. AB - Mice lacking NHE3, the major absorptive Na(+)/H(+) exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-gamma-responsive genes. The latter finding is of particular interest, since interferon-gamma has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-gamma was elevated 5-fold in NHE3 deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-gamma mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivo data suggest that elevated interferon gamma, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract. PMID- 12370193 TI - Identification of Dp71 isoforms in the platelet membrane cytoskeleton. Potential role in thrombin-mediated platelet adhesion. AB - Utrophin is a component of the platelet membrane cytoskeleton and participates in cytoskeletal reorganization (Earnest, J. P., Santos, G. F., Zuerbig, S., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 27259-27265). Although platelets do not contain dystrophin, the identification of smaller C-terminal isoforms of dystrophin, including Dp71, which are expressed in a wide range of nonmuscle tissues and cell lines, has not been investigated. In this report, we have identified Dp71 protein variants of 55-60 kDa (designated Dp71Delta(110)) in the membrane cytoskeleton of human platelets. Both Dp71Delta(110) and utrophin sediment from lysed platelets along with the high speed detergent-insoluble pellet, which contains components of the membrane cytoskeleton. Like the membrane cytoskeletal proteins vinculin and spectrin, Dp71Delta(110) and utrophin redistributed from the high speed detergent-insoluble pellet to the integrin-rich low speed pellet of thrombin-stimulated platelets. Immunoelectron microscopy provided further evidence that Dp71Delta(110) was localized to the submembranous cytoskeleton. In addition to Dp71Delta(110), platelets contained several components of the dystrophin-associated protein complex, including beta dystroglycan and syntrophin. To better understand the potential function of Dp71Delta(110), collagen adhesion assays were performed on platelets isolated from wild-type or Dp71-deficient (mdx(3cv)) mice. Adhesion to collagen in response to thrombin was significantly decreased in platelets isolated from mdx(3cv) mice, compared with wild-type platelets. Collectively, our results provide evidence that Dp71Delta(110) is a component of the platelet membrane cytoskeleton, is involved in cytoskeletal reorganization and/or signaling, and plays a role in thrombin-mediated platelet adhesion. PMID- 12370194 TI - Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism. AB - Atherosclerotic lesions preferentially develop in areas of the vasculature exposed to nonlaminar blood flow and low fluid shear stress, whereas laminar flow and high fluid shear stress are athero-protective. We have identified a set of genes including NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), ferritin (heavy and light chains), microsomal epoxide hydrolase, glutathione S transferase, and gamma-glutamylcysteine synthase, whose expression is induced by exposure to prolonged physiological levels of steady laminar flow (shear stress = 20 dyn/cm(2)) in endothelial cells (EC). These genes contain an antioxidant response element (ARE) or ARE-like transcriptional regulatory sequence in their promoters and generally function to protect cells against oxidant stress. We demonstrate that exposure of EC to laminar flow activates ARE-mediated transcriptional activity. Mutation of the ARE from either the NQO1 or HO-1 promoter abolished laminar flow-induced NQO1 and HO-1 transcriptional activation. Expression of antisense Nrf2 (a transcriptional factor for ARE), a dominant negative Nrf2, or the cytoplasmic inhibitor of Nrf2 (Keap1/INrf2) inhibited laminar flow-induced NQO1 promoter activation in EC. In addition, expression of NQO1 or Nrf2 inhibited tumor necrosis factor-alpha-induced activation of VCAM-1 (vascular cell adhesion molecule-1) gene expression in EC. These data define the ARE as a novel endothelial shear stress response element. Furthermore, laminar flow activation of antioxidant genes via an ARE-dependent transcriptional mechanism may represent a novel athero-protective and anti-inflammatory mechanism in the vasculature. PMID- 12370195 TI - Multiple mechanisms contribute to the activation of RNA polymerase III transcription in cells transformed by papovaviruses. AB - RNA polymerase (pol) III transcription is abnormally active in fibroblasts transformed by polyomavirus (Py) or simian virus 40 (SV40). Several distinct mechanisms contribute to this effect. In untransformed fibroblasts, the basal pol III transcription factor (TF) IIIB is repressed through association with the retinoblastoma protein RB; this restraint is overcome by large T antigens of Py and SV40. Furthermore, cells transformed by these papovaviruses overexpress the BDP1 subunit of TFIIIB, at both the protein and mRNA levels. Despite the overexpression of BDP1, the abundance of the other TFIIIB components is unperturbed following papovavirus transformation. In contrast, mRNAs encoding all five subunits of the basal factor TFIIIC2 are found at elevated levels in fibroblasts transformed by Py or SV40. Thus, both papovaviruses stimulate pol III transcription by boosting production of selected components of the basal machinery. Py differs from SV40 in encoding a highly oncogenic middle T antigen that localizes outside the nucleus and activates several signal transduction pathways. Middle T can serve as a potent activator of a pol III reporter in transfected cells. Several distinct mechanisms therefore contribute to the high levels of pol III transcription that accompany transformation by Py and SV40. PMID- 12370197 TI - A tree full of the fruits of opportunity. A new paradigm for education, training and career paths in the natural sciences. PMID- 12370196 TI - Do the maths. PMID- 12370198 TI - Challenges for European science. An interview with Ernst-Ludwig Winnacker, president of the DFG, Germany's largest scientific funding agency. Interviewed by Holger Breithaupt and Susan R. Owens. PMID- 12370199 TI - The law is not enough. The publication of the Helsinki report on the situation of women in European academia shows that more than legal hurdles have to be overcome on the way to equal representation. PMID- 12370200 TI - Movies for teaching science. The first public database of scientific films and images for educational use went online this year. PMID- 12370201 TI - Sex matters. In sickness and in health, men and women are clearly different. PMID- 12370204 TI - Viruses in control of the immune system. Workshop on molecular mechanisms of immune modulation: lessons from viruses. PMID- 12370205 TI - Building limb buds. Workshop on limb development. PMID- 12370206 TI - ABC transporters: one, two or four extracytoplasmic substrate-binding sites? AB - Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator. PMID- 12370207 TI - Structural organization of the endoplasmic reticulum. AB - The endoplasmic reticulum (ER) is a continuous membrane system but consists of various domains that perform different functions. Structurally distinct domains of this organelle include the nuclear envelope (NE), the rough and smooth ER, and the regions that contact other organelles. The establishment of these domains and the targeting of proteins to them are understood to varying degrees. Despite its complexity, the ER is a dynamic structure. In mitosis it must be divided between daughter cells and domains must be re-established, and even in interphase it is constantly rearranged as tubules extend along the cytoskeleton. Throughout these rearrangements the ER maintains its basic structure. How this is accomplished remains mysterious, but some insight has been gained from in vitro systems. PMID- 12370209 TI - Does leptin cause vascular disease? PMID- 12370210 TI - Angina pectoris without chest pain: clinical implications of silent ischemia. PMID- 12370211 TI - Missed opportunities to treat atherosclerosis in patients undergoing peripheral vascular interventions: insights from the University of Michigan Peripheral Vascular Disease Quality Improvement Initiative (PVD-QI2). AB - BACKGROUND: Peripheral vascular disease is a manifestation of systemic atherosclerosis and is associated with an increased risk of cardiovascular morbidity and mortality. METHODS AND RESULTS: We examined clinical outcomes in 66 consecutive patients undergoing peripheral vascular interventions at our institution between January 2001 and October 2001. At hospital discharge and at 6 months, lifestyle modifications and use of evidence-based therapy was suboptimal. At 6 months, a significant proportion continued to smoke (22.7%) and only half of the patients exercised, controlled their weight, or modified their diet for lipid control. The use of antiplatelet therapy was 77.2%; of angiotensin-converting enzyme, 35.9%; of beta-blockers, 42.5%; and of statins, 50%. Twelve of the 66 patients (18.2%) had a clinical event of death, myocardial infarction, or stroke. An appropriateness algorithm for use of secondary prevention measures was created with the use of evidence-based therapy guidelines, and a composite appropriateness variable was also created. The use of evidence-based therapy was associated with a significant reduction of the composite of death, myocardial infarction, and stroke at 6 months (OR 0.02, 95% CI 0.01 to 0.44, P=0.01). CONCLUSIONS: Atherosclerosis risk factors are very prevalent in patients with peripheral vascular disease, but these patients receive less than optimal treatment after a predominantly technical vascular intervention. Effective secondary prevention with appropriate lifestyle interventions and evidence-based medical therapy needs to be strongly encouraged and implemented in these patients. PMID- 12370212 TI - Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. AB - BACKGROUND: Experimental data suggest that bone marrow-derived cells may contribute to the healing of myocardial infarction (MI). For this reason, we analyzed 10 patients who were treated by intracoronary transplantation of autologous, mononuclear bone marrow cells (BMCs) in addition to standard therapy after MI. METHODS AND RESULTS: After standard therapy for acute MI, 10 patients were transplanted with autologous mononuclear BMCs via a balloon catheter placed into the infarct-related artery during balloon dilatation (percutaneous transluminal coronary angioplasty). Another 10 patients with acute MI were treated by standard therapy alone. After 3 months of follow-up, the infarct region (determined by left ventriculography) had decreased significantly within the cell therapy group (from 30+/-13 to 12+/-7%, P=0.005) and was also significantly smaller compared with the standard therapy group (P=0.04). Likewise, infarction wall movement velocity increased significantly only in the cell therapy group (from 2.0+/-1.1 to 4.0+/-2.6 cm/s, P=0.028). Further cardiac examinations (dobutamine stress echocardiography, radionuclide ventriculography, and catheterization of the right heart) were performed for the cell therapy group and showed significant improvement in stroke volume index, left ventricular end systolic volume and contractility (ratio of systolic pressure and end-systolic volume), and myocardial perfusion of the infarct region. CONCLUSIONS: These results demonstrate for the first time that selective intracoronary transplantation of autologous, mononuclear BMCs is safe and seems to be effective under clinical conditions. The marked therapeutic effect may be attributed to BMC associated myocardial regeneration and neovascularization. PMID- 12370213 TI - Influence of leptin on arterial distensibility: a novel link between obesity and cardiovascular disease? AB - BACKGROUND: The mechanisms by which obesity increases the risk of atherosclerotic cardiovascular disease (CVD) are poorly understood. In experimental models, leptin, a hormone produced by adipose tissue, has been shown adversely to affect vascular health. Therefore, we tested the hypothesis that high leptin concentrations are associated with lower arterial distensibility, an index of circulatory function relevant to the atherosclerotic process. METHODS AND RESULTS: Noninvasive, high-resolution, vascular ultrasound was used to measure brachial artery distensibility in 294 healthy adolescents (aged 13 to 16 years) who had a broad range of body mass indexes. Fat mass was measured by bioelectric impedance analysis; fasting serum leptin concentration by radioimmunoassay; and lipid profile, fasting insulin, glucose, and C-reactive protein concentrations by standard laboratory techniques. Higher leptin concentrations were associated with impaired arterial distensibility (regression coefficient, -1.3% change in arterial distension per 10% increase in leptin; 95% CI, -1.9% to -0.8%; P<0.001). This association was independent of fat mass, blood pressure, and C-reactive protein, fasting insulin, or LDL cholesterol concentrations. CONCLUSIONS: Elevation in leptin was associated with impaired vascular function, independent of the metabolic and inflammatory disturbances associated with obesity. Our observations are consistent with data from experimental models and suggest that high leptin concentration is an important mechanism for the adverse influence of body fatness on CVD. PMID- 12370214 TI - Supplementation with conjugated linoleic acid causes isomer-dependent oxidative stress and elevated C-reactive protein: a potential link to fatty acid-induced insulin resistance. AB - BACKGROUND: Conjugated linoleic acids (CLAs), a group of fatty acids shown to have beneficial effects in animals, are also used as weight loss supplements. Recently, we reported that the t10c12 CLA-isomer caused insulin resistance in abdominally obese men via unknown mechanisms. The aim of the present study was to examine whether CLA has isomer-specific effects on oxidative stress or inflammatory biomarkers and to investigate the relationship between these factors and induced insulin resistance. METHODS AND RESULTS: In a double-blind placebo controlled trial, 60 men with metabolic syndrome were randomized to one of 3 groups receiving t10c12 CLA, a CLA mixture, or placebo for 12 weeks. Insulin sensitivity (euglycemic clamp), serum lipids, in vivo lipid peroxidation (determined as urinary 8-iso-PGF(2alpha) [F2-isoprostanes]), 15-ketodihydro PGF(2alpha), plasma vitamin E, plasma C-reactive protein, tumor necrosis factor alpha, and interleukin-6 were assessed before and after treatment. Supplementation with t10c12 CLA markedly increased 8-iso-PGF(2alpha) (578%) and C reactive protein (110%) compared with placebo (P<0.0001 and P<0.01, respectively) and independent of changes in hyperglycemia or dyslipidemia. The increases in 8 iso-PGF(2alpha), but not in C-reactive protein, were significantly and independently related to aggravated insulin resistance. Oxidative stress was related to increased vitamin E levels, suggesting a compensatory mechanism. CONCLUSIONS: t10c12 CLA supplementation increases oxidative stress and inflammatory biomarkers in obese men. The oxidative stress seems closely related to induced insulin resistance, suggesting a link between the fatty acid-induced lipid peroxidation seen in the present study and insulin resistance. These unfavorable effects of t10c12 CLA might be of clinical importance with regard to cardiovascular disease, in consideration of the widespread use of dietary supplements containing this fatty acid. PMID- 12370215 TI - Low-density lipoprotein particle concentration and size as determined by nuclear magnetic resonance spectroscopy as predictors of cardiovascular disease in women. AB - BACKGROUND: Nuclear magnetic resonance (NMR) offers an alternative, spectroscopic means of quantifying LDL and of measuring LDL particle size. METHODS AND RESULTS: We conducted a prospective nested case-control study among healthy middle-aged women to assess LDL particle size (NMR) and concentration (NMR) as risk factors for future myocardial infarction, stroke, or death of coronary heart disease. Median baseline levels of LDL particle concentration (NMR) were higher (1597 vs 1404 nmol/L; P= 0.0001) and LDL particle size (NMR) was lower (21.5 vs 21.8 nm; P=0.046) among women who subsequently had cardiovascular events (n=130) than among those who did not (n= 130). Of these 2 factors, LDL particle concentration (NMR) was the stronger predictor (relative risk for the highest compared with the lowest quartile=4.17, 95% CI 1.96-8.87). This compared with a relative risk of 3.11 (95% CI 1.55-6.26) for the ratio of total cholesterol to HDL cholesterol and a relative risk of 5.91 (95% CI 2.65-13.15) for C-reactive protein. The areas under the receiver operating characteristic curves for LDL particle concentration (NMR), total cholesterol to HDL cholesterol ratio, and C-reactive protein were 0.64, 0.64, and 0.66, respectively. LDL particle concentration (NMR) correlated with several traditionally assessed lipid and nonlipid risk factors, and thus adjustment for these tended to attenuate the magnitude of association between LDL particle concentration (NMR) and risk. CONCLUSIONS: In this cohort, LDL particle concentration measured by NMR spectroscopy was a predictor of future cardiovascular risk. However, the magnitude of predictive value of LDL particle concentration (NMR) was not substantively different from that of the total cholesterol to HDL cholesterol ratio and was less than that of C-reactive protein. PMID- 12370216 TI - Altered fibrin clot structure in the healthy relatives of patients with premature coronary artery disease. AB - BACKGROUND: A family history of premature coronary artery disease (CAD) is an independent cardiovascular risk factor. Fibrin clots composed of dense fiber networks are found in young CAD patients and may occur in the relatives of such individuals. METHODS AND RESULTS: The ex vivo fibrin structure of 100 healthy male relatives of patients with premature CAD and 100 age-matched control subjects was assessed by measurement of permeability (K(s)), fiber mass-length ratio ( micro ), and turbidity (lag phase and maximum absorbency [max DeltaAbs]). Scanning electron microscopy was performed on selected samples. Relatives and controls shared similar levels of conventional cardiovascular risk factors. K(s) was lower in relatives than in controls, 12.2 (11.1 to 13.3) versus 15.2 (14.0 to 16.5) x10(-9) cm(2) (P<0.001), associated with a smaller decrease in micro, 8.5 (7.7 to 9.2) versus 9.7 (8.9 to 10.5) x 10(13) Da/cm (P<0.05), respectively. Lag phase was shorter in relatives than in controls, 39 (37 to 41) versus 47 (44 to 50) seconds (P<0.001), and max DeltaAbs was higher in relatives, 0.78 (0.74 to 0.82) versus 0.71 (0.67 to 0.74) in controls (P=0.02), which indicates the presence of thicker fibers in relatives. After adjustment for fibrinogen levels, lag phase and K(s) remained significantly different between relatives and control subjects. Scanning electron microscopy images confirmed increased fiber diameter in relatives, possibly of reduced density. Factor XIII Val34Leu and fibrinogen Aalpha Thr312Ala and Bbeta -455 G/A showed no association with clot structure. CONCLUSIONS: The male relatives of patients with premature CAD form fibrin clots that begin polymerization more quickly, have thicker fibers, and are less permeable than those of control subjects. PMID- 12370217 TI - Inhibition of intestinal cholesterol absorption by ezetimibe in humans. AB - BACKGROUND: Ezetimibe has been shown to inhibit cholesterol absorption in animal models, but studies on cholesterol absorption in humans have not been performed thus far. METHODS AND RESULTS: The effect of ezetimibe (10 mg/d) on cholesterol absorption and synthesis, sterol excretion, and plasma concentrations of cholesterol and noncholesterol sterols was investigated in a randomized, double blind, placebo-controlled, crossover study in 18 patients with mild to moderate hypercholesterolemia. Treatment periods lasted 2 weeks with an intervening 2-week washout period. Fractional cholesterol absorption rates averaged 49.8+/-13.8% on placebo and 22.7+/-25.8% on ezetimibe, indicating a reduction of 54% (geometric mean ratio; P< 0.001). Cholesterol synthesis increased by 89% from 931+/-1027 mg/d on placebo to 1763+/-1098 mg/d on ezetimibe (P<0.001), while the ratio of lathosterol-to-cholesterol, an indirect marker of cholesterol synthesis, was increased by 72% (P<0.001). Bile acid synthesis was insignificantly increased (placebo: 264+/-209 mg/d, ezetimibe: 308+/-184 mg/d; P=0.068). Mean percent changes from baseline for LDL and total cholesterol after ezetimibe treatment were -20.4% and -15.1%, respectively (P<0.001 for both), whereas campesterol and sitosterol were decreased by -48% and - 41%, respectively. CONCLUSION: In humans, ezetimibe inhibits cholesterol absorption and promotes a compensatory increase of cholesterol synthesis, followed by clinically relevant reductions in LDL and total cholesterol concentrations. Ezetimibe also reduces plasma concentrations of the noncholesterol sterols sitosterol and campesterol, suggesting an effect on the absorption of these compounds as well. PMID- 12370218 TI - Angiographic findings of the multicenter Randomized Study With the Sirolimus Eluting Bx Velocity Balloon-Expandable Stent (RAVEL): sirolimus-eluting stents inhibit restenosis irrespective of the vessel size. AB - BACKGROUND: Restenosis remains the major limitation of coronary catheter-based intervention. In small vessels, the amount of neointimal tissue is disproportionately greater than the vessel caliber, resulting in higher restenosis rates. In the Randomized Study With the Sirolimus-Eluting Bx Velocity Balloon-Expandable Stent (RAVEL) trial, approximately 40% of the vessels were small (<2.5 mm). The present study evaluates the relationship between angiographic outcome and vessel diameter for sirolimus-eluting stents. METHODS AND RESULTS: Patients were randomized to receive either an 18-mm bare metal Bx VELOCITY (BS group, n=118), or a sirolimus-eluting Bx VELOCITY stent (SES group, n=120). Subgroups were stratified into tertiles according to their reference diameter (RD; stratum I, RD <2.36 mm; stratum II, RD 2.36 mm to 2.84 mm; stratum III, RD >2.84 mm). At 6-month follow-up, the restenosis rate in the SES group was 0% in all strata (versus 35%, 26%, and 20%, respectively, in the BS group). In stent late loss was 0.01+/-0.25 versus 0.80+/-0.43 mm in stratum I, 0.01+/-0.38 versus 0.88+/-0.57 mm in stratum II, and -0.06+/-0.35 versus 0.74+/-0.57 mm in stratum III (SES versus BS). In SES, the minimal lumen diameter (MLD) remained unchanged (Delta -0.72 to 0.72 mm) in 97% of the lesions and increased (=late gain, DeltaMLD <-0.72 mm) in 3% of the lesions. Multivariate predictors for late loss were treatment allocation (P<0.001) and postprocedural MLD (P= 0.008). CONCLUSIONS: Sirolimus-eluting stents prevent neointimal proliferation and late lumen loss irrespective of the vessel diameter. The classic inverse relationship between vessel diameter and restenosis rate was seen in the bare stent group but not in the sirolimus-eluting stent group. PMID- 12370219 TI - Short- and long-term neuroadrenergic effects of moderate dietary sodium restriction in essential hypertension. AB - BACKGROUND: In essential hypertension, marked restrictions in dietary sodium intake cause in the short-term period an increase in muscle sympathetic nerve traffic (MSNA) and a baroreflex impairment. The present study was set out to assess on a long-term basis the neuroadrenergic and reflex effects of moderate sodium restriction. METHODS AND RESULTS: In 11 untreated mild to moderate essential hypertensive patients (age 42.0+/-2.6 years, mean+/-SEM), we measured beat-to-beat blood pressure (Finapres), heart rate (ECG), and MSNA (microneurography) at rest and during stepwise intravenous infusions of phenylephrine and nitroprusside. Measurements were performed at regular sodium intake, after 1 and 8 weeks of low-sodium diet (80 mmol NaCl/d), and repeated again at regular sodium intake. After 1 week, urinary sodium excretion was markedly reduced. This was accompanied by a slight blood pressure reduction, no heart rate change, and a significant increase in plasma renin activity, aldosterone, and MSNA (+23.0+/-4.6% P<0.05). Whereas baroreflex heart-rate control was unchanged, baroreflex modulation of MSNA was reduced by 46.8+/-5.1% (P<0.01). At the end of the 8-week low-sodium diet, the neurohumoral and baroreflex responses were similar to the ones observed after 1 week of the dietary intervention. All changes disappeared when regular sodium diet was restored. CONCLUSIONS: Thus, a moderate dietary sodium restriction triggers a sympathetic activation and a baroreflex impairment. Maintenance of low-sodium diet for several weeks does not attenuate these adverse adrenergic and reflex effects. PMID- 12370220 TI - Plasma von Willebrand factor and soluble p-selectin as indices of endothelial damage and platelet activation in 1321 patients with nonvalvular atrial fibrillation: relationship to stroke risk factors. AB - BACKGROUND: Epidemiological studies have identified clinical and echocardiographic factors associated with increased stroke risk in atrial fibrillation (AF), but mechanisms linking these factors to stroke in AF are incompletely understood. We hypothesized that stroke risk factors may be associated with increased endothelial damage/dysfunction and platelet activation among patients with AF. METHODS AND RESULTS: We measured plasma levels of von Willebrand factor (vWF, a marker of endothelial damage/dysfunction) and soluble P selectin (sP-sel, a marker of platelet activation) by ELISA in 1321 participants in the Stroke Prevention in Atrial Fibrillation (SPAF) III study and related these indices to the presence of stroke risk factors and cardiovascular disease. Age (P<0.001), prior cerebral ischemia (P<0.01), recent heart failure (P<0.001), diabetes (P<0.001), and body mass index (P<0.001) were independently associated with increased vWF (r(2) adjusted=9%). Independent associates of increased sP-sel were diabetes (P=0.01), peripheral vascular disease (P<0.001), and current smoking (P=0.01), whereas prior cerebral ischemia (P=0.002) and female sex (P<0.001) were associated with reduced sP-sel (r(2) adjusted=4%). Using prospectively validated stroke risk stratification criteria, we observed a significant stepwise increase in vWF from low- to moderate- to high-risk groups (r(2) adjusted=3%, P<0.001), whereas sP-sel remained constant (P= 0.24). CONCLUSIONS: Four recognized risk factors for stroke in AF (advancing age, prior cerebral ischemia, recent heart failure, and diabetes) were independently associated with raised plasma vWF (or endothelial damage/dysfunction), whereas only 1 (diabetes) was associated with increased sP-sel (platelet activation). Further longitudinal studies are now needed to confirm relationships between endothelial damage/dysfunction, platelet activation, and stroke in AF. PMID- 12370221 TI - Alternans of atrial action potentials during atrial flutter as a precursor to atrial fibrillation. AB - BACKGROUND: The mechanisms underlying the transition of typical atrial flutter (Afl) to fibrillation (AF) remain unclear. We set out to test the hypothesis that Afl disorganizes to AF via alternans of atrial action potentials. METHODS AND RESULTS: In 38 patients with Afl, monophasic action potentials (MAPs) were recorded at the isthmus and either high or low right atrium (HRA, LRA) during overdrive pacing to 160 ms or to the initiation of AF, whichever came first. MAP duration measured at 90% repolarization was longer at the isthmus in all patients, and failed to shorten with rate, compared with the HRA (n=38) or LRA (n=5). In 20 patients who developed AF, progressive pacing first caused alternans of isthmus MAP duration and amplitude at mean cycle length of 219+/-45 ms, followed by AF at a mean onset cycle length of 184+/-38 ms. Subsets of this group showed spontaneous action potential duration alternans at the isthmus (11 of 20 patients) and 2:1 isthmus conduction block immediately preceding AF (4 of 20 patients). In the 18 patients who did not develop AF, MAP alternans was less common (9 of 18 patients; P<0.0003), and occurred only at faster pacing (cycle length=169+/-25 ms; P<0.05). CONCLUSIONS: In patients with typical Afl, action potential duration rate maladaptation at the isthmus may lead to action potential duration alternans and conduction block preceding the transition to AF. These isthmus characteristics may enable the spontaneous initiation of AF through wavefront fractionation and may explain the benefits of isthmus ablation in preventing AF recurrence. PMID- 12370222 TI - Sympathetic nerve activity in end-stage renal disease. AB - BACKGROUND: Uremia is proposed to increase sympathetic nerve activity (SNA) in hemodialysis patients. The aims of the present study were to determine whether reversal of uremia by successful kidney transplantation (RTX) eliminates the increased SNA and whether signals arising in the diseased kidneys contribute to the increased SNA in renal failure. METHODS AND RESULTS: We compared muscle sympathetic nerve activity (MSNA) in 13 hemodialysis patients wait-listed for RTX and in renal transplantation patients with excellent graft function treated with cyclosporine (RTX-CSA, n=13), tacrolimus (RTX-FK, n=13), or without calcineurin inhibitors (RTX-Phi, n=6), as well as in healthy volunteers (CON, n=15). In addition to the above patients with present diseased native kidneys, we studied 16 RTX patients who had undergone bilateral nephrectomy (RTX-NE). Data are mean+/ SEM. MSNA was significantly elevated in hemodialysis patients (43+/-4 bursts/min), RTX-CSA (44+/-5 bursts/min), RTX-FK (34+/-3 bursts/min), and RTX-Phi (44+/-5 bursts/min) as compared with CON (21+/-3 bursts/min), despite excellent graft function after RTX. RTX-NE had significantly reduced MSNA (20+/-3 bursts/min) when compared with RTX patients. MSNA did not change significantly with RTX in 4 hemodialysis patients studied before and after RTX (44+/-6 versus 43+/-5 bursts/min, P=NS). In contrast, nephrectomy resulted in reduced MSNA in all 6 RTX patients studied before and after removal of the second native kidney. CONCLUSIONS: Despite correction of uremia, increased SNA is observed in renal transplant recipients with diseased native kidneys at a level not significantly different from chronic hemodialysis patients. The increased SNA seems to be mediated by signals arising in the native kidneys that are independent of circulating uremia related toxins. PMID- 12370223 TI - Anatomic variability in coronary arterial distribution with regard to the arterial switch procedure. AB - BACKGROUND: We investigated the coronary arterial origins and course and the position of the great arteries in hearts with discordant ventriculoarterial connections. At the same time, we sought to evaluate the practicality of alphanumeric classifications in accounting for surgically relevant features of the coronary arteries. METHODS AND RESULTS: We studied 200 postmortem hearts, noting the patterns of coronary arterial branching, the vertical and horizontal location of the arterial orifices within the aortic sinuses, the course of the proximal coronary arteries in relation to the aortic wall, and the relations of the great arteries and their respective commissures. All hearts examined had concordant atrioventricular and discordant ventriculoarterial connections. We found 7 of the 8 predicted patterns for sinusal origin of the 3 major coronary arteries and identified 5 different positions of the arterial trunks relative to each other. A correlation was found between less frequent relationships of the arterial trunks and unusual patterns of coronary arterial branching, as well as with mismatch between the valvar commissures. CONCLUSIONS: The surgically relevant features of the coronary arteries in hearts with discordant ventriculoarterial connections are best described rather than classified. Correlations exist between certain, less frequent relations of the great arteries and unusual patterns of branching of the coronary arteries. The presence of unusual great arterial positions should alert the surgeon to potentially complicated arrangements of the origin and distribution of the coronary arteries. PMID- 12370224 TI - In vivo evidence for a role of toll-like receptor 4 in the development of intimal lesions. AB - BACKGROUND: Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model. METHODS AND RESULTS: Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF kappaB and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area+/-SEM, 9134+/-1714 versus 2353+/-1076 microm(2), P<0.01). In TLR4-defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wild-type mice (intimal area, 3859+/-904 microm(2), P=0.02 versus wild type). CONCLUSIONS: A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation. PMID- 12370225 TI - Vascular endothelin-B receptor system in vivo plays a favorable inhibitory role in vascular remodeling after injury revealed by endothelin-B receptor-knockout mice. AB - BACKGROUND: Two subtypes of endothelin (ET) receptors, ET(A) and ET(B), are distributed in vascular smooth muscle cells to cause contraction and proliferation. Vascular endothelial cells express only ET(B) receptors, which cause NO release. Although ET(A) receptor blockade is reported to be effective in ameliorating vascular remodeling, there is no report on the long-term effect of ET(B) receptor blockade on vascular remodeling after injury. METHODS AND RESULTS: ET(B) receptor-knockout (KO) mice, which were genetically rescued from lethal intestinal aganglionosis, and wild-type (WT) mice underwent complete ligation of the right common carotid artery, ie, a blood flow cessation model of vascular remodeling. Fourteen days after ligation, the intimal area, the ratio of intimal to medial areas, and the stenotic ratio in the ligated artery of KO mice were significantly increased compared with those of WT mice. The expression level of ET-1 mRNA in the ligated artery of KO mice was increased similarly to that of WT mice, whereas tissue NO(x) levels in lesions of KO mice were significantly lower than those of WT mice. Long-term treatment with the ET(A) receptor antagonist TA 0201 (0.5 mg x kg(-1) x d(-1)) significantly ameliorated vascular stenosis in both groups. Long-term treatment with the ET(B) receptor antagonist A-192621 (30 mg x kg(-1) x d(-1)) worsened vascular remodeling in WT mice. CONCLUSIONS: We demonstrated that inhibition of the ET(B) receptor system is harmful for vascular remodeling after injury, the mechanism of which is partly attributed to decreased NO release, in KO mice. These results suggest that the overall effect of vascular ET(B) receptors is antiproliferative in the injured artery. PMID- 12370226 TI - Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon-denuded rabbit aorta. AB - BACKGROUND: Restenosis is a frequent problem after invasive treatment of atherosclerotic vessels and is associated with intimal hyperplasia, which is primarily a result of proliferation and migration of smooth muscle cells, leading to the formation of neointima. Because there is no effective conventional medication for restenosis, gene therapy is a potential new treatment to prevent neointima formation. METHODS AND RESULTS: In the present study, we analyzed the effects of adenovirus-mediated extracellular superoxide dismutase (EC-SOD) gene transfer (3x10(9) pfu/kg AdEC-SOD versus AdLacZ control virus) on neointima formation in balloon-denuded rabbit aortas. Local catheter-mediated gene transfer to the arterial wall reduced restenosis (P<0.001) and decreased the number of macrophages in the transduced segment (P<0.001) 2 weeks and 4 weeks after the gene transfer compared with AdLacZ controls. Transgene expression was detected in the arterial wall by RT-PCR 2 weeks after the procedure, and the production of superoxide anion was reduced after the gene transfer. Recovery of the endothelial layer was enhanced in EC-SOD-transduced rabbits compared with LacZ controls (P<0.001) 2 weeks after the gene transfer. The therapeutic effect was found to be extended, affecting the gene transfer site and flanking aortic segments from the renal arteries to the bifurcation. However, systemic AdEC-SOD gene transfer to liver did not have any effects on restenosis. CONCLUSIONS: The results suggest that EC-SOD gene transfer reduces restenosis and may be useful for the prevention of intimal hyperplasia after vascular manipulations. PMID- 12370227 TI - Ionic and cellular basis for the predominance of the Brugada syndrome phenotype in males. AB - BACKGROUND: The Brugada syndrome displays an autosomal dominant mode of transmission with low penetrance. Despite equal genetic transmission of the disease, the clinical phenotype is 8 to 10 times more prevalent in males than in females. The basis for this intriguing sex-related distinction is unknown. The present study tests the hypothesis that the disparity in expression of the Brugada phenotype is a result of a more prominent I(to)-mediated action potential notch in the right ventricular (RV) epicardium of males versus females. METHODS AND RESULTS: We studied epicardial tissue slices, arterially perfused wedge preparations, and dissociated epicardial myocytes isolated from male and female canine hearts. RV epicardium action potential phase 1 amplitude was 64.8+/-2.0% of that of phase 2 in males compared with 73.8+/-4.4% in females (P<0.05) at a cycle length of 2000 ms. I(to) density was 26% smaller and time constant for inactivation 17% smaller at +40 mV in female versus male RV epicardial cells (P<0.05). The other functional characteristics of I(to), including the voltage dependence of inactivation and time course of reactivation, were no different between the sexes. Pinacidil caused loss of action potential dome in male, but not female, RV epicardial tissue slices. Terfenadine (5 micromol/L) induced phase 2 reentry in 6 of 7 male but only 2 of 7 female arterially perfused wedge preparations. Two of 6 male and 1 of 2 female preparations developed polymorphic ventricular tachycardia/ventricular fibrillation. CONCLUSIONS: Our results suggest that the predominance of the Brugada phenotype in males is a result of the presence of a more prominent I(to) in males versus females. PMID- 12370228 TI - Ionic mechanisms of acquired QT prolongation and torsades de pointes in rabbits with chronic complete atrioventricular block. AB - BACKGROUND: The ionic basis of acquired QT prolongation and torsade de pointes (TdP) unrelated to drugs is not fully understood. METHODS AND RESULTS: We created a rabbit model with chronic complete atrioventricular block (AVB) (n=34), which showed prominent QT prolongation (by 120%), high incidence of spontaneous TdP (71%), and cardiac hypertrophy. Patch-clamp experiments were performed in left ventricular myocytes from 9 rabbits (8 with TdP, 1 without TdP) at approximately 21 days of AVB and from 8 sham-operated controls with sinus rhythm. Action potential duration was prolonged in AVB myocytes compared with control (+61% at 0.5 Hz, +21% at 3 Hz). Both rapidly and slowly activating components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in AVB myocytes were significantly smaller than in control by 50% and 55%, respectively. There was no significant difference in Ca(2+)-independent transient outward current (I(to1)). L-type Ca(2+) current (I(Ca,L)) in control and AVB myocytes was similar in peak amplitude, but the half voltage for activation was shifted to the negative direction (5.9 mV) in AVB myocytes. Voltage dependence of I(Ca,L) inactivation was not different in control and AVB myocytes. The inward rectifier K(+) current (I(K1)) significantly increased in AVB myocytes compared with control. CONCLUSIONS: In the rabbit, chronic AVB leads to prominent QT prolongation and high incidence of spontaneous TdP. Downregulation of both I(Kr) and I(Ks) in association with altered I(Ca,L) activation kinetics may underlie the arrhythmogenic ventricular remodeling. PMID- 12370229 TI - Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. AB - BACKGROUND: Peripheral blood mononuclear cells (PBMNCs), platelets, and polymorphonuclear leukocytes (PMNs) contain various angiogenic factors and cytokines. METHODS AND RESULTS: Unilateral hindlimb ischemia was surgically induced in athymic nude rats, and fluorescence-labeled human blood cells (PBMNCs [10(7) cells]+platelets [10(9)] or PBMNCs [10(7)]+platelets [10(9)]+PMNs [10(7)]) were intramuscularly implanted into the ischemic limbs. Laser Doppler imaging revealed markedly increased blood perfusion in PBMNC+platelet-implanted limbs (44% increase, P<0.001) compared with control implantation of human umbilical vein vascular endothelial cells. The addition of PMNs to PBMNCs+platelets attenuated blood perfusion (27% decrease, P<0.01). Neocapillary densities were increased by implantation of PBMNCs+platelets or platelets alone (3.5-fold and 2.4-fold, respectively; P<0.001), whereas PMNs inhibited (32%, P<0.05) PBMNC+ platelet-mediated capillary formation. There was no incorporation of implanted PBMNCs into neocapillaries, whereas PBMNCs and platelets accumulated around arterioles after implantation. Cellular extract from PBMNCs+platelets, in which vascular endothelial growth factor (VEGF), basic fibroblast growth factor, platelet-derived growth factor-AB, and transforming growth factor-beta were detected, markedly stimulated tubule formation of human umbilical vein vascular endothelial cells. Anti-VEGF neutralizing antibody markedly inhibited tubule formation and in vivo vessel formation. Neutrophil elastase inhibitor blocked the antiangiogenic action of PMNs, whereas inhibitors of oxygen metabolites had no effect. CONCLUSIONS: This study demonstrated that implantation of PBMNCs and platelets into ischemic limbs effectively induces collateral vessel formation by supplying angiogenic factors (mainly VEGF) and cytokines, suggesting that this cell therapy is useful as a novel strategy for therapeutic angiogenesis. PMID- 12370230 TI - Computed tomography and magnetic resonance imaging for noninvasive coronary angiography and plaque imaging: current and potential future concepts. PMID- 12370231 TI - Images in cardiovascular medicine. Feeding artery of a left atrial myxoma demonstrated by three-dimensional volume-rendering images with multislice computed tomography. PMID- 12370232 TI - Effect of azithromycin on endothelial function of patients with coronary artery disease and evidence of Chlamydia pneumoniae infection. PMID- 12370233 TI - Computed tomographic imaging of anomalous coronary arteries. PMID- 12370234 TI - Randomized trial of a distal embolic protection device during percutaneous intervention of saphenous vein aorto-coronary bypass grafts. PMID- 12370235 TI - Shortened head-up tilting test guided by systolic pressure reductions in neurocardiogenic syncope. PMID- 12370236 TI - Utility of B-natriuretic peptide in detecting diastolic dysfunction: comparison with Doppler velocity recordings. PMID- 12370237 TI - Stress single photon emission computed tomography in patients with normal electrocardiograms. PMID- 12370238 TI - Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy. PMID- 12370240 TI - The LIM-only protein FHL2 interacts with beta-catenin and promotes differentiation of mouse myoblasts. AB - FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified beta-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of beta-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts beta-catenin-mediated activation of a TCF/LEF dependent reporter gene in a dose-dependent and muscle cell-specific manner. After injection into Xenopus embryos, FHL2 inhibited the beta-catenin-induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with beta-catenin. PMID- 12370241 TI - An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. AB - Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LTB4) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants. p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy. PMID- 12370243 TI - DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals. AB - Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway. PMID- 12370242 TI - TGFbeta induces GDNF responsiveness in neurons by recruitment of GFRalpha1 to the plasma membrane. AB - We have previously shown that the neurotrophic effect of glial cell line-derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)beta. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFbeta promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFbeta, pretreatment with TGFbeta confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFbeta-induced recruitment of the glycosyl phosphatidylinositol-anchored GDNF receptor (GFR)alpha1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRalpha1 can promote survival in the absence of TGFbeta. Our data suggest that TGFbeta is involved in GFRalpha1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects. PMID- 12370244 TI - Influence of cargo size on Ran and energy requirements for nuclear protein import. AB - Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC. PMID- 12370245 TI - Gab1 and SHP-2 promote Ras/MAPK regulation of epidermal growth and differentiation. AB - In epidermis, Ras can influence proliferation and differentiation; however, regulators of epidermal Ras function are not fully characterized, and Ras effects on growth and differentiation are controversial. EGF induced Ras activation in epidermal cells along with phosphorylation of the multisubstrate docking protein Gab1 and its binding to SHP-2. Expression of mutant Gab1Y627F deficient in SHP-2 binding or dominant-negative SHP-2C459S reduced basal levels of active Ras and downstream MAPK proteins and initiated differentiation. Differentiation triggered by both Gab1Y627F and SHP-2C459S could be blocked by coexpression of active Ras, consistent with Gab1 and SHP-2 action upstream of Ras in this process. To study the role of Gab1 and SHP-2 in tissue, we generated human epidermis overexpressing active Gab1 and SHP-2. Both proteins stimulated proliferation. In contrast, Gab1Y627F and SHP-2C459S inhibited epidermal proliferation and enhanced differentiation. Consistent with a role for Gab1 and SHP-2 in sustaining epidermal Ras/MAPK activity, Gab1-/- murine epidermis displayed lower levels of active Ras and MAPK with postnatal Gab1-/- epidermis, demonstrating the hypoplasia and enhanced differentiation seen previously with transgenic epidermal Ras blockade. These data provide support for a Ras role in promoting epidermal proliferation and opposing differentiation and indicate that Gab1 and SHP-2 promote the undifferentiated epidermal cell state by facilitating Ras/MAPK signaling. PMID- 12370246 TI - MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells. AB - Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells. PMID- 12370247 TI - The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. AB - Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family. PMID- 12370248 TI - Both midzone and astral microtubules are involved in the delivery of cytokinesis signals: insights from the mobility of aurora B. AB - To address the mechanism that coordinates cytokinesis with mitosis, we have studied the dynamics of aurora B, a chromosomal passenger protein involved in signaling cytokinesis. Photobleaching analyses indicated dynamic exchange of aurora B between a centromeric and a cytoplasmic pool before anaphase onset, and stable associations with microtubules after anaphase onset. Bleaching near centromeres upon anaphase onset affected the subsequent appearance of fluorescence along midzone microtubules, but not that near the lateral equatorial cortex, suggesting that there were centromeric-dependent and -independent pathways that transported aurora B to the equator. The former delivered centromeric aurora B along midzone microtubules, whereas the latter delivered cytoplasmic aurora B along astral microtubules. We suggest that cultured cells use midzone microtubules as the primary signaling pathway for cytokinesis, whereas embryos, with their stockpile of cytoplasmic proteins and large sizes, rely primarily on astral microtubules. PMID- 12370249 TI - Alpha E: no more rejection? PMID- 12370250 TI - CD103 expression is required for destruction of pancreatic islet allografts by CD8(+) T cells. AB - The mechanisms by which CD8 effector populations interact with epithelial layers is a poorly defined aspect of adaptive immunity. Recognition that CD8 effectors have the capacity to express CD103, an integrin directed to the epithelial cell specific ligand E-cadherin, potentially provides insight into such interactions. To assess the role of CD103 in promoting CD8-mediated destruction of epithelial layers, we herein examined the capacity of mice with targeted disruption of CD103 to reject pancreatic islet allografts. Wild-type hosts uniformly rejected islet allografts, concomitant with the appearance of CD8(+)CD103(+) effectors at the graft site. In contrast, the majority of islet allografts transplanted into CD103(-/-) hosts survived indefinitely. Transfer of wild-type CD8 cells into CD103(-/-) hosts elicited prompt rejection of long-surviving islet allografts, whereas CD103(-/-) CD8 cells were completely ineffectual, demonstrating that the defect resides at the level of the CD8 cell. CD8 cells in CD103(-/-) hosts exhibited normal effector responses to donor alloantigens in vitro and trafficked normally to the graft site, but strikingly failed to infiltrate the islet allograft itself. These data establish a causal relationship between CD8(+)CD103(+) effectors and destruction of graft epithelial elements and suggest that CD103 critically functions to promote intragraft migration of CD8 effectors into epithelial compartments. PMID- 12370251 TI - A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. AB - The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE(-)(/)(-) mice before the development of manifest atherosclerotic lesions. Platelet-endothelial cell interaction involved both platelet glycoprotein (GP)Ibalpha and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE(-)(/)(-) mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process. PMID- 12370252 TI - Modulation of Kv channel expression and function by TCR and costimulatory signals during peripheral CD4(+) lymphocyte differentiation. AB - Ionic signaling pathways, including voltage-dependent potassium (Kv) channels, are instrumental in antigen-mediated responses of peripheral T cells. However, how Kv channels cooperate with other signaling pathways involved in T cell activation and differentiation is unknown. We report that multiple Kv channels are expressed by naive CD4(+) lymphocytes, and that the current amplitude and kinetics are modulated by antigen receptor-mediated stimulation and costimulatory signals. Currents expressed in naive CD4(+) lymphocytes are consistent with Kv1.1, Kv1.2, Kv1.3, and Kv1.6. Effector CD4(+) cells generated by optimal TCR and costimulation exhibit only Kv1.3 current, but at approximately sixfold higher levels than naive cells. CD4(+) lymphocytes anergized through partial stimulation exhibit similar Kv1.1, Kv1.2, and/or Kv1.6 currents, but approximately threefold more Kv1.3 current than naive cells. To determine if Kv channels contribute to the distinct functions of naive, effector, and anergized T cells, we tested their role in immunoregulatory cytokine production. Each Kv channel is required for maximal IL-2 production by naive CD4(+) lymphocytes, whereas none appears to play a role in IL-2, IL-4, or IFN-gamma production by effector cells. Interestingly, Kv channels in anergized lymphocytes actively suppress IL-4 production, and these functions are consistent with a role in regulating the membrane potential and calcium signaling. PMID- 12370253 TI - The D0 domain of KIR3D acts as a major histocompatibility complex class I binding enhancer. AB - In contrast to the KIR2D:HLA-C interaction, little is known of KIR3DL1's interaction with HLA-B or the role of D0, the domain not present in KIR2D. Differences in the strength and specificity for major histocompatibility complex class I of KIR3DL1 and its common chimpanzee homologue Pt-KIR3DL1/2 were exploited to address these questions. Domain-swap, deletion, and site-directed mutants of KIR3DL1 were analyzed for HLA-B binding using a novel, positively signaling cell-cell binding assay. Natural 'deletion' of residues 50 and 51 from its D0 domain causes Pt-KIR3DL1/2 to bind Bw4(+) HLA-B allotypes more avidly than does KIR3DL1. Deletion of these residues from KIR3DL1, or their substitution for alanine, enhanced binding of Bw4(+) HLA-B. None of 15 different point mutations in D0 abrogated KIR3DL1 binding to Bw4(+) HLA-B. In contrast point mutations in the D1 and D2 domains of KIR3DL1, made from knowledge of KIR2D:HLA-C interactions, disrupted binding to Bw4(+) HLA-B. The results are consistent with a model in which D1 and D2 make the principal contacts between KIR3DL1 and HLA-B while D0 acts through a different mechanism to enhance the interaction. This modulatory role for D0 is compatible with natural loss of expression of the D0 domain, a repeated event in the evolution of functional KIR genes. PMID- 12370254 TI - Regulation of the subcellular localization of tumor necrosis factor receptor associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling. AB - Tumor necrosis factor receptor-associated factor (TRAF)2 is a critical adaptor molecule for tumor necrosis factor (TNF) receptors in inflammatory and immune signaling. Upon receptor engagement, TRAF2 is recruited to CD40 and translocates to lipid rafts in a RING finger-dependent process, which enables the activation of downstream signaling cascades including c-Jun NH(2)-terminal kinase (JNK) and nuclear factor (NF)-kappaB. Although TRAF1 can displace TRAF2 and CD40 from raft fractions, it promotes the ability of TRAF2 activate signaling over a sustained period of time. Removal of the RING finger of TRAF2 prevents its translocation into detergent-insoluble complexes and renders it dominant negative for signaling. TRAF1(-/-) dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation. Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-kappaB activation. These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2. PMID- 12370255 TI - Interleukin 15 controls both proliferation and survival of a subset of memory phenotype CD8(+) T cells. AB - Previous work has shown that memory-phenotype CD44(hi) CD8(+) cells are controlled by a cytokine, interleukin (IL)-15. However, the dependency of CD44(hi) CD8(+) cells on IL-15 is partial rather than complete. Here, evidence is presented that CD44(hi) CD8(+) cells comprise a mixed population of IL-15 dependent and IL-15-independent cells. The major subset of CD122(hi) CD44(hi) CD8(+) cells is heavily dependent on IL-15 by three different parameters, namely (1) "bystander" proliferation induced via IFN-induced stimulation of the innate immune system, (2) normal "background" proliferation, and (3) T cell survival; IL 15 dependency is most extreme for the Ly49(+) subset of CD122(hi) CD44(hi) CD8(+) cells. In contrast to CD122(hi) cells, the CD122(lo) subset of CD44(hi) CD8(+) cells is IL-15 independent; likewise, being CD122(lo), CD44(hi) CD4(+) cells are IL-15 independent. Thus, subsets of memory-phenotype T cells differ radically in their sensitivity to IL-15. PMID- 12370256 TI - Peripheral deletion of autoreactive CD8 T cells by cross presentation of self antigen occurs by a Bcl-2-inhibitable pathway mediated by Bim. AB - By transgenic expression of ovalbumin (OVA) as a model self antigen in the beta cells of the pancreas, we have shown that self tolerance can be maintained by the cross-presentation of this antigen on dendritic cells in the draining lymph nodes. Such cross-presentation causes initial activation of OVA-specific CD8 T cells, which proliferate but are ultimately deleted; a process referred to as cross-tolerance. Here, we investigated the molecular basis of cross-tolerance. Deletion of CD8 T cells was prevented by overexpression of Bcl-2, indicating that cross-tolerance was mediated by a Bcl-2 inhibitable pathway. Recently, Bim, a pro apoptotic Bcl-2 family member whose function can be inhibited by Bcl-2, was found to play a critical role in the deletion of autoreactive thymocytes, leading us to examine its role in cross-tolerance. Bim-deficient T cells were not deleted in response to cross-presented self-antigen, strongly implicating Bim as the pro apoptotic mediator of cross-tolerance. PMID- 12370257 TI - CD4 effector T cell subsets in the response to influenza: heterogeneity, migration, and function. AB - The immune response of naive CD4 T cells to influenza virus is initiated in the draining lymph nodes and spleen, and only after effectors are generated do antigen-specific cells migrate to the lung which is the site of infection. The effector cells generated in secondary organs appear as multiple subsets which are a heterogeneous continuum of cells in terms of number of cell divisions, phenotype and function. The effector cells that migrate to the lung constitute the more differentiated of the total responding population, characterized by many cell divisions, loss of CD62L, down-regulation of CCR7, stable expression of CD44 and CD49d, and transient expression of CCR5 and CD25. These cells also secrete high levels of interferon gamma and reduced levels of interleukin 2 relative to those in the secondary lymphoid organs. The response declines rapidly in parallel with viral clearance, but a spectrum of resting cell subsets reflecting the pattern at the peak of response is retained, suggesting that heterogeneous effector populations may give rise to corresponding memory populations. These results reveal a complex response, not an all-or-none one, which results in multiple effector phenotypes and implies that effector cells and the memory cells derived from them can display a broad spectrum of functional potentials. PMID- 12370258 TI - Interleukin 21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naive Th cells into interferon gamma-producing Th1 cells. AB - The cytokine potential of developing T helper (Th) cells is directly shaped both positively and negatively by the cytokines expressed by the effector Th cell subsets. Here we find that the recently identified cytokine, interleukin (IL)-21, is preferentially expressed by Th2 cells when compared with Th1 cells generated in vitro and in vivo. Exposure of naive Th precursors to IL-21 inhibits interferon (IFN)-gamma production from developing Th1 cells. The repression of IFN-gamma production is specific in that the expression of other Th1 and Th2 cytokines is unaffected. IL-21 decreases the IL-12 responsiveness of developing Th cells by specifically reducing both signal transducer and activator of transcription 4 protein and mRNA expression. These results suggest that Th2 cell derived IL-21 regulates the development of IFN-gamma-producing Th1 cells which could serve to amplify a Th2 response. PMID- 12370259 TI - A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis. AB - We generated a mouse line in which the src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2 binding site of gp130, tyrosine 759, was mutated to phenylalanine (gp130(F759/F759)). The gp130(F759/F759) mice developed rheumatoid arthritis (RA)-like joint disease. The disease was accompanied by autoantibody production and accumulated memory/activated T cells and myeloid cells. Before the disease onset, the T cells were hyperresponsive and thymic selection and peripheral clonal deletion were impaired. The inhibitory effect of IL-6 on Fas ligand expression during activation-induced cell death (AICD) was augmented in gp130(F759/F759) T cells in a manner dependent on the tyrosine residues of gp130 required for signal transducer and activator of transcription 3 activation. Finally, we showed that disease development was dependent on lymphocytes. These results provide evidence that a point mutation of a cytokine receptor has the potential to induce autoimmune disease. PMID- 12370260 TI - Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide Synthase 2-independent defense in mice. AB - Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)-gamma and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to understanding the basis of Mtb virulence, the ability of gene-deleted mice incapable of making NOS2 (NOS2(-/-)), gp91(Phox) subunit of the respiratory burst NADPH-oxidase complex (Phox(-/-)), or either enzyme (NOS2/Phox(-/-)), to control airborne infection with the avirulent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with the virulent H37Rv strain. NOS2(-/-), Phox( /-), and NOS2/Phox(-/-) mice showed no deficiency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2(-/-) mice, but not Phox(-/ ) mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as virulent Mtb, depended on the synthesis of IFN-gamma, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN-gamma and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1-dependent, NOS2-independent mechanism of defense. PMID- 12370261 TI - Interferon gamma is required for activation-induced death of T lymphocytes. AB - The effector cytokine interferon gamma (IFN-gamma) may play a role in T cell homeostasis. We have examined the requirement for IFN-gamma in one mechanism that regulates T cell expansion and survival, activation-induced cell death (AICD). CD4(+) T cells lacking IFN-gamma or the Stat1 transcription factor are resistant to AICD. IFN-gamma is required for the production of caspases, and retrovirus mediated expression of caspase-8 restores the sensitivity of Stat1-deficient T cells to AICD. In vitro, IFN-gamma limits the expansion of T cells that are stimulated through their antigen receptors. Thus, IFN-gamma may function to control the expansion and persistence of T cells by promoting caspase-8-dependent apoptosis. PMID- 12370262 TI - Lessons from viral manipulation of protein disposal pathways. PMID- 12370263 TI - High-flux mitochondrial cholesterol trafficking, a specialized function of the adrenal cortex. PMID- 12370264 TI - Intracellular cholesterol transport. PMID- 12370265 TI - Regulation and mechanisms of macrophage cholesterol efflux. PMID- 12370266 TI - Consequences of cellular cholesterol accumulation: basic concepts and physiological implications. PMID- 12370267 TI - Endothelial integrins and angiogenesis: not so simple anymore. PMID- 12370268 TI - Anxiolytic drug targets: beyond the usual suspects. PMID- 12370269 TI - Antineutrophil cytoplasmic antibody and vasculitis: much more than a disease marker. PMID- 12370270 TI - PPARgamma ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis. AB - Several drugs approved for a variety of indications have been shown to exhibit antiangiogenic effects. Our study focuses on the PPARgamma ligand rosiglitazone, a compound widely used in the treatment of type 2 diabetes. We demonstrate, for the first time to our knowledge, that PPARgamma is highly expressed in tumor endothelium and is activated by rosiglitazone in cultured endothelial cells. Furthermore, we show that rosiglitazone suppresses primary tumor growth and metastasis by both direct and indirect antiangiogenic effects. Rosiglitazone inhibits bovine capillary endothelial cell but not tumor cell proliferation at low doses in vitro and decreases VEGF production by tumor cells. In our in vivo studies, rosiglitazone suppresses angiogenesis in the chick chorioallantoic membrane, in the avascular cornea, and in a variety of primary tumors. These results suggest that PPARgamma ligands may be useful in treating angiogenic diseases such as cancer by inhibiting angiogenesis. PMID- 12370271 TI - Inhibition of endothelial cell survival and angiogenesis by protein kinase A. AB - Receptors for the provisional ECM are important regulators of angiogenesis. One of these receptors, integrin alpha5beta1, plays a critical role in tumor- and growth factor-induced angiogenesis, because antagonists of this integrin potently inhibit angiogenesis and tumor growth. Here we show that the integrin alpha5beta1 promotes endothelial cell survival during angiogenesis in vivo by suppressing the activity of protein kinase A (PKA). Antagonists of integrin alpha5beta1 activate PKA, which then leads to the activation of caspase-8 and induction of apoptosis. Direct activation of PKA by cAMP or by expression of the PKA catalytic subunit also induces endothelial cell apoptosis, resulting in angiogenesis inhibition in vivo. Our studies indicate that ligation of integrin alpha5beta1 during angiogenesis suppresses an apoptotic program that is dependent on PKA. These studies also indicate that induction of endothelial cell apoptosis in vivo by genetic or pharmacological activation of PKA may be a useful strategy to inhibit angiogenesis. PMID- 12370272 TI - Pivotal role of CEACAM1 protein in the inhibition of activated decidual lymphocyte functions. AB - Lymphocytes in direct contact with embryonic extravillous trophoblasts constitute more than 40% of decidual cells and appear to play major roles in implantation and early gestation. A unique subset of NK cells, making up 70-80% of decidual lymphocytes, express high levels of CD56 but lack CD16. We have recently demonstrated a novel class I MHC-independent inhibitory mechanism of NK cell cytotoxicity that is mediated by CEACAM1 homotypic interactions. This mechanism is used by some melanoma cells to avoid attack, mainly by CD16(-) NK cells. We now demonstrate that CEACAM1 is expressed on primary extravillous trophoblasts and is upregulated on the vast majority of IL-2-activated decidual lymphocytes, including NK, T, and NKT cells. Importantly, we present evidence that CEACAM1 interactions inhibit the lysis, proliferation, and cytokine secretion of activated decidual NK, T, and NKT cells, respectively. In vivo analysis of decidual lymphocytes isolated from cytomegalovirus-infected (CMV-infected) pregnant women revealed a dramatic increase in the expression of CEACAM1. Finally, we suggest that a novel ligand for this adhesion molecule is present on the surface of CMV-infected fibroblasts. These combined results demonstrate a major role for the CEACAM1 protein in controlling local decidual immune responses. PMID- 12370273 TI - Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. AB - Antineutrophil cytoplasmic autoantibodies (ANCAs) are identified in the circulation of approximately 80% of patients with pauci-immune necrotizing and crescentic glomerulonephritis and systemic small vessel vasculitis, such as microscopic polyangiitis and Wegener granulomatosis. The most common antigen target for ANCAs is myeloperoxidase (MPO), which is found in neutrophils and monocytes. We report definitive experimental animal evidence that ANCAs are pathogenic. MPO knockout (Mpo(-/-)) mice were immunized with mouse MPO. Splenocytes from these mice or from control mice were injected intravenously into recombinase-activating gene-2-deficient (Rag2(-/-)) mice, which lack functioning B lymphocytes and T lymphocytes. All mice that received splenocytes developed mild to moderate glomerular immune deposits, but only mice that received 1 x 10(8) or 5 x 10(7) anti-MPO splenocytes developed severe necrotizing and crescentic glomerulonephritis, granulomatous inflammation, and systemic necrotizing vasculitis, including necrotizing arteritis and hemorrhagic pulmonary capillaritis. To test the pathogenic potential of antibodies alone, purified anti MPO IgG or control IgG was injected intravenously into Rag2(-/-) mice and wild type mice. Mice that received anti-MPO IgG but not mice that received control IgG developed focal necrotizing and crescentic glomerulonephritis with a paucity of glomerular Ig deposition. Thus, anti-MPO IgG alone was able to cause pauci-immune glomerular necrosis and crescent formation in the absence of functional T or B lymphocytes in Rag2(-/-) mice and in the presence of an intact immune system in wild-type C57BL/6J mice. This animal model offers strong support for a direct pathogenic role for ANCA IgG in human glomerulonephritis and vasculitis. PMID- 12370274 TI - The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II. AB - PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II-associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep. PMID- 12370275 TI - Lack of prolactin receptor signaling in mice results in lactotroph proliferation and prolactinomas by dopamine-dependent and -independent mechanisms. AB - Hypothalamic dopamine inhibits pituitary prolactin secretion and proliferation of prolactin-producing lactotroph cells by activating lactotroph dopamine D2 receptors (D2Rs). Conversely, prolactin (PRL) stimulates hypothalamic dopamine neurons via PRL receptors (PRLRs) in a short-loop feedback circuit. We used Drd2( /-) and Prlr(-/-) mutant mice to bypass this feedback and investigate possible dopamine-independent effects of PRL on lactotroph function. The absence of either receptor induced hyperprolactinemia and large prolactinomas in females. Small macroadenomas developed in aged Prlr(-/-) males, but only microscopic adenomas were found in Drd2(-/-) male mice. Pharmacologic studies in Prlr(-/-) mice with D2R agonists and antagonists demonstrated a significant loss of endogenous dopamine tone, i.e., constitutive inhibitory signaling by the D2R, in the pituitary. However, Prlr(-/-) mice exhibited more profound hyperprolactinemia and larger tumors than did age-matched Drd2(-/-) mice, and there were additive effects in compound homozygous mutant male mice. In vitro, PRL treatment markedly inhibited the proliferation of wild-type female and male Drd2(-/-) lactotrophs, but had no effect on female Drd2(-/-) lactotrophs, suggesting a downregulation or desensitization of PRLR in response to chronic hyperprolactinemia. We conclude that PRL inhibits lactotrophs by two distinct mechanisms: (a) indirectly by activation of hypothalamic dopamine neurons and (b) directly within the pituitary in a dopamine-independent fashion. PMID- 12370276 TI - IL-12 enhances the natural killer cell cytokine response to Ab-coated tumor cells. AB - The anti-tumor activity of recombinant mAb's directed against tumor cell growth receptors has generally been considered to result from direct antiproliferative effects, the induction of apoptosis, or possibly Ab-dependent cellular cytotoxicity mediated against tumor targets. However, it remains unclear to what degree these mechanisms actually aid in the clearance of Ab-coated tumor cells in vivo. We show here that NK cells secrete a distinct profile of potent immunostimulatory cytokines in response to dual stimulation with Ab-coated tumor cells and IL-12. This response could not be duplicated by costimulation with other ILs and was significantly enhanced in the presence of monocytes. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIII) and the IL-12 receptor expressed on NK cells. Coadministration of Ab-coated tumor cells and IL-12 to BALB/c mice resulted in enhanced circulating levels of NK cell-derived cytokines with the capacity to augment anti-tumor immunity. These findings suggest that, in addition to mediating cellular cytotoxicity and apoptosis, the anti-tumor activity of mAb's might also result from activation of a potent cytokine secretion program within immune effectors capable of recognizing mAb-coated targets. PMID- 12370277 TI - Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. AB - Under conditions of limited oxygen availability (hypoxia), multiple cell types release adenine nucleotides in the form of ATP, ADP, and AMP. Extracellular AMP is metabolized to adenosine by surface-expressed ecto-5'-nucleotidase (CD73) and subsequently activates surface adenosine receptors regulating endothelial and epithelial barrier function. Therefore, we hypothesized that hypoxia transcriptionally regulates CD73 expression. Microarray RNA analysis revealed an increase in CD73 and ecto-apyrase CD39 in hypoxic epithelial cells. Metabolic studies of CD39/CD73 function in intact epithelia revealed that hypoxia enhances CD39/CD73 function as much as 6 +/- 0.5-fold over normoxia. Examination of the CD73 gene promoter identified at least one binding site for hypoxia-inducible factor-1 (HIF-1) and inhibition of HIF-1alpha expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible CD73 expression. Studies using luciferase reporter constructs revealed a significant increase in activity in cells subjected to hypoxia, which was lost in truncated constructs lacking the HIF-1 site. Mutagenesis of the HIF-1alpha binding site resulted in a nearly complete loss of hypoxia-inducibility. In vivo studies in a murine hypoxia model revealed that hypoxia-induced CD73 may serve to protect the epithelial barrier, since the CD73 inhibitor alpha,beta-methylene ADP promotes increased intestinal permeability. These results identify an HIF-1-dependent regulatory pathway for CD73 and indicate the likelihood that CD39/CD73 protects the epithelial barrier during hypoxia. PMID- 12370278 TI - Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cepsilon. AB - Mice lacking protein kinase Cepsilon (PKCepsilon) are supersensitive to positive allosteric modulators of gamma aminobutyrate type A (GABA(A)) receptors. Since many of these compounds are anxiolytic, we examined whether anxiety-like behavior is altered in these mice. PKCepsilon-null mice showed reduced anxiety-like behavior and reduced levels of the stress hormones corticosterone and adrenocorticotrophic hormone (ACTH). This was associated with increased sensitivity to neurosteroid modulators of GABA(A) receptors. Treatment of PKCepsilon-null mice with the GABA(A) receptor antagonist bicuculline restored corticosterone levels and anxiety-like behavior to wild-type levels. These results suggest that increased GABA(A) receptor sensitivity to neurosteroids contributes to reduced anxiety-like behavior and stress hormone responses in PKCepsilon-null mice. The findings also suggest PKCepsilon as a possible therapeutic target for development of anxiolytics. PMID- 12370279 TI - Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor. AB - Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion. PMID- 12370280 TI - Anti-peptide autoantibodies and fatal anaphylaxis in NOD mice in response to insulin self-peptides B:9-23 and B:13-23. AB - There is evidence that amino acids 9-23 of the insulin B chain are a major target of anti-islet autoimmunity in type 1 diabetes. Administration of this peptide to NOD mice prevents diabetes, and phase I trials of an altered peptide ligand of B:9-23 are underway in humans. We were interested in long-term subcutaneous therapeutic administration of B:9-23 without adjuvant. To our initial surprise, the peptide consistently induced fatal anaphylaxis in NOD mice after 6 weeks of administration. Anaphylaxis could be blocked by a combination of antihistamine and platelet-activating factor antagonist (but neither alone) or by a combination of anti-IgG receptor and anti-IgE antibodies. High titers of anti-B:9-23 antibodies were induced within 3-4 weeks of immunization with the peptide. Peptide B:13-23 also induced anaphylaxis and was more potent than peptide B:9-23. Antibodies induced by peptide B:9-23 and peptide B:13-23 did not cross-react with each other. Thus, the insulin peptides B:9-23 and B:13-23, even when administered subcutaneously in the absence of adjuvant, can induce a dramatic humoral response leading to fatal anaphylaxis in NOD mice. PMID- 12370281 TI - Clinical and molecular analysis of patients with defects in micro heavy chain gene. AB - Autosomal recessive disorders of B cell development are rare and heterogeneous. To determine the proportion of affected patients who have defects in the micro heavy chain (IGHM) gene, we used single-stranded conformational polymorphism analysis to screen genomic DNA from 40 unrelated patients with early onset infections, profound hypogammaglobulinemia, and absent B cells. All of the patients were genotypically normal in BTK, the gene that underlies X-linked agammaglobulinemia. Eight different mutations in the micro heavy chain were identified in 19 members of 12 unrelated families. Four of the mutations were large deletions that removed more than 40 kb of DNA in the IGHM locus. In six of the 12 families, the affected patients had an identical single base pair substitution, a G-->A, at the -1 position of the alternative splice site. Immunoglobulin haplotype analysis showed that this mutation occurred on at least three different haplotypes, indicating that this is a hot spot for mutations. Compared with patients with mutations in Btk, patients with defects in the micro heavy chain had an earlier onset of disease and more complications. Our study indicates that at least 20-30% of patients with autosomal recessive defects in B cell development have mutations in the micro heavy chain. PMID- 12370282 TI - The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. AB - The present study was aimed at determining whether hepcidin, a recently identified peptide involved in iron metabolism, plays a role in conditions associated with both iron overload and iron deficiency. Hepcidin mRNA levels were assessed in two models of anemia, acute hemolysis provoked by phenylhydrazine and bleeding provoked by repeated phlebotomies. Hepcidin response to hypoxia was also studied, both ex vivo, in human hepatoma cells, and in vivo. Anemia and hypoxia were associated with a dramatic decrease in liver hepcidin gene expression, which may account for the increase in iron release from reticuloendothelial cells and increase in iron absorption frequently observed in these situations. A single injection of turpentine for 16 hours induced a sixfold increase in liver hepcidin mRNA levels and a twofold decrease in serum iron. The hyposideremic effect of turpentine was completely blunted in hepcidin-deficient mice, revealing hepcidin participation in anemia of inflammatory states. These modifications of hepcidin gene expression further suggest a key role for hepcidin in iron homeostasis under various pathophysiological conditions, which may support the pharmaceutical use of hepcidin agonists and antagonists in various iron homeostasis disorders. PMID- 12370283 TI - Deletion of phosphodiesterase 4D in mice shortens alpha(2)-adrenoceptor-mediated anesthesia, a behavioral correlate of emesis. AB - A combination of pharmacological and genetic approaches was used to determine the role of type 4 cAMP-specific cyclic nucleotide phosphodiesterase 4 (PDE4) in reversing alpha(2)-adrenoceptor-mediated anesthesia, a behavioral correlate of emesis in non-vomiting species. Among the family-specific PDE inhibitors, PDE4 inhibitors reduced the duration of xylazine/ketamine-induced anesthesia in mice, with no effect on pentobarbital-induced anesthesia. The rank order of the PDE4 inhibitors tested was 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (PMNPQ) > (R)-rolipram > (S)-rolipram >> (R)-N-[4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2 (4-pyridyl)ethyl]phenyl]N'-ethylurea (CT-2450). The specific roles of PDE4B and PDE4D in this model were studied using mice deficient in either subtype. PDE4D deficient mice, but not PDE4B-deficient mice, had a shorter sleeping time than their wild-type littermates under xylazine/ketamine-induced anesthesia, but not under that induced with pentobarbital. Concomitantly, rolipram-sensitive PDE activity in the brain stem was decreased only in PDE4D-deficient mice compared with their wild-type littermates. While PMNPQ significantly reduced the xylazine/ketamine-induced anesthesia period in wild-type mice and in PDE4B-null mice, it had no effect in PDE4D-deficient mice. These findings strongly support the hypothesis that inhibition of PDE4D is pivotal to the anesthesia-reversing effect of PMNPQ and is likely responsible for emesis induced by PDE4 inhibitors. PMID- 12370284 TI - Differential requirement of SAGA components for recruitment of TATA-box-binding protein to promoters in vivo. AB - The multisubunit Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is required to activate transcription of a subset of RNA polymerase II dependent genes. However, the contribution of each SAGA component to transcription activation is relatively unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay, we have systematically analyzed the role of SAGA components in the recruitment of TATA box binding protein (TBP) to SAGA-dependent promoters. We show that recruitment of TBP is diminished at a number of SAGA-dependent promoters in ada1delta, spt7delta, and spt20delta null mutants, consistent with previous biochemical data suggesting that these components maintain the integrity of the SAGA complex. We also find that Spt3p is generally required for TBP binding to SAGA-dependent promoters, consistent with biochemical and genetic experiments, suggesting that Spt3p interacts with and recruits TBP to the core promoter. By contrast, Spt8p, which has been proposed to be required for the interaction between Spt3p and TBP, is required for TBP binding at only a subset of SAGA-dependent promoters. Ada2p and Ada3p are both required for TBP recruitment to Gcn5p-dependent promoters, supporting previous biochemical data that Ada2p and Ada3p are required for the histone acetyltransferase activity of Gcn5p. Finally, our results suggest that TBP-associated-factor components of SAGA are differentially required for TBP binding to SAGA-dependent promoters. In summary, we show that SAGA-dependent promoters require different combinations of SAGA components for TBP recruitment, revealing a complex combinatorial network for transcription activation in vivo. PMID- 12370285 TI - Regulation of gene expression by internal ribosome entry sites or cryptic promoters: the eIF4G story. AB - As an alternative to the scanning mechanism of initiation, the direct-internal initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5' untranslated region (5'-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5'-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5'-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPbeta. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5'-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5' UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs. PMID- 12370287 TI - Early postnatal death and motor disorders in mice congenitally deficient in calnexin expression. AB - Calnexin is a ubiquitously expressed type I membrane protein which is exclusively localized in the endoplasmic reticulum (ER). In mammalian cells, calnexin functions as a chaperone molecule and plays a key role in glycoprotein folding and quality control within the ER by interacting with folding intermediates via their monoglucosylated glycans. In order to gain more insight into the physiological roles of calnexin, we have generated calnexin gene-deficient mice. Despite its profound involvement in protein folding, calnexin is not essential for mammalian-cell viability in vivo: calnexin gene knockout mice were carried to full term, although 50% died within 48 h and the majority of the remaining mice had to be sacrificed within 4 weeks, with only a very few mice surviving to 3 months. Calnexin gene-deficient mice were smaller than their littermates and showed very obvious motor disorders, associated with a dramatic loss of large myelinated nerve fibers. Thus, the critical contribution of calnexin to mammalian physiology is tissue specific. PMID- 12370286 TI - Induction of extracellular matrix-remodeling genes by the senescence-associated protein APA-1. AB - Human fibroblasts undergo cellular senescence after a finite number of divisions, in response to the erosion of telomeres. In addition to being terminally arrested in the cell cycle, senescent fibroblasts express genes that are normally induced upon wounding, including genes that remodel the extracellular matrix. We have identified the novel zinc finger protein APA-1, whose expression increased in senescent human fibroblasts independent of telomere shortening. Extended passage, telomerase-immortalized fibroblasts had increased levels of APA-1 as well as the cyclin-dependent kinase inhibitor p16. In fibroblasts, APA-1 was modified by the ubiquitin-like protein SUMO-1, which increased APA-1 half-life, possibly by blocking ubiquitin-mediated degradation. Overexpression of APA-1 did not cause cell cycle arrest; but, it induced transcription of the extracellular matrix remodeling genes MMP1 and PAI2, which are associated with fibroblast senescence. MMP1 and PAI2 transcript levels also increased in telomerase-immortalized fibroblasts that had high levels of APA-1, demonstrating that the matrix remodeling phenotype of senescent fibroblasts was not induced by telomere attrition alone. APA-1 was able to transactivate and bind to the MMP1 promoter, suggesting that APA-1 is a transcription factor that regulates expression of matrix-remodeling genes during fibroblast senescence. PMID- 12370288 TI - Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha. AB - Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER) resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response. PMID- 12370289 TI - Brevican-deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory. AB - Brevican is a brain-specific proteoglycan which is found in specialized extracellular matrix structures called perineuronal nets. Brevican increases the invasiveness of glioma cells in vivo and has been suggested to play a role in central nervous system fiber tract development. To study the role of brevican in the development and function of the brain, we generated mice lacking a functional brevican gene. These mice are viable and fertile and have a normal life span. Brain anatomy was normal, although alterations in the expression of neurocan were detected. Perineuronal nets formed but appeared to be less prominent in mutant than in wild-type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation (LTP). However, no obvious impairment of excitatory and inhibitory synaptic transmission was found, suggesting a complex cause for the LTP defect. Detailed behavioral analysis revealed no statistically significant deficits in learning and memory. These data indicate that brevican is not crucial for brain development but has restricted structural and functional roles. PMID- 12370290 TI - The rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin. AB - The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells. PMID- 12370291 TI - Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element. AB - ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-gamma) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFN-gamma-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-gamma-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-gamma-mediated reporter activity through the ISRE. This repression may provide a negative feedback mechanism operating after the initial transcriptional activation by IFN gamma. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-gamma-dependent gene regulation in an immune system-specific manner. PMID- 12370292 TI - Dynamic interplay between adhesive and lateral E-cadherin dimers. AB - E-cadherin, an adhesive transmembrane protein of epithelial adherens junctions, forms two types of detergent-resistant dimers: adhesive dimers consisting of cadherin molecules derived from two neighboring cells and lateral dimers incorporating cadherins of the same cell. Both dimers depend on the integrity of the same residue, Trp156. While the relative amounts of these complexes are not certain, we show here that in epithelial A-431 cells, adhesive dimers may be a prevalent form. Inactivation of the calcium-binding sites, located between successive cadherin ectodomains, drastically reduced the amount of adhesive dimers and concomitantly increased the amount of lateral dimers. A similar interdependence of adhesive and lateral dimers was observed in digitonin permeabilized cells. In these cells, adhesive dimers immediately disassembled after lowering the Ca2+ concentration below 0.1 mM. The disappearance of adhesive dimers was counterbalanced by an increase in Trp156-dependent lateral dimers. Increasing the calcium concentration to a normal level rapidly restored the original balance between adhesive and lateral dimers. We also present evidence that E-cadherin dimers in vivo have a short lifetime. These observations suggest that cadherin-mediated adhesion is based on the dynamic cycling of E-cadherin between monomeric and adhesive dimer states. PMID- 12370294 TI - Pleiohomeotic can link polycomb to DNA and mediate transcriptional repression. AB - Polycomb group (PcG) proteins function through cis-acting DNA elements called PcG response elements (PREs) to stably silence developmental regulators, including the homeotic genes. However, the mechanism by which they are targeted to PREs remains largely unclear. Pleiohomeotic (PHO) is a sequence-specific DNA-binding PcG protein and therefore may function to tether other PcG proteins to the DNA. Here, we show that PHO can directly bind to a Polycomb (PC)-containing complex as well as the Brahma (BRM) chromatin-remodeling complex. PHO contacts the BRM complex through its zinc finger DNA-binding domain and a short N-terminal region. A distinct domain of PHO containing a conserved motif contacts the PcG proteins PC and Polyhomeotic (PH). With mobility shift assays and DNA pulldown experiments, we demonstrated that PHO is able to link PC, which lacks sequence specific DNA-binding activity, to the DNA. Importantly, we found that the PC binding domain of PHO can mediate transcriptional repression in transfected Drosophila Schneider cells. Concomitant overexpression of PC resulted in stronger PHO-directed repression that was dependent on its PC-binding domain. Together, these results suggest that PHO can contribute to PRE-mediated silencing by direct recruitment of a PC complex to repress transcription. PMID- 12370293 TI - Coupling of DNA synthesis and histone synthesis in S phase independent of cyclin/cdk2 activity. AB - DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin. PMID- 12370295 TI - hSWI/SNF-catalyzed nucleosome sliding does not occur solely via a twist-diffusion mechanism. AB - Nucleosome remodeling by the hSWI/SNF complex and other chromatin remodeling complexes can cause translocation (sliding) of the histone octamer in cis along DNA. Structural and biochemical evidence suggest that sliding involves a DNA twist-diffusion process whereby the DNA rotates about the helical axis without major displacement from the surface of the nucleosome and that this process may be driven by torsional stress within the DNA. We report that hSWI/SNF efficiently catalyzes sliding of nucleosomes containing branched DNAs as steric blocks to twist-diffusion and a nick to allow dissipation of torsional stress within the nucleosome. These results suggest that SWI/SNF-catalyzed nucleosome sliding does not occur exclusively via a simple twist-diffusion mechanism and support models in which the DNA maintains its rotational orientation to and is at least partially separated from the histone surface during nucleosome translocation. PMID- 12370296 TI - Identification, cDNA cloning, and targeted deletion of p70, a novel, ubiquitously expressed SH3 domain-containing protein. AB - In a screen for proteins that interact with Jak2, we identified a previously uncharacterized 70-kDa protein and cloned the corresponding cDNA. The predicated sequence indicates that p70 contains an SH3 domain and a C-terminal domain with similarities to the catalytic motif of phosphoglycerate mutase. p70 transcripts were found in all tissues examined. Similarly, when an antibody raised against a C-terminal peptide to analyze p70 protein expression was used, all murine tissues examined were found to express p70. To investigate the in vivo role of p70, we generated a p70-deficient mouse strain. Mice lacking p70 are viable, develop normally, and do not display any obvious abnormalities. No differences were detected in various hematological parameters, including bone marrow colony forming ability, in response to cytokines that utilize Jak2. In addition, no impairment in B- and T-cell development and proliferative ability was detected. PMID- 12370297 TI - Circadian regulation of nocturnin transcription by phosphorylated CREB in Xenopus retinal photoreceptor cells. AB - Although CLOCK/BMAL1 heterodimers have been implicated in transcriptional regulation of several rhythmic genes in vitro through E-box sequence elements, little is known about how the circadian clock regulates rhythmic genes with diverse phases in vivo. The gene nocturnin is rhythmically transcribed in Xenopus retinal photoreceptor cells, which contain endogenous circadian clocks. Transcription of nocturnin peaks in these cells in the middle of the night, while CLOCK/BMAL1 activity peaks during the early morning. We have identified a novel protein-binding motif within the nocturnin promoter, which we designated the nocturnin element (NE). Although the NE sequence closely resembles an E-box, our data show that it functions as a cyclic AMP response element (CRE) by binding CREB. Furthermore, phosphorylated CREB (P-CREB) levels are rhythmic in Xenopus photoreceptors, with a phase similar to that of nocturnin transcription. Our results suggest that P-CREB controls the rhythmic regulation of nocturnin transcription and perhaps that of other night phase genes. The NE may be an evolutionary intermediate between the E-box and CRE sequences, both of which seem to be involved in the circadian control of transcription, but have evolved to drive transcription with different phases in these clock-containing cells. PMID- 12370298 TI - Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis. AB - Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events. PMID- 12370299 TI - High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2. AB - The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA. PMID- 12370300 TI - Generation and characterization of ecto-ADP-ribosyltransferase ART2.1/ART2.2 deficient mice. AB - This is the first study reporting the inactivation of a member of the mouse gene family of toxin-related ecto-ADP-ribosyltransferases (ARTs). Transfer of the ADP ribose moiety from NAD onto extracellular arginine residues on T-cell membrane proteins is mediated by glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to ecto-NAD blocks T-cell activation and induces T-cell apoptosis. To determine a possible role of ecto-ART2.1 and ART2.2 in these processes, we generated ART2.1/ART2.2 double-knockout mice. ART2-deficient mice were healthy and fertile and showed normal development of lymphoid organs. ART2 deficient T cells showed a dramatically reduced capacity to ADP-ribosylate cell surface proteins, indicating that most if not all ART activity on the T-cell surface can be attributed to the ART2s. Moreover, ART2-deficient T cells were completely resistant to NAD-induced apoptosis and partially resistant to NAD mediated suppression of proliferation. These results demonstrate that the ART2 ectoenzymes are an essential component in the regulation of T-cell functions by extracellular NAD, e.g., following release of NAD upon lysis of cells in tissue injury and inflammation. PMID- 12370302 TI - Centromere targeting element within the histone fold domain of Cid. AB - Centromeres require specialized nucleosomes; however, the mechanism of localization is unknown. Drosophila sp. centromeric nucleosomes contain the Cid H3-like protein. We have devised a strategy for identifying elements within Cid responsible for its localization to centromeres. By expressing Cid from divergent Drosophila species fused to green fluorescent protein in Drosophila melanogaster cells, we found that D. bipectinata Cid fails to localize to centromeres. Cid chimeras consisting of the D. bipectinata histone fold domain (HFD) replaced with segments from D. melanogaster identified loop I of the HFD as being critical for targeting to centromeres. Conversely, substitution of D. bipectinata loop I into D. melanogaster abolished centromeric targeting. In either case, loop I was the only segment capable of conferring targeting. Within loop I, we identified residues that are critical for targeting. Most mutations of conserved residues abolished targeting, and length reductions were deleterious. Taken together with the fact that H3 loop I makes numerous contacts with DNA and with the adaptive evolution of Cid, our results point to the importance of DNA specificity for targeting. We suggest that the process of deposition of (Cid.H4)2 tetramers allows for discriminating contacts to be made between loop I and DNA, providing the specificity needed for targeting. PMID- 12370301 TI - FCP1, a phosphatase specific for the heptapeptide repeat of the largest subunit of RNA polymerase II, stimulates transcription elongation. AB - FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second largest subunit of RNAP II, which had been shown previously to affect transcription elongation. PMID- 12370303 TI - DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms. AB - The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53 dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF. PMID- 12370304 TI - DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. AB - DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation. PMID- 12370305 TI - Cytosolic retention of phosphorylated extracellular signal-regulated kinase and a Rho-associated kinase-mediated signal impair expression of p21(Cip1/Waf1) in phorbol 12-myristate-13- acetate-induced apoptotic cells. AB - In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21(Cip1/Waf1) was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21(Cip1/Waf1) induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21(Cip1/Waf1) requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21(Cip1/Waf1) promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21(Cip1/Waf1) expression. Based on these findings, we propose that a ROCK mediated signal is involved in interfering with the process of ERK-mediated p21(Cip1/Waf1) induction in PMA-induced proapoptotic TF-1 and D2 cells. PMID- 12370306 TI - Identification of a novel mitogen-activated protein kinase kinase activation domain recognized by the inhibitor PD 184352. AB - Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 micro M). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding. PMID- 12370307 TI - Targeted disruption of NFATc3, but not NFATc4, reveals an intrinsic defect in calcineurin-mediated cardiac hypertrophic growth. AB - A calcineurin-nuclear factor of activated T cells (NFAT) regulatory pathway has been implicated in the control of cardiac hypertrophy, suggesting one mechanism whereby alterations in intracellular calcium handling are linked to the expression of hypertrophy-associated genes. Although recent studies have demonstrated a necessary role for calcineurin as a mediator of cardiac hypertrophy, the potential involvement of NFAT transcription factors as downstream effectors of calcineurin signaling has not been evaluated. Accordingly, mice with targeted disruptions in NFATc3 and NFATc4 genes were characterized. Whereas the loss of NFATc4 did not compromise the ability of the myocardium to undergo hypertrophic growth, NFATc3-null mice demonstrated a significant reduction in calcineurin transgene-induced cardiac hypertrophy at 19 days, 26 days, 6 weeks, 8 weeks, and 10 weeks of age. NFATc3-null mice also demonstrated attenuated pressure overload- and angiotensin II-induced cardiac hypertrophy. These results provide genetic evidence that calcineurin-regulated responses require NFAT effectors in vivo. PMID- 12370308 TI - Mammalian copper chaperone Cox17p has an essential role in activation of cytochrome C oxidase and embryonic development. AB - Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(-/-), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(-/-) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS) deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(-/-). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation. PMID- 12370309 TI - Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis. AB - Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debre-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis. PMID- 12370310 TI - Identification of mZnf8, a mouse Kruppel-like transcriptional repressor, as a novel nuclear interaction partner of Smad1. AB - To identify novel genes that play critical roles in mediating bone morphogenetic protein (BMP) signal pathways, we performed a yeast two-hybrid screen using Smad1 as bait. A novel mouse Kruppel-type zinc finger protein, mZnf8, was isolated. Interactions between mZnf8 and Smad proteins were further analyzed with various in vitro and in vivo approaches, including mammalian two-hybrid, in vitro glutathione S-transferase pulldown, and copurification assays. Results from functional analysis indicate that mZnf8 is a nuclear transcriptional repressor. Overexpression of mZnf8 represses activity of BMP and transforming growth factor beta (TGF-beta) reporters. Silencing the expression of endogenous mZnf8 with an RNA interference approach caused a significant increase in the expression of one BMP reporter. These results suggest that mZnf8 negatively regulates the TGF beta/BMP signaling pathway in vivo. Transcription of mZnf8 is ubiquitous in mouse embryos, but high levels are specifically observed in adult mouse testes, with the same cell- and stage-specific transcription pattern as Smad1. Our data support the hypothesis that mZnf8 plays critical roles in mediating BMP signaling during spermatogenesis. PMID- 12370311 TI - Rac2, a hematopoiesis-specific Rho GTPase, specifically regulates mast cell protease gene expression in bone marrow-derived mast cells. AB - Rho family GTPases activate intracellular kinase cascades to modulate transcription of multiple genes. Previous studies have examined the roles of the ubiquitously expressed Rho GTPase, Rac1, in regulation of gene expression in cell lines and implicated NF-kappaB, serum response factor, and kinase signaling pathways in this regulation. To understand the role of the closely related but hematopoiesis-specific Rho GTPase, Rac2, in regulation of gene transcription, we compared the gene expression profiles between wild-type and Rac2(-/-) bone marrow derived mast cells. Our data demonstrate remarkable specificity in the regulation of gene expression by Rac2 versus Rac1. Microarray analysis demonstrated that expression of 38 known genes was significantly altered in Rac2(-/-) mast cells after cytokine stimulation compared with those in wild-type cells. Of these, the expression of the mouse mast cell protease 7 (MMCP-7) gene in wild-type cells was highly induced at the transcriptional level after stimulation with stem cell factor (SCF). In spite of compensatorily increased expression of Rac1 in Rac2 deficient cells, SCF-induced MMCP-7 transcription did not occur. Surprisingly, the loss of MMCP-7 induction was not due to decreased activation of NF-kappaB, a transcription factor postulated to lie downstream of Rac1 and known to play a critical role in hematopoietic cell differentiation and proliferation. However, the activities of c-Jun N-terminal kinases (JNKs) were markedly decreased in Rac2(-/-) mast cells. Our results suggest that cytokine-stimulated activation of MMCP-7 gene transcription is selectively regulated by a Rac2-dependent JNK signaling pathway in primary mast cells and imply a remarkable specificity in the regulation of transcriptional activity by these two highly related Rho GTPases. PMID- 12370312 TI - Drosophila PDZ-GEF, a guanine nucleotide exchange factor for Rap1 GTPase, reveals a novel upstream regulatory mechanism in the mitogen-activated protein kinase signaling pathway. AB - PDZ-GEF is a novel guanine nucleotide exchange factor for Rap1 GTPase. Here we isolated Drosophila melanogaster PDZ-GEF (dPDZ-GEF), which contains the all conserved domains of mammalian and nematode PDZ-GEF including cyclic nucleotide monophosphate-binding, Ras exchange motif, PDZ, RA, and GEF domains. dPDZ-GEF loss-of-function mutants were defective in the development of various organs including eye, wing, and ovary. Many of these phenotypes are strikingly similar to the phenotype of the rolled mutant, implying that dPDZ-GEF functions upstream of the mitogen-activated protein (MAP) kinase pathway. Indeed, we found that dPDZ GEF is specifically involved in photoreceptor cell differentiation, facilitating its neuronal fate via activation of the MAP kinase pathway. Rap1 was found to link dPDZ-GEF to the MAP kinase pathway; however, Ras was not involved in the regulation of the MAP kinase pathway by dPDZ-GEF and actually had an inhibitory function. The analyses of ovary development in dPDZ-GEF-deficient mutants also demonstrated another role of dPDZ-GEF independent of the MAP kinase signaling pathway. Collectively, our findings identify dPDZ-GEF as a novel upstream regulator of various morphogenetic pathways and demonstrate the presence of a novel, Ras-independent mechanism for activating the MAP kinase signaling pathway. PMID- 12370313 TI - Defective associations between blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking alphav integrins. AB - Mouse embryos genetically null for the alphav integrin subunit develop intracerebral hemorrhages at midgestation and die shortly after birth. A key question is whether the hemorrhage arises from primary defects in vascular endothelial cells or pericytes or from other causes. We have previously reported normal initiation of cerebral vessels comprising branched tubes of endothelial cells. Here we show that the onset of hemorrhage is not due to defects in pericyte recruitment. Additionally, most alphav-null vessels display ultrastructurally normal endothelium-pericyte associations and normal interendothelial cell junctions. Thus, endothelial cells and pericytes appear to establish their normal relationships in cerebral microvessels. However, by both light and electron microscopy, we detected defective associations between cerebral microvessels and the surrounding brain parenchyma, composed of neuroepithelial cells, glia, and neuronal precursors. These data suggest a novel role for alphav integrins in the association between cerebral microvessels and central nervous system parenchymal cells. PMID- 12370314 TI - Transformation of bone marrow B-cell progenitors by E2a-Hlf requires coexpression of Bcl-2. AB - The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2, however, resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts, unlike the leukemic properties and cytokine independence of preB I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However, bone marrow cells expressing E2a-Hlf in combination with Bcl-2, but not E2a-Hlf alone, induced leukemias in irradiated recipients with long latencies, demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a Hlf, but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus, E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth, which are nevertheless abrogated during progression to a leukemogenic phenotype. PMID- 12370315 TI - Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. AB - The molecular mechanisms by which Notch receptors induce diverse biological responses are not fully understood. We recently cloned a mammalian homologue of the Mastermind gene of Drosophila melanogaster, MAML1 (Mastermind-like-1 molecule) and determined that it functions as a transcriptional coactivator for Notch receptors. In this report, we characterize two additional genes in this Mastermind-like gene family: MAML2 and MAML3. The three MAML genes are widely expressed in adult tissues but exhibit distinct expression patterns in mouse early spinal cord development. All MAML proteins localize to nuclear bodies, share a conserved basic domain in their N termini that binds to the ankyrin repeat domain of Notch, and contain a transcriptional activation domain in their C termini. Moreover, as determined by using coimmunoprecipitation assays, each MAML protein was found to be capable of forming a multiprotein complex with the intracellular domain of each Notch receptor (ICN1 to -4) and CSL in vivo. However, MAML3 bound less efficiently to the ankyrin repeat domain of Notch1. Also, in U20S cells, whereas MAML1 and MAML2 functioned efficiently as coactivators with each of the Notch receptors to transactivate a Notch target HES1 promoter construct, MAML3 functioned more efficiently with ICN4 than with other forms of ICN. Similarly, MAML1 and MAML2 amplified Notch ligand (both Jagged2 and Delta1)-induced transcription of the HES-1 gene, whereas MAML3 displayed little effect. Thus, MAML proteins may modify Notch signaling in different cell types based on their own expression levels and differential activities and thereby contribute to the diversity of the biological effects resulting from Notch activation. PMID- 12370318 TI - The National Down Syndrome Cytogenetic Register (NDSCR). PMID- 12370316 TI - A human mitochondrial GTP binding protein related to tRNA modification may modulate phenotypic expression of the deafness-associated mitochondrial 12S rRNA mutation. AB - Human mitochondrial 12S rRNA A1555G mutation has been found to be associated with deafness. However, putative nuclear modifier gene(s) has been proposed to regulate the phenotypic expression of this mutation. In yeast cells, mutant alleles of MSS1, encoding a mitochondrial GTP-binding protein, manifest a respiratory-deficient phenotype only when coupled with mitochondrial 15S rRNA P(R)(454) mutation corresponding to human A1555G mutation. This suggests that an MSS1-like modifier gene may influence the phenotypic expression of the A1555G mutation. We report here the identification and characterization of human MSS1 homolog, GTPBP3, the first identified vertebrate gene related to mitochondrial tRNA modification. The Gtpbp3 is the mitochondrial GTPase evolutionarily conserved from bacteria to mammals. Functional conservation of this protein is supported by the observation that isolated human GTPBP3 cDNA can complement the respiratory-deficient phenotype of yeast mss1 cells carrying P(R)(454) mutation. GTPBP3 is ubiquitously expressed in various tissues as multiple transcripts, but with a markedly elevated expression in tissues of high metabolic rates. We showed that Gtpbp3 localizes in mitochondrion. These observations suggest that the human GTPBP3 is a structural and functional homolog of yeast MSS1. Thus, allelic variants in GTPBP3 could, if they exist, modulate the phenotypic manifestation of human mitochondrial A1555G mutation. PMID- 12370317 TI - Sequential and ordered assembly of E1 initiator complexes on the papillomavirus origin of DNA replication generates progressive structural changes related to melting. AB - Multiple binding sites for an initiator protein are a common feature of replicator sequences from various organisms. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA by largely unknown mechanisms. Here we analyze origin of DNA replication (ori) binding by the E1 initiator and show sequential binding to a set of overlapping binding sites. The assembly of these initiator complexes is controlled by a gradual reduction in the dependence of interactions between the initiator and DNA and a gradual increase in the reliance on interactions between initiator molecules, providing a mechanism for sequential and orderly assembly. Importantly, the binding of the initiator causes progressive structural alterations both in the sites and in the sequences flanking the sites, eventually generating severe structural alterations. These results indicate that the process of template melting may be incremental, where binding of each initiator molecule serves as a wedge that upon binding gradually alters the template structure. This mechanism may explain the requirement for multiple initiator binding sites that is observed in many ori's. PMID- 12370319 TI - Basic variables at different positivity thresholds of a quantitative immunochemical test for faecal occult blood. AB - OBJECTIVES: Screening by faecal occult blood testing (FOBT) is effective in decreasing mortality and incidence of colorectal cancer (CRC). Immunochemical tests have proved to be more cost effective than guaiac FOBTs. The latex agglutination test (LAT) has the advantage of being a fully automated, quantitative test. The aim of this study is to interpret the overall experience with LAT according to different positivity thresholds. SETTING: A population based screening programme is currently running involving subjects aged 50-70, invited every 2 years to have an FOBT. LAT is the standard screening test and has a positivity threshold for further diagnostic tests of 100 ng haemoglobin/ml of sample solution. METHODS: Positivity rates, detection rates for CRC high risk adenomas, and positive predictive values for CRC, high risk adenomas, and low risk adenomas were calculated for several positivity thresholds. RESULTS: 19,132 attendances at screening were recorded (11,774 at first screening, 7358 at subsequent screenings). Progressively increasing the positivity threshold from 100 to 200 ng/ml showed (a) a decrease in positivity rate; (b) a decrease in detection rates for CRC or high risk adenomas; (c) an increase in positive predictive values for cancer; (d) an increase in positive predictive value for high risk adenomas. CONCLUSIONS: Increasing the positivity threshold of the LAT reduces recall rate and improves positive predictive value for cancer or high risk adenomas but substantially decreases the detection rate of CRC and high risk adenomas. For this reason increasing the positivity cut off for LATs is not advisable. On the other hand decreasing the positivity threshold would increase recall rate and sensitivity of screening. Careful evaluation of sensitivity of the quantitative results of the LAT for interval cancers is needed to definitively assess the optimal positivity threshold for LATs in population based screening programmes. PMID- 12370320 TI - Socioeconomic variation in participation in colorectal cancer screening. AB - OBJECTIVES: To investigate socioeconomic variation in participation in flexible sigmoidoscopy (FS) screening for colorectal cancer. DESIGN: A prospective study nested within a multicentre randomised controlled trial of the efficacy of FS screening for the prevention and early detection of colorectal cancer (the UK flexible sigmoidoscopy trial). SETTING: Glasgow, Scotland. PARTICIPANTS: 55-64 year old adults, registered with general practitioners participating in the FS trial. MAIN OUTCOME MEASURES: Screening participation measured at three levels: questionnaire return; interest in screening; attendance at screening. RESULTS: Socioeconomic deprivation was a strong predictor of participation. Return of the screening questionnaire, expression of interest in screening, and attendance at the test, were all lower in more deprived groups. CONCLUSIONS: These results highlight the need to consider ways to reduce inequalities in screening uptake, in parallel with the introduction of any new screening programmes, to avoid exacerbating social gradients in cancer mortality. PMID- 12370321 TI - Informed choice to undergo prenatal screening: a comparison of two hospitals conducting testing either as part of a routine visit or requiring a separate visit. AB - BACKGROUND: It is not known which of two common methods of conducting prenatal screening best facilitate women making informed choices. OBJECTIVE: To describe rates of informed choice in two hospitals: one where screening for Down's syndrome was conducted at a routine visit; the other where screening was conducted as part of a separate visit. DESIGN: Prospective descriptive study. SETTING: Two hospitals in England. PARTICIPANTS: 1499 pregnant women offered screening for Down's syndrome. OUTCOME MEASURE: A multidimensional measure of informed choice derived from measures of (a) consistency between attitudes towards undergoing the test and uptake and (b) knowledge about the screening test. RESULTS: The proportion of women making an informed choice to accept the test was higher at the routine visit hospital than at the separate visit hospital (41% v 21%, 95% confidence interval (95% CI) of the difference 16% to 25%). The proportions of women making an informed choice to decline the test were similar at the two hospitals (23% at both, 95% CI of the difference -5% to 4%). These results reflect the finding that women with negative attitudes were equally likely to decline the test at each of the two hospitals, whereas women with positive attitudes were more likely to accept the test at the routine visit hospital than at the separate visit hospital. This finding held after adjusting for parity, socioeconomic status, age, and ethnicity. At both hospitals, women with good knowledge were slightly more likely to undergo the test than were women with poor knowledge. This difference disappeared after a similar adjustment. CONCLUSION: Screening conducted as part of a routine visit may be associated with higher levels of informed choice than screening conducted at a separate visit. This finding constitutes a hypothesis for experimental investigation. PMID- 12370322 TI - Early outcome of mammography screening in Copenhagen 1991-99. AB - OBJECTIVES: To evaluate the early outcome of an organised mammography screening programme in an area with little opportunistic screening. SETTING: The municipality of Copenhagen, Denmark, during four invitation rounds 1991-99. METHODS: The following outcome measures were used: rates of participation, recall, false positive, and cancer detection. Benign biopsy, distribution of tumour size, lymph node status, and malignancy grade. RESULTS: A total of 106,933 screens were undertaken, and 824 invasive breast carcinomas or CIS were detected. The detection rate was 11.9 per 1000 participants in the first invitation round, and it continued to be high in subsequent rounds. The percentage of CIS cases was 11%. Coverage declined from 71% in the first round to 62% in the fourth, although 91% of those participating in the previous three rounds attended. The programme operated with a high recall rate. The false positive rate was also high, being 5.6% at first screen, and 1.8% later on. However, 90% of false positives were sorted out already at assessment. The percentage of screen detected invasive breast cancers with a tumour diameter < or = 10 mm was 39% compared with 16% of all invasive breast cancers in these age groups in Copenhagen before screening. CONCLUSION: Copenhagen is an area with a high incidence of breast cancer and with relatively little opportunistic screening. The start of a screening programme with a high recall rate in this area resulted in a detection rate above 1%. The Copenhagen programme met or exceeded most of the interim measures recommended in the European Guidelines. PMID- 12370323 TI - Rising incidence of breast cancer after completion of the first prevalent round of the breast cancer screening programme. AB - OBJECTIVES: After completion of the prevalent screening round of the breast cancer screening programme in Limburg, The Netherlands, incidences started rising once again. This increase was contrary to expectations, which had predicted a slightly higher, but stable, incidence after the first screening round. The trends in incidence were studied to find explanations for the observed rise in incidence. SETTING: Breast cancer screening programme in mid-Limburg and southern Limburg, the Netherlands. METHODS: The data files of the breast cancer screening programme and the Maastricht cancer registry were linked to evaluate the effect of breast cancer screening. Only the first primary breast tumour was included in the evaluation. RESULTS: The second peak of incidence after the prevalent screening round was 45% higher than the incidences before the start of the screening. Also, the decrease in incidence of large and node positive tumours was interrupted. Compared with national detection rates, the number of screen detected cancers was lower before 1995 and higher after that year. After 1997, incidence decreased again of all breast cancers, but also of large and node positive tumours. The incidence of node positive tumours showed large fluctuations, probably due to the introduction of the sentinel node procedure and immunohistochemistry. In 1999, incidence of large tumours and node positive tumours was 18% and 28%, respectively, lower than before the start of the screening. CONCLUSIONS: An increase in the background incidences and improved detection in the screening programme most likely explain this trend. The improved detection after 1995, and the lower than desirable decrease in large tumours, indicate that the screening performance was not optimal before 1996. The incidence of node positive tumours cannot be used any more as an indicator of the success of the screening programme because of detection bias. PMID- 12370324 TI - Accuracy of serial screening for abdominal aortic aneurysms by ultrasound. AB - OBJECTIVES: To assess the accuracy of screening for abdominal aortic aneurysms (AAAs) by ultrasound (US). SETTING: An aneurysm screening programme in Huntingdon. METHODS: False negative tests were identified by tracing all patients with a ruptured aneurysm who were screened and then finding the number classified as normal on US. False positive tests were identified by calculating the number of aneurysmal aortas on US that were classified as normal on CT. Measurement variability of the infrarenal aortic diameter between US and CT was estimated. RESULTS: 14 out of 93 patients with a ruptured AAA since 1991 had been screened. No ruptured aneurysm had been classified as normal on US. All 64 patients with an AAA larger than 4.5 cm on US had their aneurysm confirmed on CT. The mean difference between CT and US measurements was 4 mm. The limit of variability between CT and US was 12 mm. CONCLUSION: No false negative scans were found using a cut off point of 3 cm as abnormal. No false positives were found if subjects with an AAA exceeding 4.5 cm were referred for further procedures. A serial US screening policy has excellent screening performance, justifying its use as a screening tool. PMID- 12370325 TI - A randomised trial of skin photography as an aid to screening skin lesions in older males. AB - OBJECTIVES: We have previously shown that photographs assist in detection of change in skin lesions and designed the present randomised population based trial to assess the feasibility of photographs as an aid to management of skin cancers in older men. SETTING: 1899 men over fifty, identified from the electoral roll in two regions in New South Wales (NSW), Australia, were invited by mail to participate. METHODS: A total of 973 of 1037 respondents were photographed and randomised into intervention (participants given their photographs) or control groups (photographs withheld by investigators). At one and two years from the time of photography, all participants were advised to see their primary care practitioner for a skin examination. Those in the intervention group were examined with their photographs and those in the control group without their photographs. RESULTS: The results indicated that the practitioners were more likely to leave suspicious lesions in place for follow up observation (37% v 29%) (p=0.006) and less likely to excise benign non pigmented lesions (20 v 32%). There was little difference in excision rates for benign pigmented lesions (21% v 23%). Lesions excised were more likely to be non-melanoma skin cancer (58% v 42%) from patients who had photographs compared to those without photographs (p=0.005). The use of skin photography resulted in a substantial savings due to the reduced excision of benign lesions. CONCLUSIONS: These results suggest that it would be feasible to conduct a large scale randomised trial to evaluate the value of photography in early detection of melanoma and that such a trial could be cost effective due to the reduced excision of benign skin lesions. PMID- 12370326 TI - Baseline tests or screening: what tests do family physicians order routinely on their healthy patients? AB - OBJECTIVE: The purpose of this study was to survey the attitudes of family doctors to the performance of baseline tests and to determine which doctors perform these tests. SETTING: Family physicians in a continuing medical education programme in Tel Aviv, Israel. METHOD: An anonymous questionnaire was distributed focusing on performance of tests by doctors in healthy patients and not as part of a screening programme. RESULTS: Questionnaires were returned by 147 of 165 physicians surveyed (89% response rate). Baseline tests were performed by 98% of respondents: not routinely by 54%, 7% at the patient's request, and 2% did not perform tests. The decision to perform baseline tests was influenced by the presence of other risk factors of disease (86%), patient age (61%), family history (59%), patient request for tests (24%), and patient sex (20%). The tests performed were blood counts, glucose, renal function tests, urinalysis, liver function tests, and electrocardiograms. Baseline tests were useful in case finding of new illnesses for 49% of physicians and 40% said the tests had proved useful during a subsequent illness. The remainder of the physicians found no use for baseline tests. Physicians from the former Soviet Union were more likely to favour baseline tests. CONCLUSION: Almost all of the physicians in this study reported that they perform baseline tests on most of their patients. Evidence based guidelines for these tests and education of physicians are needed. PMID- 12370327 TI - Congenital toxoplasmosis in the United Kingdom: to screen or not to screen? AB - The United Kingdom National Screening Committee recently reviewed the evidence for prenatal and neonatal screening for toxoplasma infection and concluded that there was insufficient evidence to recommend screening in the United Kingdom. This issue will need to be revisited as new information or treatments become available. In this paper, the extent to which the research evidence on toxoplasma infection meets the criteria that need to be fulfilled for the introduction of screening in the United Kingdom is reviewed. PMID- 12370331 TI - Cutting edge: mouse pellino-2 modulates IL-1 and lipopolysaccharide signaling. AB - Pellino is a Drosophila protein originally isolated in a two-hybrid screen for proteins interacting with the serine/threonine kinase, pelle. Although mammalian homologs have been identified in mouse and man, the function of pellino is as yet unknown. In this study, the cloning, expression pattern, and a preliminary characterization of mouse pellino-2 is described. These studies reveal that mouse pellino-2 is expressed during embryogenesis and in a tissue-restricted manner in the adult. IL-1 induces the association of mouse pellino-2 with the mouse pelle like kinase/IL-1R-associated kinase protein, a mammalian homolog of pelle. Ectopic pellino-2 expression did not result in NF-kappaB activation. However, ectopic expression of a mouse pellino-2 antisense construct inhibited IL-1 or LPS induced activation of NF-kappaB-dependent IL-8 promoter activity. Our data reveal that mouse pellino-2 is a tissue-restricted component of a signaling pathway that couples the mouse pelle-like kinase/IL-1R-associated kinase protein to IL-1- or LPS-dependent signaling. PMID- 12370332 TI - Cutting edge: murine UL16-binding protein-like transcript 1: a newly described transcript encoding a high-affinity ligand for murine NKG2D. AB - Murine NKG2D is known to recognize H60 and five RAE1 variants. The human homologue recognizes both inducible MHC class I chain-related gene and constitutive (UL16-binding protein (ULBP)) ligands. Widely expressed, the latter are thought to mark transformed or infected cells for destruction by NK cells in the context of down-regulated cell surface class I (i.e., the "missing self" response). Unlike MIC and ULBP however, mRNA for the murine ligands appears only in very limited contexts in the mature animal. In this study, we describe a NKG2D ligand termed "murine ULBP-like transcript 1 (MULT1) whose mRNA appears to be widely expressed in adult parenchyma. This molecule possesses MHC class I-like alpha1 and alpha2 domains as well as a large cytoplasmic domain. Recombinant MULT1 binds NKG2D with relatively high affinity (K(D) approximately 6 nM) and low k(off) (approximately 0.006s(-1)). Expression of MULT1 by normally resistant RMA cells results in their susceptibility to lysis by C57BL/6 splenocytes. PMID- 12370333 TI - Cutting edge: suppression of T cell chemotaxis by sphingosine 1-phosphate. AB - Murine CD4 and CD8 T cells express predominantly types 1 and 4 sphingosine 1 phosphate (S1P) G protein-coupled receptors (designated S1P1 and S1P4 or previously endothelial differentiation gene-encoded 1 and 6) for S1P, which has a normal plasma concentration of 0.1-1 microM. S1P now is shown to enhance chemotaxis of CD4 T cells to CCL-21 and CCL-5 by up to 2.5-fold at 10 nM to 0.1 microM, whereas 0.3-3 microM S1P inhibits this chemotaxis by up to 70%. Chemotaxis of S1P(1), but not S1P(4), transfectants to CXCL1 and CXCL4 was similarly affected by S1P. Activation of CD4 T cells, which decreases S1P receptor expression, suppressed effects of S1P on chemotaxis. Pretreatment of labeled CD4 T cells with S1P before reintroduction into mice inhibited by a maximum of 75% their migration into chemokine-challenged s.c. air pouches. The S1P-S1P(1) receptor axis thus controls recruitment of naive T cells by maintaining their response threshold to diverse lymphotactic factors. PMID- 12370334 TI - Cutting edge: CATERPILLER: a large family of mammalian genes containing CARD, pyrin, nucleotide-binding, and leucine-rich repeat domains. AB - Large mammalian proteins containing a nucleotide-binding domain (NBD) and C terminal leucine-rich repeats (LRR) similar in structure to plant disease resistance proteins have been suggested as critical in innate immunity. Our interest in CIITA, a NBD/LRR protein, and recent reports linking mutations in two other NBD/LRR proteins to inflammatory disorders have prompted us to perform a search for other members. Twenty-two known and novel NBD/LRR genes are spread across eight human chromosomes, with multigene clusters occurring on 11, 16, and 19. Most of these are telomeric. Their N termini vary, but most have a pyrin domain. The genomic organization demonstrates a high degree of conservation of the NBD- and LRR-encoding exons. Except for CIITA, all the predicted NBD/LRR proteins are likely ATP-binding proteins. Some have broad tissue expression, whereas others are restricted to myeloid cells. The implications of these data on origins, expression, and function of these genes are discussed. PMID- 12370335 TI - Cutting edge: a crucial role for B7-CD28 in transmitting T help from APC to CTL. AB - Although APC activation via CD40-CD40L signaling plays a critical role in enabling CD4(+) T cells to provide the "help" necessary for cross-priming of naive CTL, it is unclear how this makes the APC competent for priming. We have investigated the roles of B7-1/B7-2 and their receptors [corrected] CD28/CTLA-4 in cross-priming of CD4-dependent CTL in vivo. We find that both CD28 and B7-1/B7 2 are required for CD40-activated APC to cross-prime CTL, and that priming by CD40-activated APC was prevented by blockade of CD28. Conversely, augmenting CD28 signals with an agonistic Ab bypassed the requirement for CD4(+) T help or CD40 activation. Interestingly, blockade of the negative regulatory B7 receptor CTLA-4 failed to prime CTL in the absence of T help. These results support a model in which activation-induced up-regulation of B7 molecules on APC leads to increased CD28 signaling and a commitment to cross-priming of CD4-dependent CTL. PMID- 12370336 TI - Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding. AB - The immunoreceptor NKG2D stimulates tumor immunity through activation of CD8 T cells and NK cells. Its ligand MICA has been shown to be broadly expressed on human tumors of epithelial origin. MICA expression correlates with an enrichment of Vdelta1 T cells in tumor tissue. We report that human tumor cells spontaneously release a soluble form of MICA encompassing the three extracellular domains, which is present at high levels in sera of patients with gastrointestinal malignancies, but not in healthy donors. Release of MICA from tumor cells is blocked by inhibition of metalloproteinases, concomitantly causing accumulation of MICA on the cell surface. Shedding of MICA by tumor cells may modulate NKG2D-mediated tumor immune surveillance. In addition, determination of soluble MICA levels may be implemented as an immunological diagnostic marker in patients with epithelial malignancies. PMID- 12370337 TI - Cutting edge: the differential involvement of the N-finger of GATA-3 in chromatin remodeling and transactivation during Th2 development. AB - The development of Th subset is accompanied by subset-specific chromatin remodeling of cytokine gene loci. In this study, we show that the C-terminal, but not the N-terminal zinc finger (N-finger) of GATA-3 mediates the association with the IL-4/IL-13 intergenic DNase I hypersensitive site and the induction of an extended DNase I hypersensitivity on the IL-4/IL-13 locus. Consistently, deletion of the transactivation domains or the C-finger, but not the N-finger, abrogated the induction of IL-4 and IL-13 as well as the down-regulation of IFN-gamma. In contrast, the N-finger of GATA-3 was indispensable for the binding to the IL-5 promoter and the induction of IL-5. The selective use of the N-finger may underlie the differential roles of GATA-3 in the induction of IL-4, IL-13, and IL 5. PMID- 12370338 TI - Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor. AB - The P2X7 receptor (P2X(7)R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X(7)R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X(7)R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X(7)R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X(7)R. These P2X(7)R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation. PMID- 12370339 TI - Subunits of IgM reconstitute defective contact sensitivity in B-1 cell-deficient xid mice: kappa light chains recruit T cells independent of complement. AB - The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway. PMID- 12370340 TI - Neonatal tolerance in the absence of Stat4- and Stat6- dependent Th cell differentiation. AB - Neonatal tolerance to specific Ag is achieved by nonimmunogenic exposure within the first day of life. The mechanism that regulates this tolerance may provide the basis for successful organ transplantation and has recently been thought to be immune deviation from the inflammatory Th1 response to a Th2 response. To test the importance of Th2 cells in the establishment of neonatal tolerance, we examined neonatal tolerance in Stat4- and Stat6-deficient mice, which have reduced Th1 and Th2 cell development, respectively. Neonatal tolerance of both the T and B cell compartments in Stat4- and Stat6-deficient mice was similar to that observed in wild-type mice. Cytokine production shifted from a Th1 to a Th2 response in wild-type mice tolerized as neonates. In contrast, tolerance was observed in Stat6-deficient mice despite maintenance of a Th1 cytokine profile. These results suggest that cells distinct from Stat6-dependent Th2 cells are required for the establishment of neonatal tolerance. PMID- 12370341 TI - The influence of lysophosphatidic acid on the functions of human dendritic cells. AB - Lysophosphatidic acid (LPA) is a bioactive lipid mediator which is generated by secretory phospholipase A(2). In this study, we studied the biological activity of LPA on human dendritic cells (DCs), which are specialized APCs characterized by their ability to migrate into target sites and secondary lymphoid organs to process Ags and activate naive T cells. We show that immature and mature DCs express the mRNA for different LPA receptors such as endothelial differentiation gene (EDG)-2, EDG-4, and EDG-7. In immature DCs, LPA stimulated pertussis toxin sensitive Ca(2+) increase, actin polymerization, and chemotaxis. During the maturation process, DCs lost their ability to respond toward LPA with Ca(2+) transients, actin polymerization, and chemotaxis. However, LPA inhibited in a pertussis toxin-insensitive manner the secretion of IL-12 and TNFalpha as well as enhanced secretion of IL-10 from mature DCs. Moreover, LPA did not affect the endocytic or phagocytic capacities and the surface phenotype of DCs, although it increased the allostimulatory function of mature DC and inhibited their capacity to induce Th1 differentiation. In summary, our study implicates that LPA might regulate the trafficking, cytokine production, and T cell-activating functions of DCs. PMID- 12370342 TI - Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway. AB - FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases. PMID- 12370343 TI - Antisense cyclic adenosine 5'-monophosphate response element modulator up regulates IL-2 in T cells from patients with systemic lupus erythematosus. AB - The cAMP response element modulator (CREM) has been shown to bind specifically to the -180 site of the IL-2 promoter in vitro. CREM protein is increased in T cells of patients with systemic lupus erythematosus (SLE), and it has been considered responsible for the decreased production of IL-2. In this work we show that transcriptional up-regulation is responsible for the increased CREM protein levels and that CREM binds to the IL-2 promoter in live SLE T cells. Suppression of the expression of CREM mRNA and protein by an antisense CREM plasmid, which was force expressed in SLE T cells by electroporation, resulted in decreased CREM protein binding to the IL-2 promoter and increased expression of IL-2 mRNA and protein. Our data demonstrate that antisense constructs can be used to effectively eliminate the expression of a transcriptional repressor. This approach can be used therapeutically in conditions where increased production of IL-2 is desired. PMID- 12370344 TI - Enforced expression of Bcl-2 restores the number of NK cells, but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes, in IL-2/IL 15 receptor beta-chain-deficient mice. AB - IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development. PMID- 12370345 TI - The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases. AB - The proteasome produces MHC class I-restricted antigenic peptides carrying N terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities. PMID- 12370346 TI - Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells. AB - Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination. PMID- 12370347 TI - Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors. AB - Previously we reported that TGF-beta has an important role in the generation and expansion of human "professional" CD4(+)CD25(+) regulatory T cells in the periphery that have a cytokine-independent mechanism of action. In this study we used low-dose staphylococcal enterotoxin to induce T cell-dependent Ab production. We report that TGF-beta induces activated CD4(+)CD25(-) T cells to become Th3 suppressor cells. While stimulating CD4(+) cells with TGF-beta modestly increased expression of CD25 and intracellular CTLA-4 in primary cultures, upon secondary stimulation without TGF-beta the total number and those expressing these markers dramatically increased. This expansion was due to both increased proliferation and protection of these cells from activation-induced apoptosis. Moreover, adding as few as 1% of these TGF-beta-primed CD4(+) T cells to fresh CD4(+) cells and B cells markedly suppressed IgG production. The inhibitory effect was mediated by TGF-beta and was also partially contact dependent. Increased TGF-beta production was associated with a decreased production of IFN-gamma and IL-10. Depletion studies revealed that the precursors of these TGF-beta-producing CD4(+) suppressor cells were CD25 negative. These studies provide evidence that CD4(+)CD25(+) regulatory cells in human blood consist of at least two subsets that have TGF-beta-dependent and independent mechanisms of action. TGF-beta has an essential role in the generation of both of these T suppressor cell subsets from peripheral T cells. The ability to induce CD4(+) and CD8(+) cells to become regulatory cells ex vivo has the potential to be useful in the treatment of autoimmune diseases and to prevent transplant rejection. PMID- 12370348 TI - Requirement for the p75 TNF-alpha receptor 2 in the regulation of airway hyperresponsiveness by gamma delta T cells. AB - In a recent study, we found that TNF-alpha negatively regulates airway responsiveness through the activation of gammadelta T cells. The biological activities of TNF-alpha are mediated by two structurally related but functionally distinct receptors, p55 (TNFR1) and p75 (TNFR2), which are independently expressed on the cell surface. However, the relative importance of either TNFR in airway hyperresponsiveness (AHR) is unknown. To investigate the importance of these TNFRs in the development of allergen-induced AHR, p55-deficient and p75 deficient mice were sensitized to OVA by i.p. injection and subsequently challenged with OVA via the airways; airway responsiveness to inhaled methacholine was monitored. p75-deficient mice developed AHR to a similar degree as control mice. In contrast, p55-deficient mice, which were sensitized and challenged with OVA, failed to develop AHR. In p55-deficient mice, both the numbers of eosinophils and levels of IL-5 in bronchoalveolar lavage fluid were significantly lower than in sensitized/challenged control mice (p < 0.05). However, depletion of gammadelta T cells resulted in significant increases in AHR in the p55-deficient mice, whereas no significant effect of gammadelta T cell depletion was evident in the p75-deficient mice. These data indicate that, in the absence of TNFR1 (p55), where TNF-alpha uses the p75 pathway exclusively, the development of AHR is regulated by gammadelta T cells. PMID- 12370349 TI - B cell positive selection by soluble self-antigen. AB - It is well established that autoreactive B cells undergo negative selection. This stands in paradox with the high frequency of so-called natural autoreactive B cells producing low affinity polyreactive autoantibodies with recurrent specificities, suggesting that these B cells are selected on the basis of their autoreactivity. We previously described two transgenic mouse lines (with and without IgD) producing a human natural autoantibody (nAAb) that binds ssDNA and human Fcgamma. In the absence of human IgG, nAAb-transgenic B cells develop normally. By crossing these mice with animals expressing knockin chimeric IgG with the human Fcgamma, we now show that the constitutive expression of chimeric IgG promotes the increase of nAAb-expressing B cells. This positive selection is critically dependent on the presence of IgD, occurs in the spleen, and concerns all mature B cell subsets, with a relative preferential enrichment of marginal zone B cells. These data support the view that soluble self-Ags can result in positive clonal selection. PMID- 12370350 TI - A minimal IFN-gamma promoter confers Th1 selective expression. AB - Th1 and Th2 cells differentiate from naive precursors to effector cells that produce either IFN-gamma or IL-4, respectively. To identify transcriptional paths leading to activation and silencing of the IFN-gamma gene, we analyzed transgenic mice that express a reporter gene under the control of the 5' IFN-gamma promoter. We found that as the length of the promoter is increased, -110 to -225 to -565 bp, the activity of the promoter undergoes a transition from Th1 nonselective to Th1 selective. This is due, at least in part, to a T box expressed in T cells responsive unit within the -565 to -410 region of the IFN-gamma promoter. The 225 promoter is silent when compared with the -110 promoter and silencing correlates with Yin Yang 1 binding to the promoter. The p38 mitogen-activated protein kinase signaling pathway, which also regulates IFN-gamma gene transcription, regulates the -70- to -44-bp promoter element. Together, the results demonstrate that a minimal IFN-gamma promoter contains a T box expressed in T cells responsive unit and is sufficient to confer Th1 selective expression upon a reporter. PMID- 12370351 TI - The role of bone marrow-derived stromal cells in the maintenance of plasma cell longevity. AB - Protective circulating Abs originate primarily from long-lived plasma cells in the bone marrow. However, the molecular and cellular basis of plasma cell longevity is unknown. We investigated the capacity of primary bone marrow-derived stromal cells to maintain plasma cell viability in vitro. Plasma cells purified from the bone marrow or lymph nodes died rapidly when plated in media, but a subpopulation of plasma cells survived and secreted high levels of Ab for up to 4 wk when cocultured with stromal cells. Ab secretion was inhibited by the addition of anti-very late Ag-4 to plasma cell/stromal cell cocultures indicating that direct interactions occur and are necessary between stromal cells and plasma cells. The addition of rIL-6 to plasma cells cultured in media alone partially relieved the sharp decline in Ab secretion observed in the absence of stromal cells. Moreover, when stromal cells from IL-6(-/-) mice were used in plasma cell/stromal cell cocultures, Ab levels decreased 80% after 7 days as compared with wild-type stromal cells. Further, IL-6 mRNA message was induced in stromal cells by coculture with plasma cells. These data indicate that bone marrow plasma cells are not intrinsically long-lived, but rather that plasma cells contact and modify bone marrow stromal cells to provide survival factors. PMID- 12370352 TI - Direct priming and cross-priming contribute differentially to the induction of CD8+ CTL following exposure to vaccinia virus via different routes. AB - To explore the relative importance of direct presentation vs cross-priming in the induction of CTL responses to viruses and viral vectors, we generated a recombinant vaccinia vector, vUS11, expressing the human CMV (HCMV) protein US11. US11 dislocates most allelic forms of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum into the cytosol, where they are degraded by proteasomes. Expression of US11 dramatically decreased the presentation of viral Ag and CTL recognition of infected cells in vitro without significantly reducing total cell surface MHC class I levels. However, because US11 is an endoplasmic reticulum resident membrane protein, it cannot block presentation by non-infected cells that take up Ag through the cross-priming pathway. We show that the expression of US11 strongly inhibits the induction of primary CD8(+) CTLs when the infection occurs via the i.p. or i.v. route, demonstrating that direct priming is critical for the induction of CTL responses to viral infections introduced via these routes. This effect is less dramatic following i.m. infection and is minimal after s.c. or intradermal infection. Thus, classic MHC class I Ag presentation and cross-priming contribute differentially to the induction of CD8(+) CTLs following exposure to vaccinia virus via different routes. PMID- 12370353 TI - Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. AB - NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell dependent. In this study, we report that NK cells could be induced by IL-2 and IL 15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137 stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL 2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity. PMID- 12370354 TI - T lymphocytes potentiate murine dendritic cells to produce IL-12. AB - IL-12 is mainly produced by CD8alpha(+) dendritic cells (DCs) and induces Th1 polarization of the immune response. We investigated the influence of lymphocytes on splenic DC (SDC) and thymic DC (TDC) development and on their IL-12 production capacity. First, CD3epsilon(-/-) mice, lacking T cells, and RAG-2(-/-) mice, lacking T and B cells, possess numbers of SDCs, TDCs, and CD8alpha(+) SDCs similar to wild-type (WT) mice. Second, SDCs and TDCs from CD3epsilon(-/-) mice do not secrete IL-12 in vitro after different stimulations, whereas DCs from pTalpha(-/-) mice, possessing reduced T cell number, and RAG-2(-/-) mice, produce an IL-12 level similar to that of WT DCs. We show that T lymphocytes restore the capacity of DCs to produce IL-12 after stimulation in vivo by reconstitution of CD3epsilon(-/-) mice with WT T cells and in vitro by coculture of CD3epsilon(-/-) DCs with WT T cells. The regulation of IL-12 production occurred at the transcriptional level, with an increase of IL-12p35 transcripts and a decrease of IL-12p40 transcripts. Although IL-4 restores IL-12 production by CD3epsilon(-/-) SDCs, anti-IL-4 Abs inhibited only partially the IL-12 production in coculture of CD3epsilon(-/-) DCs and WT T cells. Taken together, these data show that T lymphocytes potentiate IL-12 production by DCs and that IL-4 is not solely involved in this regulation. In conclusion, B and T cells exert balanced actions on DCs by respectively inhibiting or promoting IL-12 production. PMID- 12370355 TI - T cell-independent regulation of IgE antibody production induced by surface linked liposomal antigen. AB - Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis. PMID- 12370356 TI - Crucial role of DNA methylation in determination of clonally distributed killer cell Ig-like receptor expression patterns in NK cells. AB - Human NK cells are characterized by the expression of surface receptors of the killer cell Ig-like receptor (KIR) family, which are involved in the specific recognition of pathogenic target cells. Each NK cell expresses and maintains an individual subset of inhibitory and stimulatory KIR and in this way contributes to a diversified NK cell repertoire. To date, the molecular basis for generation of clonally distributed KIR expression patterns has been elusive. Here, analyses of DNA methylation patterns of KIR genes in NK cell lines as well as in NK cells, freshly isolated from peripheral blood, demonstrated that a small CpG island surrounding the transcriptional start site of each KIR gene is consistently demethylated in expressed KIR and methylated in unexpressed KIR. DNA demethylating treatment resulted in a rapid and stable induction of transcription and cell surface expression of all formerly unexpressed KIR in NK cell lines, NK cell clones, and freshly isolated NK cells, but not in other cell types. In vitro methylation of KIR CpG islands repressed reporter gene expression in NK cells. We conclude that clonal patterns of KIR expression are mainly epigenetically determined and maintained through DNA methylation. PMID- 12370357 TI - Pentoxifylline functions as an adjuvant in vivo to enhance T cell immune responses by inhibiting activation-induced death. AB - Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants. PMID- 12370358 TI - The Notch ligand Jagged-1 is able to induce maturation of monocyte-derived human dendritic cells. AB - Notch receptors play a key role in several cellular processes including differentiation, proliferation, and apoptosis. This study investigated whether the activation of Notch signaling would affect the maturation of dendritic cells (DCs). Direct stimulation of Notch signaling in DCs with a peptide ligand induced DC maturation, similar to LPS: DCs up-regulated maturation markers, produced IL 12, lost endocytosis capacity, and became able to activate allogeneic T cells. Furthermore, coculture of DCs with cells expressing Notch ligand Jagged-1 induced up-regulation of maturation markers, IL-12 production, T cell proliferative responses, and IFN-gamma production. Our data suggest that activation of Notch by Jagged-1 plays an important role in maturation of human DCs. Additionally, they reveal a novel role for Notch signaling in cell maturation events distal to the cell fate decision fork. These data may have important medical implications, since they provide new reagents to induce DC activity, which may be beneficial as adjuvants in situations where an immune response needs to be elicited, such as tumor immunotherapy. PMID- 12370359 TI - Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. AB - NK cell cytotoxicity, IFN-gamma expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-gamma expression, whereas IFN-alphabeta/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-alphabeta/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-alphabeta, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors' nonredundant functions under in vivo physiological conditions. PMID- 12370360 TI - IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha. AB - IL-10 is an immunosuppressive cytokine in the immune system. It was in clinical trial as an anti-inflammatory therapy for inflammatory bowel disease and various autoimmune diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiation-associated gene (MDA-7, IL-24), and AK155 (IL 26). Despite a partial homology in their amino acid sequences, they are dissimilar in their biologic functions. Little is known about the biologic function and gene regulation of IL-19. To understand the gene regulation of human IL-19, we identified a human IL-19 genomic clone and analyzed its promoter region. Five fusion genes containing different regions upstream of exon 1 linked to a luciferase reporter gene were expressed in the canine kidney epithelial-like Madin-Darby canine kidney cells. A fusion gene containing 394 bp showed luciferase activity 7- to 8-fold higher than the negative control of the promoterless fusion gene. We also isolated a full-length mouse cDNA clone. Mouse IL-19 shared 71% amino acid identity with human IL-19. Treatment of monocytes with mouse IL-19 induced the production of IL-6 and TNF-alpha. It also induced mouse monocyte apoptosis and the production of reactive oxygen species. Taken together, our results indicate that mouse IL-19 may play some important roles in inflammatory responses because it up-regulates IL-6 and TNF-alpha and induces apoptosis. PMID- 12370361 TI - Isotype switching by human B cells is division-associated and regulated by cytokines. AB - Isotype switching by murine B cells follows a pattern whereby the proportion of cells undergoing switching increases with division number and is regulated by cytokines. Here we explored whether human B cells behaved in a similar manner. The effect of IL-4, IL-10, and IL-13, alone or in combination, on Ig isotype switching by highly purified naive human CD40 ligand (CD40L)-activated B cells was measured against division number over various harvest times. Switching to IgG was induced by IL-4 and, to a lesser extent, IL-13 and IL-10. The combination of IL-10 with IL-4, but not IL-13, induced a higher percentage of cells to undergo switching. Isotype switching to IgG by human CD40L-activated naive B cells was found to be linked to the division history of the cells: IgG(+) cells appeared in cultures of B cells stimulated with CD40L and IL-4 after approximately the third cell division, with the majority expressing IgG1, thus revealing a predictable pattern of IgG isotype switching. These results reveal a useful quantitative framework for monitoring the effects of cytokines on proliferation and isotype switching that should prove valuable for screening Ig immunodeficiencies and polymorphisms in the population for a better understanding of the regulation of human humoral immune responses. PMID- 12370362 TI - P-, E-, and L-selectin mediate migration of activated CD8+ T lymphocytes into inflamed skin. AB - P- and E-selectin mediate CD4+ Th1 cell migration into the inflamed skin in a murine contact hypersensitivity model. In this model, not only CD4+ T cells but also CD8+ T cells infiltrate the inflamed skin, and the role of CD8+ type 1 cytotoxic T (Tc1) cells as effector cells has been demonstrated. Here we show that in mice deficient in both P- and E-selectin, the infiltration of CD8+ T cells in the inflamed skin is reduced, suggesting the role of these selectins in CD8+ T cell migration. We directly studied the role of selectins using in vitro generated Tc1 cells. These cells are able to migrate into the inflamed skin of wild-type mice. This migration is partially mediated by P- and E-selectin, as shown by the reduced Tc1 cell migration into the inflamed skin of mice deficient in both P- and E-selectin or wild-type mice treated with the combination of anti P-selectin and anti-E-selectin Abs. During P- and E-selectin-mediated migration of Tc1 cells, P-selectin glycoprotein ligand-1 appears to be the sole ligand for P-selectin and one of the ligands for E-selectin. P- and E-selectin-independent migration of Tc1 cells into the inflamed skin was predominantly mediated by L selectin. These observations indicate that all three selectins can mediate Tc1 cell migration into the inflamed skin. PMID- 12370363 TI - BLyS and APRIL form biologically active heterotrimers that are expressed in patients with systemic immune-based rheumatic diseases. AB - BLyS and APRIL are two members of the TNF superfamily that are secreted by activated myeloid cells and have costimulatory activity on B cells. BLyS and APRIL share two receptors, TACI and BCMA, whereas a third receptor, BAFF-R, specifically binds BLyS. Both BLyS and APRIL have been described as homotrimeric molecules, a feature common to members of the TNF superfamily. In this study, we show that APRIL and BLyS can form active heterotrimeric molecules when coexpressed and that circulating heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases. These findings raise the possibility that active BLyS/APRIL heterotrimers may play a role in rheumatic and other autoimmune diseases and that other members of the TNF ligand superfamily may also form active soluble heterotrimers. PMID- 12370364 TI - Synthesis and surface expression of hyaluronan by dendritic cells and its potential role in antigen presentation. AB - Hyaluronan (HA) is a large glycosaminoglycan consisting of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is known to act as a filling material of extracellular matrices and as an adhesive substrate for cellular migration. Here we report that dendritic cells (DC) express mRNAs for HA synthases and hyaluronidases, actively synthesize HA, and display HA on their surfaces. Interestingly, HA expression levels on DC were not significantly altered by their maturation states. With respect to physiological function, three specific HA inhibitors, i.e., bovine proteoglycan, a 12-mer HA-binding peptide (GAHWQFNALTVR) termed Pep-1, and an oligomeric Pep-1 formulation, all interfered with DC-induced activation of CD4(+) T cells isolated from DO11.10 TCR transgenic mice. For example, Pep-1 oligomer efficiently inhibited DC-dependent cluster formation, IL-2 and IFN-gamma production, and proliferation by DO11.10 T cells in vitro without affecting the viabilities of DC or T cells, DC function to uptake exogenous proteins, or DC-T cell conjugate formation at earlier time points. These observations suggest a paracrine mechanism by which DC-associated HA facilitates some of the late changes in T cell activation. Although T cells constitutively expressed mRNAs for HA synthases and hyaluronidases, their surface HA expression became detectable only after activation. Oligomeric Pep-1 and bovine proteoglycan both inhibited mitogen-triggered T cell activation in the absence of DC, suggesting an autocrine mechanism by which HA expressed by T cells assists their own activation processes. Finally, adoptively transferred DO11.10 T cells showed progressive mitosis when stimulated with Ag-pulsed DC in living animals, and this clonal expansion was inhibited significantly by administration of Pep-1 oligomer. Our findings may introduce a new concept that relatively simple carbohydrate moieties expressed on DC and perhaps T cells play an important immunomodulatory role during Ag presentation. PMID- 12370365 TI - Amelioration of collagen-induced arthritis by blockade of inducible costimulator B7 homologous protein costimulation. AB - B7 homologous protein (B7h)/B7-related protein 1 (B7RP-1) is a new member of the B7 family of costimulatory molecules that specifically interacts with inducible costimulator (ICOS) expressed on activated T cells. Collagen type II (CII) induced arthritis (CIA) is an experimental model of arthritis that has been used to dissect the pathogenesis of human rheumatoid arthritis. In this study, we have investigated the effect of neutralizing anti-B7h mAb on the development and disease progression of CIA. Administration of anti-B7h mAb significantly ameliorated the disease as assessed by clinical arthritis score and histology in the joints, and a beneficial effect was also obtained by a delayed treatment after the onset of disease. Expression of ICOS and B7h was observed in the inflamed synovial tissue as well as in the draining lymph nodes (LNs) and expansion of ICOS(+) T cells in the LN was reduced by the anti-B7h mAb treatment. Expression of mRNA for proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 in the joints was inhibited by the treatment. Proliferative responses and production of IFN-gamma and IL-10 upon restimulation with CII in vitro were significantly inhibited in LN cells from the anti-B7h mAb-treated mice. Serum anti-CII IgG1, IgG2a, and IgG2b levels were also reduced. Our present results showed a beneficial effect of the B7h blockade on CIA through anti-inflammatory actions and inhibition of both Th1- and Th2-mediated immune responses, suggesting that the ICOS-B7h interaction plays an important role in the pathogenesis of CIA and thus the blockade of this pathway may be beneficial for the treatment of human rheumatoid arthritis. PMID- 12370366 TI - Transcriptional regulation of Fcgr2b gene by polymorphic promoter region and its contribution to humoral immune responses. AB - FcgammaRIIB1 molecules serve as negative feedback regulator for B cell Ag receptor-elicited activation of B cells; thus, any impaired FcgammaRIIB1 function may possibly be related to aberrant B cell activation. We earlier found deletion polymorphism in the Fcgr2b promoter region among mouse strains in which systemic autoimmune disease-prone NZB, BXSB, MRL, and autoimmune diabetes-prone nonobese diabetic, but not NZW, BALB/c, and C57BL/6 mice have two identical deletion sites, consisting of 13 and 3 nucleotides. In this study, we established congenic C57BL/6 mice for NZB-type Fcgr2b allele and found that NZB-type allele down regulates FcgammaRIIB1 expression levels in germinal center B cells and up regulates IgG Ab responses. We did luciferase reporter assays to determine whether NZB-type deletion polymorphism affects transcriptional regulation of Fcgr2b gene. Although NZW- and BALB/c-derived segments from position -302 to +585 of Fcgr2b upstream region produced significant levels of luciferase activities, only a limited activity was detected in the NZB-derived sequence. EMSA and Southwestern analysis revealed that defect in transcription activity in the NZB derived segment is likely due to absence of transactivation by AP-4, which binds to the polymorphic 13 nucleotide deletion site. Our data imply that because of the deficient AP-4 binding, the NZB-type Fcgr2b allele polymorphism results in up regulation of IgG Ab responses through down-regulation of FcgammaRIIB1 expression levels in germinal center B cells, and that such polymorphism may possibly form the basis of autoimmune susceptibility in combination with other background contributing genes. PMID- 12370367 TI - Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice. AB - Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells. PMID- 12370368 TI - Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex. AB - During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus. PMID- 12370369 TI - Distinct roles for c-Myb and core binding factor/polyoma enhancer-binding protein 2 in the assembly and function of a multiprotein complex on the TCR delta enhancer in vivo. AB - Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to Edelta within wild-type and mutant versions of a human TCR delta minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Edelta enhanceosome in the context of chromatin in vivo. PMID- 12370370 TI - Src homology 2 domain-containing inositol polyphosphate phosphatase regulates NF kappa B-mediated gene transcription by phagocytic Fc gamma Rs in human myeloid cells. AB - FcgammaR-mediated phagocytosis is accompanied by the generation of tissue damaging products such as inflammatory cytokines and reactive oxygen species. Hence, the phagocytic response must be a tightly regulated process. Recent studies have established that clustering FcgammaR on human myeloid cells causes tyrosine phosphorylation of Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP). However, it is not known how these immunoreceptor tyrosine-based activation motif (ITAM)-bearing phagocytic FcgammaR activate SHIP, or whether the activation of SHIP by ITAMs has any functional relevance. Experiments addressing the mechanism of SHIP association with ITAMs have been done in in vitro systems using phosphopeptides. In this study we undertook to dissect the molecular mechanism by which SHIP associates with the native ITAM-FcgammaR and becomes phosphorylated. In this report we provide evidence that first, SHIP is indeed phosphorylated by ITAM-FcgammaR, using cell systems that lack FcgammaRIIb expression; second, coimmunoprecipitation experiments demonstrate that SHIP associates with native ITAM-bearing FcgammaRIIa in vivo; and third, phosphorylation of SHIP by FcgammaRIIa is inhibited by overexpressing either the SHIP Src homology 2 domain or a dominant negative mutant of Shc. In contrast, SHIP phosphorylation was not inhibited by a dominant negative mutant of Grb2. We extend these observations to show that SHIP activation by ITAM-FcgammaR down-regulates NF-kappaB-induced gene transcription. These findings both provide a molecular mechanism for SHIP association with native ITAM-bearing receptors and demonstrate that SHIP association with ITAM FcgammaR serves to regulate gene expression during the phagocytic process. PMID- 12370371 TI - Uniquely conformed peptide-containing beta 2-microglobulin-free heavy chains of HLA-B2705 on the cell surface. AB - The human class I MHC molecules are known to generally exist on the cell surface either as peptide-containing complexes of H chain (alpha-chain) and beta(2) microglobulin (beta(2)m) or as beta(2)m-free H chains incapable of binding peptides. In this study, a uniquely conformed peptide-containing beta(2)m-free HLA-B2705 H chain has been isolated using the recently described highly efficient perfusion-affinity chromatography system for purification of class I MHC protein molecules. This form recognized by the mAb MARB4 is very closely associated with the remainder of the peptide containing HLA-B2705/beta(2)m complex reactive with mAb ME1 and is present to approximately 1-10% of mAb ME1 reactive forms on the cell surface. Also, HLA-B2705 purified using the mAb ME1 affinity column includes this unique mAb MARB4-reactive, unusually stable peptide-containing beta(2)m-free form. A peptide nonamer GRWRGWYTY was isolated and identified from this beta(2)m free HLA-B2705 H chain and was used to assemble the mAb MARB4 reactive form efficiently on the surface of cells expressing HLA-B2705. The discovery of this form opens new avenues for further investigation of the role of HLA-B27 in spondyloarthropathies. PMID- 12370372 TI - Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling. AB - In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL 12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation. PMID- 12370373 TI - Modification of a tumor-derived peptide at an HLA-A2 anchor residue can alter the conformation of the MHC-peptide complex: probing with TCR-like recombinant antibodies. AB - A common assumption about peptide binding to the class I MHC complex is that each residue in the peptide binds independently. Based on this assumption, modifications in class I MHC anchor positions were used to improve the binding properties of low-affinity peptides (termed altered peptide ligands), especially in the case when tumor-associated peptides are used for immunotherapy. Using a new molecular tool in the form of recombinant Abs endowed with Ag-specific MHC restricted specificity of T cells, we show that changes in the identity of anchor residues may have significant effects, such as altering the conformation of the peptide-MHC complex, and as a consequence, may affect the TCR-contacting residues. We herein demonstrate that the binding of TCR-like recombinant Abs, specific for the melanoma differentiation Ag gp100 T cell epitope G9-209, is entirely dependent on the identity of a single peptide anchor residue at position 2. An example is shown in which TCR-like Abs can recognize the specific complex only when a modified peptide, G9-209-2 M, with improved affinity to HLA-A2 was used, but not with the unmodified natural peptide. Importantly, these results demonstrate, using a novel molecular tool, that modifications at anchor residues can dramatically influence the conformation of the MHC peptide groove and thus may have a profound effect on TCR interactions. Moreover, these results may have important implications in designing modifications in peptides for cancer immunotherapy, because most such peptides studied are of low affinity. PMID- 12370374 TI - Artiodactyl IgD: the missing link. AB - IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig delta genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cdelta gene appears to have been duplicated from the Cmu gene >300 million yr ago. The ruminant mu CH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, approximately 20 million yr ago, inserted upstream of the delta gene hinge regions, and later modified by gene conversion. A short Sdelta (switch delta) sequence resulting from the second duplication, is located immediately upstream of the bovine Cdelta gene and directs regular mu-delta class switch recombination in the cow. The presence of Cdelta genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage. PMID- 12370376 TI - Fc gamma R-mediated phagocytosis stimulates localized pinocytosis in human neutrophils. AB - Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of FcgammaR at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. FcgammaR-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca(2+) concentration were prevented. Because stimulation of FcgammaR also induces secretion, which is similarly calcium and phosphatidylinositol 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform FcgammaR-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca(2+) dependence of secretion and pinocytosis suggests that primary (lysosomal) granule exocytosis is the main determinant of pinocytosis during FcgammaR stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation. PMID- 12370375 TI - Role of STAT6 and mast cells in IL-4- and IL-13-induced alterations in murine intestinal epithelial cell function. AB - Gastrointestinal nematode infections generally invoke a type 2 cytokine response, characterized by the production of IL-4, IL-5, IL-9, and IL-13. Among these cytokines, IL-4 and IL-13 exhibit a functional overlap that can be explained by the sharing of a common receptor or receptor component (IL-4Ralpha). Binding of IL-4 by either the type 1 or 2 IL-4R, or of IL-13 by the type 2 IL-4R, initiates Jak-dependent tyrosine phosphorylation of the IL-4Ralpha-chain and the transcription factor, STAT6. In the present study, we investigated: 1) whether IL 13 has effects on intestinal epithelial cells similar to those observed with IL 4, and 2) whether the effects of IL-4 and IL-13 depend on STAT6 signaling and/or mast cells. BALB/c, STAT6(-/-), and mast cell-deficient W/W(v) mice or their +/+ littermates were treated with a long-lasting formulation of recombinant mouse IL 4 (IL-4C) or with IL-13 for seven days. Segments of jejunum were mounted in Ussing chambers to measure mucosal permeability; chloride secretion in response to PGE(2), histamine, 5-hydroxytryptamine, or acetylcholine; and Na(+)-linked glucose absorption. IL-4C and IL-13 increased mucosal permeability, decreased glucose absorption, and decreased chloride secretion in response to 5 hydroxytryptamine. These effects were dependent on STAT6 signaling. Responses to PGE(2) and histamine, which were dependent on mast cells and STAT6, were enhanced by IL-4C, but not by IL-13. The effects of IL-4 and IL-13 on intestinal epithelial cell function may play a critical role in host protection against gastrointestinal nematodes. PMID- 12370377 TI - Enhancement of complement activation and opsonophagocytosis by complexes of mannose-binding lectin with mannose-binding lectin-associated serine protease after binding to Staphylococcus aureus. AB - Human mannose-binding lectin (MBL) is a serum protein of the innate immune system that circulates as a complex with a group of so-called MBL-associated serine proteases (MASP-1, MASP-2, and MASP-3). Complexes of MBL-MASP2 are able to activate the complement system in an Ab and C1-independent fashion after binding of the lectin to appropriate microbial sugar arrays. We have evaluated the additive effect of the lectin pathway relative to other complement activation pathways and the subsequent effect on neutrophil phagocytosis. Complement activation in the sera of MBL-deficient individuals was studied with and without the addition of exogenous MBL-MASP. Flow cytometry was used to measure the deposition of C4, factor B, C3b, and iC3b on Staphylococcus aureus. Deposition of the first cleavage product of the lectin pathway, C4b, was increased using the sera of three different MBL-deficient individuals when exogenous MBL-MASP was added. Factor B was deposited in association with C4, but there was no evidence of independent alternative pathway activation. Similar enhancement of C3b deposition was also observed, with evidence of elevated amounts of C3b processed to iC3b. The increase in opsonic C3 fragments mediated by MBL was associated with a significant increase in the uptake of organisms by neutrophils. We also observed significant increases in phagocytosis with MBL-MASPs that were independent of complement activation. We conclude that MBL-MASP makes a major contribution to complement-mediated host defense mechanisms. PMID- 12370378 TI - IFN-gamma production from liver mononuclear cells of mice in burn injury as well as in postburn bacterial infection models and the therapeutic effect of IL-18. AB - Hosts after severe burn injury are known to have a defect in the Th1 immune response and are susceptible to bacterial infections. We herein show that liver NK cells are potent IFN-gamma producers early after burn injury. However, when mice were injected with LPS 24 h after burn injury, IFN-gamma production from liver mononuclear cells (MNC; which we previously showed to be NK cells) was suppressed, and the serum IFN-gamma concentration did not increase, while serum IL-10 conversely increased compared with control mice. Interestingly, a single injection of IL-18 simultaneously with LPS greatly restored the serum IFN-gamma concentration in mice with burn injury and also increased IFN-gamma production from liver MNC. Nevertheless, a single IL-18 injection into mice simultaneously with LPS was no longer effective in the restoration of serum IFN-gamma and IFN gamma production from the liver MNC at 7 days after burn injury, when mice were considered to be the most immunocompromised. However, IL-18 injections into mice on alternate days beginning 1 day after burn injury strongly up-regulated LPS induced serum IFN-gamma levels and IFN-gamma production from liver and spleen MNC of mice 7 days after burn injury and down-regulated serum IL-10. Furthermore, similar IL-18 therapy up-regulated serum IFN-gamma levels in mice with experimental bacterial peritonitis 7 days after burn injury and greatly decreased mouse mortality. Thus, IL-18 therapy restores the Th1 response and may decrease the susceptibility to bacterial infection in mice with burn injury. PMID- 12370379 TI - Retroviral interference on STAT activation in individuals coinfected with human T cell leukemia virus type 2 and HIV-1. AB - Human T cell leukemia virus (HTLV) type-2 is a human retrovirus whose infection has not been tightly linked to human diseases. However, the fairly high prevalence of this infection among HIV-1-positive individuals indicates the importance of better understanding the potential interference of HTLV-2 infection on HIV-1 infection and AIDS. We previously demonstrated that one signature of PBMC freshly derived from HIV-1-infected individuals is the constitutive activation of a C-terminal truncated STAT5 (STAT5Delta). Therefore, we analyzed the potential activation of STATs in HTLV-2 monoinfected and HTLV-2/HIV-1 dually infected individuals. We observed that PBMC of HTLV-2-infected individuals do not show STAT activation unless they are cultivated ex vivo, in the absence of any mitogenic stimuli, for at least 8 h. The emergence of STAT activation, namely of STAT1, in culture was mostly related to the secretion of IFN-gamma. Of note, this phenomenon is not only a characteristic feature of HTLV-2-infected individuals but also occurred with PBMC of HIV-1(+) individuals. Surprisingly, HTLV-2/HIV-1 coinfection resulted in low/absent STAT activation in vivo that paralleled a diminished secretion of IFN-gamma after ex vivo cultivation. Our findings indicate that both HTLV-2 and HIV-1 infection prime T lymphocytes for STAT1 activation, but they also highlight an interference exerted by HTLV-2 on HIV-1 induced STAT1 activation. Although the nature of such a phenomenon is unclear at the present, these findings support the hypothesis that HTLV-2 may interfere with HIV-1 infection at multiple levels. PMID- 12370380 TI - The innate immune response differs in primary and secondary Salmonella infection. AB - This study examines innate immunity to oral Salmonella during primary infection and after secondary challenge of immune mice. Splenic NK and NKT cells plummeted early after primary infection, while neutrophils and macrophages (Mphi) increased 10- and 3-fold, respectively. In contrast, immune animals had only a modest reduction in NK cells, no loss of NKT cells, and a slight increase in phagocytes following secondary challenge. During primary infection, the dominant sources of IFN-gamma were, unexpectedly, neutrophils and Mphi, the former having intracellular stores of IFN-gamma that were released during infection. IFN-gamma producing phagocytes greatly outnumbered IFN-gamma-producing NK cells, NKT cells, and T cells during the primary response. TNF-alpha production was also dominated by neutrophils and Mphi, which vastly outnumbered NKT cells producing this cytokine. Neither T cells nor NK cells produced TNF-alpha early during primary infection. The TNF-alpha response was reduced in a secondary response, but remained dominated by neutrophils and Mphi. Moreover, no significant IFN-gamma production by Mphi was associated with the secondary response. Indeed, only NK1.1(+) cells and T cells produced IFN-gamma in these mice. These studies provide a coherent view of innate immunity to oral Salmonella infection, reveal novel sources of IFN-gamma, and demonstrate that immune status influences the nature of the innate response. PMID- 12370381 TI - An increase in the susceptibility of burned patients to infectious complications due to impaired production of macrophage inflammatory protein 1 alpha. AB - Sepsis is a major mortality concern with burned patients, who have an increased susceptibility to infectious complications. PBMC from 41 of 45 severely burned patients (91%) failed to produce macrophage inflammatory protein 1alpha (MIP 1alpha) in cultures, while 2355-6900 pg/ml MIP-1alpha were produced by healthy donor PBMC, stimulation with anti-human CD3 mAb. Healthy chimeras (SCID mice inoculated with healthy donor PBMC) treated with anti-human MIP-1alpha mAb and patient chimeras (SCID mice reconstituted with burned patient PBMC) were susceptible (0% survival) to infectious complications induced by well-controlled cecal ligation and puncture. In contrast, patient chimeras treated with human recombinant MIP-1alpha and healthy chimeras were resistant ( approximately 77-81% survival). Similarly, after anti-mouse CD3 mAb stimulation, splenic mononuclear cells from burned mice (6 h to 3 days after thermal injury) did not produce significant amounts of MIP-1alpha in their culture fluids. Normal mice treated with anti-murine MIP-1alpha mAb and burned mice were susceptible to cecal ligation- and puncture-induced infectious complications, while burned mice treated with murine recombinant MIP-1alpha and normal mice were resistant. Burned patients seemed to be more susceptible to infectious complications when the production of MIP-1alpha was impaired. PMID- 12370382 TI - Synergistic engagement of an ineffective endogenous anti-tumor immune response and induction of IFN-gamma and Fas-ligand-dependent tumor eradication by combined administration of IL-18 and IL-2. AB - IFN-gamma is a critical component of the endogenous and many cytokine-induced antitumor immune responses. In this study we have shown that the combination of IL-18 and IL-2 (IL-18/IL-2) synergistically enhances IFN-gamma production both in vitro and in vivo, and synergizes in vivo to induce complete durable regression of well-established 3LL tumors in >80% of treated mice. We have observed a nascent, but ineffective, host immune response against 3LL that depends on endogenous IFN-gamma and IL-12 production and the Fas/Fas ligand (Fas-L) pathway. The combined administration of IL-18/IL-2 engages this endogenous response to induce tumor regression via a mechanism that is independent of NK and NKT cells or IL-12, but is critically dependent on CD8(+) T cells, IFN-gamma, and the Fas/Fas-L pathway. These studies demonstrate the importance of IFN-gamma as well as the Fas/Fas-L pathway in both endogenous and cytokine-driven antitumor immune responses engaged by IL-18/IL-2 and provide preclinical impetus for clinical investigation of this potent anti-tumor combination. PMID- 12370383 TI - Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice. AB - The induction of an adaptive immune response to a previously unencountered pathogen is a time-consuming process and initially the infection must be held in check by the innate immune system. In the case of an i.p. infection with Salmonella typhimurium, survival requires both CD14 and LPS-binding protein (LBP) which, together with Toll-like receptor 4 and myeloid differentiation protein 2, provide a sensitive means to detect bacterial LPS. In this study, we show that in the first hours after i.p. infection with Salmonella a local inflammatory response is evident and that concomitantly neutrophils flood into the peritoneum. This rapid neutrophil influx is dependent on TNF since it is 1) abolished in TNF KO mice and 2) can be induced by i.p. injection of TNF in uninfected animals. Neutrophil influx is not strictly dependent on the presence of either LBP or CD14. However, in their absence, no local inflammatory response is evident, neutrophil migration is delayed, and the mice succumb to the infection. Using confocal microscopy, we show that the neutrophils which accumulate in CD14 and LBP null mice, albeit with delayed kinetics, are nevertheless fully capable of ingesting the bacteria. We suggest that the short delay in neutrophil influx gives the pathogen a decisive advantage in this infection model. PMID- 12370384 TI - Differential requirement for NF-kappa B family members in control of helminth infection and intestinal inflammation. AB - The NF-kappaB family of transcription factors is critical in controlling the expression of a wide range of immune response genes. However, whether individual family members perform specific roles in regulating immunity and inflammation remains unclear. Here we investigated the requirement for NF-kappaB1, NF-kappaB2, and c-Rel in the expression of Th2 cytokine responses, development of host protective immunity, and regulation of intestinal inflammation following infection with the gut-dwelling helminth parasite Trichuris muris. While mice deficient in c-Rel mounted sufficient Th2 responses to expel infection, NF kappaB1 knockout (KO) and NF-kappaB2 KO mice developed chronic infections associated with elevated production of Ag-specific IFN-gamma. However, only infected NF-kappaB1 KO mice exhibited polarized IFN-gamma responses associated with the loss of intestinal goblet cells and the development of destructive colitis-like pathology. Furthermore, blockade of IL-12 (previously shown to confer resistance in susceptible strains) recovered Ag-specific IL-13 responses and resistance to infection in NF-kappaB2 KO, but not NF-kappaB1 KO mice. Therefore, unique infection, immunological, and pathological outcomes were observed in different NF-kappaB KO strains. Taken together, these results provide direct evidence of nonoverlapping functions for NF-kappaB family members in the development of Th2 cytokine-mediated resistance to T. muris and the control of infection-induced intestinal inflammation. PMID- 12370385 TI - Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma mediates its restoration and bacterial killing. AB - The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-gamma restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN gamma was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-gamma mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever. PMID- 12370386 TI - The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD4+ T cells. AB - Amebic colitis is an important worldwide parasitic disease for which there is not a well-established animal model. In this work we show that intracecal inoculation of Entamoeba histolytica trophozoites led to established infection in 60% of C3H mice, while C57BL/6 or BALB/c mice were resistant, including mice genetically deficient for IL-12, IFN-gamma, or inducible NO synthase. Infection was a chronic and nonhealing cecitis that pathologically mirrored human disease. Characterization of the inflammation by gene chip analysis revealed abundant mast cell activity. Parasite-specific Ab and cellular proliferative responses were robust and marked by IL-4 and IL-13 production. Depletion of CD4(+) cells significantly diminished both parasite burden and inflammation and correlated with decreased IL-4 and IL-13 production and loss of mast cell infiltration. This model reveals important immune factors that influence susceptibility to infection and demonstrates for the first time the pathologic contribution of the host immune response in amebiasis. PMID- 12370387 TI - Morphologic detection and functional assessment of reconstituted normal alveolar macrophages in the lungs of SCID mice. AB - Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for approximately 50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense. PMID- 12370388 TI - Differences between T cell epitopes recognized after immunization and after infection. AB - Evidence suggests that cellular immune responses play a crucial role in the control of HIV and SIV replication in infected individuals. Several vaccine strategies have therefore targeted these CD8(+) and CD4(+) responses. Whether vaccination induces the same repertoire of responses seen after infection is, however, a key unanswered question in HIV vaccine development. We therefore compared the epitope specificity induced by vaccination to that present postchallenge in the peripheral blood. Intracellular cytokine staining of PBMC stimulated with overlapping 15/20-mer peptides spanning the proteins of SIV were measured after DNA/modified vaccinia Ankara vaccination of eight rhesus macaques. Lymphocytes from 8 animals recognized a total of 39 CD8 epitopes and 41 CD4 epitopes encoded by the vaccine. T cell responses were again monitored after challenge with SIVmac239 to investigate the evolution of these responses. Only 57% of all CD8(+) T cell responses and 19% of all CD4(+) T cell responses present after vaccination were recalled after infection as measured in the peripheral blood. Interestingly, 29 new CD8 epitopes and 5 new CD4 epitopes were recognized by PBMC in the acute phase. These new epitopes were not detected after vaccination, and only some of them were maintained in the chronic phase (33% of CD8 and no CD4 responses). Additionally, 24 new CD8 epitopes and 7 new CD4 epitopes were recognized by PBMC in the chronic phase of infection. The repertoire of the immune response detected in the peripheral blood after immunization substantially differed from the immune response detected in the peripheral blood after infection. PMID- 12370389 TI - Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta. AB - The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF alpha treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1alpha and beta. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa induced inflammation. PMID- 12370390 TI - Comparison of the pro-oxidative and proinflammatory effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages. AB - Inhaled diesel exhaust particles (DEP) exert proinflammatory effects in the respiratory tract. This effect is related to the particle content of redox cycling chemicals and is involved in the adjuvant effects of DEP in atopic sensitization. We demonstrate that organic chemicals extracted from DEP induce oxidative stress in normal and transformed bronchial epithelial cells, leading to the expression of heme oxygenase 1, activation of the c-Jun N-terminal kinase cascade, IL-8 production, as well as induction of cytotoxicity. Among these effects, heme oxygenase 1 expression is the most sensitive marker for oxidative stress, while c-Jun N-terminal kinase activation and induction of apoptosis necrosis require incremental amounts of the organic chemicals and increased levels of oxidative stress. While a macrophage cell line (THP-1) responded in similar fashion, epithelial cells produced more superoxide radicals and were more susceptible to cytotoxic effects than macrophages. Cytotoxicity is the result of mitochondrial damage, which manifests as ultramicroscopic changes in organelle morphology, a decrease in the mitochondrial membrane potential, superoxide production, and ATP depletion. Epithelial cells also differ from macrophages in not being protected by a thiol antioxidant, N-acetylcysteine, which effectively protects macrophages against cytotoxic DEP chemicals. These findings show that epithelial cells exhibit a hierarchical oxidative stress response that differs from that of macrophages by more rapid transition from cytoprotective to cytotoxic responses. Moreover, epithelial cells are not able to convert N acetylcysteine to cytoprotective glutathione. PMID- 12370391 TI - Leukocyte rolling velocities and migration are optimized by cooperative L selectin and intercellular adhesion molecule-1 functions. AB - Selectin family members largely mediate initial tethering and rolling of leukocytes on vascular endothelium, whereas integrin and Ig family members are essential for leukocyte firm adhesion. To quantify functional synergy between L selectin and Ig family members during leukocyte rolling, the EA.hy926 human vascular endothelial line was transfected with either fucosyltransferase VII (926 FtVII) cDNA to generate L-selectin ligands alone or together with ICAM-1 cDNA (926-FtVII/ICAM-1). The ability of transfected 926 cells to support human leukocyte interactions was assessed in vitro using parallel plate flow chamber assays. Lymphocyte rolling on 926-FtVII cells was increased by approximately 70% when ICAM-1 was expressed at physiological levels. Although initial tether formation was similar for both cell types, lymphocyte rolling was 26% slower on 926-FtVII/ICAM-1 cells. Pretreatment of lymphocytes with an anti-CD18 mAb eliminated the increase in rolling, and all rolling was blocked by anti-L selectin mAb. In addition, rolling velocities of lymphocytes from CD18 hypomorphic mice were 48% faster on 926-FtVII/ICAM-1 cells, with a similar reduction in rolling frequency relative to wild-type lymphocytes. CD18 hypomorphic lymphocytes also showed an approximately 40% decrease in migration to peripheral and mesenteric lymph nodes during in vivo migration assays compared with wild-type lymphocytes. Likewise, wild-type lymphocyte migration to peripheral lymph nodes was reduced by approximately 50% in ICAM-1(-/-) recipient mice. Similar to human lymphocytes, human neutrophils showed enhanced rolling interactions on 926-FtVII/ICAM-1 cells, but also firmly adhered. Thus, in addition to mediating leukocyte firm adhesion, CD18 integrin/ICAM-1 interactions regulate leukocyte rolling velocities and thereby optimize L-selectin-mediated leukocyte rolling. PMID- 12370392 TI - Thrombin induces mast cell adhesion to fibronectin: evidence for involvement of protease-activated receptor-1. AB - Thrombin activates mast cells to release inflammatory mediators through a mechanism involving protease-activated receptor-1 (PAR-1). We hypothesized that PAR-1 activation would induce mast cell adhesion to fibronectin (FN). Fluorescent adhesion assay was performed in 96-well plates coated with FN (20 microg/ml). Murine bone marrow cultured mast cells (BMCMC) were used after 3-5 wk of culture (>98% mast cells by flow cytometry for c-Kit expression). Thrombin induced beta hexosaminidase, IL-6, and matrix metalloproteinase-9 release from BMCMC. Thrombin and the PAR-1-activating peptide AparafluoroFRCyclohexylACitY-NH(2) (cit) induced BMCMC adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive peptide FSLLRY-NH(2) had no effect. Thrombin and cit induced also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was inhibited by anti-alpha(5) integrin Ab (51.1 +/- 6.7%; n = 5). The combination of anti-alpha(5) and anti alpha(4) Abs induced higher inhibition (65.7 +/- 7.1%; n = 5). Unlike what is known for FcepsilonRI-mediated adhesion, PAR-1-mediated adhesion to FN did not increase mediator release. We then explored the signaling pathways involved in PAR-1-mediated mast cell adhesion. Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 +/- 7.3% (n = 4), indicating that G(i) proteins are involved. Wortmannin and calphostin almost completely inhibited PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and protein kinase C are involved. Adhesion was partially inhibited by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5 +/- 3.3%; n = 3) and the p38 inhibitor SB203580 (25.1 +/- 10.4%; n = 3). The two inhibitors had additive effects. Therefore, thrombin mediates mast cell adhesion through the activation of G(i) proteins, phosphoinositol 3-kinase, protein kinase C, and mitogen-activated protein kinase pathways. PMID- 12370393 TI - HIV-1 envelope gp41 peptides promote migration of human Fc epsilon RI+ cells and inhibit IL-13 synthesis through interaction with formyl peptide receptors. AB - We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1(MN) envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 x 10(-7) M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10(-4) M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10(-8)-10(-6) M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils. PMID- 12370394 TI - Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells. AB - Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1alpha, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1beta (IL 1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3 morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation. PMID- 12370395 TI - House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease activated receptor (PAR)-2 and inactivates PAR-1. AB - In previous studies, we demonstrated that allergenic house dust mite proteases are potent inducers of proinflammatory cytokines from the respiratory epithelium, although the precise mechanisms involved were unclear. In this study, we investigated whether this was achieved through activation of protease-activated receptor (PAR)-1 or -2. Pretreatment of A549 respiratory epithelial cells with the clinically important cysteine protease allergen, Der p 1, ablated subsequent PAR-1, but not PAR-2 agonist peptide-induced IL-6 and IL-8 release. HeLa cells transfected with the plasmid coding for PAR-2, in contrast to PAR-1, released significant concentration of IL-6 after exposure to Der p 1. Exposure of HeLa cells transfected with either PAR-1/enhanced yellow fusion protein or PAR 2/enhanced yellow fusion protein to Der p 1 caused receptor internalization in the latter cells only, as judged by confocal microscopy with re-expression of the receptor within 120-min postenzyme exposure. Der p 1-induced cytokine release from both A549 and transfected HeLa cells was accompanied by changes in intracellular Ca(2+) concentrations. Desensitization studies showed that Der p 1 pretreatment of the A549 cells resulted in the abolition of both trypsin- and PAR 2 agonist peptide-induced Ca(2+) release, but not that induced by subsequent exposure to either thrombin or PAR-1 agonist peptide. These data indicate for the first time that the house dust mite allergen Der p 1-induced cytokine release from respiratory epithelial cells is, in part, mediated by activation of PAR-2, but not PAR-1. PMID- 12370396 TI - P-selectin, and not E-selectin, negatively regulates murine megakaryocytopoiesis. AB - To assess the role of P-selectin and E-selectin in megakaryocytopoiesis, in vitro assays were performed in animal models deficient in both adhesion receptors. There was a significantly greater number of IL-3-responsive megakaryocyte progenitors CFU (CFU-MK) and an increase in immature megakaryoblasts in response to IL-6 in the P-selectin-null mice compared with the wild-type controls. Furthermore, P-selectin-null mice showed a greater number of CFU-MK colonies derived from CD34(+) cells in response to IL-3 or IL-3 plus stem cell factor. A significant shift in baseline ploidy with a reduction in 8N cells and an increase in 32N cells was also observed in the P-selectin-null mice. Secretion of the inhibitory growth factor TGF-beta1 and not TGF-beta2 was significantly lower in the supernatants of cultures containing bone marrow cells from P-selectin deficient mice as compared with those from the wild-type control bone marrow cells. No differences in the responsiveness of murine CFU-MK, immature megakaryocytes, or 5-fluorouracil-selected stem cells to cytokines were observed in E-selectin-null mice as compared with the control mice. These studies indicate that the absence of P-selectin, and not E-selectin, resulted in an altered adhesion environment with subsequent expansion of megakaryocyte progenitors and immature megakaryoblasts, enhanced secretion of TGF-beta1, and apparent increased responsiveness to inflammatory cytokines. PMID- 12370397 TI - Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production. AB - Mast cells are implicated in the pathogenesis of a broad spectrum of immunological disorders. These cells release inflammatory mediators in response to a number of stimuli, including IgE-Ag complexes. The degranulation of mast cells is modified by PGs. To begin to delineate the pathway(s) used by PGs to regulate mast cell function, we examined bone marrow-derived mast cells (BMMC) cultured from mice deficient in the EP(1), EP(2), EP(3), and EP(4) receptors for PGE(2). Although BMMCs express all four of these PGE(2) receptors, potentiation of Ag-stimulated degranulation and IL-6 cytokine production by PGE(2) is dependent on the EP(3) receptor. Consistent with the coupling of this receptor to G(alphai), PGE(2) activation of the EP(3) receptor leads to both inhibition of adenylate cyclase and increased intracellular Ca(2+). The magnitude of increase in intracellular Ca(2+) induced by EP(3) activation is similar to that observed after activation of cells with IgE and Ag. Although PGE alone is not sufficient to initiate BMMC degranulation, stimulation of cells with PGE along with PMA induces degranulation. These actions are mediated by the EP(3) receptor through signals involving Ca(2+) mobilization and/or decreased cAMP levels. Accordingly, these studies identify PGE(2)/EP(3) as a proinflammatory signaling pathway that promotes mast cell activation. PMID- 12370398 TI - Activation of human oral epithelial cells by neutrophil proteinase 3 through protease-activated receptor-2. AB - Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein coupled PAR-2 and actively participates in the process of inflammation such as periodontitis. PMID- 12370399 TI - The relationship between allergen-induced tissue eosinophilia and markers of repair and remodeling in human atopic skin. AB - Several in vitro studies suggest that eosinophils may play a role in fibrosis, remodeling, and repair processes associated with IgE-mediated hypersensitivity. However, the relationship in vivo, between allergen-induced tissue eosinophilia and markers of repair has yet to be established in human atopic subjects. Using the allergen-induced cutaneous late-phase reaction as a model of allergic inflammation, we have tested the hypothesis that eosinophil-derived TGF-beta1 and IL-13 are temporarily associated with myofibroblast formation and deposition of tenascin and procollagen I. Biopsies were taken from atopic volunteers at 1, 3, 6, 24, 48, and 72 h after intradermal allergen challenge and were examined by immunohistochemistry. Following the peak of the late-phase reaction (6 h) there were persisting TGF-beta1(+) eosinophils, alpha-smooth muscle actin(+) myofibroblasts, tenascin immunoreactivity, and procollagen-I(+) cells 24-48 h postchallenge. Direct evidence of generation of repair markers was obtained by coculture of eosinophils and fibroblasts. This resulted in alpha-smooth muscle actin immunoreactivity that was inhibitable by neutralizing Abs to TGF-beta as well as production of tenascin transcripts and protein product. TGF-beta1 and IL 13 also induced tenascin expression. We conclude that TGF-beta1 and IL-13, provided partially by eosinophils, contribute to repair and remodeling events in allergic inflammation in human atopic skin. PMID- 12370400 TI - TGF-beta and IL-13 synergistically increase eotaxin-1 production in human airway fibroblasts. AB - Chronic diseases may involve an "innate" response followed by an adaptive immune response, of a Th1 or Th2 variety. Little is known regarding the interactions of these responses. We hypothesized that TGF-beta1 (innate response factor associated with wound repair) in combination with IL-13 (Th2 factor) might augment inflammatory processes associated with asthma. Airway fibroblasts were cultured from asthmatic subjects and normal controls. These fibroblasts were exposed to TGF-beta1 and IL-13 alone or in combination, and eotaxin-1 expression and production were evaluated. At 48 h, eotaxin-1 production was markedly increased with the combination of TGF-beta1 and IL-13 (p < 0.0001) compared with either stimulus alone. mRNA increased slightly at 1 h with IL-13 or TGF-beta1 plus IL13, peaked, and became significantly increased over IL-13 alone at 24 h. Protein was measurable from 6 h with IL-13 and TGF-beta1 plus IL-13, but greater levels were measured over time with the combination. Actinomycin ablated the increase in mRNA and protein seen with IL-13 alone and with TGF-beta1 plus IL-13. Cycloheximide blocked the increase in mRNA at 6 h in both conditions, but also blocked the increase at 24 h with TGF-beta1 plus IL-13. STAT-6 was rapidly activated with both IL-13 and the combination, without difference. Finally, eotaxin-1-positive fibroblasts were identified in severe asthma biopsies in greater numbers than in normals. These results support the concept that interactions of innate and adaptive immune systems may be important in promoting the tissue eosinophilia of asthma, particularly in those with more severe disease. PMID- 12370401 TI - Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection. AB - The lung is known to be particularly susceptible to complement-mediated injury. Both C5a and the membrane attack complex (MAC), which is formed by the terminal components of complement (C5b-C9), can cause acute pulmonary distress in nontransplanted lungs. We used C6-deficient rats to investigate whether MAC causes injury to lung allografts. PVG.R8 lungs were transplanted orthotopically to MHC class I-incompatible PVG.1U recipients. Allografts from C6-sufficient (C6(+)) donors to C6(+) recipients were rejected with an intense vascular infiltration and diffuse alveolar hemorrhage 7 days after transplantation (n = 5). Ab and complement (C3d) deposition was accompanied by extensive vascular endothelial injury and intravascular release of von Willebrand factor. In contrast, lung allografts from C6-deficient (C6(-)) donors to C6(-) recipients survived 13-17 days (n = 5). In the absence of C6, perivascular mononuclear infiltrates of ED1(+) macrophages and CD8(+) T lymphocytes were present 7 days after transplantation, but vascular endothelial cells were quiescent, with minimal von Willebrand factor release and no evidence of alveolar hemorrhage or edema. Lung allografts were performed from C6(-) donors to C6(+) recipients (n = 5) and from C6(+) donors to C6(-) recipients (n = 5) to separate the effects of systemic and local C6 production. Lungs transplanted from C6(+) donors to C6(-) recipients had increased alveolar macrophages and capillary injury. C6 production by lung allografts was demonstrated at the mRNA and protein levels. These results demonstrate that MAC causes vascular injury in lung allografts and that the location of injury is dependent on the source of C6. PMID- 12370402 TI - Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases. AB - The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2 deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity. PMID- 12370403 TI - MHC susceptibility genes to IgA deficiency are located in different regions on different HLA haplotypes. AB - Familial predisposition to IgA deficiency (IgAD) suggests that genetic factors influence susceptibility. Most studies support a polygenic inheritance with a susceptibility locus (designated IGAD1) in the MHC, but its exact location is still controversial. This study aimed to map the predisposing IGAD1 locus (or loci) within the MHC by investigating the pattern of association of the disease with several markers in the region. DNA-based techniques were used to type individual alleles of four polymorphic HLA genes (HLA-DR, -DQA1, -DQB1, and HLA B), six microsatellites (all located between HLA-DR and HLA-B), and three single nucleotide polymorphisms on the TNF gene. The frequencies of these alleles were compared among ethnically matched populations comprising 182 patients and 343 controls. Additionally, we investigated parents and siblings of 100 of these patients. All four parental haplotypes were established in each family (n = 400), and transmission disequilibrium tests were performed. Surprisingly, our results did not support the hypothesis of a unique susceptibility gene being shared by all MHC susceptibility haplotypes. On HLA-DR1 and -DR7-positive haplotypes IGAD1 mapped to the class II region, whereas on haplotypes carrying HLA-DR3 the susceptibility locus mapped to the telomeric end of the class III region, as reported previously. Our results show how, in complex diseases, individuals may be affected for different genetic reasons and a single linkage signal to a region of a chromosome may actually be the result of disease-predisposing alleles in different linked genes in different pedigrees. PMID- 12370404 TI - A transgenic mouse model of autoimmune glomerulonephritis and necrotizing arteritis associated with cryoglobulinemia. AB - Mice implanted with hybridoma secreting 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) with cryoglobulin activity develop acute glomerulonephritis and cutaneous leukocytoclastic vasculitis. As the RF activity is implicated in the skin, but not glomerular lesions, it is still unclear whether the renal pathogenicity is determined by 6-19 H chains alone or their combination with L chains. To address this question, we have generated transgenic mice expressing only the H chain gene or both H and L chain genes of the 6-19 IgG3 anti-IgG2a RF and determined the development of glomerular and vascular lesions. H-single and H/L-double transgenic mice displayed comparable high amounts of IgG3 cryoglobulins, but only H/L-double transgenic mice having 10-fold higher levels of IgG3 anti-IgG2a RF progressively developed chronic, lethal glomerulonephritis. The severe glomerular lesions observed at 8-10 mo of age were very heterogeneous (membranoproliferative changes, crescents, and sclerosis); in addition, one-third of them had necrotizing arteritis in the kidneys and skeletal muscles. These renal and vascular changes were very different from those observed in the acute cryoglobulinemia, characterized by mainly "wire-loop" glomerular lesions and a cutaneous leukocytoclastic form of vasculitis. Thus, our data demonstrate the importance of a unique combination of the H and L chains for the expression of the pathogenic activity of IgG3 cryoglobulins and that a single autoantibody is able to induce different types of glomerular and vascular complications, depending on its production levels and kinetics. PMID- 12370405 TI - Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses. AB - Adenoviral (AdV) vectors can be used to transduce a wide range of human cells and tissues. However, pre-existing immunity to AdV, and enhancement of this immunity after repeated administration, limits their clinical application. This may be especially relevant when vectors are loaded into APCs. Helper-dependent AdV (Hd AdV), in which viral coding regions are replaced by human stuffer DNA, offers a new approach for limiting antiviral responses. To evaluate their immunogenicity, human dendritic cells (DCs) were infected with either an Hd-AdV or a conventional replication-deficient E1-deleted AdV (E1-AdV) and were evaluated for their capacity to stimulate antiviral T cell responses. Hd-AdV proved to be 50- to 275 fold more effective than E1-AdV at expressing the lacZ transgene in human DCs. PCR demonstrated similar transduction efficiencies, but RT-PCR revealed much higher expression of transgene mRNA after transduction with Hd-AdV. Functionally, DCs transduced with Hd-AdV stimulated the proliferation of autologous T cells to the same level as DCs transduced with E1-AdV. Identical viral-specific T cell responder frequencies were observed and T cells stimulated with either type of AdV-transduced DC lysed viral-infected target cells. Disrupting transcription of vector-based genes had no effect on T cell activation, suggesting that responses against both vectors were directed against preformed components of the viral capsid. We conclude that Hd-AdV vectors can be used to obtain higher transgene expression in human DCs but that they still evoke a vector-related immune response similar to that generated by E1-AdV. PMID- 12370406 TI - Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection. AB - We quantified T cell proliferation and thymic function in primary HIV infection (PHI; n = 19) and chronic HIV infection (CHI; n = 14) by measuring Ki67 staining and TCR excision circle (TREC) number. After antiretroviral therapy of PHI there is a profound decrease in the number and percentage of Ki67(+) T cells (<6% Ki67(+)) with no significant increase in TREC per million cells and a transient increase in TREC per milliliter. In contrast, after antiretroviral therapy of CHI there is a reduction in the percentage but little change in the total number of Ki67(+)CD4(+) T cells associated with increases in both TREC per million cells and TREC per milliliter. Using a mathematical model that accounts for proliferation, death, and redistribution of T cells, we find that redistribution is consistent with the TREC changes observed during treatment of PHI and that an increase in thymic output is needed to explain the increase in TREC during treatment of CHI. Consideration of TREC per milliliter shows that changes in proliferation alone cannot explain the changes in TREC. In addition, although increased proliferation of memory cells in HIV infection has been established, we find no difference in TREC per million CD45RA(-) "memory" T cells between healthy and infected individuals (p = 0.154 for CD4(+); p = 0.383 for CD8(+)). Finally, although the number of TREC per million cells is always much lower in memory T cells than in naive T cells, in the setting of HIV infection, given that memory cells make up a larger proportion of total T cells, we find that 50% of TREC per milliliter in CD4(+) T cells is harbored in the CD45RA(-) "memory" subset of our infected subjects. PMID- 12370407 TI - Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients. AB - We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance. PMID- 12370408 TI - Reciprocal developmental regulation of presynaptic ionotropic receptors. AB - Activation of ionotropic glycine receptors potentiates glutamate release in mature calyceal nerve terminals of the rat medial nucleus of the trapezoid body, an auditory brainstem nucleus. In young rats, glycine and its receptors are poorly expressed. We therefore asked whether GABA (gamma-aminobutyric acid) might play a larger role than glycine in the regulation of glutamate release in the absence of glycine receptors. Indeed, in rats younger than postnatal day 11 (P11), and before the onset of hearing, calyces expressed high levels of ionotropic GABA(A) receptors but few glycine receptors. Isoguvacine, a selective agonist at GABA(A) receptors, strongly enhanced excitatory postsynaptic currents in young rats but had little effect in rats older than P11. Down-regulation of presynaptic GABA(A) receptors did not reflect global changes in receptor expression, because the magnitude of GABA and glycine responses was similar at P13 in the parent-cell bodies of the calyces, the bushy cells of the cochlear nucleus. In outside-out patches excised from the nonsynaptic face of calyces, GABA and glycine evoked single-channel currents consistent with the properties of postsynaptic GABA(A) and glycine receptors. Inhibitory GABA(B) receptors were present on the calyx at all developmental stages examined. Thus, GABA initially acts on two receptor subtypes, both promoting and inhibiting glutamate release. With age, the former role is transferred to the glycine receptor during the period in which postsynaptic glycinergic transmission is acquired. PMID- 12370409 TI - The tomato fer gene encoding a bHLH protein controls iron-uptake responses in roots. AB - Iron deficiency is among the most common nutritional disorders in plants. To cope with low iron supply, plants with the exception of the Gramineae increase the solubility and uptake of iron by inducing physiological and developmental alterations including iron reduction, soil acidification, Fe(II) transport and root-hair proliferation (strategy I). The chlorotic tomato fer mutant fails to activate the strategy I. It was shown previously that the fer gene is required in the root. Here, we show that fer plants exhibit root developmental phenotypes after low and sufficient iron nutrition indicating that FER acts irrespective of iron supply. Mutant fer roots displayed lower Leirt1 expression than wild-type roots. We isolated the fer gene by map-based cloning and demonstrate that it encodes a protein containing a basic helix-loop-helix domain. fer is expressed in a cell-specific pattern at the root tip independently from iron supply. Our results suggest that FER may control root physiology and development at a transcriptional level in response to iron supply and thus may be the first identified regulator for iron nutrition in plants. PMID- 12370410 TI - Structure of the single-strand annealing domain of human RAD52 protein. AB - In eukaryotic cells, RAD52 protein plays a central role in genetic recombination and DNA repair by (i) promoting the annealing of complementary single-stranded DNA and (ii) stimulation of the RAD51 recombinase. The single-strand annealing domain resides in the N-terminal region of the protein and is highly conserved, whereas the nonconserved RAD51-interaction domain is located in the C-terminal region. An N-terminal fragment of human RAD52 (residues 1-209) has been purified to homogeneity and, similar to the full-size protein (residues 1-418), shown to promote single-strand annealing in vitro. We have determined the crystal structure of this single-strand annealing domain at 2.7 A. The structure reveals an undecameric (11) subunit ring with extensive subunit contacts. A large, positively charged groove runs along the surface of the ring, readily suggesting a mechanism by which RAD52 presents the single strand for reannealing with complementary single-stranded DNA. PMID- 12370411 TI - Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides. AB - Epithelial carcinoma and leukemia cells express sialyl Lewis x oligosaccharides as tumor-associated carbohydrate antigens. To determine the role of sialyl Lewis x oligosaccharides in tumor dissemination, human melanoma MeWo cells, which do not express sialyl Lewis x, were transfected with alpha1,3-fucosyltransferase III (FTIII), and cell lines expressing different amounts of sialyl Lewis x were isolated. When these cells were injected into the tail vein of nude mice, cells expressing moderate amounts of sialyl Lewis x (MeWo-FTIII.M) produced a significantly greater number of lung tumor foci than did parental MeWo cells. In contrast, cells expressing large amounts of sialyl Lewis x (MeWo-FTIII.H) produced few lung tumor foci in nude mice but were highly tumorigenic in beige mice, which have defective natural killer (NK) cells. In vitro assays demonstrated that MeWo-FTIII.H cells are much more sensitive to NK cell-mediated cytotoxicity than are MeWo-FTIII.M cells or parental MeWo cells and the susceptibility of MeWo-FTIII.H cells to NK cell-mediated cytolysis can be inhibited by preincubating MeWo-FTIII.H cells with anti-sialyl Lewis x antibody. Moreover, we discovered that NK cell-mediated cytolysis of MeWo-FTIII.H cells can be inhibited by the addition of an antibody against the NK cell receptor CD94 or sialyl Lewis x oligosaccharides. These results, combined with structural analysis of MeWo-FTIII.H cell carbohydrates, indicate that moderate amounts of sialyl Lewis x lead to tumor metastasis, whereas expression of high levels of sialyl Lewis x leads to an NK cell attack on tumor cells, demonstrating that expression of different amounts of sialyl Lewis x results in entirely different biological consequences. PMID- 12370412 TI - Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. AB - By using a cre-lox conditional knockout strategy, we report here the generation of androgen receptor knockout (ARKO) mice. Phenotype analysis shows that ARKO male mice have a female-like appearance and body weight. Their testes are 80% smaller and serum testosterone concentrations are lower than in wild-type (wt) mice. Spermatogenesis is arrested at pachytene spermatocytes. The number and size of adipocytes are also different between the wt and ARKO mice. Cancellous bone volumes of ARKO male mice are reduced compared with wt littermates. In addition, we found the average number of pups per litter in homologous and heterozygous ARKO female mice is lower than in wt female mice, suggesting potential defects in female fertility and/or ovulation. The cre-lox ARKO mouse provides a much-needed in vivo animal model to study androgen functions in the selective androgen target tissues in female or male mice. PMID- 12370413 TI - Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR). AB - The nuclear receptor PXR (pregnane X receptor) protects the body from hepatotoxicity of secondary bile acids such as lithocholic acid (LCA) by inducing expression of the hydroxylating cytochrome P450 enzyme CYP3A and promoting detoxification. We found that activation of PXR also increases the activity and gene expression of the phase II conjugating enzyme dehydroepiandrosterone sulfotransferase (STD) known to sulfate LCA to facilitate its elimination. This activation is direct and appears to extend to other xenobiotic sulfotransferases as well as to 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2), an enzyme that generates the donor cofactor for the reaction. Because sulfation plays an important role in the metabolism of many xenobiotics, prescription drugs, and toxins, we propose that PXR serves as a master regulator of the phase I and II responses to facilitate rapid and efficient detoxification and elimination of foreign chemicals. PMID- 12370414 TI - Genomewide analysis of the Drosophila tetraspanins reveals a subset with similar function in the formation of the embryonic synapse. AB - Tetraspanins encode a large conserved family of proteins that span the membrane four times and are expressed in a variety of eukaryotic tissues. They are part of membrane complexes that are involved in such diverse processes as intracellular signaling, cellular motility, metastasis, and tumor suppression. The single fly tetraspanin characterized to date, late bloomer (lbm), is expressed on the axons, terminal arbors, and growth cones of motoneurons. In embryos lacking Lbm protein, motoneurons reach their muscle targets, but initially fail to form synaptic terminals. During larval stages, however, functional contacts are formed. The newly available genomic sequence of Drosophila melanogaster indicates the existence of 34 additional members of the tetraspanin family in the fly. To address the possibility that other tetraspanins with functions that might compensate for a lack of lbm exist, we determined the expression domains of the Drosophila tetraspanin gene family members by RNA in situ analysis. We found two other tetraspanins also expressed in motoneurons and subsequently generated a small chromosomal deletion that removes all three motoneuron-specific tetraspanins. The deletion results in a significant enhancement in the lbm phenotype, indicating that the two additional motoneuron-expressed tetraspanins can, at least in part, compensate for the absence of lbm during the formation of the embryonic synapse. PMID- 12370415 TI - Amino acid deletions are introduced into the V2 region of gp120 during independent pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV) infections of rhesus monkeys generating variants that are macrophage tropic. AB - Highly pathogenic simian immunodeficiency virus/HIV chimeric viruses (SHIVs) cause extremely rapid, irreversible, and systemic depletions of CD4(+) T lymphocytes in inoculated rhesus monkeys. In the absence of this T cell subset, virus production can be sustained for several months by tissue macrophage. During independent infections of seven animals with uncloned virus stocks, SHIV variants emerged bearing amino acid deletions that affected specific residues of the gp120 V2 loop. Some of these macrophage-phase SHIVs replicated to high levels in alveolar macrophage. PMID- 12370416 TI - Error catastrophe and antiviral strategy. PMID- 12370417 TI - Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy. AB - Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and dihexanoylphosphatidylcholine (DHPC) provided a detailed description of protein-detergent interactions. The NOEs were measured in 3D (15)N- and (13)C-resolved [(1)H,(1)H]-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly (2)H,(13)C,(15)N-labeled OmpX in micelles of DHPC at natural isotope abundance. In these mixed micelles the NMR structure of OmpX consists of an eight-stranded antiparallel beta-barrel. The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 A in height, which is centered about the middle of the long axis through the beta-barrel. In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket. The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the beta-barrel in a double-layer lipid membrane. PMID- 12370418 TI - B cells develop in the zebrafish pancreas. AB - The zebrafish, with its transparent free-living embryo, is a useful organism for investigating early stages in lymphopoiesis. Previously, we showed that T cells differentiate in the thymus by day 4, but no sites for B cell differentiation were seen until 3 weeks. We report here that on day 4, we detect rearrangements of genes encoding B cell receptors in DNA extracted from whole fish. Also by day 4, rag1 transcripts are seen in the pancreas, an organ not previously associated with lymphopoiesis; by day 10, Igmu transcripts are detected here. Thus, in zebrafish, the pancreas assumes the role of both the liver in fetal mice and the spleen in neonatal mice. PMID- 12370419 TI - DBC2, a candidate for a tumor suppressor gene involved in breast cancer. AB - A previously uncharacterized gene, DBC2 (deleted in breast cancer), was cloned from a homozygously deleted region at human chromosome 8p21. DBC2 contains a highly conserved RAS domain and two putative protein interacting domains. Our analyses indicate that DBC2 is the best candidate tumor suppressor gene from this region. It lies within the epicenter of the deletions and is homozygously deleted in 3.5% (7/200) of breast tumors. Mutation analysis of DBC2 led to discovery of two instances of somatic missense mutations in breast tumor specimens, whereas no missense mutations were found in other candidates from the region. Unlike other genes in the region, expression of DBC2 is often extinguished in breast cancer cells or tissues. Moreover, our functional analysis revealed that DBC2 expression in breast cancer cells lacking DBC2 transcripts causes growth inhibition. By contrast, expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancer cells. PMID- 12370420 TI - Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes. AB - We report on preparation of rhodopsin proteoliposomes with the cytoplasmic domain of rhodopsin facing the exterior of the proteoliposomes. Rhodopsin purified from rod outer segments of bovine retinae by immunoaffinity chromatography in octyl glucoside was reconstituted into liposomes prepared from soybean phospholipids by detergent dialysis. The orientation of rhodopsin in the liposomes was determined by susceptibility of its C terminus to papain and the endoproteinase, Asp-N, followed by SDS/PAGE, which showed that the cytoplasmic domain in at least 90% of rhodopsin faced the exterior of the proteoliposomes. By using escape of (32)P KP(i) encapsulated in the proteoliposomes as the assay, the half-life of the proteasomes was approximately 8 days. After light activation, rhodopsin in proteoliposomes showed the rate of decay of metarhodopsin II and the initial rate of transducin activation comparable with the rates of rhodopsin in rod outer segment membranes. This finding demonstrates the functional capability of rhodopsin in proteoliposomes for kinetic studies of protein-protein interactions. PMID- 12370421 TI - Interruption of antiretroviral therapy to augment immune control of chronic HIV-1 infection: risk without reward. PMID- 12370422 TI - Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. AB - Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H(2)L(d) promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium. PMID- 12370424 TI - Climate, changing phenology, and other life history traits: nonlinearity and match-mismatch to the environment. PMID- 12370423 TI - Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. AB - An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI(-) HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI(-) stable cell line showed it to be Man(5)GlcNAc(2). In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI(-) inducible cell line at levels comparable with those obtained with WT rhodopsin. PMID- 12370425 TI - Mice with chronic norepinephrine deficiency resemble amphetamine-sensitized animals. AB - Acute pharmacological blockade of alpha1 adrenoreceptors (ARs) attenuates the locomotor response to amphetamine (LRA). We took a genetic approach to study how norepinephrine (NE) signaling modulates psychostimulant responses by testing LRA in dopamine beta-hydroxylase knockout (Dbh-/-) mice that lack NE. Surprisingly, Dbh-/- animals were hypersensitive to the behavioral effects of amphetamine. Amphetamine (2 mg/kg) elicited greater locomotor activity in Dbh-/- mice compared to controls, whereas 5 mg/kg caused stereotypy in Dbh-/- mice, which is only observed in control mice at higher doses. Prazosin, an alpha1AR antagonist, attenuated LRA in Dbh+/- mice but had no effect in Dbh-/- mice. Changes in the sensitivity of dopamine (DA)-signaling pathways may contribute to the altered amphetamine responses of Dbh-/- mice because they were relatively insensitive to a D1 agonist and hypersensitive to a D2 agonist. Daily amphetamine administration resulted in behavioral sensitization in both Dbh+/- and Dbh-/- mice, demonstrating that NE is not required for the development or expression of behavioral sensitization. Daily prazosin administration blunted but did not completely block locomotor sensitization in Dbh+/- mice, suggesting that alpha1AR signaling contributes to, but is not required for sensitization in Dbh+/- control animals. We conclude that in contrast to acute alpha1AR blockade, chronic NE deficiency induces changes similar to sensitization, perhaps by altering DA signaling pathways. PMID- 12370426 TI - Long-term systemic therapy of Fabry disease in a knockout mouse by adeno associated virus-mediated muscle-directed gene transfer. AB - Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, alpha-galactosidase A (alpha-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the alpha-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that alpha-gal A activity in plasma was increased to approximately 25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-alpha-gal A antibodies. The alpha-gal A activity in various organs of treated Fabry mice remained 5-20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease. PMID- 12370427 TI - The origin of a developmentally regulated Igh replicon is located near the border of regulatory domains for Igh replication and expression. AB - The 3' Ig heavy chain locus (Igh) regulatory region is the most downstream known element of the murine Igh gene cluster. We report here that the nearest non-Igh genes-Crip, Crp2, and Mta1-are located approximately 70 kb further downstream and are beyond the end of the domain of Igh transcriptional regulation. We have localized an origin of replication in MEL cells to a 3-kb segment located between the 3' Igh regulatory region and Crip. Sequences downstream of this origin are replicated by forks that move in both directions. Sequences upstream of this origin (Igh-C, -D, and -J) are replicated in a single direction through a 500-kb segment in which no active bidirectional origins can be detected. We propose that this origin may lie at or near the end of the Igh regulation domain. PMID- 12370428 TI - An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta,17beta diol, and CYP7B1, regulates prostate growth. AB - Epithelial proliferation of the ventral prostate in rodents peaks between 2 and 4 weeks of age, and by week 8, proliferating cells are rare. We have used ERbeta(-/ ) and CYP7B1(-/-) mice to investigate the role of ERbeta and one of its ligands, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), in growth of the ventral prostate. Before puberty, ERbeta was found in quiescent but not in proliferating cells, and proliferating cells occurred more frequently in ventral prostates of ERbeta(-/-) mice than in wild-type littermates. Treatment with 3betaAdiol decreased proliferation in wild-type but not in ERbeta(-/-) mice. In rats, treatment with 3betaAdiol from postnatal day 2 to 28 resulted in reduction in growth of ventral prostates. The prostates of CYP7B1(-/-) mice were hypoproliferative before puberty and smaller than those of their wild-type littermates after puberty. Because CYP7B1 represents the major pathway for inactivating 3betaAdiol in the prostate, we suggest that ERbeta, 3betaAdiol, and CYP7B1 are the components of a pathway that regulates growth of the rodent ventral prostate. In this pathway, ERbeta is an antiproliferative receptor, 3betaAdiol is an ERbeta ligand, and CYP7B1 is the enzyme that regulates ERbeta function by regulating the level of 3betaAdiol. PMID- 12370429 TI - APC-dependent suppression of colon carcinogenesis by PPARgamma. AB - Activation of PPARgamma by synthetic ligands, such as thiazolidinediones, stimulates adipogenesis and improves insulin sensitivity. Although thiazolidinediones represent a major therapy for type 2 diabetes, conflicting studies showing that these agents can increase or decrease colonic tumors in mice have raised concerns about the role of PPARgamma in colon cancer. To analyze critically the role of this receptor, we have used mice heterozygous for Ppargamma with both chemical and genetic models of this malignancy. Heterozygous loss of PPARgamma causes an increase in beta-catenin levels and a greater incidence of colon cancer when animals are treated with azoxymethane. However, mice with preexisting damage to Apc, a regulator of beta-catenin, develop tumors in a manner insensitive to the status of PPARgamma. These data show that PPARgamma can suppress beta-catenin levels and colon carcinogenesis but only before damage to the APC/beta-catenin pathway. This finding suggests a potentially important use for PPARgamma ligands as chemopreventative agents in colon cancer. PMID- 12370430 TI - Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. AB - Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug. PMID- 12370431 TI - Natural alleles at a tomato fruit size quantitative trait locus differ by heterochronic regulatory mutations. AB - fw2.2 is a major quantitative trait locus that accounts for as much as 30% of the difference in fruit size between wild and cultivated tomatoes. Evidence thus far indicates that fw2.2 alleles modulate fruit size through changes in gene regulation rather than in the FW2.2 protein itself. To investigate the nature of these regulatory changes and the manner in which they may affect fruit size, a pair of nearly isogenic lines has been subjected to detailed developmental, transcriptional, mitotic, and in situ hybridization studies. The results indicate that the large- and small-fruited alleles of fw2.2 differ in peak transcript levels by approximately 1 week. Moreover, this difference in timing of expression is associated with concomitant changes in mitotic activity in the early stage of fruit development. The changes in timing of gene expression (heterochronic allelic variation), combined with overall differences in total transcript levels, are sufficient to account for a large portion phenotypic differences in fruit weight associated with the two alleles. PMID- 12370432 TI - Identification of a localization factor for the polar positioning of bacterial structural and regulatory proteins. AB - Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90 kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a DeltapodJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system. PMID- 12370433 TI - Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. AB - Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group A Streptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component regulatory system, designated covRS (cov, control of virulence; csrRS), negatively controls expression of five proven or putative virulence factors (capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase). Inactivation of covRS results in enhanced virulence in mouse models of invasive disease. Using DNA microarrays and quantitative RT-PCR, we found that CovR influences transcription of 15% (n = 271) of all chromosomal genes, including many that encode surface and secreted proteins mediating host-pathogen interactions. CovR also plays a central role in gene regulatory networks by influencing expression of genes encoding transcriptional regulators, including other two-component systems. Differential transcription of genes influenced by covR also was identified in mouse soft-tissue infection. This analysis provides a genome-scale overview of a virulence gene network in an important human pathogen and adds insight into the molecular mechanisms used by group A Streptococcus to interact with the host, promote survival, and cause disease. PMID- 12370434 TI - Stimulation of HIV-specific cellular immunity by structured treatment interruption fails to enhance viral control in chronic HIV infection. AB - Potent antiretroviral therapy (ART) suppresses HIV-1 viral replication and results in decreased morbidity and mortality. However, prolonged treatment is associated with drug-induced toxicity, emergence of drug-resistant viral strains, and financial constraints. Structured therapeutic interruptions (STIs) have been proposed as a strategy that could boost HIV-specific immunity, through controlled exposure to autologous virus over limited time periods, and subsequently control viral replication in the absence of ART. Here, we analyzed the impact of repeated STIs on virological and immunological parameters in a large prospective STI study. We show that: (i) the plateau virus load (VL) reached after STIs correlated with pretreatment VL, the amount of viral recrudescence during the treatment interruptions, and the off-treatment viral rebound rate; (ii) the magnitude and the breadth of the HIV-specific CD8(+) T lymphocyte response, despite marked interpatient variability, increased overall with STI. However, the quantity and quality of the post-STI response was comparable to the response observed before any therapy; (iii) individuals with strong and broad HIV-specific CD8(+) T lymphocyte responses at baseline retained these characteristics during and after STI; (iv) the increase in HIV-specific CD8(+) T lymphocyte frequencies induced by STI was not correlated with decreased viral set point after STI; and (v) HIV-specific CD4(+) T lymphocyte responses increased with STI, but were subsequently maintained only in patients with low pretreatment and plateau VLs. Overall, these data indicate that STI-induced quantitative boosting of HIV specific cellular immunity was not associated with substantial change in viral replication and that STI was largely restoring pretherapy CD8(+) T cell responses in patients with established infection. PMID- 12370435 TI - Two regulatory levels of transcriptional gene silencing in Arabidopsis. AB - In mammals, some fungi, and plants, DNA methylation plays a central role in the epigenetic control of gene transcription. Recently, however, a subclass of Arabidopsis mutants revealed that the release of transcriptional gene silencing (TGS) does not necessarily require DNA demethylation. Here, we address the fundamental question of whether these mutants delineate a previously uncharacterized, methylation-independent level of epigenetic regulation, or whether they just act downstream of DNA methylation signals. Two mutants described earlier, ddm1 and mom1, reactivate previously silent loci: ddm1 impairs TGS by reducing chromosomal DNA methylation, and mom1 releases TGS without affecting DNA methylation. We examined the epistatic relationship between ddm1 and mom1 by constructing double mutant strains. The synergistic release of TGS revealed by gene expression patterns from silent loci, drastic developmental abnormalities, and characteristic changes in nuclear architecture in these double mutants implies that DDM1 and MOM are likely to operate at independent levels in TGS control. Our results indicate that the methylation-independent silencing mechanism reinforces the methylation-based system and prevents extremely rapid epigenetic deregulation in plants with DNA methylation deficiencies. PMID- 12370436 TI - Computer assisted cloning of human neutral alpha-glucosidase C (GANC): a new paralog in the glycosyl hydrolase gene family 31. AB - The exponential expansion of the publicly available human DNA sequence database has increasingly facilitated cloning by homology of genes for biochemically defined, functionally similar proteins. We hypothesized that an as-yet uncloned human alpha-glucosidase (human neutral alpha-glucosidase C or GANC) is a previously uncharacterized member of a paralogous human glycosyl hydrolase gene family 31, sharing sequence homology and related, but not identical, functions with other cloned human alpha-glucosidases. We now report both the in silico and physical cloning of two alleles of human neutral alpha-glucosidase (designated GANC on the human gene map). This cloning and correct identification and annotation as GANC was successful only because of the application of the biochemical and genetic information we had previously developed regarding this gene to the results of the in silico method. Of note, this glucosidase, a member of family 31 glycosyl hydrolases, has multiple alleles, including a "null" allele and is potentially significant because it is involved in glycogen metabolism and localizes to a chromosomal region (15q15) reported to confer susceptibility to diabetes. PMID- 12370437 TI - Natural radioactivity and human mitochondrial DNA mutations. AB - Radioactivity is known to induce tumors, chromosome lesions, and minisatellite length mutations, but its effects on the DNA sequence have not previously been studied. A coastal peninsula in Kerala (India) contains the world's highest level of natural radioactivity in a densely populated area, offering an opportunity to characterize radiation-associated DNA mutations. We sampled 248 pedigrees (988 individuals) in the high-radiation peninsula and in nearby low-radiation islands as a control population. We sequenced their mtDNA, and found that the pedigrees living in the high-radiation area have significantly (P < 0.01) increased germ line point mutations between mothers and their offspring. In each mutation case, we confirmed maternity by autosomal profiling. Strikingly, the radioactive conditions accelerate mutations at nucleotide positions that have been evolutionary hot spots for at least 60,000 years. PMID- 12370438 TI - Structures and stabilities of higher coordinate onium-boronium dications (X(+)BH(3)+ and X(+)BH(5)+; X = NH(3), PH(3), H(2)O, and H(2)S). AB - Structures of higher coordinate onium-boronium dications (X(+)BH(3)+ 1-4a and X(+)BH(5)+ 1-4d; X = NH(3), PH(3), H(2)O, and H(2)S) were calculated by using the ab initio method at the MP2/6-311+G** level. All of the dications 1-4a contain a four-coordinate boron atom with a three-center two-electron bond involving boron and two hydrogens. On the other hand, all the dications 1-4d contain a six coordinate boron atom with two three-center two-electron bonds. The thermodynamics of the complexations of 1-4a and H(2) to form 1-4d were computed. Deprotonations of 1-4d were found to be substantially endothermic. PMID- 12370439 TI - The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast. AB - The Ume6 transcription factor in yeast is known to both repress and activate expression of diverse genes during growth and meiotic development. To obtain a more complete profile of the functions regulated by this protein, microarray analysis was used to examine transcription in wild-type and ume6Delta diploids during vegetative growth in glucose and acetate. Two different genetic backgrounds (W303 and SK1) were examined to identify a core set of strain independent Ume6-regulated genes. Among genes whose expression is controlled by Ume6 in both backgrounds, 82 contain homologies to the Ume6-binding site (URS1) and are expected to be directly regulated by Ume6. The vast majority of those whose functions are known participate in carbon/nitrogen metabolism and/or meiosis. Approximately half of the Ume6 direct targets are induced during meiosis, with most falling into the early meiotic expression class (cluster 4), and a smaller subset in the middle and later classes (clusters 5-7). Based on these data, we propose that Ume6 serves a unique role in diploid cells, coupling metabolic responses to nutritional cues with the initiation and progression of meiosis. Finally, expression patterns in the two genetic backgrounds suggest that SK1 is better adapted to respiration and W303 to fermentation, which may in part account for the more efficient and synchronous sporulation of SK1. PMID- 12370440 TI - Molecular evidence for ecological speciation in tropical habitats. AB - Recent research on rainforest speciation has highlighted the importance of habitat variation in generating population diversification but lacks evidence of an associated reduction in gene flow. This paper describes a study in which molecular markers were used to examine the effects of allopatric divergence and habitat on levels of gene flow in the Caribbean lizard, Anolis roquet. Three study transects were constructed to compare variation in microsatellite allele frequencies and morphology across phylogenetic and habitat boundaries in northern Martinique. Results showed reductions in gene flow to be concordant with divergent selection for habitat type. No evidence could be found for divergence in allopatry influencing current gene flow. Morphological data match these findings, with multivariate analysis showing correlation with habitat type but no grouping by phylogenetic lineage. The results support the ecological speciation model of evolutionary divergence, indicating the importance of habitats in biodiversity generation. PMID- 12370441 TI - Predicting the effects of climate change on avian life-history traits. AB - Across North America, tree swallows have advanced their mean date of clutch initiation (lay date) by approximately 9 days over the past 30 years, apparently in response to climate change. In a sample of 2,881 nest records collected by the lay public from 1959 to 1991, we examined whether clutch size has also responded to climate change. We found that clutch size is strongly related to lay date, both within and among years, and there has been no significant temporal variation in the slopes or intercepts of the clutch-size/lay-date regressions. As a consequence, we expected increases in clutch size with advancement in lay date; however, we detected no such trend over time. The distributions of egg-laying dates were more constricted in the warmest (and earliest) years, suggesting that changes in mean clutch size might be constrained by changes in the distribution of laying dates. If spring temperatures continue to increase, we predict further reductions of variance in laying dates and relatively small increases in clutch size. Such constraints on life-history variation probably are common and need to be considered when modeling the effects of climate change on reproduction in natural populations. Predicting the long-term effects of constraints and interpreting changes in life-history traits require a better understanding of both adaptive and demographic effects of climate change. PMID- 12370442 TI - Transport of nitrated albumin across continuous vascular endothelium. AB - Because modification of plasma albumin on tyrosine residues generates nitrated albumin (NOA) that may function as a mechanism of nitrogen monoxide clearance from microcirculation, we investigated biochemicaly and morphologically the cell surface binding and the transendothelial transport of NOA. An electron microscopic study was carried out with mouse lungs and hearts perfused in situ with NOA and NOA-Au complexes. The results indicate that NOA-Au can bind to the endothelial cell surface, and its binding can be blocked by albumin plus nitrotyrosine (NO-tyrosine) or abolished by excess NOA. We detected NOA-Au into perivascular spaces as early as 30 sec after the beginning of its perfusion. NOA, unlike native albumin, leaves the vascular lumina via both endothelial caveolae and open junctions. By cross-linking and ligand blotting analysis, we showed that NOA interacted with the same albumin binding proteins of 16-18, 30-32, 60, and 74 kDa as native albumin. ELISA performed on tissue homogenates obtained from the same specimens showed that NOA transport was 2- to 4-fold greater than native albumin. The augmented transendothelial transport of NOA reflects its transcytosis as well as its exit from the microcirculation via open junctions. The increased transport of NOA may serve as an important mechanism that protects a vascular bed against the damaging effects of nitrogen monoxide and peroxynitrite. PMID- 12370443 TI - S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase. AB - The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses asymmetrically methylated arginine residues that are endogenously produced inhibitors of nitric oxide synthases (NOS). We and others have proposed that DDAH activity is a key determinant of intracellular methylarginine concentrations and that factors that regulate the activity of DDAH may modulate nitric oxide (NO) production in vivo. We recently solved the crystal structure of a bacterial DDAH and identified a Cys-His-Glu catalytic triad [Murray-Rust, J., Leiper, J. M., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P. & McDonald, N. (2001) Nat. Struct. Biol. 8, 679-683]. The presence of a reactive cysteine residue (Cys-249) in the active site of DDAH raised the possibility that DDAH activity might be directly regulated by S-nitrosylation of this residue by NO. In the present study, we demonstrate that recombinant DDAH is reversibly inhibited after incubation with NO donors in vitro. Similarly mammalian DDAH in cytosolic extracts is also reversibly inhibited by NO donors. In cultured endothelial cells, heterologously expressed human DDAH II was S-nitrosylated after cytokine induced expression of the inducible NOS isoforms. The implication of these findings is that under certain conditions when NO generation increases, S nitrosylation diminishes DDAH activity and this would be expected to lead to accumulation of asymmetric dimethylarginine and inhibition of NOS. This observation may help explain why expression of iNOS often leads to inhibition of activity of constitutively expressed NOS isozymes. We also identify Cys-His-Glu as a nitrosylation motif that is conserved in a family of arginine handling enzymes. PMID- 12370444 TI - Linkage limits the power of natural selection in Drosophila. AB - Population genetic theory shows that the efficacy of natural selection is limited by linkage-selection at one site interferes with selection at linked sites. Such interference slows adaptation in asexual genomes and may explain the evolutionary advantage of sex. Here, we test for two signatures of constraint caused by linkage in a sexual genome, by using sequence data from 255 Drosophila melanogaster and Drosophila simulans loci. We find that (i) the rate of protein adaptation is reduced in regions of low recombination, and (ii) evolution at strongly selected amino acid sites interferes with optimal codon usage at weakly selected, tightly linked synonymous sites. Together these findings suggest that linkage limits the rate and degree of adaptation even in recombining genomes. PMID- 12370445 TI - Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior. AB - By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous. We find no evidence of distinct elongation states among RNA polymerases. Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing. When transcribing a gene without strong pause sites, RNA polymerase molecules display transient pauses that are distributed randomly in both time and distance. Transitions between the active elongation mode and the paused state are instantaneous within the resolution of our measurements (<1 s). This elongation behavior is compared with that of a mutant RNA polymerase that pauses more frequently and elongates more slowly than wild type. PMID- 12370446 TI - A family of MHC class I-like genes located in the vicinity of the mouse leukocyte receptor complex. AB - Some members of the major histocompatibility complex (MHC) class I gene family are encoded outside the MHC. Here we describe a family of mouse class I-like genes mapping to the vicinity of the leukocyte receptor complex (LRC) on chromosome 7. This family, which we call Mill (MHC class I-like located near the LRC), has two members designated Mill1 and Mill2. Both genes are predicted to encode membrane glycoproteins with domain organization essentially similar to that of MHC class I heavy chains. The following features of Mill are noteworthy. (i) The deduced MILL proteins lack most of the residues known to be involved in the docking of peptides in classical MHC class I molecules. (ii) Among the known members of the class I gene family, MILL1 and MILL2 are related most closely to MICA/MICB encoded in the human MHC. (iii) Unlike all other known members of the class I gene family, Mill1 and Mill2 have an exon between those coding for the signal peptide and the alpha1 domain. (iv) Mill1 has a more restricted expression profile than Mill2. (v) The gene orthologous to Mill1 or Mill2 apparently is absent in the human. (vi) Mill1 and Mill2 show a limited degree of polymorphism in laboratory mice. The observation that the Mill family is related most closely to the MIC family, together with its apparent absence in the human, suggests its involvement in innate immunity. PMID- 12370448 TI - Cholesterol-lowering statins possess anti-inflammatory activity that might be useful for treatment of MS. PMID- 12370447 TI - Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon. AB - The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 x g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies. PMID- 12370449 TI - Painkiller headache. PMID- 12370450 TI - AAN clinical practice guidelines: above the fray. PMID- 12370451 TI - Statins as immunomodulators: comparison with interferon-beta 1b in MS. AB - BACKGROUND: Recent data suggest that statins may be potent immunomodulatory agents. In order to evaluate the potential role of statins as immunomodulators in MS, the authors studied their immunologic effects in vitro and compared them to interferon (IFN)beta-1b. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from untreated or IFN beta-1-treated patients with relapsing-remitting MS or from healthy donors (HD) and T cells were stimulated with concanavalin A, phytohemagglutinin, or antibody to CD3 in the presence of lovastatin, simvastatin, mevastatin, IFN beta-1b, or statins plus IFN beta-1b. The authors analyzed proliferative activity of T cells and B cells, cytokine production and release, activity of matrix metalloproteinases (MMP), and surface expression of activation markers, adhesion molecules, and chemokine receptors on both T and B cells. RESULTS: All three statins inhibited proliferation of stimulated PBMC in a dose-dependent manner, with simvastatin being the most potent, followed by lovastatin and mevastatin. IFN beta-1b showed a similar effect; statins and IFN beta-1b together added their inhibitory potentials. Furthermore, statins reduced the expression of activation-induced adhesion molecules on T cells, modified the T helper 1/T helper 2 cytokine balance, reduced MMP-9, and downregulated chemokine receptors on both B and T cells. Besides strong anti-inflammatory properties, statins also exhibited some proinflammatory effects. CONCLUSIONS: Statins are effective immunomodulators in vitro that merit evaluation as treatment for MS. PMID- 12370452 TI - MRI predictors of early conversion to clinically definite MS in the CHAMPS placebo group. AB - OBJECTIVE: To assess the ability of baseline MRI characteristics to predict the early development of clinically definite MS (CDMS) and combined CDMS/MRI outcomes in 190 patients with a positive MRI at the time of their first demyelinating event. METHODS: Based on individual and sets of baseline MRI characteristics, the authors evaluated the percentage of patients meeting outcomes of CDMS and various combined CDMS/MRI outcomes by 18 months. They also optimized a cutpoint for dichotomizing each baseline MRI characteristic and evaluated these variables using logistic regression to determine which MRI characteristics best predicted CDMS by 18 months. RESULTS: The presence of two or more gadolinium-enhancing lesions better predicted the development of CDMS and combined CDMS/MRI outcomes by 18 months than any other individual MRI characteristic or set of MRI characteristics. Among patients with two or more gadolinium-enhancing lesions, 52% developed CDMS compared with 24% of patients with fewer than two lesions. For those meeting the criteria of Barkhof et al., 32% of patients developed CDMS compared with 16% of those not meeting these criteria. Irrespective of individual or sets of criteria, however, the majority of patients developed either CDMS or demonstrated disease activity on brain MRI by 18 months. CONCLUSIONS: For patients with positive MRI at the time of their initial neurologic event, both gadolinium-enhancing lesions and the Barkhof criteria are predictors for development of CDMS over a short interval. However, these results, based on a combined CDMS/MRI outcome, suggest that the majority of these patients are already in the earliest stages of MS, regardless of whether any further MRI criteria are met. PMID- 12370453 TI - Early onset multiple sclerosis: a longitudinal study. AB - OBJECTIVE: To evaluate the clinical course of MS in individuals with onset of MS before age 16. METHODS: Patients with onset of MS before age 16 (n = 116) with complete clinical information on the clinical course from the MS Clinic at The University of British Columbia (UBC) Site Hospital computerized database (MS COSTAR) were included in this study. The data were compared to those from the Canadian natural history study for MS clinic attendees, regardless of age at onset. RESULTS: The mean duration of observation was 19.76 +/- 0.90 years; the mean age at MS onset was 12.73 +/- 0.25 years. Only three cases (2.6%) had a primary progressive (PP) MS course. To date, 60 (53.1%) of 113 subjects have developed secondary progressive (SP) MS. The 50% probability for SPMS was reached 23 years after onset. For patients with relapsing remitting (RR) or SPMS the mean disease duration from onset to the time of confirmed Expanded Disability Status Scale (EDSS) 3.0 was 16.03 +/- 1.17 years (at mean age 28.47 +/- 1.14); mean duration from onset to the time of EDSS 6.0 was 19.39 +/- 1.43 years (at mean age 32.32 +/- 1.44). Annual relapse rate was 0.54 +/- 0.05 per year. The correlation between the number of relapses during the first year of disease and the course of the disease was also significant. CONCLUSIONS: The prevalence of early onset MS (3.6%) in our study confirms the previous findings on early onset MS. A RR course was seen in the majority of cases of early onset MS. A high frequency of relapses, early age at permanent disability, and the presence of malignant cases raise the question of possible early use of disease-modifying therapy in patients with early onset MS. PMID- 12370454 TI - Features of medication overuse headache following overuse of different acute headache drugs. AB - OBJECTIVE: To investigate pharmacologic features such as mean critical duration until onset of medication-overuse headache (MOH) (MCDO), mean critical monthly intake frequencies (MCMIF), and mean critical monthly dosages (MCMD) as well as specific clinical features of MOH after overuse of different acute headache drugs, with a focus on newly approved triptans. METHODS: In a prospective study 98 patients with MOH according to International Headache Society (IHS) criteria underwent standardized inpatient withdrawal from their medication. Patient diaries and structured interviews were used to calculate the MCDO, MCMIF, and MCMD for each substance group. RESULTS: The MCDO was shortest for triptans (1.7 years), longer for ergots (2.7 years), and longest for analgesics (4.8 years). The MCMIF was lowest for triptans (18 single doses per month), higher for ergots (37), and highest for analgesics (114). Although patients overusing ergots and analgesics typically had a daily tension-type headache, patients with triptan induced MOH were more likely to describe a (daily) migraine-like headache or an increase in migraine frequency. CONCLUSION: Overuse of triptans leads to MOH faster and with lower dosages compared with ergots and analgesics. Clinical features of MOH depend on the type of overused headache medication. Pharmacologic and clinical characteristics of triptan-induced MOH call for the renewal of the current IHS classification. PMID- 12370455 TI - Opioids versus antidepressants in postherpetic neuralgia: a randomized, placebo controlled trial. AB - BACKGROUND: Tricyclic antidepressants (TCA) provide less than satisfactory pain relief for postherpetic neuralgia (PHN), and the role of opioids is controversial. OBJECTIVE: To compare the analgesic and cognitive effects of opioids with those of TCA and placebo in the treatment of PHN. METHODS: Seventy six patients with PHN were randomized in a double-blind, placebo-controlled, crossover trial. Each subject was scheduled to undergo three treatment periods (opioid, TCA, and placebo), approximately 8 weeks' duration each. Doses were titrated to maximal relief or intolerable side effects. The primary outcome measures were pain intensity (0 to 10 scale), pain relief (0 to 100%), and cognitive function. Analyses included patients who provided any pain ratings after having received at least a single dose of a study medication. RESULTS: Fifty patients completed two periods, and 44 patients completed all three. Mean daily maintenance doses were morphine 91 mg or methadone 15 mg and nortriptyline 89 mg or desipramine 63 mg. Opioids and TCA reduced pain (1.9 and 1.4) more than placebo (0.2; p < 0.001), with no appreciable effect on any cognitive measure. The trend favoring opioids over TCA fell short of significance (p = 0.06), and reduction in pain with opioids did not correlate with that following TCA. Treatment with opioids and TCA resulted in greater pain relief (38 and 32%) compared with placebo (11%; p < 0.001). More patients completing all three treatments preferred opioids (54%) than TCA (30%; p = 0.02). CONCLUSIONS: Opioids effectively treat PHN without impairing cognition. Opioids and TCA act via independent mechanisms and with varied individual effect. PMID- 12370456 TI - Alzheimer's disease can be accurately diagnosed in very mildly impaired individuals. AB - BACKGROUND: The growing propensity to diagnose AD in individuals with very mild cognitive impairment increases the danger of false-positive diagnostic errors. Unfortunately, there is little systematically acquired information about the accuracy of the AD diagnosis in very mildly impaired patients. OBJECTIVE: To determine the accuracy of the diagnosis of AD in very mildly impaired patients and to identify objective measures that effectively distinguish these patients from elderly normal controls (NC). METHODS: Consecutive patients with Mini-Mental State Examination scores of > or = 24 who received a clinical diagnosis of AD were evaluated annually for at least 3 years. The initial diagnosis was verified or refuted by autopsy or by information obtained in subsequent evaluations. Initial neuropsychological test scores of verified AD patients were compared with those of NC subjects to identify effective diagnostic measures. RESULTS: The diagnosis of AD was confirmed in 98 of 110 (89%) very mildly impaired patients (33/36 by autopsy, 65/74 by disease progression). The diagnosis was inaccurate in 12 patients (11%): Seven were subsequently diagnosed with other neurologic disorders, and five were ultimately found to be normal. Neuropsychological measures of delayed recall, verbal fluency, and global cognitive status (i.e., Mattis Dementia Rating Scale) provided excellent sensitivity (> or = 96%) and specificity (> or = 93%) for differentiating between very mildly impaired AD patients and NC subjects. CONCLUSIONS: When comprehensive assessment procedures are employed, AD can be diagnosed with reasonably high accuracy in very mildly impaired individuals. However, the dementia evaluation should be repeated after approximately 1 year to ensure the accuracy of the initial diagnosis. PMID- 12370457 TI - Reduced inhibition within primary motor cortex in patients with poststroke focal motor seizures. AB - BACKGROUND: Following an ischemic brain lesion, the affected cortex undergoes structural and functional changes that may lead to increased cortical excitability or decreased inhibitory neuronal activity, resulting in the occurrence of poststroke epileptic seizures in 6 to 10% of patients with stroke. METHODS: To assess motor cortical excitability, transcranial magnetic stimulation (TMS) was used to determine the silent period (SP) duration in 84 consecutive patients with ischemic stroke. RESULTS: In a subpopulation of six patients (38 to 72 years old) a significant decrease of the SP duration (mean 116 +/- 14 msec) was detected in either the arm or the leg on the affected side as compared to the corresponding unaffected limb (mean 231 +/- 32 msec). This electrophysiologic abnormality was clinically associated with focal motor seizures in five of the six patients, whereas none of the other 76 patients with normal or prolonged SP durations developed seizures or epilepsy. CONCLUSIONS: Silent period shortening in this group reflects decreased inhibitory activity that may partly be related to functional or structural impairment of GABAergic interneurons. TMS may be of value for determining patients with stroke at risk for developing poststroke seizures. PMID- 12370458 TI - Rates of progression in mild cognitive impairment and early Alzheimer's disease. AB - OBJECTIVE: To compare rates of progression in the very mildest stages of AD, including the stage currently identified as mild cognitive impairment (MCI), and to identify predictors of those rates. METHODS: Demented (n = 289) and nondemented (n = 230) individuals enrolled in longitudinal studies at an Alzheimer Disease Research Center received annual clinical and psychometric examinations for up to 20 years. In order of increasing dementia severity, demented individuals were diagnosed with incipient, very mild, or mild dementia; the incipient stage is equivalent to MCI. Outcome measures included death, nursing home placement, and psychometric scores. RESULTS: Rate of progression increased with dementia severity as staged by the Clinical Dementia Rating at entry into the study. With respect to the dichotomous outcomes, the increase with dementia severity was more dramatic for the endpoint of nursing home entry than it was for the endpoint of death. Increased rates of cognitive decline with increased dementia severity were also obtained for psychometric scores. There was limited evidence of other predictors of progression. CONCLUSIONS: The lack of effective predictors of the rate of dementia progression extends to the very earliest stages of the disease, including what is often called MCI. A new approach to the identification of correlates of rates of progression is needed. PMID- 12370459 TI - Mild cognitive impairment can be detected by multiple assessments in a single day. AB - BACKGROUND: Reliable detection of mild cognitive impairment (MCI), in many cases preceding AD, is important in determining the efficacy of emerging treatments. The operational definition of MCI is currently imprecise and would be improved by objective criteria. Inherent in the transition from MCI to AD is cognitive decline, which can be detected using multiple assessments over several years. OBJECTIVE: To determine whether multiple assessments on the same day could also differentiate well-studied subjects with very mild MCI from normal control subjects. METHODS: This study utilized a novel 15- to 18-minute computerized cognitive battery designed for frequent serial use, administered four times within 3 hours. Subjects were participants in a longitudinal healthy aging study (20 with MCI, 40 control subjects matched for age, gender, and education). RESULTS: The MCI group showed significantly attenuated learning performance with repetition on accuracy and reaction time tasks. Discriminant function analysis correctly classified 95% of subjects and 80% of those with MCI. CONCLUSIONS: Multiple assessments with standardized, repeatable cognitive measures is a promising method for reliably differentiating patients with early MCI in a single test session and deserves further study for refining patient selection in trials of therapeutic agents for MCI. PMID- 12370460 TI - Exercise tolerance in carnitine palmitoyltransferase II deficiency with IV and oral glucose. AB - BACKGROUND: Patients with carnitine palmitoyltransferase II (CPT II) deficiency often experience muscle pain and myoglobinuria during prolonged exercise because of impaired oxidation of long-chain fatty acids. OBJECTIVE: To investigate whether IV or oral glucose can improve exercise tolerance in CPT II deficiency. METHODS: Five patients with CPT II deficiency and healthy matched volunteers were investigated on a cycle ergometer at a constant workload of 60% of VO(2max). Perceived exertion, heart rate, and venous plasma glucose and insulin levels were monitored. The study was randomized, placebo controlled, single blind, and crossover. Glucose and placebo were administered both orally and IV in patients and IV in healthy subjects. RESULTS: In patients with CPT II, exercise duration was prolonged by 28 +/- 8% (p = 0.02), and perceived exertion (p = 0.05) and heart rate (p = 0.09) were lowered by glucose infusion. In contrast, IV glucose resulted in higher heart rate during exercise in healthy subjects. Oral glucose and placebo resulted in the same exercise duration, perceived exertion, and heart rate in patients. Plasma glucose and insulin were consistently elevated during exercise by oral and IV glucose vs placebo, but plasma glucose was higher and insulin lower in IV vs oral glucose studies in patients (p = 0.02). CONCLUSION: Exercise tolerance is markedly improved by a glucose infusion in patients with CPT II deficiency, but because of lower glucose availability and higher insulin levels that inhibit muscle glycogenolysis, the patients cannot achieve this effect themselves by oral glucose ingestion. PMID- 12370461 TI - Midlife adiposity and the future risk of Parkinson's disease. AB - BACKGROUND: Evidence suggests that nigrostriatal system disorders are associated with PD and adiposity. Whether patterns of adiposity coexist or predate clinical PD is unknown. This report examines the relation between midlife adiposity and the risk of PD. METHODS: Measurement of adiposity occurred from 1965 to 1968 in 7,990 men in the Honolulu Heart Program (aged 45 to 68 years and without PD). Adiposity measures included body mass index (BMI), subscapular skinfold thickness (SSF), and triceps skinfold thickness (TSF). Follow-up for incident PD occurred over a 30-year period. RESULTS: During the course of follow-up, PD was observed in 137 men. Among the measures of adiposity, age-adjusted incidence of PD increased threefold from 3.7/10,000 person-years in the bottom quartile of TSF (1 to 5 mm) to 11.1/10,000 person-years in the top quartile (11 to 32 mm, p < 0.001). Effects of TSF on PD were independent of cigarette smoking, coffee consumption, physical activity, daily caloric and fat intake, and the other measures of adiposity (p < 0.001). Whereas rates of PD were lowest in the bottom quartile of BMI and SSF vs higher quartiles, associations with PD were weaker than they were for TSF. The effect of TSF on clinical onset before age 65 years was similar to the effect that was observed in later life. CONCLUSIONS: Increased triceps skinfold thickness measured in midlife is associated with an elevated risk of future PD. Whether patterns of adiposity reflect a unique metabolic pathology in individuals at a high risk of PD warrants further study. PMID- 12370462 TI - Neuroanatomy of holoprosencephaly as predictor of function: beyond the face predicting the brain. AB - BACKGROUND: Despite advances in neuroimaging and molecular genetics of holoprosencephaly (HPE), the clinical spectrum of HPE has remained inadequately described. OBJECTIVE: To better characterize the clinical features of HPE and identify specific neuroanatomic abnormalities that may be useful predictors of neurodevelopmental function. METHODS: The authors evaluated 68 children with HPE in a multicenter, prospective study. Neuroimaging studies were assessed for the grade of HPE (lobar, semilobar, and alobar), the degree of nonseparation of the deep gray nuclei, and presence of dorsal cyst or cortical malformation. RESULTS: In general, the severity of clinical problems and neurologic dysfunctions correlated with the degree of hemispheric nonseparation (grade of HPE). Nearly three-quarters of the patients had endocrinopathies, with all having at least diabetes insipidus. The severity of endocrine abnormalities correlated with the degree of hypothalamic nonseparation (p = 0.029). Seizures occurred in approximately half of the children with HPE. The presence of cortical malformations was associated with difficult-to-control seizures. The presence and degree of dystonia correlated with the degree of nonseparation of the caudate and lentiform nuclei and the grade of HPE (p < 0.05). Hypotonia correlated with the grade of HPE (p < 0.05). Mobility, upper extremity function, and language correlated with the degree of nonseparation of the caudate, lentiform and thalamic nuclei, and grade of HPE (p < 0.01). CONCLUSIONS: Patients with HPE manifest a wide spectrum of clinical problems and neurologic dysfunction. The nature and severity of many of these problems can be predicted by specific neuroanatomic abnormalities found in HPE. PMID- 12370463 TI - The first sign of Babinski. PMID- 12370464 TI - Severity of sports-related concussion and neuropsychological test performance. AB - Concussion severity grades according to the Cantu, Colorado Medical Society, and American Academy of Neurology systems were not clearly related to the presence or duration of impaired neuropsychological test performance in 21 professional rugby league athletes. The use of concussion severity guidelines and neuropsychological testing to assist return to play decisions requires further investigation. PMID- 12370465 TI - Multiple sclerosis-associated retrovirus and MS prognosis: an observational study. AB - MS-associated retrovirus (MSRV) in the CSF may have gliotoxic properties and could be associated with a more disabling MS. The authors tested this hypothesis in 15 untreated patients with MS: 6 MSRV- and 9 MSRV+ at the time of CSF withdrawal. After a 3-year mean follow-up, MSRV- patients showed a stable MS course, whereas MSRV+ patients had a progressive course (p = 0.01). PMID- 12370466 TI - Treatment of steroid-unresponsive tumefactive demyelinating disease with plasma exchange. AB - The authors describe a patient with an isolated, gadolinium-enhancing, biopsy proven focus of tumefactive demyelination. There was marked clinical improvement with plasma exchange after failure of high-dose i.v. corticosteroids. The post treatment clinical course correlated with decreasing enhancement and lesion size on MRI. This patient's rapid clinical and MRI response suggests that plasma exchange may be beneficial in this disorder, and could perhaps serve as a diagnostic tool to avoid the need for brain biopsy. PMID- 12370467 TI - The overlap of amyotrophic lateral sclerosis and frontotemporal dementia. AB - Patients with frontotemporal dementia (FTD) with no known diagnosis of ALS or family history of ALS were clinically and electrophysiologically assessed for the presence of ALS. Of 36 patients studied, five met criteria for a definite diagnosis of ALS and two had EMG findings suggestive of denervation in one limb. An additional five patients had prominent fasciculations and six other patients had trouble swallowing but all had normal results on EMG studies. One of the patients with fasciculations and a normal EMG study progressed to definite ALS over the course of 1 year. PMID- 12370468 TI - Familial dementia with Lewy bodies: clinicopathologic analysis of two kindreds. AB - Familial cases of dementia with Lewy bodies (DLB) are rare. The authors describe two small kindreds with familial DLB: one with pure DLB meeting consensus criteria for DLB and one with coexistent AD pathology that did not fulfill DLB criteria. The authors call attention to the diverse features of DLB and suggest that current clinical criteria may not detect all cases. Familial DLB is clinically heterogeneous and occurs with or without coexistent AD, suggesting the relevance of LB pathology for the developing dementia. PMID- 12370469 TI - Main trunk tibial neuropathies. AB - The authors present a retrospective study of 52 patients with main trunk tibial neuropathy. They found trauma and ischemia to be the most frequent causes, followed by tumors. These etiologic groups are underrepresented in the literature. Electrodiagnostic examination was helpful for localizing the lesion as well as for excluding S1 radiculopathies, with which tibial neuropathies can be confused clinically. PMID- 12370470 TI - Postexercise facilitation of reflexes is not common in Lambert-Eaton myasthenic syndrome. AB - Postexercise facilitation (PEF) with clinical reflexes, H-reflex, and T-reflexes at the ankle and knee was systematically studied in 16 patients with Lambert Eaton myasthenic syndrome (LEMS). PEF was observed in ankle and knee deep tendon reflexes in five patients, in H-reflex in three patients, and in T-reflexes in six patients. When all reflex tests were combined, 7 (43.7%) of 16 patients showed PEF by at least one test. The authors conclude that the PEF of reflexes, the most helpful diagnostic clinical marker for LEMS, is not common. PMID- 12370471 TI - Tick paralysis in children: electrophysiology and possibility of misdiagnosis. AB - The authors report six patients with tick paralysis seen over 5 years. Clinical and electrodiagnostic findings failed to adequately distinguish tick paralysis from Guillain-Barre syndrome in these patients. Finding a tick attached to the scalp or the nape of the neck and removing it resulted in rapid clinical improvement. PMID- 12370472 TI - A novel murine myotonia congenita without molecular defects in the ClC-1 and the SCN4A. AB - The authors report a new murine model for myotonia congenita designated as B6MT. This line spontaneously arose from breeding of transgenic C57BL/6CrSlc mice, irrelevant of the transgene. The B6MT mouse showed moderate to severe action myotonia, and electromyography revealed myotonic discharge. The phenotype was transmitted with autosomal recessive inheritance. Molecular genetic study of the ClC-1 and the SCN4A genes revealed polymorphism with no functional consequences. PMID- 12370473 TI - Cutaneous innervation in chronic inflammatory demyelinating polyneuropathy. AB - The authors evaluated epidermal nerve density (END) and thermal thresholds in 18 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). END of patients with CIDP were lower than those of controls (4.5 +/- 2.9 vs 10.5 +/- 3.9 fibers/mm, p < 0.001). Reduced END were associated with autonomic symptoms. Thermal thresholds of patients with CIDP were elevated (88.2% for warm stimuli and 70.6% for cold stimuli). Patients with CIDP have small-fiber sensory and autonomic neuropathies. PMID- 12370474 TI - Migraine with aura is not linked to the FHM gene CACNA1A or the chromosomal region, 19p13. AB - Two microsatellite markers, tightly linked to CACNA1A, were genotyped in migraine with aura (MA) families to determine if this gene, which underlies the 19p13 linked forms of familial hemiplegic migraine, is also linked to MA. Two-point parametric lod and nonparametric linkage scores did not support linkage. Transmission disequilibrium testing provided no evidence for linkage of MA to CACNA1A. In a large dataset of 64 Canadian MA families, the authors did not find evidence to support an MA susceptibility gene in the region of 19p13. PMID- 12370475 TI - Neuroleptic malignant syndrome with prolonged catatonia in a dopa-responsive dystonia patient. AB - The authors describe a patient with dopa-responsive dystonia who developed neuroleptic malignant syndrome with prolonged catatonia following treatment with neuroleptic agents. Use of these agents probably expanded the patient's neuronal dysfunction beyond the nigrostriatal system to involve multiple dopaminergic systems. Electroconvulsive treatment alleviated the prolonged catatonia. PMID- 12370476 TI - Heterogeneity of presenile dementia with bone cysts (Nasu-Hakola disease): three genetic forms. AB - Nasu-Hakola disease (NHD) is an autosomal recessive disorder characterized by presenile dementia and bone cysts. Finnish patients revealed a large deletion in DAP12 gene encoding a key element for transducing activation signal. The authors examined six Japanese cases for DAP12 alleles. Five of the six had loss-of function mutation, either a single-base deletion or a novel point mutation. The single patient without mutation normally expressed DAP12 protein. Japanese NHD has at least three genetic forms regarding DAP12. PMID- 12370477 TI - Presenilin-1 mutation (E280G), spastic paraparesis, and cranial MRI white-matter abnormalities. AB - The authors report unusual presentations of members of an Irish family with familial AD due to an E280G mutation in exon 8 of presenilin-1. One had spastic paraparesis and white matter abnormalities on cranial MRI. A sibling had an internuclear ophthalmoplegia, spastic-ataxic quadriparesis, and "cotton-wool plaques" with amyloid angiopathy on brain biopsy. Another affected sibling also had MRI white matter abnormalities. The MRI findings may reflect an ischemic leukoencephalopathy due to amyloid angiopathy affecting meningocortical vessels. PMID- 12370478 TI - A "bunch of grapes" intracranial tuberculoma. PMID- 12370479 TI - The absence of survival motor neuron 2 gene may play a role in multifocal motor neuropathy. PMID- 12370480 TI - Severe sensory neuronopathy responsive to infliximab in primary Sjogren's syndrome. PMID- 12370481 TI - Possible gluten sensitivity in multiple system atrophy. PMID- 12370482 TI - Unusual presentation of CADASIL with reversible coma and confusion. PMID- 12370483 TI - Transient repetitive movements of the limbs in patients with acute basilar artery infarction. PMID- 12370484 TI - Recent advances in the genetics and pathogenesis of Parkinson's disease. PMID- 12370485 TI - Hypotension and cognitive impairment: selective association in patients with heart failure. PMID- 12370486 TI - Seizure-associated brain injury in term newborns with perinatal asphyxia. PMID- 12370487 TI - "Atraumatic" Sprotte needle reduces the incidence of post-lumbar puncture headaches. PMID- 12370488 TI - Clinical features and changing patterns of neurodegenerative disorders on Guam, 1997-2000. PMID- 12370489 TI - Oxygen deprivation induced cell death: an update. AB - Mammalian cells have multiple responses to low or zero oxygen concentrations. In the complete absence of oxygen, cells undergo cell death through apoptosis, and not necrosis. Apoptotic signaling during oxygen deprivation occurs through the release of cytochrome c and apaf-1 mediated caspase-9 activation. The upstream regulators of cytochrome c release are the Bcl-2 family members. Pro-apoptotic Bcl-2 family members such as bax or bak are clearly required to initiate cytochrome c/apaf-1/caspase-9 mediated cell death during oxygen deprivation. Here we review what is currently known oxygen deprivation induced cell death and speculate about initiating mechanisms resulting in the activation of pro apoptotic Bcl-2 family members. PMID- 12370490 TI - Heregulin-induced apoptosis. AB - Heregulins (HRGs) are a group of polypeptide factors that are encoded by four different HRG genes that can express multiple isoforms through alternate RNA splicing. A number of HRG isoforms possess both growth stimulatory and growth inhibitory functions that are necessary for their important role in the development and maintenance of the heart, nervous system and epithelial cells in multiple organs including the breast. Growth inhibition by HRG relates to its ability to induce apoptosis, differentiation, and cell cycle G(2) arrest. Current studies suggest that HRGs can induce a unique form of apoptosis. In this article, we review recent progress in characterizing and understanding HRG-induced apoptosis. Particular attention has been given to: (1). the activation of caspases-7 and -9; (2). the role of the anti-apoptotic Bcl-2 protein; and (3). the signaling molecules and pathways that regulate HRG-induced apoptosis, including the p38, JNK, mTOR kinase, and PKC alpha kinase. PMID- 12370491 TI - Mechanosensitive induction of apoptosis in fibroblasts is regulated by thrombospondin-1 and integrin associated protein (CD47). AB - Fibroblasts are cultured in three-dimensional collagen matrices to investigate the effect of mechanical tension on the regulation of apoptosis. Under the influence of mechanical loading, the cells show little apoptosis whereas releasing of tension leads to an increase up to tenfold during the first 24 h and remains constant for further 48 h. An autocrine loop of the integrin alpha(V)beta(3)/CD47 receptor complex and thrombospondin-1 is identified as the molecular coupling device between mechanical loading and apoptosis: The integrin alpha(V)beta(3) is expressed under mechanical loading as well as unloading whereas the CD47 could only be identified after the release of tension. The secreted thrombospondin binds to the active receptor and induces apoptosis. The presented mechanosensitive regulation of apoptosis in fibroblast cultures could be an essential mechanism for the regression of the granulation tissue by apoptosis in the process of wound healing. PMID- 12370492 TI - Hyperbaric oxygen enhances apoptosis in hematopoietic cells. AB - Hyperbaric oxygen (HBO) is 100% oxygen administered at elevated atmospheric pressure. In this study, we examined the effect of HBO on hematopoietic cell apoptosis. Cells exposed to HBO were incubated in a chamber containing 97.9% O(2) and 2.1% CO(2) at 2.4 atmospheres absolute (ATA). HBO enhanced spontaneous HL-60 cell apoptosis in a time-dependent manner; a 12 h exposure increased apoptosis by 42%. Exposing these cells to hyperoxia at standard atmospheric pressure (95% O(2), 5% CO(2) at 1 ATA) or increased pressure alone (8.75% O(2), 2.1% CO(2) at 2.4 ATA) had minimal effect on apoptosis. HBO also enhanced stimulus-induced apoptosis. HL-60 cells stimulated to die using gamma radiation underwent 33% more apoptosis than cells exposed to radiation alone. HBO enhanced melphalan, camptothecin, and chlorambucil-induced apoptosis by 22%, 13%, and 8%, respectively. Jurkat cells stimulated to die with anti-Fas antibody underwent 44% more apoptosis when exposed to HBO. Spontaneous apoptosis was increased by 15% in HBO-exposed murine thymocytes. HBO's effect on apoptosis did not require new protein synthesis. As expected, HBO exposure increased the intracellular concentration of H(2)O(2). Incubating HL-60 cells in the presence of dehydroascorbic acid partially abrogated HBO-induced increases in intracellular H(2)O(2) and apoptosis. In summary, HBO enhances spontaneous and stimulus-induced apoptosis in hematopoietic cells, at least in part, by enhancing the intracellular accumulation of H(2)O(2). PMID- 12370493 TI - Intracellular superoxide induces apoptosis in VSMCs: role of mitochondrial membrane potential, cytochrome C and caspases. AB - Apoptosis of vascular smooth muscle cells (VSMCs) is an integral part of cardiovascular diseases including atherosclerosis, hypertension and restenosis. Here we studied the fate of VSMCs in response to intracellular superoxide stimulation. Diethyldithiocarbamic acid (DDC) was used to inhibit copper-zinc superoxide dismutase thereby increasing intracellular superoxide levels. The results show that DDC at a dose from 25-100 micro M is able to induce VSMC apoptosis. Superoxide was found to be responsible for DDC-induced apoptosis. In the apoptotic process mitochondrial membrane potential was decreased and caspase 3, -8 and -9 were activated. Surprisingly, neither cytochrome c release nor Bid cleavage could be observed. These data suggest a role for intracellular superoxide in the regulation of VSMCs apoptosis. PMID- 12370494 TI - Imidazole-induced cell death, associated with intracellular acidification, caspase-3 activation, DFF-45 cleavage, but not oligonucleosomal DNA fragmentation. AB - Intracellular acidification is known to be involved in the initiation phase of apoptosis. However, the necessity of intracellular acidic conditions in the execution phase of apoptosis remains unknown. In this study, we found that in HL 60 cells imidazole induces cell death, associated with intracellular acidification, caspase-3 activation and DFF-45 cleavage, but not oligonucleosomal DNA fragmentation. A caspase inhibitor prevented cell death but not intracellular acidification. When pHi was neutralized by changing from imidazole-containing medium to fresh medium, oligonucleosomal DNA fragmentation and increased caspase 3 activity was observed in the imidazole-treated HL-60 cells. Furthermore, the DNA fragmentation induced by intracellular neutralization was inhibited by caspase inhibitor treatment. These results indicate that imidazole induces caspase-dependent cell death, and suggest that maintaining pHi in the neutral range is essential for the induction of oligonucleosomal DNA fragmentation in the execution phase of apoptosis. PMID- 12370495 TI - Protective effect of adrenomedullin in mannitol-induced apoptosis. AB - Mannitol therapy is widely used for reducing brain edema, and ischemic brain swelling. However, mannitol at clinical concentrations induces apoptosis in endothelial cells. Because apoptosis may be a pathogenic mechanism in vascular injury, antiapoptotic agents may have a protective role in mannitol-induced apoptosis. In this study, we examined whether adrenomedullin (AM) prevents mannitol-induced apoptosis and also evaluated the associated signaling pathway of AM in human umbilical vein endothelial cells. AM prevented mannitol-induced apoptosis in a dose-dependent manner. Pretreatment with wortmannin blocked the AM induced antiapoptotic effect. AM stimulated Akt at Ser473, and wortmannin inhibited the AM-induced Akt phosphorylation. These findings indicate that phosphatidylinositol 3'-kinase/Akt pathway transmits the survival signal from AM. The potency of antiapoptotic effect of AM is stronger than that of vascular endothelial growth factor and angiopoietin-1 in mannitol-induced apoptosis. AM can have a protective role not only in umbilical vein, but also in pulmonary, coronary, and aortic endothelial cells. These findings indicate that AM has a potent protective role in mannitol-induced apoptosis, through phosphatidylinositol 3'-kinase/Akt pathway. Therefore, pretreatment with AM might help to maintain normal endothelial integrity during systemic mannitol therapy. PMID- 12370496 TI - Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis. AB - We investigated the mode of cell death induced by the anthracyclines, aclarubicin, doxorubicin and daunorubicin in the human leukemia cell lines, HL60 and Jurkat. The cells were incubated with drug concentrations up to 500 nM for periods between 3 and 24 hours, followed by morphological and biochemical analyses. All three substances induced DNA fragmentation, evident as DNA laddering and appearance of cells with hypodiploid DNA content, externalization of phosphatidyl serine, activation of caspases and degradation of the apoptosis specific endonuclease inhibitor DFF45. However, concentrations and times necessary for these effects to occur were different, aclarubicin being the quickest acting drug with a lag phase of 3 h, followed by daunorubicin with 6 h and doxorubicin with 24 h. More importantly, aclarubicin induced these effects while the cell membrane was intact, whereas doxorubicin and daunorubicin led to immediate loss of membrane integrity. Programmed cell death is characterised by preservation of membrane integrity in order to allow removal of apoptotic bodies, whereas cell rupture is an early event in necrosis. We therefore suggest that, in our experimental settings, doxorubicin- and daunorubicin-induced cell death occurs by necrosis, while aclarubicin induces programmed cell death. PMID- 12370497 TI - A review of clinical trials to prevent mother-to-child HIV-1 transmission in Africa and inform rational intervention strategies. PMID- 12370498 TI - Lack of Epstein-Barr virus- and HIV-specific CD27- CD8+ T cells is associated with progression to viral disease in HIV-infection. AB - OBJECTIVE: Despite readily detectable virus-specific CD8+ T cells in most HIV infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein-Barr virus (EBV)-specific CD8+ T cells was performed. DESIGN: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV associated non-Hodgkin lymphoma's (NHL), both number and phenotype of virus specific CD8+ T cells was studied longitudinally. METHODS: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells. RESULTS: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27- T cells, in contrast to rising numbers of EBV-specific CD27- CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS related NHL had very low numbers of EBV-specific CD27- CD8+ T cells. Higher numbers of HIV-specific CD27- CD8+ T cells were associated with delayed disease progression. Virus-specific CD27- T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function. CONCLUSIONS: Taken together, our data indicate that virus-specific CD27- T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27- effector T cells may lead to progression of viral disease. PMID- 12370499 TI - TNF-alpha promoter region gene polymorphisms in HIV-positive patients with lipodystrophy. AB - OBJECTIVE: The pathogenic mechanisms underlying lipodystrophy in HIV-positive patients are largely unknown. TNF-alpha has many actions that are consistent with the features of lipodystrophy; therefore, an analysis was carried out to determine whether functionally active polymorphisms in the promoter region of the TNF-alpha gene are associated with the development of lipodystrophy. DESIGN: Genetic case-control association study. METHODS: Individuals were genotyped for the -238 and -308 polymorphisms in the TNF-alpha gene using polymerase chain reaction-restriction fragment length polymorphism analysis. The genotype and allele frequencies for 61 HIV-positive patients with lipodystrophy were compared with (a) 35 HIV-positive patients with no evidence of lipodystrophy and (b) 239 healthy HIV-negative individuals. RESULTS: The frequency of the variant rare -238 allele was significantly different (P = 0.01) in HIV-positive patients with lipodystrophy than in those without lipodystrophy. At the genotype level, a trend towards a difference between patients with and without lipodystrophy was observed (chi2 for linear trend 5.2, P = 0.02). For the -308 polymorphism, no difference was found in genotype and allele frequencies between HIV patients with and without lipodystrophy. CONCLUSIONS: The data suggest that the -238 (but not the 308) promoter region TNF-alpha gene polymorphism is a determinant in the development of HIV-related lipodystrophy. However, the results need to be confirmed in larger numbers of patients as well as in an ethnically diverse population. PMID- 12370500 TI - Antiviral activity of the neutralizing antibodies 2F5 and 2G12 in asymptomatic HIV-1-infected humans: a phase I evaluation. AB - BACKGROUND: The human monoclonal antibodies (MAbs) 2F5 and 2G12 were identified to be two of the most potent neutralizing antibodies against HIV-1. In a first human study they have been shown to be safe after repeated intravenous infusions to asymptomatic HIV-1-infected individuals. However, the antiviral effects of antibody treatment have not been fully analyzed in this first clinical trial. METHODS: The aim of the present study was to gain a preliminary insight into the antiviral effects of 2F5 and 2G12 in humans. For this purpose, plasma samples obtained from the previous phase I study were studied for RNA copy numbers by reverse transcriptase-polymerase chain reaction. As a measure for activation of complement levels of the major complement factor C3 were measured by enzyme linked immunosorbent assay. Flow cytometry was used to study T-lymphocyte counts and the amount of infected peripheral blood mononuclear cells (PBMC) was determined by co-culture with uninfected donor PBMC. Virus escape from antibody neutralization was determined in vitro in a PBMC neutralization assay. RESULTS: Transient reduction in viral loads was observed in five of seven patients. Vigorous complement activation was observed directly after HIV-specific antibody infusions. The number of infective peripheral blood mononuclear cells was reduced in some patients whereas CD4+ T-lymphocyte counts and CD4+/CD8+ ratios were transiently increased in all patients. Virus escape occurred only against 2G12. CONCLUSIONS: Analysis of disease progression markers indicate that antibody therapy may have antiviral effects. These findings suggest that neutralizing antibodies should be further evaluated as an alternative therapeutic approach in HIV-1 disease. PMID- 12370501 TI - Interleukin-2 accelerates CD4 cell reconstitution in HIV-infected patients with severe immunosuppression despite highly active antiretroviral therapy: the ILSTIM study--ANRS 082. AB - BACKGROUND: Despite effective highly active antiretroviral therapy (HAART), some patients infected with HIV have persistently low CD4 cell counts with risk of HIV disease progression. The addition of interleukin-2, a cytokine that stimulates CD4 T lymphocyte helper cells, may benefit patients with discordant responses. METHODS: A total of 72 HIV-infected patients with CD4 cell counts of 25-200 x 10(6) cells/l (median 145) and plasma HIV RNA < 1000 copies/ml were randomized in a multicentre study to receive open-label 4.5 x 10(6) IU interleukin-2 subcutaneously twice daily for 5 days every 6 weeks plus their ongoing HAART or were maintained on HAART alone (control group). After 24 weeks, all patients received interleukin-2 therapy plus HAART up to week 80. Primary end-point was the CD4 T cell area under the curve minus baseline up to week 24. RESULTS: After four cycles of interleukin-2, in an intent-to-treat analysis, the respective median CD4 cell area under the curve minus baseline values were +51 and +11 cells in the interleukin-2 (n = 34) and the control group (n = 36) (P < 0.0001). The percentage of patients in the two groups with CD4 cell counts > 200 x 10(6) cells/l was 81% and 33%, respectively (P < 0.0001). At week 80, the median CD4 cell counts in the two groups were 380 and 270 x 10(6) cells/l, respectively. Interleukin-2 treatment was reasonably well tolerated and did not result in sustained increases in plasma HIV RNA levels. CONCLUSIONS: Administration of interleukin-2 produces significant and sustained increase in CD4 cell counts in HAART-treated patients with persistent CD4 cell counts < 200 x 10(6) cells/l. PMID- 12370502 TI - Prevalence and clinical correlates of HIV viremia ('blips') in patients with previous suppression below the limits of quantification. AB - OBJECTIVE: To examine the prevalence and clinical correlates of subsequently measurable viremia in HIV-infected patients who have achieved viral suppression below the limits of quantification (< 50 copies/ml). DESIGN: Non-randomized dynamic cohort study of ambulatory HIV patients in nine HIV clinics in eight cities. PATIENTS: Patients had two consecutive HIV-1 RNA levels < 50 copies/ml (minimum, 2 months apart) that were followed by at least two more viral level determinations while remaining on the same antiretroviral therapy (ART) between January 1997 and June 2000 (median 485 days). Transiently viremic patients were defined having a subsequently measurable viremia but again achieved suppression < 50 copies/ml. RESULTS: Of the 448 patients, 122 (27.2%) had transient viremia, 19 (4.2%) had lasting low-level viremia and 33 (7.4%) had lasting high-level viremia (defined as 50-400 and > 400 copies/ml, respectively). Only 16 (13.1%) of those who had transient viremia later had persistent viremia > 50 copies/ml. The occurrence of transient viremia did not vary with whether the patient was ART naive or experienced (P = 0.31), or currently taking protease inhibitors or not (P = 0.08). On consistent ART, the median percentage increase in CD4 cell count was statistically different between subgroups of our cohort (Kruskal-Wallis, P = 0.002). CONCLUSIONS: Transiently detectable viremia, usually 50-400 copies/ml, was frequent among patients who had two consecutive HIV-1 RNA levels below the limits of quantification. In this analysis, such viremia did not appear to affect the risk of developing lasting viremia. Caution is warranted before considering a regimen as 'failing' and changing medications. PMID- 12370503 TI - Polymorphisms in cytokine genes define subpopulations of HIV-1 patients who experienced immune restoration diseases. AB - OBJECTIVE: To further elucidate the immunopathogenesis of immune restoration diseases (IRD) in HIV patients responding to antiretroviral therapy and determine whether IRD associated with different opportunistic pathogens involve distinct immunopathological mechanisms. DESIGN: DNA samples from patients with a range of IRD were typed for polymorphic loci in genes encoding immune-mediators. METHODS: PCR-restriction fragment length polymorphism assays were used to type loci in the and genes. Alleles of a microsatellite in the CD30 promoter were determined by capillary electrophoresis. RESULTS: Only 8% of patients with IRD associated with a herpesvirus infection carried IL12B-3'UTR*2, compared with 42-54% of patients with other or no IRD. Patients with IRD arising from mycobacterial infection rarely carried IL6-174*C (36% versus 61-71%) and never carried TNFA-308*2 (0% versus 23-52%). TNFA-308*2 was carried by 52% of patients who experienced IRD associated with a herpesvirus infection, as several patients with exacerbations of cytomegalovirus retinitis carried this as part of a HLA-A2,B44 haplotype. Polymorphisms in and showed no distinct patterns. CONCLUSIONS: Distinct cytokine mediated mechanisms contribute to IRD initiated by herpesvirus and mycobacterial infections. PMID- 12370504 TI - Virological and immunological effects of short-course antiretroviral therapy in primary HIV infection. AB - BACKGROUND: National and international guidelines call for the treatment of primary HIV infection (PHI) with combination antiretroviral therapy, although the ideal timing and duration of this intervention is unknown. Recent immunological studies of antiretroviral therapy on small numbers of patients with PHI have reported preservation of HIV-specific CD4 T-helper responses, ordinarily lost in the absence of intervention. We sought to investigate whether a short course of antiretroviral therapy (SCART) at PHI was sufficient to preserve HIV-specific cellular immunity. METHODS: Forty-five subjects with confirmed PHI were offered SCART at diagnosis. HIV specific cellular immune responses and virological parameters were assessed at monthly intervals. RESULTS: Thirty-seven of the subjects chose SCART at PHI, and achieved a plasma viral load < 50 RNA copies/ml by a median of 10 weeks (range, 4-32 weeks). Two of the 45 individuals had evidence of genotypic HIV drug resistance at baseline, and none developed new mutations following therapy. All patients who received SCART at PHI showed preservation of HIV-specific CD4 T-helper responses up to 64 weeks off SCART. CONCLUSION: SCART at PHI was safe, did not induce the development of drug resistance, and appeared sufficient to preserve HIV-specific CD4 T-helper responses. However, PHI is highly heterogeneous, and a large-scale randomized trial of SCART at PHI is now needed. PMID- 12370505 TI - Detection of HIV-1 subtypes, recombinants, and dual infections in east Africa by a multi-region hybridization assay. AB - OBJECTIVE: To enable more rapid and efficient genotyping of HIV-1 in East Africa, where subtypes A, C, and D and their recombinants are co-circulating. DESIGN: Full-genome sequencing of HIV-1 provides complete discrimination of subtypes and recombinant forms but is costly and low-throughput compared to other genotyping approaches. Here we describe the development and evaluation of a Multi-region Hybridization Assay (MHA) for the efficient determination of HIV-1 subtypes A, C, D, recombinants, and dual infections. METHODS: Five genome regions containing clustered mutations distinguishing subtypes A, C, and D were identified and used to design subtype-specific probes. DNA from primary peripheral blood mononuclear cells was used as template for real-time PCR using the fluorescent, subtype specific probes. RESULTS: A panel of 45 clinical samples from Uganda, Kenya, and Tanzania, previously characterized by full-genome sequencing and including 26 pure subtypes and 19 recombinant strains, was evaluated by MHA. The MHA provided 90% sensitivity and 98% specificity for the three subtypes, efficiently discriminated subtypes from recombinant forms, and detected several dual infections. CONCLUSIONS: Accurate and efficient genotyping of HIV-1 strains in vaccine trial populations in East Africa, ascertainment of dual infections, and elucidation of the genesis of recombinant forms in individuals can be facilitated by the application of MHA. PMID- 12370506 TI - Socioeconomic status, access to triple therapy, and survival from HIV-disease since 1996. AB - BACKGROUND: In the era before highly active antiretroviral therapy (HAART), socioeconomic status was associated with survival from HIV disease. We have explored socioeconomic status, access to triple therapy (HAART), and mortality in the context of a universal healthcare system. METHODS: We evaluated 1408 individuals who initiated double or triple therapy between 1 August 1996 and 31 December 1999, and were followed until 31 March 2000. Cumulative HIV-related mortality rates were estimated using Kaplan-Meier methods and Cox proportional hazards regression. RESULTS: In the overall Cox model, we found that adherence [risk ratio (RR) 0.83; per 10% increase], CD4 cell count (RR 1.53; per 100 cell decrease), and lower socioeconomic status (RR 2.19; high versus low), were associated with HIV-related mortality. However, socioeconomic status was not significant among patients prescribed triple therapy in a stratified analysis, or in a sub-analysis restricted to patients prescribed HAART in the initial regimen. When we investigated if inequitable access to HAART by socio-economic status could explain the discrepancy, we found that persons in the lower socio-economic strata were less likely to be prescribed triple therapy even after adjustment for clinical characteristics. CONCLUSION: In a universal healthcare system, socioeconomic status was strongly associated with HIV-related mortality. When we investigated possible explanations for this association, we found that individuals of lower socioeconomic status were less likely to receive triple therapy after adjustment for clinical characteristics. Our findings highlight the need for the monitoring of therapeutic guidelines to ensure equitable access, as treatment strategies are updated. PMID- 12370507 TI - Correlates of human herpesvirus 8 seropositivity among heterosexual men in Kenya. AB - BACKGROUND: Several studies have suggested that sexual transmission of human herpesvirus 8 (HHV-8) occurs among homosexual men in developed countries. However, few studies have examined heterosexual HHV-8 transmission, especially among African populations in which HHV-8 is endemic. OBJECTIVES: To determine the seroprevalence and correlates of HHV-8 infection among heterosexual African men. DESIGN: Cross-sectional study. METHODS: Participants were 1061 men enrolled in a prospective cohort study of risk factors for HIV-1 acquisition among trucking company employees in Mombasa, Kenya. Stored frozen sera from the study baseline visit were tested for antibodies to HHV-8 by whole-virus lysate ELISA. RESULTS: HHV-8 seroprevalence was 43%. In multivariate logistic regression analysis, HHV-8 infection was independently associated with older age [for men aged 30-39 years: odds ratio (OR), 1.5; 95% confidence interval (CI), 1.1-2.0; for men aged > or = 40 years: OR, 1.7; 95% CI, 1.1-2.7, compared with men aged < 30 years], Christian religion (OR, 1.6; 95% CI, 1.2-2.1), being uncircumcised (OR, 1.5; 95% CI, 1.0 2.2), and ever having syphilis (OR, 2.2; 95% CI, 1.4-3.5). Ever having used condoms was associated with decreased likelihood of infection (OR, 0.7; 95% CI, 0.6-1.0). Seropositivity was not significantly related to other sexual behaviors characterized or to HIV-1 status. CONCLUSIONS: HHV-8 seropositivity is common in this population and increases with age, suggesting on-going transmission during adulthood. Infection was more common among men who were uncircumcised or who had ever had syphilis and was less common among those who had ever used condoms, suggesting that sexual factors may play a role in HHV-8 transmission. Prospective studies of HHV-8 acquisition in heterosexual African populations are needed to demonstrate whether safer sexual practices can reduce transmission. PMID- 12370508 TI - Effect of dietary intervention on highly active antiretroviral therapy-related dyslipemia. AB - Changes in cholesterol and triglyceride levels after prescribing a lipid-lowering diet were assessed in 230 HIV-infected patients with dyslipemia associated with antiretroviral therapy. Lipid levels decreased significantly in subjects having good diet compliance. The reduction in triglyceride levels was greater than in cholesterol levels. Patients on protease inhibitor-containing regimens experienced a slightly greater decline in lipid levels compared with the rest. PMID- 12370509 TI - Clinical use of lopinavir/ritonavir in a salvage therapy setting: pharmacokinetics and pharmacodynamics. AB - Lopinavir/ritonavir was administered to 35 HIV-infected patients after therapeutic failure with other protease inhibitors. The pharmacokinetics (trough concentrations) and baseline viral genotype were determined, together with the immunovirological outcome. The 22 responders had significantly higher mean lopinavir concentrations and lower baseline numbers of mutations. On multivariate analysis, a lopinavir concentration of 5.7 microg/ml or greater was an independent predictor of viral suppression over a 9-month follow-up period. PMID- 12370510 TI - Pilot study of interferon alpha high-dose induction therapy in combination with ribavirin for chronic hepatitis C in HIV-co-infected patients. AB - Twenty-three HIV/hepatitis C virus (HCV)-co-infected patients received dose escalated IFN-alpha (5 MIU/day) induction therapy for 10 weeks, followed by 36 weeks of thrice-weekly IFN-alpha treatment (5 MIU), both in combinations with ribavirin. Sustained HCV clearance was observed in three patients. Nine patients discontinued the study aas a result of adverse reactions such as anaemia, pancreatitis and depression. In HIV/HCV-co-infected patients, the therapeutic benefit of high-dose IFN-alpha therefore seems to be limited by its poor tolerability. PMID- 12370511 TI - HIV-1-specific CD8 T cell responses in a pediatric slow progressor infected as a premature neonate. AB - We describe the long-term survival of an individual infected with HIV-1 during extrauterine life as a premature newborn. In the absence of viral attenuation in the Nef/LTR structure or significant co-receptor polymorphisms, slow progression was associated with the strong HIV-1-specific broadly cross-reactive CD8 T cell responses. HIV-1 infection as early as 25 weeks' gestation may thus results in the development of immune responses that control viral replication and lead to prolonged survival. PMID- 12370512 TI - The prevalence and determinants of the K65R mutation in HIV-1 reverse transcriptase in tenofovir-naive patients. AB - The K65R mutation in HIV-1 reverse transcriptase is associated with reduced susceptibility to abacavir and tenofovir. We established its prevalence within a large clinical database, and investigated correlations with other resistance associated mutations and antiretroviral history. The presence of K65R is associated with previous abacavir use. Although rare, it is preferentially selected within non-thymidine analogue-containing regimens, compared with concurrent zidovudine or stavudine use, which is associated with thymidine analogue mutations. Both genetic routes may compromise abacavir and tenofovir activity. PMID- 12370513 TI - Risk factors for indinavir-related renal colic in HIV patients: predictive value of indinavir dose/body mass index. AB - In a prospective study evaluating risk factors for indinavir-related renal colic in 555 HIV-infected patients receiving highly active antiretroviral therapy, followed-up fir 24 months, 23.6% developed one or more renal colic episodes, and 50 patients stopped indinavir. No correlation was observed between renal colic onset and sex, age, CD4 cell count, history, and hepatitis B or C virus co infection, but baseline anthropometric values were significantly related to the onset of renal colic. PMID- 12370514 TI - The radiographic appearance of pneumococcal pneumonia in adults is unaltered by HIV-1-infection in hospitalized Kenyans. PMID- 12370515 TI - Intravenous drug users risk group should also benefit from simpler highly active antiretroviral therapy. PMID- 12370516 TI - Rash-associated severe neutropenia as a side-effect of indinavir in HIV postexposure prophylaxis. PMID- 12370518 TI - Lack of 'occult' hepatitis B virus infection in HIV-infected patients. PMID- 12370519 TI - Population-level risk factors for HIV transmission and 'the 4 Cities Study': temporal dynamics and the significance of sexual mixing patterns. PMID- 12370521 TI - Hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in HIV-1: clinical, phenotypic and genotypic correlates. AB - OBJECTIVE: The routine use of phenotypic drug resistance testing in patient management has revealed that many HIV-1 strains possess significantly increased drug sensitivity, or 'hypersusceptibility' compared with wild-type viruses. This study describes hypersusceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) and was designed to determine the prevalence of and viral characteristics associated with NNRTI hypersusceptibility in patient-derived viruses. METHODS: Retrospective analyses were performed on a large clinical laboratory dataset containing phenotypic drug susceptibility and genotypic sequence results from HIV-1 patient isolates. Genetically engineered viruses were used to confirm the role of certain nucleoside reverse transcriptase inhibitor (NRTI)-resistance mutations in NNRTI hypersusceptibility. RESULTS: Hypersusceptibility to delavirdine, efavirenz and nevirapine was detected in 10.7, 10.8 and 8.0% of more than 17,000 consecutive plasma samples submitted for phenotypic susceptibility testing. In analyses limited to a subset of viruses derived from patients with known treatment histories, NNRTI hypersusceptibility was observed significantly more frequently among viruses from NRTI experienced/NNRTI-naive patients compared with viruses from NRTI/NNRTI-naive patients. Significant inverse correlations between NRTI and NNRTI susceptibility exist among the viruses from NRTI-experienced patients. Analyses of viruses classified according to their NNRTI susceptibility identified 18 positions in reverse transcriptase where substitutions were significantly associated with NNRTI hypersusceptibility. CONCLUSIONS: NNRTI hypersusceptibility is common among patient HIV-1 isolates, especially in NRTI-resistant viruses. Genotypic correlates of hypersusceptibility are complex and not easily defined by a simple analysis of NRTI-associated resistance mutations. NNRTI hypersusceptibility may provide an explanation for the superior virologic response to NNRTI-containing salvage regimens observed in NRTI-experienced patients in several clinical trials. PMID- 12370520 TI - The clinical relevance of non-nucleoside reverse transcriptase inhibitor hypersusceptibility: a prospective cohort analysis. AB - OBJECTIVE: To evaluate the clinical significance of hypersusceptibility to non nucleoside reverse transcriptase inhibitors (NNRTI). DESIGN: Analysis of a prospective clinical trial cohort. PATIENTS: NNRTI-naive patients failing a stable antiretroviral regimen. MEASUREMENTS: HIV phenotype, HIV RNA, and CD4 cell counts were prospectively collected after patients changed to a new regimen. Hypersusceptibility to NNRTI was defined as a fold-change (FC) in IC50 (inhibitory concentration of 50%) of < 0.4. RESULTS: The 177 patients had a mean HIV RNA of 4.1 log10 copies/ml, CD4 cell count of 322 x 10(6) cells/l and 41 months of prior antiretroviral treatment. Hypersusceptibility to one or more NNRTI was present in 29%. Both longer duration and reduced susceptibility to nucleoside reverse transcriptase inhibitors were associated with efavirenz hypersusceptibility (P < 0.05). NNRTI-containing regimens were initiated in 106 patients at baseline. The mean change in log HIV RNA after 6 months was greater for patients with hypersusceptibility (-1.2 log10 copies/ml; n = 21) than in patients without (-0.8 log10 copies/ml; n = 77) (P = 0.016). Differences persisted to month 12 (P = 0.023). Multiple linear regression models confirmed that hypersusceptibility to NNRTI was a significant independent predictor of the magnitude of early (months 1-4) HIV RNA reduction, after accounting for the baseline HIV RNA and the number of drugs to which the patient's virus was susceptible (P < 0.02). CD4 cell increases (months 4-10) were 28- 60 x 10(6) cells/l greater in patients with hypersusceptible virus (P < or = 0.1). CONCLUSION: NNRTI hypersusceptibility occurred in more than 20% of nucleoside experienced patients and was associated with greater reduction of HIV RNA and increase in CD4 cells. PMID- 12370522 TI - The etiology and management of genital ulcers in the Dominican Republic and Peru. AB - BACKGROUND: Clinical diagnosis of genital ulcers is difficult, and diagnostic tests are least available in settings where rates of disease are highest. The World Health Organization (WHO) has developed protocols for the syndromic management of genital ulcers in resource-poor settings. However, because risk factors, patterns and causes of disease, and antimicrobial susceptibilities differ from region to region and over time, they must be adapted to local situations. GOAL: The goal of this study was to determine etiologic factors, evaluate syndromic management, and compare polymerase chain reaction (PCR) testing with other diagnostic alternatives for genital ulcers among patients attending sexually transmitted disease clinics in the Dominican Republic and Peru. STUDY DESIGN: Eighty-one men with genital ulcers in the Dominican Republic and 63 in Peru underwent identical interviews and identical multiplex PCR (M-PCR) tests of genital lesion specimens for etiologic diagnoses. Algorithms for managing genital ulcers were developed. RESULTS: In the Dominican Republic, 5% were M-PCR-positive for, 26% for, and 43% for herpes simplex virus (HSV); in Peru, 10%, 5%, and 43%, respectively, were positive. The WHO algorithm for treating syphilis and chancroid had a sensitivity of 100%, a positive predictive value (PPV) of 24%, and an overtreatment rate of 76%. A modified algorithm for treating only those without vesicular lesions had 88% sensitivity and a 27% PPV, and the overtreatment rate was reduced to 58%. CONCLUSION: HSV caused 43% of genital ulcers in these populations. The modified algorithm had lower sensitivity but a reduced overtreatment rate. M-PCR testing was more sensitive than standard tests and more specific and sensitive than clinical diagnosis. PMID- 12370523 TI - Methods to reduce social desirability bias in sex surveys in low-development settings: experience in Zimbabwe. AB - BACKGROUND: Social desirability bias hampers measurement of risk behavior for acquiring STDs and evaluation of control interventions. More confidential data collection methods reduce this bias in Western countries but generally require technology not available in less developed settings. GOAL The goal of this report was to describe and evaluate an informal, confidential, low-technology method Informal Confidential Voting Interviews (ICVIs)-for collecting sexual behavior data in less developed settings. STUDY DESIGN: Reports of multiple sex partners by sexually active, basic-literate, population-based survey participants in rural Zimbabwe randomly assigned to ICVIs and face-to-face interviews (FTFIs) were compared. RESULTS: Ninety-two percent of respondents (n = 7,823) were sufficiently literate for ICVIs. Error rates were low but higher than in FTFIs. More male and female ICVI respondents interviewed reported multiple current sex partners (OR = 1.33 and 5.24, respectively) and multiple partners in the past month (OR = 1.71 and 2.92) and the past year (OR = 1.35 and 1.97). CONCLUSION: The ICVI method appears to reduce bias but requires further evaluation to assess viability and effect in alternative settings. PMID- 12370524 TI - Evaluation of the Hybrid Capture 2 CT/GC DNA tests and the GenProbe PACE 2 tests from the same male urethral swab specimens. AB - BACKGROUND: A previous study demonstrated that Digene's Hybrid Capture 2 (HC2) DNA tests for detection of and (CT/GC) could be performed using cervical swab specimens collected in GenProbe transport media with significantly greater sensitivity for the detection of than with the GenProbe PACE 2 system. GOAL: The goal was to assess the performance of HC2 tests in comparison with GenProbe PACE 2 tests for the detection of CT/GC in male urethral swab specimens. STUDY DESIGN: A total of 1,202 male urethral swab specimens were collected in GenProbe PACE transport medium. All specimens were first tested with the PACE 2 system, followed by masked HC2 CT/GC testing. The GenProbe AMPLIFIED CT Assay (AMP CT) and PCR/SHARP Signal System (SHARP) were used for adjudication of discrepant results. RESULTS: The prevalence rates for this population were 8.4% for and 14.6% for, based on the adjudicated results. The relative sensitivity and specificity for the detection of were 97.0% and 99.8% for HC2 and 69.3% and 98.3% for PACE 2, respectively. The relative sensitivities for the detection of were 98.9% for HC2 and 99.4% for PACE 2, with the same specificity of 99.9% for both tests. Agreement between the two testing methods was 95.4% for and 99.6% for. CONCLUSION: The HC2 test is compatible with the GenProbe collection medium, with significantly greater sensitivity than the GenProbe PACE 2 test for detecting and similar sensitivities for detecting. PMID- 12370525 TI - Rectal applications of nonoxynol-9 cause tissue disruption in a monkey model. AB - BACKGROUND: Efforts to develop topical microbicide products have all but ignored evaluation for rectal use. GOAL: The goal of this study was to assess the effects of multiple rectal applications of Conceptrol (containing 4% nonoxynol-9) on flora and mucosal tissues in the pig-tailed macaque model. STUDY DESIGN: Monkeys (8 per group) received daily rectal applications of Conceptrol, placebo gel, or no product, for 3 days. At each visit, a preapplication rectal lavage specimen and swab specimen for microbiology and pH determination were collected. Conceptrol or placebo gel (2.5 ml) was then administered intrarectally. Fifteen minutes after application, samples were again collected. RESULTS: Gross observation of rectal lavage indicated sheets of epithelium 15 minutes after application of the nonoxynol-9 product. Histopathology of these samples revealed epithelial sheets with stroma attached. The presence of H(2)O(2)-producing lactobacilli remained relatively constant, whereas that of H(2)O(2)-producing viridans streptococci diminished in all nonoxynol-9-exposed animals in which these organisms were detected at baseline. CONCLUSIONS: Repeated applications of nonoxynol-9 disrupts the rectal mucosa of the pig-tailed macaque. The disruption of these tissues could have serious implications for an increase in likelihood of acquisition of sexually transmitted infection/HIV in humans. PMID- 12370526 TI - NAAT-identified and self-reported gonorrhea and chlamydial infections: different at-risk population subgroups? AB - BACKGROUND: Information on the characteristics and behaviors of persons at high risk for gonorrhea and chlamydial infection has typically been derived from studies of sexually transmitted disease (STD) clinic populations. The Baltimore STD and Behavior Survey (BSBS) used urine-based nucleic acid amplification testing (NAAT) to assess the prevalence and behavioral correlates of gonorrhea and chlamydial infection in a population-based cross-sectional survey of adults in Baltimore, Maryland. GOAL: The goal of this study was to examine the demographic characteristics and behavioral markers of gonorrhea and chlamydial infection as reported by adults with a self-reported history of gonorrhea and chlamydial infection and to compare these to the characteristics and behaviors of individuals with current NAAT-identified gonorrhea and/or chlamydial infection. STUDY DESIGN: A probability sample of adults aged 18 to 35 years residing in Baltimore was evaluated with collection of urine specimens and administration of a health and behavior survey. Data and specimens were collected between January 1997 and September 1998. RESULTS: Respondents with NAAT-detected gonorrhea and/or chlamydial infection (7.9%) did not report a history of high-risk behaviors or more recent occurrences of those behaviors, and the majority were asymptomatic. However, adults in our study who self-reported a history of infection (26.0%) were more likely than those with no history of infection to report multiple partners, paid sex, partners with prior STDs, and STD symptoms-a pattern consistent with findings described in previous clinic-based reports. CONCLUSION: The risk profile generated from studies of clinic populations, with a focus on symptomatic disease, may not characterize the broader population with current, untreated, largely asymptomatic infection. PMID- 12370527 TI - Entry of inflammatory cells into the mouse vagina following application of candidate microbicides: comparison of detergent-based and sulfated polymer-based agents. AB - BACKGROUND: Because topical microbicides designed to prevent the spread of sexually transmitted diseases may be applied frequently, it is important to ensure product safety as well as efficacy. A murine model was developed to test for induction of inflammatory responses following application of candidate microbicides. GOAL: A comparison was made of the induction of inflammation following vaginal application of detergent-based and sulfated polymer-based microbicides. STUDY DESIGN: Vaginal leukocytes were collected, identified, and quantified following microbicide application to detect the entry of inflammatory leukocytes into the vaginal lumen. RESULTS: Large numbers of neutrophils and macrophages entered the vaginal lumen following a single application of detergent based microbicides. No significant increase in vaginal leukocytes was detected following a single or repeated application of sulfated polymer-based microbicides. CONCLUSION: Application of sulfated polymer-based microbicides was less likely to result in inflammatory responses than was application of detergent based compounds. This murine model should prove useful as part of a screening process to prioritize candidate microbicides before clinical trial. PMID- 12370528 TI - Streptococcus pyogenes tuboovarian abscess: a potential sexually transmitted disease? PMID- 12370529 TI - Projection of the future dimensions and costs of the genital herpes simplex type 2 epidemic in the United States. AB - BACKGROUND: Infection with herpes simplex virus type 2 (HSV-2) currently affects approximately 22% of adult Americans and increased markedly in prevalence between the late 1970s and early 1990s. Although some estimates of the costs of prevalent disease due to HSV-2 are available, selection of interventions to prevent HSV-2 infection, as well as evaluation of their potential cost-effectiveness, should take into account projected future costs that will result if the epidemic is left unchecked. GOAL: The goal was to estimate the future health and economic consequences attributable to the HSV-2 epidemic in the absence of interventions to slow the epidemic. STUDY DESIGN: A mathematical model was constructed to project future increases in HSV-2 seroprevalence in the United States. The probability of heterosexual transmission of HSV-2 was estimated from cross sectional estimates of infection prevalence reported by the National Health and Nutrition Examination Survey (NHANES). Per-infection expected costs were calculated on the basis of data obtained from the published medical literature. RESULTS Without intervention, the prevalence of HSV-2 infection among individuals aged 15 to 39 years was projected to increase to 39% among men and 49% among women by 2025. Annual incidence was projected to increase steadily between 2000 and 2025, from 9 to 26 infections per 1,000 men and from 12 to 32 infections per 1,000 women in this age group. The cost of incident infections in the year 2000 were estimated to be $1.8 billion; the cost of incident infections was predicted to rise to $2.5 billion by 2015 and $2.7 billion by 2025. The projected cumulative cost of incident HSV-2 infections occurring over the next 25 years was estimated to be $61 billion; at a 3% discount rate, this sum has a present value of $43 billion. CONCLUSION: The costs of incident HSV-2 infection in the United States are substantial and can be expected to increase as both the incidence and prevalence of this disease increase in the first half of the century. The level of resource allocation for HSV-2 prevention strategies should reflect the economic benefits that would result from control of this epidemic. PMID- 12370530 TI - Controversies in clinical pancreatology: intraductal papillary-mucinous tumor (IPMT): American Pancreatic Association Clinical Symposium. PMID- 12370531 TI - Role of endoscopic ultrasound (EUS) and EUS-guided fine needle aspiration in the diagnosis and treatment of cystic lesions of the pancreas. AB - INTRODUCTION AND AIMS: Cystic neoplasms of the pancreas may be inadvertently treated as benign pseudocysts in clinical practice, often without the use of cytology, cyst tumor markers, or histopathology. We assessed the utility of EUS guided fine-needle aspiration (EUS-FNA) to assist in the diagnosis and management of pancreatic cysts. METHODOLOGY: All patients who had pancreatic cysts detected by EUS over a 24-month period were analyzed. Preoperative diagnosis was derived from an algorithm combining clinical history and endosonographic features. In selected cases, EUS-FNA was performed and cyst fluid aspirates were analyzed. Surgical specimens served as diagnostic standard. RESULTS: A total of 43 patients with pancreatic cysts underwent 45 EUS examinations. Surgical specimens were obtained from 9 patients (mucinous cystadenocarcinoma, 3; adenocarcinoma, 3; pancreatic endocrine tumor, 2; and benign cyst, 1); diagnostic EUS correctly predicted malignant cysts in 8/9 (88.9%). One case inaccurately interpreted by EUS as cystic neoplasm turned out to be a benign cyst on resection. Twenty-one patients underwent EUS-FNA. The cytologic interpretation was adenocarcinoma in 9.5% (2/21); suspicious for malignancy or atypical cells in 19.0% (4/21); benign in 66.6% (14/21); and insufficient cells in 4.8% (1/21). CONCLUSION: The information gathered from clinical history and EUS, complemented by fluid analysis after EUS-guided FNA, predicts neoplastic pancreatic cysts and assists in decision-making for medical or surgical approach. PMID- 12370532 TI - Is severity of necrotizing pancreatitis increased in extended necrosis and infected necrosis? AB - INTRODUCTION: We previously reported that organ failure occurs in 50% of patients with necrotizing pancreatitis, that extended pancreatic necrosis (greater than 50% necrosis) is not associated with an increased prevalence of organ failure or infected necrosis, and that the prevalence of organ failure is similar in sterile necrosis and infected necrosis. AIMS To analyze these relations in a larger group of patients and to evaluate other factors that might have prognostic significance. METHODOLOGY: We reviewed 1,110 consecutive cases of acute pancreatitis between January 1, 1995, and January 1, 2000. Necrosis was documented by contrast-enhanced CT. A value less than 0.05 was considered significant. RESULTS: Ninety-nine patients (9%) had necrotizing pancreatitis; 52% had organ failure. Patients with extended pancreatic necrosis did not have increased prevalence of organ failure or infected necrosis but did have an increased need for intubation and an increased mortality rate associated with multiple organ failure. Patients with infected necrosis did not have an increased prevalence of organ failure but did have a marginally increased prevalence of multiple organ failure and increased need for intubation. Overall mortality was 14% and was markedly increased among patients with organ failure at admission (47%) and among patients who had multiple organ failure during the hospitalization (49%). CONCLUSION: Although severity of necrotizing pancreatitis was somewhat increased in extended pancreatic necrosis and infected necrosis, mortality was more strongly linked to organ failure at admission and multiple organ failure during hospitalization. PMID- 12370533 TI - Expression of clusterin in human pancreatic cancer. AB - INTRODUCTION: Clusterin, also known as apolipoprotein J, has been implicated in numerous processes, including active cell death. Clusterin is reported to be overexpressed in breast and prostate cancers. However, its expression in pancreatic cancer is yet to be reported. AIM AND METHODOLOGY: To examine clusterin expression and apoptosis in 52 pancreatic tissues, including specimens from 33 cases of pancreatic cancer, by immunohistochemistry. RESULTS: Clusterin was expressed in 49% (16) of 33 cases of pancreatic cancer, 50% (13) of 26 cases of chronic pancreatitis, and 67% (4) of 6 cases of mucinous cystadenoma. It was not expressed in normal pancreas. Clusterin mRNA was also expressed in the pancreatic cancer cell lines. Clusterin was significantly more highly expressed at stage I and II (well-differentiated and moderately differentiated cancers). Clusterin and apoptosis were localized in the same cells in pancreatic cancer. However, clusterin expression was not significantly associated with apoptosis in any pancreatic disease. Clusterin-positive patients with pancreatic cancer survived significantly longer. CONCLUSION: These results suggest that clusterin expression is induced in pancreatic cancer as well as chronic pancreatitis and that downregulation of clusterin may be involved in the progression of pancreatic cancer. PMID- 12370534 TI - Regulation of carbachol-induced c-fos mRNA expression in AR42J cells by somatostatin receptor subtypes 1, 2, and 3. AB - INTRODUCTION: Somatostatin is an inhibitory peptide that exerts its effects tissue-specifically by activating one or more of five receptors (SSTR 1-5). Although several studies have examined which SSTR subtypes control gastrointestinal function, effects of somatostatin on pancreatic gene expression are not well defined. AIM: To examine the effects of somatostatin and newly synthesized selective SSTR agonists on the cholinergically stimulated expression of the immediate early response gene METHODOLOGY AND RESULTS: In pancreatic acinar AR42J cells, polymerase chain reaction analysis revealed that mRNAs for SSTR 1, 2, and 3 were expressed. SSTR 4 and 5 were not detected. When AR42J cells were exposed to the cholinergic agonist carbachol in the presence of somatostatin or selective SSTR agonists, significant and dose-dependent reductions in agonist induced levels of mRNA were noted. Pretreatment with agonists specific for SSTR 4 or 5 had no inhibitory effects. The inhibitory actions of somatostatin were pertussis toxin-sensitive. In addition, since somatostatin did not affect intracellular calcium homeostasis, the inhibitory actions of somatostatin are independent of calcium signaling. CONCLUSION: The current studies demonstrate that somatostatin inhibits carbachol-induced increases in expression by interacting with somatostatin receptor subtypes 1, 2, and 3. In addition, because somatostatin did not affect intracellular calcium homeostasis, it can be concluded that SSTR actions are independent of carbachol-stimulated calcium signaling. PMID- 12370535 TI - Human leukocyte antigen-DR expression on peripheral monocytes as a predictive marker of sepsis during acute pancreatitis. AB - INTRODUCTION: The mortality associated with severe acute pancreatitis is still high, and death in the later stage of the disease is chiefly due to bacterial infection and sepsis. However, objective parameters for the risk of sepsis in acute pancreatitis have not been established. AIM: To investigate the value of human leukocyte antigen-DR (HLA-DR) on peripheral monocytes for predicting the development of sepsis during acute pancreatitis. METHODOLOGY: The expression of HLA-DR on peripheral monocytes was measured in 64 patients by flow cytometry at admission and 7 and 14 days after the onset of acute pancreatitis. Twenty-eight patients with severe acute pancreatitis and 36 with mild acute pancreatitis, as determined by the Atlanta classification, were enrolled. RESULTS: Six patients had sepsis, and two of them died during the hospital stay. At admission, the percentage of HLA-DR-expressing cells in the monocyte population was significantly lower in the patients who had sepsis in the later course than in the patients who did not have sepsis. A percentage lower than 80% at admission was observed in 17 patients, and the patients who had persistently low percentages of HLA-DR-expressing monocytes throughout the observation period had sepsis in the later clinical course, whereas the patients in whom expression recovered to the normal range were spared the development of sepsis. CONCLUSION: In acute pancreatitis, the low percentage of HLA-DR-expressing cells in the monocyte population is a reliable predictor of the development of sepsis. Monitoring of monocyte HLA-DR expression may be a useful marker for identifying the patients who are at high risk of sepsis in acute pancreatitis. PMID- 12370536 TI - Cerulein-induced acute pancreatitis in the rat is significantly ameliorated by treatment with MEK1/2 inhibitors U0126 and PD98059. AB - INTRODUCTION: Both cerulein and cholecystokinin activate mitogen-activated protein (MAP) kinase (ERK1/2) in vivo and in isolated pancreatic acini. AIMS AND METHODOLOGY: ERK1/2 in pancreas homogenates was activated in rats rendered pancreatitic by subcutaneous injections of cerulein (5 microg/kg per hour). To determine if blocking ERK1/2 activity might rescue cerulein-induced acute pancreatitis, the "MAP kinase kinase" (also known as MEK1/2) inhibitors PD98059 and U0126 were administered in vivo. RESULTS: In rats pretreated with PD98059 (10 mg/kg per i.v. injection) or U0126 (5 mg/kg per i.v. injection) 30 minutes before and then together with hourly cerulein injections for 3 hours, pancreatitis was significantly attenuated on the basis of pancreatic wet weight and histology. Serum amylase concentration was significantly reduced when PD98059 was administered intraperitoneally (10 mg/kg per intraperitoneal injection). PD98059 also ameliorated pancreatitis over a 6-hour cerulein time course. The phosphorylation of pancreatic ERK1/2 was attenuated in PD98059- and U0126-treated animals at both 30 minutes and 3 hours after cerulein injection. Rats rendered neutropenic with vinblastine and pretreated with U0126 still showed attenuated manifestations of cerulein-induced acute pancreatitis, a finding suggesting that pancreatic ERK1/2 is mostly responsible for the effect, rather than infiltrating neutrophils. CONCLUSIONS: Inhibition of pancreatic ERK1/2 in vivo affords significant protection against inflammatory sequelae following cerulein-induced acute pancreatitis. PMID- 12370537 TI - Expression of the chemokines MCP-1/JE and cytokine-induced neutrophil chemoattractant in early acute pancreatitis. AB - INTRODUCTION: Inflammatory mediators play a critical role in acute pancreatitis. The precise role played by members of the chemokine family remains unclear. AIMS: To investigate the expression of the CC chemokine monocyte chemotactic protein (MCP)-1/JE and the CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) in early acute pancreatitis. METHODOLOGY: Pancreatitis was induced in rats, either by intraperitoneal injection of cerulein or by infusion of 5% sodium taurocholate into the pancreatic duct. Expression of MCP-1/JE and CINC in pancreas and plasma was determined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Northern analysis, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Following induction of acute pancreatitis, MCP-1/JE and CINC immunoreactivity was seen in acinar cells. Infiltrating neutrophils were strongly immunolabeled with an anti MCP-1/JE antibody, whereas macrophages reacted strongly with an antibody to CINC. Northern analysis and quantitative real-time RT-PCR demonstrated upregulation of MCP-1/JE and CINC mRNA levels in pancreatic tissue. Plasma MCP-1 levels were significantly increased after 6 hours in the cerulein hyperstimulation model (2,444 +/- 93 microg/mL versus control, 1,853 +/- 262 microg/mL; < 0.05). Plasma CINC levels were significantly increased after 6 hours in the cerulein hyperstimulation model (1,680 +/- 134 microg/mL versus control, 725 +/- 128 microg/mL; < 0.005) and after 3 hours in the bile salt infusion model (6,663 +/- 1,405 microg/mL versus control, 2,339 +/- 800 microg/mL; < 0.05). CONCLUSION: CINC and MCP-1/JE may be early mediators of the inflammatory response in acute pancreatitis. PMID- 12370538 TI - Experimental pancreatitis causes acute perturbation of energy metabolism in the intestinal wall. AB - INTRODUCTION: The systemic inflammatory response syndrome (SIRS) may be initiated by a number of underlying conditions such as acute pancreatitis. The association between the local inflammatory reaction, the systemic response, and potential concomitant dysfunction of remote organs is not quite clear. AIM: To evaluate whether severe acute pancreatitis in the rat affects energy metabolism in the pancreas and whether the focal inflammation also causes biochemical deterioration in remote organs such as the liver and intestine. METHODOLOGY: With the patient under general anesthesia, microdialysis probes were inserted in the pancreas, liver, and small intestine. Two groups of eight rats each were studied: the sham (control) group and the pancreatitis group. Acute pancreatitis was induced by intraductal injection of 5% sodium taurodeoxycholate, and the animals were studied for 3 hours thereafter. The microdialysis fluid was analyzed for glucose, lactate, and pyruvate. RESULTS: In the pancreatitis group we found significant increases in glucose concentration in the pancreas and lactate levels in the pancreas and intestinal wall, and the lactate/pyruvate ratio was significantly higher in the intestine than in the sham group. CONCLUSION: Induction of severe acute pancreatitis results in immediate metabolic alterations in the pancreas. In the intestinal wall a severe perturbation of energy metabolism is observed after only 1 hour. This implies a rapid onset of metabolic disturbances, not only in the local, challenged organ (pancreas) but also in remote organs. PMID- 12370539 TI - The role of nitric oxide in edema formation in L-arginine-induced acute pancreatitis. AB - INTRODUCTION: Nitric oxide (NO) has been implicated in the regulation of the pancreatic circulation, the promotion of the capillary integrity, and the inhibition of leukocyte adhesion. AIMS: To investigate the rates of changes in the pancreatic constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities and the role of NO in the vascular permeability changes during the development of L-arginine (Arg)-induced acute pancreatitis. METHODOLOGY: Acute pancreatitis was induced in male Wistar rats by injecting 250 mg/100 g body weight of Arg i.p. twice at an interval of 1 hour, as a 20% solution in 0.15 NaCl (group I). The control rats received the same quantity of glycine (group II). In group III, 30 mg/kg N -nitro-L-arginine methyl ester (L-NAME) was injected i.p. 19 hours after the first Arg injection. The rats were killed at 6, 12, 24, or 48 hours following Arg administration, and the plasma amylase concentration and the pancreatic weight/body weight (pw/bw) ratios were evaluated. NOS activity was determined via the conversion of L- C-Arg monohydrochloride to C-citrulline. The vascular permeability was examined by means of the extravasation of Evans blue dye (20 mg/kg bw) into the pancreatic tissue. RESULTS: The serum amylase level was already increased at 6 hours in group I animals, peaked at 12 hours after the Arg injection (11.800 +/- 590 versus 6.618 +/- 252 U/L in group II), and returned to the control level at 48 hours. The pw/bw ratio peaked at 24 hours in group I (6.63 +/- 0.52 versus 4.02 +/- 0.22 mg/g in group II) and returned to the control level at 48 hours. The cNOS activity was depleted at 6 hours in group I (0.02 +/- 0.003 versus 0.23 +/- 0.02 pmol/min/mg protein in group II); it then gradually increased to a level significantly higher than that in group II and decreased thereafter (0.45 +/- 0.03 and 0.13 +/- 0.01 pmol/min/mg protein at 24 and 48 hours). The iNOS activity was significantly increased at 24 and 48 hours versus that in group II (0.15 +/- 0.05 and 0.07 +/- 0.01 versus 0.04 +/- 0.01 pmol/min/mg protein). The pancreatic concentration of Evans blue dye was significantly higher in group I than in group II (138.59 +/- 11.04 versus 43.57 +/- 2.67 (g/dry weight). Treatment with L-NAME significantly reduced the amylase activity, pw/bw, Evans blue concentration, and cNOS activity of the pancreas but did not exert any beneficial effect on the histologic score at 24 hours after the onset of pancreatitis, as compared with those values in group I (6.528 +/- 673 U/L, 4.56 +/- 0.65 mg/g, 86.84 +/- 3.9 (g/dry weight, 0.14 +/- 0.04 pmol/min/mg protein). CONCLUSION: Endogenous NO is involved in the formation of pancreatic edema in Arg-induced acute pancreatitis by increasing the vascular permeability and protein extravasation. L-NAME treatment decreased the cNOS activity and edema formation but did not prevent the histologic damage in Arg-induced acute pancreatitis. PMID- 12370540 TI - Clinical study of chronic pancreatitis with focal irregular narrowing of the main pancreatic duct and mass formation: comparison with chronic pancreatitis showing diffuse irregular narrowing of the main pancreatic duct. AB - INTRODUCTION: Main pancreatic duct (MPD)-narrowed chronic pancreatitis (CP) may be an autoimmune abnormality. It also has been called autoimmune pancreatitis and sclerosing pancreatitis. It is unclear whether cases with focal pancreatographic changes are part of the same clinical entity as cases with diffuse MPD changes. AIM AND METHODOLOGY: We reviewed seven cases of chronic pancreatitis (CP) with focal narrowing of the main pancreatic duct (MPD), evidenced by endoscopic retrograde cholangiopancreatography (ERCP), and swelling of one or two segments of the pancreas, evidenced by ultrasonography (US) /computed tomography (CT), and indicated the clinicopathologic features of focal-type MPD-narrowed CP. RESULTS: The patient group comprised six men and one woman, and their age range was 28-75 years, with a mean of 63.7 years. Affected sites were in the head in two patients, the body in one patient, the tail in one patient, and the body and tail in three patients; ERP showed narrowing in six patients and obstruction in one. Stricture of the lower portion of the common bile duct (CBD) that caused obstructive jaundice was shown by ERC in two cases in which the pancreas head was affected. In all six patients, a dynamic study by CT or MRI homogeneously showed delayed enhancement of involved segments of the pancreas. Serum levels of pancreatic enzyme were elevated in five patients, but only one subject had pancreatitis-like epigastric pain. Serological evidence suggestive of autoimmune abnormality was detected in only three patients with hypergammaglobulinemia (> or =2.0 g/dL) or positive titers of antinuclear antibody (ANA; > or =80). Histological assessment was available for five patients, who characteristically had dense lymphocytic or plasmocytic infiltration with severe fibrosis that caused luminal narrowing. The clinical, serologic, and histologic findings as described above were comparable to those for 12 CP patients with diffuse narrowing of the MPD, diagnosed during the same period. Surgical resection was performed in 5 patients, in 2 of whom a similar inflammatory process recurred in the remnant head of the pancreas, whereas pancreatitis no longer developed in the other 3 patients. One patient was initially treated with steroids, with clinical remission, although there was neither hypergammaglobulinemia nor positive ANA. CONCLUSION: These results indicate that CP with focal narrowing of the MPD is part of the same clinical spectrum as CP with diffuse narrowing of the MPD, and whether the distribution is diffuse or focal seems to be related to the stage or the extent of the disease. It is therefore important to recognize the possible existence of this focal variant to avoid unnecessary surgery. PMID- 12370541 TI - Angiotensin II evokes calcium-mediated signaling events in isolated dog pancreatic epithelial cells. AB - INTRODUCTION: Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells. AIMS: To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells. METHODS: Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 microM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca+2]i) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels. RESULTS: Western blots demonstrated presence of both AT and AT AngII receptors in DPDE and CFPAC cells; the density of AT receptors appeared lower than that of AT receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 +/- 11 nM) and CFPAC (103 +/- 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 +/- 98 nM) and CFPAC (879 +/- 207 nM) cells, as did ATP (DPDE = 1722 +/ 228 nM; CFPAC = 1522 +/- 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na+ channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the 11pS NSC and the 3pS channels. CONCLUSION: In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca ]. The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion. PMID- 12370542 TI - Effects of chronic hypoxia on the circulating and pancreatic renin-angiotensin system. AB - INTRODUCTION: The circulating renin-angiotensin system (RAS) plays a crucial role in the regulation of blood pressure, electrolytes, and fluid homeostasis. In contrast to the circulating RAS, the presence of an intrinsic RAS has been demonstrated in different tissues/organs, which may affect both local and global functions of a biologic system. Our previous studies provided solid evidence of the existence of a local RAS in rat pancreas. Our further investigation showed that such a pancreatic RAS could be activated by experimental models of chronic hypoxia and chemically induced pancreatitis. These previous findings formed the basis for the current study. METHODOLOGY: Adult Sprague-Dawley rats were exposed to isobaric hypoxia (10% O2), and the effects on the circulating and pancreatic RAS were documented. RESULTS: The current study shows that exposure of rats to isobaric hypoxia caused a time-dependent increase in plasma renin activity. The activation of circulating RAS by hypoxia was associated with a parallel upregulation of local RAS components, including the mRNA expression of angiotensinogen and angiotensin II receptor types I and II in the pancreas. CONCLUSION: The upregulation of local pancreatic RAS, along with its counterpart circulating RAS, may be responsible for both physiologic and pathophysiologic aspects of a biologic system under chronic hypoxic stress. PMID- 12370543 TI - Activation of raphe pallidus neurons increases insulin through medullary thyrotropin-releasing hormone (TRH)-vagal pathways. AB - INTRODUCTION: Pancreatic insulin secretion is regulated by the vagus nerve. Medullary thyrotropin-releasing hormone (TRH) containing projections from the raphe pallidus (Rpa) neurons innervate vagal preganglionic motor neurons in the dorsal vagal complex (DVC) and are involved in vagal regulation of gastric functions. AIM: To investigate whether chemical stimulation of Rpa neurons influences circulating insulin levels through brain medullary TRH-vagal pathways. METHODOLOGY: In fasted, pentobarbital-anesthetized rats, kainic acid (10 ng/50 nL) was microinjected into the Rpa, and serum insulin levels were measured. Gastric acid secretion was monitored as a control of vagally mediated visceral response. RESULTS: Chemical stimulation of Rpa neuronal cell bodies significantly increased serum insulin levels. Values before and at 30, 60, and 90 minutes after the microinjection of kainic acid were 0.34 +/- 0.02, 0.54 +/- 0.06, 0.60 +/- 0.06, and 0.99 +/- 0.13 ng/mL, respectively. In the same rats, gastric acid secretion was stimulated (basal, 2.3 +/- 0.6, versus 26.1 +/- 8.6 micromol/15 min at 30 minutes). Microinjections outside of the Rpa had no effect. The Rpa stimulation-induced increase in serum insulin could be mimicked by DVC microinjection of TRH analog, completely prevented by bilateral cervical vagotomy, and significantly reduced by bilateral microinjection of TRH antibody into the DVC. CONCLUSION: Chemical activation of Rpa neurons increases pancreatic insulin release through medullary TRH and vagal-mediated pathways. PMID- 12370544 TI - Effects of gamma-aminobutyric acid on action of gastrin-releasing peptidergic neurons in exocrine secretion of isolated, perfused rat pancreas. AB - INTRODUCTION: gamma-Aminobutyric acid (GABA) has been reported to enhance exocrine secretion evoked by intrinsic neuronal excitation in the pancreas. AIM: To see the effect of GABA on the action of gastrin-releasing peptide (GRP)ergic neurons in exocrine secretion of the pancreas. METHODOLOGY: Pancreatic neurons were excited by electrical field stimulation (EFS) in the isolated, perfused rat pancreas. GRP in the pancreatic circulation was neutralized by an anti-GRP antiserum to block GRPergic neuronal action on pancreatic exocrine secretion. RESULTS: GABA (3, 10, 30 microM), given intra-arterially, elevated the EFS-evoked pancreatic secretions of fluid and amylase dose-dependently. An anti-GRP antiserum (10 microL/mL: titer of 1:66,000) reduced the GABA (10 microM)-enhanced EFS-evoked pancreatic secretions. Synthetic porcine GRP-27 (30, 100, 300 p ) increased the pancreatic secretions dose-dependently, and these were further elevated by GABA (10 microM). The anti-GRP antiserum also reduced the GABA enhanced GRP (100 p )-induced pancreatic secretions. Bicuculline (10 microM) reduced the enhancing effect of GABA on pancreatic secretions evoked by EFS as well as GRP. CONCLUSION: GABA enhances pancreatic secretions evoked by EFS as well as GRP, which is reduced by the anti-GRP antiserum. The enhancing effects of GABA on the EFS- and GRP-induced pancreatic secretions are diminished by bicuculline. The results indicate that GABA enhances intrinsic GRPergic neuronal action on exocrine secretion via the GABA(A) receptors in the rat pancreas. PMID- 12370545 TI - Annular pancreas with obstructive jaundice: beware of underlying neoplasm. AB - INTRODUCTION: Annular pancreas is a rare congenital abnormality, and in adult patients it presents with clinical features that differ from those seen in newborns. Features in the adult patient include peptic ulceration, duodenal obstruction, acute pancreatitis, and obstructive jaundice. Treatment strategies for annular pancreas with obstructive jaundice remain controversial. AIM: To present three cases involving adult patients with annular pancreas and obstructive jaundice, due to carcinoma of the ampulla of Vater in two patients and chronic pancreatitis in the third. METHODOLOGY AND RESULTS: Pancreaticoduodenectomy was performed on all patients, and the postoperative courses were uneventful. CONCLUSION: Our experience suggests that for adult patients with annular pancreas presenting with obstructive jaundice, it is necessary to consider the possibility of associated or coexisting periampullary malignancy. PMID- 12370546 TI - Osteoclast-like giant cell tumor of the pancreas with ductal adenocarcinoma: case report with novel data on histogenesis. PMID- 12370547 TI - Malignant fibrous histiocytoma of the pancreas. PMID- 12370548 TI - Fas and Fas ligand expression in pancreatic adenocarcinoma. AB - INTRODUCTION: Fas and Fas ligand (FasL) mediate apoptosis of tumor cells in immune surveillance, and expression of FasL by tumors may mediate their counterattack on cytotoxic lymphocytes. Both proteins are expressed in most if not all pancreatic carcinoma cell lines, but their study in primary human tumors has been limited. AIM: We performed Fas and FasL immunohistochemical staining on 81 primary pancreaticobiliary or ampullary ductal adenocarcinomas of patients in our institutional database to determine the extent and strength of staining. METHODOLOGY: The expression of Fas and FasL was compared with regard to clinicopathologic variables, K- mutations, and immunoexpression of HER2, p21, p27, and p53. RESULTS AND CONCLUSION: Fas was expressed in 19% of patients with strong or intermediate intensity but with variable percentages of tumor cell staining. FasL was expressed in 49% of patients, usually with diffuse expression but variable intensity. Fas expression was more common in women than men, as women had 93% of Fas-positive tumors but only 55% of Fas-negative tumors ( = 0.007), and was associated with strong HER2 expression (67% of Fas-positive versus 18% of Fas-negative patients; = 0.04). Fas expression tended to be less common in blacks (4% had Fas-positive tumors) than whites (22% had Fas-positive tumors; = 0.052). FasL expression tended to be associated with stage 4 disease at diagnosis (24% versus 0%; = 0.07). Neither Fas expression nor FasL expression was associated with survival, a circumstance suggesting that their role, if any, in contributing to the aggressiveness of these tumors is complex. PMID- 12370549 TI - Improving function and survival of porcine islet xenografts using microencapsulation and culture preconditioning. AB - INTRODUCTION AND AIMS: Porcine pancreatic islets have been difficult to preserve because isolated porcine islets tend to disaggregate to single cells and lose function under culture conditions. In the current study, the influence of agarose microencapsulation on the maintenance of the number and function of islets in culture preservations and the effect of culture preconditioning of microencapsulated porcine islets on xenogenic transplantation were investigated. METHODOLOGY: Porcine islets were isolated and then microencapsulated in 5% agarose membrane. The percentage of naked and microencapsulated islets remaining in the culture preservations was assessed. The effect of microencapsulation and culture on secretory function was investigated in vitro. The survival of overnight-cultured and 7-days-cultured microencapsulated islets in xenogenic transplantations was examined. RESULTS: A good percentage of microencapsulated islets remained in the culture preservations. They could maintain good secretory functions in vitro after 7 days of culture. In addition, we observed a significant prolongation of mean islet survival by culture preconditioning. CONCLUSIONS: The present findings suggest that microencapsulation is one of the useful preserving methods for maintenance of the number and function of cultured isolated porcine islets. Moreover, culture preconditioning is effective for improving islet survival and might be a good option leading to clinical success. PMID- 12370550 TI - Differences in receptor binding and stability to enzymatic digestion between CCK 8 and CCK-58. AB - INTRODUCTION AND AIMS: It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus. METHODOLOGY: Canine CCK-58 was purified from intestinal extracts and CCK-8 was purchased. Their amounts were determined by amino acid analysis. The effect of tertiary structure on receptor binding at CCK-A receptors and CCK-B receptors was evaluated using membrane preparations from mouse pancreas and brain. The influence of C-terminal tertiary structure on stability to enzymatic digestion was evaluated by reacting CCK-8 and CCK-58 with endopeptidase 24:11. RESULTS: CCK-58 was three times more potent than CCK-8 for binding mouse pancreatic membrane CCK-A receptors and equipotent to CCK-8 for binding mouse brain CCK-B receptors. CCK-8 was readily digested by endopeptidase 24:11, whereas CCK-58 was not. CONCLUSIONS: The results strongly support the hypothesis that differences in tertiary structure of the carboxyl terminus of CCK 8 and CCK-58 influence receptor binding and stability to enzymatic digestion. PMID- 12370551 TI - Gender verification of female Olympic athletes. AB - Gender verification of female athletes has long been criticized by geneticists, endocrinologists, and others in the medical community. Problems include invalid screening tests, failure to understand the problems of intersex, the discriminatory singling out of women based only on laboratory results, and the stigmatization and emotional trauma experienced by individuals screened positive. Genuine sex-impostors have not been uncovered by laboratory-based genetic testing; however, gender verification procedures have resulted in substantial harm to a number of women athletes born with relatively rare genetic abnormalities. Individuals with sex-related genetic abnormalities raised as females have no unfair physical advantage and should not be excluded or stigmatized, including those with 5-alpha-steroid-reductase deficiency, partial or complete androgen insensitivity, and chromosomal mosaicism. In 1990, the International Amateur Athletics Federation (IAAF) called for ending genetic screening of female athletes and in 1992 adopted an approach designed to prevent only male impostors from competing. The IAAF recommended that the "medical delegate" have the ultimate authority in all medical matters, including the authority to arrange for the determination of the gender of the competitor if that approach is judged necessary. The new policy advocated by the IAAF, and conditionally adopted by the International Olympic Committee, protects the rights and privacy of athletes while safeguarding fairness of competition, and the American Medical Association recommends that it become the permanent approach. PMID- 12370553 TI - A controlled trial of hospital versus home-based exercise in cardiac patients. AB - BACKGROUND: Large numbers of patients who stand to benefit from the exercise training component of cardiac rehabilitation are not being served due to access issues. Home-based exercise training may be a potentially useful alternative to training in institutional environments. PURPOSE: The purpose of this study was to examine the benefit of 6 months of hospital-based exercise training versus 6 months of monitored, home-based exercise training with respect to physical, quality of life, and social support outcomes in patients after coronary artery bypass graft (CABG) surgery. METHODS: Randomized controlled trial of "direct-to home" (Home; = 120) versus "direct-to-hospital" (Hosp; = 122) exercise training, 35-49 d post CABG surgery. The primary outcome was peak exercise capacity, measured by peak oxygen consumption (VO(2)) on a symptom-limited cycle ergometer exercise test. Secondary outcomes were health-related quality of life (measured by the SF-36) and social support (measured by the ISEL). Measurements were taken at baseline and after 3 and 6 months of exercise training. RESULTS: The study groups had similar demographic and health profiles at baseline. Peak VO(2) improved significantly in both groups after 6 months of exercise training; 36% in the Hosp group (1,222.1 +/- 269.0 mL x min(-1) to 1,497.2 +/- 594.3 mL x min(-1); < 0.0001) and 31% in the Home group (1,260.3 +/- 306.5 mL x min(-1) to 1,433.4 +/ 589.7 mL x min(-1); < 0.05). The Home group reported greater total social support than the Hosp group at 3 (36.2 +/- 4.5 vs 34.0 +/- 6.7; < 0.0001) and 6 months (36.0 +/- 4.9 vs 34.6 +/- 6.4; = 0.05). The Home group demonstrated a greater improvement in health-related quality of life (physical) by 6 months in comparison to the Hosp patients (51.2 +/- 6.4 vs 48.6 +/- 7.1; = 0.004). CONCLUSION: This study suggests that low-risk CABG surgery patients may be served as well or better with a monitored, home-based exercise program than with an institution-based program. PMID- 12370554 TI - Peak exercise capacity of electrically induced ambulation in persons with paraplegia. AB - INTRODUCTION: Persons with spinal cord injury (SCI) are generally limited to exercise activities using the relatively smaller, less productive upper extremities with limited benefits as compared with leg exercise training. Functional electrical stimulation (FES) assisted ambulation has previously been demonstrated to allow persons with paraplegia to stand and ambulate limited distances. PURPOSE: This study compared the peak physiological responses of persons with paraplegia during FES ambulation and voluntary arm exercise. METHODS: Fifteen subjects (T -T ) previously habituated to FES ambulation, completed peak testing of both arm cranking (AC) and FES walking to the point of exhaustion. The AC tests were performed using a graded incremental protocol to exhaustion in 3-min stages and 10-W power output increments. The FES walking test consisted of successive 10-m walking bouts, each trial progressively increased in pace. Metabolic activity was continuously monitored via open-circuit spirometry with heart rate (HR) determined by a 12-lead electrocardiograph for AC and by direct palpation during FES. RESULTS: Peak VO(2) did not differ between AC (22.9 +/- 3.8 mL x kg x min(-1)) and FES (22.7 +/- 3.9 mL x kg x min(-1)). FES ambulation elicited significantly greater peak values of HR (191 beats x min(-1) versus 179 beats x min(-1)) and lower peak values of respiratory exchange ratio (1.06 vs 1.12) compared with AC. There were no significant differences in peak values of any other variables. CONCLUSION: This study indicates that FES ambulation performance, in persons with paraplegia, elicits similar exercise capacity, as indicated by similar peak oxygen consumption, as voluntary arm exercise. PMID- 12370555 TI - Onset of electrical stimulation leg cycling in individuals with paraplegia. AB - PURPOSE: This study investigated cardiovascular and hemodynamic responses during the transition from rest to electrical stimulation-induced leg cycling exercise (ES-LCE) in individuals with chronic paraplegia (PARA). METHODS: Ten PARA (T(4) T(9); ASIA A) participated in this study. Heart rate (HR), mean arterial pressure (MAP), stroke volume (SV), and cardiac output (Q) were measured on a beat-to-beat basis at rest and during the first 60 s of ES-LCE. RESULTS: PARA exhibited two discrete MAP responses during ES-LCE. Those with high thoracic lesions (HIGH: T(4) -T(6), = 5) responded to ES-LCE with a significant rise in MAP (maxdelta 8.3 +/- 3.6 mm Hg), whereas MAP did not exhibit any sustained change from resting values during ES-LCE in those subjects with lower lesions (LOW: T -T, = 5). In HIGH PARA, the immediate increase in MAP corresponded to a decrease in HR (maxdelta 6.8 +/- 3.1 b x min(-1)), which returned toward resting levels by the end of 60 s. In contrast, for LOW PARA there was no change in HR from resting levels during transition to ES-LCE. In both subgroups, SV and Q were not significantly increased during ES-LCE. CONCLUSION: These results suggest that the on-transient responses of MAP during ES-LCE in HIGH PARA elicited reflex changes in HR via the arterial baroreflex, whereas in LOW PARA, an unchanged HR from rest was likely due to a constant MAP during ES-LCE. PMID- 12370556 TI - Comparison of oxygen uptake on-kinetic calculations in heart failure. AB - PURPOSE: The analysis of oxygen (O(2)) uptake on-kinetics during steady-rate is gaining interest in the heart failure (HF) population. The rate change in O(2) at the initiation of exercise can be assessed via nonlinear regression time constant (TC) or an algebraic equation (mean response time [MRT]). These calculations are presumed to be interchangeable, but research supporting this claim is limited. This investigation compares and contrasts two of the more commonly used O(2) uptake on-kinetic calculations. METHOD: Twenty-eight subjects diagnosed with compensated HF and 19 age, sex, and activity-matched controls underwent a symptom limited exercise test and a steady-rate exercise session (6 min). Peak O(2) uptake, O(2) uptake at ventilatory threshold, the O(2) uptake TC (TC), and the O(2) uptake mean response time (MRT) were calculated for each subject. RESULTS: O(2) uptake on-kinetics was significantly faster for the control group ( < 0.05) regardless of calculation method. There was a significant difference between the O(2) uptake TC and MRT for the HF group. All O(2) uptake on-kinetic calculations were significantly correlated with aerobic capacity. CONCLUSIONS: O(2) uptake TC and MRT values may not be interchangeable in the HF population. All O(2) uptake on-kinetic calculations did produce a significant difference between experimental and control groups and correlated with indicators of aerobic capacity. The 10-s O(2) uptake on-kinetic calculations may be preferable secondary to expired gas fluctuations associated with breath-by-breath measures. Further work is, however, needed to determine which averaged O(2) uptake on-kinetic expression is optimal given the significant difference between TC and MRT. A mechanism for this difference may be the oscillatory ventilatory expired gas pattern demonstrated by some patients with HF. PMID- 12370557 TI - Arm mechanical efficiency and arm exercise capacity are relatively preserved in chronic obstructive pulmonary disease. AB - PURPOSE: Previous studies indicate that energy expenditure related to physical activity is enhanced and that mechanical efficiency of leg exercise is reduced in patients with chronic obstructive pulmonary disease (COPD). However, it is yet unclear whether an inefficient energy expenditure is also present during other activities in COPD. This study was carried out to examine arm efficiency and peak arm exercise performance relative to leg exercise in 33 (23 male) patients with COPD ((mean +/- SEM) age: 61 +/- 2 yr; FEV : 40 +/- 2% of predicted) and 20 sex- and age-matched healthy controls. METHODS: Body composition, pulmonary function, resting energy expenditure (REE), and peak leg and arm exercise performance were determined. To calculate mechanical efficiency, subjects performed submaximal leg and arm ergometry at 50% of achieved peak loads. During exercise testing, metabolic and ventilatory parameters were measured. RESULTS: In contrast to a reduced leg mechanical efficiency in patients compared with controls (15.6 +/- 0.6% and 22.5 +/- 0.6%, respectively; < 0.001), arm mechanical efficiency was comparable in both groups (COPD: 18.3 +/- 0.9%, controls: 21.0 +/- 1.2%; NS). Arm efficiency was not related to leg efficiency, pulmonary function, work of breathing, or REE. Also, arm exercise capacity was relatively preserved in patients with COPD (ratio arm peak work rate/leg peak work rate in patients: 89% vs 53% in controls; < 0.001). CONCLUSION: Mechanical efficiency and exercise capacity of the upper and lower limbs are not homogeneously affected in COPD, with a relative preservation of the upper limbs. This may have implications for screening of exercise tolerance and prescription of training interventions in patients with COPD. Future studies need to elucidate the mechanism behind this observation. PMID- 12370558 TI - Muscle power increases after resistance training in growth-hormone-deficient adults. AB - PURPOSE: To measure the effects of a resistance training (RT) program over muscle function and body composition of adults with GH deficiency without replacement. METHODS: 11 GH-deficient patients (39 +/- 11 yr) were evaluated in four occasions (two pretraining and at 6 and 12-wk of training). We performed anthropometric measurements and physical tests. Muscle power was measured by a specific tensiometer (Fitro, Bratislava, Slovakia) in five different exercises: seated chest press, rear lat pull-down, knee extension, standing upright row, and triceps press down. Muscle endurance was assessed by maximum number of sit-ups and maximum static strength by measurement with a handgrip dynamometer. A 12-wk home-based RT program was individually prescribed and consisted of 13 exercises, performed each other day, using simple material. RESULTS: No significant differences occurred in body weight or limb circumferences ( > 0.05), although the sum of central skinfolds decreased with RT (111 +/- 9 vs 100 +/- 9 mm; < 0.05). RT induced significant gains in four of five exercises: rear lat pull-down (141 +/- 19 vs 198 +/- 20 W), standing upright row (134 +/- 22 vs 157 +/- 24 W), triceps press down (85 +/- 14 vs 123 +/- 21 W), and seated chest press (114 +/- 20 vs 143 +/- 21 W; < 0.05). Sit-up results also showed significant improvements, while handgrip did not ( > 0.05). CONCLUSION: GH-deficient adults without GH replacement may improve their maximum muscle power when submitted to an individualized, simple, and short home-based RT program. Considering that limb girths did not significantly change, the gains were most likely due to improvements in neuromuscular components. PMID- 12370559 TI - Patellofemoral stress during walking in persons with and without patellofemoral pain. AB - OBJECTIVE: To determine whether individuals with patellofemoral pain (PFP) demonstrate elevated patellofemoral joint (PFJ) stress compared with pain-free controls during free and fast walking. DESIGN: A cross-sectional study utilizing an experimental and a control group. BACKGROUND: Although the cause of PFJ pathology is believed to be related to elevated joint stress (force per unit area), this hypothesis has not been adequately tested and causative mechanisms have not been clearly defined. METHODS: Ten subjects with a diagnosis of PFP and 10 subjects without pain participated. All subjects completed two phases of data collection: 1) magnetic resonance imaging (MRI) assessment to determine PFJ contact area and 2) comprehensive gait analysis during self-selected free and fast walking velocities. Data obtained from both phases were required as input variables into a biomechanical model to quantify PFJ stress. RESULTS: On the average, PFJ stress was significantly greater in subjects with PFP compared with control subjects during level walking. The observed increase in PFJ stress in the PFP group was attributed to a significant reduction in PFJ contact area, as the PFJ reaction forces were similar between groups. CONCLUSION: Our results are consistent with the hypothesis that increased patellofemoral joint stress may be a predisposing factor with respect to development of PFP. Clinically, these findings indicate that treatments designed to increase the area of contact between the patella and the femur may be beneficial in reducing the PFJ stress during functional activities. RELEVANCE: Patellofemoral pain affects about 25% of the population, yet its etiology is unknown. Knowledge of the biomechanical factors contributing to patellofemoral joint pain may improve treatment techniques and guide development of prevention strategies. PMID- 12370560 TI - Decrease in serum leptin after prolonged physical activity in men. AB - PURPOSE: This study was designed to determine whether serum leptin levels were affected by a 5-d military course after 3 wk of combat training. METHODS: 26 male soldiers (mean age = 21 +/- 2 yr) were examined at the beginning of the training program and just at the end of the 5-d course. The combination of continuous heavy physical activity and sleep deprivation led to energy deficiency. Blood samples were analyzed for serum leptin, insulin, cortisol, adrenocorticotropin (ACTH), and testosterone; plasma was analyzed for free fatty acids (FFA), glycerol, glucose, and catecholamines. RESULTS: At the end of the 5-d course, there was a significant reduction in serum leptin (0.40 +/- 0.04 ng x mL(-1) versus 1.47 +/- 0.14 ng x mL(-1), < 0.001), i.e., a mean decrease of 67.00 +/- 3.75%. Plasma norepinephrine and dopamine rose significantly from 296 +/- 17 ng x L(-1) to 672 +/- 48 ng x L(-1) and 23 +/- 3 ng x L(-1) to 40 +/- 5 ng x L(-1) ( < 0.001 and < 0.01, respectively), whereas epinephrine remained unchanged. Serum concentrations of the anabolic hormone, insulin, fell from 31.17 +/- 3.03 microU x mL(-1) to 17.79 +/- 1.58 microU x mL(-1) ( < 0.001), whereas plasma FFA and glycerol were increased ( < 0.001, < 0.05, respectively). A statistically significant correlation appeared between the changes in leptin and insulin (r = 0.5306, < 0.01). Serum testosterone decreased significantly ( < 0.001), whereas serum cortisol, ACTH, and plasma glucose were unchanged at the end of the course. The training program had no significant effect on mean body mass index. CONCLUSION: A 4-wk strenuous military training program, which induced an energy deficiency, reduced serum leptin to a third of normal levels. The decrease in serum leptin was attributed to the exercise-induced elevation in catecholamines and hypoinsulinemia. PMID- 12370561 TI - Effect of prior exercise on VO(2) slow component is not related to muscle temperature. AB - INTRODUCTION: It has been widely reported that the VO(2) slow component is reduced in the second of two bouts of heavy exercise. It has also been shown that an increase in muscle temperature (Tm) produced by wearing hot-water-perfused pants causes a reduction in the VO(2) slow component. Therefore, the aim of this study was to investigate whether the effect of prior heavy exercise on the VO(2) slow component of subsequent heavy exercise is related to the warming-up of the exercising limbs. METHODS: Six male subjects completed an exercise protocol consisting of two constant-load exercise bouts (EX-1 and EX-2) at 90% VO(2peak), separated by 6 min of rest. The Tm of the m. vastus lateralis was measured with an indwelling thermistor. Seven days later, the subjects completed a second exercise protocol consisting of a passive warming-up of the upper legs until the same Tm was reached as after EX-1, followed by a constant-load work bout (EX-3) identical to EX-1 and EX-2. RESULTS: Tm reached comparable levels at the start of EX-2 and EX-3 (37.3 +/- 0.6 degrees C and 37.2 +/- 0.3 degrees C, respectively). The VO(2) slow component (measured as deltaVO(2)(6-2 min)) was reduced by 57% after prior heavy exercise ( < 0.05), whereas no significant reduction was observed after prior passive warming-up. CONCLUSIONS: The results of this study indicate that the reduction in VO(2) slow component observed after prior heavy exercise cannot be explained by an increase in muscle temperature of the upper legs. PMID- 12370562 TI - The effects of fish oil and isoflavones on delayed onset muscle soreness. AB - INTRODUCTION/PURPOSE: Fish oils (FO) have been shown to modulate the inflammatory response through alteration of the eicosanoid pathway. Isoflavones (ISO) appear to reduce the inflammatory pathway through their role as a tyrosine kinase inhibitor. Delayed onset muscle soreness (DOMS) develops after intense exercise and has been associated with an inflammatory response. Therefore, we hypothesized that physical parameters associated with DOMS could be decreased via the modulation of the inflammatory response by supplementing subjects with either FO or ISO. METHODS: 22 subjects were recruited and randomly assigned to one of three treatment groups: FO (1.8 g of omega-3 fatty acids x d(-1)), ISO (120 mg soy isolate x d(-1)), or placebo (PL) (Western fat blend and/or wheat flour). All treatment groups received 100-IU vitamin E x d(-1) to minimize lipid peroxidation of more highly unsaturated fatty acids. Subjects were supplemented 30 d before the exercise and during the week of testing and were instructed to refrain from unusual exercise. DOMS was induced by 50 maximal isokinetic eccentric elbow flexion contractions. Strength parameters, pain, arm circumference, and relaxed arm angle (RANG) were measured at 48, 72, and 168 h post exercise. Cortisol, creatine kinase (CK), interleukin-6 (IL-6), tumor necrosis factor (TNFalpha), malondialdehyde (MDA), and serum iron were measured before supplementation, after supplementation, and post exercise. RESULTS: Significant decreases were observed in RANG and strength 48 h postexercise among all groups, and there were significant increases in pain and arm circumference. There were no significant changes among all groups from baseline at 168 h (7 d) post exercise. There were no significant treatment effects between groups for the physical parameters or for cortisol, CK, IL-6, TNFalpha, MDA, or serum iron. CONCLUSION: These data indicate FO or ISO, at the doses supplemented, were not effective in ameliorating DOMS with the above-cited protocol. PMID- 12370563 TI - Muscle deoxygenation as related to work rate. AB - PURPOSE: The kinetics of the decrease in venous O(2) content in response to constant work rate exercise below the lactic acidosis threshold (LAT) is very rapid, reaching a constant value by approximately 1 min. However, for work rates above the LAT, a slow further decrease in venous O(2) content takes place that is attributable to the Bohr effect rather than further decrease in end capillary PO. We hypothesized that similar differences, with respect to the LAT, will be observed in muscle deoxygenation kinetics when studied with near-infrared spectroscopy (NIRS). METHODS: Twelve normal subjects performed three constant work rate tests from unloaded cycling at 60% of LAT, 80% LAT, each with four repetitions, and above LAT (LAT + 35% between LAT and VO(2max) three times, on a cycle ergometer for 6 min. We measured tissue deoxygenation with NIRS, with the probe over the vastus lateralis muscle, time-averaging the repetitions. Gas exchange and heart rate (HR) were measured breath-by-breath and beat-by-beat. RESULTS: Tissue deoxygenation kinetics were significantly faster than VO(2) and HR at 60%- and 80%-LAT work rates. By 1 min of exercise, deoxygenation was constant for the work rate below the LAT. At 30 s, tissue deoxygenation was 70 95% complete, whereas VO(2) and HR were only 30-60% complete. For the work rate above the LAT, a steady state for muscle deoxygenation was not reached during the 6 min of exercise. After 1 min of above-LAT exercise, either one of two patterns of slow change in tissue oxygenation developed, deoxygenation or reoxygenation. It is postulated that these different responses might be due to effects of the exercise lactic acidosis. H accompanying lactate increase might cause further deoxygenation due to the Bohr effect, and acidosis-induced vasodilatation might cause reoxygenation after the initial deoxygenation. CONCLUSION: 1) The kinetics of tissue deoxygenation are significantly more rapid than VO(2) and HR kinetics at all work rates studied, and 2) steady-state in tissue deoxygenation is seen by 1 min of constant work rate exercise below the LAT, but this is much delayed for work rates above the LAT. PMID- 12370564 TI - Plasma compartment filling after exercise or heat exposure. AB - PURPOSE: The present study was assessed to study the restoration of the vascular compartment by rehydration after heat exposure or exercise. METHODS: Eight subjects completed four trials in a randomized order: 2.7% dehydration of body mass by passive controlled hyperthermia once with rehydration and once without rehydration during recovery, and 2.7% dehydration of body mass by treadmill exercise once with rehydration and once without rehydration during recovery. An isotonic glucose electrolyte beverage was provided twice during the recovery period for a total volume, which was equivalent to the target value of body mass loss during dehydration procedures. Plasma volume (PV) was measured using Evans Blue dilution technique, and PV changes (deltaPV) were determined using hematocrit and hemoglobin measurements. RESULTS: PV was better maintained during exercise than during heat exposure, and the difference in deltaPV between the two patterns of dehydration was maintained during the first 3 h of recovery. Plasma protein seemed to be accountable for the difference in deltaPV during heat exposure and exercise but not during the 270 min of recovery. Rehydration partly restored body fluid losses, but the plasma compartment was privileged, because 26 30% of the net fluid gain was found in the plasma compartment (about 300 mL). Rehydration restored plasma osmolality and diminished the drive for arginin vasopressin response. CONCLUSION: The similar selective retention of water in the plasma compartment might essentially be explained by osmotic factors provided by the beverage. As PV was completely restored by rehydration after exercise and only partly restored after heat exposure, the volume of ingested beverage should be higher after heat exposure to completely restore the plasma compartment. PMID- 12370565 TI - Salivary IgA response to prolonged exercise in a cold environment in trained cyclists. AB - PURPOSE: The purpose of the present study was to determine the effect of a prolonged bout of exercise in freezing cold conditions on saliva immunoglobulin A (s-IgA) responses in endurance-trained males. METHODS: Using a randomized cross over design, 15 trained male cyclists cycled for 2 h on a stationary ergometer at 70% VO(2max) in an environmental chamber on one occasion at a temperature of -6.4 +/- 0.1 degrees C (cold) and on another occasion at a temperature of 19.8 +/- 0.2 degrees C (control). Trials began at 12:30 h to avoid the fall in s-IgA concentration that occurs during the morning hours. Unstimulated whole-saliva samples were collected over a 2-min period at preexercise, postexercise, and 2-h postexercise. The s-IgA concentration was determined using a sandwich-type ELISA method. RESULTS: Saliva flow rate decreased postexercise by 31%, returning to preexercise levels by the 2-h postexercise collection (main effect of time: < 0.01). The decrease in saliva flow rate postexercise in the control trial (39% compared with 22% on cold trial) approached significance (interaction: = 0.08) and may have accounted for the corresponding increase in s-IgA concentration postexercise in the control trial (s-IgA concentration: control preexercise; 91 +/- 12; postexercise; 110 +/- 13 mg x L(-1); < 0.05). Saliva IgA secretion rate decreased postexercise by 19.5% returning to preexercise levels by 2-h postexercise measure (main effect of time: < 0.05). CONCLUSIONS: These data show that performing a bout of prolonged exercise results in a reduction in s-IgA secretion rate. Additionally, these data demonstrate that performing prolonged exercise in freezing cold conditions does not influence saliva flow rate or s-IgA secretion rate responses to prolonged exercise. PMID- 12370566 TI - Effect of combined electrostimulation and plyometric training on vertical jump height. AB - PURPOSE: This study investigated the influence of a 4-wk combined electromyostimulation (EMS) and plyometric training program on the vertical jump performance of 10 volleyball players. METHODS: Training sessions were carried out three times weekly. Each session consisted of three main parts: EMS of the knee extensor muscles (48 contractions), EMS of the plantar flexor muscles (30 contractions), and 50 plyometric jumps. Subjects were tested before (week 0), during (week 2), and after the training program (week 4), as well as once more after 2 wk of normal volleyball training (week 6). Different vertical jumps were carried out, as well as maximal voluntary contraction (MVC) of the knee extensor and plantar flexor muscles. RESULTS: At week 2, MVC significantly increased (+20% knee extensors, +13% plantar flexors) as compared to baseline ( < 0.05). After the 4-wk training program, different vertical jumps considered were also significantly higher compared to pretraining ( < 0.001), and relative gains were comprised between 8-10% (spike-counter movement jump) and 21% (squat jump). The significant increases in maximal strength and explosive strength produced by the present training program were subsequently maintained after an additional 2 wk of volleyball training. CONCLUSION: EMS combined with plyometric training has proven useful for the improvement of vertical jump ability in volleyball players. This combined training modality produced rapid increases (approximately 2 wk) of the knee extensors and plantar flexors maximal strength. These adaptations were then followed by an improvement in general and specific jumping ability, likely to affect performance on the court. In conclusion, when EMS resistance training is proposed for vertical jump development, specific work out (e.g., plyometric) must complement EMS sessions to obtain beneficial effects. PMID- 12370567 TI - Level ground and uphill cycling efficiency in seated and standing positions. AB - PURPOSE: This study was designed to examine the effects of cycling position (seated or standing) during level-ground and uphill cycling on gross external efficiency (GE) and economy (EC). METHODS: Eight well-trained cyclists performed in a randomized order five trials of 6-min duration at 75% of peak power output either on a velodrome or during the ascent of a hill in seated or standing position. GE and EC were calculated by using the mechanical power output that was measured by crankset (SRM) and energy consumption by a portable gas analyzer (Cosmed K4b(2)). In addition, each subject performed three 30-s maximal sprints on a laboratory-based cycle ergometer or in the field either in seated or standing position. RESULTS: GE and EC were, respectively, 22.4 +/- 1.5% (CV = 5.6%) and 4.69 +/- 0.33 kJ x L(-1) (CV = 5.7%) and were not different between level seated, uphill seated, or uphill standing conditions. Heart rate was significantly ( < 0.05) higher in standing position. In the uphill cycling trials, minute ventilation was higher ( < 0.05) in standing than in seated position. The average 30-s power output was higher ( < 0.01) in standing (803 +/- 103 W) than in seated position (635 +/- 123 W) or on the stationary ergometer (603 +/- 81 W). CONCLUSION: Gradient or body position appears to have a negligible effect on external efficiency in field-based high-intensity cycling exercise. Greater short-term power can be produced in standing position, presumably due to a greater force developed per revolution. However, the technical features of the standing position may be one of the most determining factors affecting the metabolic responses. PMID- 12370568 TI - Relationship of physical activity to eating behaviors and weight loss in women. AB - PURPOSE: To examine whether change in physical activity is associated with compliance to changes in dietary intake and eating behaviors in an 18-month behavioral weight loss program, and to examine the contribution of exercise to weight loss when these other weight loss behaviors are also considered. METHODS: Data from 104 subjects who completed an 18-month behavioral weight loss program were analyzed in this study. All subjects were prescribed a reduced energy (1,200 1,500 kcal x d(-1)) and fat (20 to 30%) diet, and exercise progressed from 100 to 200 min x wk(-1). Subjects attended group behavioral lessons throughout the study. Weight, physical activity, energy intake, and weight loss eating behaviors were assessed at 0 and 18 months. RESULTS: Body weight decreased 7.8 +/- 7.5 kg and body mass index decreased 2.8 +/- 2.7 kg x m(-2) from 0 to 18 months ( < 0.05). Total energy intake (kcal x d(-1)) and macronutrient intake (g x d(-1)) decreased, whereas physical activity and eating behaviors associated with weight loss increased from 0 to 18 months ( < 0.05). Change in physical activity was significantly correlated with weight loss (r = 0.33), reductions in energy intake (r = 0.20), and improvements in eating behaviors associated with weight loss (r = 0.24) ( < 0.05). Regression analysis indicated that change in physical activity significantly improved weight loss after changes in energy intake and weight loss eating behaviors were considered in the analysis, with R(2)significantly improving by approximately 0.04 ( < 0.05). However, results from multiple regression showed weight loss was influence more by changes in eating behaviors than changes in physical activity (R(2) = 0.17 vs R(2) = 0.04). CONCLUSIONS: The combination of changes in eating and physical activity behaviors can improve long term weight loss compared with either behavior alone. Interventions targeting both behaviors are recommended for improving long-term weight loss. PMID- 12370569 TI - Autonomic adaptations to intensive and overload training periods: a laboratory study. AB - PURPOSE: Looking for practical and reliable markers of fatigue is of particular interest in elite sports. One possible marker might be the autonomic nervous system activity, known to be well affected by physical exercise and that can be easily assessed by heart rate variability. METHODS: We designed a laboratory study to follow six sedentary subjects (32.7 +/- 5.0 yr) going successively through 2 months of intensive physical training and 1 month of overload training on cycloergometer followed by 2 wk of recovery. Maximal power output over 5 min (Plim5'), VO(2) and standard indices of heart rate variability were monitored all along the protocol. RESULTS: During the intensive training period, physical performance increased significantly VO(2peak) : +20.2%, < 0.01; Plim5': +26.4%, < 0.0001) as well as most of the indices of heart rate variability (mean RR, Ptot, HF, rMSSD, pNN50, SDNNIDX, SDNN, all < 0.05) with a significant shift in the autonomic nervous system toward a predominance of its parasympathetic arm (LF/HF, LFnu, HFnu, < 0.01). During the overload training period, there was a stagnation of the parasympathetic indices associated to a progressive increase in sympathetic activity (LF/HF, < 0.05). During the week of recovery, there was a sudden significant rebound of the parasympathetic activity (mean RR, HF, pNN50, rMSSD, all < 0.05). After 7 wk of recovery, all heart rate variability indices tended to return to the prestudy values. CONCLUSION: Autonomic nervous system status depends on cumulated physical fatigue due to increased training loads. Therefore, heart rate variability analysis appears to be an appropriate tool to monitor the effects of physical training loads on performance and fitness, and could eventually be used to prevent overtraining states. PMID- 12370570 TI - Field evaluation of energy expenditure in women using Tritrac accelerometers. AB - PURPOSE: To investigate the use of Tritrac accelerometers to measure energy expenditure (EE) of various activities for women in the field setting, as compared with portable indirect calorimetry. METHODS: Twenty women (age 20-29) performed a choreographed routine of six activities (walking, jogging, stair climbing, walking on an incline, stationary cycling, and arm ergometry) while wearing a Tritrac-R3D accelerometer (Hemokinetics Inc., Madison WI) and the Cosmed K4b(2) portable metabolic cart (Cosmed, Rome, Italy). RESULTS: Comparing the mean error scores (K4b(2) - Tritrac), the Tritrac overestimated the EE (kcal x min(-1)) of walking (-1.45) and jogging (-1.75), whereas underestimating the EE of stair climbing (2.76), stationary cycling (2.75), and arm ergometry (1.20). Walking on an incline showed the lowest mean error score (-0.11). Intraclass correlations were moderate for walking (r = 0.568, < 0.05), jogging (r = 0.666, < 0.05), and stairs (r = 0.503, < 0.05) but for the other activities ranged from r = 0.290 ( > 0.05) to r = 0.480 ( < 0.05). The raw data from the Tritrac was applied to a previously developed nonlinear model to adjust the Tritrac scores to the standard of whole-room indirect calorimetry. This resulted in statistically significant improvements in the agreement between the adjusted Tritrac value and the K4b for walking, jogging, and walking on an incline ( < 0.05). CONCLUSION: When compared with portable indirect calorimetry, the Tritrac overestimates the EE of walking and jogging, whereas underestimating that of stair climbing, stationary cycling, and arm ergometry. This limits the use of such a technique to measure EE in the field. The main issues appear to be the type and intensity of the activity and the need for movement in order for the Tritrac to register EE. Activity specific linear regression equations are proposed as a tool to improve the measurement of EE using the Tritrac in the field. PMID- 12370571 TI - Batting performance of wood and metal baseball bats. AB - INTRODUCTION/PURPOSE: Although metal baseball bats are widely believed to outperform wood bats, there are few scientific studies which support this. In a batting cage study, Greenwald et al. found that baseballs hit with a metal bat traveled faster than those hit with a wood bat, but the factors responsible for this difference in bat performance remain unidentified. The purpose of this study was to determine the effects of swing speed, impact location, and elastic properties of the bat on batted ball speeds. METHODS: The pitched ball, batted ball, and swings of two wood and five metal baseball bats by 19 different players were tracked in three dimensions at 500 Hz using a passive infrared motion analysis system. RESULTS: Increases in the batted ball speeds of metal bats over those of wood bats resulted from faster swing speeds and higher elastic performance with an apparent increase in the ball-bat coefficient of restitution. The contribution of these variables to batted ball speed differed with metal bat model. The "sweet spot" associated with maximum batted ball speeds was located approximately the same distance from the tip of wood bats as it was from metal bats. CONCLUSIONS: The variables that correlated with differences between metal and wood bat performance, and most notably differences in the percentage of faster batted balls, were identified using a novel kinematic analysis of the ball and bat. These variables and their correlation with bat performance should be applicable to other players and bats, although more skilled players and higher performing bats would likely result in even faster batted ball speeds. PMID- 12370572 TI - Symposium: conference on the science and policy of performance-enhancing products. PMID- 12370573 TI - Chronic fatigue syndrome, deconditioning, and graded exercise therapy. PMID- 12370575 TI - Non-specific back pain in children and adolescents: a prospective study until maturity. AB - Fifty-eight children with non-specific back pain lasting for at least 3 months were included in this prospective study. Not included were children with radicular pain or with abnormal findings on clinical examination, radiography, bone scan, computed tomography or magnetic resonance imaging. They were all re examined after skeletal maturity, on average 7.6 years later. Thirty-one children had pain in the lumbar spine, 20 in the thoracic region and seven over the entire spine. Sixty-two percent of the children were still in pain at follow-up. Female sex and pain in the thoracic region were associated with an increased risk of the pain remaining chronic. PMID- 12370577 TI - Failures of open reduction in developmental dislocation of the hip. AB - Eight patients with the same number of developmental dislocated hips were initially treated with open reduction through an anterolateral approach. The hips then became dislocated again. There were six girls, with a mean age at first open reduction of 13.3 months. We treated the hips with a new open reduction through an anteromedial approach. A constricted anteromedial capsule was always found as the main factor; all had an intact anteromedial capsule, there was an inverted transverse ligament in three cases and a very tight psoas tendon in another three cases. All were reduced, without complications and with only one simultaneous bone procedure. Risk of avascular necrosis and residual dysplasia could not be evaluated with this follow-up. We conclude that in any open reduction for developmental dislocation of the hip the surgeon must consider a release of the anteromedial capsule, which we have found to be the most important factor in technical failures. PMID- 12370576 TI - Dynamic magnetic resonance guided treatment of developmental dysplasia of the hip. AB - This study demonstrates the feasibility and advantages of near real-time, multiplanar, dynamic magnetic resonance image-assisted treatment of patients with developmental dysplasia of the hip. Pathoanatomy and dynamic blocks to reduction are visualized with anatomic clarity not otherwise possible. Continuous imaging allows accurate assessment and maintenance of optimum positioning throughout the casting procedure. Patient charges for this new technique are less than standard methods of treatment, and the child receives no ionizing radiation. PMID- 12370578 TI - Developmental dysplasia of the hip incidence in Ethiopian Jews revisited: 7-year prospective study. AB - We describe the sonographic and true incidence of developmental dysplasia of the hip among Ethiopian Jews compared with the general Israeli population. Previous results were based on a small number of Jewish Ethiopian children; this study is based on over 7 years of experience, during which 34,048 newborns, 768 of whom were Ethiopian, were examined clinically and sonographically. The incidence of sonographic developmental hip dysplasia was 5.5% in the general Israeli population, compared with 1.24% for the Ethiopian Jews. True developmental hip dysplasia incidence for Arabs and other Jews was 0.51%, as compared with 0.15% in the Ethiopians. Our data support the theory that either a single unknown gene or a multiple gene system plays a major role in the incidence of the dysplasia. PMID- 12370579 TI - Synovial hemangioma of the knee in young children. AB - Synovial hemangiomas are relatively rare tumors. Clinicians are inclined to delay treatment in most cases. We encountered three cases, in which there was a delay before the patients were operated on. During the relatively long-term postoperative follow up, none of the three cases showed a recurrence of either hemoarthrosis or knee pain. However, limitations in motion or osteoarthritic changes in the affected knee joint remained. We therefore consider that synovial hemangiomas of the knee, even if found in young children, could possibly result in postoperative limitations in motion or osteoarthritic changes. PMID- 12370580 TI - Isolated congenital pseudoarthrosis of the fibula. AB - Congenital pseudarthrosis of the limb most commonly involves the tibia, although various combinations of bones including fibula, radius, ulna, clavicle and humerus have all been described. Isolated congenital pseudarthrosis of the fibula is a very rare entity with only 12 cases reported in the English literature. We report three cases of this condition treated in our institution. The first child had a varus ankle deformity at the age of 4 months. The other two children presented with valgus ankle deformity after they started to walk. Two patients were treated conservatively while the third had a distal tibio-fibular fusion in view of severe valgus deformity. All three patients showed good early results after 1 to 2 years. We advocate early distal tibio-fibular fusion to prevent valgus deformity in these children. PMID- 12370581 TI - Tibial lengthening: does the fibula migrate? AB - A retrospective study of 32 patients who underwent tibial lengthening was performed in order to establish the need for distal tibio-fibular fixation. In 16 patients stabilization of the inferior tibio-fibular joint was carried out and in the other 16 no stabilization was performed. Three established and one new radiographic index of the tibio-fibular relationship at the ankle were used to assess proximal fibular migration. All patients showed proximal migration of the distal fibula, but those without stabilization demonstrated marked migration of the fibula associated with a valgus tendency. The difference between the groups was statistically significant ( <0.001), confirming the need for fibular fixation. PMID- 12370582 TI - Effect on knee flexion of a modification to the surgical technique of pin placement during femoral lengthening. AB - Loss of knee movement is a common problem in femoral lengthening. Two groups of 10 children were compared: one group lengthened by the Ilizarov technique using a standard method and one group in whom the technique was modified to incorporate a different method for determining the pin placement. Loss of knee flexion was compared between the two groups. A significant difference in the total loss of flexion ( <0.002), and in the amount of knee flexion, at the end of lengthening ( <0.001) and at 6 months after frame removal ( <0.004) was observed. This simple modification to surgical technique appears to decrease the knee flexion lost in children undergoing femoral lengthening by the Ilizarov method. PMID- 12370583 TI - An aetiological classification for developmental synostoses at the elbow. AB - Synostoses at the elbow joint are rare. The literature divides them into three groups based on the nature of bony ankylosis; the commonest are humeroradial synostoses. Approximately 150 cases have been reported. There are 29 reported cases of humeroradioulnar synostosis and five of humeroulnar synostosis. An anatomical classification was previously described for humeroradial synostoses. Due to significant phenotypic variability we believe a classification based solely on anatomical characteristics will in some cases be misleading. No classification exists for humeroradioulnar and humeroulnar synostosis. By re examining the literature we have produced a combined classification for all elbow synostoses which more accurately predicts causes. Congenital elbow synostoses often cause little functional disability. Treatment by soft tissue release and osteotomy has been attempted, but although range of movement is initially, improved re-ossification is the norm. Investigation is more complicated and may be helped by classification which identifies syndrome association, risk of organ anomaly, and inheritance pattern. PMID- 12370584 TI - Fractures of the olecranon in children. Long-term follow-up of 39 cases. AB - Thirty-nine patients, who had received a fracture of the olecranon at an average age of 7.4 years, were reviewed at an average age of 32 years, in order to evaluate the results of treatment. All patients had reached skeletal maturity at follow-up. Thirty-four fractures were treated conservatively and five, surgically. Of the 34 fractures treated conservatively, six also had surgical treatment of associated fractures. We identified five patterns of fracture on the basis of the anatomic site of the fracture line, the interfragmentary displacement and the presence of an associated lesion. According to our grading scale, 34 patients had a good result, two a fair result and three a poor result. We observed poor results in only 7.6% of cases, even though 85% of the patients had received an intraarticular fracture. We believe that the long-term prognosis of olecranon fractures in children is related to the anatomic site of the fracture line, to the interfragmentary displacement and to the presence of an associated lesion. Conservative treatment may be indicated when the interfragmentary displacement is less than 2 mm. The presence of an associated lesion is a negative prognostic factor. PMID- 12370585 TI - Hangman's fracture caused by suspected child abuse. A case report. AB - This report highlights the difficulties associated with diagnosing cervical spine injuries in children especially as the history and mechanism of injury may often be unclear and the normal variations in roentgenographic appearance may be confusing. As far as we are aware this is only the second case of traumatic Hangman's fracture in a child under the age of 3 years and the only case where there is a strong probability of child abuse. A female child aged 23 months was admitted with a 5-day history of irritability and general malaise. Her father reported noticing that she was reluctant to move her neck. He denied any possibility of trauma. On admission she had neck stiffness with a temperature of 37 degrees C and supported her neck with her hands. There was evidence of otitis media of her right ear. Her physical examination was otherwise normal. A full blood count and lumbar puncture were within normal limits. Cervical spine x rays suggested a Hangman's fracture of C2 with slight anterior subluxation of C2 on C3 and a kyphus at that level. Computerized Tomography demonstrated no significant canal encroachment. An isotope bone scan was non-diagnostic. She was treated in a moulded cervical collar with neck held in slight extension. Her symptoms resolved and further radiographs showed improved alignment. Repeat CT scans seven weeks post admission showed callus formation. At follow-up at one year she remains asymptomatic. Hangman's fracture is very rare in children under 3 years and the considerable normal variations further complicate diagnosis. Swischuk described the posterior cervical line connecting the spinous process of C1-C3 vertebrae on the lateral projection to differentiate a true fracture dislocation from physiological anterior displacement. A detailed history, roentgenograms, bone scans, CT scans and MRI scans are often required for accurate diagnosis. PMID- 12370586 TI - Nail patella syndrome. A 55-year follow-up of the original description. AB - The long-term skeletal changes and the lack of significant clinical complaints in a 77-year-old woman with nail patella syndrome are described. Fifty-five years previously she was one of the first reported patients. These early patients came from two families with involvement of multiple individuals with the variable constellation of deformities. We reviewed her skeletal natural history and her family history as it related to nail patella syndrome involvement and treatment, and correlated the original premolecular biology description and subsequent long term follow-up with the current molecular and genetic concepts of the cause of the variable expression of nail patella syndrome. PMID- 12370587 TI - Progressive osseous heteroplasia. A case report and review of the literature. AB - Progressive osseous heteroplasia is a rare childhood disorder that is characterized by ectopic progressive ossification of skin, muscle, and connective tissue. We report a 5 year-old female patient with familial transmission. She developed cutaneous calcifications and ossifications within the first 2 months of life. Her father and father's aunt had subcutaneous nodules. At the age of 5 years, physical examination of the patient revealed ossified subcutaneous nodules and plaques on both upper limbs, the right side was predominantly affected. All the joints of the upper limbs were ankylosed except the left shoulder. Biopsy specimens of the patient and her father revealed islands of bone in the reticular dermis and deep dermis, respectively. We suggest that all family members of progressive osseous heteroplasia patients are carefully investigated for ossified nodules because of autosomal dominant inheritance. PMID- 12370588 TI - Spontaneous bone regeneration in surgically induced bone defects in young rabbits. AB - Defects of the femoral shaft (15%, 20%, 25%, and 30% of the femoral length) with intact periosteum were made in young rabbits to investigate differences in union time. The mean union time was 7.3, 7.1, 7.4, and 7.0 weeks, respectively and there was no significant difference ( >0.05) between the groups. The mean healing indices were 6.7, 4.6, 3.9, and 3.1 weeks/cm, respectively and these were significantly different ( <0.05) between the 15% bone defect group and the other bone defect groups. These results suggested that union time was not affected by the amount of bone defect. Femoral lengthening of 20% was performed to compare the quality of callus with those of the femoral defect of 20%. The mean healing indices of defect and lengthening were 4.6 and 5.7, respectively and the difference was significant ( = 0.004). The callus stiffness of femoral defect was higher ( = 0.02) than that after femoral lengthening whereas the bone mineral density of the callus showed no significant difference ( = 0.37) between two groups. The method of filling bone defect with callus generated from the intact periosteum may be a new therapeutic option for the reconstruction of large bone defects in children when other treatments are not available. PMID- 12370589 TI - Anatomic variations of the coracoacromial ligament in neonatal cadavers: a neonatal cadaver study. AB - One of the most common causes of pain and disability in the upper limb is inflammation of the rotator cuff tendons. When no significant bony abnormality exists in the surrounding structures, the coracoacromial ligament has been implicated as a possible cause of impingement on the cuff tendons and various morphological variants of the ligament have so far been claimed to be either the cause or the result of impingement. In this study, 110 shoulders from 60 neonatal cadavers that were preserved in a preparation of formaldehyde were dissected. Anatomic variations of coracoacromial ligaments were investigated with metric and histologic analysis. Three main ligament types were identified: quadrangular, broad band and U-shaped. The multiple banded ligament was not found. Histologic analysis showed that in U-shaped ligaments a thin tissue existed in the central part of the ligament close to the coracoid. Comparing our data with the adult measurements of a previous study we suggest that the primordial ligament is broad shaped, but assumes a quadrangular shape due to the different growth rates of the coracoid and acromial ends. We also suggest that broad and U-shaped ligaments account for the primordial and quadrangular and Y-shaped ligaments account for the adult types of the single or double banded anatomic variants respectively. Our results show that various types of the coracoacromial ligament are present at the neonatal period and that the final shape of the ligament should be defined by developmental factors, rather than degenerative changes. PMID- 12370590 TI - The flexion-adduction test: an early sign of hip disease. PMID- 12370591 TI - Influence of venous function on exercise tolerance in chronic heart failure. AB - PURPOSE: The clinical phase of chronic heart failure (HF) includes a marked decline in exercise tolerance, in part due to impaired skeletal muscle blood flow delivery. Interestingly, the role of the venous system on exercise tolerance in patients with HF has not received much attention, despite evidence of changes in venous structure and function. The purpose of this study was to examine the relationship between forearm arterial and venous function, and exercise tolerance in patients with HF and age-matched controls. METHODS: Vascular function and exercise tolerance was examined in 20 patients with HF (age 59 +/- 13 years) and 10 control subjects (age 51 +/- 16 years). Nondominant forearm arterial inflow, vascular resistance, venous capacitance, and venous outflow were evaluated at rest and after 5 minutes of upper arm occlusion, using strain gauge plethysmography. Exercise tolerance was measured as the maximum walking distance achieved on a 6-minute walking test. RESULTS: Maximum walking distance (HF: 178 +/- 65 m; controls: 562 +/- 136 m, P=.0001), and forearm vascular function after occlusion were significantly different between groups (forearm arterial inflow: HF 15.3 +/- 6; controls 22 +/- 6.7; forearm venous capacitance: HF 1.4 +/- 0.5; controls 2.0 +/- 0.4; forearm venous outflow: HF 24.5 +/- 9.4; controls: 33 +/- 10 mL x 100 mL tissue(-1) x min(-1); and forearm vascular resistance: HF 7.8 +/- 3; controls 4.6 +/- 1.4 U). Correlation analysis revealed significant associations between all forearm vascular measurements after occlusion and maximum walking distance. CONCLUSION: These data confirm previous studies indicating the importance of arterial reactivity on exercise tolerance in patients with HF. Additionally, the results suggest the importance of venous function as a contributing factor to exercise performance. PMID- 12370592 TI - Venous function in chronic heart failure: the neglected part of circulatory regulation. PMID- 12370593 TI - Outcomes in cardiac rehabilitation programs across Illinois. PMID- 12370594 TI - The utility and viability of outcome measurement and monitoring in cardiac rehabilitation. PMID- 12370595 TI - Pulmonary rehabilitation compared with brief advice given for severe chronic obstructive pulmonary disease. AB - PURPOSE: The objective of this study was to compare the effectiveness of a short term pulmonary rehabilitation program with brief advice given to patients with severe ventilatory impairment due to chronic obstructive pulmonary disease (COPD). METHODS: One hundred three patients with severe COPD, defined as having forced expiratory volume in 1 second < 40% predicted, were randomly assigned to rehabilitation or to brief advice. Fifty-four patients attended a rehabilitation program twice a week for 6 weeks. Forty-nine patients attended a single session during which they were given printed educational materials and verbal advice and guidance about exercise. Subjects were reassessed at 3 months. RESULTS: The shuttle walking distance increased significantly in the rehabilitation group by 43 meters. The increase of 23 meters in the brief advice group was significantly less than in the rehabilitation group. Improvements in quality of life in the rehabilitation group were small and not clinically significant. CONCLUSIONS: In these patients with severe COPD, a short outpatient rehabilitation program of low intensity achieved small but significant improvement in shuttle walking distance, compared with brief advice. The improvements in quality of life were modest and did not reach statistical significance, although in some instances the confidence limits include differences that approach clinical significance. The relatively small effect may be due to the low intensity of the program or to the severity of the subjects' ventilatory impairment. PMID- 12370600 TI - New genetic principles. PMID- 12370602 TI - Cytogenetics and molecular cytogenetics. PMID- 12370601 TI - Molecular diagnostics in obstetrics and gynecology. PMID- 12370603 TI - Preimplantation genetic diagnosis. PMID- 12370605 TI - Principles of screening. PMID- 12370604 TI - Prenatal diagnosis using fetal cells and cell-free fetal DNA in maternal blood: what is currently feasible? PMID- 12370606 TI - Screening for chromosomal defects by fetal nuchal translucency at 11 to 14 weeks. PMID- 12370607 TI - Inherited risk of women's cancers: what's changed for the practicing physician? PMID- 12370608 TI - Genetic therapies for the fetus. PMID- 12370609 TI - Newborn screening: the role of the obstetrician. PMID- 12370610 TI - Application of the human genome to obstetrics and gynecology. PMID- 12370612 TI - Diagnosis and treatment of nongynecologic cancer. PMID- 12370613 TI - Breast cancer: introduction. PMID- 12370614 TI - Mammography: screening and diagnostic. PMID- 12370615 TI - Breast mass evaluation. PMID- 12370616 TI - Breast cancer and masses in women 22 years of age and younger. PMID- 12370617 TI - Breast cancer: adaptation of fine-needle aspiration to office practice. PMID- 12370618 TI - Treatment of invasive breast carcinoma. PMID- 12370619 TI - Diagnosis and treatment of invasive breast cancer during pregnancy and lactation. PMID- 12370620 TI - In situ breast carcinoma: diagnosis and treatment. PMID- 12370621 TI - Breast cancer prevention and surveillance. PMID- 12370622 TI - Lung and bronchus cancer in women: a 21st-century epidemic. PMID- 12370623 TI - Role of the obstetrician-gynecologist in reducing the incidence of and death rate from colorectal cancer. PMID- 12370624 TI - Non-Hodgkin's lymphomas. PMID- 12370625 TI - Cutaneous malignant melanoma. PMID- 12370626 TI - Urinary bladder cancer. PMID- 12370627 TI - Pancreatic cancer: evidence-based diagnosis and treatment. PMID- 12370628 TI - Leukemias. PMID- 12370629 TI - Thyroid cancer. PMID- 12370630 TI - Cancer of the kidney. PMID- 12370631 TI - Gastric cancer. PMID- 12370632 TI - Brain neoplasms in women: a review. PMID- 12370633 TI - Multiple myeloma. PMID- 12370634 TI - Liver and hepatic duct cancer. PMID- 12370635 TI - Mouth cancer. PMID- 12370636 TI - Endoscopically assisted transconjunctival approach in orbital medial wall fractures. AB - Full exposure of the medial orbital wall for fracture repair poses difficulty with traditional approaches except coronal incision, especially in cases of wide fracture. The endoscopic-assisted approach with limited incision has been introduced. The authors used the endoscopically assisted transconjunctival approach in 21 cases: 15 isolated medial orbital wall fractures and 6 cases of concomitant floor fractures. Through the medial transconjunctival slit incision, repair of the fracture using calvarial bone graft was undertaken with the aid of an endoscope. All patients recovered without any eye symptoms including clinically notable enophthalmos, but one immediate revisional operation was needed because of a displaced bone graft. Otherwise, the desired position of the graft was confirmed via computed tomography. The endoscopically assisted transconjunctival approach to the orbital medial wall provides improved surgical exposure of the most posterior and superior aspects of the fracture site, enabling more accurate reduction of orbital soft tissue and placement of bone grafts. PMID- 12370637 TI - Bilateral side finger transposition flaps in the treatment of chronic postburn flexion contractures of the fingers. AB - The authors present a surgical method of releasing postburn flexion contracture of the finger by two separate transverse incisions and covering the skin defects by transposing two random-pattern flaps from both sides of the finger. One of the proximally based flaps was transposed from one side of the proximal phalanx and the other flap was transposed from the opposite side of the middle phalanx. Because the flaps were raised from different sides of different phalanxes, the donor sites were closed primarily. A total of 37 fingers (14 hands, 11 patients) were treated with this method. The patients were all men aged 20 to 24 years old. The mean follow-up period was 9 months. The lack of extension of the proximal interphalangeal joints of the fingers was improved by approximately 45 deg using the described method. The authors conclude that this method can be used effectively in the treatment of mild to moderate postburn flexion contractures of the fingers. PMID- 12370638 TI - Closed-catheter irrigation is as effective as open drainage for treatment of pyogenic flexor tenosynovitis. AB - Surgical treatment of pyogenic flexor tenosynovitis traditionally consists of open drainage (OD) and irrigation of the flexor tendon sheath. An alternative, less traumatic method of closed-catheter irrigation (CCI) has been advocated, but no comparative studies have been reported. The authors reviewed 47 cases of pyogenic flexor tenosynovitis to determine whether a difference in outcomes exists between these two methods. OD was used in 32 patients and CCI was used in 15 patients. Complications appeared to be more common in the OD group (N = 9) compared with the CCI group (N = 3), but this difference was not significant. This study supports the use of CCI as the preferred treatment for pyogenic flexor tenosynovitis because it provides thorough mechanical tendon sheath irrigation and causes smaller wounds with less scarring, which may offer a more rapid return to function. PMID- 12370639 TI - Arterial grafts in microscope-assisted pedal bypass for limb salvage. AB - Leg amputations are associated with marked morbidity and mortality in the atherosclerotic population. The survival in patients undergoing lower extremity amputations is 50% at 3 years and 30% at 5 years. Despite excellent reported results with pedal bypass, some patients are "nonbypassable" with traditional techniques because of very small, short target vessels in the pedal arch. The authors report six cases of microscope-assisted inframalleolar bypass using autogenous artery in 5 patients presenting with threatened limb loss, with 83% graft patency at 52 months average follow-up. They hypothesize that the success in these patients is the result of less surgical trauma and less compliance mismatch at the distal anastomosis, and perhaps the result of vasoactive substances secreted by the arterial grafts. Microscope-assisted pedal bypass with autogenous artery should be considered for "nonbypassable" patients with tissue necrosis or rest pain who do not appear to have sufficient inflow to heal a forefoot amputation. PMID- 12370640 TI - Microscopic and immunohistochemical analysis of the skin changes of free forearm flaps in intraoral reconstruction. AB - In the literature, few studies based on clinical and histological evaluation analyze skin structural changes after transplantation to the oral cavity. Ten patients affected by squamous cell carcinoma of the oral cavity who were reconstructed with a free forearm flap were included in the current study to analyze skin alterations. The authors performed a histological and ultrastructural evaluation of skin samples from the free forearm flap before transplantation and 18 months after intraoral reconstruction. They analyzed cytokeratin and involucrin distribution, which represent markers of maturation and differentiation of epithelia. The aim of this study was to demonstrate whether skin "mucosalization" occurs. They found that the skin undergoes some morphological changes induced by the intraoral environment. Cytokeratin and involucrin distribution is mostly unchanged. This aspect is in favor of skin structure preservation. Thus, they found that "mucosalization" of the skin is not evident after 18 months. PMID- 12370641 TI - Retrospective study of the management of chemotherapeutic extravasation injury. AB - Despite the now widespread experience with the administration of chemotherapeutic agents in oncology, extravasation injuries still occur. Furthermore, the most appropriate management of such injuries is not known. The authors examined the current treatment options for extravasation injury and the incidence of this problem. All cases of extravasation referred to the plastic surgery service at one institution from 1994 through 1996 were examined. During a 6-year period there were 44 cases of extravasation injury identified in 42 patients. Comparison with a previous study conducted 15 years before at the same institution revealed a significant reduction in the incidence of extravasation injuries during that time (0.01% vs. 0.1%; = 0.00). The site of extravasation was peripheral in 32 cases and central in 12. Paclitaxel and doxorubicin were the two most common drugs involved. The local infusion of antidotes was not performed routinely. Only 26 of the 42 patients were referred to the plastic surgery service for care. Only 10 of those 26 patients required local ulcer excision and closure to achieve a healed wound. The mean time between injury and referral was 40 days. This time did not predict the subsequent need for a surgical procedure. Most patients, including the remaining 16 referred to the plastic surgery service, did not require surgical intervention. All were watched expectantly, and their injuries healed spontaneously. In conclusion, the incidence of extravasation is decreasing, most likely as a result of the diligence in the administration and identification of extravasation injuries as well as the result of the use of more central infusion sites. Most cases can be managed conservatively, with directed surgical treatment of the ulceration when appropriate. PMID- 12370642 TI - Reversed sural island flap supplied by the lower septocutaneous perforator of the peroneal artery. AB - The current study was conducted to document the vascular anatomy of the distally based superficial sural artery flap and to study the vascular anastomoses between the superficial sural artery and the septocutaneous perforator of the peroneal artery. Five fresh human cadavers were injected with lead oxide, gelatin, and water. Ten legs were then dissected and an overall map of the cutaneous vasculature by source vessel was constructed. Vascular communication between the superficial sural artery and the lowest septocutaneous perforator of the peroneal artery was evaluated to determine the cutaneous vascular territory of the superficial sural flap. This anatomic information enhances our understanding of flap design. The authors' clinical experience with the usefulness of the distally based island flap as a method of reconstruction in the lower leg and foot in a series of 26 patients is presented. PMID- 12370643 TI - Neovaginal reconstruction with the modified McIndoe technique: a review of 32 cases. AB - The authors reviewed 32 patients who underwent vaginal reconstruction using a modified McIndoe procedure during the past 15 years. This technique consists of the application of split-thickness skin grafts into a new cavity created between the rectum, bladder, and urethra. The grafts are placed previously on a mold of Optosil, which is a silicon-based condensation curing impression material used by dentists. The mold is kept for 3 months 24 hours each day. During the next 3 to 4 weeks it is applied 12 hours per day. Later, according to sexual activity, the mold can be removed completely. In case of no sexual activity it should be used 1 hour per week. Parameters assessed during the follow-up were mold management, grade of pseudomucinous metaplasia of the skin grafts, sensation of the neovagina, neovagina size changes, sexual satisfaction, and complications. Postoperative complications included partial take of skin grafts (N = 3), postoperative anxiety (N = 2), donor site cheloids (N = 1), and neovaginal stricture in 3 patients who used the mold for 1 month only without having any further sexual activity. Patients who managed the mold correctly or who had constant sexual activity obtained satisfactory dimensions of the neovagina in terms of length, diameter, and elasticity. PMID- 12370644 TI - Primary cutaneous mucormycosis: guide to surgical management. AB - Mucormycosis is the most acute, fulminate, and fatal of all fungal infections in humans. It presents most frequently in immunocompromised patients, but can occur in healthy patients in the presence of often-insignificant trauma. Surgical management of primary cutaneous mucormycosis is almost always required. Case reports of surgical treatment for primary cutaneous mucormycosis are reported in the literature; however, the extent of debridement required for cure is unclear and no uniform plan of treatment has been suggested. To date, no clinical guidelines exist to assist the clinician in the surgical management of this disease. This article reviews the literature, reports on two clinical cases, and submits clinical guidelines designed to assist the clinician in the surgical management of primary cutaneous mucormycosis. Because of the infrequent and potentially fatal nature of the diagnosis, a high index of suspicion and a low threshold for wound biopsy must be maintained. Wound cultures are grossly inadequate and should not be relied on for a false sense of security. It is recommended that, for the early diagnosis of cutaneous mucormycosis, chemotherapy and surgical debridement of grossly necrotic tissue be performed at the earliest possible time. The debrided wound is monitored for the resolution of surrounding erythema and induration before definitive reconstruction. In the case of delayed diagnosis and/or advanced or rapidly progressive disease, surgical debridement of all involved tissue, in addition to chemotherapy, is warranted. PMID- 12370645 TI - Paraffin oil injection in the body: an obsolete and destructive procedure. AB - Injection of foreign materials, such as paraffin oil, is an old and obsolete procedure. The authors describe previous uses for this procedure that had been used since the 19th century and the treatment of patients affected by such a disease. PMID- 12370646 TI - Conventional versus epineural sleeve neurorrhaphy technique: functional and histomorphometric analysis. AB - Three methods of nerve repair involving the epineural sleeve technique were compared with conventional nerve repair using the rat sciatic nerve transection model in four groups. In group 1, the sciatic nerve was repaired using the conventional epineural technique by placing four sutures. In groups 2, 3, and 4, the epineural sleeve technique was combined with two sutures, fibrin glue, and two sutures with fibrin glue, respectively. Functional recovery was evaluated using walking track analysis, limb circumference, and the severity of toe contracture. Diameter of the sciatic nerve fibers, total number of myelinated fibers, diameter of the myelin sheath, and the axon-to-fiber diameter ratio were measured at 12 weeks. The results showed better functional recovery as well as a higher number of myelinated fibers in groups using the epineural sleeve technique compared with conventional technique ( < 0.05). The addition of fibrin glue, however, did not make any significant difference. The epineural sleeve technique was found to be superior when compared with conventional nerve repair, providing faster functional recovery and improved nerve regeneration. PMID- 12370647 TI - Effect of cryopreservation on patency and histological changes of arterial isogeneic and allogeneic grafts in the rat model. AB - Vascular grafting is used frequently in the management of length discrepancies between blood vessels. Cryopreservation permits vascular graft storage and aids availability; however, long-term patency of cryopreserved arterial allografts is not well established. Fifty Fisher and 55 Wistar rats were used in the study. Thirty-eight cryopreserved Fisher femoral arterial grafts were transplanted into the femoral arteries of 15 Fisher (cryoisografts) and 23 Wistar rats (cryoallografts). Thirty-two fresh Fisher arterial grafts were implanted into 32 Wistar femoral arteries (fresh allografts). The animals were killed at 1, 4, and 8 months in each group, and graft patency was assessed. One-month graft patency was 100% in all groups. At 4 months, graft patencies were 86%, 100%, and 75% in the cryoisografts, cryoallografts, and fresh allografts respectively. All cryoisografts and fresh allografts were patent, whereas all the cryoallografts were occluded at 8 months ( < 0.01). Cryopreserved rat arterial allografts offered satisfactory graft patency up to 4 months after implantation and may therefore be applicable clinically in selected cases. PMID- 12370648 TI - Pyoderma gangrenosum after reduction mammaplasty in an otherwise healthy patient. AB - Pyoderma gangrenosum (PG) is a rare postoperative complication of plastic surgery of the breast. Initial signs and symptoms resemble those of infection, and antimicrobial therapy is usually initiated and fails before considering PG as a diagnosis. Therapy consists of immune modulators, and use of corticosteroids is frequent, as is local wound care. Sufficiently small wounds are allowed to heal secondarily, but larger wounds require coverage with either skin grafts or flaps. Long-term (1 year or more) postoperative surveillance is necessary because late failure of the graft or flap can occur. PMID- 12370649 TI - Arteriovenous malformation involving the thumb and hand: radical excision and reconstruction of multiple components. AB - Arteriovenous malformation of the fingers is not an uncommon presentation in daily practice. The lesions may or may not become clinically evident from birth to adulthood. Treatment of the arteriovenous malformation includes conservative treatment, selective embolization, partial excision, and radical excision. However, recurrence, repeat operations, and even deformity requiring amputation are common problems. The excision is difficult because it is easy to damage the nutrient vessels of the digit, and ischemia or necrosis develop subsequently. Embolization and partial excision are prone to recurrence as well. Radical excision and flap reconstruction are beneficial for some patients, as demonstrated by the authors. In the treatment of digital arteriovenous malformation, it is always important to maintain a balance of cure, function, and aesthetic result. PMID- 12370650 TI - Correction of blepharoptosis in oculopharyngeal muscular dystrophy. AB - Oculopharyngeal muscular dystrophy is a hereditary, autosomal dominant, slowly progressive disorder with onset that occurs during middle age. Major symptoms are ptosis and dysphagia resulting primarily from selectively involved levator palpebrae and the pharyngeal muscles. Progressive, usually symmetrical blepharoptosis, with or without dysphagia, appears during middle age. Muscular weakness in the limbs can be noted in some patients. The guidelines for surgery in myopathic ptosis are conservative in view of the increased risk of postoperative corneal complications. However, orbicularis function remains intact in oculopharyngeal muscular dystrophy; therefore, corrective surgery is performed in most patients. This report describes four cases of ptosis correction in patients with oculopharyngeal muscular dystrophy in one family. The frontalis action was very poor to qualify for frontalis transfer; therefore, the authors performed moderate to large levator resection in all patients. The follow-up results 5 years postoperatively are promising to date and all the patients are satisfied with the results. PMID- 12370652 TI - Technique to re-establish continuity of the recipient artery after end-to-end anastomoses in cross-leg free flap procedure. AB - In this report, a simple technique is described to restore the continuity of the recipient artery in cross-leg free flap procedure after end-to-end anastomoses. In the first stage, the latissimus dorsi flap was revascularized by end-to-end anastomosis between the posterior tibial artery of the noninjured leg and the thoracodorsal artery of the flap. After 4 weeks of neovascularization period, in the second stage when the pedicle was to be divided, the thoracodorsal artery was dissected until its bifurcation in the muscle, transected, and rerouted to the distal ligated end of the posterior tibial artery, accomplishing a simple end-to end anastomosis between these two vessel ends. Thus, the continuity of the recipient artery was re-accomplished by replacing the normally discarded segment of the flap's arterial pedicle as an arterial graft, the patency of which was demonstrated clinically and by Doppler examination. The authors believe that this technique provides further benefit to the patient by accomplishing vascular flow through a major lower limb artery when the reconstructive pathway involves an end to-end anastomosis in a cross-leg free flap procedure. PMID- 12370651 TI - Hard palate mucosal grafts for defects of the nail bed. AB - The authors present 2 children in whom a hard palate mucosal graft was used for a defect of the nail bed after resecting subungual exostosis. After the tumor was resected with the overriding nail bed, hard palate mucosa without periosteum was grafted to the nail bed defect. In both patients the graft took completely, and within 2 weeks after the operation the patients were able to enjoy activities of daily life, including athletic movement, without any symptoms. Nail growth was uneventful and was complete in 4 or 5 months after the operation without any complications. The authors think that a hard palate mucosal graft is a valid choice for a defect of the nail bed, and the mucosa does not need to be harvested with periosteum. The grafting of hard palate mucosa without periosteum to a defect of the nail bed contributes to a shorter healing time, resulting in a reduction in the period of restriction of movement in activities of daily life, and this is a great advantage in children. PMID- 12370653 TI - Four parallel flaps for correction of a wide forehead defect. AB - A simple method for closure of a defect in the forehead area by four parallel flaps along the forehead wrinkle lines is illustrated. The main advantage of this method lies in its simple design, which enables the surgeon to close large defects with no elevation of the eyebrow, while preserving the hairline, and most of the suture lines are parallel to the forehead wrinkle lines. PMID- 12370655 TI - The professional portfolio. PMID- 12370654 TI - Role of physicians and patients in the diagnostic delay of cutaneous malignant melanoma. AB - The incidence of cutaneous malignant melanoma is increasing constantly. The most accurate prognostic factor of primary melanoma is thickness of the lesion according to Breslow. Information and screening campaigns for early diagnosis of melanoma are based on the assumption that tumor thickness is the consequence of a delay in diagnosis. However, the correlation of delay in diagnosis with prognosis remains controversial. In this report, the authors investigated the role of the physician and the patients in diagnostic delay in melanoma and areas in which improvement is needed. The reduction of the time to diagnosis in specific population groups may improve the prognosis of melanoma. PMID- 12370656 TI - K-wire: a simple and safe method for internal stabilization of costal cartilage in L-strut grafts. PMID- 12370657 TI - The current status and future outlook of intestinal transplantation. AB - Recently, the clinical reality of intestinal, combined liver-intestinal, and multivisceral transplantation qualified the procedure by the American Health Care Financing Administration (HCFA) as the standard of care for patients with irreversible intestinal failure. The decision was supported by a decade of clinical experience with cumulative improvement in survival. Prior to tacrolimus, the worldwide experience was plagued with uncontrolled rejection, graft versus host disease (GVHD), and fatal infection. These undefeated barriers stemmed from the large gut lymphoid mass and heavy microbial load contained in the intestinal lumen with the absence of an effective immunosuppressive and antimicrobial therapy. With the emerge of small bowel and multivisceral transplantation in 1990, multiple factors, in addition to the clinical introduction of tacrolimus, have sustained and increased these efforts including evolution in surgical techniques and improvements in postoperative care. The most valuable achievement, however, has been the effective control of rejection and treatment of life threatening opportunistic infections. This chapter outlines the common current practice, surgical techniques and postoperative management of the three different types of intestinal transplantation. In addition, new strategies to overcome some of the current immunologic and biologic challenges are defined with the aim of raising the level of such a creative surgery to be a better therapy for TPN dependent patients. PMID- 12370658 TI - The molecular genetics of pancreatic ductal carcinoma. AB - Pancreatic ductal carcinoma remains the 4th leading cause of cancer death in both men and women in the United States, with an overall 5-year survival of less than 3%. Over the last decade, significant advances have been made in our understanding of the molecular biology of pancreatic ductal carcinoma, with pancreatic cancer now considered one of the better characterized neoplasms at the genetic level. The advances in the understanding of the molecular genetics of pancreatic cancer initially focused on events that occur in the development and early genetic progression of the disease. This progression has been associated with the accumulation of multiple genetic alterations in various cancer-causing genes, leading to the development of a histological and genetic progression model. In the model, pancreatic cancer develops from non-invasive intraepithelial precursor lesions termed pancreatic intraepithelial neoplasias, with each progressive stage associated with accumulated mutations in oncogenes, tumor suppressor genes, and mismatch repair genes. Other aspects of the development of pancreatic ductal carcinoma, such as tumor invasion, tumor-stromal interaction, metastasis, and chemotherapeutic resistance are more poorly understood. Recent studies utilizing global gene expression methodologies have provided insight into some of these processes and have allowed for the development of potential tumor markers which could be used for early detection and diagnosis of this difficult disease. In order to improve the survival of patients with pancreatic carcinoma, we need to better understand the fundamental changes that occur in pancreatic ductal carcinoma. The following article reviews the genetic mutations and syndromes known to be associated with pancreatic ductal carcinoma as well as recent advances in the study of global gene expression. PMID- 12370659 TI - Liver transplantation for hepatocellular carcinoma. AB - Hepatocellular carcinoma (HCC) is one of the commonest malignancies worldwide, and accounts for more than 1 million deaths annually. Identification of tumors early in the course of disease appears to be important for treatment, yet remains difficult to accomplish. Without treatment the prognosis is dismal with a median survival of 6-9 months. Partial hepatic resection is generally accepted as the treatment of choice for HCC with reported survival rates of up to 50% at 5 years. Unfortunately poor underlying liver function as well as tumor number or location preclude traditional hepatic resection in many cases. Total hepatectomy with transplantation (LT) has been advocated such cases, but the results have been variable. LT offers the advantage of radical tumor removal even in patients with multifocal disease or severe cirrhosis. Additionally, LT removes the possibility of metachronous lesions developing in the liver remnant and restores normal liver function. The critical limitation to advocating LT as primary oncotherapy in patients with HCC is the severe shortage of donor livers. Until organ availability improves, transplantation for HCC can only be offered to patients whose survival is predicted to be similar to that in patients transplanted for benign disease. This report reviews the current role and indications for liver transplantation as therapy for hepatocellular carcinoma. PMID- 12370660 TI - [The role of apoptosis in hepatic graft rejection]. AB - In 1965, Kerr described a type of death, apoptosis, with different characteristics from necrosis. Apoptosis has an important role in the development and cell homeostasis. Excessive or insufficient apoptosis contributes to the pathogenesis of pathology like ischemia, neurodegeneration, autoimmunity, viral infection, and tumor growth or regression. Apoptosis is subdivided into four sequential phases: order of death; death of cell; phagocytosis of apoptotic bodies and degradation of apoptotic bodies. Death programs converge on sequential activation of a proteases family, caspases. Some aspects of graft rejection can be interpreted as failure of apoptosis in host immunity cells; sometimes rejection involves induction of apoptosis. Apoptotic-type lesions were found in early vascular occlusions, one of the cause of graft failure. Then, an augmented apoptosis in hepatic graft biopsy can be used like a signal of early vascular occlusion. In hepatic transplantation, apoptosis is followed by a proteolytic cascade, which causes sequential activation of caspases. Synthetic inhibitor of caspases can be used, then, in the prevention and/or treatment of pathologies with implication of apoptosis due to ischemia-reperfusion. These inhibitors are not enough for prevention of hepatic lesions, even if caspases inhibitor can be a strategy for treatment of hepatic graft rejection. PMID- 12370661 TI - [Cytoreductive surgery and intraperitoneal hyperthermic-antiblastic therapy (HAPP) in peritoneal carcinomatosis]. AB - Peritoneal carcinosis often occurs during the evolution of many neoplasias either abdominal or extra-abdominal. The free time survival of the patients affected by carcinosis is poor (about 6 months) as regards gastric and colorectal cancer. In the last ten-year period a combined surgical technique aiming at the total removal of parietal and visceral peritoneal lesions (peritonectomy) and at the perfusion of peritoneal cavity with chemo-drugs in hyper-thermia had developed. This method is based on the presence of the peritoneal-plasmatic barrier that holds back high molecular weight drugs, keeping from passing at the systemic circulation; in this way it is possible to use higher and more concentrate chemo drug doses in a very limited area than in the systemic chemotherapy. The association between chemotherapy and hyperthermia produces a synergic effect: hyperthermia, infarct, makes chemo-drugs more effective and selective, improving their capability of penetration in tumoral masses; heat has furthermore an intrinsic anti-neoplastic action, being altered the reparation mechanisms of the tumoral cells. A WEB research on Medline site has been conducted choosing especially those articles referable at the 1999-2000 period. The selected articles have been briefly analysed in the "Clinical experience" section. Authors' experiences have been divided, as far as possible, on the basis of the tumors treatable with cytoreduction and HAPP: 1) Colon-rectal Cancer; 2) Pseudomyxoma peritonei/Appendicular Adenocarcinoma; 3) Gastric Cancer; 4) Ovarian Cancer; 5) Peritoneal Mesothelioma. The determinant variables in the analysis of the results are basically three: 1) Selection of the patients, 2) Characteristics of the surgical operation, 3) Characteristics of the HAPP. Colon-rectal carcinoma: the survival time of the recurrent disease, obtained by some authors like P.H. Sugarbaker, is surely remarkable (50% at 5 years in the patients where a complete cytoreduction was possible to perform), but the result obtained in patients affected by peritoneal carcinosis, subjected to this technique at the first clinical presentation (100% at 5 years) is much more interesting. For this type of neoplasia, it is important to underline that not all the authors report the same results, with a median survival time lower than that of the American author. Pseudomyxoma peritonei: about this rare neoplasia, the "golden standard" treatment consists on cytoreduction of all visceral and peritoneal macroscopical lesions, with a homogeneous distribution of the data obtained by most authors: median survival time included among 70% and 90% at 5 years. Gastric cancer: there is a considerable difference between the data of Japanese authors and others. In the treatment of peritoneal carcinosis the results are, on average, rather poor; better results have been obtained using this technique as an adjuvant presidium for the prevention of the onset of peritoneal carcinosis. Ovarian cancer: the results about the recurrent ovarian cancer are good; in the future, it will be useful to start a phase III study to render effective the use of this technique in the ovarian cancer at the beginning of its clinical story. Peritoneal mesothelioma: till now, a standardized alternative, approaching this neoplasia, does not exist; the results are encouraging, with good median and free time survival. PMID- 12370662 TI - [Video-assisted surgery in oncology. From diagnostic applications to therapeutic possibilities]. AB - The growing use of laparoscopic techniques in general surgery over the last decade has also involved the oncological sector where it is not only used for diagnostic purposes but also for therapeutic procedures, unfortunately in the latter instance, with controversial results owing to its indiscriminate use. Where there are no contraindications, the mini-invasive approach has undoubtedly become the gold standard for the treatment of benign pathologies, such as biliary lithiasis or gastroesophageal reflux. However, video-assisted surgery is not yet sufficiently radical, and in some cases lacks the feasibility to legitimate its unqualified use in oncological practice as a valid alternative to open surgery. In the case of malignant tumours its role is hampered by major drawbacks which restrict its use to carefully selected patients. The persistent lack of randomised clinical trials in many cases still exposes the comparison between traditional laparotomy and mini-invasive procedures open to the risk of subjective opinion, unless backed up by immediate clinical evidence. It is certainly a very exciting field of surgery and this review aims to offer an extremely concise panorama of the application of these methods to oncological pathologies affecting individual organs. PMID- 12370663 TI - Reduction pneumoplasty for severe emphysema. Does the debate await a neat sentence? AB - The aim of this study is to review the literature regarding reduction pneumoplasty (RP) or lung volume reduction surgery in order to assess the state of the art of this topic. Reduction pneumoplasty is a palliative surgical therapy that is offered to selected patients with severe non-bullous emphysema not responding to maximized medical therapy. The use of staple excision or plication of the most destroyed target areas of the lung appeared to be more effective than laser ablation. Currently, a one-stage bilateral procedure is the standard of care although a unilateral reduction can be preferable in patients with asymmetric emphysema and/or if a staged bilateral treatment strategy is planned. Randomized studies have suggested that RP is superior to medical therapy including respiratory rehabilitation for improving subjective dyspnea, exercise capacity, respiratory function and quality of life for up to 1 year. In addition, few long-term studies have suggested that the improvements obtained with RP can be maintained for several years in properly selected patients. Although several issues still await a definitive answer, the available literature data and our current experience have clearly indicated that RP works well and is a safe and effective procedure for palliating symptoms and improving respiratory function in severely disabled emphysematous patients. PMID- 12370664 TI - [Techniques and outcomes of laparoscopic surgery in gastroesophageal reflux disease]. AB - BACKGROUND: The purpose of this study is to report personal experience in laparoscopic antireflux surgery and to analyze the clinical and functional outcomes of this procedure, also in relation to the different techniques used. METHODS: From 1996 to 2000, 20 patients with gastroesophageal reflux disease associated with hiatal hernia underwent laparoscopic surgery. The indication for surgery was failure of long-term medical therapy. All patients had severe acid reflux on 24 hrs-pH monitoring, endoscopic evidence of esophagitis and hiatal hernia, and defective lower esophageal sphincter. A Nissen fundoplication was performed in 13 patients with normal esophageal body motility, and a 270 degrees posterior fundoplication in seven patients with low esophageal motility. RESULTS: Mortality and conversion rate were 0. Mean operative time was 135 min and mean postoperative hospital stay 5 days. Operative morbidity was 15%. All the patients were completely cured of reflux symptoms; transient mild postoperative dysphagia occurred in two patients (10%). There was a significantly improvement of the results in postoperative esophageal manometry and 24 hrs-pH monitoring. CONCLUSIONS: This preliminary experience suggests that laparoscopic surgery represents a safe and effective procedure for the treatment of gastroesophageal reflux disease. Precise selection of patients and adequate surgical technique are essential. PMID- 12370665 TI - [Superextended lymphadenectomy (D4) in the treatment of gastric adenocarcinoma]. AB - BACKGROUND: The incidence of paraaortic lymph node metastasis (N4) in relation with the site of the tumour, and survival in patients with gastric cancer who underwent gastric resection and superextended lymphadenectomy (D4), have been analyzed. METHODS: The frequency of paraaortic lymph node metastasis was studied in 132 patients who underwent gastrectomy with D4 lymphadenectomy during the period June 1988 - December 2000. Six patients with plastic linitis and 3 with carcinoma of the gastric stump were excluded from the analysis. RESULTS: In personal experience the most frequent postoperative morbidity were respiratory complication (7.6%) and pancreatic fistula (6.8%). Among the 132 patients the total number of dissected nodes was 6362 and the mean number of dissected nodes per case was 48.2. The total number of retrieved lymph nodes from the paraaortic station was 755 with a mean number 5.7 per patients. N4 nodal involvement was found in 25 (19%) of 132 patients: 14 (36%) patients with carcinoma located in the proximal third, 5 (13%) with tumour located in the middle third and 6 (11%) with carcinoma of the distal third of the stomach. The median survival time and the overall cumulative 5-year survival rate for curatively (R0) resected patients were 74 months and 52% respectively. CONCLUSIONS: The presence of metastasis in paraaortic lymph nodes in 19% of our patients, the low morbidity and mortality, the good survival after superextended lymphadenectomy, suggest that this lymphadenectomy should be considered in the curative surgical treatment of advanced gastric cancer, especially if located in the proximal third of the stomach (N4 in 36% of cases). PMID- 12370666 TI - [Helicobacter pylori and gastric cancer. The incidence of infection in personal experience]. AB - BACKGROUND: Helicobacter pylori is an identified carcinogen for gastric cancer, but the underlying mechanisms remain to be defined. The aim of this study is to analyze the incidence of Hp infection in our series of patients with gastric carcinoma. METHODS: Between 1988 and 1998, 60 patients with diagnosis of gastric adenocarcinoma underwent partial or total gastrectomy. Forty-one were males and 19 females with an average age of 62 years (range 36-79). Twenty-seven cancers (45%) were localized in the lower third of the stomach, 17 (28%) in the middle third and eight (13%) in the upper third or cardias. In six patients (10%) the tumor was multicentric, while a recurrence on gastric stump after subtotal gastrectomy was present in two cases (3%). According to Lauren's criteria 39 cancers (65%) were of intestinal type, 16 (27%) of diffuse type and five (8%) of mixed type. The histologic preparations have been re-examined in order to verify the presence or not of Hp on gastric mucosa around neoplasm. RESULTS: Hp was found in 35 (58%) of the analyzed specimens and therefore a significant percentage of patients was Hp-positive at the time of diagnosis and surgery. Between 35 Hp-positive samples, 24 were adenocarcinomas of intestinal type, nine of diffuse type and two of mixed type, with a prevalence of Hp in intestinal type cancer. CONCLUSIONS: This study confirmed the high incidence of Hp infection in patients with gastric carcinoma, particularly in those with intestinal type cancer. PMID- 12370667 TI - [The use of surgical endo-prosthesis in the treatment of choledocolithiasis in the elderly. Personal experience]. AB - BACKGROUND: The usefulness of biliary endoprostheses in some selected elderly patients affected by hepato-choledocolithiasis is described. METHODS: In the Department of Surgery, University of Perugia, 119 elderly patients were surgically treated for choledocholithiasis from January 1999 to December 2000. In 44 selected cases#151;oldest patients with thinner hypovascularized ectasic choledochal wall#151; a permanent transpapillary polyurethane prosthesis was placed, after choledocolithotomy with or without sphinterotomy; sometimes prosthesis was placed under the duodenal mucosa. RESULTS: Endoprosthesis had a long duration and gave good results in canalization and periprosthetic flow, in absence of biliary stasis and/or angiocholitis. Only one patient had prostheses displacement. CONCLUSIONS: On the basis of personal experience and considering physiopathology and pathogenesis of biliary stones in the elderly, the authors underline, in selected cases, the need of stenting the hepatocholedochal lumen with the aim of avoiding collapse. PMID- 12370668 TI - Analysis of pathogenetic mechanisms of common bile duct iatrogenic lesion during laparoscopic cholecystectomy. A review of the literature. AB - BACKGROUND: Laparoscopic cholecystectomy as the new gold standard for gallstone treatment has reopened the chapter of complications due to cholecystectomy. METHODS: The present work refers to the period between 1988-2001 and analyses 277,121 cholecystectomies, carried out in some European and extra-European Countries. 1,353 CBD lesions with an incidence rate of 0.48%, that almost doubles that of the laparotomic cholecystectomies, were found. Transection of CBD within <2 cm of bifurcation was the most frequent lesion. RESULTS: The mechanism and extension of CBD lesions is different when compared to that of laparotomic intervention. Actual damage is caused by a failure to recognise the anatomical structures, and also by errors in the surgical technique, learning curve and different visualisation of the operative field. CONCLUSIONS: Once the lesion has been diagnosed it is always necessary to define its type and extension accurately. A therapeutic approach that fails to take this aspect into account will expose the patient to repeated and increasingly complex surgical interventions, recurrent cholangitis episodes and a higher risk of suffering a secondary biliary cirrhosis. PMID- 12370669 TI - [Long-term results of endoscopic treatment of biliary stenosis from laparoscopic cholecystectomy]. AB - BACKGROUND: The outcome of endoscopic biliary stent insertion for postoperative bile duct stenosis was retrospectively evaluated. METHODS: Fifty-seven patients with biliary stenosis from laparoscopic cholecystectomy were included from February 1992 to January 2000. One to three stents were inserted for an average of 12.4 months, with stent exchange every three months to avoid cholangitis caused by obstruction. RESULTS: Successful stent insertion was achieved in 43/57 (75.4%) patients. Stent insertion failed in 10 patients with complete and four patients with incomplete biliary obstruction. Early complications occurred in four patients. Late complications occurred in 5/43 patients. Five patients experienced recurrence of stenosis. CONCLUSIONS: Endoscopic treatment should be the initial management of choice for postoperative bile duct stetiosis. PMID- 12370672 TI - [Renal stone ileus in xanthogranulomatous pyelonephritis. A case report]. AB - A peculiar case of intestinal occlusion caused by a renal stone in a patient with nephroduodenal fistula due to previous xanthogranulomatous pyelonephritis is reported. Only few cases of nephroduodenal fistula are described in the literature, generally as a single case report or in small series. A nephroduodenal fistula as a result of chronic renal inflammatory disease such as xanthogranulomatous pyelonephritis, is usually associated with renal stones, recurrent urinary tract infections or endocrine disorders. Finally, renal stone as a cause of ileus is an event rarely described in the literature. In the case described, a correct preoperative diagnosis was possible with computerized tomography. During the operation a big renal stone was found and removed from the small bowel, but a limited resection was necessary because of the vascular impairment of the tract. At 8-month follow-up from operation, the patient was in good health, and no symptoms of renal or intestinal diseases were found. PMID- 12370670 TI - [Gynaecological pelvic mass. Emergency clinical assessment]. AB - BACKGROUND: The authors report the gynaecological pathology of surgical interest occurring in an Emergency Department in the first half of 2000 and occasionally found during an operation in women who presented a pelvic mass or abdominal pain. METHODS: Six women, average age of 50 years, reached the Emergency Department: five of them were operated with an emergency procedure. RESULTS: Of the 6 cases, 1 patient was affected by uterine mass, 1 by uterine-ovarian mass and 3 by ovarian mass; in one patient, affected by ovarian tumour, another intervention was necessary for intestinal metastasis. CONCLUSIONS: The authors have considered the clinical aspect of the abdominal masses, the age of the patients, the diagnostic laboratory and instrumental implications, the surgical approach and the histological result, referring to literature. Their contribution is related to the need for an appropriate surgical approach in emergency where it may, sometimes, solve or, at least, stabilize an uncertain clinical case, thanks to the implicit basic knowledge of multidisciplinary surgical technique. PMID- 12370671 TI - [Endoscopic treatment of intestinal anastomotic leakage in low anterior resection of the rectum by using fibrin adhesive. Our experience]. AB - BACKGROUND: Personal experience about treatment of anastomotic leakage in low anterior resection of the rectum by using human fibrin adhesive "Tissucol" is reported. METHODS: Eight cases of anastomotic leakage treated with using human fibrin adhesive "Tissucol", are analyzed in a retrospective study. Patients had three/six months-one year follow up. Treatment with human fibrin adhesive "Tissucol" was performed in our Endoscopic ambulatory. Six cases had either an immediate resolution or an ambulatorial follow-up; in 2 cases only, general complications forced to a prolonged hospital stay. The study concerns 58 patients subjected to low anterior resection of the rectum and endoscopic treatment of 8/58 patients with anastomotic leakage. Fistulas were sealed with human fibrin adhesive "Tissucol" by using flexible endoscope. Anastomotic leakage identification leakage was made and low anterior resection of the rectum and sealing with human fibrin adhesive "Tissucol" were performed. RESULTS: Complete sealing of fistula and rectum patent. CONCLUSIONS: The excellent results obtained with this non invasive and fast treatment, easily practicable even in ambulatorial regimen, lead the authors to consider it effective and as first choice treatment of this dangerous complication. The cost/benefit ratio is favorable if compared with the long hospital stay required for other treatments, which also present loaded high morbidity and mortality. PMID- 12370673 TI - [Primary adenocarcinoma of the appendix. Case report and review of the literature]. AB - A rare case of primary adenocarcinoma of the vermiform appendix (less than 250 cases described in the literature) in a 36 year-old female patient presenting signs and symptoms of an acute appendicitis is reported. Adeno-carcinoma of the vermiform appendix is a rare neoplasm of the gastrointestinal tract with an incidence of about 0,01-0,2%. Usually the diagnosis of adenocarcinoma of the vermiform appendix is difficult because symptoms and signs are not pathognomonic. In the case described, the diagnosis was intra and postoperative and confirmed by the pathological examination of the surgical specimens. At laparotomy, performed under suspicion of an acute appendicitis, disseminated disease was discovered, characterized by the involvement of the two ovaries, the left colon, the cecum, the vermiform appendix, with a peritoneal carcinosis and a hydroureteronephrosis. According to the dissemination of the disease, the surgical treatment was right hemicolectomy, anterior resection of left colon, bilateral oophorectomy and omentectomy. The post-operative course was regular. Adjuvant therapy was performed for 6 cycles, with 5FU and oxaliplatinum. The patient is still alive after 6 months and there is no sign of progression of the disease. A mild left hydroureteronephrosis is persistent. PMID- 12370674 TI - [Adenomas of the nipple]. AB - A case of adenoma of the nipple is reported. Although this is a comparatively rare condition, knowledge of it is extremely important because clinically it simulates Paget's disease. A failure to recognise the pathology could therefore lead to mistaken diagnosis and consequent surgery which would create serious damage to the patient. According to almost all authors, it is a benign pathology and can therefore be treated with an intervention limited to removing the complete tumour. This for two reasons: the first is that if the tumour grows it can become painful, create a sense of tension and bleed in the erosive phase; the second, more important, is that malignant degeneration has been hypothesised. For these reasons it is essential to examine the other breast immediately and follow the patient over time to prevent unpleasant surprises. PMID- 12370675 TI - [Bochdaleck diaphragmatic hernia in adults. Case report]. AB - The case of a 27-year-old woman, admitted to our surgical ward with symptoms of epigastric-ache, postmeal vomiting and significant weight loss, is reported. Clinical and radiographic suspicion of mesenterium commune, with duodenal compression due to bands, requested an explorative laparatomy that confirmed the mesenterium commune presence with left caecum and colon adhesion and left Bochdaleck hernia, which is rare in adult age. PMID- 12370676 TI - [Use of the "flat mesh" T4r in the Trabucco inguinal hernioplasty. Technical note]. AB - Many of the surgical techniques proposed over the years for inguinal hernia repair have been associated with a high number of recurrences due to the presence of great tension on the suture line and to a lack of consideration for the alteration of the collagen metabolism at the fascia trasversalis level. The advent of the new "tension-free" techniques, among which that described by Trabucco, has represented a turning point in inguinal hernia surgery. In this article, the characteristics, indications and use of the T4r "flat mesh" in this hernioplasty are described. The T4r is not a real "plug" but a "flat mesh", a 5 cm-diameter-round pre-shaped polypropylene mesh with an intermediate rigidity grade with a 1 cm diameter hole in an eccentric position for the passage of the elements of the spermatic funicle. To make its collocation inside the deep inguinal ring in the preperitoneal position easier, a Foley catheter (14 Ch) is used whose balloon is inflated with 20-30 cc of physiologic solution or air. One of the actual problems among the possible complications of prosthetic surgery of hernia is the "migration" of the plug and thus the use of "plugs" in the Trabucco inguinal hernioplasty has been reconsidered. The positioning of the T4r in place of a three-dimensional plug like T1 in particular is an elective choice to prevent the risk of compression of the loco-regional vascular structures. PMID- 12370678 TI - Minerva Anestesiologica today. PMID- 12370677 TI - [Computer-based preoperative planning for breast reconstruction in the woman with unilateral breast hypoplasia]. AB - BACKGROUND: Aim of this paper is to describe a computer program which can provide objective and quantitative data useful for the selection of the proper implant in order to obtain the symmetry with the contralateral breast in case of unilateral breast reconstruction by tissue expansion, especially for the surgeon without experience or for the occasional operator. METHODS: Our C++ program provides the final implant volume using the measurements of the semi-circumference and projection of the contralateral breast performed on the supine patient. The aim is the symmetry of the two breasts. RESULTS: According to our experience in breast reconstruction by tissue expanders, this program allows non invasive and simple measurements of the breast volume, useful to obtain the mammary symmetry. CONCLUSIONS: In case of breast reconstruction by tissue expansion, the preoperative evaluation is usually based on the surgeon's experience and on empirical observations without knowing the correct volume to reach. For this reason our program is useful to know the necessary volume for breast reconstruction, and therefore it allows the surgeon to obtain a better plastic result. PMID- 12370679 TI - Open Lung in ARDS. PMID- 12370680 TI - The use of remifentanil for bloodless surgical field during vertebral disc resection. AB - BACKGROUND: A short hospital stay is nowadays desirable and affordable for a wide range of surgical pathology, respecting safety of care and home discharge. In the present study, the Authors investigated the use of TIVA with propofol/remifentanil during microsurgical vertebral disc resection to maintain a controlled vascular hypotension for bloodless surgical field aiming to reduce the operating time and consequently recovery room length of stay and morbility related to anaesthesia. METHODS: The study took place in a 300 bed Orthopaedics hospital over a period of 3 months and 50 ASA I-II patients were enrolled in this trial; further data are presented for comparison of 50 ASA I-II patients homogeneous for age and sex to the studied population, operated under a standard TIVA with propofol and boluses of fentanyl. Duration of anaesthesia and surgery, time for awakening after cessation of TIVA, incidence of postoperative nausea and vomiting (PONV), amount and quality of postoperative analgesia, length of stay in the recovery room are reported in statistical presentation. RESULTS: Time of surgery and anaesthesia were reduced in the remifentanil group compared with the fentanyl group, thanks to an easily reachable and durable state of controlled hypotension in the first group without the use of any other drug. The recovery profile was shorter in the remifentanil group the drug being rapidly metabolised by plasma cholinesterase. CONCLUSIONS: No difference occurred between the two groups regarding quality and amount of postoperative analgesia, while PONV presented more in the fentanyl group and shivering more in the remifentanil group. PMID- 12370681 TI - Effects of remifentanil infusion bis-titrated on early recovery for obese outpatients undergoing laparoscopic cholecystectomy. AB - BACKGROUND: In obese patients functional residual capacity comes down with a possible hypoxemia in postoperative period. In fact many studies has been begun to determine optimum ventilation regulation and the best position for these patients, but the question has not been solved. As remifentanil can reduce of 50% the inhalatory anaesthetic request and reverse Trendelemburg position is extremely useful for these patients, we hypothesized that use of a continuous remifentanil infusion during balanced anaesthesia with sevoflurane, BIS-titrated, associated to reverse Trendelem-burg position could facilitate emergence from anaesthesia in obese patients undergoing laparascopic cholecystectomy. METHODS: We studied 40 patients, ASA II class, with higher than 30 kg/m2 body mass index, undergoing to laparoscopic cholecystectomy. All the patients, in operating room, received standard monitoring and BIS sensor application. All the data were continuously collected. Induction of anaesthesia has been with a refracted bolus in 120 sec of remifentanil 1 mg/kg, followed by propofol 1.5 mg/kg and cisatracurium 0.15 mg/kg. Maintenance of anaesthesia has been by balanced anaesthesia with continuous remifentanil infusion, ventilating patients with sevoflurane in oxygen and air. Patients were randomized into two homogenous groups. Into the control group has been varied sevoflurane inspiratory concentration on the ground of BIS value (from 0.3% to 3%), while into remifentanil group remifentanil infusion has been varied (from 0.25 to 2 mg/kg/min) to maintain medium pressure values which don't stray more than 25% from basal values, on the ground of BIS values. On pre-established times of operation, respiratory mechanics and blood gases were examined. RESULTS: As it was to expect, sevoflurane concentration variations resulted very high in control group compared to remifentanil group. Awakening time, extubation, orientation and transfer to PACU (postanaesthesia care unit) resulted significantly lower than remifentanil group. CONCLUSIONS: Concluding, remifentanil infusion, BIS-titrated, facilitates awakening times from balanced anaesthesia with Sevoflurane in obese patients, submitted to laparoscopic cholecystectomy. PMID- 12370682 TI - [Assessment of operating theatre procedures and quality of perioperative anaesthesia using a computer system for collecting anaesthesiological data]. AB - BACKGROUND: In order to manage the operating theatre and guarantee constant improvement in the quality of procedures, it is indispensable to be able to have available a computerised workstation to enable the team to collect data and analyse them. METHODS: We used our anaesthesia department's computer workstation to retrospectively analyse operating sessions over a 16 month period during which organisational adjustments were made to optimise patient turnover and maximise the utilisation of operating theatres. Meantime we studied and compared the trend in anaesthesiological quality through the use of certain structural, process and outcome indicators. RESULTS: During the period under study an improvement was noted in the efficiency of operating theatre procedures (reduction in entry time of the first patient into the theatre, time between one case and the next and subutilisation of the operating theatre, p<0.05). As regards data on the quality of anaesthesiological assistance to the patient, no significant differences were noted when comparing the four periods examined. CONCLUSIONS: To make the operating complex work more efficiently, it is necessary to have available a computer system that makes it possible to continuously monitor theatre operation. The improvement initiatives undertaken led to a more effective utilisation of the operating theatre but did not produce a significant increase in productivity. In spite of the increased work load, analysis of the trend in quality indicators as regards anaesthesiological procedures did not reveal any modification. PMID- 12370683 TI - General anesthesia with spontaneous ventilation without intubation for short-stay operations. AB - BACKGROUND: The choice of the anesthetic technique plays a decisive role in the use of operating protocols in day hospital or short-stay given that full compliance with surgical needs must be associated with rapid recovery and the patient's renewed autonomy leading to discharge in total safety. METHODS: The use of spontaneous ventilation general anesthesia without intubation is proposed for all operations not requiring muscular paralysis and where the patient's conditions are compatible. The authors describe the technique used and its utilisation in over 4,000 patients undergoing orthopedic surgery of the leg. RESULTS: Using rapid clearance drugs with minimum metabolic involvement this anesthetic technique allows a full postoperative recovery within a short time and with no immediate or long-term sequelae. CONCLUSIONS: It can be used for short stay or day-case surgery when concomitant with adequate surgical requirements. PMID- 12370684 TI - Post-thoracotomy analgesia: epidural vs intravenous morphine continuous infusion. AB - BACKGROUND: We compared thoracic morphine epidural analgesia (TEA) and I.V. analgesia (IVA) with morphine, in respect to the time to extubation, the quality of postoperative analgesia, side effects, complications, postoperative hospital length of stay in patients having thoracotomy lung resection. METHODS: We prospectively studied 563 consecutive patients, undergoing thoracotomy (lobectomy, bilobectomy or pneumonectomy), randomized in two groups: TEA 286 patients and IVA 277 patients. In the epidural group, before the induction of anesthesia, continuous infusion of 15 mg of morphine in 250 mL of normal saline at 5 mL/h was started. In the IVA group a continuous infusion of 30 mg of morphine associated with 180 mg ketorolac in 250 mL of normal saline at 5 mL/h was started before the induction of anesthesia. The pain degree was evaluated on an analogic scale by Keele modified at 1 (end of anesthesia) 6, 12, 24, and 48 postoperative hours, at rest and after movements. Data obtained were analysed by means of the analysis of variance for repeated measures. RESULTS: The time from the end of surgery to tracheal extubation was similar in both groups. Significantly lower numeric verbal pain scores at rest and after movements were found in the epidural group (p<0.001). Postop complications, nausea and vomiting were higher in the IVA group (p<0.05). Postoperative mean hospital length of stay was 9+/-4 days in TEA and 11+/-4 in the IVA group (p<0.05). CONCLUSIONS: In our study the epidural root was superior in terms of analgesia, side effects, length of stay and postoperative complications after thoracotomy. PMID- 12370685 TI - Pain control during diagnostic and/or therapeutic procedures in children. AB - Children's fear of pain in illness has been greatly underestimated. In cancer disease, pain characterizes most of the diagnostic and long-term therapeutic procedures. Children's psycho-emotional processing during illness has been recently recognized as characterized by loss of control, anger, and fear, to which pain adds fear of death jeopardizing the sense of survival. Illness and pain provoke a traumatic condition affecting psycho-emotional maturation. The aim of the pharmacologic and psychologic support is to help children better cope with pain and prevent the healed child having traumatic effects in his/her future personality structure and behavior. To assure the best pain control, support has to be offered right from the first intrusive procedure in order to avoid anticipatory anxiety. Respiration, relaxation, visualization, desensibilization through the "switch technique" and the "magic glove", distraction and involvement, muscular relaxation all have the common aim of focusing the child's mind and attention away from body perception of pain connected to the procedure. Different medical methods are utilized depending on the child's age and the level of consciousness required for the procedure. Local anaesthesia and conscious sedation are used. Children are particularly sensitive in this critical condition and need as serene and comfortable an environment as possible. Relief of pain and suffering in cancer treatments that cause further discomfort to the patient is a fundamental ethics in the treatment of clinical neoplasias. PMID- 12370687 TI - Emergency care management. PMID- 12370686 TI - [Cornea donation: role of the local coordinator in monitoring and implementation]. AB - BACKGROUND: In order to contribute to the dissemination of an organ and tissue donation culture, the authors report the results of a retrospective investigation into the donation of corneas for transplantation relative to the period from January 1997 to December 2001 at a leading hospital in the Milan area. METHODS: All cases of intra-hospital death (n=2137) were considered. The adoption of a selection protocol for potential donors, in accordance with the indications of the North Italy Transplant program, and constant monitoring on the part of the local coordinator, led to the collection of 348 corneal flaps using the procedure whereby all deaths have to be notified to the Health Department. RESULTS: Over this period, the donors/deaths ratio increased from 2.1 to 17.6%, while the collection index (real donors/potential donors including those wrongly excluded) increased from 40.8% in 1999-2000 to 67.8% in 2001. Most involved in the donor selection activity were the intensive care centres with an increase for Resuscitation of 12.7% and for First Aid of 33%, to a lesser extent non-intensive centres (increase of 11.2%). The causes of exclusion from cornea collection in the last three-year period were clinical contraindications in 51.2% of cases (sepsis, blood transfusions, diseases of the central nervous system of unknown aetiology), failure to activate the procedures in 17.4% of cases, opposition in 17%, unsuitability of the corneal tissues in 11.3%, inadequate age in 3%. CONCLUSIONS: The results obtained indicate good prospects for technical implementation and for the development of a donation culture in respect of other organs and tissues. PMID- 12370689 TI - Risk factors associated with acute lower extremity ischemia after coronary revascularization. AB - Acute lower extremity ischemia (ALEI) is a recognized complication of coronary revascularization that can lead to emergent lower extremity revascularization, amputation, and death. Patients with correctable coronary artery disease have a high incidence of lower extremity arterial occlusive disease (AOD). But, despite the known high correlation between AOD and coronary artery disease, the status of the lower extremity vasculature in patients undergoing coronary revascularization may be overlooked until the lower extremity becomes profoundly ischemic. Data from a retrospective review of 35,000 coronary revascularization procedures identified 55 patients who developed ALEI, subsequent to their cardiac procedures. Risk factors for ALEI included femoral artery instrumentation, previous coronary revascularization, hemodynamic instability, and documented AOD. Means of identifying patients at risk for ALEI are discussed. PMID- 12370690 TI - Endovascular stent-graft repair of descending thoracic aortic aneurysms: the nursing implications for care. AB - Endovascular repair of descending thoracic aortic aneurysms is a minimally invasive procedure performed with the patient under epidural or spinal anesthesia as an alternative to the conventional left thoracotomy repair. A Dacron graft, similar to the one used in the conventional repair, is placed in the thoracic aorta with fluoroscopic guidance via the femoral or iliac artery. Once the graft is in place, the aneurysm is excluded from the general circulation, thereby preventing rupture. Endovascular repair is currently being offered at selected sites to patients who otherwise would not be candidates for surgical repair due to severe comorbidities such as cardiac, pulmonary, or renal disease. As both the technique and the devices become perfected, endovascular stent-graft repair of descending thoracic aortic aneurysms will most likely be offered as a method of treatment in both high- and low-risk patients who are anatomic candidates for the procedure. This article describes the conventional repair and the endovascular repair of descending thoracic aneurysms. It discusses the implications for nursing care in the preoperative and postoperative settings and defines guidelines for the long-term follow-up of patients who undergo endovascular repair. PMID- 12370691 TI - An exploration of health beliefs and attitudes of smokers with vascular disease who participate in or decline a smoking cessation program. AB - The descriptive cross-sectional aspect of this study compared a group of smokers with peripheral arterial disease who chose to participate in a smoking intervention with a group of smokers with peripheral arterial disease who declined to participate. The longitudinal aspect of this study used Ajzen and Fishbein's(1) Theory of Reasoned Action, Keeney's(2) Expected Utility Decision Theory, and Prochaska and DiClemente's(3) Transtheoretical Model of Change to describe the influence of this smoking cessation program on the beliefs and attitudes about smoking in group 1. Smokers completed a smoking beliefs questionnaire with vascular disease at baseline and after 13 weeks of smoking cessation intervention. Smokers who did not want to participate in the smoking cessation program also completed this questionnaire for comparison. Statistically significant differences were found to differentiate people who enrolled in the smoking cessation program from those who did not. Subjects in group 2 smoked less per day, were less educated, were less often diagnosed as having peripheral arterial disease, were found to be more in the precontemplation stage of change in smoking cessation, cared more about what their physician and family thought they should do, and perceived themselves to be at less risk for developing more severe circulatory problems if they did not quit smoking. After 13 weeks, participants in both groups 1 and 2 were found to smoke significantly less per day. No support was found for the expectation that the smoking intervention would influence stage of change in smoking behavior or attitudes and beliefs about the risks of smoking to the participants' health after 13 weeks. PMID- 12370692 TI - Supermarket model for vascular disease care. AB - A supermarket model for vascular patient care proposes an interdisciplinary group of health care teams such as vascular nurses, interventional radiologists, vascular surgeons, angiologists, internists, cardiologists, and neurologists and facilities such as diagnostic testing laboratories, subcenters such as wound care and foot care centers, atherosclerotic risk prevention centers, rehabilitation centers, vein centers, and socioeconomic follow-up centers that would provide health care of vascular disease in a comprehensive manner in terms of quality care, convenience for patients, 1-stop shopping, education and training, and research and development. PMID- 12370693 TI - A clinical practice update--implications for vascular nursing. PMID- 12370694 TI - Geriatric best practices in nursing: helping the patient feel valued. PMID- 12370695 TI - NTP Technical Report on the metabolism, toxicity and predicted carcinogenicity of diazoaminobenzene (CAS No. 136-35-6). AB - Diazoaminobenzene is used as an intermediate, complexing agent, and polymer additive. It is also an impurity in certain color additives used in cosmetics, food products, and pharmaceuticals. Diazoaminobenzene was selected for metabolism and toxicity studies based on the potential for worker exposure from its use in laboratories, positive Salmonella typhimurium gene mutation data, its presence as an impurity in foods and cosmetics, and the lack of adequate toxicity data. Several structural analogues and presumed metabolites of diazoaminobenzene are carcinogenic, providing evidence for the possible carcinogenicity of diazoaminobenzene. The chemical structure of diazoaminobenzene suggested that it would be metabolized into aniline and benzene; therefore, metabolism and disposition studies were performed in male and female F344/N rats and male B6C3F1 mice administered a single oral, dermal, or intravenous dose of diazoaminobenzene. Electron spin resonance (ESR) studies were conducted to assess the possible formation of a phenyl radical from the reduction of diazoaminobenzene by components of the cytochrome P450 mixed-function oxidase (P450) system in microsomes or by gut microflora in anaerobic cecal incubations. Bile duct-cannulated male F344/N rats were administered diazoaminobenzene and 5,5 dimethyl-1- pyrroline-N-oxide (DMPO) for in vivo determination of the DMPO-phenyl radical. 16-Day toxicity studies were performed to identify target organs of diazoaminobenzene following dermal application to male and female F344/N rats and B6C3F1 mice. In the disposition and metabolism studies, oral doses of 20 mg/kg to male and female rats and male mice were readily absorbed and excreted mainly in the urine, with exhalation of volatile organics accounting for about 1% of the dose. The only volatile metabolite detected in the breath was benzene, and all the metabolites in the urine were those previously shown to result from the metabolism of benzene and aniline in rats and mice. While dermal doses to rats and mice (2 and 20 mg/cm2) were only slightly absorbed, benzene and aniline metabolites were nonetheless detected in the urine. High circulating levels of benzene, aniline, and their metabolites were detected in the blood of rats administered 20 mg/kg diazoaminobenzene as early as 15 minutes after exposure. At 24 hours after dosing, diazoaminobenzene was detected at low levels (<1%) in the adipose tissue, blood, kidney, liver, muscle, skin, and spleen. Metabolites of benzene and aniline were also formed in an in vitro study using human liver slices. In the ESR spin-trapping experiments, the ESR spectrum of the DMPO-phenyl radical was detected when diazoaminobenzene was incubated with microsomes or P450 reductase, DMPO, and NADPH, or when incubated with cecal contents and DMPO. The DMPO-phenyl radical spectrum was not attenuated by the P450 inhibitor, 1 aminobenzotriazole, or carbon monoxide suggesting that P450s were not required. In in vivo experiments in which rats were administered diazoaminobenzene and DMPO, the DMPO-phenyl radical adduct was detected in bile within 1 hour after treatment. In the 16-day toxicity studies, groups of five male and five female F344/N rats and B6C3F1 mice received dermal applications of 0, 12.5, 25, 50, 100, or 200 mg diazoaminobenzene/kg body weight. Animals were evaluated for absolute and relative organ weights, for hematological effects, and for gross and microscopic lesions. No mortality occurred in rats. However, most male mice exposed to concentrations of 50 mg/kg or greater and female mice exposed to 200 mg/kg died. Body weights of male and female rats and female mice were less than those of the vehicle controls. Similar chemical-related toxicities were observed in both species. Clinical pathology data indicated a chemical-related methemoglobinemia and Heinz body formation in male and female rats and mice. Analysis of organ weights indicated possible chemical-related effects in the thymus, heart, spleen, kidney, and liver of rats and/or mice. Increases in the incidences of several skin lesionseral skin lesions, including hyperplasia of the epidermis and hair follicles, and inflammation in rats and mice and ulceration in female mice were observed. Other nonneoplastic lesions that were considered to be related to diazoaminobenzene administration were atrophy of the thymus, mandibular and/or mesenteric lymph nodes, and white pulp of the spleen, as well as splenic hematopoietic cell proliferation in rats and mice. In mice, there were increased incidences of atrial thrombosis, and necrosis was observed in the renal tubules and liver. Diazoaminobenzene was mutagenic in S. typhimurium strains TA98, TA100, and TA1537 with induced rat or hamster liver S9 enzymes; no activity was noted in strain TA1535, with or without S9. In vivo, two gavage administrations of either diazoaminobenzene or benzene induced highly significant increases in micronucleated polychromatic erythrocytes in bone marrow of male B6C3F1 mice at all doses tested. Diazoaminobenzene is metabolized to the known carcinogens benzene and aniline. Further evidence of this metabolism is that some toxic effects associated with aniline (methemoglobinemia) and benzene (atrophy of the lymphoid tissue) were identified. Based on these results, it is predicted that diazoaminobenzene is a carcinogen. PMID- 12370696 TI - Reflections of a health policy advocate: the natural extension of nursing activities. PMID- 12370697 TI - Caregivers' descriptions of sleep changes and depressive symptoms. AB - PURPOSE/OBJECTIVES: To describe caregiver sleep and depression using caregiver narratives. To compare qualitative descriptions with quantitative scores. DESIGN: Descriptive, one-time, open-ended interview followed by structured sleep and depression questions. SETTING: Interview conducted in person or via telephone at caregiver's preference. SAMPLE: 47 caregivers of patients with advanced stage cancer. Caregivers had a mean age of 54 years, and most were female (81%), Caucasian (82%), and spouses (61%). They provided care for a mean of 24 months. Patients' diagnoses were lung cancer (36%), colorectal cancer (13%), or recurrences (51%). METHODS: Two cancer care sites in southern California provided participants. After consent, the researcher conducted interviews. The Pittsburgh Sleep Quality Index (PSQI) and Center for Epidemiological Studies-Depression (CES D) instruments were administered following interviews. MAIN RESEARCH VARIABLES: Sleep pattern changes and depression levels over time as defined by caregivers. FINDINGS: Caregivers described severe fluctuations in sleep patterns over time and how these changes affected caregiver depressive symptoms. PSQI and CES-D scores matched narrative comments. CONCLUSIONS: Caregivers' narratives suggest they suffer progressive sleep deprivation that affects their emotions and ability to continue as caregivers. IMPLICATIONS FOR NURSING: Nurses must recognize the severe sleep problems experienced by caregivers and respond with interventions to increase sleep quality and decrease depression. PMID- 12370698 TI - Breast cancer lymphedema: pathophysiology and risk reduction guidelines. AB - PURPOSE/OBJECTIVES: To review the normal physiology of the blood capillary interstitial-lymphatic vessel interface, describe the pathophysiology of lymphedema secondary to treatment for breast cancer, and summarize the physiologic bases of the current National Lymphedema Network (NLN) risk reduction guidelines. DATA SOURCES: Journal articles, anatomy and physiology textbooks, published research data, and Web sites. DATA SYNTHESIS: Lymphedema occurring after treatment for breast cancer significantly affects physical, psychological, and sexual functioning. About 28% of breast cancer survivors develop lymphedema. When arterial capillary filtration exceeds lymphatic transport capacity, lymphedema occurs. NLN risk reduction guidelines may decrease lymphedema risk. CONCLUSION: Lymphedema is chronic and disfiguring. Most NLN risk reduction guidelines, although not evidence-based, are based on sound physiologic principles. Evidence-based research of the effectiveness of NLN risk reduction guidelines is indicated. IMPLICATIONS FOR NURSING: Until evidence-based research contradicts NLN's risk reduction guidelines, nurses should inform patients with breast cancer about their risk for lymphedema, guidelines to reduce that risk, and the physiologic rationale for the guidelines. PMID- 12370699 TI - Postmenopausal breast cancer survivors at risk for osteoporosis: physical activity, vigor, and vitality. AB - PURPOSE/OBJECTIVES: To test a multicomponent intervention to prevent and treat osteoporosis in breast cancer survivors. DESIGN: Descriptive, correlational. SETTING: Midwestern urban and rural sites. SAMPLE: 27 postmenopausal breast cancer survivors between the ages of 42-65 who had completed treatment, except for tamoxifen, and were not candidates for hormone replacement therapy. METHODS: Bone mineral density (BMD) of the hip, spine, and forearm was measured using dual energy x-ray absorptiometry. Physical activity was recorded using the Seven-Day Physical Activity Recall-Adapted, which classifies activities as light, moderate, hard, or very hard. Vigor was measured with the eight-item subscale of the Profile of Mood State based on the previous week. Vitality was measured using the four-question subscale of the Medical Outcomes Study 36-Item Short Form Health Survey. MAIN RESEARCH VARIABLES: Physical activity, vigor, vitality, and BMD. FINDINGS: More than half reported no very hard physical activity, and 37% reported no hard activity. The association of vigor with total metabolic equivalents for combined moderate, hard, and very hard activities was significant (r = 0.536, p = 0.007), as were the hours spent in the combined moderate to very hard activities. No relationship was found between vigor, vitality, or any level of activity and BMD. CONCLUSIONS: Survivors reported high levels of perceived vigor and vitality but spent more time engaged in light versus hard or very hard activities. Positive correlations between higher levels of vitality and vigor with metabolic equivalents support the idea that activity promotes perceptions of energy and positive feelings. IMPLICATIONS FOR NURSING: Breast cancer survivors are at risk for osteoporosis. Nurses should be aware of increased risk, recommend screening for bone health, and encourage physical activity. PMID- 12370700 TI - The usefulness of a daily pain management diary for outpatients with cancer related pain. AB - PURPOSE/OBJECTIVES: To describe the usefulness of daily pain management diaries to outpatients with cancer who participated in a randomized clinical trial of the PRO-SELF Pain Control Program. DESIGN: Randomized clinical trial in which a daily pain management diary was used for data collection in the control group and for data collection and nurse coaching regarding the pain management program in the intervention group. SETTING: Seven outpatient oncology settings. SAMPLE: 155 patients with pain from bone metastases and 90 family caregivers. METHODS: Content and statistical analysis of audiotaped answers to a semistructured questionnaire. MAIN RESEARCH VARIABLES: Patients' and family caregivers' perceptions of the usefulness of a daily pain management diary; specific ways in which the diary was used. FINDINGS: Patients in both the intervention (75%) and control groups (73%) found the diary useful. The diary was used to heighten awareness of pain, guide pain management behavior, enhance a sense of control, and facilitate communication. Family caregivers in both groups also reported that the diary was useful. CONCLUSIONS: The completion of a daily pain management diary is useful to patients and family caregivers and may function as an intervention for self-care. IMPLICATIONS FOR NURSING: Research-based evidence supports the importance of using a daily pain management diary in clinical practice. PMID- 12370701 TI - Sources of social support: adolescents with cancer. AB - PURPOSE/OBJECTIVES: To evaluate how a cancer diagnosis affects adolescents' perceived sources of social support, amount of support needed, and level of satisfaction with support compared to an age-matched, healthy, adolescent group. DESIGN: Cross-sectional, comparative, nonrandom survey. SETTING: Summer camp for adolescents with cancer and a rural high school in the southeastern United States. SAMPLE: Adolescents with a diagnosis of cancer (n = 64) and age-matched, healthy adolescents (n = 115). METHODS: Subjects completed the Social Support Questionnaire, Perceived Social Support From Family Scale, Perceived Social Support From Friends Scale, and demographic information forms. MAIN RESEARCH VARIABLES: Sources of social support, amount of support perceived, and level of satisfaction with support. FINDINGS: Adolescents with cancer perceived social support coming from both friends and family and reported high levels of support satisfaction from each source. Compared to healthy adolescents, those with cancer reported similar support sources and satisfaction levels; however, adolescents with cancer perceived parental relationships as more supportive. CONCLUSIONS: Similarities between healthy adolescents and those with cancer regarding social support were more prevalent than differences. The social benefits of camp settings for chronically ill children should be explored further. IMPLICATIONS FOR NURSING: Nurses and other healthcare professionals should allow adolescents in the healthcare setting every opportunity to maintain their social networks of friends and family by encouraging visitation, providing social opportunities in the hospital, and emphasizing the importance of attending school when medically able. PMID- 12370702 TI - Core competencies in cancer genetics for advanced practice oncology nurses. AB - PURPOSE/OBJECTIVES: To determine core competencies in cancer genetics for advanced practice nurses (APNs) in oncology. DESIGN: Survey. SAMPLE: Expert panel of 9 nursing educators or researchers, 9 general genetics experts, 9 genetics experts with specialties in oncology, and 10 oncology APN nurse consumers (N = 37). METHODS: Utilizing the Delphi Technique, two rounds of surveys were conducted. Round 1's survey required open-ended responses to identify skills, attitudes, and competencies specific to cancer genetics. Round 2 requested ranking of the importance of identified competencies. MAIN RESEARCH VARIABLES: Skills, attitudes, and competencies specific to cancer genetics. FINDINGS: Recommended genetics competencies and knowledge for oncology APNs were identified for the categories of direct caregiver (6 items), coordinator (6 items), consultant (7 items), educator (6 items), researcher (8 items), and professional attitudes (16 items). CONCLUSIONS: Identified competencies provide a foundation and direction for development of the education curriculum recommended for all practicing oncology APNs. IMPLICATIONS FOR NURSING: Integrating genetic concepts into clinical practice is essential. Oncology APNs must have an expanded knowledge base in genetics to enable them to incorporate advances in genetics into practice to ensure quality outcomes. Development of genetics education is crucial to ensure future competency. Research that determines the impact of such education is warranted PMID- 12370703 TI - The trajectory of fatigue in adult patients with breast and ovarian cancer receiving chemotherapy. AB - PURPOSE/OBJECTIVES: To describe the trajectory of fatigue and determine the feasibility of exploring physiologic mechanisms of fatigue in adult patients receiving chemotherapy for breast and ovarian cancer. DESIGN: Descriptive, longitudinal, repeated measures. SETTING: Outpatient ambulatory cancer centers within two large, academic, teaching hospitals with overnight hospital stays in general clinical research centers. SAMPLE: Seventeen adult participants with either early-stage breast or ovarian cancer receiving chemotherapy for the first time. METHODS: Demographic questionnaire; Piper Fatigue Scale (PFS); hemoglobin, bilirubin, melatonin, and weight change were measured at baseline, three months, and approximately six months. PFS also was collected at three additional two-week nadir, post-treatment, measurement points. Descriptive statistics and repeated analysis of variance measures were used to analyze data. MAIN RESEARCH VARIABLES: Fatigue, hemoglobin, bilirubin, melatonin, and presence of other comorbid disease. FINDINGS: Subjective fatigue was experienced by the majority of patients receiving chemotherapy. It was irregular over time, intensified at three months, and continued after treatment ended. The physiologic trajectory of fatigue from baseline to three months indicated a significant change over time in hemoglobin in the breast cancer group (p = 0.02) and in nighttime melatonin levels for both breast and ovarian cancer groups (p = 0.03). Although not significant, daytime melatonin levels changed over time from baseline to six months. CONCLUSIONS: Fatigue fluctuates during the course of chemotherapy treatment and does not cease after treatment ends. Preliminary findings suggest that fatigue mechanisms may have an undetermined physiologic basis. IMPLICATIONS FOR NURSING: Assessment of cancer treatment-related fatigue must be ongoing, even after treatment ends. Findings suggest an awareness of the importance of understanding fatigue mechanisms to enable future testing of research-based interventions. PMID- 12370704 TI - Early detection of breast cancer by self-examination: the influence of perceived barriers and health conception. AB - PURPOSE/OBJECTIVES: To discover the factors that influence the decision to perform breast self-examination (BSE). DESIGN: Quantitative, correlational. SETTING: Institutional; urban and suburban. SAMPLE: A nonrandomized convenience sample of 93 women. METHODS: Willing participants were asked to complete by mail a short demographic form, the Reduced Laffrey's Health Conception Scale, Champion's Health Belief Instrument, and a BSE questionnaire. MAIN RESEARCH VARIABLES: Health conception, barriers to BSE, frequency and thoroughness of BSE practice. FINDINGS: A wellness conception of health and frequency were not significantly related, nor did a significant relationship exist between a wellness conception of health and thoroughness of BSE. A negative relationship between barriers and thoroughness was highly significant. A statistically significant relationship did not exist between barriers and frequency of BSE. CONCLUSIONS: Those with a clinical conception of health practiced BSE less frequently. If health is viewed as the absence of disease, BSE may be perceived as looking for trouble. Subjects with greater barriers were less thorough when they practiced BSE. The specific barriers tested in this study (i.e., feelings about practicing BSE, worry about breast cancer, embarrassment, time, unpleasantness of procedure, lack of privacy) interfered more with the thoroughness of the behavior than with the frequency of the behavior. The most reported barrier was worry about breast cancer. The data suggest that worry may interfere with performing BSE thoroughly. IMPLICATIONS FOR NURSING: This work offers insight into the thoughts and behavior of women to promote a behavior that could save their lives. Potential implications for nursing practice could include issues related to better education and assessment of barriers to practicing BSE in women. PMID- 12370705 TI - The determinants of breast cancer screening behavior: a focus group study of women in the United Arab Emirates. AB - PURPOSE/OBJECTIVES: To explore perceptions, knowledge, attitudes, and beliefs about breast cancer and its screening among Emirati national women in Al Ain, United Arab Emirates. DESIGN: A qualitative study using focus group methods. SETTING: Primary healthcare centers and a community-based women's association in the United Arab Emirates. SAMPLE: 41 women, aged 25-45 years. METHODS: Four 90 minute focus group discussions exploring perceptions, knowledge, attitudes, beliefs, and practices regarding breast cancer were audiotaped, transcribed, translated, and analyzed. MAIN RESEARCH VARIABLES: Social and cultural themes related to breast cancer and its screening. FINDINGS: Focus group methodology worked well in this setting. The women's perceptions, knowledge, attitudes, and beliefs regarding cancer and screening, together with aspects of the healthcare system and social milieu, appeared to strongly influence the women's preventive practices. Some of these factors had an encouraging effect on the women's practices, and others had a deterring effect. The encouraging factors included feelings of susceptibility, high levels of knowledge in some women, attitudes and beliefs about personal responsibility for health, and a supportive social milieu. Deterring factors included anxiety and fear leading to denial; lack of knowledge about cancer and the screening program; fear, embarrassment, and mistrust of health care; and belief in predestination. CONCLUSIONS: Health planners and healthcare providers must capitalize on encouraging factors and minimize deterring factors to optimize breast cancer screening practices among these women. IMPLICATIONS FOR NURSING: Identifying and accounting for the factors that encourage or deter women in their breast cancer screening practices will help to optimize screening programs. PMID- 12370706 TI - ONS 2002 environmental scan: a basis for strategic planning. AB - PURPOSE/OBJECTIVES: To analyze information about the environments in which the Oncology Nursing Society (ONS) operates as a basis for strategic planning. DATA SOURCES: Published reports and ONS internal surveys. DATA SYNTHESIS: Analysis of internal and external trends resulted in a list of implications with regard to managing change, avoiding mistakes, and identifying critical issues for ONS leadership. The team presented ONS leaders with a tool that helped to guide the development of the 2003-2006 Strategic Plan. CONCLUSIONS: The continuing vitality of professional nursing societies such as ONS is critical to the vitality of the profession of nursing itself. Monitoring the environment in which these organizations operate--and effectively using the knowledge that is gained- contributes to their long-term viability and growth. A stronger ONS is in a position to better serve its members, who ensure high-quality care to people with cancer. PMID- 12370707 TI - Revising the blueprint for the Oncology Certified Nurse (OCN) examination: a role delineation study. AB - PURPOSE/OBJECTIVES: To conduct a role delineation study of basic oncology nursing practice as a basis for revision of the blueprint for the Oncology Certified Nurse (OCN) examination. DESIGN: Three-phase study of oncology nurses' practice. SAMPLE: 735 oncology nurses randomly chosen from all nurses who are OCN certified. METHODS: A pilot survey was mailed to a small group to allow refinement of the survey instrument. The revised survey then was e-mailed to a total sample of 3,000 OCNs. The results and input from experts on the subject matter were used to revise the test blueprint. MAIN RESEARCH VARIABLES: Frequency and importance of 223 oncology nursing activities previously identified by the group of experts in oncology nursing. FINDINGS: The highest ranked items for the combined frequency and importance scales pertained to the subscales Professional Performance, Patient/Family Education, Comfort, Protective Mechanisms, and Coping. The lowest ranked activities pertained to subscales Research, Detection, Sexuality, and Prevention. CONCLUSIONS: The blueprint for the OCN examination reflects entry-level oncology nursing practice and includes eight domains of practice: Quality of Life (36%), Protective Mechanisms (13%), Gastrointestinal and Urinary Function (10%), Cardiopulmonary Function (8%), Oncologic Emergencies (7%), Scientific Basis for Practice (12%), Health Promotion (3%), and Professional Performance (11%). IMPLICATIONS FOR NURSING: Because oncology nursing is changing, reconfirming and updating the blueprint for the certification examination is necessary. Certification examinations beginning in April 2003 will be based on the revised blueprint. PMID- 12370708 TI - Acute chest syndrome. AB - Acute chest syndrome is an acute pulmonary illness in patients with sickle cell disease. It is a common problem, causing significant morbidity and mortality. Many factors may cause this syndrome. Treatment is primarily supportive. Therapy includes hydration, analgesia, supplemental oxygen, antibiotics, blood transfusion and mechanical ventilation. Early detection and aggressive management may limit its severity and prevent its complications. This article reviews the current information for its definition, frequency, pathogenesis, clinical features, complications, investigations, management and prevention. Recent advances in management of acute and recurrent attacks will be discussed. PMID- 12370709 TI - The insulin resistance syndrome among type II diabetics. AB - OBJECTIVE: The aim of this study is to determine the prevalence of insulin resistance syndrome among type-II Saudi diabetics. METHODS: The study involved type-II Saudi diabetics followed at the Out-patient Clinic of King Abdul-Aziz University Hospital, Jeddah, Kingdom of Saudi Arabia, from January 1997 to December 1998. Their age, sex and body mass index was recorded. Serum samples were analyzed for glucose. Insulin C-peptide level and insulin glucose index was calculated. Serum cholesterol, high density lipoproteins, low density lipoproteins, triglyceride, and uric acid were measured. RESULTS: A total of 109 patients were studied, (67 females and 42 males) with an age range from 28 to 105 years. Median body mass index was 27 in males and 30.2 in females. Percentage of male and female patients with the following abnormalities were as follows: Total cholesterol >5.3 mmol/L (47.6% males, 40.9% females), high density lipoproteins cholesterol <1.2 mmol/L (71.4% males, 40.9% females), low density lipoproteins cholesterol >3.4 mmol/L (42.8% males, 37.9% females), triglyceride >2.3 mmol/L (40.5% males, 31.8% females), insulin >24 mIU/l (23.8% males, 29.7% females), C peptide >1324 pmol/L (21.2% males, 13% females) and hypertension (33.3% males, 43.8% females). Uric acid >420 umol/L was found in 35.5% males and >390 umol/L in 25.6% females. Body mass index >27.8 was found in males (35.7%) and >27.3 in females (67.7%). Insulin resistance as defined by insulin glucose index >5.4 was found in 19.8% of the study group (23.8% males, 15.9% females). Insulin resistance syndrome was found in 16.5% (17.1% males, 15.9% females). CONCLUSION: Insulin resistance syndrome is common among type-II Saudi diabetics. PMID- 12370710 TI - Radioactive iodine in the treatment of Graves' disease. AB - OBJECTIVES: This study was performed to evaluate the efficacy of radioactive iodine 131I therapy of Graves' hyperthyroidism at Sultan Qaboos University Hospital, Oman and to determine the optimal dose of 131I needed to achieve the euthyroid or hypothyroid status. METHODS: The medical records of 366 patients with Graves hyperthyroidism who received a single dose of 131I at Sultan Qaboos University Hospital, Oman between 1991 and 1999 were reviewed. The diagnosis was based on clinical, biochemical grounds and 99mTc thyroid scintigraphy. The patients were followed up for a minimum period of 12 months. For the analysis, the patients were divided into 6 groups according to the 131I dose administered: Dose one (350-399), dose 2 (400-449), dose 3 (450-499), dose 4 (500-549), dose 5 (550-599) and dose 6 (> or = 600) MBq. RESULTS: Fifty-eight percent of all the patients were hypothyroid after 3 months. Three hundred and twenty two patients (88%) were treated by a single dose of 131I in 12 months (85.5% hypothyroid and 2.5% euthyroid). Forty-one patients (11.2%) required a 2nd 131I dose and only 3 patients required 3 doses of 131I. The best cure rate (93%) was observed in group dose 5 (574.0 +/- 16.4 MBq) which however, was not significantly different from other dosage levels. The female to male ratio was 2:1 and the cure rates were not gender or age related. CONCLUSION: Treatment of Graves' hyperthyroidism from a single 131I dose is our aim, rather than avoidance of hypothyroidism. Our results indicate that cure rates are higher with larger doses of 131I except in group dose 6 (special category of patients). In the future, fixed doses would be adopted in our radioactive iodine treatment practice guidelines. As the majority of our patients were hypothyroid at 3 months regular monthly follow-up is essential. Whenever appropriate, physicians are encouraged to consider early referral of Graves' hyperthyroidism patients for radioactive iodine treatment as it is cheap, effective, easy to administer and free from serious side effects. PMID- 12370711 TI - Lipid profile in patients with coronary artery disease. AB - OBJECTIVE: To determine the lipid profile and to identify and stratify risk factors in diabetic and non-diabetic patients with proven coronary artery disease at King Hussein Medical Center, Amman, Jordan. METHODS: One hundred and ninety two patients who were admitted to Queen Alia Heart Institute, Amman, Jordan, proving to have coronary artery disease (CAD) by angiogram, with a mean age of 54 +/- 22 years were studied. Seventy-seven patients were diabetics and 115 non diabetics. Their lipid profiles (T-Cholesterol, high density lipoprotein level (HDL-C), low density lipoprotein level (LDL-C), triglyceride, glucose, glycosylated hemoglobin) and thyroid function test were compared to a control group of 162 individuals with no cardiac events or diabetes, mean age 48.9 +/- 18 years. Prevalence of hyperlipidemia was calculated. Patients with high thyroid stimulating hormone were excluded. RESULTS: The mean (+/- standard deviation) plasma cholesterol for the group with CAD is 231.43 +/- 57.99 mg/dl versus 202.8 +/- 36.58 in the control group (p<0.0003). High density lipoprotein 35.98 +/- 9.37 versus 44.43 +/- 8.34 (p=0.00011). Low density lipoprotein 146.75 +/- 50.93 versus 118.97 +/- 45.9 (p=0.003). Triglyceride level 246.95 +/- 142.1 versus 164 mg/l +/- 93.78 (p=0.0002). Thyroid stimulating hormone level was 1.55 +/- 0.9 versus 1.51 +/- 0.89 ng/l in control group (p=0.35 NS), HbA1c in diabetic group 7 +/- 2.3%. The prevalence of high plasma cholesterol, triglycerides (TG), LDL-C and low HDL-C was 60.9%, 68.3%, 63.5% and 48.4%. Inter-group comparison of patients with CAD (diabetics versus non-diabetics) revealed higher TG level in the diabetic group and statistically significant difference of the HDL and LDL levels between the 2 groups in favor of diabetic group explained by higher percentage of patients on anti-hyperlipidemic drugs than non-diabetics. More females with CAD were found in the diabetic group versus non-diabetic group (16.9% versus 6.1%. z=2.4027 p=0.00820). CONCLUSION: Jordanian patients with CAD have higher cholesterol, LDL-C, Triglyceride and lower HDL-C levels than the control group which comes in accordance of other studies. Hyperlipidemia remains the strongest risk factor for CAD. Diabetic females are at higher risk for CAD versus non-diabetics with the same lipid profile. Aggressive treatment of hyperlipidemia is of paramount importance to reduce the morbidity and mortality of cardiac events in diabetic and non-diabetic patients. PMID- 12370712 TI - Incidence of post cesarean section wound infection in a tertiary hospital, Riyadh, Saudi Arabia. AB - OBJECTIVE: To measure the rate of wound infection after cesarean section and assess risk factors for such infection. METHODS: A prospective surveillance was conducted at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia, during the period January 2000 through to December 2000. This included a total of 754 cesarean sections. The following risk factors, which were studied included, age, parity, gravida, gestational age, antenatal care, type of cesarean section emergency or elective, previous history of cesarean section, duration of operation, rank of surgeon, use of antibiotics, wound infection, complications and length of stay in the hospital. Post discharge surveillance was carried out 5 10 days later to check for wound infection. RESULTS: The overall wound infection rate was 4.5% (95% confidence interval [CI], 3-6%). In the multivariate analysis, the independent risks factors for wound infection were age of the mother less than 20 years (Odds ratio (OR) = 12.13: 95% CI 1.43-118.50: P = 0.039), the duration of surgery, more than one and a half hour (OR = 23.9: 95% CI 10.36 55.78: P = < .001) and medical complications namely diabetes mellitus (OR = 2.28: 95% CI 1.01-5.05: P = 0.03). There was a statistical significant relationship between wound infection and length of stay (P < 0.001). CONCLUSION: A protocol for prophylactic antibiotics is needed, in addition to a new strategy to reduce the nosocomial infection, in order to decrease the incidence of wound infection after cesarean section. PMID- 12370713 TI - Causative pathogens of severe diarrhea in children. AB - OBJECTIVES: To investigate the enteropathogens in children with diarrhea attending Salmaniya Medical Complex, Bahrain. METHODS: Fecal samples from 805 children up to 15 years were examined for parasites, ova and cysts by direct wet preparation, formol-ether concentration and modified Ziehl-Neelsen stain, during the period November 1998 through to June 2000. Samples were cultured for Salmonella, Shigella, Campylobacter and Enteropathogenic Escherichia coli. Antibiotic sensitivity tests were performed on the relevant clinical isolates by agar disk diffusion method. All stools from children below 3 years of age (653 samples) were processed for adenovirus and rotavirus using a commercially available latex agglutination test (Diarlex). In addition, reverse transcription polymerase chain reaction was performed on 200 randomly selected samples using oligonucleotide primers for Rotavirus A, B and C. RESULTS: Four subjects were found positive for parasites. Eighty-three (10.3%) samples were found positive for Salmonella (46 isolates), Shigella (26 isolates), Campylobacter jejuni (7 isolates), and Enteropathogenic Escherichia coli (4 isolates). Rotavirus was found in 91 (13.9%) samples and 4 samples (0.6%) were found positive for adenovirus. Out of 200 samples examined by reverse transcription-polymerase chain reaction, 73 (36.5%) were positive for group A rotavirus. CONCLUSIONS: Rotavirus type A appeared to be the most common single agent in our pediatric population, followed by the classical bacterial pathogens. Adenovirus and parasites appeared to play a very minor role in diarrhea. Thus, we suggest the introduction of rotavirus diagnostic tests in microbiological examination of diarrheic stools of children below 3 years of age. PMID- 12370714 TI - Experience of liver disease at a University Hospital in Western Saudi Arabia. AB - OBJECTIVE: The aim of this retrospective study is to review the pattern of hepatic diseases seen in our setting at King Abdul-Aziz University Hospital, Jeddah, Kingdom of Saudi Arabia and to assist us in appreciating the hepatic prototype in our region. METHODS: Our study consisted of 246 consecutive liver biopsies. Lesions were studied considering histological type, age and gender of the patients and anatomic location. Distribution of hepatic lesions were classified into 4 categories as 1. Chronic inflammatory lesions, 2. Cirrhotic lesions, 3. Neoplastic hepatic lesions and 4. Pediatric and hereditary hepatic lesions. RESULTS: Chronic inflammatory lesions comprised of 123 (50%) cases (mean age 38.1), most commonly reported being chronic hepatitis 82 cases (33.3%, mean age 42). Among these patients with chronic hepatitis, 59 cases were positive for hepatitis C virus (HCV), 21 cases were positive for hepatitis B virus (HBV) and 2 cases had both HCV and HBV. Twenty cases were diagnosed with cirrhosis (mean age 38.2). Among these cirrhotic lesions 16 cases were positive for HCV, 4 cases were positive for HBV. Neoplastic lesions were mostly malignant and comprised of 41 (15.5%) cases (mean age 44.7), with only one benign lesion diagnosed as benign hemangioma (age 48 years). Among malignant lesions, the majority were metastatic lesions, 18 were adenocarcinoma metastasis, all with primary from the gastrointestinal tract (mean age 46.6) and 12 with lymphomatous metastasis (mean age 41.2). There were 10 cases of hepatocellular carcinoma (mean age 43). Pediatric hepatic lesions comprised of 35 (14.2%) cases with the most common lesion being extrahepatic bile duct obstruction in 6 cases (mean age 2 months). CONCLUSION: Chronic active hepatitis was the most common inflammatory lesion, metastatic carcinoma was the most common neoplastic lesion and extrahepatic bile duct obstruction was the most common pediatric lesion of the liver. PMID- 12370715 TI - Spasmocanulase in irritable bowel syndrome. AB - OBJECTIVES: Irritable bowel syndrome is a functional gastro intestinal disorder, with various symptomatology and difficult to treat using several medications. Spasmocanulase, which has an anti-spasmodic and anti-flatulence effect and contains several ingredients, was tried in patients with irritable bowel syndrome, who had been on other medications previously without improvement. METHODS: At the gastroenterology out-patient clinic, Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia, 21 patients who were diagnosed with irritable bowel syndrome for more than 2 years on treatment and did not benefit from these medications, received spasmocanulase one tablet 3 times a day and followed in the gastroenterology out-patient clinic, every 6 weeks for 6 months. Their previous medications were discontinued which, had been used for different durations and in different combinations included Mebeverine, Colpermin, Normacol, Importal, Librax. The main symptoms were different types of abdominal pain, bloating, flatulence, diarrhea, constipation or both. RESULTS: There was improvement or disappearance of the symptoms in more than 50% of the patients who were not improved on previous medication used for more than 2 years. The overall improvement in symptoms ranged between 43-75% in these patients, when followed up in the clinic at 6 weeks and 3 months. Spasmocanulase caused improvement in abdominal pain, flatulence and bloating compared to their previous medications. Only 43% of patients with diarrhea, showed improvement on spasmocanulase. CONCLUSION: Although the number of patients, we studied is small, this study has shown that spasmocanulase is beneficial in improving symptoms of irritable bowel syndrome. PMID- 12370716 TI - Congenital malformation of the gastrointestinal tract in Aseer region, Saudi Arabia. AB - OBJECTIVE: The aim of this prospective study was to evaluate the prevalence and pattern of congenital malformations of the gastrointestinal tract among the Saudi newborn population in Aseer region, Kingdom of Saudi Arabia. METHODS: Every consecutive newborn admitted to the neonatal intensive care unit of Aseer Central Hospital, Kingdom of Saudi Arabia with features of gastrointestinal tract anomaly during the period January 1995 to December 2000 had relevant data obtained and entered into a program form. RESULTS: During the 6 year period, a total of 1386 Saudi infants were admitted into the neonatal intensive care unit of Aseer Central Hospital. Of these, 12.4% were confirmed to have congenital malformation of the gastrointestinal tract; male/female ratio of 1.7:1. The total number of live births by Saudi mothers in Aseer region during the period was 128,093, giving an incidence rate of 1.3 per 1000 live births. The 172 newborns presented with 174 anomalies of the gastrointestinal tract. The leading malformations were imperforate anus (78 cases or 44.8%), tracheosophageal fistula/atresia (42 cases or 24.1%) and intestinal atresia (37 cases or 21.3%). Other lesions included Hirschsprung's disease (14 cases or 8%) and stenosis (2 pyloric and one duodenal) (1.7%). Some patients had more than one defect within the tract (1%) and multisystemic defects (23%). The overall fatality rate was (12%), due largely to post-operative infection (75% of cases) and multiple anomalies (25% of cases). CONCLUSION: The prevalence of congenital defect of the gastrointestinal tract in Aseer region appears to be high. The incidence of associated multisystemic anomalies is also high. Fatality incidence is influenced by post-operative sepsis and associated multiple defects. A high incidence of consanguineous marriage in the region may be the underlying etiological factor hence genetic counseling may be helpful. PMID- 12370717 TI - The outcome and analysis of 40 cases of fetal gastroschisis. AB - OBJECTIVES: This collaborative retrospective study was undertaken at Royal Medical Services Hospital, Amman, Jordan, between 1993 through to 2000. Its purpose was to assess the difference in terms of morbidity and mortality in neonates with gastroschisis delivered by cesarean section versus vaginal delivery. METHODS: The records of all neonates born with gastroschisis (n=40, 26 females, 14 males) from 1993-2000 were analyzed. The mode of delivery was noted. Those babies delivered by cesarean section (n=22) were labelled group one, while those delivered vaginally (n=18) were labelled group 2. The mean maternal age was 26.45 +/- 5.97 and 28.30 +/- 4.22 years for groups one and 2 (P<0.05). Statistical analysis was carried out using standard deviation (SD), Fisher's exact test and Mann-Whitney U test. RESULTS: The mean gestational age at diagnosis was 17.59 +/- 1.58 for group one, and 17.95 +/- 1.44 weeks for group 2 (P>0.05). The mean gestational age at cesarean section was 36.04 +/- 1.02, and for vaginal delivery, 38.40 +/- 1.10 (P>0.05). The mean maternal age was 26.45 +/ 5.97 for group one, and 28.30 +/- 4.22 years for group 2 (P<0.05). The mean birth weight was 2.39 +/- 0.39 kg for group one, and 3.10 +/- 0.20 for group 2 (P<0.05). Surgical repair was immediate after delivery and the mean neonatal age was 41.04 +/- 6.40 minutes for both groups. The mean days of stay at hospital were 24.26 +/- 8.75 for group one, and 34.20 +/- 7.30 days for group 2 (P<0.05). CONCLUSION: Our study demonstrated that complications and morbidity were less in the cesarean section group compared to the vaginal delivery group. Immediate surgery for the neonate, in either group, was performed either by primary or secondary closure. However, a large multicenter, prospective randomised study is needed to ascertain the suitable route for delivery in gastroschisis fetuses. PMID- 12370718 TI - Delays in primary vaccination of infants living in Western Saudi Arabia. AB - OBJECTIVES: Vaccination is one of the most cost-effective means of preventing serious infectious diseases. Several studies from developing and developed countries documented considerable delays in the administration of primary vaccinations. Our objectives were to study the circumstances and contributing factors to such delays in order to design preventative measures. METHODS: Parents of consecutive infants seen during a routine vaccination visit at King Abdul-Aziz University Hospital, Jeddah, Kingdom of Saudi Arabia were included prospectively between September 2000 and February 2001. Structured interviews were performed using a 20-item questionnaire. Vaccinations were considered delayed if they took place 4 or more weeks after the designated time. RESULTS: During the study period, 227 structured interviews were conducted. All approached parents agreed to participate. The mother was interviewed in 97% of cases. Infant's ages ranged between 2-52 months (mean 3.4, standard deviation (SD) 5.1). The majority of the parents were married (98%), and 83% of the mothers were housewives. Most families (79%) had other older children. In most infants (91%), the primary vaccinations were given on time. In the remaining 9%, vaccinations were 1-38 months late (mean 3.8, SD 8.1). The most common reasons for such delays were difficulties with the appointment (30%) and non-febrile upper respiratory tract illness (20%). In only 3 (15%) infants, was the delay based on physician's advice, and only 2 (10%) had a real contraindication. Most of these parents (65%) were not concerned at all regarding the vaccination delay, and only 2 (12%) were highly concerned. CONCLUSIONS: Delays of primary vaccination of infants, although uncommon, continue to occur in our region. Improved parental education and timely scheduling of follow-up appointments can easily prevent such delays. PMID- 12370719 TI - Prevalence of hepatitis B and hepatitis C in blood donors and high risk groups in Hajjah, Yemen Republic. AB - OBJECTIVE: To determine the prevalence of hepatitis B antigen and anti-hepatitis C virus (HCV) antibodies in blood donors, hospital employees, patients suspected to have liver disease, and hemodialysis patients. METHODS: This study was conducted from April 1997 through to September 1999 as a hospital based study in Hajjah, Republic of Yemen. All healthy blood donors, hospital employees, suspected hepatitis patients and patients in the hemodialysis unit were included in this study. The hepatitis B antigen (HbsAg) measured in IMX system (Abott) using the monoclonal anti-HBs assays. The Hepatitis C screened by the same system using HCV version 3.0 [Third generation (Recombinant HCr43, c200, c100-3, NS5)]. RESULTS: The screened blood donors for HbsAg and HCV were 7868 and 2434 with a prevalence of 9.8% for hepatitis B antigen and 1.1% for anti-hepatitis C virus. Two hundred of the hospital employees were screened with a prevalence rate of 1.5% and 0.5% for hepatitis antigen and anti-hepatitis C virus. The patients referred selectively for testing the hepatitis B antigen and anti-hepatitis C virus were 1229 and 749, the prevalence rate of HbsAg was 14.9% and 8.8% for anti HCV, double infection (both hepatitis B virus and HCV) recorded in 8 patients forming 3.2% of the positives (in 0.4% of the total). CONCLUSION: The prevalence was high in Hajjah governorate, Republic of Yemen in both the healthy blood donors and in the risky groups except the hospital employees. PMID- 12370720 TI - Stage IV oral cavity carcinoma. Is conventional radical treatment an option? AB - OBJECTIVE: To evaluate the outcome of radical treatment for patients with stage IV squamous cell carcinoma of the oral cavity. METHODS: Using head and neck tumor database, 57 patients with stage IV non-metastatic invasive squamous cell carcinoma of the oral cavity treated with curative intent at King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia, between July 1992 and June 1998, were identified and retrospectively reviewed. RESULTS: Our cohort of patients consisted of 33 males and 24 females, with a median age of 65 years. The primary sites were alveolus (26), tongue (22), buccal mucosa (6), floor of mouth (2) and retromolar trigone (one). Definitive radiotherapy was used in 7 patients, surgery in 17 and combined modality in 33. With a median follow-up for surviving patients of 53-months, the actuarial 5-year overall survival and relapse free survival was 20% and 14%. Tumors arising from the alveolus showed a better outcome as compared to the rest of oral cavity sites with an overall survival and relapse free survival of 32% and 26% compared to 8% and 4% (p value=0.0057 and 0.0038). CONCLUSION: Advanced oral cavity tumors are aggressive neoplasms with a poor outcome to conventional treatment modalities. New approaches like neoadjuvant or concurrent chemoradiotherapy with or without surgery need to be considered and evaluated in prospective studies. PMID- 12370721 TI - Discharging umbilicus. AB - OBJECTIVE: Umbilical discharge is a symptom of varied pathology. Treatment policies of this clinical problem vary among different institutions. Our experience in the management of 44 patients with umbilical discharge is presented. METHODS: This is a retrospective study of the 44 patients treated at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia over a period of 19 years from 1982 to 2001. RESULTS: With the exception of one patient, all patients were treated in the outpatient clinic. General anesthesia was not employed but instead local anesthesia was used in some cases. One patient required admission for surgical excision, diagnosed to have an ulcerating dermoid cyst. Most patients had hair tuft in the infected umbilicus (pilonidal sinus of umbilicus), 2 patients had concrete like material inside the infected umbilicus. CONCLUSION: We propose a conservative approach to treat this problem and preserve surgical excision only for selected cases. PMID- 12370722 TI - Levamisole treatment in steroid sensitive nephrotic syndrome. AB - OBJECTIVE: To evaluate the effectiveness of levamisole in maintaining remission in children with steroid-sensitive nephrotic syndrome (SSNS) who had a frequent relapsing or steroid-dependent course. METHODS: All children with SSNS who had a frequent relapsing or steroid-dependent course and were treated with levamisole between 1997 and 2001 at King Abdul-Aziz University Hospital, Jeddah, Kingdom of Saudi Arabia were reviewed. All patients were treated by the same steroid protocol used in our unit. Levamisole was considered effective if the patient successfully remained in remission on Prednisolone 0.5 mg/kg/48 hours or less. RESULTS: Nine children were treated with levamisole (3 mg/kg/48 hours) with median (range) age of 6 (3.5-10) years. Seven received levamisole for more than 6 (6-24) months and 2 were excluded because they did not adhere to treatment. Levamisole was effective in 4 patients (57%) with remarkable reduction in the number of relapses and the steroid maintenance dose. Renal biopsy was performed in 4 patients: 2 responders with biopsy findings of minimal change disease (MCD) and mesangioproliferative glomerulonephritis and another 2 non responders with biopsy findings of MCD and focal segmental glomerulosclerosis. No significant side effect was observed. CONCLUSION: Levamisole is effective in maintaining remission in steroid SSNS in Arab children and has few side effects. PMID- 12370723 TI - Sleep problems among pupils in Benghazi, Libya. AB - OBJECTIVE: To find the prevalence of sleep problems among pupils, its possible causes and associated behaviors. METHODS: This study was carried out during 3-24 February 1996. We randomly selected a sample of (277) 5th graders (181 boys and 96 girls), from 3 public primary schools in 3 different socio-economic areas in Benghazi, Libya. We interviewed the pupils and the teachers by a pretested questionnaire concerning sleep, school achievement and other behaviors. Those pupils who sleep after 10:00 pm, or those having bad sleep characters, or both, twice or more a week in the last month, were considered suffering from sleep problems. According to their performance in the first semester in both Arabic and Mathematics, pupils were classified in to 3 groups (>70% in both the good group, 50-70% in both subjects or in one provided that the other was above 70% the average group, <50% in one or both subjects the poorly achieving group). RESULTS: This study shows high prevalence of sleep problems among pupils (28.9%), especially in a high-density area, with non-significant gender difference. Associated behaviors are bad school achievement, sleepiness and mood fluctuation. CONCLUSION: Parental ignorance, unawareness and bad child rearing and follow-up are evident, and considered to be the main cause of these problems, that needs intensive education towards better sleep hygiene. PMID- 12370724 TI - Parents' attitude towards children's first dental visit in the College of Dentistry, Riyadh, Saudi Arabia. AB - OBJECTIVES: The objectives of this study were to evaluate parents' awareness about the timing of the first dental visit for their children, the parents' attitude toward behavior modification for their children at the first dental visit, and to determine the main reasons for bringing the child to the dentist in the first visit among the Saudi parents attending the dental school at the College of Dentistry, King Saud University, Riyadh, Kingdom of Saudi Arabia. METHODS: A self-administered questionnaire consisting of 12 items was distributed to any adult patient reporting at the Registration Appointments and Records Division of King Saud University, College of Dentistry, Riyadh, Kingdom of Saudi Arabia, during the year 2001. The questionnaire response rate was 96.5%. RESULTS: Some parents reported that their child's first dental visit should be in the 3rd year (42%) while others thought it should be in the 6th year (34.4%). Seventy three percent of the parents prefer behavior modification during the first dental visit. Regular visit (40.3%) and emergencies (28.1%) were the main reasons to bring the children to the dentist. CONCLUSION: These data indicate that there is a relatively low level of parents' knowledge about the timing of a child's first dental visit. The dental profession and pediatricians have major responsibilities to change this concept. PMID- 12370725 TI - Can we predict prognosis using mortality probability model IIo? AB - OBJECTIVE: To evaluate Mortality Probability Model (MPM) IIo as a tool to predict very poor prognosis after intensive care unit admission. METHODS: The study was conducted as a prospective observational study in a medical-surgical intensive care unit in a tertiary care teaching hospital, Riyadh, Kingdom of Saudi Arabia. Data necessary to calculate MPM IIo predicted mortality was collected from March 1999 through to February 2000 on all intensive care unit admissions. The hospital outcome was documented. We calculated the sensitivity, specificity, positive predictive value and negative predictive value of MPM IIo using cutoff points of 90% and 95%. RESULTS: Data was complete on 557/569 patients (98%). Thirty-one patients had predicted mortality of >95% and all died yielding a specificity of 100% and positive predictive value of 100%. However, sensitivity was only 18% and negative predictive value 73%. Forty-four patients had predicted mortality of >90% of whom only one survived yielding a specificity of 99.7% and a positive predictive value of 97.7%. Sensitivity was only 25% and negative predictive value of 75%. CONCLUSIONS: Using a decision-cutoff of 95% predicted mortality using MPMI IIo had a very high specificity in predicting death after intensive care unit admission, although with a low sensitivity. This information can be used to support clinical judgment regarding the very ill patients who are unlikely to benefit from intensive care unit admission. PMID- 12370726 TI - Enterococcus faecalis endocarditis. AB - The increasing usage of cephalosporins, to which the enterococci are resistant, has resulted in the rising number of enterococcal infections worldwide. Enterococci are a normal part of the flora of the human gastrointestinal tract, buccal cavity, perineal skin, vagina, urethra and gallbladder, but may occur as pathogens in a number of sites causing urinary tract infections, intra-abdominal infections, fatal bacteremia, meningitis and endocarditis. A Saudi male who developed enterococcal endocarditis with vegetations on both aortic and mitral valves required mitral and aortic valve replacement. The attention of physicians is drawn to the increasing frequency of enterococcus as a cause of nosocomial infections, the risk factors, and antibiotic resistance pattern including resistance to vancomycin as well as its potential for virulence. PMID- 12370727 TI - Extrarenal retroperitoneal angiomyolipoma. AB - Angiomyolipoma of the retroperitoneum is extremely rare with only 7 cases reported to date in the literature. Spontaneous hemorrhage and shock is the most common presentation that requires emergency intervention. We report a case of large extrarenal retroperitoneal angiomyolipoma that illustrates a different form of presentation for such a rare tumor mimicking a large locally advanced renal parenchymal tumor. The diagnosis was made by histopathology after performing radical nephrectomy. Although rare, angiomyolipoma of the perinephric fat may have variable presentations and should be considered in the differential diagnosis of large renal tumors particularly in view of possible kidney sparing management. PMID- 12370728 TI - Group B streptococcal endocarditis. AB - Group B Streptococcus can cause early onset neonatal disease. Beyond neonatal life, group B Streptococci are unusual pathogens. It can cause septicemia, epiglottis, fascitis, and endocarditis. A male Saudi child with group B endocarditis who has congenital heart disease is discussed. PMID- 12370729 TI - Tuberculosis of the knee. AB - Tuberculosis is a worldwide problem that has persisted in developing countries and is re-emerging in developed ones. Infectious complications with Mycobacterium tuberculosis have also been reported with increasing frequency. Among these, skeletal and intra-articular infections continue to affect patients and pose a difficult diagnostic problem to physicians and orthopedic surgeons. This is so, as the non-specific clinical presentation which, overlaps with several infectious and non-infectious diseases, and the latency period of this bacteria that can persist up to several years after the initial infection, contribute to diagnosis and management delay. Thus due to the paucity of the disease, physicians should heighten their index of suspicion for diagnosing tuberculosis of the joints especially when faced with recurrent symptoms of what they think is a common problem. In this context, we present a case of a protracted recurrent illness of a knee joint, which was proved by culture to be due to M. tuberculosis. Moreover, the aspects of this disease entity that poses a diagnostic challenge to the treating physician are highlighted together with those that differentiate it from other overlapping diseases such as brucellosis, especially in endemic areas. PMID- 12370730 TI - Septic postpartum ovarian vein thrombosis. AB - This report describes the clinical findings and outcome of a patient suffering from septic postpartum ovarian vein thrombosis. Treatment modalities are well described and range from hysterectomy and thrombectomy to the use of vena cava filters in combination with anticoagulation and antibiotics. Defervescence with a combination infusion of tissue plasminogen and heparin were used. This treatment approach has been found particularly successful in cases of ilio-femoral, hepatic, renal and vena caval thromboses. PMID- 12370731 TI - An anemic patient who presented with abdominal pain due to trichobezoar. PMID- 12370732 TI - Evaluation of current management of homozygous beta-thalassemia in Eastern Saudi Arabia. PMID- 12370733 TI - The role of antenatal corticosteroid in an extremely preterm fetus with reversed end diastolic flow velocity in umbilical artery. PMID- 12370734 TI - Antiphospholipid antibodies associated with different presentations at a University Hospital. PMID- 12370735 TI - Precipitating factors for diabetic ketoacidosis. PMID- 12370736 TI - Neuroleptic malignant syndrome revisited! PMID- 12370737 TI - The homeobox gene BARX2 can modulate cisplatin sensitivity in human epithelial ovarian cancer. AB - Epithelial ovarian cancer (EOC) is the most common cause of death from gynaecological malignancy. Resistance to platinum chemotherapy is a major reason for treatment failure and poor prognosis. The human homeobox gene BARX2 is located within a minimal region at 11q25 that is associated with frequent loss of heterozygosity (LOH) and adverse survival in EOC. BARX2 is a transcription factor known to regulate transcription of specific cell adhesion molecules in the mouse. We have previously shown that BARX2 expression is low in clear cell/endometrioid and high in serous adenocarcinomas of the ovary, histologic variants that are less and more sensitive, respectively to platinum chemotherapy. The aim of this study was to define whether BARX2 could modulate sensitivity to cisplatin in human epithelial ovarian cancer. In two cell line series sequentially derived from ovarian cancer patients pre- and post-cisplatin chemotherapy, BARX2 expression was downregulated in the cell lines established upon tumor recurrence after platinum therapy. Transfection of BARX2 into a platinum resistant cell line significantly reversed cisplatin resistance compared with its isogenic platinum sensitive parent, in both growth inhibition and clonogenic assays. Taken together, our data demonstrate that the homeobox gene BARX2 may be a biological factor involved in determining sensitivity or resistance to the cytotoxic effects of cisplatin. PMID- 12370738 TI - Distribution and expression of CD44 isoforms and Ezrin during prostate cancer endothelium interaction. AB - CD44 is a multifunctional cell surface adhesion molecule that has been implicated in tumour cell invasion and metastasis. Many cancer cell types as well as their metastases express high levels of CD44. Furthermore, the expression of certain CD44 variants has been linked with metastasis and tumour progression. It is known that ezrin, a member of the ERM family of proteins, can bind to CD44 and thus raises the possibility that it is involved in cell migration and metastasis. Therefore we examined the expression and distribution of CD44, its co localisation and translocation with ezrin in prostate cancer cell lines as they interact with endothelial cells. Experimental results indicate prostate cancer cells express multiple CD44 isoforms that co-localise with ezrin in DU-145 and PC 3 prostate cancer cells. Treatment with hepatocyte growth factor (HGF/SF) resulted in up-regulation of CD44 and its co-translocation with ezrin during tumour-endothelial cell interactions. In addition, tumour cell adhesion to endothelial cells and their invasiveness was increased after exposure to HGF/SF, and can be blocked by the presence of anti-CD44 antibodies. It is concluded that CD44 and ezrin interact in endothelial cells and that they co-localise in the areas of tumour-endothelial contact. The CD44/ezrin complex plays a pivotal role in the capture and invasion of endothelial cells by prostate cancer cells. PMID- 12370739 TI - Transforming growth factor alpha, amphiregulin and cripto-1 are frequently expressed in advanced human ovarian carcinomas. AB - The expression of transforming growth factor alpha (TGFalpha), amphiregulin (AR) and cripto-1 (CR-1) was assessed by immunohistochemistry in 83 specimens (59 primary ovarian tumors and 24 extra-ovarian carcinomas) that were obtained from 68 ovarian carcinoma patients. Within the 59 primary tumors, 54 (92%) expressed immunoreactive TGFalpha, 45 (76%) expressed AR, and 28 (47%) expressed CR-1. The expression of AR and CR-1 mRNAs in the ovarian carcinomas was also demonstrated by RT-PCR analysis. Seventeen extra-ovarian specimens (71%) were found to express CR-1, whereas AR and TGFalpha were expressed respectively in 21 (87%) and 22 (92%) extra-ovarian tissues. In 15 cases for whom both ovarian and extra-ovarian tissues were available, a statistically significant higher expression of CR-1 was found in extra-ovarian specimens. A statistically significant correlation was found between AR expression in the ovarian carcinomas and both low grade and low proliferative activity. Finally, expression of TGFalpha was predictive of longer progression-free survival. These data strongly suggest that the EGF-related peptides might be involved in the pathogenesis and outcome of human ovarian cancer. PMID- 12370740 TI - Allelic imbalance and microsatellite instability in plasma DNA released from polyclonal pancreatic adenocarcinoma. AB - Evidence of circulating soluble DNA in blood-stream of cancer patients has emerged. Because the plasma DNA is largely derived from cancer cells, genetic analysis of plasma DNA is important to understand the molecular events occurred in cancer patient. Seven microsatellite markers in the soluble plasma DNA from patients with pancreatic adenocarcinoma and other pancreato-biliary malignant tumors were examined for microsatellite instability (MSI) and allelic imbalance (AI). A variety of genetic alterations including MSI and AI were detected in the plasma DNA. Some alterations were detected before recurrence of the tumor was verified. Analysis of five primary pancreatic adenocarcinomas by microdissection revealed that the heterogeneous nature of pancreatic tumors is associated with both MSI and AI in the same tumor. The presence of altered plasma DNA including MSI and/or AI from the same pancreatic cancer patient may be important evidence for the presence of these alterations in heterogeneous primary tumors. Analysis of plasma DNA could become one of the diagnostic or therapeutic measures for this type of pancreatic adenocarcinoma. PMID- 12370741 TI - Overexpression of heterogeneous nuclear ribonucleoprotein B1 in lymphoproliferative disorders: high expression in cells of follicular center origin. AB - It is reported that overexpression of hnRNP A2 and B1 proteins is useful for detecting early cancers, and that B1, a splicing minor isoform of A2, is more specific than A2. The B1 expression is still undetermined in human lymphoid tissues. We quantitatively studied the B1 expression in 85 lymph node specimens, comprising reactive lymphoid hyperplasia (RLH; n=8), B-cell lymphoma (n=23), T cell lymphoma (n=22), and metastatic carcinoma (n=32). Immunostaining and immunoblotting analyses with an anti-B1 monoclonal antibody, 2B2 were performed, and the two sets of results correlated with each other (p<0.05). In RLH specimens, B1 expression rate was significantly higher in follicular centers (FC; 44%) than in mantle zone (MZ; 15%) and paracortex (16%) (p<0.01). B1 expression was statistically higher in B-cell lymphoma than in T-cell lymphoma (p<0.01). In B-cell lymphomas, B1 expression rates were 51% in diffuse large B-cell lymphoma (DLBL; n=5) and 45% in follicular lymphoma (FL; n=16), and they were almost the same as that of the FC. Especially in DLBLs, CD10+ FC-origin lymphomas expressed greater amount of B1 than CD10- non-FC-origin lymphomas. B1 expression rate was low in mantle cell lymphoma (MCL; n=2) and similar to that of MZ in RLH. These results suggest that B1 expression is associated with differentiation in lymphoid tissue rather than transformation. B1 expression increases during the process of B-cell differentiation in the FC, and that high B1 expression is maintained in B cell lymphomagenesis, especially in cells of FC-origin DLBL. PMID- 12370742 TI - Interactions of alpha2-HS-glycoprotein (fetuin) with MMP-3 and murine squamous cell carcinoma cells. AB - Alpha2-HS glycoprotein (fetuin) is a major plasma glycoprotein predominantly synthesized by the liver. We have previously demonstrated that human fetuin produced by hepatocellular carcinoma cells can activate the matrix metalloproteinase MMP-9 (gelatinase-B). Stromelysin-1 (MMP-3) is over-expressed in murine skin tumors and is associated with a metastatic cell phenotype. We hypothesize that fetuin plays a role in tumor progression of cell types which by themselves do not have the ability to express fetuin. Immunohistochemical staining revealed that fetuin surrounds the tumor cells in murine squamous cell carcinoma tumor specimens, similar to the expression pattern previously seen for MMP-3. The physical association of fetuin and MMP-3 was demonstrated by immunoprecipitation of radiolabeled fetuin by antibodies to MMP-3. Fetuin facilitates the conversion of pro-MMP-3 to its active form, although this effect is indirect. The association of iodinated fetuin to the cell surface of intact cultured cells derived from a murine tumor with squamous (B9) and spindle (A5) morphologies was determined by binding experiments and Scatchard analysis. Fetuin binds with Bmax values in the range of 1.26-2.1 (mean = 1.7) fmol/1 x 10(5) cells for A5 cells, and 1.5-1.7 (mean = 1.6) fmol/1 x 10(5) cells for B9 cells. The mean KD was 0.46+/-0.19 nmol for both A5 and B9 cells. Our data therefore are consistent with the model that fetuin binds to the cell surface of tumor cells and acts to localize and anchor other molecules important during tumor progression to the plasma membrane. PMID- 12370743 TI - Immunohistological analysis of immune cell infiltration of a human colon tumor xenograft after treatment with Stealth liposome-encapsulated tumor necrosis factor-alpha and radiation. AB - The toxicity associated with tumor necrosis factor-alpha (TNF-alpha) has limited its usefulness as an anticancer agent. However, encapsulation of TNF-alpha in Stealth (SL) liposomes can minimize risk for toxicity and thus increase its potential as an adjuvant treatment. Our recent studies have shown that SL-TNF alpha plus radiation is more effective at inhibiting LS174T colon tumor growth than either radiation alone or free TNF-alpha plus radiation. This increase in efficacy was coincident with a modulation of immune parameters in blood and spleen. The aim of this study was to determine if infiltration of natural killer (NK) cells, macrophages, and neutrophils into LS174T tumors was altered by SL-TNF alpha treatment and whether any observed changes could potentially contribute to the enhanced antitumor efficacy seen with SL-TNF-alpha plus radiation treatment. Sections of excised tumors were examined histologically and quantitative analysis was performed using laser scanning cytometry. The data showed that the group receiving multiple treatments with SL-TNF-alpha plus radiation had the smallest tumors, but yet the level of necrosis was similar to that found in groups with much larger tumors. Furthermore, the necrotic areas in the SL-TNF-alpha plus radiation group had signs of recent and/or continuing cell death and the highest levels of NK cell and macrophage infiltrates. In time course experiments, a single injection of SL-TNF-alpha (but not free TNF-alpha) induced fluctuations in leukocyte infiltration into tumors that correlated inversely with our previous findings in blood and spleen. Overall, the data indicate that the mechanisms underlying the increased efficacy of SL-TNF-alpha compared to free TNF-alpha include a rapid and relatively sustained recruitment of NK cells, macrophages, and neutrophils. PMID- 12370744 TI - Overexpression of c-erbB-2 oncoprotein in muscle-invasive bladder carcinoma: relationship with gene amplification, clinicopathological parameters and prognostic outcome. AB - Amplification of the c-erbB-2 oncogene and protein overexpression are well-known in breast cancer and a basis for therapy with the monoclonal antibody trastuzumab, which binds to the receptor encoded by c-erbB-2. Regarding bladder carcinoma, several studies have examined c-erbB-2 expression, but their results are quite heterogeneous. In the present study, we evaluated the expression of this oncoprotein immunohistochemically in 203 muscle-invasive urothelial bladder carcinomas using the HercepTest. Additionally, 42 cases were studied for gene amplification by fluorescence in situ hybridization (FISH) using the PathVysion kit. Follow-up was known in 147 patients. The results were compared with pathologic characteristics and disease-related survival. Immunohistochemical c erbB-2 overexpression was observed in 37% of the tumors (76/203). However, only 5% (2/42) showed amplification of the oncogene, indicating that predominantly other mechanisms than gene amplification may cause protein overexpression in bladder cancer. C-erbB-2 protein overexpression was significantly associated with high tumor grade (p=0.004) and infiltrative growth pattern (p=0.0001), and tendentiously associated with the presence of lymph node metastases (p=0.077). Regarding tumor stage, sex and age, no significant correlation was registered. Kaplan-Meier curves showed a significantly worse disease-related survival for patients with c-erbB-2 overexpressing tumors (p=0.0346 by log-rank test). Multivariate analysis revealed that, besides nodal status (p=0.0001) and tumor stage (p=0.028), c-erbB-2 overexpression was an independent predictor of disease related survival (p=0.030). Thus, our results suggest that immunohistochemical c erbB-2 detection might represent an additional tool in determining bladder cancer prognosis. Clinical trials evaluating the efficacy of trastuzumab therapy in bladder cancer patients are warranted. PMID- 12370745 TI - Loss of heterozygosity at microsatellite markers from region p11-21 of chromosome 8 in microdissected breast tumor but not in peritumoral cells. AB - Alterations of chromosomal region 8p11-21 are very frequent in human cancers, and especially in breast cancer; yet, most of the genes involved have not been identified. We performed laser capture microdissection in a series of 52 consecutive breast tumor samples to obtain pure tumor cells without surrounding normal breast. To determine genomic subregions in which some of the cancer genes may be located, we conducted a search for loss of heterozygosity (LOH) at 13 microsatellite markers from this region. Two-thirds of the tumors showed LOH at least at one marker. Microdissection of pure tumor samples was helpful to precisely define four LOH subregions. No LOH was observed in the corresponding peritumoral tissues. We studied by immunohistochemistry (IHC) on tissue microarrays the expression in the same tumors, of the protein product of three potential tumor genes lying close to or within the subregions of LOH. In most samples, the TACC1 gene product was downregulated in tumor cells as compared to normal cells. Our results show that the centromeric portion of chromosome arm 8p is frequently altered in breast tumor cells. PMID- 12370746 TI - Inactivation of the PTEN gene by mutation, exonic deletion, and loss of transcript in human oral squamous cell carcinomas. AB - PTEN, a tumor suppressor gene, has been found to be inactivated by structural abnormalities or epigenetic changes in several types of human cancers. Recently, several studies have also suggested the possibility that the PTEN gene is a target of genomic instability in human cancers displaying microsatellite instability (MSI). To investigate the role of PTEN in human oral squamous cell carcinomas, we screened the entire coding region sequences and examined the expression of the PTEN gene in 81 oral cancers displaying microsatellite stability (MSS) and 5 oral cancers displaying MSI. Mutation of the PTEN gene was identified in one MSS cancer (1/81; 1.2%) and three MSI cancers (3/5; 60%). The MSS cancer harbored a missense mutation from Ala (GCA) to Val (GTA) at codon 137. Of the MSI cancers containing the PTEN mutation, case 36 had a missense mutation from Lys (AAA) to Glu (GAA) at codon 254, case 43 contained a frameshift mutation (one A deletion) in a 6 bp poly(A) tract affecting codon 265-267, and case 64 harbored two missense mutations from Val (GTG) to Ala (GCG) at codon 222, and from Gly (GGA) to Arg (AGA) at codon 230 indicating biallelic mutation of PTEN. Genomic deletion of exon 5, resulting in loss of PTEN mRNA, was observed in two MSS cancers. In spite of an intact PTEN gene, one MSS and one MSI cancer lacked PTEN mRNA. These findings suggest that the inactivation of PTEN by either mutation or loss of transcript plays a role in the pathogenesis of some oral cancers (8/86; 9.3%). Furthermore, inactivation of PTEN was far more frequent in MSI oral cancers (4/5; 80%) than in MSS oral cancers (4/81; 4.9%). PMID- 12370747 TI - Macro-microscopic fluorescence imaging of human NPC xenografts in a murine model using topical vs intravenous administration of 5-aminolevulinic acid. AB - The use of 5-aminolevulinic acid to induce endogenous porphyrins for the purpose of detection of epithelial cancers is being studied extensively in many centres around the world. The challenge is to prepare an efficacious formulation of 5-ALA for the purpose of cancer detection. In this study, we compared two formulations of topical 5-ALA applications with intravenous administration in NPC/CNE-2 xenografts on balb/c nude mice. One of the formulations was a gantrez muco adhesive patch and the other was a polyvinyl-pyrolidone muco-adhesive patch. The Karl Storz fluorescence endoscopy system was used to obtain macroscopic fluorescence images. Microscopic fluorescence imaging was done by laser confocal microscopy. The macroscopic images were further analysed for fluorescence intensity distribution. It was found that between the two formulations of topical application of 5-ALA; there was very little difference in the fluorescence biodistribution. When the topical applications were compared with the intravenous administration, the tumour to normal differential in biodistribution was significantly higher with the topical application compared to the intravenous application. PMID- 12370748 TI - Ultrastructural localization of binding sites for PNA and VVA-B(4) lectins in human breast cancer cell lines detected by confocal fluorescence microscopy. AB - Three cancer cell lines (MCF-7, HBL-100, MDA-MB 231) and subnormal breast epithelial cell line MCF-10A were labeled with FITC-conjugated VVA-B4 lectin, specific for D-GalNAcalpha-O-ser/thr, matching the structure of Tn antigen sugar residues, and with RTIC-conjugated PNA lectin, specific for DGalbeta1-3GalNAc-O ser/thr, corresponding to the structure of T antigen. Simultaneous expression of Tn and T antigens on the same cells (but in widely differing proportions) led to their large heterogeneity and occurrence of numerous cell subpopulations within each of the studied cell lines. This observation proved that the changes leading to the formation of Tn antigen are not caused by an irreversible genetic mutation of beta1-3-galactosyltransferase. Expression of Tn antigen on MCF-10A cells with normal (or subnormal) karyotype suggests that the process of malignant transformation of the cell begins with the changes in molecular structure of glycoconjugates. PMID- 12370749 TI - Overexpression of E2F-1 leads to bax-independent cell death in human glioma cells. AB - Gliomas are highly resistant to any kind of treatment. Multiple genetic abnormalities exist in gliomas indicating that effective gene therapy should be directed towards replacement of multiple rather than single genes. Bax is a protein of the Bcl-2 family that promotes apoptosis and functions as a tumor suppressor gene. The E2F family of transcription factors plays a pivotal role in the regulation of cell-cycle and cell-death related genes in gliomas. We examined the therapeutic potential of the simultaneous transfer of Bax and E2F molecules (1, 2 or 4) to gliomas. We used first generation E1A-deleted adenoviral vectors to transduce the E2Fs and Bax cDNAs. The recombinant adenoviral vector encoding bax uses the inducible Cre-loxP system to transduce the protein expression. Western blot analysis and immunofluorescence assays demonstrated high level of expression of the exogenous proteins. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated an additive effect of these molecules to induce cell death via apoptosis. Western blot analysis showed that the ectopic expression of E2F-1 decreased the level of expression of Bax. These results indicate that E2F-1 and Bax have an additive anti-glioma effect when expressed simultaneously at high levels. Our data also suggest that Bax is not involved in the E2F-1-mediated apoptosis. PMID- 12370750 TI - Reversal of drug resistance using hammerhead ribozymes against multidrug resistance-associated protein and multidrug resistance 1 gene. AB - We examined the effects of suppressing multidrug resistance-associated protein (MRP) and multidrug resistance 1 (MDR1) gene expression in HCT-8DDP human colon cancer cell lines, which showed both cisplatin and multidrug resistance. Hammerhead ribozymes, designed to cleave MRP mRNA (anti-MRP Rz) and MDR1 mRNA (anti-MDR1 Rz), were transfected into the HCT-8DDP cells. Drug sensitivity was estimated by MTT assay in vitro. The HCT-8DDP/anti-MRP Rz cells were more sensitive to doxorubicin (DOX) and etoposide (VP-16) by 2.5- and 4.1-fold, respectively, compared with HCT-8DDP cells. The HCT-8DDP/anti-MDR Rz cells were more sensitive to DOX and VP-16 by 2.3- and 3.8-fold, respectively. The anti-MRP Rz and anti-MDR1 Rz significantly down-regulated resistance to DOX and VP-16, while anti-MRP Rz and anti-MDR1 Rz did not affect resistance to cisplatin, methotrexate and 5-fluorouracil. The hammerhead ribozyme-mediated specific suppression of MRP or MDR1 was sufficient to reverse multidrug resistance in the human colon cancer cell line. PMID- 12370751 TI - Ribozyme mediated cleavage of cell-associated isoform of vascular endothelial growth factor inhibits liver metastasis of a pancreatic cancer cell line. AB - Stromal angiogenesis is an important factor for progression of malignant neoplasms. We used hammerhead ribozymes against vascular endothelial growth factor (VEGF) gene transcripts to down-regulate cell-associated VEGF189 isoform function in the pancreatic cancer cell line MIA PaCa2. MIA PaCa2 transfected with anti-VEGF189 ribozyme did not show any alteration of growth rate under tissue culture. When the transformants were subcutaneously transplanted, tumour volume of the ribozyme-transfected MIA PaCa2 xenografts was significantly smaller (P<0.01). No metastasis of MIA PaCa2 transfected with anti-VEGF189 was apparent, while disabled ribozyme-transfected MIA PaCa2 showed significant liver metastasis (P<0.05). These results suggested that VEGF189 plays an important role in growth and metastatic potential through alteration of angiogenic balance in cancer. PMID- 12370752 TI - Activity of E2F-dependent promoters in bladder carcinoma cells and their use for tumour-specific targeting of p53-induced apoptosis. AB - Inactivation of P53 and RB functions are crucial changes in bladder cancer (TCC). High-level re-expression of P53 elicits apoptosis in TCC cell lines, but also--as shown here--in normal uroepithelial cells. Compromised RB function is thought to cause increased activity of E2F-dependent promoters in carcinoma cells. Indeed, several, but not all E2F-dependent promoters were stronger in TCC lines than in normal cells, with the highest activities in cell lines lacking RB rather than p16INK4A. Re-expression of p53 from an E2F-dependent promoter suppressed clone formation and induced apoptosis in TCC lines as efficiently as expression from the stronger RSV-LTR or LINE-1 promoters. In normal cells, p53 expression from an E2F-dependent promoter was tolerated, whereas expression from both stronger promoters was lethal. Thus, specific E2F-dependent promoters allow adjustment of p53 expression to selectively induce apoptosis in TCC vs. normal uroepithelial cells. This approach could be useful in targeting apoptosis to TCC and other carcinomas lacking p53 and RB function. PMID- 12370753 TI - Isolated trisomy of chromosomes 8, 11, 13 and 21 is an adverse prognostic factor in adults with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B 8461. AB - Isolated trisomy is a relatively common cytogenetic abnormality in acute myeloid leukemia (AML), but with uncertain prognostic significance. We studied a large cohort of newly diagnosed de novo AML patients karyotyped on CALGB 8461 from 1984 1999, where trisomy was the sole abnormality. The common isolated trisomies (IT(C)), +8, +11, +13 and +21, comprised 90% of all sole trisomies. The outcome of 101 IT(C) patients was compared to that of 976 with normal and "poor risk" cytogenetics. The overall survival (OS) for IT(C) patients was unsatisfactory with 10% [95% confidence interval (CI), 3-17%] alive at 5 years. Repeated cycles of I/HDAC intensification did not improve outcome. However, SCT significantly improved relapse-free survival (RFS). Among IT(C) patients <60 years in first remission, only 1 of 7 receiving SCT relapsed, compared to 16 of 19 patients treated with chemotherapy only. The prognosis of IT(C) was dependent on SCT. For non-transplanted patients, the 5-year OS for IT(C) was 5% (95% CI, 0-11%), compared to 20% (95% CI, 16-23%) for 640 normal cytogenetics patients. IT(C) was an independent adverse prognostic factor for OS in non-transplanted patients. In those receiving SCT, however, the 5-year OS for IT(C) patients (69%, 95% CI, 32 100%) was not different to that of transplanted normal cytogenetics patients (60%, 95% CI, 38-81%). We conclude that in de novo adult AML patients not receiving SCT, IT(C) appears to independently predict a poor outcome that may be improved with SCT in first remission. Prospective studies are required to confirm this hypothesis. PMID- 12370754 TI - Frequent polymorphic changes but rare tumor specific mutations of the LATS2 gene on 13q11-12 in esophageal squamous cell carcinoma. AB - We previously reported that loss of heterozygosity on 13q12-13 is related with poor prognosis of esophageal cancer. However, a target tumor-suppressor gene on this region is not yet identified. Recently, LATS2, a new human homologue of the Drosophila tumor suppressor gene (lats/warts) was identified on 13q11-12. We therefore screened esophageal tumor cell lines and tumor tissues to detect tumor specific mutations of the LATS2 gene. Although we found 5 different polymorphisms in this gene (4 kinds of single-base changes and a 6-bp insertion), a tumor specific mutation was identified in only one out of 60 tumor tissues. These results indicated that the LATS2 gene is inactivated in only a small part of esophageal tumors, if any. PMID- 12370755 TI - Phosphorylation of chk1 at serine-345 affected by topoisomerase I poison SN-38. AB - Human head and neck squamous carcinoma cell lines, A253 and FaDu, were utilized to identify mediators associated with response to topoisomerase I poison, SN-38, a metabolite of irinotecan. The drug sensitivity of FaDu cells to SN-38 was significantly higher than that of the A253 cells. In A253 cells, G2/M arrest following drug treatment (0.35 microM SN-38, 2-h exposure) was accompanied by DNA fragmentation in the 50-300 kb range, but FaDu cells accumulated in S-phase concurrently with induction of smaller DNA fragmentation in the 4-80 kb range. Because the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of Tyrosine 15 (Tyr15), we examined the Tyr15 phosphorylation status of cdc2 in both cell lines. Slightly increased levels of cdc2 phosphorylation was observed in the A253 cells, while reduced levels of cdc2 phosphorylation was noted in the FaDu cells, corresponding to the abrogation of the G2-phase arrest. Increased chk1 phosphorylation at Ser345 induced by SN-38 was accompanied by the observed G2 phase arrest in the A253 cell line, while significant downregulation of chk1 and cdc25C phosphorylation, which resulted in the abrogation of G2/M checkpoint arrest, was noted in FaDu cells at this timepoint. These results suggest that alterations of chk1 signaling are associated with the response to topoisomerase I poison SN-38. Furthermore, A253 cells possess higher levels of endogenous hMLH1, compared to FaDu cells. A deficiency in G2 arrest was observed in FaDu cells, suggesting endogenous hMLH1 protein expression is associated with the abrogation of G2/M arrest, subsequently with the response to topoisomerase I poison SN-38. PMID- 12370756 TI - Genetic alterations in adenoma-carcinoma sequencing of intraductal papillary mucinous neoplasm of the pancreas. AB - We investigated the adenoma-carcinoma sequence in intraductal papillary-mucinous neoplasm from the aspect of genetic changes. The formalin-fixed paraffin-embedded tumors and surrounding normal pancreatic tissues from patients with 16 intraductal papillary-mucinous adenoma of the pancreas (IPMA) and 10 intraductal papillary-mucinous carcinoma of the pancreas (IPMC) were provided for DNA extraction after microdissection. SSCP-DNA sequencing analysis demonstrated K-ras mutations at codon 12 in 75% of IPMA and 70% of IPMC, while those at codon 13 were observed neither in IPMA nor IPMC. There were no characteristic K-ras mutation types in IPMA and IPMC and no significant differences in incidence of K ras mutations between the two categories. The frequencies of p53 mutations analyzed by SSCP-DNA sequencing were not high in IPMA (18.8%) and IPMC (30%), showing no significant difference between them. LOHs of APC in IPMA and IPMC were infrequent (6.3 and 20%, respectively) and showed no significant difference in incidence between the two categories. The LOH frequencies of DCC in IPMA and IPMC were 31.3 and 40%, respectively, and were not statistically different from each other. Taken together, genetic changes such as K-ras, p53, APC and DCC mutations may not be associated with adenoma-carcinoma sequence in intraductal papillary mucinous neoplasm of pancreas. PMID- 12370757 TI - Aberrant expression of integrin and erbB subunits in breast cancer cell lines. AB - Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells. PMID- 12370758 TI - Correlation between nuclear action of anthracycline anticancer agents and their binding affinity to the proteasome. AB - 4'-O-tetrahydropyranyladriamycin (THP) showed an approximately 10-fold greater inhibitory effect on DNA synthesis in L1210 mouse leukemia cells than adriamycin (ADM). The intracellular transfer rate and nuclear accumulation of THP were approximately 5-fold higher than those of ADM. The intensity of in vitro inhibition of topoisomerase II activity by ADM was almost the same as that by THP. There were no significant differences between the uptake of either of these agents by the isolated nuclei of L1210 cells. The nuclear uptake of both agents in the presence of the cytosolic fraction of L1210 cells consisted of both simple diffusion and carrier-mediated components, and the carrier-mediated component of THP was approximately 2-fold higher than that of ADM. THP showed approximately 5 fold higher affinity to the proteasome than ADM, and interfered with ADM binding in a competitive manner. These results suggest that the binding affinity of these anticancer agents to the proteasome is an important factor in their transport to the nucleus and determines their specificity of action for the nuclear DNA. PMID- 12370760 TI - Cancer-related fatigue (review). AB - Fatigue is one of the most common complaints of people with cancer. It affects the majority of patients actively undergoing cancer related therapies, but also a meaningful number of those who successfully completed therapy and are disease free and potentially cured at the end of the treatments. In cancer setting, fatigue is to be defined as a chronic form of tiredness, which is perceived by the patient as being unusual or abnormal, and absolutely disproportionate with respect to the amount of exercise or activity he/she has carried out and which is not removed by resting or sleeping. The exact cause of fatigue is not known. In cancer setting there are many contributing or associated factors, such as cancer itself, cancer treatment (chemotherapy, radiation therapy, immunotherapy and surgery), depression or anxiety, some medications, pain, nausea, vomiting or diarrhea, poor nutrition, anemia, infections, insomnia. There is no standard of care for the assessment or treatment of fatigue in patients with cancer. The evaluation of fatigue is intrinsically multidimensional, even though the lack of objective measurement methods makes it difficult to draw up worldwide-accepted guidelines; nonetheless, a number of methods have been developed to assess it. Treatment of fatigue should depend on its cause, but presently it is still addressed against the associated symptoms rather than fatigue itself. Useful approaches includes erythropoietin alpha, psychostimulants, medications to treat pain, depression, nausea and difficult sleeping, physical therapy for reconditioning exercises or energy saving techniques, health education. In this report some of the crucial issues related to fatigue in people with cancer are reviewed. PMID- 12370759 TI - Inhibition of human telomerase enhances the effect of chemotherapeutic agents in lung cancer cells. AB - Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. Earlier studies have reported that the presence of telomerase activity in tumors of patients with non-small cell lung cancer patients correlates with a high proliferation rate and advanced pathological stage. Thus, the modification of telomerase activity may be a potential therapeutic modality for the treatment of lung and other cancers. We introduced vectors encoding dominant negative (DN)-hTERT, or wild-type (WT) hTERT, or a control vector expressing only a drug-resistance marker, into the A549 lung cancer cell line, and assessed the biological effect of telomerase inhibition on cellular immortality. Ectopic expression of DN-hTERT resulted in complete inhibition of telomerase activity and reduction of telomere length. The entire population of telomerase-inhibited A549 cells exhibited cytoplasmic blebbling and chromatin condensation, which are features of apoptosis. In contrast, A549 cells expressing wild-type hTERT, which differs from the mutants by only two amino acids, exhibited normal morphology. Evidence for apoptosis in the telomerase-inhibited cells was provided by flow cytometric analysis with APO2.7 monoclonal antibody. We also observed enhanced induction of apoptosis by chemotherapeutic reagents, including cisplatin, docetaxel and etoposide, in DN hTERT-expressing A549 cells, as compared with WT-hTERT-expressing cells. These results demonstrate that disruption of telomere maintenance limits the cellular lifespan of lung cancer cells, and show that the combined use of chemotherapeutic agents and telomere maintenance inhibition may be effective in the treatment of patients with non-small cell lung cancer. PMID- 12370761 TI - Surgical decision making in upper aerodigestive tract cancer patient follow-up. AB - The objective was to analyze the impact of clinical beliefs on surgical decision making in the posttreatment follow-up of patients with upper aerodigestive tract cancer. Clinical beliefs, defined as perceived benefits and risks of surveillance, were examined. All 824 members of the Society of Head and Neck Surgeons (SHNS) and 522 members of the American Society for Head and Neck Surgery, who were not SHNS members, were surveyed using TNM stage-specific clinical vignettes to measure surgical decision making in the posttreatment follow-up of patients with upper aerodigestive tract cancer. Controlling for physician demographic and practice characteristics, the relationship between clinical beliefs and diagnostic test ordering practices of surgeons was examined using Poisson and negative binomial regression analysis. Age 50 and over and South Central U.S. practice location were significant predictors of the frequency of surveillance testing in at least three TNM stage I models as was the clinical belief that no survival benefit results from the follow-up of patients with TNM stage I cancers. Less than 15% of the variability in follow-up intensity was explained by the TNM stage I models. Predictive ability was substantially improved for the TNM stage II-IV models by including lower TNM stage practice patterns as an independent variable. Most models predicted at least 50% of the variation in follow-up testing. The two clinical beliefs with the greatest impact on surgical decision making in the posttreatment follow-up of patients with upper aerodigestive tract cancer are that surveillance: i) permits palliative treatment and improves quality of life and ii) provides no survival benefit for patients with TNM stage I cancers. Knowledge of lower TNM stage practice patterns can be used to further improve predictive ability for higher stage models. PMID- 12370762 TI - Monitoring expression of HER-2 on circulating epithelial cells in patients with advanced breast cancer. AB - Nineteen breast cancer patients with measurable metastatic disease who were starting an initial or new line of therapy were evaluated for circulating epithelial cells (CECs) a minimum of 4 times over the course of treatment. In 7 of the 10 CEC+ patients, HER-2 expression was detected on the CECs. CECs expressing HER-2 varied among patients and in serial samples from the same patient including a shift from HER-2- to HER-2+ CECs. These results demonstrate that it is possible to quantify receptors essential for rationally designed therapy using CECs and that reliance on the immunophenotype of the primary tumor can be misleading. PMID- 12370763 TI - Evaluation of NQO1 gene expression and variant allele in human NSCLC tumors and matched normal lung tissue. AB - NQO1 gene expression was evaluated by RT-PCR and SNP status by RFLP in matched samples of lung tumors and adjacent normal tissue. NQO1 was found to be overexpressed in lung tumors when compared to matched normal lung tissue. The mean expression in normal lung tissue was 28.26 x 10(-14) ng/microl +/- 44.9 SD and 61.46 x 10(-14) ng/microl +/- 103.2 SD in lung tumors. NQO1 gene expression was higher in the tumor than in the matched normal lung tissue in 27/50 (59%) patients (p=0.014). In the normal samples, 25 (50%) were wild-type, 16 were heterozygotes (32%) and 8 had the SNP (16%). In the matched tumor samples 14 were wild-type (28%), 16 were heterozygous (32%) and 19 (38%) had the SNP (p=0.0043). The genomic NQO1 mutation was associated with survival in a pilot study of stage II/III NSCLC patients. Patients with a homozygous SNP genotype had a significantly shorter survival (median 12 months), than heterozygous or homozygous wild-type patients (median 41 months) (p=0.007), suggesting NQO1 may be important in chemosensitivity as well as the pathogenesis of lung cancer and NQO1 genotyping may be a useful component of pharmacogenetic strategies for the treatment of NSCLC. PMID- 12370764 TI - O(6)-methylguanine-DNA methyl transferase gene expression and prognosis in breast carcinoma. AB - O(6)-methylguanine-DNA methyl transferase (MGMT) in human carcinomas has been associated with tumor resistance to alkylating agents. The aims of this study were: i) to correlate tumor MGMT expression and patient and tumor characteristics in malignant breast carcinomas treated with induction chemotherapy including cyclophosphamide (CPM) and ii) to study the predictive and prognostic values of tumor MGMT gene expression. We used RT-PCR to measure the levels of tumor MGMT expression in 107 patients with breast carcinomas prior to neoadjuvant chemotherapy. Sixty patients (56%) received anthracyclines and CPM and 47 (44%) received only anthracyclines. Low levels of MGMT expression correlated with Scarff-Bloom-Richardson grade III (p<0.005), elevated S-phase (p<0.05), negative estrogen receptors (p<0.05), metastatic status (p<0.05) and occurrence of death (p=0.01). MGMT expression was not predictive of treatment response. Unexpectedly, survival was longer when tumor MGMT expression was high (p<0.005). The 4-year survival rate was 76% for high level MGMT patients and only 55% for others. This difference is also significant using the COX model (p<0.05). In breast cancer, tumor MGMT expression was not predictive of response to CPM. A low MGMT expression was significantly related to poor survival. PMID- 12370765 TI - Interferon-gamma inhibits growth of human neuroendocrine carcinoma cells via induction of apoptosis. AB - Although biotherapy of gastroenteropancreatic neuroendocrine tumors (NET) provides excellent control for the hypersecretion syndrome, tumor regression is rarely observed, implying the need for novel antiproliferative strategies. Here, we demonstrate that human pancreatic QGP-1 NET cells express functionally intact interferon-gamma (IFN-gamma) receptors and downstream effectors, including the putative tumor suppressor interferon regulatory factor-1 (IRF-1). IFN-gamma treatment profoundly inhibited anchorage-dependent and anchorage-independent growth of QGP-1 cells. Concomitant with the onset of growth inhibition, apoptotic cells were detected in cell cycle analyses of IFN-gamma treated cultures. Apoptosis was confirmed by evaluation of DNA fragmentation and PARP cleavage. Immunoblots of IFN-gamma treated QGP-1 cells revealed a substantial upregulation of caspase-1, followed by the appearance of active proteolytic fragments of caspase-3, suggesting that autocatalytic activation of caspase-1 might initiate the caspase cascade. Apoptosis induction by IFN-gamma was also observed in two of four primary cultures established from tumors of patients with for- and midgut NETs, respectively. Taken together our results characterize IFN-gamma as a potent proapoptotic stimulus in a subset of gastrointestinal NETs and suggest an IRF-1 mediated induction of caspase-1 as a relevant underlying mechanism. Based on these results, the potential of IFN-gamma in experimental biotherapeutic treatment of NETs can be further explored. PMID- 12370766 TI - Genetic profile, PTEN mutation and therapeutic role of PTEN in glioblastomas. AB - New therapeutic strategies are needed to improve survival in glioblastoma (GBM) the most malignant astrocytic tumor. We evaluated: a) the genetic status of 22 GBMs by comparative genomic hybridization (CGH); b) the specific role of mutation and/or homozygous deletion of PTEN in the genesis of GBM; and c) the possible therapeutic role of PTEN against GBM, in vitro. CGH demonstrated that the most frequent region of gain was at chromosome 7p, whereas the most frequent losses occurred at chromosomes 10q and 13q. Losses at chromosome 10 were found in 36% of patients, and PTEN was mutated in 27% of the 22 GBMs, including 4 point mutations and 2 homozygous deletions. The possible therapeutic role of PTEN in GBM was also studied in a system based on retroviral infection of the GBM cell line A172, homozygously deleted at the PTEN locus. A172 growth and proliferation rate were reduced by 50% after PTEN transduction. Moreover, we showed that inhibition of cell growth occurred through the PI3K/Akt/p27 pathway. Our findings suggest that PTEN participates in the genesis of GBM, and might be further studied as a candidate therapeutic agent in other testing systems. PMID- 12370767 TI - Homozygous proline at codon 72 of p53 as a potential risk factor favoring the development of undifferentiated thyroid carcinoma. AB - The p53 tumor suppressor gene is altered in human cancer. A common polymorphism occurs at codon 72 of exon 4, with two alleles encoding either arginine (CGC) or proline (CCC). No data exist about the association of a distinct codon 72 variant with the histological subtypes of thyroid carcinoma. We developed a new one-step real-time PCR assay on the LightCycler to detect codon 72 polymorphism in the p53 gene. We studied 21 papillary, 18 follicular and 22 anaplastic thyroid carcinomas and compared them with 15 adenomas and 36 normal thyroid tissues (controls); moreover, we compared the cases for histological, clinical and demographic variables and genotype prevalence. In controls, the frequency of the three genotypes Arg/Arg, Arg/Pro, and Pro/Pro was 41.7, 50.0 and 8.3%, respectively. The homozygous proline was not found in benign thyroid adenomas and differentiated thyroid carcinomas. In contrast, all undifferentiated thyroid carcinomas (100%) had the homozygous proline phenotype. The frequency of the two other genotypes Arg/Arg and Arg/Pro was 66.7% and 33.3% in adenomas, 81.0% and 19.0% in papillary thyroid carcinomas, and 83.3% and 16.7% in follicular thyroid carcinomas, respectively. Comparing the genotypes with tumor stage, no correlation was found. However, lymph node and distant metastases status showed a statistically significant prevalence for the homozygous phenotypes Arg/Arg and Pro/Pro. There was no association between a special genotype and age and sex. We conclude that homozygous proline is a potential risk factor favoring the development of an undifferentiated thyroid carcinoma, and that the homozygous phenotypes at codon 72 of p53 are associated with a poorer prognosis of thyroid carcinoma. PMID- 12370768 TI - Quality of placental pathology reports. AB - The surgical report is an important means of documenting normal and abnormal findings, and for distilling such information into a meaningful clinico pathologic correlation. An audit of the quality of placental reports from four laboratories was performed using an arbitrary numerical scoring scheme that examined the gross, histologic, and commentary components of each report. The mean scores from the four laboratories were not statistically different from each other. Three (2%) and 48 (33%) of the 147 singleton placentas scored less than 50 and 75%, respectively, on this scoring scheme. None and 14 (41%) of the placentas from 34 multiple pregnancies scored less than 50 and 75%, respectively. Different aspects of the gross and histologic examination were reported variably by the laboratories. Commentaries on gross or histologic abnormalities, and in relation to clinical indications, were inconsistently reported. The standards of placental surgical reporting can be improved. The use of templates and checklists for reporting of placentas may be considered. PMID- 12370769 TI - Aberrant immunohistochemical expression in nonrhabdomyosarcoma soft tissue sarcomas of infancy: retrospective review of clinical material. AB - Malignant soft tissue tumors other than rhabdomyosarcoma (RMS) are uncommon in infancy, representing approximately 5% of pediatric sarcomas. The pathological categorization of non-RMS soft tissue malignancies from these young patients is complicated by variation in both morphologic and immunohistochemical features. A search covering an 11-year period identified 19 patients presenting at birth or in infancy with a clinical or referral diagnosis of soft tissue sarcoma. After histologic and immunohistochemical review, nine of these tumors were classified as primitive neuroectodermal tumor (PNET), three as infantile hemangiopericytoma (HPC), two as infantile fibrosarcoma (FS), and five as undifferentiated sarcoma. Those identified as undifferentiated sarcomas showed an atypical spindle and ovoid cell morphology, with cellular pleomorphism and high mitotic rate, but lacking the fascicular growth pattern of classic infantile fibrosarcoma. Immunohistochemical staining in this group showed variable weak positivity for a range of markers (desmin, smooth muscle actin, Myo-D1, PGP, NSE, S100, CO56, cytokeratin, and CD99), and did not fit readily into any distinct diagnostic category. In this series, tumors classified as soft tissue PNETs had a poor prognosis despite aggressive treatment. However, once RMS, PNET, and other rare specific lesions are excluded, the remaining undifferentiated sarcomas, despite their unusual morphology and immunohistochemistry, appear to behave in a similar favorable manner to infantile fibrosarcoma. PMID- 12370770 TI - Limited reliability of lipid-laden macrophage index restricts its use as a test for pulmonary aspiration: comparison with a simple semiquantitative assay. AB - The lipid-laden macrophage index (LLMI) is a semiquantitative evaluation of alveolar macrophage lipid content used in diagnosis of pulmonary aspiration. To date, there are no published reports regarding the reliability of LLMI. We sought to evaluate the interobserver and intraobserver variability and validity of LLMI and to compare it to a simpler macrophage lipid content index (LCI). To evaluate reliability we compared both the LLMI and LCI of 26 bronchoalveolar lavage (BAL) specimens from 14 aspirators and 12 non-aspirators on 10 separate occasions by two observers. The ranges of means and standard deviations (SD) of LLMI for observer 1 (Obs 1) were 19-160 (5-31) for aspirators, and 0-48 (0-15) for non aspirators; and those of observer 2 (Obs 2) were 77-249 (13-33) for aspirators and 47-170 (8-37) for non-aspirators. The ranges of means and SD of LCI for Obs 1 were 2-8 (0-2) for aspirators and 0-4 (0-1) for non-aspirators, compared with 2-9 (0-2) for aspirators and 1-6 (0-2) for non-aspirators for Obs 2. No statistical significance was found between LLMI and LCI by comparing coefficients of variation (CV) in either groups or observers. Poor agreement between the two observers was found using a Bland Altman analysis, with the difference of the two observations mostly exceeding zero and becoming larger as the average of the two observations became bigger. The combined sensitivity, specificity, and positive and negative predictive value (PPV and NPV) for both observers of the LLMI were 57%, 75%, 84%, and 69% and those of LCI were 58%, 92%, 93%, and 69%. We conclude that there is poor reliability for both methods. The LCI is simpler and appears to be at least as good as the LLMI. PMID- 12370773 TI - Appearances may be deceiving: does aggressive histology correlate with behavior of infantile sarcomas? PMID- 12370774 TI - Progressive liver fibrosis in late-onset argininosuccinate lyase deficiency. AB - A 4-month-old boy, with late-onset argininosuccinate lyase (ASL) deficiency with hepatomegaly, was treated by protein restricted diet and arginine supplementation; he was followed for 3 years. Hepatomegaly and mild liver dysfunction persisted without significant hyperammonemia. He maintained normal psychomotor development to the age of 12 months, but, at 3 years of age, his developmental status is in the borderline normal range. Liver biopsy performed at 12 months of age demonstrated swollen and pale hepatocytes with abnormal glycogen deposition and mild periportal fibrosis. A subsequent liver biopsy at 3 years of age showed progressive liver fibrosis in the periportal and central areas, which extended into the liver lobule. These findings suggest that liver impairment in ASL deficiency may advance without significant hyperammonemia and underline the importance of repeated liver biopsy in this disorder, even when the plasma ammonia level is well controlled. PMID- 12370775 TI - Tailored approach to Zenker's diverticula. AB - BACKGROUND: Zenker's diverticula (ZD) can be treated by diverticulostomy or open surgery (upper esophageal sphincter myotomy and diverticulectomy or diverticulopexy). The aim of this study was to compare the outcome of the two alternative treatments. METHODS: Fifty eight patients were scored for symptoms and upper esophageal sphincter (UES) pressure; relaxations and intrabolus pressures were recorded by manometry. Treatment depended on operative risk and ZD size. Twenty four patients with high surgical risk and/or a <3-cm or >5-cm pouch underwent diverticulostomy; the other 34 had open surgery. RESULTS: Mortality was nil. Five patients had postoperative complications after open surgery (p<0.05). Hospital stay was shorter after diverticulostomy (p<0.001). Follow-up (41 months; range, 1-101) was obtained in 53 patients. Postoperative manometry showed a UES pressure reduction, improved UES relaxation, and lower intrabolus pressure in both groups (p<0.05). In the diverticulostomy group, three patients complained of severe dysphagia. vs none in the open surgery group (p<0.05). CONCLUSION: Diverticulostomy is safe, quick, and effective for most patients with medium sized ZD, but open surgery offers better long-term results and should be recommended for younger, healthy patients with small or very large diverticula. PMID- 12370776 TI - Laparoscopic cholecystectomy: quality of care and benchmarking. Results of a single-institution specialized in laparoscopy compared with those of a nationwide study in Switzerland. AB - BACKGROUND: Quality control is an important issue in surgery. Therefore, we assessed the outcome of laparoscopic cholecystectomies (LC) performed at our institution specialized in laparoscopic surgery in order to do a benchmarking. METHODS: The perioperative courses of the first 1000 LCs performed in Aarberg hospital were recorded, analyzed, and compared with the results of a recent study including 10, 174 patients published by the Swiss Association of Laparoscopic and Thoracoscopic Surgery (SALTS). RESULTS: The following quality indicators were compared with the corresponding SALTS rates: primary conversion rate 1.5% (SALTS 8.2%; p <0.01); conversion rate for intraoperative complications 6.5% (63.8%; p <0.01); intraoperative complication rate 22.2% (34.4%; p <0.01); postoperative morbidity rate 8.1% (10.4%; n.s.); in-hospital mortality rate 0.1% (0.2%; n.s.); and reoperation rate 0.8% (1.7%; n.s.). CONCLUSIONS: LC has reached a high quality level in its widespread use, but in a small specialized center even a higher quality level can be achieved. Favorable results seem to depend on structural advantages of a surveyable unit in association with a continuously motivated surgical team. PMID- 12370778 TI - Multiple transcription start sites and alternative splicing in the methylenetetrahydrofolate reductase gene result in two enzyme isoforms. AB - Methylenetetrahydrofolate reductase (MTHFR) reduces 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major carbon donor in the remethylation of homocysteine to methionine. Mild MTHFR deficiency, due to a common variant at nucleotide 677, has been reported to alter risk for several disorders including cardiovascular disease, neural tube defects, pregnancy complications, and certain cancers. Little is known about MTHFR regulation, since the complete cDNA and gene sequences have not been determined. In earlier work, we isolated and expressed a 2.2-kb human cDNA comprised of 11 coding exons, and we demonstrated that it encoded an active 70-kDa isoform. However, transcript sizes of approximately 7.5 kb and 9.5 kb and the presence of a second isoform of 77 kDa on Western blots suggested that cDNA sequences were incomplete. In this report, we characterized the complete cDNA and gene structure in human and mouse. Variable 5? and 3? UTR regions were identified, resulting in transcript heterogeneity. The 5? and 3? termini of the MTHFR cDNA were found to overlap with the 5? terminus of a chloride ion channel gene (CLCN-6) and the 3? terminus of an unidentified gene, respectively; this finding has resulted in finer mapping of MTHFR on Chromosome (Chr) 1p36.3. Ribonuclease protection assays identified clusters of transcriptional start sites, suggesting the existence of multiple promoters. MTHFR has several polyadenylation sites creating 3?UTR lengths of 0.2 kb-5.0 kb or 0.6 kb-4.0 kb in human and mouse, respectively. In both species, the previously reported exon 1 was redefined to approximately 3.0 kb in length and shown to be alternatively spliced. An important splice variant contains novel coding sequences; this cDNA was expressed and shown to encode the isozyme of 77 kDa. Our results, which suggest intricate regulation of MTHFR, will facilitate additional regulatory and functional studies of the different isoforms. PMID- 12370779 TI - The mouse PcG gene eed is required for Hox gene repression and extraembryonic development. AB - The Polycomb group (PcG) of genes was first identified in Drosophila as maintenance factors for long-term transcriptional repression of homeotic genes. In mice, the PcG protein Eed (Embryonic ectoderm development) is present in a distinct complex that interacts with histone deacetylase (HDAC) and the PcG member Ezh2 (Enhancer of zeste homolog 2), but not in the larger Polycomb repressive complex 1 (PRC1) formed by several other PcG proteins. eednull mutants manifest a distinct early gastrulation defect that occurs prior to homeotic gene expression. To determine whether Eed is also required for regulating homeotic genes, a later acting eedhypomorph mutation was analyzed. The anterior expression boundaries of several Hox genes were shifted rostrally by one segment, indicating that Eed is required for stable repression of homeotic genes. Furthermore, although the eednull/hypomorph compound heterozygotes die during mid-gestation stage, they did not show a more severe derepression of Hox genes than the eedhypomorph/hypomorph homozygotes. A detailed analysis of the mid-gestation lethality associated with the eednull/hypomorph compound heterozygotes revealed a novel function for eed in the development of secondary trophoblast giant cells during murine placenta formation. Tetraploid rescue experiments demonstrated that the defect is cell autonomous in the extraembryonic lineage. Mash2, a paternally imprinted gene important for trophoblast development, was ectopically expressed in the eed mutants. However, genetic crosses with a Mash2 null allele suggested that Eed was not required to maintain Mash2 imprinting, but could be required in a lineage specific fashion to suppress Mash2 expression. PMID- 12370780 TI - Reduced body growth and excessive incisor length in insertional mutants mapping to mouse Chromosome 13. AB - Phenotypic and molecular genetic examinations of a transgenic mouse line showing developmental defects caused by a recessive insertional mutation were carried out. The mutant phenotype is characterized by general retardation of postnatal body growth and by the appearance of increased incisor length in the upper and lower jaw. The mutation causing the aberrant phenotype was mapped to Chromosome 13, 40 cM. Examination of the expression of the candidate genes did not show any alterations. This mutant mouse line provides a reproducible model for the identification and examination of gene(s) involved in growth and in the craniofacial development, including that of the jaws and teeth. PMID- 12370781 TI - Identification of transcription factor binding sites in the human genome sequence. AB - The identification of transcription factor binding sites (TFBS) is an important initial step in determining the DNA signals that regulate transcription of the genome. We tested the performance of three distinct computational methods for the identification of TFBS applied to the human genome sequence, as judged by their ability to recover the location of experimentally determined, and uniquely mapped, TFBS taken from the TRANSFAC database. These identification methods all attempt to filter the quantity of TFBS identified by aligning positional weight matrices that describe the binding site and employ either (i) a P-value threshold for accepting a site, (ii) an over-representation measure of neighboring sites, or (iii) conservation with the mouse genome and application of P-value thresholds. The results show that the best recognition of TFBS is achieved by combining the identification of TFBS in regions of human-mouse conservation and also by applying a high stringency P-value to the TFBS identified in non-coding regions that are not conserved. Additionally, we find that only half of the 481 experimentally mapped sites can be found in sequence regions conserved with mouse, but the predictive power of the binding site identification method is up to threefold higher in the conserved regions. PMID- 12370782 TI - Differential gene expression analysis during porcine hepatocyte spheroid formation. AB - Primary porcine hepatocytes cultured in suspension self-assemble into multicellular aggregates or spheroids that display enhanced liver-specific functional capability and remain viable for an extended period of time in vitro. The molecular events underlying the process of spheroid formation were explored by differential gene expression analysis. Critical time points in spheroid formation were first identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of stress-related gene expression levels at different stages of spheroid formation. Suppression subtractive hybridization was used to identify transcripts up- or down-regulated at different stages of spheroid formation. Subsequently, three sets of reciprocal subtractions, comparing freshly isolated hepatocytes, spheroid-forming hepatocytes, and mature spheroids were carried out, and differentially expressed transcripts were isolated, cloned, sequenced, and annotated. A total of 65 genes and 14 novel transcripts were identified as differentially expressed, and very high sequence conservation between pig and human transcripts was observed. The resultant expressed sequence tags (ESTs) revealed a rapid decrease in the transcript levels of a subset of liver-specific genes, cytochrome P450s, and enzymes involved in heme biosynthesis, as well as up-regulation of genes involved in calcium-dependent vesicle trafficking and a number of acute-phase proteins in mature spheroids. Previous morphological and functional data on hepatocyte spheroid formation support cellular polarization of the hepatocyte into apical and basolateral domains in spheroids. This is important for the re-emergence of differentiated functions in vitro and is reflected by differences in gene expression patterns. PMID- 12370783 TI - Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome. AB - The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo FISH on ECA7 and ECA13p. PMID- 12370784 TI - Genetic mapping of the (G)-locus, responsible for the coat color phenotype "progressive greying with age" in horses (Equus caballus). PMID- 12370785 TI - Comparison of human and mouse Fuc-TX and Fuc-TXI genes, and expression studies in the mouse. PMID- 12370786 TI - Harbinger or hermit? Pancreatic anatomy and surgery through the ages--part 3. PMID- 12370787 TI - Reconstructive procedure after distal gastrectomy for gastric cancer that best prevents duodenogastroesophageal reflux. AB - Billroth I and II reconstructions are commonly performed after distal gastrectomy. Both may cause duodenogastric and duodenogastroesophageal reflux, conditions reported to have carcinogenetic potential. The aim of this study was to investigate which reconstructive procedure would most effectively prevent bile reflux into the gastric remnant and esophagus after distal gastrectomy. A group of 92 patients who underwent curative distal gastrectomy for gastric cancer were subjected and classified into three groups retrospectively by the reconstructive procedure undertaken: group A, Roux-en-Y (Roux-Y) reconstruction (n = 29); group B, Billroth I reconstruction (n = 41); group C, Billroth II reconstruction (n = 22). The bile reflux periods (percent time) for the gastric remnant and esophagus were measured with the Bilitec 2000 under standardized conditions. The percent time for the gastric remnant was significantly less in group A than in group B or C. In 61% of all patients, bile reflux into the esophagus was found to be more than 5.0% of the time; it was less in group A than in group B or C (p = 0.057). A questionnaire revealed a good correlation between the incidence of reflux symptoms and the percent time for the gastric remnant and esophagus. Roux-Y reconstruction is superior to either Billroth I or II reconstruction for preventing bile reflux into the gastric remnant and esophagus after distal gastrectomy. PMID- 12370790 TI - Aortic aneurysm after patch aortoplasty for coarctation: analysis of patch size and wall growth. AB - Aortic aneurysm may develop after surgery for coarctation of aorta especially patch aortoplasty. The size of patch and of adjacent native aortic wall was analyzed to determine whether aortic dilatation represents a true aneurysm. Electron beam tomography (EBT) was done on 19 patients, three months to 17.5 years after patch aortoplasty. Tomograms of aorta were obtained in 6-mm slices, and maximal cross-sectional area was digitized to obtain: total circumference (Ct), patch component (Cp), and aortic wall component (Cw). Ct, Cp, and Cw were normalized to the circumference of distal aorta (Cda) as: isthmus/distal aorta (Ct/Cda), patch segment/distal aorta (Cp/Cda), wall segment/distal aorta (Cw/Cda). Ct/Cda ranged from 109% to 260%. In 12 patients (group A), it varied from 168% to 260%; and in seven (group B), 109% to 133%. There was strong correlation (r = 0.92) between Ct/Cda and Cp/Cda. Ct/Cda, Cp/Cda, and Cp/Cw were higher in group A than B (p <0.001) but Cw/Cda did not differ. Cw/Cda was greater than the coarctation/distal aorta diameter ratios of preoperative angiograms, consistent with accelerated aortic wall growth postsurgery. No definite aneurysm was seen. Localized dilatation of aorta following patch aortoplasty in children is primarily due to a large synthetic patch and, partly, to increased aortic wall growth. Serial EBT or magnetic resonance imaging is indicated to monitor aortic wall growth and occurrence of aneurysm. PMID- 12370788 TI - Role of nitric oxide in the internal anal sphincter of Hirschsprung's disease. AB - It is not clear what contribution the internal anal sphincter (IAS) makes to the impaired motility observed in patients with Hirschsprung's disease (HD). Nitric oxide (NO) has recently been shown to be a neurotransmitter in the nonadrenergic noncholinergic (NANC) inhibitory nerves in the human gut. To clarify the physiologic significance of NO in the IAS of HD (aganglionosis), we investigated the enteric nerve responses on lesional (aganglionic) and normal IAS muscle strips above the dentate line. Lesional and normal IAS muscle strips above the dentate line were derived from patients with HD (10 cases) and patients who underwent rectal amputation for low rectal cancer (12 cases). A mechanographic technique was used to evaluate in vitro muscle responses to electrical field stimulation (EFS) before and after treatment with various autonomic nerve blockers, N(G)-L-nitroarginine, and L-arginine. The following results were obtained: (1) Cholinergic nerves are mainly involved in the regulation of enteric nerve responses to EFS in the normal IAS. (2) The aganglionic IAS of patients with HD was more strongly innervated by cholinergic nerves than the normal IAS (p < 0.05). (3) NANC inhibitory nerves were found to act on the normal IAS but had no effect on the enteric nerves in patients with aganglionosis. (4) NO was found to act on normal IAS, but no effect was observed in the aganglionic IAS. These findings suggest that innervation of the cholinergic nerves and a loss of NO mediation of NANC inhibitory nerves play an important role in the impaired motility observed in the IAS with HD. PMID- 12370791 TI - Balloon dilatation of aortic stenosis in infants younger than 6 months of age: intermediate outcome. AB - The objectives of this study were to review the outcome of balloon dilatation of critical/severe aortic valve stenosis in patients younger than 6 months of age, with particular emphasis on subdivisions of age at intervention, and to identify factors that might influence outcome. From 1988 to 1998, 42 patients underwent dilatation. Patients were divided into three groups (group 1: 1-7 days, n = 16; group 2: 8-30 days; n = 10; group 3: 1-6 months, n = 16). Medical records and echocardiograms were reviewed retrospectively for presentation, clinical course, and left ventricular, aortic valve, and Doppler flow parameters. Median follow-up was 53 months (range, 6 months to 10 years). Of 16 group 1 patients, 11 (70%) had, respiratory distress requiring ventilator support, 12 (80%) received prostaglandin, and 5 (30%) received inotropic support. Nine (56.2%) patients died and 7 (44%) required reintervention. Of 10 group 2 patients, 4 (40%) were ventilated, 2 (20%) received prostaglandin, and 3 (30%) received inotropic support. Three (30%) patients died and 5 (50%) required reintervention. Of 16 group 3 patients, only 1 had symptoms (respiratory distress) at presentation. One (6%) patient died and 4 (15%) required reintervention. The overall actuarial survival rate at 10 years was 72% (88% at 10 years for indexed aortic annulus > 25 mm/m2. Freedom from reintervention was 70% and 21% at 5 and 10 years, respectively (80 and 33% at 5 and 10 years, respectively, for indexed aortic annulus > 25 mm/m2). The actuarial survival rates at 10 years for groups 1, 2, and 3 were 42%, 65%, and 93%, respectively. Predictors of death included young age at presentation, and multivariate analysis of left heart measures yielded an 83% positive prediction of outcome. An improved chance of survival was associated with indexed aortic valve annulus > 25 mm/m2. Patients with critical aortic stenosis who require balloon dilatation within the first month of life, but especially within the first week, have a poorer outcome than those requiring the procedure later, and this can be accounted for by a tendency toward less favorable anatomical features. Many will require repeat intervention. PMID- 12370792 TI - Silent and audible persistent ductus arteriosus: an angiographic study. AB - Persistent ductus arteriosus (PDA) murmurs become silent probably due to the direction of the jet across the ductus arteriosus when entering the pulmonary artery. Out of 15 children with silent PDA, 14 demonstrated a ductal flow not contacting and away from the anterior wall of the main pulmonary artery. In 15 children with a continuous murmur caused by a PDA, 12 exhibited a ductal flow toward and reaching the anterior wall of the MPA. There was no correlation between the presence of a murmur and the size of the arterial duct in this study. PMID- 12370793 TI - A new on-line method for predicting aortic root dilatation during two-dimensional echocardiography in pediatric patients with Marfan syndrome using the sinus of valsalva to annulus ratio. AB - The objective of this study was to determine if calculation of the sinus of Valsalva to annulus ratio (SOV/Ann) effectively screens for root dilatation (ARD) during two-dimensional (2D) echocardiography in young patients (1 month-15 years) with suspected or confirmed Marfan syndrome. The aortic root was imaged using 2D echocardiography (parasternal long-axis view) and the SOV/Ann ratio calculated/analyzed in 27 Marfan patients and 45 age-matched controls. All controls had a SOV/Ann ratio <1.45, and 82% of Marfan's with ARD had a SOV/Ann ratio 1.45. Five of the Marfan patients with ARD had a SOV/Ann ratio < 1.45 due to normalization of the SOV/Ann ratio because of pathologic annular enlargement. Determination of the SOV/Ann ratio allows rapid on-line screening for aortic root dilatation during 2D echocardiography. A SOV/Ann ratio 1.45 predicted ARD in young Marfan patients with a high degree of accuracy (sensitivity = 0.82, specificity = 1.00). Screening for ARD using the SOV/Ann ratio fails when annular dilatation has progressed to the degree that the SOV/Ann ratio normalizes (SOV/Ann < 1.45). In this subgroup of patients, determination of aortic root dilatation will have to be evaluated by standard methods. PMID- 12370794 TI - Use of intravenous amiodarone for postoperative junctional ectopic tachycardia in children. AB - To assess the efficacy and safety of intravenous (IV) amiodarone for the treatment of postoperative junctional ectopic tachycardia (JET) in children, we retrospectively reviewed 11 patients treated with IV amiodarone for JET between 1/92 and 2/00. Data included heart rate and hemodynamics pre- and post amiodarone, drug dosage, duration of therapy, and effect. Success was defined as reversion to sinus rhythm or slowing to a hemodynamically stable rate. The mean heart rate prior to amiodarone was 203 bpm, and the mean systolic blood pressure was 64 mmHg. Mean IV amiodarone loading dose was 8.2 +/- 4.0 mg/kg, followed by an infusion in 7 patients at a dose of 12.9 +/- 3.9 mg/kg/day for a duration of 74.3 +/- 46.9 hours. At 1 hour post-load, mean heart rate was 147 bpm and mean systolic blood pressure was 88 mmHg for the group. Three patients were in sinus rhythm, 4 in intermittent sinus rhythm with accelerated junctional rhythm, and 4 patients solely accelerated junctional rhythm. Control of JET persisted in 9 patients. Of the two patients requiring additional treatment, both had received a 5 mg/kg load and neither was on an infusion. Five patients were paced at some point following amiodarone: four to improve hemodynamics and one for late sinus bradycardia. Side effects included hypotension with loading (1) and late sinus bradycardia (1). One patient was discharged on oral amiodarone. Intravenous amiodarone given in doses of 10 mg/kg in two 5 mg/kg increments, followed by an infusion of 10-15 mg/kg/day for 48-72 hours, appears to be safe and effective for postoperative JET in patients who fail conventional therapy or who are hemodynamically unstable. Long-term oral therapy is usually not necessary. PMID- 12370795 TI - Estrogen and "exercise" have a synergistic effect in preventing bone loss in the lumbar vertebra and femoral neck of the ovariectomized rat. AB - This study was designed to study the individual or combined effects of estrogen and bipedal stance "exercise" on the lumbar vertebral body (LVB) and femoral neck (FN). At 6 months of age, six rats were sacrificed as baseline controls and all the others were either bilateral sham-ovariectomized or ovariectomized (OVX). Groups of OVX rats were housed in normal height cage (NC, 28 cm) or raised height cages (RC, 33 cm) and received biweekly s.c. injections of 10 microg/kg 17 beta estradiol (E2) or vehicle for 4 and 8 weeks. Histomorphometric measurements were performed on the undecalcified mid-transverse sections of the 4th LVB and FN. Ovariectomy alone induced cancellous bone loss by 21% and 39% in the LVB and FN, respectively; intracortical porosity area of the FN increased by 108% while total bone area did not change significantly because of the periosteal expansion following OVX. E2 alone partially prevented cancellous bone loss in the LVB and FN and prevented increased intracortical porosity area in the FN by reducing eroded surface and activation frequency. RC alone partially prevented the decrease of cancellous bone in the LVB and FN by reducing the bone-eroded surface but increased wall width. E2 plus RC completely preserved cancellous bone by having an additive effect on reducing eroded surface and activation frequency. RC helped to partially prevent decreased periosteal bone formation after estrogen administration. In conclusion, apart from inducing cancellous bone loss in the LVB and FN, OVX also increased intracortical remodeling in the FN. Estrogen prevented the overall activation of remodeling space induced by OVX. Apart from having similar effects as estrogen on remodeling space, RC induced positive bone balance within each remodeling unit. Combination treatment increased total bone mass beyond that of sham-control level by having an additive effect on lowering bone remodeling and increasing wall in both the LVB and FN. PMID- 12370796 TI - Magnesium deficiency: effect on bone and mineral metabolism in the mouse. AB - Insufficient dietary magnesium (Mg) intake has been associated in humans with low bone mass. Mg deficiency in the rat has suggested bone loss is due to increased bone resorption and/or inadequate bone formation during remodeling. The purpose of this study was to assess the effect of a low Mg diet on bone and mineral metabolism in the young and mature BALB/c mouse and explore the hypothesis that inflammatory cytokines may contribute to Mg deficiency-induced osteoporosis. Using an artificial diet, we induced targeted Mg depletion (0.002% Mg) with all other nutrients maintained at the normal level. In all Mg-depleted mice, hypomagnesemia developed and skeletal Mg content fell significantly. The serum Ca in Mg-deficient mice was higher than in control mice; however, serum PTH levels were not significantly different. Osteoprotegerin (OPG) in dosages that inhibit osteoclastic bone resorption did not prevent hypercalcemia in Mg-deficient animals. No significant difference in serum Ca was observed between groups when dietary Ca was reduced by 50%, suggesting that a compensatory increase in intestinal absorption might account for the hypercalcemia. Growth plate width decreased 33% in young Mg-deficient animals and chondrocyte columns decreased in number and length, suggesting that Mg deficiency reduced bone growth. Trabecular bone volume in the metaphysis of the tibia in these animals was decreased and osteoclast number was increased by 135%. Osteoblast number was significantly reduced. Immunohistochemistry revealed that substance P increased 230% and 200% in megakaryocytes and lymphocytes, respectively, after 1 day of Mg depletion. IL 1 increased by 140% in osteoclasts by day 3 and TNF alpha increased in osteoclasts by 120% and 500% in megakaryocytes on day 12. This study demonstrates a profound effect of Mg depletion on bone characterized by impaired bone growth, decreased osteoblast number, increased osteoclast number in young animals, and loss of trabecular bone with stimulation of cytokine activity in bone. PMID- 12370797 TI - A new bone-healing material: a hyaluronic acid-like bacterial exopolysaccharide. AB - Critical size defect (CSD) technique was used to evaluate the bone regeneration capacity of a newly discovered hyaluronic acid-like exopolysaccharide synthesized by a bacteria originating from a deep sea hydrothermal vent. A 5 mm-diameter hole was made on each parietal bone of male rats. The right hole was filled with either a new bacterial exopolysaccharide referenced HE 800 or with collagen used as negative control, while the left hole remained free of any treatment. After 15 days, the holes and surrounding tissues were examined by direct examination, X ray films, and histological staining. Using HE 800, bone healing was almost complete after only 15 days, with osteoblasts onto lying external bone surfaces and enhancing osteocyte inclusion. Neovascularization was also observed along with an organized trabecular bone. No abnormal bone growth or conjunctival abnormalities were noticed. At the end of the experiment, 95.9% (+/-6.2) bone healing (n = 20) was observed. Conversely, the collagen-treated animals did not demonstrate significant healing-17.8% (+/-18.1). PMID- 12370798 TI - Randomized, double-blind, clinically controlled trial of intranasal calcitonin treatment in patients with hip fracture. AB - The objective of this study was to evaluate the short-term outcome of intranasal calcitonin treatment of elderly hip fracture patients on pain, bone loss, functional recovery, and length of hospital stay. In addition, we wanted to compare the effect of calcitonin with placebo on fusion of hip fractures treated with internal fixation using a screw or a nail. In a randomized, double-blind, clinically controlled trial, 260 independently living patients (aged 65 years or older) with acute hip fracture were randomly assigned to intranasal calcitonin 200 IU daily for 3 months or matching placebo nasal spray. Analyses were completed on an intention-to-treat basis. Three months after the operation, the median intensity of pain in visual analog scale was 0 mm (IQR 0.20) in the calcitonin group and 4 mm (IQR 0.33) in the placebo group (P = 0.15). The mean change in calcaneal bone mineral density from baseline to 3 months was not statistically significant between the groups -0.004 (95% CI -0.008 to -0.001) in the calcitonin group and -0.007 (95% CI -0.012 to -0.003) in the placebo group (P = 0.28). There were no significant differences in mortality, side effects, length of hospital stay, and functional recovery. Among patients with internal fixation using a screw or a nail (n = 99), fusion of the fracture was observed in an X-ray 3 months after the operation in 84% in the calcitonin group and in 63% in the placebo group (P = 0.029, difference 20% [95% CI 2 to 39]). We conclude that intranasal calcitonin might be useful for hip fracture patients but the clinical significance of this finding needs to be confirmed by studies with more participants, a longer treatment period, a longer follow-up, and perhaps a higher dose of calcitonin. PMID- 12370799 TI - Structure analysis of high resolution magnetic resonance imaging of the proximal femur: in vitro correlation with biomechanical strength and BMD. AB - The purpose of this study was to use high resolution magnetic resonance imaging (HR-MRI) combined with structure analysis to investigate the trabecular structure of the human proximal femur and to compare this technique with bone mineral density (BMD) using dual energy X-ray absorptiometry (DXA) in the prediction of bone strength in vitro. Thirty-one fresh human proximal femur specimens were examined with HR-MRI using a T1-weighted 3D spinecho-sequence in a coronal plane (voxel size: 0.195 x 0.195 x 0.9 mm and 0.195 x 0.195 x 0.3 mm). In these images structure parameters analogous to standard bone histomorphometry were obtained in a femoral head, neck, and trochanteric region of interest (ROI). In addition, BMD measurements were obtained using DXA and finally, all specimens were tested biomechanically in a materials testing machine, and maximum compressive strength (MCS) was determined. Correlations between BMD and MCS were significant (p <0.01) with R-values up to 0.74. Correlating structure parameters and MCS R-values up to 0.69 (P <0.01) were obtained. Using multivariate regression analysis, combining structure parameters and BMD, improved correlations versus MCS substantially (up to R = 0.93; P <0.01). In conclusion, this study showed that in an experimental setting, structure parameters determined in high resolution MR images of the proximal femur correlated significantly with bone strength. The highest correlations, however, were obtained combining BMD and structure measures. PMID- 12370800 TI - Effect of alendronate on periprosthetic bone loss after total knee arthroplasty: a one-year, randomized, controlled trial of 19 patients. AB - Undesired bone loss around implants is considered to occur mainly because of a stress-shielding phenomenon. Bone surrounding the total knee arthroplasty (TKA) adjusts its mineral density and structure to meet new mechanical demands. Immobilization, in combination with local operative trauma to the bone and soft tissues, has an additional impact on bone loss. The clinical survival of TKA is associated with the quality and quantity of the surrounding bone environment. Poor bone quality and quantity may predispose to aseptic implant loosening and periprosthetic fractures. We investigated the efficacy of oral bisphosphonate (alendronate, Fosamax) with calcium (Calcichew) for the inhibition of early bone mineral density (BMD) loss after TKA in a prospective, randomized, one-year follow-up study. Periprosthetic BMD changes were measured with fan-beam dual energy X-ray absorptiometry (DXA) in 19 patients with knee osteoarthrosis. Patients (n = 8) treated with 10 mg alendronate and 500 mg calcium daily maintained distal femoral BMD values close to the baseline values (P > 0.04), while patients receiving only 500 mg of calcium daily (n = 11) showed significant bone loss during the one-year follow-up (P < 0.015). The treatment groups differed significantly in metaphyseal anterior, posterior, diaphyseal, and metaphyseal total regions of interest (ROIs) (repeated measures ANOVA analyses, P = 0.019, P = 0.010, P = 0.022, and P = 0.024, respectively). Our results indicate that oral alendronate reduces early postoperative periprosthetic bone loss significantly. This therapeutic strategy may improve the results and longevity of primary total knee arthroplasties. PMID- 12370801 TI - Ultrastructure of basement membranes in developing shark tooth. AB - Based on studies of the tooth of largely mammalian species, the dental basement membranes are shown to be specialized for various roles significant in the development and maintenance of the tooth. Comparative studies with the nonmammalian tooth will facilitate further clarification of the mechanisms of mammalian tooth formation. In this study, basement membranes of the shark tooth in successive developmental stages was ultrastructurally examined for elucidation of their roles in odontogenesis. Teeth of a shark, Cephaloscyllium umbratile, were processed for thin section electron microscopy. Throughout the developmental stages the lamina densa of the basement membrane was made up of a fine network of "cords," irregular anastomosing strands known to be the major component of mammalian basement membranes. In the presecretory stage of the shark tooth, dental papilla cells were immobilized for their differentiation into odontoblasts by means of the binding of their processes to numerous narrow extensions of the lamina densa of the inner dental epithelium. In the secretory stage, a number of cords of the widened lamina densa were extended towards and bound to tubular vesicles of the forming enameloid. During the mineralization stage, fragments of the degrading enameloid matrix appeared to be moving through the lamina densa to the epithelial cells for processing. In the maturation stage, half of the lamina densa facing the enameloid was mineralized forming an advancing edge of mineralization of the enameloid. It provided strong binding and smooth transition of organic to mineral phase which may allow transportation of substances across the phases for enameloid maturation in a way similar to that reported in the mammalian tooth. These observations indicate that basement membranes of the developing shark tooth, as those in the mammalian tooth, play various roles, including anchoring, firm binding, and possible mediation of the transport of substances that are known to be vital for the development of the tooth. PMID- 12370802 TI - The role of prenylated small GTP-binding proteins in the regulation of osteoclast function. AB - The Ras superfamily of small GTP-binding proteins (also known as small GTPases) comprises more than 80 highly conserved proteins of the Ras, Rho, and Rab subfamilies that are involved in multiple intracellular signalling pathways. These proteins are able to function as molecular switches in the transduction of signals from membrane receptors by cycling between an inactive, GDP-bound state and an active, GTP-bound state, which can then interact with a number of different effector molecules (Fig. 1). The activity of small GTPases is regulated by three classes of regulatory proteins: guanine nucleotide exchange factors (GEFs), which catalyse the exchange of GDP for GTP, thereby activating the small GTPase; GTPase-activating proteins (GAPs), which enhance the intrinsic ability of small GTPases to hydrolyse GTP, resulting in reversion to the inactive GDP-bound state; and guanine nucleotide dissociation inhibitors (GDIs), which preferentially bind to the GDP-bound GTPases in the cytoplasm, thereby inhibiting the release of GDP and maintaining the GTPase in the inactive state [1]. GDIs have not been identified for all small GTPases, but play an important role in the control of the Rho family GTPases. PMID- 12370803 TI - Jak2 is involved in c-Myc induction by Bcr-Abl. AB - We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of Bcr-Abl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by Bcr-Abl. We treated the 32Dp210 Bcr Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bbeta is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bbeta. Expression of the SH2-Bbeta R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in c-Myc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC RNA expression but also interferes with proteasome-dependent degradation of c-Myc protein. PMID- 12370804 TI - A role for TGF-beta in estrogen and retinoid mediated regulation of the nuclear receptor coactivator AIB1 in MCF-7 breast cancer cells. AB - AIB1 (amplified in breast cancer 1) is a nuclear receptor coactivator gene amplified and overexpressed in breast cancer. However, the mechanisms by which AIB1 is regulated are unclear. Here we show that 17beta-estradiol represses AIB1 mRNA and protein expression in MCF-7 human breast cancer cells primarily by suppressing AIB1 gene transcription. Estrogen levels present in fetal calf serum are sufficient to maintain AIB1 mRNA and protein at low basal levels, and this repression is reversed by the addition of antiestrogens or all-trans retinoic acid. Interestingly, cycloheximide inhibition experiments revealed that secondary protein synthesis was necessary to induce AIB1 expression by antiestrogens and retinoids. Experiments with TGF-beta and TGF-beta blocking antibodies demonstrated that this growth factor modulates AIB1 expression and showed that the antiestrogen and retinoid induction of AIB1 gene expression is mediated at least in part through TGF-beta. These data reveal a mechanism of estrogen-induced down-modulation of the overall hormone sensitivity of cells through feedback inhibition of coactivator gene expression. These data also suggest that antiestrogens can shift the sensitivity of cells to non-estrogenic proliferative signaling by increasing cellular levels of AIB1. This effect may play a role in breast cancer progression and resistance to drug treatment. PMID- 12370805 TI - Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. AB - The Runx family of transcription factors plays pivotal roles during normal development and in neoplasias. In mammals, Runx family genes are composed of Runx1 (Pebp2alphaB/Cbfa2/Aml1), Runx2 (Pebp2alphaA/Cbfa1/Aml3) and Runx3 (Pebp2alphaC/Cbfa3/Aml2). Runx1 and Runx3 are known to be involved in leukemogenesis and gastric carcinogenesis, respectively. Runx2, on the other hand, is a common target of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) and plays an essential role in osteoblast differentiation. Runx2 is induced by the receptor-activated Smad; Runx2 mediates the blockage of myogenic differentiation and induces osteoblast differentiation in C2C12 pluripotent mesenchymal precursor cells. However, Smad does not directly induce Runx2 expression; an additional step of de novo protein synthesis is required. Here we report that Smad-induced junB functions as an upstream activator of Runx2 expression. Furthermore, not only the Smad pathway but also the mitogen-activated protein kinase (MAPK) cascades are involved in the induction of Runx2 by TGF-beta1 and BMP-2. Our results demonstrate that following TGF-beta and BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. PMID- 12370806 TI - Mathematical modeling of noise and discovery of genetic expression classes in gliomas. AB - The microarray array experimental system generates noisy data that require validation by other experimental methods for measuring gene expression. Here we present an algebraic modeling of noise that extracts expression measurements true to a high degree of confidence. This work profiles the expression of 19 200 cDNAs in 35 human gliomas; the experiments are designed to generate four replicate spots/gene with switching of probes. The validity of the extracted measurements is confirmed by: (1) cluster analysis that generates a molecular classification differentiating glioblastoma from lower-grade tumors and radiation necrosis; (2) By what other investigators have reported in gliomas using paradigms for assaying molecular expression other than gene profiling; and (3) Real-time RT-PCR. The results yield a genetic analysis of gliomas and identify classes of genetic expression that link novel genes to the biology of gliomas. PMID- 12370807 TI - Paracrine cyclooxygenase-2-mediated signalling by macrophages promotes tumorigenic progression of intestinal epithelial cells. AB - In human colorectal adenomas or polyps, cyclooxygenase-2 is expressed predominantly by stromal (or interstitial) macrophages. Therefore, we tested the hypothesis that macrophage cyclooxygenase-2 has paracrine pro-tumorigenic activity using in vitro models of macrophage-epithelial cell interactions. We report that macrophages can promote tumorigenic progression of intestinal epithelial cells (evidenced by decreased cell-cell contact inhibition, increased proliferation and apoptosis, gain of anchorage-independent growth capability, decreased membranous E-cadherin expression, up-regulation of cyclooxygenase-2 expression, down-regulation of transforming growth factor-beta type II receptor expression and resistance to the anti-proliferative activity of transforming growth factor-beta(1)) in a paracrine, cyclooxygenase-2-dependent manner. Pharmacologically relevant concentrations (1-2 microM) of a selective cyclooxygenase-2 inhibitor had no detectable, direct effect on intestinal epithelial cells but inhibited the macrophage-epithelial cell signal mediating tumorigenic progression. Cyclooxygenase-2-mediated stromal-epithelial cell signalling during the early stages of intestinal tumorigenesis provides a novel target for chemoprevention of colorectal cancer (and other gastro-intestinal epithelial malignancies, which arise on a background of chronic inflammation, such as gastric cancer) and may explain the discrepancy between the concentrations of cyclooxygenase inhibitors required to produce anti-neoplastic effects in vitro and in vivo. PMID- 12370808 TI - Functional interaction of Sam68 and heterogeneous nuclear ribonucleoprotein K. AB - Sam68 is a target of the c-Src tyrosine kinase. We previously showed that overexpression of Sam68 functionally substitutes for, as well as synergies with, HIV-1 Rev in Rev-response element (RRE)-mediated gene expression and virus replication. Here we describe the identification of heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a protein that specifically interacts with Sam68 in vitro and in vivo. HnRNP K did not bind to RRE-RNA directly, but formed a super complex with Sam68 and RRE in vitro. RNase treatment did not change the strength of binding of hnRNP K to Sam68. We demonstrated that hnRNP K significantly inhibited Sam68-mediated, but not Rev-mediated, RRE-dependent gene expression. We further showed that Sam68, but not a non-functional mutant Sam68p21, inhibited transcriptional activation of CT element by hnRNP K. Interestingly, the Sam68p21 with a single amino acid substitution in the nuclear localization domain exhibited less affinity for hnRNP K in vitro. We propose that the direct interaction of Sam68 and hnRNP K adversely affect the activities of both proteins in signal transduction pathways of both transcriptional and post transcriptional events. PMID- 12370809 TI - The ferredoxin reductase gene is regulated by the p53 family and sensitizes cells to oxidative stress-induced apoptosis. AB - The p53 tumor suppressor protein, a transcription factor, induces cell cycle arrest and apoptosis via the upregulation of downstream target genes. Ferredoxin Reductase (protein, FR; gene, FDXR) transfers electron from NADPH to cytochrome P450 via ferredoxin in mitochondria. Here, we identified FDXR as a target gene of the p53 family, that is, p53, p63, and p73. We found that FDXR can be induced by DNA damage in cells in a p53-dependent manner and by a mutated form of p53 that is competent in inducing apoptosis. In addition, we identified a p53 response element located within the FDXR promoter that is responsive to wild-type p53, p63alpha, p63gamma, p73alpha, and p73beta. Furthermore, we showed that p53, p63alpha and p73alpha directly bind to the p53 response element in vivo and promote the accessibility of the FDXR promoter by increasing the acetylation of histones H3 and H4. To determine the role of FR in p53 tumor suppression, we generated cell lines that express FR using a tetracycline-regulated promoter. We found that over-expression of FR in lung H1299, breast MCF7, and colorectal HCT116 carcinoma cells have no effect on cell proliferation. However, we showed that FR increases the sensibility of H1299 and HCT116 cells to 5-fluorouracil-, doxorubicin- and H(2)O(2)- mediated apoptosis. Our data support a model of feed forward loop for p53 activity, that is, various cellular stresses, including reactive oxygen species (ROS), activate p53, which induces the expression of FDXR; and the FDXR gene product, FR, in turn sensitizes cells to ROS-mediated apoptosis. PMID- 12370810 TI - The distinct capacity of Fyn and Lck to phosphorylate Sam68 in T cells is essentially governed by SH3/SH2-catalytic domain linker interactions. AB - Sam68 phosphorylation correlates with Fyn but not Lck expression in T cells. This substrate has been used here to explore the possible basis of the specificity of Fyn versus Lck. We show that this specificity is not based on a spatial segregation of the two kinases, since a chimeric Lck molecule containing the membrane anchoring domain of Fyn does not phosphorylate Sam68. Moreover, a Sam68 molecule targeted to the plasma membrane by the farnesylation signal of c-Ha-Ras remains poorly phosphorylated by Lck. In T cells, Fyn appears to be the active Src kinase in rafts, but Sam68 is not expressed in rafts, and its distinct phosphorylation by Fyn and Lck is not affected by raft dispersion. The Fyn/Lck specificity does not reflect a higher kinase activity of Fyn in general, as both Fyn and Lck are similarly recognized by an anti-active Src antibody. Both also strongly phosphorylate another Src substrate in vivo. Mainly, Lck phosphorylates Sam68 when the interaction between the SH3 domain and the SH2-catalytic domain linker is altered in heterologous Src molecules or after mutating key residues in the linker that increase the accessibility of the SH3 domain. Thus, the distinct potential of Fyn and Lck to phosphorylate Sam68 is likely controlled by the interaction of the kinase SH3 domain with the linker and Sam68, possibly on a competitive binding basis. PMID- 12370811 TI - Functional analysis of cyclin D2 and p27(Kip1) in cyclin D2 transgenic mouse mammary gland during development. AB - Two mammary gland phenotypes were detected in pregnant MMTV-cyclin D2 transgenic mice; line D2-53 exhibited a lack of alveologenesis and failure to nurse, whereas line D2-58 featured a reduction in alveologenesis, but retained normal nursing behavior. In pregnant mammary glands, cyclin D2 protein levels were twofold (P<0.107) and 3.8-fold (P<0.0076) higher in line D2-58 and D2-53, respectively, compared to wild type. Concomitantly with the increase in cyclin D2 was a fivefold decrease in cyclin D1 hyper-phosphorylated isoform in mammary glands of pregnant cyclin D2-58 mice. Because cyclin D1 is a critical molecule in normal mammary lobuloalveolar development, these data suggest that overexpression of cyclin D2 may block mammary lobuloalveolar development through inhibition of cyclin D1 phosphorylation. During mammary gland development, p27(kip1) protein level oscillated in a similar profile in wild type and cyclin D2 transgenic mice, but was consistently higher in the cyclin D2 mice suggesting that p27(kip1) functions downstream of cyclin D2. The ratio of p27(kip1)-cdk4/p27(kip1)-cdk2 was 6.5-fold (P<0.0003) higher in cyclin D2 mammary glands compared to wild type in pregnant animals. This ratio reversed to 2.2-fold (P<0.005) higher in wild type compared to cyclin D2 mammary glands in involution suggesting that overexpression of cyclin D2 moderately induced apoptosis during pregnancy but accelerated involution. Collectively, the effects of cyclin D2 overexpression on mammary gland development during pregnancy and involution are attributed to two major factors, altered p27(kip1) protein level and inhibition of cyclin D1 phosphorylation. PMID- 12370812 TI - Stabilization of p53 and transactivation of its target genes in response to replication blockade. AB - Although it is clear that p53 plays a pivotal role in G1/G2 checkpoints to conserve genomic integrity, its role in S phase checkpoint is less well understood. Recently, it has been reported that p53 is transcriptionally impaired even though it is stabilized during replication blockade. However, the mechanisms underlying this phenomenon are not known. In the present study, it has been shown that p53 accumulates and transactivates its target genes such as p21, gadd45 and bax in response to replication blockade in normal and cancer cells. Lack of transcriptional activation under similar conditions in cells lacking p53 shows that p53-target gene activation during replication blockade is indeed p53 dependent. Further, transactivation of p21 in response to replication blockade by hydroxyurea and aphidicolin is similar to that in response to ionizing radiation except that the latter is more immediate compared to the response to replication blockade. These findings suggest that impairment of transcriptionally active p53 in response to replication blockade is not a general phenomenon. PMID- 12370813 TI - Genomic instability driven by the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax. AB - The importance of maintaining genomic stability is evidenced by the fact that transformed cells often contain a variety of chromosomal abnormalities such as euploidy, translocations, and inversions. Gene amplification is a well characterized hallmark of genomic instability thought to result from recombination events following the formation of double-strand, chromosomal breaks. Therefore, gene amplification frequency serves as an indicator of genomic stability. The PALA assay is designed to measure directly the frequency with which a specific gene, CAD, is amplified within a cell's genome. We have used the PALA assay to analyse the effects of the human T-cell leukemia virus type I (HTLV I) oncoprotein, Tax, on genomic amplification. We demonstrate that Tax-expressing cells are five-times more likely to undergo gene amplification than control cells. Additionally, we show that Tax alters the ability of cells to undergo the typical PALA-mediated G(1) phase cell cycle arrest, thereby allowing cells to replicate DNA in the absence of appropriate nucleotide pools. This effect is likely the mechanism by which Tax induces gene amplification. These data suggest that HTLV-I Tax alters the genomic stability of cells, an effect that may play an important role in Tax-mediated, HTLV-I associated cellular transformation. PMID- 12370814 TI - Genomic instability in mouse Burkitt lymphoma is dominated by illegitimate genetic recombinations, not point mutations. AB - lambda-MYC-induced mouse Burkitt lymphoma (BL) harboring the shuttle vector pUR288, which includes a lacZ reporter gene to study mutagenesis, was employed to assess genomic instability associated with MYC deregulation. The frequency of lacZ mutations in lymphomas was elevated only 1.75-fold above that in normal tissue, indicating that mouse BL does not exhibit a phenotype of hypermutability. However, the nature of lacZ mutations was strikingly different in normal tissues and lymphomas. While point mutations comprised approximately 75% of the mutations found in normal tissues, apparent translocations, deletions and inversions constituted the majority of mutations ( approximately 65%) in lymphomas. Genomic instability in mouse BL thus seems characterized by a preponderance of illegitimate genetic rearrangements in the context of near-background mutant frequencies. SKY analyses of cell lines from primary BL tumors revealed substantial changes in chromosomal structure, confirming the lacZ studies. Bi allelic deletions of the tumor suppressor p16(Ink4a) were detected in six out of 16 cell lines, illustrating cellular selection of advantageous mutations. Together, these approaches indicate that MYC may contribute to lymphomagenesis through the dominant mutator effect of inducing chromosomal instability. The results further suggest that a phenotype of hypermutability (elevated mutant frequency) may not always be required for oncogenesis to occur. PMID- 12370815 TI - The interaction of HTLV-1 Tax with HDAC1 negatively regulates the viral gene expression. AB - Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are known to interact with several transcription factors and regulate their transcriptional activities. The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein activates transcription from its long terminal repeat (LTR) through interaction with cellular factors such as CREB and a transcriptional coactivator CBP/p300. However, little is known about the interaction between Tax and transcriptional repressors. Here, we demonstrate the physical and functional interaction between Tax and HDAC1. We found that HDAC1 represses the trans-activation function of Tax in 293T and MT4 cells. However, this repression was restored by treatment with an HDAC inhibitor, Trichostatin A. We also observed physical interaction between Tax and HDAC1 both in vitro and in vivo. The N-terminal region of HDAC1 (amino acid residues 28-97) was required for this binding. Moreover, HDAC1 inhibited the synergistic trans-activation of Tax observed on ectopic expression of CBP. However, this repression was relieved by overexpression of CBP. Thus, HDAC1 is likely to compete with CBP in binding with Tax and functions as a negative regulator for the transcriptional activation by Tax. PMID- 12370816 TI - Large-scale identification of novel potential disease loci in mouse leukemia applying an improved strategy for cloning common virus integration sites. AB - The identification of common virus integration sites (cVIS) in retrovirally induced tumors in mice provides a powerful strategy to isolate novel transforming genes. Applying virus LTR-specific inverse-PCR and RT-PCR combined with automated sequencing on CasBr-M Murine Leukemia Virus (MuLV) induced myeloid leukemias, 126 virus integration sites were cloned. Using locus- and LTR-specific primers, a nested-PCR/Southern-blotting procedure was developed on genomic DNA from a large panel of MuLV-induced leukemias, to analyse whether a particular virus insertion represented a cVIS. In fact 39 out of 41 integrations analysed this way appeared to represent a common virus integration. We recognized six previously cloned cVISs, i.e. Evi1, Hoxa7, c-Myb, Cb2/Evi11, Evi12, and His1 and 33 novel common insertions, designated Cas-Br Virus Integration Site (Casvis). Among this group we found integrations in or near genes encoding nuclear proteins, e.g. Dnmt-2, Nm23-M2, Ctbp1 or Erg, within receptor genes, e.g. Cb2 or mrc1, novel putative signaling or transporter genes, the ringfinger-protein gene Mid1 and a panel of genes encoding novel proteins with unknown function. The finding that 39 out of 41 integrations analysed represented a cVIS, suggests that the majority of the other virus insertions that were not yet analysed by the PCR/Southern-blotting method are located in a cVIS as well and may therefore also harbor novel disease genes. PMID- 12370817 TI - Identification of Src transformation fingerprint in human colon cancer. AB - We used a classical rodent model of transformation to understand the transcriptional processes, and hence the molecular and cellular events a given cell undergoes when progressing from a normal to a transformed phenotype. Src activation is evident in 80% of human colon cancer, yet the myriad of cellular processes effected at the level of gene expression has yet to be fully documented. We identified a Src 'transformation fingerprint' within the gene expression profiles of Src-transformed rat 3Y1 fibroblasts demonstrating a progression in transformation characteristics. To evaluate the role of this gene set in human cancer development and progression, we extracted the orthologous genes present on the Affymetrix Hu95A GeneChip (12k named genes) and compared expression profiles between the Src-induced rodent cell line model of transformation and staged colon tumors where Src is known to be activated. A similar gene expression pattern between the cell line model and staged colon tumors for components of the cell cycle, cytoskeletal associated proteins, transcription factors and lysosomal proteins suggests the need for co-regulation of several cellular processes in the progression of cancer. Genes not previously implicated in tumorigenesis were detected, as well as a set of 14 novel, highly conserved genes with here-to-fore unknown function. These studies define a set of transformation associated genes whose up-regulation has implications for understanding Src mediated transformation and strengthens the role of Src in the development and progression of human colon cancer. Supportive Supplemental Data can be viewed at http://pga.tigr.org/PGApubs.shtml. PMID- 12370818 TI - Cloning and characterization of the common fragile site FRA6F harboring a replicative senescence gene and frequently deleted in human tumors. AB - The common fragile site FRA6F, located at 6q21, is an extended region of about 1200 kb, with two hot spots of breakage each spanning about 200 kb. Transcription mapping of the FRA6F region identified 19 known genes, 10 within the FRA6F interval and nine in a proximal or distal position. The nucleotide sequence of FRA6F is rich in repetitive elements (LINE1 and LINE2, Alu, MIR, MER and endogenous retroviral sequences) as well as in matrix attachment regions (MARs), and shows several DNA segments with increased helix flexibility. We found that tight clusters of stem-loop structures were localized exclusively in the two regions with greater frequency of breakage. Chromosomal instability at FRA6F probably depends on a complex interaction of different factors, involving regions of greater DNA flexibility and MARs. We propose an additional mechanism of fragility at FRA6F, based on stem-loop structures which may cause delay or arrest in DNA replication. A senescence gene likely maps within FRA6F, as suggested by detection of deletion and translocation breakpoints involving this fragile site in immortal human-mouse cell hybrids and in SV40-immortalized human fibroblasts containing a human chromosome 6 deleted at q21. Deletion breakpoints within FRA6F are common in several types of human leukemias and solid tumors, suggesting the presence of a tumor suppressor gene in the region. Moreover, a gene associated to hereditary schizophrenia maps within FRA6F. Therefore, FRA6F may represent a landmark for the identification and cloning of genes involved in senescence, leukemia, cancer and schizophrenia. PMID- 12370819 TI - Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer. AB - The 3p21.3 tumour suppressor gene (TSG) RASSF1A is inactivated predominantly by promoter methylation and rarely by somatic mutations. Recently we demonstrated that epigenetic inactivation of RASSF1A is frequent in both clear cell and papillary adult renal cell carcinomas (even though 3p21.3 allele loss is rare in papillary tumours). Wilms' tumour is the most common childhood kidney tumour, but relatively little is known about its molecular pathogenesis. Thus TSGs such as WT1, p16(CDKN2a) and p53 are inactivated in only a minority of cases. In view of the involvement of RASSF1A in adult renal cancers we investigated RASSF1A as a candidate Wilms' TSG. We detected RASSF1A hypermethylation in 21 of 39 (54%) primary Wilms' tumours. 3p21.3 allele loss was not detected in nine informative Wilms' tumours (five with RASSF1A methylation). In contrast to RASSF1A, only a minority (10.3%) of Wilms' tumours demonstrated p16 promoter methylation. As chromosome 3p allele loss is frequent in colorectal cancer, we proceeded to investigate RASSF1A promoter methylation in colorectal cancer and detected RASSF1A methylation in 80% (4/5) colorectal cancer cell lines and 45% (13/29) primary colorectal cancers. There was no correlation between RASSF1A and p16 methylation in colorectal cancer. We have demonstrated that RASSF1A inactivation is the most frequent genetic or epigenetic event yet reported in Wilms' tumourigenesis and that allelotyping studies may fail to identify regions containing important TSGs. PMID- 12370820 TI - Oncogenic transformation by beta-catenin: deletion analysis and characterization of selected target genes. AB - Genetic analysis of beta-catenin-induced oncogenic transformation in chicken embryo fibroblasts (CEF) revealed the following prerequisites for oncogenicity: (1) removal of the N terminal phosphorylation sites targeted by glycogen synthase kinase 3beta (GSK3beta), (2) retention of the N terminal transactivation domain, and (3) retention of the armadillo repeats. The C terminal transactivation domain played an ancillary role in the transformation of CEF. There was a rough correlation between the transforming activity of various beta-catenin constructs and their transactivation of the TOPFLASH reporter. Expression levels of the candidate target genes of beta-catenin-LEF, cyclin D1 and myc were not correlated with each other or with the transforming activity of beta-catenin constructs. A new target gene, coding for inositol hexakisphosphate kinase 2 (IP6K2) was identified. Its expression showed concordance with the transforming activity of beta-catenin constructs. PMID- 12370821 TI - Tyr-863 phosphorylation enhances focal adhesion kinase autophosphorylation at Tyr 397. AB - Tyr-397 phosphorylation is important for focal adhesion kinase (FAK)-mediated signalling. In vitro FAK immunocomplex kinase experiments demonstrated that both FAK Tyr-576/577 and Tyr-863 phosphorylation regulated FAK Tyr-397 phosphorylation. While the former increased the intermolecular transphosphorylation activity of FAK, the latter was crucial for its cis phosphorylation. This observation was further supported by the reduced complex formation between Src and 3F-FAK (576F/577F/863F-FAK) as compared to that between Src and 576F/577F-FAK or Src and 863F-FAK. Regulation of cis- and transphosphorylation activities of FAK by such a differential tyrosyl phosphorylation mechanism is unprecedented. Furthermore, in fibronectin stimulated cells, both Tyr-576/577 and Tyr-863 phosphorylation could enhance FAK Tyr-397 phosphorylation. This observation implies that integrin-mediated FAK Tyr 397 phosphorylation was also regulated through both FAK cis- and transphosphorylation mechanisms. PMID- 12370822 TI - Roles of activated Src and Stat3 signaling in melanoma tumor cell growth. AB - Activation of protein tyrosine kinases is prevalent in human cancers and previous studies have demonstrated that Stat3 signaling is a point of convergence for many of these tyrosine kinases. Moreover, a critical role for constitutive activation of Stat3 in tumor cell proliferation and survival has been established in diverse cancers. However, the oncogenic signaling pathways in melanoma cells remain to be fully defined. In this study, we demonstrate that Stat3 is constitutively activated in a majority of human melanoma cell lines and tumor specimens examined. Blocking Src tyrosine kinase activity, but not EGF receptor or JAK family kinases, leads to inhibition of Stat3 signaling in melanoma cell lines. Consistent with a role of Src in the pathogenesis of melanoma, we show that c-Src tyrosine kinase is activated in melanoma cell lines. Significantly, melanoma cells undergo apoptosis when either Src kinase activity or Stat3 signaling is inhibited. Blockade of Src or Stat3 is also accompanied by down-regulation of expression of the anti-apoptotic genes, Bcl-x(L) and Mcl-1. These findings demonstrate that Src-activated Stat3 signaling is important for the growth and survival of melanoma tumor cells. PMID- 12370823 TI - Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo. AB - The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression. PMID- 12370824 TI - Negative regulation of urokinase-type plasminogen activator production through FGF-2-mediated activation of phosphoinositide 3-kinase. AB - Activation of phosphoinositide 3-kinase (PI3-kinase) is involved in many cellular responses. FGF-2 is one of the potent inducers of urokinase-type plasminogen activator (uPA) production in endothelial cells. However, little is known about the molecular mechanisms underlying FGF-2-mediated uPA production. Here we examined the signal transduction pathways involved in the regulation of uPA production by FGF-2-treatment. FGF-2 potently upregulated uPA production in murine brain capillary endothelial cells (IBE cells), as well as porcine aortic endothelial (PAE) cells and L6 myoblasts ectopically expressing FGFR1. PI3-kinase inhibitors, wortmannin and LY294002, both enhanced FGF-2-dependent uPA production by these cells. Stable expression of activated mutant p110alpha catalytic subunit of PI3-kinase into IBE cells decreased FGF-2-mediated uPA production, suggesting that PI3-kinase exhibited the negative regulatory effect on uPA production. No increase in FGF-2-induced PI3-kinase activity was observed in proteins immunoprecipitated by anti-phosphotyrosine antibody. Although stable expression of deleted mutant p85alpha regulatory subunit, which lacks association with p110 catalytic subunit, in IBE cells showed no dominant negative effect, transient expression of dominant negative Ras inhibited FGF-2-mediated PI3-kinase activation. These results suggest that only activated Ras contributed the FGF-2 mediated PI3-kinase activation. In cells stably expressing mutant p85alpha subunit, FGF-2 efficiently induced uPA production. Taken together, activation of PI3-kinase by FGF-2 is Ras-dependent and results in down-regulation of uPA production. PMID- 12370825 TI - Increased CpG methylation of the estrogen receptor gene in BRCA1-linked estrogen receptor-negative breast cancers. AB - A distinctive feature of BRCA1-linked breast cancers is that they typically do not express estrogen receptor-alpha (ER(alpha)). Previous investigation suggests that methylation of CpGs within the ER(alpha) promoter mediates repression of gene expression in some ER(alpha)-negative breast cancers. To determine if methylation of CpGs within the ER(alpha) promoter is associated with BRCA1-linked breast cancers, we evaluated methylation in exon 1 of the ER(alpha) gene in 40 ER(alpha)-negative breast cancers, 20 of which were non BRCA1-linked and 20 BRCA1 linked. CpG methylation was evaluated by either methylation-sensitive restriction digest (HpaII), methylation-sensitive PCR (MSP), or direct sequencing of bisulfite-treated genomic DNA. Results from HpaII digests and MSP documented a high degree of methylation, the MSP data showing slightly higher methylation in the BRCA1-linked group. CpGs analysed by direct sequencing showed an overall average methylation of 25% among non BRCA1-linked cancers and 40% among BRCA1 linked cancers (P=0.0031). The most notable difference was found at five particular CpGs, each of which exhibited a greater than twofold increase in methylation in the BRCA1-linked group compared to the non BRCA1-linked group (P<0.03 for each CpG). Methylation of certain critical CpGs may represent an important factor in transcriptional repression of the ER(alpha) gene in BRCA1 linked breast cancers. PMID- 12370826 TI - Localization of p0071-interacting proteins, plakophilin-related armadillo-repeat protein-interacting protein (PAPIN) and ERBIN, in epithelial cells. AB - PAPIN has six PDZ domains and interacts with p0071, a catenin-related protein. Recent studies have revealed that catenins determine the subcellular localization of some PDZ proteins. We have examined whether the localization of PAPIN is determined by p0071 in epithelial cells. PAPIN was localized not only on the lateral membrane but also on the apical membrane, where p0071 was absent. The targeting to both membranes was mediated by the middle region of PAPIN and did not require the p0071-interacting PDZ domain. In cells that came into contact, PAPIN was diffusely distributed on the plasma membrane, while p0071 was concentrated at immature cell-cell contacts. When epithelial cells were exposed to the low concentration of calcium, p0071 was internalized, whereas PAPIN remained on the plasma membrane. We also confirmed that the interaction with p0071 was not essential for the membrane targeting of ERBIN, a recently identified p0071- and ErbB2-binding protein. PAPIN, p0071, and ERBIN formed a complex in 293T cells. Furthermore, ERBIN and ErbB2 were colocalized with PAPIN on the lateral membrane. These findings suggest that PAPIN, p0071, and ERBIN come to the cell-cell contacts independently and interact with each other on the lateral membrane. PMID- 12370827 TI - Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-1 transfected C33A human cervical carcinoma cell line. AB - BAG-1 protein can be expressed as four isoforms of 50, 46, 33 and 29 kDa with different subcellular localizations, which may have different functions in anti apoptosis, but the exact mechanism remains unclear. We constructed BAG-1 full length and deletion mutated plasmids in a pCR3.1 vector and established stable transfections of BAG-1 isoforms in low BAG-1 expressing C33A cells. Treatment of the transfected cells with cisplatin, staurosporine, paclitaxel and doxorubicine showed that BAG-1 p50, p46 and p33 isoforms enhanced the resistance to apoptosis. BAG-1 p50, p46 and p33 exhibited different degrees of apoptosis inhibition in the transfected cells and BAG-1 p46 isoform had the most pronounced effect on anti apoptosis. BAG-1 p29 failed to protect the transfected cells from apoptosis. Resistance to apoptosis by BAG-1 isoforms was correlated with decreased caspase-3 activation. We also detected the expression of Bax, Bak, p53, Bcl-2, Bcl-X(L), AIF and MRP1 by Western blots. Bcl-2 protein expression was significantly increased in p50, p46 and p33 transfected cells, while the expression of Bax, Bak, p53, Bcl-X(L) and MRP1 was essentially unchanged. These in vitro results suggest that distinct isoforms of BAG-1 have different anti-apoptotic functions and their functions may be correlated to increased Bcl-2 expression. PMID- 12370828 TI - Light Chain 3 associates with a Sos1 guanine nucleotide exchange factor: its significance in the Sos1-mediated Rac1 signaling leading to membrane ruffling. AB - A 19 kDa protein was identified to associate with the Dbl oncogene homology domain of Sos1 (Sos-DH) and was purified from rat brains by GST-Sos-DH affinity chromatography. Peptide sequencing revealed that the protein is identical to light chain 3 (LC3), a microtubule-associated protein. LC3 coimmunoprecipitated with Sos1, and GST-LC3 was capable of forming complexes with Sos1 in in vitro GST pull down assay. Furthermore, LC3 was colocalized with Sos1 in cells, as determined by immunohistochemistry. While Sos1 stimulated the guanine nucleotide exchange reaction on Rac1, LC3 suppressed the ability of Sos1 to activate Rac1 in in vitro experiments using COS cell lysates. Consistent with this, overexpression of LC3 decreased the level of active GTP-bound Rac1 in COS cells. Sos1 expression induced membrane ruffling, a downstream target for Rac1, but LC3 expression inhibited this biological effect of Sos1. These findings suggest that LC3 interacts with Sos1 and thereby negatively regulates the Sos1-dependent Rac1 activation leading to membrane ruffling. PMID- 12370829 TI - The transmembrane receptor protein tyrosine phosphatase DEP1 interacts with p120(ctn). AB - The receptor-like protein tyrosine phosphatase DEP1, also known as CD148, is expressed predominantly in epithelial cells, in a variety of tumor cell lines, and in lymphocytes. Expression of DEP1 is enhanced at high cell density, and this observation suggests that DEP1 may function in the regulation of cell adhesion and possibly contact inhibition of cell growth. In order to investigate the function of DEP1, substrate-trapping mutants of the phosphatase were used to identify potential substrates. GST-fusion proteins containing the DEP1 catalytic domain with a substrate-trapping D/A mutation were found to interact with p120(ctn), a component of adherens junctions. DEP1 also interacted with other members of the catenin gene family including beta-catenin and gamma-catenin. The interaction with p120(ctn) is likely to be direct, as the interaction occurs in K562 cells lacking functional adherens junctions and E-cadherin expression. Catalytic domains of the tyrosine phosphatases PTP-PEST, CD45, and PTPbeta did not interact with proteins of the catenin family to detectable levels, suggesting that the interaction of DEP1 with these proteins is specific. DEP1 expression was concentrated at sites of cell-cell contact in A549 cells. p120(ctn) was found to colocalize with these structures. Together these data suggest an important role for DEP-1 in the function of cell-cell contacts and adherens junctions. PMID- 12370830 TI - Transcriptional upregulation of SPARC, in response to c-Jun overexpression, contributes to increased motility and invasion of MCF7 breast cancer cells. AB - Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotype changes related to malignant progression including increased motility and invasion. Concurrent with these phenotypic effects are changes in the expression of multiple gene targets. We previously demonstrated that expression of the SPARC/osteonectin gene, while undetectable in the MCF7 cell line, is highly induced in response to stable c-Jun overexpression (c Jun/MCF7). Because the SPARC gene product is associated with tumor cell invasion in a variety of different cancers, we have examined its role in mediating the phenotypic changes induced by c-Jun in MCF7 cells. We found that antisense mediated suppression of SPARC dramatically inhibits both motility and invasion in this c-Jun/MCF7 model. In contrast, stable overexpression of SPARC in the parental MCF7 cell line is not sufficient to stimulate cell motility or invasion. Examination of the promoter region of the human SPARC gene reveals three non canonical AP-1 sites. We demonstrate that one of these sites binds c-Jun/Fra1 heterodimers in vitro, but that this and the other AP-1 like sites are dispensable with respect to c-Jun stimulated SPARC promoter activation. Deletion analysis identified a region between -120 and -70 as a c-Jun responsive element sufficient to induce maximal promoter activation. This region does not contain any AP-1 sites but does mediate binding by SP1 'like' complexes. Furthermore, this region is necessary for SP1/SP3 responsiveness in Drosophila SL2 cells. These results demonstrate that SPARC plays an important role in stimulating motility and the invasive behavior of c-Jun/MCF7 cells and that SPARC promoter activation by c-Jun appears to occur through an indirect mechanism. PMID- 12370831 TI - The role of p38 in UVA-induced cyclooxygenase-2 expression in the human keratinocyte cell line, HaCaT. AB - We examined the expression of cycloxygenase-2, the rate-limiting enzyme in the production of prostaglandins, in the UVA-irradiated human keratinocyte cell line, HaCaT. UVA induced a dose-dependent increase in COX-2 at the protein level at 2 and 4 h post-irradiation and at the mRNA level at 1 and 2 h post-irradiation. Experiments using semi-quantitative RT-PCR demonstrate that UVA increased the half-life of the COX-2 message by more than fourfold in the presence of Actinomycin D (with a half life between 4 and 8 h post-irradiation), suggesting that UVA induction of COX-2 is post-transcriptionally regulated. Through the use of the specific p38 inhibitor, SB202190, increases in COX-2 message and protein levels were abrogated in UVA-irradiated cells. In UVA-irradiated cells treated with SB202190, the half-life of the COX-2 message was decreased to basal levels (between 1 and 2 h post-irradiation), indicating that p38 was responsible for the stabilization of the message. Luciferase activity was increased in UVA-irradiated cells transfected with reporter constructs containing the 3' UTR of COX-2, a region containing AU-rich elements (AREs). These regulatory sequences of AUUUA have been proposed as one mechanism of post-transcriptional regulation. Increases observed in luciferase activity could be decreased using a p38 dominant-negative construct. We report for the first that UVA can induce COX-2 expression in the human keratinocyte cell line, HaCaT. Additionally, p38 appears to play a critical role in the UVA-induced expression of COX-2 in these keratinocytes and may serve as a potential drug target in the chemoprevention of skin cancer. PMID- 12370832 TI - Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21. AB - Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21(Waf1/Cip1) (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha-helical conformation within the transactivation domain. Mutagenesis-derived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF(II)31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF(II)31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF(II)31. Consistently, p53 null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF(II)31 recruitment. PMID- 12370833 TI - Characterization of KLK4 expression and detection of KLK4-specific antibody in prostate cancer patient sera. AB - The ability to identify prostate tumor or prostate tissue specific genes that are expressed at high levels and use their protein products as targets could greatly aid in the diagnosis and treatment of prostate cancer. Using a polymerase chain reaction (PCR)-based subtraction technique, we have recovered the recently described KLK4 (prostase) gene from human prostate cDNA. In this study, KLK4 gene expression in human prostate tumors was further characterized using cDNA quantitative PCR and immunohistochemistry, demonstrating that the gene is specifically expressed at both the mRNA and protein levels in normal human prostate tissue, and in both primary and metastatic prostate tumor samples. Quantitative mRNA analysis also demonstrated low level expression including adrenal gland, salivary gland and thyroid. Finally, it was demonstrated that prostate cancer patient sera contain antibodies that bind specifically to recombinant KLK4 protein. This antibody has been used to detect KLK4-specific peptides in epitope mapping experiments. The relatively specific expression profile and elevated level of KLK4 mRNA and protein in both tumor and normal prostate tissues, in addition to detectable KLK4-specific antibody in cancer patient sera, supports additional efforts to determine if KLK4 can play a role in the diagnosis of prostate cancer, the monitoring of residual disease, or act as a target for immunotherapy. PMID- 12370834 TI - Mutational analysis defines a minimum level of telomerase activity required for tumourigenic growth of human cells. AB - A hallmark of cancer cells is the ability to proliferate indefinitely. This acquisition of an immortal lifespan usually requires the activation of telomerase, the enzyme that elongates telomeres. Human telomerase is minimally composed of the reverse transcriptase subunit hTERT, and the RNA subunit hTR. While hTR is ubiquitously expressed in human cells, the hTERT subunit is generally transcriptionally repressed in most normal somatic cells, but is illegitimately activated to restore telomerase activity in cancer cells. Indeed, in the thousands of different human tumours assayed, 85% were scored positive for telomerase activity. However, the levels of telomerase activity detected in tumour samples can vary substantially and even some normal somatic cells have been found to have low levels of enzyme activity. As the functional significance of low levels of telomerase activity is unclear, we investigated whether there is a minimum level of telomerase activity required for tumourigenesis. Using mutants of hTERT that induce varying levels of telomerase activity, we show that there does indeed exist a threshold of activity required for the processes of immortalization, transformation and tumourigenesis. Thus, low levels of activity detected in certain somatic cells would not be expected to contribute to tumourigenesis, nor does the mere detection of telomerase in cancer cells necessarily signify an immortal lifespan. PMID- 12370835 TI - Elevated mutant frequencies and predominance of G:C to A:T transition mutations in Msh6(-/-) small intestinal epithelium. AB - The DNA mismatch repair (MMR) system is primarily responsible for purging newly synthesized DNA of errors incurred during semi-conservative replication. Lesion recognition is initially carried out by one of two heterodimeric protein complexes, MutS(alpha) or MutS(beta). While the former, comprised of MSH2 and MSH6, recognizes mispairs as well as short (1-2 nucleotide) insertions/deletions (IDLs), the latter, made up of MSH2 and MSH3, is primarily responsible for recognizing 2-6 nucleotide IDLs. As most of the functional information on these heterodimers is derived from in vitro studies, it was of interest to study the in vivo consequences of a lack of MutS(alpha). To this end, Big Blue( trade mark ) mice, that carry a lacI(+) transgenic lambda shuttle-phage mutational reporter, were crossed with Msh6(-/-) mice to evaluate the specific contribution of MutS(alpha) to genome integrity. Consistent with the importance of MutS(alpha) in lesion surveillance, small intestine epithelial cell DNA derived from lacI(+) Msh6(-/-) mice exhibited striking increases (average of 41-fold) in spontaneous mutant frequencies. Furthermore, the lacI gene mutation spectrum was dominated by G:C to A:T transitions, highlighting the critical importance of the MutS(alpha) complex in suppressing this frequently observed type of spontaneous mutation. PMID- 12370836 TI - Modulation of PI3K/Akt pathway by E1a mediates sensitivity to cisplatin. AB - In order to investigate the molecular mechanisms implicated in the induction of chemo sensitivity by adenovirus E1a gene expression, we decided to investigate which signal transduction pathways could be affected by the E1a gene in Human Normal Fibroblast (IMR90). No effect was observed in SAPK pathways (p38MAPK and JNK), but E1a was able to affect the Akt activation mediated by insulin. This result was confirmed by transient transfection experiments performed in Cos-7 cells and also observed in other transformed cell lines such as A431. Furthermore, E1a expression induces a decrease in the basal status of Akt activity. Finally we demonstrated that E1a is able to block the Akt activation mediated by cisplatin and correlates with a sensitive phenotype. In summary, our data demonstrate that specific inhibition of the PI3K/Akt pathway mediates some of the biological properties of E1a such as induction of chemosensitivity. PMID- 12370837 TI - The DNA-based structure of human chromosome 5 in interphase. AB - In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells. PMID- 12370838 TI - Improved glucose control decreases the interaction of plasma low-density lipoproteins with arterial proteoglycans. AB - The entrapment and retention of plasma low-density lipoproteins (LDL) by arterial proteoglycans (PG) is a process central to atherogenesis. We postulated therefore that accelerated atherosclerosis of diabetic individuals may result from hyperglycemia-associated modifications in LDL that enhance their interaction with arterial PG. To evaluate the role of clinical treatment on this process, LDL-PG binding was evaluated in uncontrolled type 2 diabetic subjects on monotherapy followed by combination therapy. After a 4-week washout (baseline), subjects were randomized to receive glipizide GITS monotherapy (20 mg/d, n = 12) or metformin monotherapy (2.5 g/d, n = 8) for 6 weeks followed by combination treatment with both agents for 12 weeks. Fasting blood glucose and fructosamine were significantly reduced with monotherapy and further reduced with combination therapy P <.01). With combination therapy, glycated hemoglobin (GHb) levels were significantly reduced from baseline for the group initially treated with glipizide GITS (8.5% v 10.5%, P <.0005), or initially with metformin (6.7% v 9.7%, P <.0005). No significant changes in total plasma cholesterol (TPC), LDL, high-density lipoprotein (HDL), or triglycerides (TG) were measured after monotherapy or combination therapy. Plasma LDL was isolated by differential ultracentrifugation. In an in vitro binding assay, LDL from subjects on combination glipizide GITS/metformin demonstrated significantly less binding to arterial PG than LDL obtained after monotherapy with either agent (7.6 +/- 0.5 v 10.7 +/- 0.9 microg LDL cholesterol/microg PG [mean +/- SEM], P <.05). These data demonstrate that combination treatment with glipizide GITS/metformin will further improve glucose control in type 2 diabetic subjects and may favorably improve diabetes-associated modification of LDL-arterial PG binding. PMID- 12370839 TI - A calcium-deficient diet caused decreased bone mineral density and secondary elevation of estrogen in aged male rats-effect of menatetrenone and elcatonin. AB - In view of the fact that a deficient calcium (Ca) intake results in osteoporosis in elderly males, we conducted an animal experiment on aged male Wistar rats given a Ca-deficient diet. The rats were divided into 2 groups according to diet: a Ca-deficient diet group (Ca content, 0.08% to 0.1%) and a regular diet group (Ca content, 0.8% to 1.2%). The Ca-deficient diet reduced bone mineral density (BMD) by approximately 12%. Administration of menatetrenone or elcatonin was able to reverse the reduction in BMD induced by Ca deficiency. The mean estradiol level in sera of rats fed the Ca-deficient diet was significantly increased to 4.3 times that in the regular diet group. However, the increased estradiol concentration was reduced after the administration of menatetrenone or elcatonin. The estrone concentrations in sera of menatetrenone- or elcatonin-treated rats fed the Ca-deficient diet decreased to a level lower than that of animals fed the regular diet. Testicular aromatase cytochrome P450 (P450(arom); estrogen synthetase) activity was significantly increased by 2.4-fold in the Ca-deficient diet group compared to that in the regular diet group, and the aromatase mRNA level was also significantly increased 1.45-fold. Testicular aromatase activity was strongly correlated with aromatase mRNA level and serum estradiol level. These data suggest that the change in testicular aromatase expression might be, in part, a compensatory mechanism for the bone mineral deficiency induced by the Ca-deficient diet in aged male rats. PMID- 12370840 TI - Post-methionine-load hyperhomocysteinemia and increased lipoprotein(a) are associated with renal metabolic dysfunction: a hypothesis. AB - Previous studies have shown that homocysteine influences the structure of lipoprotein(a) [Lp(a)] and its affinity to fibrin, and that there is an increased risk of vascular disease when both homocysteine and Lp(a) are elevated. The aim of this study was to determine whether there is a correlation between increased total homocysteine (tHCY) and high Lp(a) concentrations, and whether increased concentrations of tHCY affect the concentration of unbound serum apolipoprotein(a) [Apo(a)]. Forty-seven male subjects recruited from a primary prevention screening program with normal serum creatinine and Lp(a) concentrations above 30 mg/dL were included and underwent a standardized oral methionine-loading test to increase the plasma tHCY concentration. This increase might lead to a modification of the Apo(a) structure, thus possibly influencing the serum concentration of unbound Apo(a). Fasting blood samples were taken before the tests and after 6 hours. The median values of tHCY increased about 4 fold after the methionine-loading test. Fasting tHCY did not show an association with Apo(a) and a post-methionine load increase of unbound Apo(a) was not observed. Backward multiple linear regression analysis, however, revealed that only post-load tHCY was independently and significantly influenced by Lp(a). Furthermore, Lp(a) correlated significantly with post-load tHCY, but not with fasting tHCY. Subdividing the subjects according to the Lp(a) concentration showed a significantly higher median concentration of tHCY after methionine load in subjects with Lp(a) over 50 mg/dL compared to subjects with Lp(a) under 50 mg/dL (P =.009). A similar cut-off was seen for post-load Apo(a) at 7.3 mg/dL (P =.04). Factors such as age, C677T-methylene-tetrahydrofolate-reductase (MTHFR) mutation, folate, vitamin B(12), and creatinine showed no significant influence on post-load tHCY in the different subgroups. The reasons for our findings remain partially unclear. However, considering our results and the current knowledge on the association of tHCY and Lp(a) concentration with the renal function, we hypothesize that both parameters may be linked by commencing renal metabolic dysfunction. It should be stressed that our hypothesis is speculative and that further studies will be necessary to improve the understanding of the interrelation of tHCY and Lp(a) concentration. PMID- 12370841 TI - Effect of diet-induced obesity on ovalbumin-specific immune response in a murine asthma model. AB - Some epidemiologic surveys have demonstrated that asthma is more prevalent in obese children and adults. However, the mechanism of association between obesity and asthma has not been fully clarified. This report investigates a murine model for antigen-induced asthma and diet-induced obesity from an immunologic perspective. For the induction of obesity, C57BL/6J mice were fed a high-fat diet supplemented with lard or soybean oil. Mice were then sensitized and challenged with ovalbumin (OVA) to induce allergic lung inflammation. OVA-specific serum immunoglobulin levels were lower in obese mice compared with non-obese control mice. The decline of OVA-specific IgE in the soybean oil group was found to be especially pronounced. However, obese mice with OVA-induced asthma showed a higher sensitivity of antigen-induced T-cell responses, and increased gamma interferon (IFN-gamma) production of splenocytes with phytohemagglutinin (PHA) stimulation. Furthermore, mast cell numbers in the tracheal mucosa were increased in obese mice upon sensitization by OVA. These results suggest that obesity induced changes in T-cell function may be partly involved in the pathophysiology of asthma in human obesity, rather than Ig E-mediated allergic responses. PMID- 12370842 TI - Relationships of the systolic blood pressure response during exercise with insulin resistance, obesity, and endurance fitness in men with type 2 diabetes mellitus. AB - The purpose of the present study was to investigate the relationships among the resting systolic (SBP) and diastolic blood pressure (DBP) or SBP response during exercise with insulin resistance evaluated by a homeostasis model (HOMA-IR), abdominal fat accumulation (visceral fat area [VFA], subcutaneous fat area [SFA]) by computed tomography (CT), and an estimation of the maximal oxygen uptake (V*O2max) in 63 Japanese middle-aged male patients with type 2 diabetes mellitus (type 2 DM). Body mass index (BMI) and waist-to-hip ratio (WHR) in type 2 DM subjects were significantly higher than in age-matched healthy male control subjects (n = 135) with normal glucose tolerance. Resting SBP (127.7 +/- 16.2 mm Hg v 119.4 +/- 13.0 mm Hg) and DBP (82.2 +/- 11.9mmHg v 76.8 +/- 9.4 mm Hg) levels, and the percentage of hypertension (20.6% v 1.5%) in type 2 DM subjects were significantly higher than in the control subjects (P <.05). According to a multiple regression analysis for resting blood pressure in type 2 DM, VFA was found to be an independent predictor of SBP, while V*O2max and HOMA-IR were independent predictors of DBP. In the controls, however, HOMA-IR was not found to be a significantly independent predictor for either resting SBP or resting DBP. Measurement of the SBP response during graded exercise using a ramp test was performed by an electrical braked cycle ergometer in 54 patients with type 2 DM only. The SBP was measured at 15-second intervals during exercise. The exercise intensity at the double product breaking point (DPBP), which strongly correlated with the exercise intensity at the lactate threshold, was used as an index for the SBP response to standardized exercise intensity. The SBP corresponding to exercise intensity at DPBP (SBP@DPBP) was evaluated as an index of the SBP response to standardized exercise intensity. The change in SBP (deltaSBP = SBP@DPBP - resting SBP) was significantly and positively associated with log area under the curve for glucose (log AUCPG) during a 75-g oral glucose tolerance test (OGTT). In addition, deltaSBP significantly and negatively correlated with the log area under the curve for insulin (log AUCIRI) and log AUCIRI/log AUCPG. Based on these results, insulin resistance was suggested to be independently associated with the resting DBP and SBP response to standardized exercise intensity in type 2 DM patients. PMID- 12370843 TI - Effect of alpha-linolenic acid-rich Camelina sativa oil on serum fatty acid composition and serum lipids in hypercholesterolemic subjects. AB - Camelina sativa-derived oil (camelina oil) is a good source of alpha-linolenic acid. The proportion of alpha-linolenic acid in serum fatty acids is associated with the risk of cardiovascular diseases. We studied the effects of camelina oil on serum lipids and on the fatty acid composition of total lipids in comparison to rapeseed and olive oils in a parallel, double-blind setting. Sixty-eight hypercholesterolemic subjects aged 28 to 65 years were randomly assigned after a 2-week pretrial period to 1 of 3 oil groups: camelina oil, olive oil, and rapeseed oil. Subjects consumed daily 30 g (actual intake, approximately 33 mL) of test oils for 6 weeks. In the camelina group, the proportion of alpha linolenic acid in fatty acids of serum lipids was significantly higher (P <.001) compared to the 2 other oil groups at the end of the study: 2.5 times higher compared to the rapeseed oil group and 4 times higher compared to the olive oil group. Respectively the proportions of 2 metabolites of alpha-linolenic acid (eicosapentaenoic and docosapentaenoic acids) increased and differed significantly in the camelina group from those in other groups. During the intervention, the serum low-density lipoprotein (LDL) cholesterol concentration decreased significantly by 12.2% in the camelina oil group, 5.4% in the rapeseed oil group, and 7.7% in the olive oil group. In conclusion, camelina oil significantly elevated the proportions of alpha-linolenic acid and its metabolites in serum of mildly or moderately hypercholesterolemic subjects. Camelina oil's serum cholesterol-lowering effect was comparable to that of rapeseed and olive oils. PMID- 12370844 TI - Erythrocyte membrane phospholipid composition is related to hyperinsulinemia in obese nondiabetic women: effects of weight loss. AB - The complex mechanisms by which obesity predisposes to insulin resistance are not clearly understood. According to a cell membrane hypothesis of insulin resistance, the defects in insulin action could be related to changes in membrane properties. The purpose of this work was to examine the relationship between 2 markers of insulin resistance (fasting plasma insulin [FPI] and homeostasis model assessment [HOMA IR]) and erythrocyte membrane lipid composition. In the first cross-sectional study, 24 premenopausal nondiabetic overweight women (body mass index [BMI], 32.5 +/- 0.9 kg/m(2); age, 35.7 +/- 2.2 years) were compared to 21 lean healthy women (BMI, 21 +/- 0.4 kg/m(2); age, 35.4 +/- 2.2 years). The second study examined whether a 3-month diet-induced weight loss, which usually improves insulin resistance, could also affect the membrane phospholipid (PL) composition and fluidity in the overweight group. Overweight women had significantly higher FPI levels (P <.0001), HOMA IR (P <.0001), membrane sphingomyelin (SM) (P <.05), and cholesterol (P <.05) contents than lean women. Baseline FPI and HOMA IR were positively correlated with membrane SM (P <.005), phosphatidylethanolamine (PE) (P <.005), and phosphatidylcholine (PC) (P <.05) contents, and negatively with phosphatidylinositol (PI) (P <.05) contents in the whole population. Multivariate regression analyses showed that 2 membrane parameters, PE and SM, were among the independent predictors of FPI or HOMA IR in the whole population, but also in the lean and the obese groups separately. Intervention induced a significant reduction in body weight (-5.7% +/- 0.7%), fat mass (-11.3% +/- 1.4%), and FPI ( 10.2% +/- 5.4%). An improvement in membrane lipid composition was only observed in the insulin resistant subgroup (FPI > 9.55 mU/L). The reduction in FPI or HOMA IR was directly associated with reduction in SM and PE contents, a finding independent of the reduction in fat mass. A stepwise multiple regression analysis indicated that the changes in SM accounted for 26.6% of the variance in the changes in FPI as an independent predictor, with the changes in fat mass and PE as other determinants (27.8% and 20%, respectively, adjusted r(2) =.704, P <.0001). These results suggest that the abnormalities in the membrane PL composition could be included in the unfavorable lipid constellation of obesity which correlated with impaired insulin sensitivity. PMID- 12370846 TI - Increase in postprandial serum insulin levels in epileptic patients with valproic acid therapy. AB - A significant weight gain and increase in serum leptin levels in the course of antiepileptic treatment with valproic acid (VPA) has been described in several clinical studies. With respect to the long treatment period in antiepileptic therapy, these side effects might increase insulin resistance and metabolic risk factors. We have studied clinical and laboratory effects of VPA treatment in a cohort of female patients (n = 22) and in patients treated with carbamazepine (CBZ) or lamotrigine monotherapy (n = 21). All study participants underwent an oral glucose tolerance test (OGTT) with 75 g glucose. Body mass index (BMI) in the VPA group was higher (28.1 +/- 3.6 kg/m(2)) than in the control group (23.9 +/- 3.7 kg/m(2)) (P <.039). While plasma glucose, serum leptin, insulin, and C peptide levels did not differ significantly between the study groups in the fasting state, postprandial (pp) insulin and proinsulin levels were found to be significantly higher in the VPA than in the control group. In the course of the OGTT, serum insulin levels reached their peak values 1 hour postprandially with 68.8 +/- 10.0 microU/mL in the VPA group and 49.8 +/- 11.2 microU/mL in the control group (P <.042). After 2 hours, the corresponding serum insulin levels were 48.5 +/- 25.2 microU/mL and 34.1 +/- 17.2 microU/mL (P <.048) and the proinsulin levels 52.5 +/- 30.2 pmol/L and 29.5 +/- 12.0 pmol/L (P <.017). While BMI values in the non-VPA group showed a significant correlation only with the fasting values of insulin, proinsulin, and C-peptide, the BMI values of the VPA treated group were also positively related to the 2-hour pp levels of insulin (R =.690; P <.001), proinsulin (R =.667; P <.001) and C-peptide (R =.502; P <.017). VPA is a fatty acid derivative, competes with free fatty acids (FFA) for albumin binding, and acts as a gamma aminobutyric acid (GABA)-ergic agonist, mechanisms, which are known to be involved in pancreatic beta-cell regulation and insulin secretion. Therefore, it might be suspected that VPA therapy is associated with increased glucose stimulated pancreatic secretion and thus a higher body weight in the VPA group. PMID- 12370845 TI - L-Arginine supplementation enhances diabetic wound healing: involvement of the nitric oxide synthase and arginase pathways. AB - Diabetes impairs wound healing and there are few therapeutic options to reverse it. Previous work has demonstrated the importance of nitric oxide for successful wound healing. In diabetes, NO synthesis is reduced in the wound milieu. The amino acid L-arginine is the only substrate for NO synthesis. We hypothesized that L-arginine supplementation would enhance wound healing by restoring NO synthesis. Thirty-six male Sprague-Dawley rats (body weight, 225 to 250 g) were separated in 4 groups: 20 rats were rendered diabetic 7 days prior to wounding by intraperitoneal streptozotocin (STZ) injection (70 mg/kg). Sixteen rats served as controls. Half of the animals of each group received 1 g/kg supplemental L arginine administered by gavage twice daily. Control rats were gavaged with water. Treatment was started 3 days before wounding. All rats underwent a dorsal skin incision and subcutaneous implantation of polyvinyl alcohol (PVA) sponges. The rats were killed 10 days post wounding and wound breaking strength, hydroxyproline content of the sponges, nitrite/nitrate (NO(x)) concentration, arginase activity, and amino acid composition of the wound fluid and plasma were analyzed. Wound fluid NO(x) concentrations and wound breaking strength were significantly reduced in the diabetic group compared to the controls. L-Arginine treatment restored diabetic NO(x) levels toward normal values and significantly enhanced wound breaking strength. Wound fluid arginase activity and ornithine concentrations were significantly lower in the diabetic animals but unaffected by treatment. The data demonstrate that the impaired NO synthesis in the diabetic wound milieu can at least partially be reversed by arginine supplementation. In view of previous results on the importance of NO for wound healing, the data suggest that arginine supplementation restores impaired healing in this acute wound model by normalizing the NO pathway but without affecting arginase activity. PMID- 12370847 TI - Application of quantitative competitive polymerase chain reaction for measurements of mRNA from antioxidative enzymes in the diabetic rat retina and kidney. AB - This present study applied quantitative competitive polymerase chain reaction (QC PCR) in the analyses of mRNA expression of the endogenous antioxidative enzymes CuZn superoxide dismutase (SOD), MnSOD, catalase, and glutathione peroxidase in tissue samples from the retina and kidney cortex of diabetic rats. RNA was extracted from snap-frozen retinas and pieces of the kidney cortex of male Wistar rats with streptozotocin (STZ)-induced diabetes and control rats. The mRNA levels were analyzed using QC-PCR. The animals were kept in the laboratory for 1 and 6 months, respectively, and fed a normal or probucol- (1% wt/wt) enriched diet. By using QC-PCR, relative mRNA levels of all antioxidative enzymes could be estimated in the retina and kidney cortex. In the retina, the relative catalase mRNA concentration was about 1/10 that of the other enzymes. After 6 months of diabetes, there was a 100% increase of the catalase (median, 0.012 [range, 0.008 to 0.017] v 0.006 [0.005 to 0.010]; P =.011) and a 50% increase of the glutathione peroxidase mRNA levels (0.88 [0.44 to 1.12] v 0.52 [0.31 to 0.79]; P =.044). In the kidney cortex, the relative glutathione peroxidase mRNA level was 10 to 15 times higher, and catalase mRNA level about half of those of CuZnSOD and MnSOD. After 1 month of diabetes, there was an increase only of the glutathione peroxidase mRNA levels, by 170% (17.59 [6.19 to 29.49] v 6.96 [2.34 to 9.04]; P =.047). We conclude that quantification of mRNA can provide difficulties when the amount of sample RNA is limited and/or the gene expression is low. The present study shows QC-PCR to be useful as a tool for measuring expression of mRNA not only in the kidney cortex but also in small tissue samples like the retina. Our results indicate modestly increased mRNA expression of catalase and glutathione peroxidase in the retina and likewise modestly increased mRNA expression of glutathione peroxidase in the kidney cortex of rats with STZ-induced diabetes. Extended studies, also including enzyme activities, are needed before any effect of hyperglycemia on the overall enzyme activity can be established. PMID- 12370848 TI - Alcohol increases c-myc mRNA and protein in skeletal and cardiac muscle. AB - The pathogenic mechanisms responsible for alcohol-induced muscle disease are unknown, although it is possible that increased proto-oncogene expression may be the causative process. Therefore, we investigated the responses of skeletal muscle c-myc protein and mRNA to a standard acute ethanol dosage regimen (75 mmol/kg/body weight [BW]) for 2.5 to 24 hours. Comparative studies were made on the heart. Acute ethanol administration in vivo led to an increase in c-myc proto oncogene mRNA in rat skeletal and cardiac muscle. The changes in c-myc mRNA were mirrored by increases in the c-myc protein as demonstrated by immunohistochemistry. The changes in the c-myc protein were localized to the myonuclei, with no corresponding changes seen in the interstitial cell nuclei. This is the first report of altered proto-oncogene expression in muscle in response to ethanol. Increased c-myc mRNA and protein may reflect adaptive changes, a stress response, or another uncharacterized cellular adaptation. PMID- 12370849 TI - Effect of macronutrient composition of the diet on the regulation of lipolysis in adipose tissue at rest and during exercise: microdialysis study. AB - The aim of the present study was to elucidate, using a microdialysis technique, whether modifications in the proportion of fat in the diet influence lipid mobilization from adipose tissue in situ. Nine healthy volunteers (age, 23.4 +/- 0.2 years; body mas index [BMI], 23.5 +/- 1.6 kg/m(2)) were fed, in random order, with a high-fat diet (HFD) (65% of energy content fat, 15% protein, 20% carbohydrate) or a high-carbohydrate diet (HCD) (70% carbohydrate, 15% protein, 15% fat) for 5 days, with a washout period of 10 days between the diets. Subjects were studied in the fasting state on the morning following days 4 and 5 of each diet. We measured the concentration of extracellular glycerol (EGC) in adipose tissue in response to (1) pharmacologic stimulation with isoprenaline (1 and 10 micromol/L) in situ, (2) stimulation with intravenous infusion of epinephrine (0.0375 microg/min/kg body weight), and (3) submaximal aerobic exercise (50% V*O2max, 60-minute duration). No effect of the diet composition was found in the increases of EGC in response to isoprenaline (area under the curve [AUC]: HFD, 1,534 +/- 370 micromol/90 min; HCD, 1,108 +/- 465 micromol/90 min; not significant [NS]) or epinephrine stimulations (AUC: HFD, 190 +/- 92 micromol/30 min; HCD, 251 +/- 298 micromol/30 min; NS). The exercise-induced increase in EGC was higher during the HFD (AUC: HFD, 1,641 +/- 181 micromol/60 min; HCD, 963 +/- 156 micromol/60 min; P <.05) and was associated with a higher exercise-induced response of norepinephrine (P <.05) and epinephrine (P =.056) and lower insulinemia during exercise. The results suggest that macronutrient composition of diet does not affect the beta-adrenergic responsiveness of adipose tissue to catecholamine action at rest. During exercise, the HFD promotes higher lipolysis in adipose tissue and this is associated with a higher catecholamine response and lower insulinemia. PMID- 12370850 TI - Common variants in the lipoprotein lipase gene, but not those in the insulin receptor substrate-1, the beta3-adrenergic receptor, and the intestinal fatty acid binding protein-2 genes, influence the lipid phenotypic expression in familial combined hyperlipidemia. AB - Familial combined hyperlipidemia (FCHL) is a common, atherogenic lipid disorder characterized by a variable phenotypic expression of hyperlipidemia. Variations in genes regulating fatty acid metabolism must be considered in the search for factors affecting the lipid phenotypic expression of FCHL. Therefore, we have evaluated the association of the common variants in the lipoprotein lipase (LPL) (D9N, N291S, and S447X), insulin receptor substrate-1 (IRS-1) (G972R), fatty acid binding protein-2 (FABP-2) (A54T), and beta3-adrenergic receptor (beta3-AR) (W64R) genes with lipid and lipoprotein levels in 30 Italian FCHL families (195 individuals). The transmission disequilibriun test (TDT) was used to evaluate the association between these variants and the FCHL trait. No significant differences were observed in the frequencies of the common LPL variants between affected and nonaffected FCHL family members. A significantly lower frequency of the LPL447X allele was noted only when members of the FCHL families were compared with normolipemic controls (.06 v.142, respectively; P <.01) suggesting a reduced representation of this LPL variant in FCHL families. The frequencies of variants in the IRS-1, FABP-2, and beta3-AR genes were not significantly different between affected and nonaffected FCHL family members and normolipemic controls. The TDT did not demonstrate any significant association of these gene variants with the FCHL trait. FCHL individuals carrying the LPL N291S gene showed higher plasma lipids and apolipoprotein B (apoB) levels compared with affected noncarriers. Only a marginal effect of the LPL D9N and S447X variants on lipid levels in FCHL individuals was observed. Conversely, the variants in the IRS-1, FABP2, and beta3 AR genes did not show any major influence on lipid and lipoprotein levels in FCHL family members. In conclusion, these results confirmed that none of the investigated genes were major loci for FCHL. Nevertheless, variations in genes affecting the removal rate of triglycerides (TG) from plasma, such as the LPL gene, significantly influence the lipid phenotypic expression of FCHL. Conversely, genetic variants in the IRS-1, FABP-2, and the beta3-AR gene appear not to have a major role as modifier genes in FCHL. PMID- 12370851 TI - Release of cholesterol from cell membranes to postprandial plasma from mildly hypercholesterolemic subjects: the effect of meals rich in olive and safflower oils. AB - Triglyceride-rich lipoproteins increase net transport of cell cholesterol to postprandial plasma from healthy subjects after a meal rich in fat and cholesterol. The aim of the present study was to determine the effect of meals rich in polyunsaturated fats (PUFA) and monounsaturated fats (MUFA) and low in cholesterol on net in vitro transport of cholesterol from red blood cells (RBCs) to postprandial plasma from 21 men with mild to moderate hypercholesterolemia in a randomized, crossover trial. Cholesterol concentration increased by 12% due to accumulation of cell cholesterol in fasted hypercholesterolemic plasma incubated with a 2/1 (vol/vol) excess of RBCs at 37 degrees C for 18 hours. The increase in cell cholesterol in plasma was mainly localized in the low-density lipoprotein (LDL) fraction (64%) and the remainder was approximately equally divided between the very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) fractions. Accumulation of cell cholesterol in the LDL fraction prevented the significant decrease in LDL cholesterol in plasma incubated alone. When RBCs were incubated with postprandial plasma isolated 4 hours and 6 hours after liquid meals rich in safflower and olive oils, the accumulation of cell cholesterol in plasma increased significantly (11%, P <.004) above values for fasted plasma and irrespective of the type of fat in the meal. Also, the content of cell cholesterol increased significantly (70%, P <.001) in triglyceride (TG)-rich lipoproteins and decreased significantly (P =.006) in the LDL fraction, which remained the main ultimate destination of cell cholesterol in postprandial plasma. The increased loss of cell cholesterol to fasted and postprandial plasma was closely correlated (r > 0.823, P <.001) with the concomitant increase in plasma cholesteryl esters (CE) generated by lecithin cholesterol acyltransferase (LCAT) activity. There was a small (5%), significant (P <.001) increase in plasma cholesterol esterification in postprandial plasma. These data suggest that high fat meals rich in MUFA and PUFA and low in cholesterol may produce a small postprandial increase in the capacity of plasma to accept cell membrane cholesterol that is limited by a concomitant small increase in plasma cholesterol esterification, in hypercholesterolemic subjects. Thus, low-fat, lipid-lowering diets may have a minimal effect on this capacity but will reduce levels of atherogenic LDL cholesterol that appear to be maintained by diffusion of cell cholesterol to plasma. PMID- 12370852 TI - Cigarette smoking, high-density lipoprotein cholesterol subfractions, and lecithin: cholesterol acyltransferase in young women. AB - Much of the published data on the relationship of cigarette smoking (CS) with serum lipids and lipoproteins is based on studies of middle-aged individuals. Data on young women are scarce. This study examined the relationship of CS with high-density lipoprotein cholesterol (HDL-C) subfractions and lecithin:cholesterol acyltransferase (LCAT) activity in Japanese collegiate women. Twenty-three current smokers were individually matched for physical activity scores, age, and body mass index (BMI) with 23 nonsmokers. There were no significant differences between smokers and nonsmokers in the mean nutrient intakes. Smokers had significantly lower mean HDL-C, HDL2-C, total cholesterol, and LCAT activity than nonsmokers. In univariate analyses, BMI significantly negatively correlated with HDL-C and HDL2-C. LCAT activity significantly positively correlated with HDL3-C, LDL-C, total cholesterol (TC) and triglycerides (TG). In multiple regression analyses, the number of CS was positively related to TG. BMI was negatively related to TC. LCAT activity was positively related to LDL-C, TC, and TG. These results suggest that the known associations in older adults of CS with HDL-C subfractions and LCAT activity are already apparent in young women. PMID- 12370853 TI - Effect of beer on the plasma concentrations of uridine and purine bases. AB - We conducted the present study to determine whether beer, both with and without ethanol content, increases the plasma concentration and urinary excretion of purine bases and uridine. Because 10 mL of regular beer (with ethanol) was found to contain 0.34 g of freeze-dried beer (without ethanol) and 0.5 mg of uridine, 5 healthy males were given regular beer (10 mL/kg of body weight) and freeze-dried beer (0.34 g/kg of body weight) or uridine (0.5 mg/kg of body weight). The plasma concentrations of hypoxanthine, xanthine, and uridine increased by 3.5-fold (P <.05), 4.7-fold (P <.05), and 1.8-fold (P <.05), respectively, 30 minutes after regular beer ingestion, and the urinary excretion of hypoxanthine, xanthine, and uridine increased by 4.0-fold (P <.05), 4.5-fold (P <.01), and 1.7-fold (P <.05), respectively, when measured 1 hour after ingestion. The plasma concentrations of uric acid and total purine bases increased by 6.5% (P <.05) and 7.6% (P <.05), respectively, 30 minutes after regular beer ingestion, whereas the urinary excretion of uric acid did not increase, while that of total purine bases increased by 1.3-fold (P <.05) when measured 1 hour after ingestion. As for freeze-dried beer, the plasma concentrations of uric acid total purine bases increased by 4.4% (P <.05) and 4.6% (P <.05), respectively, and that of uridine by 1.5-fold (P <.01) 30 minutes after ingestion, while the urinary excretion of uridine increased by 1.4-fold (P <.01) 1 hour after ingestion. However, the plasma concentrations and urinary excretion of hypoxanthine and xanthine and the urinary excretion of uric acid and total purine bases did not change significantly. As for uridine ingestion, the plasma concentration of uridine increased by 1.37-fold (P <.01) 30 minutes after ingestion, and the urinary excretion of uridine increased by 1.3-fold (P <.01) 1 hour after ingestion. However, the plasma concentrations and urinary excretion of hypoxanthine, xanthine, uric acid, and total purine bases did not change significantly. These results suggest that the purines in beer played a major role in the increase in the plasma concentration of uric acid, while both uridine and ethanol in beer had a significant effect on the increase in plasma concentration of uridine. PMID- 12370854 TI - Portal glucose infusion exerts an incretin effect associated with changes in pancreatic neural activity in conscious dogs. AB - We sought to determine whether an incretin effect could be observed when glucose was infused via the hepatic portal (Po) vein versus a peripheral (Pe) vein. Identical hyperglycemia (155 +/- 7 and 154 +/- 8 mg/dL, respectively) was produced in 2 groups (n = 9 each) of conscious dogs by Po or Pe glucose infusion. During glucose infusion, arterial plasma insulin levels increased by 28 +/- 5 and 16 +/- 3 microU/mL in Po and Pe, respectively (P <.05 between groups). Pancreatic insulin output increased by 10.4 +/- 3.2 and 6.7 +/- 2.3 mU/min in Po and Pe, respectively (P =.12 between groups). Arterial plasma glucagon levels and pancreatic glucagon output were similarly suppressed by Po and Pe glucose infusion. Pancreatic polypeptide (PP) output and norepinephrine (NE) spillover were measured as indices of pancreatic parasympathetic and sympathetic neural activity, respectively. During Pe, pancreatic PP output decreased from basal (delta -4.8 +/- 2.5 ng/min, P <.05), with no significant change in NE spillover (delta +4.4 +/- 4.0 ng/min). The PP output:NE spillover ratio decreased by 65% (P <.05), suggesting a shift toward a dominance of sympathetic tone. During Po, there were no significant changes in PP output (delta -1.4 +/- 3.1 ng/min) or NE spillover (delta +1.6 +/- 1.2 ng/min), and consequently there was no significant change in the PP output:NE spillover ratio. Thus, activation of the Po glucose signal appears to inhibit the shift toward sympathetic dominance that would otherwise result, thereby causing an incretin effect. PMID- 12370855 TI - Magnesium, sodium, and potassium content and [3H]ouabain binding capacity of skeletal muscle in relatives of patients with type 2 diabetes: effect of dexamethasone. AB - Theoretically, disturbancies in sodium (Na) and potassium (K) homeostasis and a magnesium (Mg) deficit could be possible factors in the development of obesity, type 2 diabetes, and hypertension. Therefore, we measured electrolyte content and [3H]ouabain binding capacity of skeletal muscle in 20 relatives of type 2 diabetic patients and in 20 controls before and after glucose infusion and before and after treatment with dexamethasone, which decreases insulin sensitivity. Muscle electrolyte content and [3H]ouabain binding capacity did not differ between groups. Infusion of glucose increased muscle Na content 25%, decreased muscle potassium content 9%, and muscle Mg content 5%. Muscle potassium/Mg ratio decreased only in relatives. Treatment with dexamethasone increased muscle Na content 15% and decreased muscle Mg content 7%, whereas muscle potassium/Na ratio decreased 17%. Dexamethasone increased muscle [3H]ouabain binding capacity by 42% in both groups. Basal and 1-hour intramuscular glucose content correlated inversely with basal muscle potassium/Na ratio in relatives only. In conclusion, persons who were predisposed to the development of type 2 diabetes exhibited an increased interdependency between glucose, Na, and potassium handling in skeletal muscle. Muscle Na content and [3H]ouabain binding capacity increased during treatment with dexamethasone, and muscle potassium/Na ratio decreased. Intravenous (IV) glucose injection decreases muscle Mg content, as does a decrease in insulin sensitivity, without any differences between relatives and controls. PMID- 12370856 TI - Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. AB - We investigated the effect of a chronic exposure to high levels of free fatty acid (FFA; 2 mmol/L oleate/palmitate 2:1) or glucose (16.7 mmol/L) on islet cell apoptosis. Apoptosis was detected using 4 different methods: (1) cell staining with annexin-V fluorescien isothiocyanate (FITC) conjugate and propidium iodide (PI); (2) quantification of cytoplasmatic DNA fragments by an enzyme-linked immunosorbent assay (ELISA); (3) assay of caspase 3 activity; and (4) TdT mediated dUTP nick-end labeling (TUNEL). Islet cells were also costained with an anti-insulin antibody to identify apoptotic beta cells. We also evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR) the expression of bax, bcl-2, and caspas 3, genes involved in apoptosis. In islets cultured for 7 days in the presence of high FFA or for 3 days in the presence of high glucose levels, we observed: (1) a 2- to 3-fold increase of apoptotic cells conjugated with annexin-V FITC and PI; (2) a 4- to 6-fold increase of cytoplasmatic DNA fragments; (3) a 3- to 4-fold increase of caspase 3 activity; and (4) a significant increase of insulin positive apoptotic cells as detected with the TUNEL method. RT-PCR analysis indicated in islets exposed to high FFA or glucose levels an increase of bax (proapoptotic gene), a reduction of bcl-2 (antiapoptotic gene), and a slight (although not significant) increase in caspase 3 expression. Western blot analysis also showed an increase of Bax protein levels in islets exposed to high FFA or glucose. The simultaneous presence of both metabolic abnormalities did not further increase the amount of apoptotic cells, although the time-course of the cellular damage induced by FFA was accelerated by the contemporary presence of high glucose. To elucidate the mechanism by which FFA and glucose may induce pancreatic beta-cell damage, we examined whether nicotinamide prevents apoptosis in pancreatic islets cultured for 7 days with high FFA or for 3 days with high glucose. Nicotinamide was able to prevent beta cell damage by significantly reducing apoptosis in both experimental conditions. Also, the increase of Bax protein level was prevented by nicotinamide. These data indicate that chronic exposure to elevated FFA or glucose levels increases apoptosis in rat pancreatic islets and these cytotoxic effects could be mediated by oxidative stress. This may contribute to the beta-cell failure that occurs in most in type 2 diabetic patients few years after clinical diabetes onset. PMID- 12370857 TI - Cilostazol, a potent phosphodiesterase type III inhibitor, selectively increases antiatherogenic high-density lipoprotein subclass LpA-I and improves postprandial lipemia in patients with type 2 diabetes mellitus. AB - Low levels of high-density lipoproteins cholesterol (HDL-C) as well as impaired postprandial lipemia are known to be associated with the increased risk for coronary artery disease (CAD) in patients with type 2 diabetes mellitus (type 2 DM). HDL are heterogeneous in size and apolipoprotein composition. Recent evidence indicates that among the 2 major HDL subclasses, those without apolipoprotein A-II (LpA-I) are more antiatherogenic compared with those with apoA-II (LpA-I:A-II). Cilostazol, a novel selective phosphodiesterase type III inhibitor, has been shown to inhibit platelet activation and is also a potent vasodilator. Additionally, cilostazol has been shown to modulate lipoprotein profiles by raising HDL-C and lowering plasma triglyceride (TG) levels. The present study investigated the effect of cilostazol on HDL composition (LpA-I and LpA-I:A-II levels) and postprandial lipemia in patients with type 2 DM. Seventeen patients were given cilostazol 200 mg twice daily for 12 weeks. At weeks 0 and 12, fat tolerance tests (30 g/m(2)) were performed to assess postprandial lipemia. Plasma TG and remnant-like lipoprotein particles cholesterol (RLP-C) were significantly decreased by 17% and 26%, respectively (P <.05), and HDL-C was significantly increased by 14% (P <.01). LpA-I was significantly increased by 23% (P <.01) from the mean value of 45 mg/dL to 55 mg/dL. In contrast, LpA-I:A-II remained unchanged, resulting in significantly increased %LpA-I (apoA-I on LpA I/total apoA-I x 100) from 35% to 40% (P <.01). Areas under the curve for TG and RLP-C after the fat meal were both nonsignificantly decreased by 17%. Patients with higher plasma TG levels had a greater benefit from the treatment with cilostazol as revealed by fasting TG levels and fat tolerance tests. HDL-C responses to cilostazol were independent of baseline plasma TG levels or percentage changes in TG, indicating that the underlying mechanisms for raising HDL and reducing TG levels are distinct. In conclusion, cilostazol selectively increased LpA-I, thus favorably altering HDL towards a more antiatherogenic composition. This finding, together with the improved postprandial lipemia, indicates that cilostazol has a potent antiatherogenic function by modulating HDL and remnant metabolism in patients with type 2 DM. PMID- 12370858 TI - Comparison in patients with type 2 diabetes of fibric acid versus hepatic hydroxymethyl glutaryl-coenzyme a reductase inhibitor treatment of combined dyslipidemia. AB - Patients with combined dyslipidemia are at greatly increased coronary heart disease (CHD) risk. The threat of rhabdomyolysis with dual pharmacologic treatment (hepatic hydroxymethyl glutaryl coenzyme A [HMG-CoA] reductase inhibitors plus fibric acid derivatives) has tended to limit therapy in patients with combined dyslipidemia to a choice of one or the other drug. Judgment of the potential benefits of either agent has rarely taken into account their effect on the postprandial accumulation of highly atherogenic, triglyceride (TG)-rich, remnant lipoprotein particles (RLPs). Because this information could be of substantial clinical relevance, we addressed this question in patients with type 2 diabetes and combined dyslipidemia by comparing the effects of gemfibrozil versus HMG-CoA reductase inhibitors (statins) on both fasting and postprandial lipid and lipoprotein concentrations. For this purpose, 22 patients with type 2 diabetes and combined dyslipidemia were randomized to treatment with either a statin or gemfibrozil for 3 months. Glycemic control was similar in both groups at baseline and did not change in response to treatment. Baseline lipid and lipoprotein concentrations were also similar in the 2 treatment groups, but the responses to therapy were quite different. Statin-treated patients had a statistically significant decrease in low-density lipoprotein (LDL) cholesterol concentration (156 mg/dL to 96 mg/dL, P <.001), whereas there was no change in patients treated with gemfibrozil. In contrast, there was a statistically significant decrease (P <.05) in plasma TG concentrations (116 mg/dL) in gemfibrozil-treated individuals, without any change in subjects treated with statins. However, the decrease in total integrated postprandial plasma RLP response measured hourly from 8 AM to 4 PM was not different in patients treated with either gemfibrozil (-43%) or statins (-34%). These results indicate that statin treatment, in addition to its beneficial effect on hypercholesterolemia, was as effective as gemfibrozil in reducing postprandial accumulation of triglyceride-rich, atherogenic RLPs in patients with type 2 diabetes and combined dyslipidemia. As such, the clinical utility of statin monotherapy in the treatment of combined dyslipidemia may have been underestimated. PMID- 12370859 TI - Effect of oxidative stress on glutathione pathway in red blood cells from patients with insulin-dependent diabetes mellitus. AB - Recently, increased oxidative stress and impaired antioxidant defense have been suggested as a contributory factor for initiation and progression of complications in diabetes. Although glutathione (GSH) and the enzymes included by glutathione redox cycle have an important role for protection of cells against free radical-mediated damage, they may be susceptible to oxidation themselves. We examined the susceptibility of the GSH pathway to oxidation and inactivation in subjects with well-controlled and poorly controlled insulin-dependent diabetes mellitus (IDDM) versus controls and the effect of glycemic control on this susceptibility. Red blood cells (RBCs) were isolated, RBC level of GSH, activity of glutathione peroxidase (G-Px), and glutathione reductase (G-Red) were measured at the baseline and after a 2-hour incubation with hydrogen peroxide. Significant decreases were observed in the GSH level and in the activity of GSH peroxidase and GSH reductase in all the groups after the incubation with hydrogen peroxide. Maximum decrease was observed in the poorly controlled diabetic group for all parameters. This result indicates that the GSH pathway is susceptible to oxidation; and this susceptibility increases in poorly controlled diabetics. Therefore, insufficient antioxidant defense by the GSH pathway may be one of the factors responsible for development of complications in patients with IDDM. PMID- 12370860 TI - Evidence for integrity of the growth hormone/insulin-like growth factor-1 axis in patients with severe head trauma during rehabilitation. AB - Severe traumatic head injury has been recognized to be associated with hypothalamo-hypophyseal impairment and subsequent abnormalities in hormone secretion, which can contribute to a prolonged clinical course and to hampered recovery in many head-injured patients. Most of the data on the growth hormone/insulin-like growth factor -1 (GH/IGF-1) axis function have been obtained early after head injury, whereas GH secretory pattern has not been fully elucidated after patients had left the intensive care unit. We examined the activity of the GH/IGF-1 axis in 16 severely closed head-injured (CHI) patients (14 males; age range, 17 to 47 years; body mass index [BMI], 21.4 +/- 0.8 kg/m(2)) during the rehabilitation period at least 1 month after leaving the intensive care unit and in 12 sex-, age-, and weight-matched healthy controls. The severity of trauma was assessed by the Glasgow Coma Scale (GCS) score (8 or less), posttraumatic amnesia (PTA, more than 24 hours), and initial computed tomography (CT) scan. The clinical picture at time of the study was evaluated by the Rancho Los Amigos Scale of Cognitive Functioning (CFS) and the Functional Independence Measure (FIM). In all subjects, we evaluated basal levels of anterior pituitary hormones, IGF-1, insulin-like growth factor-binding protein (IGFBP)-3, and IGFBP-1, as well as the GH responses to intravenous (IV) infusion of growth hormone-releasing hormone (GHRH) alone, GHRH plus arginine (ARG), and the GH release evoked by somatostatin (SRIH) infusion withdrawal, which is related to endogenous GHRH tone. In all subjects, nutritional parameters and nitrogen balance were normal. Basal plasma concentrations of GH, IGF-1, IGFBP-3, and IGFBP-1 did not significantly differ between CHI patients and controls. The GH responses to GHRH and GHRH plus ARG did not significantly differ between CHI patients (GH peak, 10.7 +/- 3.0 microg/L; area under the curve [AUC], 5.9 +/- 1.5 microg/L. min; and GH peak, 34.7 +/- 6.1 microg/L; AUC, 20.25 +/- 3.3 microg/L. min, respectively) and normal subjects (GH peak at 30 minutes, 7.23 +/- 1.35 microg/L; AUC, 4.7 +/- 0.8 microg/L. min; and GH peak at 60 minutes, 41.0 +/- 5.1 microg/L; AUC, 24.3 +/- 1.7 microg/L. min, respectively). SRIH withdrawal resulted in an unequivocal increase in plasma GH concentrations both in CHI patients and in controls, without any significant difference between the 2 groups. A negative correlation was found between the GH response (deltaGH peak) to SRIH withdrawal and CFS (r = -.615, P <.005). In conclusion, our study indicates that patients receiving rehabilitation after leaving the intensive care unit for severe traumatic head injury have no significant changes of GH secretion with normal central regulation of the GH-IGF-1 axis. PMID- 12370861 TI - Acute and prolonged effects of insulin-induced hypoglycemia on the pituitary thyroid axis in humans. AB - Secretory activity of the pituitary-thyroid axis and thyroid hormone metabolism show characteristic changes in response to different stressors often referred to as the euthyroid sick syndrome. Hypoglycemia is an acute metabolic stressor inducing various neuroendocrine responses, the effects of which on pituitary thyroid secretory activity so far have been entirely neglected. We performed stepwise hypoglycemic and euglycemic clamps each lasting 6 hours in 30 healthy men. To assess the potential influence of hyperinsulinemia on pituitary-thyroid hormone release, 2 different rates of insulin infusion were used for the clamps. During the hypoglycemic clamps, serum thyroid-stimulating hormone (TSH) concentration decreased in comparison to the euglycemic condition on average by 28% +/- 4% (P <.001), while serum concentration of free triiodothyronine (fT3), free thyroxine (fT4), and thyroxine-binding globulin (TBG) remained unchanged. The effect did not depend on the rate of insulin infusion. To assess the prolonged effect of acute hypoglycemia on pituitary-thyroid secretory activity, serum TSH and thyroid hormone concentrations were subsequently measured in another 15 healthy men before and 18 hours after 2 consecutive hypoglycemic clamps together lasting about 270 minutes. Compared with values before the hypoglycemic clamps, serum levels of TSH, fT3, and fT4 were found to be still reduced (by 44% +/- 6%, 12% +/- 2%, and 10% +/- 1%, respectively) 18 hours after the last hypoglycemic episode (P <.001 for all comparisons). The observed hormonal changes after hypoglycemia were not accompanied by any change in resting energy expenditure (REE). Data indicate acute as well as prolonged inhibitory influences of hypoglycemia on pituitary-thyroid secretory activity. The pattern of changes suggests that hypoglycemia exerts its influence primarily at a central, ie, pituitary and/or hypothalamic, site of the axis. PMID- 12370862 TI - Association between vitamin D receptor (VDR) polymorphism and type 2 diabetes. PMID- 12370864 TI - Dissatisfaction with medical services among Medicare beneficiaries with disabilities. AB - OBJECTIVE: To test the hypothesis that Medicare beneficiaries who have difficulties performing activities of daily living (ADLs) are more likely to report dissatisfaction with their health care than those without ADL difficulties. DESIGN: Cross-sectional study. SETTING: Sample from the 1998 Medicare Current Beneficiaries Survey. PARTICIPANTS: A population-based sample (N=19,650) of noninstitutionalized Medicare beneficiaries. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Satisfaction with overall quality and 9 specific aspects of medical services received in the last year. RESULTS: After adjusting for sociodemographic, behavioral, and system characteristics and compared with those without ADL difficulties, Medicare enrollees were more likely to report dissatisfaction with the overall quality of their health care as their number of activity restrictions increased (1-2 ADLs: odds ratio [OR]=1.5; 95% confidence interval [CI], 1.2-2.0; 3-4 ADLs: OR=1.7; 95% CI, 1.2-2.4; 5-6 ADLs: OR=1.9; 95% CI, 1.4-2.8). Analysis of satisfaction with the 9 specific aspects of care yielded similar results. CONCLUSION: Disability is a significant independent risk factor for dissatisfaction with health care in the Medicare population. Efforts should be made to identify individuals with ADL difficulties and to improve their ease and convenience of getting to a doctor, the availability of care off hours, the access to specialists, and the follow-up care received. PMID- 12370865 TI - Sensory motor retuning: a behavioral treatment for focal hand dystonia of pianists and guitarists. AB - OBJECTIVE: To evaluate the long-term effectiveness of sensory motor retuning (SMR), a new treatment for focal hand dystonia in musicians. DESIGN: Prospective case series with an (adventitious) comparison group with 3- to 25-month follow-up in piano and guitar and 0- to 4-month follow-up in flute and oboe players. SETTING: General community in Germany. PARTICIPANTS: Eleven professional musicians. INTERVENTION: Immobilization by splints of 1 or more digits other than the focal dystonic finger. This finger carried out repetitive exercises in coordination with 1 or more of the other digits for 1(1/2) to 2(1/2) hours a day for 8 consecutive days under therapist supervision. The subjects then were instructed to continue practice for 1 hour daily for 1 year. MAIN OUTCOME MEASURES: Spectral analysis of the output of a dexterity-displacement device that continuously recorded digital displacement during finger movements and a dystonia evaluation scale on which patients rated how well they had just performed dystonic movement sequences and repertoire passages. RESULTS: The 3 wind players (adventitious placebo controls) did not improve substantially. However, each pianist and guitarist showed marked and significant improvement in spontaneous repertoire performance without the splint. The first subject is now 25 months posttreatment. CONCLUSIONS: Results suggest that SMR is of value for the treatment of focal hand dystonia in pianists and guitarists. PMID- 12370866 TI - Inter- and intrarater reliability of the Ashworth Scale and the Disability Assessment Scale in patients with upper-limb poststroke spasticity. AB - OBJECTIVE: To evaluate the reliability of the Ashworth Scale and the Disability Assessment Scale (DAS) in poststroke patients with upper-limb spasticity and functional disability. DESIGN: Single-center trial. SETTING: University medical center. PARTICIPANTS: Nine patients > or = 6 months poststroke with upper-limb spasticity and impairment in the areas of hygiene, dressing, limb posture, or pain were included in the analysis. INTERVENTIONS: Ten experienced medical professionals rated each patient in randomized order twice on the same day (results based on mean of evaluations at times 1 and 2). Elbow, wrist, finger, and thumb flexion tones were assessed by using the Ashworth score (range, 0-4), and functional disability was assessed using the DAS (range, 0-3). MAIN OUTCOME MEASURES: Intra- and interrater reliability of the Ashworth Scale and DAS. RESULTS: For the Ashworth parameters, 38 of 40 evaluations indicated excellent (weighted kappa > or = .75) or good (weighted kappa > or = .4) intrarater reliability. For DAS parameters, 31 of 40 evaluations indicated excellent or good intrarater reliability. The interrater reliability was also good for both the Ashworth Scale (Kendall W=.598-.792) and DAS (Kendall W=.494-.772) with statistically significant agreement found among raters (all P<.001). CONCLUSIONS: In patients with upper-limb spasticity after stroke, the Ashworth Scale and DAS had good intra- and interrater reliability when used by trained medical professions. PMID- 12370867 TI - Regular Tai Chi Chuan exercise may retard bone loss in postmenopausal women: A case-control study. AB - OBJECTIVE: To evaluate the potential benefits of regular Tai Chi Chuan exercise on the weight-bearing bones of postmenopausal women. DESIGN: Case-control study. SETTING: University medical school in Hong Kong. PARTICIPANTS: Postmenopausal women (age range, 50-59y), including 17 self-selected regular Tai Chi Chuan exercisers (TCE) with over 4 years of regular exercise, and 17 age- and gender matched nonexercising controls (CON). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Bone mineral density (BMD) in the lumbar spine and proximal femur was measured at baseline and at follow-up 12 months later by using dual-energy x-ray absorptiometry (DXA) and in the distal tibia using multislice peripheral quantitative computed tomography (pQCT). RESULTS: Baseline results showed that the TCE group had significantly higher BMD (10.1%-14.8%, all P<.05) than the CON group in the lumbar spine, proximal femur, and the ultradistal tibia. The follow up measurements showed generalized bone loss in both groups. Although both DXA and pQCT measurements revealed decelerated rates of bone loss in the TCE group, only the more sensitive pQCT showed significantly reduced rate of bone loss in trabecular BMD of the ultradistal tibia (TCE vs CON: -1.10%+/-1.26% vs -2.18%+/ 1.60%, P<.05) and of cortical BMD of the distal tibial diaphysis (TCE vs CON: 0.90%+/-1.36% vs -1.86%+/-0.93%, P<.05). CONCLUSION: This is the first case control study to show that regular Tai Chi Chuan exercise may help retard bone loss in the weight-bearing bones of postmenopausal women. PMID- 12370868 TI - Rehabilitation after proximal femur fracture surgery in the oldest old. AB - OBJECTIVE: To assess the course and results of rehabilitation after proximal femur fracture (PFF) in patients 85 years of age or older, compared with younger elderly patients, with an emphasis on functional status. DESIGN: Prospective cohort study. SETTING: A rehabilitation geriatric ward in a tertiary university hospital in southern Israel. PARTICIPANTS: The study group included 127 elderly patients 85 years of age or older who were hospitalized for rehabilitation following surgery for PFF. The comparison group was comprised of 297 patients aged 75 to 84 years who were hospitalized for the same indication in the same time period. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Functional studies by FIM trade mark instrument, mental status by the Folstein Mini-Mental State Examination, Geriatric Depression Screening (GDS) scale, length of rehabilitation, and complications and mortality during rehabilitation. RESULTS: Compared with patients aged 75 to 84 years, the older study group was in a worse mental state (P=.00005), even though the groups did not differ in their GDS scores. There were no significant differences between the groups in rehabilitation length of stay, in the rate of most postoperative complications, or in death rates during rehabilitation. FIM values before PFF, at the beginning of rehabilitation and at its end, and the difference between the beginning and end of rehabilitation were lower in the older group (P<.00001 for all tests). CONCLUSIONS: From the functional standpoint, rehabilitation after PFF surgery is much less successful in the 85+ age group than in the 75-to-84 age group but did not differ in its duration, rates of most complications, or mortality. Nonetheless, a significant percentage of patients in this age group have successful rehabilitation so they should not be deprived the chance. PMID- 12370869 TI - Improvement of isokinetic knee extensor strength and reduction of postural sway in the elderly from long-term Tai Chi exercise. AB - OBJECTIVES: To compare isokinetic strength of leg muscles and foot center of pressure (COP) as a measure of sway between long-term Tai Chi practitioners and controls. DESIGN: Cross-sectional study. SETTING: Community setting. PARTICIPANTS: Twenty subjects in the Tai Chi group and 19 subjects in the control group (age, >55 y). INTERVENTION: Subjects in Tai Chi group had practiced Tai Chi for a minimum of 3 years. MAIN OUTCOME MEASURES: Concentric and eccentric strength of knee extensors and flexors at 60 degrees/s and 120 degrees/s, and foot COP displacement during quiet stance with eyes open or closed. RESULTS: People in the Tai Chi group had significantly higher knee extensor strength at all speeds tested (P<.013), and smaller foot COP excursions for both eyes open and eyes closed conditions (P<.05) than people in control group. No significant difference existed in knee flexors between the 2 groups (P<.713). The COP excursions correlated significantly with the eccentric strength of knee extensors (P<.07) but not with the concentric strength of knee extensors (P<.14) or with the isokinetic strength of knee flexors at most of the speeds (P<.27). CONCLUSION: These findings support the hypothesis that the maintenance of eccentric strength of postural muscles in the lower extremities, which is beneficial for maintaining good postural stability, is helped through the long term practice of Tai Chi. PMID- 12370870 TI - Long-term effect of body weight-supported treadmill training in Parkinson's disease: a randomized controlled trial. AB - OBJECTIVE: To investigate whether body weight-supported treadmill training (BWSTT) is of long-term benefit for patients with Parkinson's disease (PD). DESIGN: Randomized controlled trial. SETTING: Inpatient rehabilitation unit for neurologic diseases in Japan. PARTICIPANTS: Twenty-four patients (Hoehn and Yahr stages 2.5 or 3) who were not demented (Mini-Mental State Examination score, >27). INTERVENTIONS: Patients were randomized to receive either a 45-minute session of BWSTT (up to 20% of body weight supported) or conventional physical therapy (PT) for 3 days a week for 1 month. MAIN OUTCOME MEASURES: Outcome measures were evaluated at baseline and at 1, 2, 3, and 6 months. Measures included the Unified Parkinson's Disease Rating Scale (UPDRS), ambulation speed (s/10 m), and number of steps taken for a 10-m walk as a parameter for stride length. RESULTS: Four patients needed modification of medications in the follow up period. Twenty patients (BWSTT, n=11; PT, n=9) without modified medications were analyzed for functional outcome. Age, duration of PD, gender, and doses of medications were comparable. There was no difference in the baseline UPDRS (BWSTT=33.3; PT=32.6), speed (BWSTT=10.8; PT=11.5), and steps (BWSTT=23.4; PT=22.8). The BWSTT group had significantly greater improvement than the PT group (Mann-Whitney U test, Bonferroni adjustment for multiple comparison) in ambulation speed at 1 month (BWSTT=8.5; PT=10.8; P<.005); and in the number of steps at 1 (BWSTT=20.0; PT=22.7; P<.005), 2 (BWSTT=19.5; PT=22.4; P<.005), 3 (BWSTT=20.1; PT=23.1; P<.005), and 4 months (BWSTT=21.0; PT=23.0; P=.006). CONCLUSIONS: BWSTT has a lasting effect specifically on short-step gait in PD. PMID- 12370871 TI - Longer versus shorter daily constraint-induced movement therapy of chronic hemiparesis: an exploratory study. AB - OBJECTIVE: To evaluate and compare the effects of 3-hour versus 6-hour daily training sessions in constraint-induced movement therapy (CIMT). DESIGN: Intervention study, 2-group randomized trial; baseline, pretreatment, and posttreatment measures; 1-month follow-up (weekly measures). SETTING: University department of psychology in Germany. PARTICIPANTS: A convenience sample of 15 adults with chronic hemiparesis (13 stroke, 2 traumatic brain injury). INTERVENTION: CIMT (14 consecutive days; constraint of unaffected hand for a target of 90% of waking hours) with either 6 hours (6h/d group, n=7) or 3 hours (3h/d group, n=8) of shaping training with the affected hand per day. MAIN OUTCOME MEASURES: The Motor Activity Log and Wolf Motor Function Test. RESULTS: Significant improvements in motor function in the laboratory and increased use of the affected hand in the real-world environment were found in both groups. The beneficial effects were significantly greater in the 6h/d group than in the 3h/d group. CONCLUSION: The 3-hour CIMT training schedule significantly improved motor function in chronic hemiparesis, but it was less effective than the 6-hour training schedule. PMID- 12370872 TI - Cardiovascular stress during a contemporary stroke rehabilitation program: is the intensity adequate to induce a training effect? AB - OBJECTIVES: To investigate the level of cardiovascular stress of physical therapy (PT) and occupational therapy (OT) sessions of a contemporary stroke rehabilitation program and to identify therapeutic activities that elicit heart rate responses adequate to induce a training effect. DESIGN: A descriptive, longitudinal study with heart rate and activity monitoring of PT and OT sessions at biweekly intervals, 2 to 14 weeks poststroke. SETTING: An acute inpatient stroke unit and inpatient and outpatient stroke rehabilitation units. PARTICIPANTS: A consecutive sample of 20 patients with ischemic stroke who participated in inpatient and outpatient stroke rehabilitation. INTERVENTION: Observation of routine PT and OT sessions for patients poststroke without influencing the extent and content of the sessions. MAIN OUTCOME MEASURE: Time per session in which heart rate was within the calculated target heart rate zone. RESULTS: Time per PT session spent in target heart rate zone was low (2.8+/-0.9 min), and per OT session was negligible (0.7+/-0.2 min) over the course of rehabilitation. CONCLUSIONS: The PT and OT sessions between 2 and 14 weeks poststroke did not elicit adequate cardiovascular stress to induce a training effect. PMID- 12370873 TI - Health-related quality of life and disability in young patients with spina bifida. AB - OBJECTIVES: To assess the health-related quality of life (QOL) and disability in young patients with spina bifida and to correlate them with the clinical examination findings. DESIGN: Prospective multidimensional study by means of (1) clinical assessment, (2) self-administered questionnaire for general health, and (3) standardized disability measurements. Relationships between disability measurement, patient-oriented examination, and conventional clinical assessment were evaluated. SETTING: Pediatric department at a university hospital in Italy. PATIENTS: Twelve consecutive young patients with spina bifida (mean age, 15.2 y; range, 14-18 y). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The Medical Outcomes Study 36-Item Short-Form Health Survey, the FIM trade mark instrument, and the Barthel Index. RESULTS: As expected, disability was inversely related (r=.72, P<.02) to the physical aspect of QOL. Unexpectedly, for the mental aspects of QOL, less disability was associated (r=-.70, P<.05) with higher psychologic distress and severe role disability because of emotional problems. The findings at clinical examination, especially proximal deficit of inferior limbs (r=-.70, P<.05), were usually related to higher disability and lower physical aspects of QOL. CONCLUSION: There was no linear inverse correlation between disability and QOL in patients with spina bifida. Patients with mild disability needed as much psychologic support as patients with severe whole disability. PMID- 12370874 TI - Trends in rehabilitation after amputation for geriatric patients with vascular disease: implications for future health resource allocation. AB - OBJECTIVE: To assess the effect of demographic changes on rehabilitation of geriatric patients after amputation and the implications for future health resource allocation. DESIGN: Population-based study. SETTING: Olmsted County, MN. PARTICIPANTS: Residents over the age of 65 years who had a major lower-extremity amputation because of peripheral arterial disease between 1956 and 1995. Patients who had amputations between 1956 and 1973 (earlier cohort) were compared with those who had amputations between 1974 and 1995 (later cohort). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Demographic and clinical features, total number of amputations, amputation rates, and rate of successful prosthetic fitting over time. RESULTS: Of 292 patients, 93 had amputations between 1956 and 1973 and 199 between 1974 and 1995. Amputation rates declined after 1985, but the total number of amputations was unchanged. Patients in the later cohort were more likely to have a below-knee amputation (P<.001) and cerebrovascular disease (P=.008) and to be discharged to a nursing home (P<.001). There was no significant difference in median age at amputation, survival, or rates of successful prosthetic fitting over time. CONCLUSION: Although amputation rates have declined, the total number of amputations has increased. The rate of successful prosthetic fitting in the geriatric population has not changed significantly over 40 years. Amputations in the geriatric population in the United States will probably double from 28,000 to 58,000 per year by 2030, requiring considerable resources. PMID- 12370875 TI - Association of pain with employment status and satisfaction among amputees in Japan. AB - OBJECTIVE: To assess the relationship between residual and phantom limb pain and working life among persons with limb amputation. DESIGN: Cross-sectional study in which amputee patients completed a mailed questionnaire about their residual limb and phantom limb pain, employment status, and satisfaction with working life. SETTING: Department of rehabilitation medicine of a major hospital in Japan. PARTICIPANTS: All participants were registered at the industrial rehabilitation center of a general hospital in Japan. Responses were received from 101 of the 147 patients (response rate, 68.7%) who were sent the questionnaire. INTERVENTION: An amputation pain and employment status survey that included a standardized pain measure. MAIN OUTCOME MEASURES: A self-report questionnaire, with 1 part concerning employment status and satisfaction with working life, and the other regarding amputation-related pain, which the participant described according to the Chronic Pain Grade (CPG). RESULTS: We found (1) no statistically significant association between types of pain and the return to work rate, (2) no statistically significant association between the pain severity as graded by the CPG and return to work rate, and (3) satisfaction with working life was significantly related to the CPG categories. CONCLUSION: The severity of pain does not appear to be associated with return to work among limb amputees. However, it is associated with satisfaction with working life. Appropriate treatment of pain may therefore improve work-related satisfaction. PMID- 12370876 TI - Predictors of personal care assistance for people with spinal cord injury. AB - OBJECTIVE: To assess the predictors of personal care assistance (PCA) use in people with spinal cord injury (SCI). DESIGN: Cross-sectional. SETTING: Follow-up of individuals crossing their 1st, 5th, 10th, 15th, 20th, or 25th anniversary of injury who underwent their initial rehabilitation at a Spinal Cord Injury Model Systems center. PARTICIPANTS: A total of 2154 participants (2547 records) who met the inclusion criteria for the National Spinal Cord Injury Database and had valid values for the main outcome measures. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Daily hours of paid, unpaid, and occasional PCA services. RESULTS: Differences in an interval version of the motor portion of the FIM trade mark instrument accounted for 26.3% of the variance in total PCA hours, Model Systems differences accounted for 9.3%, and no other predictor accounted for more than 2.1% of the variance. CONCLUSION: Activities of daily living functioning, as measured by the motor portion of the FIM, was the strongest predictor of PCA use among people with SCI. PMID- 12370877 TI - Immediate effects of various physical therapeutic modalities on cervical myofascial pain and trigger-point sensitivity. AB - OBJECTIVE: To investigate the immediate effect of physical therapeutic modalities on myofascial pain in the upper trapezius muscle. DESIGN: Randomized controlled trial. SETTING: Institutional practice. PATIENTS: One hundred nineteen subjects with palpably active myofascial trigger points (MTrPs). INTERVENTION: Stage 1 evaluated the immediate effect of ischemic compression, including 2 treatment pressures (P1, pain threshold; P2, averaged pain threshold and tolerance) and 3 durations (T1, 30s; T2, 60s; T3, 90s). Stage 2 evaluated 6 therapeutics combinations, including groups B1 (hot pack plus active range of motion [ROM]), B2 (B1 plus ischemic compression), B3 (B2 plus transcutaneous electric nerve stimulation [TENS]), B4 (B1 plus stretch with spray), B5 (B4 plus TENS), and B6 (B1 plus interferential current and myofascial release). MAIN OUTCOME MEASURES: The indexes of changes in pain threshold (IThC), pain tolerance (IToC), visual analog scale (IVC), and ROM (IRC) were evaluated for treatment effect. RESULTS: In stage 1, the IThC, IToC, IVC, and IRC were significantly improved in the groups P1T3, P2T2, and P2T3 compared with the P1T1 and P1T2 treatments (P<.05). In stage 2, groups B3, B5, and B6 showed significant improvement in IThC, ItoC, and IVC compared with the B1 group; groups B4, B5, and B6 showed significant improvement in IRC compared with group B1 (P<.05). CONCLUSIONS: Ischemic compression therapy provides alternative treatments using either low pressure (pain threshold) and a long duration (90s) or high pressure (the average of pain threshold and pain tolerance) and short duration (30s) for immediate pain relief and MTrP sensitivity suppression. Results suggest that therapeutic combinations such as hot pack plus active ROM and stretch with spray, hot pack plus active ROM and stretch with spray as well as TENS, and hot pack plus active ROM and interferential current as well as myofascial release technique, are most effective for easing MTrP pain and increasing cervical ROM. PMID- 12370878 TI - Predictors of driving outcome after traumatic brain injury. AB - OBJECTIVE: To examine predictors of driving status and fitness to drive after traumatic brain injury (TBI). DESIGN: Retrospective and prospective follow-up of a cohort ranging from 4 months to 10 years post-TBI. SETTING: A Midwestern, urban university-affiliated rehabilitation hospital. PARTICIPANTS: Seventy-one pairs of adults who had sustained a TBI and their significant others. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Driving status (whether the patient resumed driving), driving frequency (estimated miles driven per week), and postinjury driving records compiled by the Department of Motor Vehicles. RESULTS: Logistic and hierarchical regression analyses indicated that the significant other's perceptions of the patient's fitness to drive were the strongest predictor of patients' driving status and driving frequency. However, years postinjury, disability at discharge, and current neuropsychologic functioning best predicted postinjury driving safety as measured by actual incidents. The relation between perception of patients' fitness and actual driving incidents, however, was modest. CONCLUSIONS: Neuropsychologic and medical information available by traditional methods showed unique value in predictive driving safety. However, caregiver perception of patients' fitness was the overwhelming determinant of whether and how much patients drive. The bases on which caregivers form their opinions affect the safety of patients and the public. The rehabilitation setting is a unique resource for family education regarding abilities essential to safe driving. PMID- 12370879 TI - The reliability of upper- and lower-extremity strength testing in a community survey of older adults. AB - OBJECTIVE: To examine the stability (test-retest reliability) of strength measures in older adults obtained by nontherapist lay examiners by using a hand held portable muscle testing device (Nicholas Manual Muscle Tester). DESIGN: A prospective relational design was used to collect test-retest data for 1 male subject by using 27 lay raters who completed intensive training in manual muscle. SETTING: Data were collected from older Mexican-American adults living in the community. PARTICIPANTS: Twenty-seven lay raters who completed intensive training in manual muscle testing for a field-based assessment and interview of older adults and 63 Mexican-American subjects completing wave 4 of the Hispanic Established Populations for the Epidemiologic Study of the Elderly. INTERVENTIONS: Training involved reviewing a manual describing each testing position followed by approximately 6 hours of instruction and practice supervised by an experienced physical therapist. Lay raters then collected test-retest information on older Mexican-American subjects. MAIN OUTCOME MEASURE: Stability (test-retest) for a portable manual muscle testing device. RESULTS: Intraclass correlation coefficients (ICCs) were computed for the 27 lay raters examining 1 male subject (2 trials) and 12 lay raters assessing 63 older Mexican-American adults (1 practice and 2 trials recorded). The ICC values for the first 27 lay raters ranged from .74 to.96. The ICC values for the latter 12 lay raters ranged from .87 to.98. No differences were found in ICC values between male or female subjects. CONCLUSIONS: Stable and consistent information for upper- and lower extremity strength was collected from the older adults participating in this study. The results suggest reliable information can be obtained by lay raters using a portable manual muscle testing device if the examiners receive intensive training. PMID- 12370880 TI - Prediction of reflex sympathetic dystrophy in hemiplegia by evaluation of hand edema. AB - OBJECTIVE: To determine the predictive value of measurements of hand edema for the development of reflex sympathetic dystrophy (RSD). DESIGN: Cohort study. SETTING: Departments of rehabilitation medicine in 3 general hospitals and 1 rehabilitation hospital in Japan. PARTICIPANTS: Thirty-four stroke patients. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Measurement of the circumference of the middle finger was used to evaluate hand edema. The degree of hand edema was expressed by the ratio of circumference of the middle finger (RCMF) in the affected side to that in the uninvolved extremity. RESULTS: Eight of 34 patients developed clinical RSD from 2 to 4 months after stroke. Hand edema showed a significant relationship to the development of RSD (ie, the patients who had an RCMF of above 1.06 at 4 weeks poststroke had significantly higher incidence of RSD than those with a lower RCMF; P=.0127). CONCLUSION: It is possible to predict the development of RSD in hemiplegia by measuring hand edema 4 weeks poststroke. PMID- 12370881 TI - Improvement of voluntary quadriceps muscle activation after total knee arthroplasty. AB - OBJECTIVE: To evaluate the maximal voluntary contraction (MVC) force and the voluntary activation of the quadriceps femoris muscle in patients with knee osteoarthritis (OA) before and after total knee arthroplasty (TKA). DESIGN: A prospective intervention study. SETTING: University hospital clinic in Germany. PATIENTS: Fifty patients (32 women, 18 men; mean age +/- standard deviation, 65.8+/-5.6 y) with knee OA and 23 healthy age- and gender-matched control subjects. INTERVENTION: Unilateral TKA without patella resurfacing. MAIN OUTCOME MEASURES: Voluntary activation, MVC, and true maximal contraction forces of the bilateral quadriceps femoris muscles, using the twitch interpolation technique before and 33+/-8 months after TKA. Assessment of postoperative knee pain by the Lewis score. RESULTS: Voluntary activation increased bilaterally after surgery (P<.01 operated side, P=.02 nonoperated side) but remained lower than the voluntary activation of the controls. MVC (P<.001) and true maximal contraction forces (P=.01) increased significantly on the operated side. MVC remained unchanged (P=.45), and true maximal contraction forces decreased significantly (P=.04) on the nonoperated side. CONCLUSION: Patients with knee OA have significant bilateral voluntary activation deficits that are, at least in part, reversible within 3 years after TKA. Rehabilitation programs immediately after TKA should focus on reduction of voluntary activation deficits. After voluntary activation improves, physical therapy should target the augmentation of quadriceps femoris muscle strength. PMID- 12370882 TI - Discriminative validity of the Dutch Pediatric Evaluation of Disability Inventory. AB - OBJECTIVE: To examine the discriminative validity of the Dutch Pediatric Evaluation of Disability Inventory (PEDI) to differentiate functional status between children with and without disabilities. DESIGN: Cross-sectional study. SETTING: A university children's hospital in the Netherlands. PARTICIPANTS: A clinical sample comprising 197 children with disabilities (infantile encephalopathy, n=40; juvenile idiopathic arthritis, n=20; neurometabolic conditions, n=36; neuromuscular disorders, n=9; skeletal disorders, n=28; spina bifida, n=41; traumatic injury, n=23), and 62 children without disabilities. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURE: Functional status was measured by using a Dutch version of the PEDI. RESULTS: Discriminant analysis established the sensitivity and specificity of the PEDI. Correct predictions of group membership (disabled vs nondisabled) were found in both children without disabilities (93.5% correctly predicted) and children with disabling conditions (91.6% correctly predicted). CONCLUSION: The discriminative validity of the Dutch PEDI between children with and without disabilities was excellent. PMID- 12370883 TI - Rasch analysis of the Rivermead Mobility Index: a study using mobility measures of first-stroke inpatients. AB - OBJECTIVE: To evaluate the validity and item unidimensionality of the Rivermead Mobility Index (RMI) by using Rasch analysis. DESIGN: Application of Rasch analysis on the RMI partial data set. SETTING: A stroke program at a rehabilitation hospital in Italy. PARTICIPANTS: A total of 308 consecutive patients (155 women, 153 men; avg age, 62.79+/-11.94 y) hospitalized between 1990 and 1996. Average interval between stroke onset and admission was 52.48+/-36.22 days. INTERVENTION: Medical inpatient rehabilitation. MAIN OUTCOME MEASURES: Patients' mobility status was assessed using the RMI administered at admission and discharge. Ratings were assigned by 4 staff members working as a team. We performed separate Rasch analyses on the RMI data, gathered from different groups of first stroke inpatients examined before and after rehabilitation treatment. RESULTS: asch analysis showed the overall good validity of the RMI, except for item 15, which did not fit the unidimensional continuum estimated through the Rasch rating model. CONCLUSION: The RMI is a unidimensional scale with a hierarchy of easy-to-hard test questions. Item difficulty level was stable when processed on different groups of patients assessed on different occasions. PMID- 12370884 TI - Effect of a speech prosthesis on electromyographic activity levels of the levator veli palatini muscle activity during syllable repetition. AB - OBJECTIVES: To examine whether repeated production of a syllable effects levator veli palatini muscle activity for speakers with velopharyngeal incompetence and whether the effect can be changed by a speech prosthesis. DESIGN: Repeated measures analysis; each subject produced the speech sample /pu/ more than 200 times in each of 2 experimental conditions. SETTING: Graduate dental school in Japan. PARTICIPANTS: Four patients with operated cleft palate with a speech prosthesis. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Electromyographic traces were highband-pass filtered at 30 Hz, rectified, and smoothed with a time constant of 30 ms. Electromyographic traces were made of each production in 2 conditions: (1) without the prosthesis and (2) with the prosthesis. The regression slope of the linear regressor line, when smoothed levator activity was the criterion variable and the number of productions was the explanatory variable, was calculated in each condition. RESULTS: The mean value of levator activity was significantly smaller with the prosthesis in than without it (P<.10, t test). With the prosthesis, the regression slope was significant for all 4 subjects, whereas it was insignificant without the prosthesis for 3 of 4 subjects (P<.10, t test). Absolute values of the regression slope were significantly smaller with the prosthesis than without it for all subjects (P<.10, t test). Comparison of the regression slopes for the 2 conditions identified a significant difference in slopes between the 2 conditions (P<.10, t test). CONCLUSION: In operated cleft palate patients with velopharyngeal incompetence, a speech prosthesis can stabilize both temporal changes in levator muscle activity and connected speech, such as conversation. PMID- 12370885 TI - The effectiveness of a hands-free environmental control system for the profoundly disabled. AB - OBJECTIVE: To investigate the effectiveness of a hands-free environmental control system (ECS) that allows profoundly disabled persons to activate and control electric devices in their home by using consciously controlled changes in their brain signals. DESIGN: A cohort study with a field trial testing of the ECS on 3 occasions. SETTING: Participants' homes. PARTICIPANTS: Ten profoundly disabled persons (mean age, 42.9 y), all of whom had very limited movement from the neck downward. Six had spinal cord injury with lesions ranging from C2 to C5-6. The other 4 had profound disability (1 each from polio, spinal muscular atrophy, multiple sclerosis, cerebral palsy). INTERVENTIONS: Participants performed tasks on each of 3 test occasions. The tasks consisted of turning a television on at the beginning of the trial, changing channels (up, down), changing volume, and turning it off at the conclusion of each trial. MAIN OUTCOME MEASURES: Time participants took to select the correct option and number of errors made in selecting the correct option. Measures were taken for each trial, so that any improvement in switching could be detected. RESULTS: All participants effectively used the ECS to operate their television sets. Selecting a correct option took about 30 seconds (with the majority of this time attributed to machine cycling time), with an error rate of 1.8 per 5 options selected. The time taken to operate the ECS reduced slightly over the 3 trials and selection errors reduced by around 50% (to less than 1 error per 5 options). CONCLUSIONS: With minimal training, profoundly disabled persons were able to use an ECS that uses changes in brain wave signals. This result demonstrates the efficacy of an additional and novel ECS in an area in which few switches are available. PMID- 12370887 TI - Constraint-induced therapy for a child with hemiplegic cerebral palsy: a case report. AB - A 12-year-old boy with hemiplegic cerebral palsy (CP) presented with decreased function in his left upper extremity. He was treated with a 3-week protocol of constraint-induced therapy (CIT) consisting of six 2-hour sessions of physical and occupational therapy, plus home practice. Improvements in upper-extremity function were found in the mean and median time for completion of the Wolf Motor Function Test immediately posttreatment and at 8-month follow-up. Also, improvements in functional use of the arm were documented with the Assessment of Motor and Process Skills and by patient self-report of use of the upper extremity at home. The results suggest that CIT may be useful in the treatment of upper extremity dysfunction in hemiplegic CP. Larger, experimentally controlled investigations of the efficacy of CIT and the mechanism of recovery in patients with CP are warranted. The effects of the duration and intensity of CIT protocols need additional study to increase its clinical application. PMID- 12370886 TI - Bilateral anterior superior iliac spine pressure ulcers: a case report. AB - The most frequent sites for pressure ulcers are the occiput, sacrum, ischial tuberosities, trochanters, lateral malleoli, and posterior heels. A 27-year-old woman with Wegener's granulomatosis was admitted to our rehabilitation unit after spending 65 days in an intensive care unit and 40 days in an internal medicine ward. She required mechanical ventilation because of respiratory failure. Adequate oxygenation was only achieved in the prone position. As a result, she developed bilateral anterior superior iliac spine pressure ulcers. Pressure ulcers in this location have not been reported in the literature. Complicating factors included variable levels of oxygenation, malnutrition, anemia, and steroid therapy. Complete healing, documented with serial photographs, occurred over 9 months. Although prone positioning can improve pulmonary gas exchange, it exposes the patient to unique complications. When it is required, specific care should be directed to the unusual weight-bearing surfaces to avoid pressure ulcers. PMID- 12370888 TI - Evaluating a written emotional disclosure homework intervention for lower-limb amputees. AB - OBJECTIVE: To evaluate the Pennebaker Emotional Disclosure paradigm with lower limb amputee patients in terms of compliance and efficacy. DESIGN: Repeated measures. SETTING: Home based. PARTICIPANTS: Low compliance, both with the initial mailed request (28%) and the subsequent writing task (48%), resulted in 23 lower-limb amputees who had been fitted with a prosthesis participating. INTERVENTIONS: Patients completed a 15-minute writing task, 6 times, over 2 weeks, with initial baseline and 2-month follow-up assessments. MAIN OUTCOME MEASURES: Cognitive processing, well-being, adjustment to an artificial limb, pain, and prosthetic use. RESULTS: Stronger emotional disclosure was associated with significant reductions in psychologic and physical aspects of amputees' satisfaction with their prosthesis, some of which were mediated by positive changes in affect immediately after the writing tasks. CONCLUSIONS: Our results failed to support previous findings with nonclinical samples; in fact, our results contradicted previous findings. We therefore caution that written emotional disclosure may be contraindicated with lower-limb amputee patients. PMID- 12370889 TI - The association of pain with aerobic fitness in patients with chronic low back pain. AB - OBJECTIVE: To investigate the association of aerobic fitness (VO(2)max) with pain intensity as reported by a sample of patients with chronic low back pain (LBP). DESIGN: Cross-sectional with partial longitudinal follow-up. SETTING: Outpatient interdisciplinary pain management program in a teaching hospital. PATIENTS: A convenience sample of 75 patients with chronic LBP. INTERVENTION: Patients reported pain intensity before and after undergoing a modified treadmill test. Peak VO(2) was measured by using indirect calorimetry. Predicted VO(2)max was determined by extrapolating peak VO(2) and heart rate values during testing to predicted maximal heart rate. MAIN OUTCOME MEASURES: Aerobic fitness and pain intensity before and after testing. RESULTS: No significant relation was found between pain intensity and predicted VO(2)max or aerobic fitness. CONCLUSION: There is no association between pain intensity and aerobic fitness. Deconditioning, defined as a lack of cardiovascular fitness levels normal for age and gender, therefore does not contribute to pain intensity in patients with chronic LBP. PMID- 12370890 TI - Effect of a functional knee brace on knee flexion and extension strength after anterior cruciate ligament reconstruction. AB - OBJECTIVES: To investigate the effect of a knee brace on knee flexion and extension muscular strength of patients after anterior cruciate ligament (ACL) reconstruction and to evaluate whether the effect of the brace depends on patient symptoms and muscular strength. DESIGN: Repeated measures. SETTING: A university based outpatient orthopedic clinic and musculoskeletal assessment laboratory. PARTICIPANTS: Twenty-seven patients (14 women, 13 men; mean age, 28+/-11 y) having undergone arthroscopically assisted ACL reconstruction by using a semitendinosus and gracilis autograft. INTERVENTION: A custom-fit ACL functional knee brace. MAIN OUTCOME MEASURES: The brace effect was calculated as the change in peak torque observed with the brace, expressed as a percentage of peak torque observed without the brace, during isokinetic concentric knee flexion and extension movements performed at 90 degrees /s. Patient symptoms were quantified by using a disease-specific health-related quality of life questionnaire. RESULTS: Knee flexion strength decreased significantly with the brace (mean brace effect=-7.3%, P<.05). The brace effect during knee flexion varied considerably ( 52% to 47%) and was significantly related to peak torque observed without the brace (r=-.50, P<.01). All other comparisons and correlations were not significant. CONCLUSIONS: These findings suggest that brace effects depend on patient strength. A brace may inhibit knee flexion strength of stronger patients, yet result in no change or even improvements in strength of weaker patients. Future research is required to further elucidate which patients may derive most benefit or detriment from bracing. PMID- 12370891 TI - The canary in the mine. AB - Many Americans are concerned about their access to health care in the future, especially their ability to pay for needed services. However, a person with a disabling condition requiring ongoing clinical vigilance, supportive care, and other assistive services or technologies faces special difficulties: that person is the "canary in the mine," warning others about fundamental problems within our health care system. Persons with disabilities who have health insurance are often unable to get items and services not covered by their plans. They experience more problems than others with follow-up care, availability of specialists, getting to doctors, and obtaining help during off hours. These problems suggest that people with disabilities fall into the "quality chasm," the metaphor used by the Institute of Medicine to describe the gap between ideal care and current reality. The Crossing the Quality Chasm report suggests 6 aims for fundamental reform, exhorting the health care system to become safe, effective, patient-centered, timely, efficient, and equitable. Each of these aims holds special resonance for persons with disabilities. Despite the compelling need to overhaul the health care system, the American public as yet seems little inclined to fundamental change. Perhaps the impetus must come from subgroups within the population who are particularly at risk from the current system, such as persons with disabilities. As solutions are crafted, people with disabilities, their families, and communities should help design and direct fundamental changes to the health care system. PMID- 12370892 TI - Gastric emptying and gastrointestinal motility abnormalities after spinal cord injury. PMID- 12370894 TI - Metalloid aluminum and gallium clusters: element modifications on the molecular scale? AB - As members of the same group in the periodic table, the industrially significant elements aluminum and gallium exhibit strong similarities in the majority of their compounds. In contrast there are significant differences in the structures of the two elemental forms: Aluminum forms a typical closest-packed metallic structure whereas gallium demonstrates a diversity of molecular bonding principles in its seven structural modifications. It can therefore be expected that differences between Al and Ga compounds will arise when, as for the elemental forms, many metal-metal bonds are formed. To synthesize such cluster compounds, we have developed the following synthesis procedure: Starting from gaseous monohalides at around 1000 degrees C, metastable solutions are generated from which the elements ultimately precipitate by means of a disproportionation reaction at room temperature. On the way to the elemental forms, molecular Al and Ga cluster compounds can be obtained by selection of suitable ligands (protecting groups), in which a core of Al or Ga atoms are protected from the formation of the solid element by a ligand shell. Since the arrangement of atoms in such clusters corresponds to that in the elements, we have designated these clusters as metalloid or elementoid. In accordance with the Greek word [see text] (ideal, prototype), the atomic arrangement in metalloid clusters represents the prototypic or ideal atomic arrangement in the elements at the molecular level. The largest clusters of this type contain 77 Al or 84 Ga atoms and have diameters of up to two nanometers. They hold the world record with respect to the naked metal-atom core for structurally characterized metalloid clusters. PMID- 12370895 TI - "Asymmetric" catalysis by lanthanide complexes. AB - Catalysis with lanthanide (Ln) complexes has been underestimated for long time, although Ln(III) complexes have great advantages as Lewis acid catalysts for "asymmetric" carbon-carbon bond-forming reactions. Lanthanide complexes are highly active in ligand-substitution reactions, especially with hard ligands. The association with substrates and dissociation of products are achieved fast enough for high catalyst efficiency. The asymmetric catalysis of organic reactions can be greatly advanced by the use of Ln complexes with chiral ligands such as binaphthol (binol). Ln(II) complexes are good reducing agents, which can be used in a wide variety of synthetically important reactions; when chiral ligands are used, many of these reactions are highly stereoselective. In the context of "green chemistry", the development of asymmetric Ln catalysts, and their recyclable use, is of increasing importance. This review gives an overview of the most recent developments in catalysis with lanthanide(II) and lanthanide(III) complexes. PMID- 12370896 TI - Relativity, gold, closed-shell interactions, and CsAu.NH3. AB - The chemical properties of gold are strongly influenced by relativistic effects. One example is the large electronegativity of Au, which qualitatively explains the stability of (solid or liquid) cesium auride, Cs(+)Au(-), and other systems with Au(-) ions. An especially impressive compound is CsAu.NH(3), the structure and bonding of which are discussed. Future possibilities for finding further aurides are outlined. PMID- 12370897 TI - Teaching the right reasons: lessons from the mistaken origin of the rotational barrier in ethane. PMID- 12370899 TI - Direct electrochemical aziridination of alkenes under metal-free conditions. PMID- 12370898 TI - Carbohydrate arrays as tools for glycomics. PMID- 12370900 TI - Microwave-assisted reactions in organic synthesis-are there any nonthermal microwave effects? Response to the highlight by N. Kuhnert. PMID- 12370901 TI - The elusive terminal imido of manganese(v). PMID- 12370902 TI - Light-harvesting dendrimers: efficient intra- and intermolecular energy-transfer processes in a species containing 65 chromophoric groups of four different types. PMID- 12370903 TI - Synthesis of amphiphilic conjugated diblock oligomers as molecular diodes. PMID- 12370904 TI - A helical array of pendant fullerenes on an optically active polyphenylacetylene. PMID- 12370906 TI - An inclusion complex with [Gd(dmf)(8)](3+) ions encapsulated in pockets of an anionic array of [(Cu(6)(CN)(9))(3-)] infinity units; a cyanide-bridged Cu-Gd layer structure. PMID- 12370905 TI - Changeable pore sizes allowing effective and specific recognition by a molybdenum oxide based "Nanosponge": en route to sphere-surface and nanoporous-cluster chemistry. PMID- 12370907 TI - An antibody that reconstitutes the "base-on" form of B(12) coenzymes. PMID- 12370908 TI - Solid-phase synthesis and biological evaluation of a pepticinnamin E library. PMID- 12370909 TI - A molecular beacon for quantitative monitoring of the DNA polymerase reaction in real-time. PMID- 12370910 TI - Using molecular symmetry to form new drugs: hydroxymethyl-substituted 3,9 diazatetraasteranes as the first class of symmetric MDR modulators. PMID- 12370911 TI - Fluorescence spectroscopic quantification of the release of cyclic nucleotides from photocleavable [bis(carboxymethoxy)coumarin-4-yl]methyl esters inside cells. PMID- 12370912 TI - Protonated benzene: IR spectrum and structure of C(6)H(7)(+). PMID- 12370913 TI - Synthesis of furoquinolines by a multicomponent domino process. PMID- 12370914 TI - An asymmetric cyanation reaction and sequential asymmetric cyanation-nitroaldol reaction using a [YLi(3)[tris(binaphthoxide)]] single catalyst component: catalyst tuning with achiral additives. PMID- 12370915 TI - A trigonal-bipyramidal ferric aqua complex with a sterically hindered salen ligand as a model for the active site of protocatechuate 3,4-dioxygenase. PMID- 12370916 TI - Micro/Nanoengineering of the self-organized three-dimensional fibrous structure of functional materials. PMID- 12370917 TI - Exceptional rate enhancements and improved diastereoselectivities through chelating diamide coordination in intramolecular alkene hydroaminations catalyzed by yttrium and neodymium amido complexes. PMID- 12370919 TI - Self-assembly using organometalloligands as spacers in the controlled formation of isomeric 1D and 2D supramolecular quinonoid networks. PMID- 12370920 TI - Reversible photoinsertion of ferrocene into a hydrophobic semiconductor surface: a chemionic switch. PMID- 12370918 TI - Bispyrene-conjugated 2'-O-methyloligonucleotide as a highly specific RNA recognition probe. PMID- 12370921 TI - Gram-scale synthesis of suspension-polymerized styrene-divinylbenzene-based resins using an oscillatory baffled reactor. PMID- 12370922 TI - An azulene dimer as a near-infrared quencher. PMID- 12370923 TI - Single-step assembly of a C(2)-symmetrical palladium(IV) spirocyclic complex. PMID- 12370924 TI - Direct assembly of large arrays of oriented conducting polymer nanowires. PMID- 12370925 TI - A new generation of air stable, highly active Pd complexes for C--C and C--N coupling reactions with aryl chlorides. PMID- 12370926 TI - Sulfur-functionalized olefins for titanacycle formation: tandem asymmetric cyclization and the pummerer reaction based on sulfoxides promoted by titanium(II)-to-titanium(IV) relay. PMID- 12370927 TI - Cyclotrehalins: cyclooligosaccharide receptors featuring a hydrophobic cavity. PMID- 12370929 TI - Perfectly straight nanostructures of metallosupramolecular coordination polyelectrolyte amphiphile complexes on graphite. PMID- 12370928 TI - Generation of libraries of pharmacophoric structures with increased complexity and diversity by employing polymorphic scaffolds. PMID- 12370930 TI - (C(2)H(10)N(2))[Cr(HPO(3))F(3)]: The First Organically Templated Fluorochromium(III) Phosphite. PMID- 12370931 TI - Tuning electronic behavior of carbonyl metal clusters by substitution of interstitial and capping atoms. PMID- 12370932 TI - Synthesis, X-ray structure, and properties of a tetrabenzannelated 1,2,4,5 cyclophane. PMID- 12370933 TI - Asymmetric, catalytic phenyl transfer to imines: highly enantioselective synthesis of diarylmethylamines. PMID- 12370934 TI - [(PPh(3))Ag(HCB(11)Me(11))]: a complex with intermolecular Ag.H3C interactions. PMID- 12370936 TI - Stereocontrolled total synthesis of apicularen A and its delta(17,18) Z isomer. PMID- 12370935 TI - A simple, reliable, catalytic asymmetric allylation of ketones. PMID- 12370937 TI - Efficient and simple solid-phase synthesis of short cyclic oligodeoxynucleotides bearing a phosphorothioate linkage. PMID- 12370938 TI - Kinetics of a reversible covalent-bond-forming reaction observed at the single molecule level. PMID- 12370939 TI - Hydroboration of coordinated dinitrogen: a new reaction for the N2 ligand that results in its functionalization and cleavage. PMID- 12370942 TI - Comparative study of metal-porphyrins, -porphyrazines, and -phthalocyanines. AB - A theoretical comparative study of complexes of porphyrin (P), porphyrazine (Pz), and phthalocyanine (Pc) with metal (M) = Fe, Co, Ni, Cu, and Zn has been carried out using a DFT method. The calculations provide a clear elucidation of the ground states for the MP/Pz/Pc molecules and for a series of [MP/Pz/Pc](x-) and [MP/Pz/Pc](y+) ions (x = 1, 2, 3, 4; y = 1, 2). There are significant differences among MP, MPz, and MPc in the electronic structure and other calculated properties. For FeP/Pz and CoP/Pz, the first oxidation occurs at the central metal, while it is the macroring of FePc and CoPc that is the site of oxidation. The smaller coordination cavity results in a stronger ligand field in Pz than in P. However, the benzo annulation produces a surprisingly strong destabilizing effect on the metal-macrocycle bonding. The effects of Cl axial bonding upon the electronic structures of the iron(III) complexes of P, Pz, and Pc were examined, as was the bonding of pyridine (py) to NiP, NiPz, and NiPc. The porphinato core size plays a crucial role in controlling the spin state of Fe(III) in these complexes. FePc(Cl) is predicted to be a pure intermediate-spin system, whereas NiPz(py)(2) and NiPc(py)(2) are metastable in high-spin (S = 1) states. The NiPz/Pc-(py)(2) binding energy curve has only a shallow well that facilitates decomposition of the complex. The NiP-(py)(2) bond energy is small, but the relatively deep well in the binding energy curve ought to make this system stable to decomposition. PMID- 12370943 TI - The treatment of solvation by a generalized Born model and a self-consistent charge-density functional theory-based tight-binding method. AB - We present a model to calculate the free energies of solvation of small organic compounds as well as large biomolecules. This model is based on a generalized Born (GB) model and a self-consistent charge-density functional theory-based tight-binding (SCC-DFTB) method with the nonelectrostatic contributions to the free energy of solvation modeled in terms of solvent-accessible surface areas (SA). The parametrization of the SCC-DFTB/GBSA model has been based on 60 neutral and six ionic molecules composed of H, C, N, O, and S, and spanning a wide range of chemical groups. Effective atomic radii as parameters have been obtained through Monte Carlo Simulated Annealing optimization in the parameter space to minimize the differences between the calculated and experimental free energies of solvation. The standard error in the free energies of solvation calculated by the final model is 1.11 kcal mol(-1). We also calculated the free energies of solvation for these molecules using a conductor-like screening model (COSMO) in combination with different levels of theory (AM1, SCC-DFTB, and B3LYP/6-31G*) and compared the results with SCC-DFTB/GBSA. To assess the efficiency of our model for large biomolecules, we calculated the free energy of solvation for a HIV protease-inhibitor complex containing 3,204 atoms using the SCC-DFTB/GBSA and the SCC-DFTB/COSMO models, separately. The computed relative free energies of solvation are comparable, while the SCC-DFTB/GBSA model is three to four times more efficient, in terms of computational cost. PMID- 12370944 TI - An improved OPLS-AA force field for carbohydrates. AB - This work describes an improved version of the original OPLS-all atom (OPLS-AA) force field for carbohydrates (Damm et al., J Comp Chem 1997, 18, 1955). The improvement is achieved by applying additional scaling factors for the electrostatic interactions between 1,5- and 1,6-interactions. This new model is tested first for improving the conformational energetics of 1,2-ethanediol, the smallest polyol. With a 1,5-scaling factor of 1.25 the force field calculated relative energies are in excellent agreement with the ab initio-derived data. Applying the new 1,5-scaling makes it also necessary to use a 1,6-scaling factor for the interactions between the C4 and C6 atoms in hexopyranoses. After torsional parameter fitting, this improves the conformational energetics in comparison to the OPLS-AA force field. The set of hexopyranoses included in the torsional parameter derivation consists of the two anomers of D-glucose, D mannose, and D-galactose, as well as of the methyl-pyranosides of D-glucose, D mannose. Rotational profiles for the rotation of the exocyclic group and of different hydroxyl groups are also compared for the two force fields and at the ab initio level of theory. The new force field reduces the overly high barriers calculated using the OPLS-AA force field. This leads to better sampling, which was shown to produce more realistic conformational behavior for hexopyranoses in liquid simulation. From 10-ns molecular dynamics (MD) simulations of alpha-D glucose and alpha-D-galactose the ratios for the three different conformations of the hydroxymethylene group and the average (3)J(H,H) coupling constants are derived and compared to experimental values. The results obtained for OPLS-AA-SEI force field are in good agreement with experiment whereas the properties derived for the OPLS-AA force field suffer from sampling problems. The undertaken investigations show that the newly derived OPLS-AA-SEI force field will allow simulating larger carbohydrates or polysaccharides with improved sampling of the hydroxyl groups. PMID- 12370945 TI - Integral algorithm and density matrix integration scheme for ab initio band structure calculations on polymeric systems. AB - A new program for band structure calculations of periodic one-dimensional systems has been constructed. It is distinguishable from other codes by the efficient two electron integral evaluation and the integration schemes of the density matrix in the first Brillouin zone. The computation of polymeric two-electron integrals is based on the McMurchie Davidson algorithm and builds batches of the different cell indices included in the polymeric system. Consequently it presents efficient scaling with respect to the number of unit cells taken into account. Our algorithm takes into account fully the polymeric symmetry rather than the molecular symmetry. A semidirect procedure where only exchange integrals are computed at each SCF cycle is proposed in order to maintain balance between computation time and disk space. In addition, the integration of the density matrix over a large number of cell indices can be performed by different methods, such as Gauss-Legendre, Clenshaw-Curtis, Filon, and Alaylioglu-Evans-Hyslop. This last scheme is able to obtain an accuracy of 10(-13) a.u. on each individual density matrix element for all cell indices with only 48 k-points. PMID- 12370946 TI - Methodology of predicting approximate shapes and size distribution of micelles: illustration for simple models. AB - We propose an efficient methodology for predicting approximate shapes and size distribution of micelles. The methodology is a judicious combination of a conventional thermodynamic approach, the reference interaction site model (RISM) theory, and the Monte Carlo (MC) simulated annealing technique. Solvent effects are fully incorporated using the RISM theory with our robust and very efficient algorithm for solving the RISM equations, and the MC technique is applied only to surfactant molecules. The methodology is potentially applicable to realistic models of surfactant and solvent molecules with all-atom potentials. As the first step, however, it is illustrated for simplified models having only essential characteristics of the amphiphiles. We estimate the critical micelle concentration, approximate shapes, and size distributions at some surfactant concentrations. Roles of the solvent and effects due to the type of the surfactant molecule are discussed in detail. PMID- 12370947 TI - DFT and ab initio direct dynamics studies on the hydrogen abstraction reactions of chlorine atoms with CH(4-n)F(n) (n = 1-3). AB - A direct dynamics study is carried out for the hydrogen abstraction reactions Cl + CH(4-n)F(n) (n = 1-3) in the temperature range of 200-1,000 K. The minimum energy paths (MEPs) of these reactions are calculated at the BH&H-LYP/6-311G(d,p) level, and the energies along the MEPs are further refined at the QCISD(T)/6 311+G(2df,2p) and QCISD(T)/6-311+G(d,p) (single-point) level. The rate constants obtained by using the improved canonical variational transition state theory incorporating small-curvature tunneling correction (ICVT/SCT) are in good agreement with the available experimental results. It is shown that the vibrational adiabatic potential energy curves for these reactions have two barriers, a situation similar to the analogous reactions CH(3)X+Cl (X=Cl, Br). The theoretical results show that for the title reactions the variational effect should not be neglected over the whole considered temperature range, while the small-curvature tunneling effect is only important in the lower temperature range. The effects of fluorine substitution on the rate of this kind of reactions are also examined. PMID- 12370948 TI - Water polarizability in condensed phase: ab initio evaluation by cluster approach. AB - The polarizability of a water molecule in liquid is evaluated via ab initio and density functional calculations for water clusters. This work has considerably improved our previous effort [J Chem Phys 1999, 110, 11987] to attain quantitative accuracy for polarizability. The calculations revealed that the water polarizability in the liquid is reduced from that in the gaseous phase by 7 9%. These results suggest significant implications for polarizable water models. PMID- 12370949 TI - Analysis of the intermolecular interaction between CH(3)OCH(3), CF(3)OCH(3), CF(3)OCF(3), and CH(4): high level ab initio calculations. AB - The intermolecular interaction energies of the CH(3)OCH(3)-CH(4), CF(3)OCH(3) CH(4), and CF(3)OCF(3)-CH(4) systems were calculated by ab initio molecular orbital method with the electron correlation correction at the second order Moller-Plesset perturbation (MP2) method. The interaction energies of 10 orientations of complexes were calculated for each system. The largest interaction energies calculated for the three systems are -1.06, -0.70, and -0.80 kcal/mol, respectively. The inclusion of electron correlation increases the attraction significantly. It gains the attraction -1.47, -1.19, and -1.27 kcal/mol, respectively. The dispersion interaction is found to be the major source of the attraction in these systems. In the CH(3)OCH(3)-CH(4) system, the electrostatic interaction (-0.34 kcal/mol) increases the attraction substantially, while the electrostatic energies in the other systems are not large. Fluorine substitution of the ether decreases the electrostatic interaction, and therefore, decreases the attraction. In addition the orientation dependence of the interaction energy is decreased by the substitution. PMID- 12370950 TI - A revised quantum chemistry-based potential for poly(ethylene oxide) and its oligomers in aqueous solution. AB - We have conducted a high-level quantum chemistry study of the interactions of 1,2 dimethoxyethane (DME) with water for complexes representing both hydrophilic and hydrophobic hydration. It was found that our previous quantum chemistry-based force field for poly(ethylene oxide) (PEO) and its oligomers in aqueous solution did a poor job in describing the hydrophobic binding of water to the ether, consistent with our recent calculations of the excess free energy and entropy of hydration of DME. Our original force field was revised to more accurately reproduce the interaction of water with the carboneous portions of DME. Molecular dynamics simulations of aqueous DME solutions using the revised quantum chemistry based potential yielded good agreement with experiment for excess free energy, enthalpy, and volume as well as excess solution viscosity and the self-diffusion of water. Comparison with our original potential revealed that the relatively hydrophobic ether-water interactions in the new potential strongly reduced the favorable excess free energy and enthalpy but have relatively little influence on the excess entropy for dilute DME solutions. Other properties of DME and PEO solutions including conformational populations and dynamics, solution viscosity, hydrogen bonding, water translational and rotational diffusion and neutron structure factor as a function of solution composition were found to be largely unchanged from those obtained using the original potential. PMID- 12370951 TI - Update of the AIM2000-program for atoms in molecules. AB - The second version of the program package AIM2000 is presented. AIM2000 makes use of the well established theory of atoms in molecules. AIM2000 analyzes the molecular structure and calculates properties of atoms in molecules as well as properties of interatomic surfaces. The program has an interactive, context sensitive help component and extensive 2D and 3D visualization components. PMID- 12370952 TI - Analytical TDHF second derivatives of dynamic electronic polarizability with respect to nuclear coordinates. Application to the dynamic ZPVA correction. PMID- 12370953 TI - A discrete fully recurrent network of max product units for associative memory and classification. AB - This paper defines the truncated normalized max product operation for the transformation of states of a network and provides a method for solving a set of equations based on this operation. The operation serves as the transformation for the set of fully connected units in a recurrent network that otherwise might consist of linear threshold units. Component values of the state vector and outputs of the units take on the values in the set [0, 0.1,..., 0.9, 1]. The result is a much larger state space given a particular number of units and size of connection matrix than for a network based on threshold units. Since the operation defined here can form the basis of transformations in a recurrent network with a finite number of states, fixed points or cycles are possible and the network based on this operation for transformations can be used as an associative memory or pattern classifier with fixed points taking on the role of prototypes. Discrete fully recurrent networks have proven themselves to be very useful as associative memories and as classifiers. However they are often based on units that have binary states. The effect of this is that the data to be processed consisting of vectors in R(n) have to be converted to vectors in [0, 1]m with m much larger than n since binary encoding based on positional notation is not feasible. This implies a large increase in the number of components. The effect can be lessened by allowing more states for each unit in our network. The network proposed demonstrates those properties that are desirable in an associative memory very well as the simulations show. PMID- 12370954 TI - Neural network based temporal video segmentation. AB - The organization of video information in video databases requires automatic temporal segmentation with minimal user interaction. As neural networks are capable of learning the characteristics of various video segments and clustering them accordingly, in this paper, a neural network based technique is developed to segment the video sequence into shots automatically and with a minimum number of user-defined parameters. We propose to employ growing neural gas (GNG) networks and integrate multiple frame difference features to efficiently detect shot boundaries in the video. Experimental results are presented to illustrate the good performance of the proposed scheme on real video sequences. PMID- 12370955 TI - A neural network approach to approximating MAP in belief networks. AB - Bayesian belief networks (BBN) are a widely studied graphical model for representing uncertainty and probabilistic interdependence among variables. One of the factors that restricts the model's wide acceptance in practical applications is that the general inference with BBN is NP-hard. This is also true for the maximum a posteriori probability (MAP) problem, which is to find the most probable joint value assignment to all uninstantiated variables, given instantiation of some variables in a BBN. To circumvent the difficulty caused by MAP's computational complexity, we suggest in this paper a neural network approximation approach. With this approach, a BBN is treated as a neural network without any change or transformation of the network structure, and the node activation functions are derived based on an energy function defined over a given BBN. Three methods are developed. They are the hill-climbing style discrete method, the simulated annealing method, and the continuous method based on the mean field theory. All three methods are for BBN of general structures, with the restriction that nodes of BBN are binary variables. In addition, rules for applying these methods to noisy-or networks are also developed, which may lead to more efficient computation in some cases. These methods' convergence is analyzed, and their validity tested through a series of computer experiments with two BBN of moderate size and complexity. Although additional theoretical and empirical work is needed, the analysis and experiments suggest that this approach may lead to effective and accurate approximation for MAP problems. PMID- 12370956 TI - Extracting the principal behavior of a probabilistic supervisor through neural networks ensemble. AB - In this paper, we propose a model of a neural network ensemble that can be trained with a supervisor having two kinds of input-output functions where the occurrence probability of each function is not even. This condition can be likened to a learning condition, in which the learning data are hampered by noise. In this case, the neural network has the impression that the learning supervisor (object) has a probabilistic behavior in which the supervisor generates correct learning data most of the time but occasionally generates erroneous ones. The objective is to train the neural network to approximate the greatest distributed input-output relation, which can be considered to be the principal nature of the supervisor, so that we can obtain a neural network that is able, to some extent, to suppress the ill effect of erroneous data encountered during the learning process. PMID- 12370957 TI - Improved backpropagation learning in neural networks with windowed momentum. AB - Backpropagation, which is frequently used in Neural Network training, often takes a great deal of time to converge on an acceptable solution. Momentum is a standard technique that is used to speed up convergence and maintain generalization performance. In this paper we present the Windowed Momentum algorithm, which increases speedup over Standard Momentum. Windowed Momentum is designed to use a fixed width history of recent weight updates for each connection in a neural network. By using this additional information, Windowed Momentum gives significant speedup over a set of applications with same or improved accuracy. Windowed Momentum achieved an average speedup of 32% in convergence time on 15 data sets, including a large OCR data set with over 500,000 samples. In addition to this speedup, we present the consequences of sample presentation order. We show that Windowed Momentum is able to overcome these effects that can occur with poor presentation order and still maintain its speedup advantages. PMID- 12370958 TI - The accurate estimation of meteorological profiles employing ANNs. AB - The lack of meteorological measurements at a location of interest (target location) constitutes a problem that is crucial for the purposes of both weather forecasting and energy system design/validation. This paper constitutes a pilot study for the accurate estimation of meteorological values at a target location employing the meteorological measurements collected at a nearby (reference) location. Artificial neural networks are investigated and compared with traditional estimation methods such as linear models of first and higher orders and the non-linear model. The significance of the improvement obtained via the estimation--and especially the artificial neural network approach--over simply considering the measurements at the reference location is demonstrated in a number of energy applications. PMID- 12370959 TI - Constructing a query-able radial basis function artificial neural network. AB - Artificial neural networks will be more widely accepted as standard engineering tools if their reasoning process can be made less opaque. This paper describes NetQuery, an explanation mechanism that extracts meaningful explanations from trained Radial Basis Function (RBF) networks. RBF networks are well suited for explanation generation because they contain a set of locally tuned units. Standard RBF networks are modified to identify dependencies between the inputs, to be sparsely connected, and to have an easily interpretable output layer. Given these modifications, the network architecture can be used to extract "Why?" and "Why not?" explanations from the network in terms of excitatory and inhibitory in puts and their linear relationships, greatly simplified by a run-time pruning algorithm. These query results are validated by creating an expert system based on the explanations. NetQuery is also able to inform a user about a possible change in category for a given pattern by responding to a "How can I...?" query. This kind of query is extremely useful when analyzing the quality of a pattern set. PMID- 12370960 TI - Stability analysis of higher-order neural networks for combinatorial optimization. AB - Recurrent neural networks with higher order connections, from here on referred to as higher-order neural networks (HONNs), may be used for the solution of combinatorial optimization problems. In Ref. 5 a mapping of the traveling salesman problem (TSP) onto a HONN of arbitrary order was developed, thereby creating a family of related networks that can be used to solve the TSP. In this paper, we explore the trade-off between network complexity and quality of solution that is made available by the HONN mapping of the TSP. The trade-off is investigated by undertaking an analysis of the stability of valid solutions to the TSP in a HONN of arbitrary order. The techniques used to perform the stability analysis are not new, but have been widely used elsewhere in the literature. The original contribution in this paper is the application of these techniques to a HONN of arbitrary order used to solve the TSP. The results of the stability analysis show that the quality of solution is improved by increasing the network complexity, as measured by the order of the network. Furthermore, it is shown that the Hopfield network, as the simplest network in the family of higher-order networks, is expected to produce the poorest quality of solution. PMID- 12370961 TI - Relating priming and repetition suppression. AB - We present a prototype of a recently proposed two stage model of the entorhinal hippocampal loop. Our aim is to form a general computational model of the sensory neocortex. The model--grounded on pure information theoretic principles--accounts for the most characteristic features of long-term memory (LTM), performs bottom up novelty detection, and supports noise filtering. Noise filtering can also serve to correct the temporal ordering of information processing. Surprisingly, as we examine the temporal characteristics of the model, the emergent dynamics can be interpreted as perceptual priming, a fundamental type of implicit memory. In the model's framework, computational results support the hypothesis of a strong correlation between perceptual priming and repetition suppression and this correlation is a direct consequence of the temporal ordering in forming the LTM. We also argue that our prototype offers a relatively simple and coherent explanation of priming and its relation to a general model of information processing by the brain. PMID- 12370962 TI - Training simultaneous recurrent neural network with resilient propagation for static optimization. AB - This paper proposes a non-recurrent training algorithm, resilient propagation, for the Simultaneous Recurrent Neural network operating in relaxation-mode for computing high quality solutions of static optimization problems. Implementation details related to adaptation of the recurrent neural network weights through the non-recurrent training algorithm, resilient backpropagation, are formulated through an algebraic approach. Performance of the proposed neuro-optimizer on a well-known static combinatorial optimization problem, the Traveling Salesman Problem, is evaluated on the basis of computational complexity measures and, subsequently, compared to performance of the Simultaneous Recurrent Neural network trained with the standard backpropagation, and recurrent backpropagation for the same static optimization problem. Simulation results indicate that the Simultaneous Recurrent Neural network trained with the resilient backpropagation algorithm is able to locate superior quality solutions through comparable amount of computational effort for the Traveling Salesman Problem. PMID- 12370964 TI - What is newsworthy? Frenzy to feed the media can be bad for patients. PMID- 12370963 TI - Unsupervised neural learning on lie group. AB - The present paper aims at introducing the concepts and mathematical details of unsupervised neural learning with orthonormality constrains. The neural structures considered are single non-linear layers and the learnable parameters are organized in matrices, as usual, which gives the parameters spaces the geometrical structure of the Euclidean manifold. The constraint of orthonormality for the connection-matrices further restricts the parameters spaces to differential manifolds such as the orthogonal group, the compact Stiefel manifold and its extensions. For these reasons, the instruments for characterizing and studying the behavior of learning equations for these particular networks are provided by the differential geometry of Lie groups. In particular, two sub classes of the general Lie-group learning theories are studied in detail, dealing with first-order (gradient-based) and second-order (non-gradient-based) learning. Although the considered class of learning theories is very general, in the present paper special attention is paid to unsupervised learning paradigms. PMID- 12370965 TI - Don't forget syphilis. Access to services for genitourinary medicine needs to be made easier. PMID- 12370966 TI - Reducing unintended pregnancy among adolescents. Changes in social, economic, and educational policy need to be taken into account. PMID- 12370967 TI - Wall between neurology and psychiatry. Some parts of the wall are thicker than others. PMID- 12370968 TI - Wall between neurology and psychiatry. Integration of mind and brain creates biopsychological understanding in psychiatry Neuropsychiatry is alive and well. PMID- 12370969 TI - aPL and stroke in young women: more fuel for the stroke risk fire. PMID- 12370970 TI - Expanded UC Davis network abets child abuse investigations. Pediatric specialists work with rural community hospitals on identifying and treating suspected cases of abuse. PMID- 12370971 TI - Home is where the healthcare is. PMID- 12370973 TI - Shift to digital spurs revisions in image quality standards. PMID- 12370972 TI - From research to practice: telemedicine for children with disabilities in rural Iowa. PMID- 12370974 TI - Telehealth testbed launches technology evaluation program. PMID- 12370976 TI - Offshore teleradiology solves manpower issues at home. PMID- 12370975 TI - Illinois network helps rural sites make the most of broadband. PMID- 12370977 TI - To determine market size, follow the money. PMID- 12370978 TI - ECU course puts home telehealth tools into hands of providers. PMID- 12370979 TI - Thinking outside the box helps break through barriers.. PMID- 12370980 TI - [Only placebo?]. PMID- 12370981 TI - [Understanding carcinogenesis]. PMID- 12370982 TI - [Interpersonal psychotherapy in the treatment of depression]. PMID- 12370983 TI - [The patients' health status, right to drive and doctor's obligation to maintain confidentiality]. PMID- 12370984 TI - [Measuring respiratory gas exchange during orthostatic test--a novel test to diagnose tendency to hyperventilation]. PMID- 12370985 TI - [Axial myopathy--an unrecognized back disease]. PMID- 12370986 TI - [Localizing the cause for unilateral sensorineural hearing loss]. PMID- 12370987 TI - [Recurrent central nervous system symptoms and elevated erythrocyte sedimentation rate in a patient with rheumatoid arthritis]. PMID- 12370988 TI - [To use the fibrinolytic treatment or not?]. PMID- 12370989 TI - [A hint about the level of Finnish emergency medicine]. PMID- 12370990 TI - [Diagnostics on the wrong track]. PMID- 12370991 TI - Nontuberculous mycobacteria in the environment. AB - It is likely that the incidence of infection by environmental opportunistic mycobacteria will continue to rise. Part of the rise will be caused by the increased awareness of these microbes as human pathogens and improvements in methods of detection and culture. Clinicians and microbiologists will continue to be challenged by the introduction of new species to the already long list of mycobacterial opportunists (see Table 3). The incidence of infection will also rise because an increasing proportion of the population is aging or subject to some type of immunosuppression. A second reason for an increase in the incidence of environmental mycobacterial infection is that these microbes are everywhere. They are present in water, biofilms, soil, and aerosols. They are natural inhabitants of the human environment, especially drinking water distribution systems. Thus, it is likely that everyone is exposed on a daily basis. It is likely that certain human activities can lead to selection of mycobacteria. Important lessons have been taught by study of cases of hypersensitivity pneumonitis associated with exposure to metalworking fluid. First, the implicated metalworking fluids contained water, the likely source of the mycobacteria. Second, the metalworking fluids contain hydrocarbons (e.g., pine oils) and biocides (e.g., morpholine) both of which are substrates for the growth of mycobacteria [53,193]. Third, outbreak of disease followed disinfection of the metalworking fluid [136,137]. Although the metalworking fluid was contaminated with microorganisms, it was only after disinfection that symptoms developed in the workers. Because mycobacteria are resistant to disinfectants, it is likely that the recovery of the mycobacteria from the metalworking fluid [137] was caused by their selection. Disinfection may also contribute, in part, to the persistence of M avium and M intracellulare in drinking water distribution systems [33,89,240]. M avium and M intracellulare are many times more resistant to chlorine, chloramine, chlorine dioxide, and ozone than are other water-borne microorganisms [141,236]. Consequently, disinfection of drinking water results in selection of mycobacteria. In the absence of competitors, even the slowly growing mycobacteria can grow in the distribution system [33]. It is likely that hypersensitivity pneumonitis in lifeguards and therapy pool attendants [139] is caused by a similar scenario. PMID- 12370992 TI - Epidemiology of human pulmonary infection with nontuberculous mycobacteria. AB - A great deal of study has gone into the assessment of the epidemiology of NTM infection and disease in many different parts of the world. Review of the available studies provides insight into the frequency of this clinical problem as well as important limitations in current data. Study methods have varied greatly, undoubtedly leading to differing biases. In general, reported rates of infection and disease are likely underestimates, with the former probably less accurate than the latter, given that people without significant symptoms are not likely to have intensive investigations to detect infection. Pulmonary NTM is a problem with differing rates in various parts of the world. North American rates of infection and disease have been reported to range from approximately 1-15 per 100,000 and 0.1-2 per 100,000, respectively (see Table 1). Rates have been observed to increase with coincident decreases in TB. MAC has been reported most commonly, followed by rapid growers and M kansasii. Generally similar rates have been reported in European studies, with the exception of extremely high rates in an area of the Czech Republic where mining is the dominant industry (see Table 2). These studies have also shown marked geographic variability in prevalence. The only available population-based studies have been in South Africa and report extremely high rates of infection, three orders of magnitude greater than studies from other parts of the world (see Table 3). This undoubtedly reflects the select population with an extremely high rate of TB and resultant bronchiectasis leading to NTM infection. Rates in Japan and Australia were similar to those reported in Europe and North America and also show significant increases over time (see Table 3). Specific risk factors have been identified in several studies. CF and HIV, mentioned above, are two important high-risk groups. Other important factors include underlying chronic lung disease, work in the mining industry, warm climate, advancing age, and male sex. Aside from HIV and CF, mining with associated high rates of pneumoconiosis and previous TB may be the most important historically, reported in studies worldwide [63]. A recurring observation is the increase in rates of infection and disease. The reason for this is unclear but may be caused by any of several contributing factors. The possibility exists that the apparent increase is either spurious or less significant than studies would suggest. Changes in clinician awareness leading to increased investigations, or laboratory methods leading to isolation and identification of previously unnoticed organisms, could play a role in this trend, and studies have been published that support [67] and refute [31] this argument. We believe such factors may contribute to but do not explain the significant increases that have been observed. A true increase could be related to the host, the pathogen, or some interaction between the two. Host changes leading to increased susceptibility could play an important role, with increased numbers of patients with inadequate defenses from diseases such as HIV infection, malignancy, or simply advanced age [31]. An increase in susceptibility could also relate to the decrease in infection with two other mycobacteria. It has been speculated that infection with TB [29,38] and Bacillus Calmette-Guerin (BCG) [19,68] may provide cross-immunity protecting against NTM infection. Many investigations have observed decreasing rates of TB concomitant with the increases in NTM. In addition, studies from Sweden [68] and the Czech Republic [19] have found that children who were not vaccinated with BCG had a far higher rate of extrapulmonary NTM infection. Potential changes in the pathogens include increases in NTM virulence, and it has been argued that this should be considered as a possible contributing factor [69]. Finally, an interaction between the host and pathogen could involve a major increase in pathogen exposure or potential inoculum size. This may be occurring secondary to the increase in popularity of showering as a form of bathing [66], a habit that greatly increases respiratory exposure to water contaminants. Several limitations of our review should be noted. We reviewed English-language reports and abstracts, probably leading to fewer data from non-English speaking regions, which may explain the paucity of studies from Africa, Eastern Europe, and most Asian nations. The heterogeneity of study methods in identifying cases and the lack of a uniformly applied definition of disease makes it difficult to compare rates between studies. Finally, the lack of systematic reporting of NTM infection in most nations limits the ability to derive accurate estimates of infection and disease. Regardless, there are more than adequate data to conclude that NTM disease rates vary widely depending on population and geographic location. NTM disease is clearly a major problem in certain groups, including patients with underlying lung disease and also in individuals with impaired immunity. The rates of NTM infection and disease are increasing, so the problem will likely continue to grow and become a far more important issue than current rates suggest. PMID- 12370993 TI - Pathogenesis of nontuberculous mycobacteria infections. AB - M avium is a microorganism well adapted to living in the environment and in different hosts. During the past 15 years, a substantial amount of information has been accumulated about the mechanisms used by M avium to cross the host's mucosal barrier, replicate inside cells, circumvent the host's immune response, and persist inside the host. It turns out that M avium is a fascinating pathogen after all. The increasing knowledge about M avium pathogenesis may one day provide means for a more effective prophylaxis as well as for treatment of the infection. PMID- 12370994 TI - Laboratory diagnosis of nontuberculous mycobacteria. AB - In conclusion, it is important to realize that there is no "stand alone" assay for the identification of NTM. Many new species may not be recognized in all assays. Newer molecular tests are more accurate for identification than phenotypic tests and have significantly improved turnaround time. Clinical significance of an isolate should be determined, however, before committing resources for the identification of a mycobacterial isolate to the species level. In addition, there are significant differences in the range and quality of services provided by different laboratories. Today, techniques and equipment are increasingly complex and costly, making it more difficult to upgrade every local laboratory to perform these assays. But because specimen delivery and communication of results can be rapidly and easily achieved, utilization of reference laboratories for rarely performed sophisticated tests is a more practical approach. PMID- 12370995 TI - Diagnosis, differentiating colonization, infection, and disease. AB - Nontuberculous mycobacteria (NTM) are found commonly in respiratory specimens. In many clinical laboratories, the majority of mycobacteria recovered from respiratory samples are NTM. Perhaps this is because NTM are common in the environment. The water that we drink or shower in often contains NTM; consequently, we often find NTM in respiratory secretions. The term colonization has been used when NTM are recovered more than once even though a specific disease cannot be demonstrated. Under these circumstances, it is never quite clear whether a low-grade infection exists or if secretions are simply contaminated by environmental organisms; however, colonization can be discerned from infection by focusing on the elements of making the diagnosis of an NTM related disease, rather than simply finding the organism in respiratory samples. PMID- 12370996 TI - Radiology of pulmonary Mycobacterium avium-intracellulare complex. AB - Although the radiographic appearance of pulmonary MAC infection in the immunocompetent host can be varied, there are several generalizations that can be made. The classic radiographic appearance is indistinguishable from that of pulmonary tuberculosis. The classic form is seen most commonly in males and is typically associated with other predisposing diseases, especially chronic obstructive pulmonary disease. Most patients have upper lobe disease with associated pleural thickening. Widespread disease is common, as is cavitation. Pleural effusions and adenopathy are uncommon. The Lady Windermere syndrome is a special form of pulmonary MAC seen primarily in middle-aged and elderly women. The radiographic findings are bronchiectasis and small nodules, predominately located within the middle lobe and lingula. The combination of bronchiectasis involving exclusively, or primarily, the right middle lobe and lingula is highly suggestive of pulmonary MAC, even in the face of negative sputum cultures. Pulmonary infection with MAC in the immunocompromised patient generally reflects a widespread systemic disease. As such, the radiographic appearance is highly variable. Diffuse pulmonary opacities and adenopathy are common features. Plain radiographs are frequently normal despite active pulmonary infection. Regardless of the clinical situation, pulmonary MAC infection is often omitted from the radiographic differential even when the appearance is characteristic. In general, when pulmonary abnormalities are identified that are consistent with a granulomatous infection, pulmonary MAC needs to be considered along with tuberculosis and fungal infection. Especially with pulmonary MAC, radiographic stability over several years does not exclude active disease. The radiographic appearance may be suggestive of the diagnosis of pulmonary MAC, but correlation with the clinical and microbiological data is necessary to confirm the diagnosis. PMID- 12370997 TI - Management of disease due to Mycobacterium kansasii. AB - Mycobacterium kansasii presents clinically in a manner most resembling tuberculosis. Diagnosis is usually not difficult; however, the significance of M kansasii isolates from some patients may be hard to determine. Usually, the presence of even one respiratory culture positive for M kansasii is sufficient to make a diagnosis, though few patients can have single respiratory culture positive for M kansasii without evidence of active disease. If not started on medication, these patients must be followed closely. Effective treatment can usually be accomplished with a rifampin-based regimen, or a rifabutin-based regimen for HIV-seropositive patients receiving antiretroviral therapy. Short course and intermittent regimens for treating M kansaii disease show promise but are not yet recommended for routine use. PMID- 12370998 TI - Pulmonary disease caused by rapidly growing mycobacteria. AB - The rapidly growing mycobacteria (RGM) differ from slow-growing mycobacteria such as Mycobacterium tuberculosis by virtue of their more rapid growth in culture media and their in vitro resistance to standard antituberculosis drugs. The RGM can produce numerous infections including chronic lung disease. The most common causes of pulmonary disease are Mycobacterium abscessus and Mycobacterium fortuitum. This article reviews the management of patients with lung disease caused by RGM. PMID- 12370999 TI - Medical management of pulmonary disease caused by Mycobacterium avium complex. AB - The current medical therapy for Mycobacterium avium complex is controversial. American Thoracic Society recommendations advocate empiric therapy with drug susceptibility testing only for clarithromycin. At National Jewish, where we mainly see cases complicated by treatment failure and drug intolerance, we use in vitro susceptibility testing and therapeutic drug monitoring to optimize efficacy and reduce toxicity. PMID- 12371000 TI - Mycobacterium avium complex pulmonary disease in patients with pre-existing lung disease. AB - Patients with MAC-PD and pre-existing lung disease are a distinct group from the more common and recently recognized group of predominantly middle- to older-aged women without pre-existing lung disease. Those with pre-existing disease are expected to have more sputum positivity and slower conversion of sputum with treatment, and they may require combined medical treatment with surgical resection for optimal results. Attention to bronchial hygiene, avoidance of unnecessary use of macrolides, and treatment of underlying esophageal and lung disease can result in marked symptomatic improvement in many cases. Appropriate consideration must be given to mycobacterial antibiotic treatment, and awareness must be maintained for other processes such as bronchogenic cancer in select groups of high-risk patients. PMID- 12371001 TI - Nontuberculous mycobacteria in the setting of cystic fibrosis. AB - Nontuberculous mycobacterial pulmonary infections are increasingly recognized in patients with CF. This may reflect the increasing longevity of this population with increased environmental exposure time, a higher clinical index of suspicion, and/or some as yet unidentified predisposing factor(s). The most common species of NTM in CF is MAC, followed by M abscessus. We recommend that adult patients with CF be screened for the presence of nontuberculous mycobacteria in pulmonary secretions on a regular basis, and that consideration be given to this diagnosis if a patient has an escalating pattern of exacerbations or admissions. Positive cultures are likely to indicate disease if they are multiple or if a patient has clinical evidence of pulmonary disease exacerbation (increased cough, increased purulence of secretions, systemic manifestations such as fever, weight loss) that is not responding to conventional antibiotic therapy. Cystic fibrosis patients who do not respond to treatment for the usual organisms should be carefully re evaluated for the presence of NTM and treated with a macrolide-containing multidrug regimen directed against the identified NTM if diagnostic criteria are met. Novel treatments with cytokines and intermittent dosing of antibiotics are currently under investigation in non-CF populations and may have applicability to CF in the future. PMID- 12371002 TI - Nontuberculous mycobacteria in the HIV infected patient. AB - The reduction in disseminated NTM infections caused by HAART is one of the success stories in the history of HIV in the developed world. Despite this success, these diseases still occur and may have atypical presentations in patients receiving HAART. Clinicians treating HIV-infected patients must remain familiar with the diagnosis and treatment of these diseases and implement prevention strategies when indicated. PMID- 12371003 TI - Nontuberculous mycobacteria in women, young and old. AB - Lady Windermere syndrome is a unique entity within the spectrum of pulmonary NTM diseases. There are differences in several clinical aspects between Lady Windermere syndrome and the classic pulmonary NTM disease, including manifestations, pathogenesis, and natural history. Recently, emerging pieces of information provide a more scientific explanation of why women are more susceptible to this form of infection and how they develop clinical disease. As the result, these patients probably require quite different diagnostic and therapeutic approaches compared with those with the classic presentation. Studies exclusive to LWS are lacking and are absolutely necessary as they will enhance our understanding of, and hence successful management strategies for, this increasingly recognized disease. PMID- 12371004 TI - Right ventricular function. AB - This article describes the importance of the right ventricle in both the normal circulation, and in the abnormal milieu of previously palliated or corrected congenital heart disease. The latter group represents natural models of abnormal right ventricular loading that do not exist in any other experimental arena, and their study has provided insights into chronic adaptation of the right ventricle, in terms of cardiopulmonary haemodynamics, right-left heart interactions and mechano-electric inter-relationships. PMID- 12371005 TI - The role of the atria in congenital heart disease. AB - The atria play an important role in adult congenital heart disease. Atrial function is often altered due to longstanding pressure or volume overload. Cardiac surgery inflicts lasting damage to the atria, which leads to loss of atrial compliance. Both the history of atrial overload and the atrial scarring form substrates for atrial tachycardias. There has been a growing interest in the interatrial septum in the past few years. There is evidence for a role of the persisting foramen ovale and atrial septal aneurysm as a causative or permissive factor in cerebral stroke. Catheter closure of the PFO may be an attractive option, especially for younger patients. PMID- 12371006 TI - How congenital heart disease originates in fetal life. AB - Knowledge of the early development of the heart has increased rapidly in recent years as microscopic techniques, experimental models using animal, avian and insect species, and various genetic techniques have been brought to bear on the mysteries of human fetal cardiac development. The development of the heart occurs rapidly from embryonic day 18 in humans to the twelfth week of fetal life. The stages include gastrulation and formation of the primitive heart tube with rhythmic contractions appearing at day 21, segmentation of the primitive heart tube, looping, realignment of inflow and outflow segments, septation of the atria, ventricles and outflow segments, formation of atrio-ventricular valves, and development of aortic and pulmonary trunks and aortic arches. The genes and factors currently known to be involved in cardiac development are reviewed, but much is still to be determined as the field is evolving with extraordinary rapidity. PMID- 12371007 TI - The genetics of congenital heart disease: a point in the revolution. AB - Extraordinary diagnostic precision and definitive therapies characterize the current management of congenital heart disease, but the state of the art is not perfect; and in spite of tremendous progress in diagnosis and treatment, our understanding of cause is rudimentary. During the past decade, advances in molecular genetics have defined disease gene loci and lead to identification of genes whose mutations cause congenital heart disease. Identification of additional genes and mutations will lead to improved understanding of pathophysiology of cardiovascular disease in the young. Better understanding of pathophysiology and identification of individuals at risk will provide an opportunity to develop preventive strategies. Taken as a whole, the prospect of understanding the genetic basis of congenital heart disease and translating it into improved diagnostic and therapeutic strategies has never been better. PMID- 12371008 TI - Late problems in tetralogy of Fallot--recognition, management, and prevention. AB - Most adults with previous repair of tetralogy of Fallot lead unrestricted lives and are asymptomatic. Residual RVOT problems such as significant PR and/or RVOT obstruction however are common and often lead to gradual RV dilation and dysfunction with consequent supraventricular or ventricular arrhythmias. Hemodynamic causes for the tachyarrhythmia should be sought and corrected, and therapy directed towards suppressing the arrhythmia (antiarrhythmics, cryoablation or AICD) should be carried out as well. Recent changes in the surgical approaches to the repair of tetralogy at the time initial repair may well translate into a reduced incidence of late complications. PMID- 12371009 TI - Transposition complexes in the adult: a changing perspective. AB - This study has shown the heterogeneous group of patients with discordant ventricular arterial relations, their management and problems encountered during follow up. Patients after surgery for transposition are still relatively young by cardiology standards and their problems continue to evolve; nevertheless the future is becoming clearer. However there are still important lessons to be learnt by continued and diligent observation and systematic, multicenter research. It is important to maintain a low threshold for thorough re-evaluation of patients whenever new symptoms are discovered. Indeed, patients should undergo regular detailed investigations at timely intervals. It is vital that this evolving group of adult patients, as with most patients emerging from a childhood with other congenital heart malformations, is managed by cardiologists fully trained in congenital heart disease. PMID- 12371010 TI - Subaortic stenosis: at risk substrates and treatment strategies. AB - Subaortic stenosis can be a complex disease of multiple anatomic etiologies. At the core is either an elongated and narrow outflow tract as compared to normal or a fully muscle-rimmed VSD used as an intraventricular routing pathway. An array of treatment modalities is needed for an effective management strategy. PMID- 12371011 TI - Ebstein's anomaly of the tricuspid valve complexities and strategies. AB - Ebstein's anomaly of the tricuspid valve is a fascinating but very complex congenital malformation with a wide spectrum of anatomic and clinical variations. A brief review is given of the clinical presentation and therapeutic options. We need further functional studies to understand the relationship between clinical severity and anatomic derangement. This will lead to better decision-making regarding the indications for and timing of surgery, which are still problematic. PMID- 12371012 TI - Pulmonary arterial hypertension in congenital heart disease. AB - Pulmonary arterial hypertension (PAH) is a recognized complication of congenital systemic to pulmonary arterial cardiac shunts. The prognosis of PAH in this situation is better than primary or other secondary forms of PAH. Our knowledge of the pathophysiology of PAH complicating congenital heart disease has evolved over the past decade. Despite differences in etiology and pathobiology, therapies that have proven successful for primary PAH may benefit this group of patients. PMID- 12371013 TI - Atrial arrhythmias in congenital heart disease. AB - Surgical scars, volume overload and pressure overload are the three main determinants of atrial arrhythmias in patients with prior congenital heart surgery. The workup should include a thorough hemodynamic assessment. Treatment modalities available include drugs, pacemakers, catheter ablation and surgery. Despite the above, arrhythmias have a significant negative impact on life expectancy and quality in these patients. Newer surgical strategies for repair of the heart defect may delay or prevent the development of arrhythmias and are being actively pursued. PMID- 12371014 TI - Role of catheter and surgical ablation in congenital heart disease. AB - The role of surgery and radiofrequency current ablation for the treatment of tachycardias in patients with congenital heart disease The use of radiofrequency current application as a treatment strategy has stimulated a revolution in our understanding of tachycardia mechanisms. The extension of its use to patients with congenital heart defects and tachyarrhythmias has opened the door to new treatments with known success rates and known risks for mortality and morbidity. Antiarrhythmic surgery aims to dissect or excavate a responsible substrate and is especially worth considering if cardiac surgery is being undertaken for other reasons. With suitable surgical skill and interest, and with strong electrophysiologic support, high success rates have been documented. Antiarrhythmic surgical incisions have the advantage of being visually controllable regarding the extent and location of damage to myocardial tissue. In other situations, radiofrequency current ablation is preferred because of its less-invasive character, its use of local anesthesia, and the avoidance of surgical trauma. Both surgery and catheter ablation require precise clarification of the tachycardia mechanism and precise localization of the underlying substrate. The importation of such techniques into the realm of open chest surgery would be difficult in light of the need for multiple intracardiac catheters and repeated fluoroscopically guided catheter positioning. Electrophysiologic studies performed during the antiarrhythmic surgical procedure cannot provide complete information, and their use is thus restricted to the arrhythmogenic myocardial target only [32,45]. In contrast, catheter-mediated electrophysiologic studies offer the option of exact diagnosis, precise substrate localization, and interventional treatment in a single session. Moreover, validation of the linear lesion's completeness has become a reliable predictor for mid- and long-term success in avoiding recurrences. As a result, the application of catheter-mediated ablation has exploded within the past 15 years. Antiarrhythmic surgery has survived as a discipline in a decreasing number of experienced hands [43,44]. As a result of recent experiences and modern technology, success rates above 90% [74-76, 81,88] for the interventional treatment of congenital tachycardias have become comparable to those reported in patients with "normal" hearts. For acquired tachycardias, acute success rates today range about 80% at the atrial level. The rate of recurrence is still relatively high at about 10-25% [73,76,77,79,91,96,102]. Further improvements are being pursued. Data on the treatment of acquired tachycardias at the ventricular level is largely anecdotal. Good early success rates are combined with a tendency to recurrence in longer-term follow-up [50,76,103-108]. Some of the late VT ablation recurrences may be explained by the fact that fibrotic, scarred, and hypertrophic myocardial tissue at the targeted site often prevents effective radiofrequency current application and lesion generation. In order to improve RF lesion depth and continuity, newly designed technologies for radiofrequency current ("cooled tip electrode", Cordis Webster, Baldwin Park, CA), and alternative energy sources (cryo-ablation, micro-wave, or ultrasound) are being readied for introduction in the very near future. For patients suffering from recurrent tachycardias and having other reasons for open-heart surgery, a hybrid concept can be created, utilizing modern 3-D electro-anatomical reconstruction as a basis for an electrophysiologically informed surgical procedure. Following such a concept, a hemodynamic catheterization can be combined with an electrophysiologic study to define critical myocardial zones for induced macro-re entry tachycardias, or of those zones expected to play an arrhythmogenic role in the future. With such information, surgical incisions for cardiac access and repair can be planned and performed. The role of surgery in antiarrhythmic treatment can become preventive. Myocardial tissue is incised for cannulation and repair in a way that can reduce the chance of later scar-associated tachycardias [109]. The extension of surgical cuts to physiologic barriers of electrical conduction is a major strategy for the primary prevention of postsurgical or incisional arrhythmias. In addition, the simultaneous treatment at heart surgery of already existing tachycardias can be offered within the same session as a secondary preventive concept. Despite the immense growth of knowledge and experience in recent years, there is still a need for more knowledge about the factors causing arrhythmogenesis and their interactions. Prospective and randomized studies are needed to show the most effective strategies to prevent arrhythmia-mediated death. The future of antiarrhythmic treatment will less be directed by the limitations of current interventional tools, which will be improved, and more by an evolutionary process in philosophy regarding the understanding of arrhythmogenesis in these patients as the basis for new concepts of arrhythmia prevention and treatment. PMID- 12371015 TI - Physician leaders of medical groups face increasing challenges. AB - Physician leadership has emerged as one of the biggest challenges and opportunities for medical group success. The environment for medical groups has become increasingly complex as the result of five major factors: 1) varying reimbursement methods, 2) growth in the size of groups, 3) technology investments, 4) sale and merger of groups, and 5) regulatory and legal issues. Striking the right balance between too little or too much physician involvement in leading medical groups is a key business decision. Most large, successful businesses view investment in their leaders as critical for success. Medical groups can learn from other businesses that investment in education, coaching, and succession planning for leaders is a key to long-term success. PMID- 12371016 TI - The development of a successful physician compensation plan. AB - Physician compensation plans are critical to the success of a physician group or may lead to the demise of the group. Essential components of the development and implementation of a successful physician compensation plan include: strategic planning, physician understanding and buy-in, appropriate incentives, objective performance measurement, and a specific funding source or mechanism. There are two basic philosophies to consider for use: the market-based model and the net economic contribution model. Advantages and disadvantages of each are discussed. Methods of incorporating these multiple aspects into a single plan are described. PMID- 12371017 TI - A comparison of the performance of hospital- and physician-owned medical group practices. AB - This study compares the financial and productivity performance of hospital- versus physician-owned medical group practices. Nineteen hospital-owned and twenty-three physician-owned family practices were matched by location (state) and size (full-time equivalent providers). The data were obtained from the 1998 Medical Group Management Association (MGMA) Cost Survey database. The focus of this study is on the "bottom-line" performance of the organizations as well as the production costs of the different type of practices. Analyses of these data consider staffing differences, charge and revenue differentials, productivity factors, and differences in patient volume and procedure volume. When comparing the hospital-owned and physician-owned family practice groups, the statistical analysis of these data suggest that the underlying distinctions are driven by differences in the volume of patients and volume of procedures. PMID- 12371018 TI - Physician office productivity improvement through operations analysis and process redesign. AB - The operation of any physician practice is a complicated process with many different functions and systems. Though frequently overlooked, operations improvements and efficiencies are possible if a participative, process-oriented approach is used. An actual case study of a "hospital system-owned" primary care physician group practice is presented. The methods of analysis and specific actions for improvement in operational efficiency and quality are described in detail. Opportunities for cost reduction, increased revenues, and increased patient and physician satisfaction are identified. PMID- 12371019 TI - A health care anomaly: a successful community hospital-affiliated group practice. AB - Many hospital-affiliated group practices have had significant problems. Failures are common. Sturdy Memorial Associates, a hospital affiliated group practice functioning out of twelve sites in the greater Attleboro area in Massachusetts, has been a success. Two senior managers discuss the history and the reasons why. PMID- 12371020 TI - Back to the future for many hospital-physician relationships: where do we go from here? AB - Many hospitals, health systems, and large physician group practices have experienced the rise and fall of "managed care," over the past decade or so. The impact has been large and has included the rapid growth and acquisition of physician practices, followed by huge financial losses, and subsequent re organization, divestiture, and bankruptcies. Regardless, physicians, hospitals, and health systems still face the burden of a rising demand for patient care services. Hospital-physician relationships are still crucial to the health care system. Suggestions with regard to how to analyze your local market and move forward from here to rebuild hospital-physician relationships and care systems are presented and discussed. PMID- 12371021 TI - Constructing powerful control charts. AB - Control charts are intended to tell a story about whether or not a process is stable, improving, or deteriorating. Effective storytelling requires more than correctly following the rules for choosing the best control chart and applying the tests for a special cause. The story will be told more effectively when control charts include all the information needed to convey the key messages and exclude information that distracts from the storyline. This article presents some graphical guidelines for making control charts tell their stories more forcefully. PMID- 12371022 TI - Physician leadership: an interview with David M. Barrett, MD. Interview by Ronald B. Goodspeed and Zach Gerbarg. PMID- 12371023 TI - Medical neutrality: another casualty of the intifada. AB - The Physicians for Human Rights (PHR) reports findings of a March 2002 investigation in Israel and the Occupied Territories. Although there were many cases of medical professionals providing unbiased care to patients and continual respect and collaboration with colleagues regardless of ethnicity, evidence shows that violations of medical neutrality are being committed by combatants on both sides. PMID- 12371024 TI - Carpal tunnel syndrome: a historical perspective. AB - Historical reports about carpal tunnel syndrome (CTS) exist from its first description in 1854 to current developments. Although now a well-recognized entity, it took nearly 100 years from its initial description before accurate pathophysiology was determined and agreed upon by the medical community. Along the way, many individuals have contributed to our present understanding of CTS. Stumbling blocks have included a delay of many years for accurate anatomical localization, as well as incorrect initial surgical treatment selection without scientific basis. By the middle of the 20th century, the standard of care for CTS had reached the standard of our modern era, based on scientific study and documentation. By the latter half of the 20th century, pathophysiology had been established, diagnostic signs and testing had been well evolved, and surgical techniques had become refined. Emerging frontiers being explored from the late 20th century into the new millennium include and incorporate advances in diagnostic and surgical technology and equipment. Patient demographics have also shown evolution. Controversies continue and more research is needed to establish more definitive criteria as to causation of CTS by various factors, including repetitive stress. PMID- 12371025 TI - Practical anatomy of the carpal tunnel. AB - The carpal tunnel is most narrow at the level of the hook of the hamate. The median nerve is the most superficial structure. It has specific relationships to surrounding structures within the carpal tunnel to the ulnar bursa, flexor tendons, and endoscopic devices placed inside the canal. The importance of the ring finger axis is stressed. Knowledge of topographical landmarks that mark the borders of the carpal tunnel, the hook of the hamate, superficial arch, and thenar branch of the median nerve ensure appropriate incision placement for endoscopic as well as open carpal tunnel release surgery. Anatomy of the transverse carpal ligament, its layers and relationships to adjacent structures including the fad pad, Guyon's canal, palmar fascia, and thenar muscles has been discussed. Fibers derived primarily from thenar muscle fascia with connections to the hypothenar muscle fascia and dorsal fascia of the palmaris brevis form a separate fascial layer directly palmar to the TCL and can be retained. This helps to preserve postoperative pinch strength. The fat pad in line with the ring finger axis overlaps the deep surface of the distal edge of the TCL and must be retracted in order to visualize the distal end of the ligament. Whereas the ulnar artery within Guyon's canal is frequently located radial to the hook of the hamate, injury to this structure has not been a problem during ECTR surgery. Variations of the median nerve and its branches, as well as the palmar cutaneous nerve distribution, have been reviewed. A rare ulnar-sided thenar branch from the median nerve, interconnecting branches between the ulnar and median nerves located just distal to the end of the TCL, and transverse ulnar-based cutaneous nerves can be injured during open or ECTR surgery. Anomalous muscles, tendons or interconnections, and the lumbricals during finger flexion may be seen within the carpal tunnel. These structures can be the cause of compression of the median nerve. The anatomy of the carpal tunnel and surrounding structures have been reviewed with emphasis on clinical applications to endoscopic and open carpal tunnel surgery. A thorough knowledge of the anatomy of the carpal tunnel is essential in order to avoid complications and to ensure optimal patient outcome. An understanding of the contents and their positions and relationships to each other allows the surgeon to perform a correct approach and accurately identify structures during procedures at or near the carpal tunnel. PMID- 12371026 TI - Pathophysiology of nerve compression. AB - Both ischemic and mechanical factors are involved in the development of compression neuropathy. Experimental studies suggest a dose response curve such that the greater the duration and amount of pressure, the more significant is neural dysfunction. With changes of axonal injury, significant neurologic dysfunction would be anticipated; however, the vast majority of patients with CTS present with symptoms in association with electrophysiologic findings of demyelination (prolonged latency). Frequently, the prolongation in latency is minimal and some patients may even present with normal electrodiagnostic studies, still complaining of significant symptomatology. This would support the concept that in the majority of patients with CTS, the symptoms relate to problems with the connective tissue "container" of the nerve rather than pathology of the nerve fiber itself. This would be in keeping with the histopathologic findings of fibrosis, with thickening of the external epineurium and perineurium. These changes would interfere with blood flow as the vessels pass through the epineurium and perineurium and produce dynamic ischemia to the nerve fibers. As well, this fibrosis would decrease the excursion of the nerve fibers, resulting in traction, and prevent the nerve fibers themselves from going through a full range of movement without traction and decreased gliding. The importance of neural gliding and movement of the nerve in the extremity has been recently emphasized in the clinical management of patients with multilevel nerve compression. Clinical maneuvers that put the nerve on stretch will provoke patients' symptoms and have been used to diagnose specific compression neuropathies (neural tension test). Similarly, physical therapy modalities to stretch the nerves and restore neural gliding are frequently successful in relieving patients' symptoms [33]. This physical therapy approach is based on the premise that the connective tissue "container" of the nerve is tight and short and needs to be mobilized. This is in keeping with the histopathologic findings of increased connective tissue at the perineurial and epineurial levels. A greater understanding of the pathophysiology of compression neuropathy will have immediate impact on our management of this problem and likely result in emphasis on conservative management and physical therapy rather than surgical intervention. PMID- 12371027 TI - Electrodiagnosis in carpal tunnel syndrome. AB - There is currently no gold standard to definitively diagnose carpal tunnel syndrome. It remains a clinical diagnosis supported by characteristic electrodiagnostic abnormalities. Properly performed electrodiagnostic studies should provide the hand surgeon with information regarding severity, progression if a previous study was performed, and a reasonable assurance that concomitant peripheral nervous system abnormalities are not present. Hand surgeons do not need to discern nuances of an electrodiagnostic evaluation; however, the ability to identify state-of-the-art techniques coupled with a thoughtful interpretation by the electrodiagnostician will improve their confidence in using this important diagnostic tool to evaluate carpal tunnel syndrome. PMID- 12371028 TI - Medical history of carpal tunnel syndrome. AB - The anatomical configuration of the carpal tunnel is that of an inelastic channel. Consequently, any increase in its volume or alteration in shape will usually result in a significant increase in interstitial pressure. At a pressure threshold of 20 mm Hg to 30 mm Hg, epineurial blood flow is compromised. When that pressure is sustained, the symptoms and physical findings associated with CTS appear. Typically, patients present with intermittent pain and paresthesias in all or part of the median nerve distribution of their hand(s). As weeks and months pass, symptoms progressively increase in frequency and severity. Eventually, thenar muscle weakness develops that initially manifests itself as "fatigue," or "tiredness." The progressive increase in symptoms and physical findings, usually accompanied by a progressive deterioration in electrodiagnostic studies, facilitates the classification of the condition into early, intermediate, and advanced stages. The increase in interstitial pressure in the carpal tunnel is in the vast majority of cases idiopathic (spontaneous). It can also be caused by a myriad of other conditions that can be classified into three other categories: intrinsic factors that increase the volume of the tunnel (outside and inside the nerve), extrinsic factors that alter the contour of the tunnel, and repetitive/overuse conditions. In addition, there is another category of neuropathic factors that affect the nerve without increasing interstitial pressure. In rare situations CTS can present as an acute problem. Far less common than the chronic form of the condition, it can follow acute wrist trauma, rheumatologic disorders, hemorrhagic problems, vascular disorders affecting a patent median artery, and high pressure injection injuries. Prompt recognition is important, followed in most cases by urgent surgical decompression of the median nerve. PMID- 12371029 TI - Examination of patients for carpal tunnel syndrome sensibility, provocative, and motor testing. AB - The value of a test for carpal tunnel syndrome (CTS) depends on the purpose of performing the test. When screening a large population with a low prevalence for CTS, a test with a high sensitivity is needed so that no possible case goes undetected. However, in order to establish a diagnosis, a more specific test is required. Using a combination of physical examination techniques, including sensibility and provocative testing, the probability of correctly diagnosing CTS without relying on electrodiagnostic studies can be very high. Because CTS is a clinical syndrome, the diagnosis should be made on clinical grounds. Electrodiagnosis is extremely important, however, in its ability to objectively document median nerve slowing and eliminate other competing differential diagnoses. PMID- 12371030 TI - Nonoperative carpal tunnel syndrome treatment. AB - Many factors influence the development of CTS; therefore, nonoperative treatment should not be limited to only one intervention. Nonoperative treatment is most effective in the early stages, prior to irreparable damage to the nerve. Early intervention combined with a comprehensive treatment plan can help improve effectiveness of treatment during this phase. We do not endorse any one particular conservative treatment/program as the solution for CTS, but our purpose is to explore potential options. Further study is needed to determine the most beneficial and cost-effective treatments. PMID- 12371031 TI - Surgical release of the carpal tunnel. AB - A thorough understanding of the normal anatomy and possible anomalies that may exist is important for the surgeon managing median nerve compression at the wrist. Given the high incidence of anatomic variability occurring in and around the carpal canal, open decompression of the median nerve is the preferred surgical technique for treating carpal tunnel syndrome. This approach provides complete visualization of the region, enabling the surgeon to decompress the nerve thoroughly, identify and treat anatomic abnormalities, and protect important neurovascular structures. Open carpal tunnel release is a safe and reliable operation with a high rate of functional improvement and patient satisfaction. PMID- 12371032 TI - Carpal tunnel release via limited palmar incision. AB - The limited incision carpal tunnel release provides an effective, reliable, and safe method for decompression of the median nerve at the wrist. The technique described above minimizes risk of complication through the design of the instruments and conceptual approach to the anatomy and surgical exposure. This method combines the reduced postoperative pain and quicker recovery of the ECTR technique with the safety and lower operative expense of the conventional open technique. PMID- 12371033 TI - Endoscopic carpal tunnel release. AB - Endoscopic carpal tunnel release is not a procedure to be taken lightly. Like many surgical procedures, it is a demanding exercise that requires exacting knowledge of the anatomy of the hand, attention to detail, and the ability to manipulate three-dimensional objects while observing them in two dimensions on a video screen. In the hands of well trained surgeons, ECTR provides patients with a safe, predictable solution to their carpal tunnel sydrome that will allow them a rapid return to normal activities with minimal postoperative discomfort. PMID- 12371034 TI - Role of ancillary procedures in surgical management of carpal tunnel syndrome: epineurotomy, internal neurolysis, tenosynovectomy, and tendon transfers. AB - The role of ancillary procedures in the treatment of carpal tunnel syndrome is controversial, especially with regard to internal neurolysis and epineurotomy. At present, there are little to no data to support their routine use in the treatment of primary carpal tunnel syndrome. Similarly, the use of tenosynovectomy in carpal tunnel surgery should be limited to those patients with clear underlying rheumatologic or inflammatory risk factors, or with gross synovitis incidentally noted at surgery. The Camitz transfer is uniquely suited to treating the thenar wasting seen in advanced carpal tunnel syndrome. It can be performed concurrently with open carpal tunnel release with minimal additional dissection and morbidity. PMID- 12371035 TI - Management of carpal tunnel syndrome in the working population. AB - CTS, which has been determined to be caused or aggravated by work, can be a complex challenge. The proper diagnosis is often elusive, as a patient may have other conditions that mimic CTS. The patient's job may be a factor in the development of symptoms, but there are a host of other risk factors that should be considered in establishing the cause of the problem. While the medical and surgical treatment of CTS is not always straightforward, dealing with the social and economic aspects of this problem can be even more complex and frustrating. Trying to coordinate vocational and psychological aspects of treatment while helping the patient to stay motivated can be far more stressful than the most difficult surgical procedure. The physician may be tempted to diagnose CTS without objective evidence or to define a problem as work related without performing the necessary investigation. Labeling a patient inappropriately may cause far more harm than good [8,17,25,27]. Do not give patients with CTS the impression that they will be "crippled for life" or totally disabled. PMID- 12371036 TI - Carpal tunnel syndrome in children. AB - CTS is uncommon in children. A number of conditions predispose children to developing CTS. These conditions include the lysosomal storage diseases, a multigenerational history of CTS, and macrodactyly. Because many case are idiopathic, isolated cases are possible even in the normal child or adolescent. Children with CTS often have modest complaints in spite of long-standing difficulties such as decreased manual dexterity in one hand or poorly localized pain. Physical findings tend to be dramatic abnormalities such as severe thenar weakness and atrophy. These findings suggest that many children are diagnosed late in the course of their nerve compression. Because some patients have profound atrophy at a young age, the condition can be confused with primary thenar hypoplasia. Provocative tests, the Tinel and Phalen tests, are often normal in children with long-standing nerve compression. Electrodiagnostic studies are essential to establishing the diagnosis in many children. Pediatric cases of CTS tend to demonstrate bilateral electrophysiologic abnormalities, even if only one hand is primarily involved clinically. Therefore, it is recommended that EMG and NCS be performed bilaterally in all suspected children. Sedation may be helpful in obtaining electrodiagnostic studies in a small child. Open operative release is the only treatment documented to be effective in the treatment of childhood CTS. PMID- 12371037 TI - Recurrent carpal tunnel syndrome. AB - Persistent or recurrent symptoms following carpal tunnel release surgery are an infrequent but challenging clinical problem. A thorough evaluation of these patients is mandatory and must confirm the accuracy of the original diagnosis and rule out the presence of concurrent conditions or disorders that may cause persistent symptoms that mimic carpal tunnel syndrome. If an alternative explanation of the patients' symptoms cannot be identified, and if conservative care is ineffective, then surgical treatment should be considered. Adequate exposure of the median nerve and carpal tunnel are mandatory. The general approach should include gentle mobilization of the nerve from adherent scar tissue and interposition of a biologic barrier between the nerve and surrounding tissues. No published data conclusively demonstrate that internal neurolysis provides superior results. Postoperative care should include early mobilization to encourage tendon and nerve gliding. PMID- 12371039 TI - Outcomes assessment in carpal tunnel syndrome. AB - Carpal tunnel syndrome, the most prevalent compressive neuropathy, is exceedingly common. The incidence of this condition has been estimated to be as low as 0.1% to as high as 10%. Direct medical costs related to carpal tunnel syndrome exceed $1 billion per year, with over 200,000 surgical procedures performed annually. Additionally, untold millions of dollars are spent on as-of-yet unproven ergonomic aides in attempts to prevent the condition. a survey of nearly 30,000 workers affected by carpal tunnel syndrome were reported to have lost a median of 25 work days that further adds to the cost of this condition. After spending time in any busy hand surgeon's office, one would think that an epidemic of "carpal tunnel" has erupted. PMID- 12371038 TI - Complications related to carpal tunnel release. AB - Complications of operative carpal tunnel release continue to occur in the clinical practice of hand surgery. Anatomic localization of nerve injury has been reviewed in the area of the palmar cutaneous nerve, the median motor branch, and in the combined sensory/motor median nerve itself. Diagnosis and appropriate treatment plans have been reviewed to facilitate early appropriate treatment which usually diminishes disability. General complications have also been discussed including recurrent scar formation which is probably the most commonly encountered complication following carpal tunnel release. Possible neurovascular complications involving the development of reflex sympathetic dystrophy have received some attention in this presentation in order to alert the clinical surgeon to the possibility of this entity providing further disability to an already injured median nerve. PMID- 12371040 TI - Informed consent: a process. PMID- 12371041 TI - Hispanic perceptions of organ donation. AB - The low rate of organ donation among Hispanics is of increasing concern to the transplant community at a time when the Hispanic population is growing rapidly, especially in Southern California. OneLegacy, the nation's largest organ procurement organization, commissioned a series of in-depth individual interviews with Spanish-language-dominant Hispanics to identify barriers and motivators to organ and tissue donation. Participants included 5 families who had consented to the donation of a loved one's organs within the past year and 7 families from the general Hispanic public who were either opposed to or ambivalent about organ donation. Individuals from both groups indicated a common reticence to speak of or make plans for either their own or a family member's death and lacked knowledge of procedures surrounding donation (whether consenting or being a donor themselves). Some respondents from the opposing group did not understand that organ donation takes place after death, expressing fears that declaring themselves donors would put them at risk of being allowed to die so that their organs could be recovered. Other attitudinal barriers included the wish to die with all body parts intact and reluctance to have another person's organ in their bodies. Consenting respondents found comfort in having helped another person to live. They wished for a personal expression of thanks from the recipients and an opportunity to learn more about and meet them. Interview findings suggest a great need to further educate the Hispanic community about organ donation, especially concerning brain death, the process for organ donation, and the protections afforded to donors and their families in the United States. Overcoming the taboos surrounding discussion of death and planning for death is an essential first step. PMID- 12371042 TI - Retrospective clinical comparison of Celsior solution to modified blood Wallwork solution in lung transplantation for cystic fibrosis. AB - OBJECTIVE: To compare the preservative effects of Celsior solution and modified blood Wallwork solution in lung transplantation. METHODS: From 1989 to 2000, 44 lung transplantations for cystic fibrosis were performed: 26 grafts were preserved with modified blood Wallwork solution and 18 with Celsior solution. RESULTS: Preoperative status of the 2 groups was similar. The ratio of arterial oxygen to fraction of inspired oxygen and the pulmonary vascular resistance on the first postoperative day did not differ significantly between the 2 groups. Early death was 4% (SD, 20%) in the Wallwork group versus 11% (SD, 32%) in the Celsior group (not significant). No death was related to graft failure. The forced expiratory volume in 1 second during the first month after transplantation was 63% (SD, 19%) in the Wallwork group versus 63% (SD, 16%) in the Celsior group (not significant). CONCLUSION: Because the solution does not need to be prepared on site and does not require blood from the donor, Celsior seems better than Wallwork solution for preserving lung grafts. PMID- 12371043 TI - Transplant coordination as the "keystone" in non-heart-beating donations. AB - The shortage of donated organs has become a problem in the transplantation world. Transplant teams are continuously looking for new ways to increase and improve the donor pool. Non--heart-beating donors could be a source for increasing the number of donors, even though in some countries legal, ethical, or logistical problems obstruct the development of that source. The good results obtained by some groups working with non--heart-beating donors should stimulate others to implement this type of policy in their hospitals. We describe the origin and development of our policy on non--heart-beating donation, which has become the main source for organ donation in our hospital. We have found that the 5-year survival rate for kidneys from these donors is similar to the survival rate for kidneys obtained from brain-dead donors, and this encourages us to continue to use kidneys from non--heart-beating donors. PMID- 12371044 TI - A multiethnic study of the relationship between fears and concerns and refusal rates. AB - This study builds on previous research that identified fears and concerns heard by procurement coordinators during the donation discussion and that classified those concerns according to the ease with which they can be addressed. In this study, 53 coordinators working for 4 procurement agencies provided data on 323 donation discussions, including fears and concerns expressed by families. The fears and concerns were analyzed by outcome (consent or refusal), race and ethnicity of the family, frequency of reports, and difficulty in addressing. This research confirms many of the findings of the earlier study. The results also indicate that the types of concerns expressed by donor and non-donor families vary somewhat by the family's race and ethnicity. The results can be used to provide training targeted at raising consent rates and to train minority requestors. PMID- 12371045 TI - Evaluating the reliability and validity of the Questionnaire for Lung Transplant Patients. AB - CONTEXT: The Questionnaire for Lung Transplant Patients was designed for clinical use by the pulmonary transplant team in the routine evaluation of lung transplant recipients. Using the self-administered questionnaire, recipients check symptoms that they have had since their last evaluation and rate their shortness of breath, cough, and activity tolerance. OBJECTIVE: To determine whether the questionnaire meets reliability and validity standards for empirical measurement. METHODS: Demographics and disease-severity characteristics were examined in a cross-sectional survey of 37 recipients. Test-retest and intraclass correlation methods were used to estimate stability, and the Cronbach alpha was used to estimate internal consistency. Criterion validity was examined by using The Modified Symptom Frequency/Symptom Distress Scale, Functional Performance Inventory, and visual analog scales for cough and shortness of breath as criterion measures. Construct validity was examined to assess the predicted negative correlation between symptoms and functional performance. RESULTS: The questionnaire and its subscales were internally consistent (Cronbach alpha = 0.82, 0.76, 0.80, and 0.96), and the questionnaire was stable (r = 0.70) and reliable (intraclass correlations = 0.80 and 0.90). Significant correlations were found between the questionnaire and all criterion measures (r = 0.50-0.93). Significant correlations in the predicted negative direction were found between the respiratory subscale and functional performance (r = -0.51) and between cough (r = -0.51) and shortness of breath (r = -0.68) ratings and functional performance. CONCLUSIONS: The Questionnaire for Lung Transplant Patients is reliable and valid, and it provides scientifically sound information for clinical and empirical evaluation of symptoms and their effects on activity tolerance after lung transplantation. PMID- 12371046 TI - The effects of information and support on individuals awaiting cadaveric kidney transplantation. AB - CONTEXT: No empirical studies exist to direct nursing interventions for individuals during the long period of waiting for a transplant. OBJECTIVE: To measure the effect of information and support on hope and uncertainty for individuals awaiting cadaveric kidney transplantation. DESIGN: Randomized, controlled study. SETTING: A university-affiliated hospital in the Midwest from 1997 to 1999. PATIENTS: Fifty participants awaiting cadaveric kidney transplantation. INTERVENTIONS: The control group received no intervention phone calls or mailings, which was the current standard of care. The treatment group received phone calls and mailings once every month for 6 months. MAIN OUTCOME MEASURES: Hope, measured by the Herth Hope Index, and uncertainty, measured by the Mishel's Uncertainty in Illness Scale for Adults, were evaluated at the beginning of the study and 6 months later. RESULTS: No statistically significant effect of the nursing intervention was found on hope and uncertainty in this sample (F = 0.5322, P = .81). Hope was found to be negatively related to uncertainty both before (r = -0.53, P = .0001) and after (r = -0.59, P = .0001) intervention. No significant change was found between hope before and after intervention, and uncertainty before and after intervention in the treatment group (F = 1.10, P = .40) or the control group. CONCLUSION: The individuals indicated that definite needs were met by the information and support intervention even though the results did not statistically support the effect of the nursing intervention. PMID- 12371047 TI - Single-center analysis of living donor nephrectomy: hand-assisted laparoscopic, pure laparoscopic, and traditional open. AB - CONTEXT: Laparoscopic living donor nephrectomy has been shown to be a safe method for removing kidneys for transplantation, but concerns have been raised regarding safety and long-term kidney function. OBJECTIVE: To compare safety and long-term kidney function in hand-assisted laparoscopic, pure laparoscopic, and traditional open living donor nephrectomy. METHOD: The charts of 48 patients with more than 1 year follow-up were reviewed. Thirty-four consecutive patients underwent laparoscopic live donor nephrectomy, and 14 had open donor nephrectomy. All kidneys functioned immediately at transplantation. In the laparoscopic group, 11 had the pure laparoscopic technique, and 23 patients had hand-assisted laparoscopic nephrectomy. RESULTS: Total operative and warm ischemic times were reduced with the hand-assisted technique when compared with pure laparoscopy. Operative and warm ischemic times were similar in open nephrectomy and hand assisted laparoscopy. Long-term follow-up of serum creatinine levels revealed no significant differences between the 3 groups. Complication rates in the 3 groups were similar. CONCLUSION: Laparoscopic donor nephrectomy appears to be comparable to open donor nephrectomy in terms of safety and long-term graft function. PMID- 12371048 TI - Education, pay, and job status: a national survey of transplant coordinators. AB - To identify education, pay, and job responsibilities, an informal 19-item survey was sent to 1661 transplant coordinators; 424 (26%) were returned. Respondents worked in all phases of transplantation. Education levels varied widely; most respondents had some formal nursing education. Full-time salaries were from $25,000 to $110,000 per year; 67% worked 40 to 50 hours and 19% worked more than 50 hours per week. Of 402 clinical coordinators, 280 (70%) took call, and 122 (30%) did not. Call frequency varied with rotations every second, third, and fourth week; 44% of those taking call did not receive additional on-call pay. Nurse-managed clinics (with physician availability) were the predominant workplace for clinical coordinators. Autonomy and contact with patients were the most liked aspects of the job, and the least-liked aspects were on-call status and paper-work. Increased salary and added support staff ranked top among desired changes in the job. Transplant coordinators appear to be committed professionals who are critical to all endeavors in organ transplantation. PMID- 12371049 TI - Benefit analysis of administrative and clinical computerization of a large transplant center. AB - PURPOSE: To evaluate the implementation of a computer system that fully integrates all activities of a transplant center and coordinates secure, live data across the continuum of care. METHODS: Our center implemented a comprehensive patient tracking solution customized at each point of patient entry. Benefits were measured by provider, patient, and staff feedback; time study; and retrospective cost analysis. RESULTS: Computerization of each patient file maintained current clinical information, which facilitated patient monitoring, expeditious evaluations and listings, marketing, automated correspondence, and regulatory reporting. Enhanced data have allowed for clinical analysis, which has improved outcomes. Data availability has promoted consistency in negotiations with commercial insurers to ensure profitability. Research capacity has been increased through standardized budgets, time study, and cost analysis and has facilitated patient recruitment. CONCLUSION: The need to integrate information is vital for data accuracy and integrity. Data integration has maximized performance, profitability, and accuracy while decreasing administrative time and costs. PMID- 12371050 TI - Impact of regulations on tissue donation: mandatory referral of hospital deaths. AB - In Buenos Aires province, there has been surprisingly less tissue procurement activity following cardiac arrest than after brain-death cases. The purpose of this study was to analyse the impact of a regulation that made referral of all irreversible cardiac arrests mandatory to our organ procurement organisation. Data were collected from 7 hospitals in La Plata city during 7 months (from June 2000 to December 2000). The results showed that the regulation was effective because there was a striking increment of tissue procured following cardiac arrest during the analysed period, despite the low efficacy of the regulation (the referrals were 48% of the dead patients). PMID- 12371051 TI - Challenges in donor care: Part 2. PMID- 12371052 TI - Transplant medications--Part 2: Top 20 tips. PMID- 12371053 TI - Semi-automation of the polymerase chain reaction for laboratory confirmation of meningococcal disease. AB - Demand for accurate high-throughput detection and characterisation of medically important bacteria has increased dramatically within research and clinical laboratories. Liquid-handling robots have been developed to achieve high levels of accuracy and reproducibility. Assay automation can play a key role in the modern diagnostic laboratory and the data presented here shows that automated PCR is comparable with manual methods. Importantly, automation is preferred when high quality results cannot be guaranteed using manual methods. This is particularly important when results are required quickly for public health management. PMID- 12371055 TI - Smoking and use of smokeless tobacco in treated hypertensive men at high coronary risk: utility of urinary cotinine determination. AB - Guidelines for the treatment of hypertension underline the central importance of strenuous efforts to reduce the prevalence of smoking, as epidemiological studies consistently have demonstrated that smoking increases the risk of cardiovascular disease and death by some two- or three-fold. Accuracy of a questionnaire is examined against the ability of urinary cotinine determination to distinguish between men exposed to tobacco (94 smokers [25%], 30 snuff users [8%]) and men not exposed (n = 257), all of whom where treated hypertensives and were associated with at least one of the following factors: smoking, diabetes mellitus, serum cholesterol > or = 6.5 mmol/L. Main outcome variables in this cross-sectional study of 381 men were cotinine concentration and cotinine:creatinine ratio in overnight urine samples (decision limits: 2 mumol/L and 1.0 mmol/mol, respectively); tobacco use according to questionnaire; and follow-up examination by questionnaire of alleged non-smokers with high urinary cotinine levels. Questionnaire sensitivity was 85%, whereas the urinary cotinine assay showed 98% sensitivity and 99% specificity. Fourteen (15%) out of 94 patients may have used tobacco without reporting it in the questionnaire. In conclusion, cotinine measurement substantially improved the discrimination between smokers and non-smokers in men with multiple risk factors for cardiovascular disease. PMID- 12371054 TI - Do catecholamines influence the level of plasma leptin in patients with phaeochromocytoma? AB - The relationship between plasma leptin and catecholamine concentrations during chronic and acute catecholamine excess is studied. Patients with phaeochromocytoma, divided according to gender, were examined under basal conditions (n = 18) and at selected time-points during surgical removal of the tumour (n = 12). Appropriate controls were used (n = 23) for the basal study. Plasma leptin was determined by radioimmunoassay (RIA) and plasma noradrenaline (NA) and adrenaline (A) by high-performance liquid chromatography (HPLC). Statistical evaluation employed Student's t-test, Wicoxon test and Spearman's correlation coefficient. Gender-related differences in plasma leptin in normal subjects was confirmed, and these were maintained in the patients. Phaeochromocytoma patients had normal plasma leptin levels in the basal state and decreased levels following the massive catecholamine surge provoked by surgery. Plasma leptin concentration did not correlate with plasma NA or A in either group studied. In the patients with phaeochromocytoma, acute but not chronic catecholamine excess affected plasma leptin, suggesting a role for sympathetic activity in modulating leptin release. PMID- 12371056 TI - Helicobacter pylori cagA and vacA cytotoxin genes in Changsha, China. AB - Cytotoxin-associated protein (cagA) and the vacuolating cytotoxin (vacA) encoded by cagA and vacA genes are virulence determinants of Helicobacter pylori. In earlier studies among Chinese patients, all H. pylori strains were cagA-positive and vacAs1a/m2 type. Here, we determine the cagA, vacA and allele status of H. pylori strains isolated from patients with upper gastrointestinal symptoms in Changsha, China. Forty strains of H. pylori isolated from patients with peptic ulcer disease between March 1997 and August 1999 were recovered from storage at 80 degrees C and studied by the polymerase chain reaction (PCR) for cagA and vacA genotypes. cagA was positive in 75% of H. pylori isolates. Patients with peptic ulcer demonstrated cagA in 83% (15/18), compared with 68% (15/22) patients with superficial gastritis. vacAs1 allele was carried in 82.5% (33/40) isolates, of which 52.5% (21/40) were subtype vacAs1a/m2 and 17.5% (7/40) were subtype vacAs1b/m2. PMID- 12371057 TI - Incidence of Blastocystis hominis in faecal samples submitted for routine microbiological analysis. AB - Over a one-year period, 1390 faecal samples were submitted to Aberystwyth Public Health Laboratory for routine microbiological examination. All were stained using a commercial trichrome method. Blastocystis hominis was detected in 96 (6.9%), making it the most common parasite found in the study. Of the B. hominis-positive specimens, 73% were missed on direct microscopy. Molecular typing of B. hominis has revealed extensive genetic diversity in morphologically identical strains and thus detection by microscopy alone may not be sufficient to confirm the role of this organism in human disease. PMID- 12371058 TI - Plasmid profiles of urease-positive thermophilic campylobacter (UPTC) strains isolated in Europe and Asia (Japan). PMID- 12371059 TI - Multilocus sequence typing and porA gene sequencing differentiates strains of Neisseria meningitidis during case clusters. PMID- 12371060 TI - Prevalence of Campylobacter species among HIV/AIDS patients in Nigeria. PMID- 12371061 TI - Pandoraea apista isolated from a patient with cystic fibrosis: problems associated with laboratory identification. PMID- 12371062 TI - Cloning and sequence analysis of the recA gene in urease-positive thermophilic campylobacter (UPTC). PMID- 12371063 TI - Clinical aspects of pneumonia. AB - Pneumonia is a serious medical condition and a major cause of morbidity and mortality worldwide. It has many infectious aetiologies, although bacterial and viral forms are most common. Our understanding of pneumonia has improved significantly during the course of the 20th century but the overall disease burden has changed little. Although antibiotics have helped to reduce the mortality associated with some types of pneumonia, the level of morbidity remains constant. Furthermore, the existence of antibiotic-resistant bacteria worldwide is becoming an increasing problem in treatment. This essay describes the different types of pneumonia from a clinical perspective and highlights the problems associated with the condition. PMID- 12371064 TI - Tumour necrosis factor blocking agents: a new therapeutic modality for inflammatory disorders. AB - Development of anti-tumour necrosis factor-alpha (anti-TNF alpha) treatment offers the potential to alter radically the course of inflammatory diseases such as rheumatoid arthritis and Crohn's disease using modalities directed against a specific inflammatory mediator. Controlled randomised trials in these diseases demonstrate clinical benefit associated with significant improvement in patients with severe active joint and intestinal disease, often when conventional therapies are unsuccessful. To date, anti-TNF alpha therapy has been well tolerated and shows a favourable safety profile. This review considers the nature of this therapy and current evidence of its clinical benefit and adverse effects. PMID- 12371065 TI - Need for improved molecular biology training for biomedical scientists in NHS microbiology laboratories. PMID- 12371066 TI - LTC providers. IT-challenged. PMID- 12371067 TI - Driving quality care from the desktop. PMID- 12371068 TI - E-mail security Keyed to compliance. PMID- 12371069 TI - Farewell to paper forms. PMID- 12371070 TI - With a little help from their friends. Not-for-profit facilities cinch already tight belts and turn to local communities for help in surviving the fiscal crisis. PMID- 12371071 TI - Getting connected. PMID- 12371072 TI - An interview with John Elliot. Regional provider seizes technology--rise in litigation impacts decision. Interview by Lynn Wagner. PMID- 12371073 TI - The insurance crisis and the law. PMID- 12371074 TI - Bedside labs for quality, savings. PMID- 12371075 TI - Procedures for no-pay billing. PMID- 12371076 TI - Preserving a tax-exempt status. PMID- 12371077 TI - HIPAA 'exception' protects endowments. PMID- 12371078 TI - [The current status of noninvasive cardiac diagnosis in women with suspected coronary heart disease]. AB - BACKGROUND AND OBJECTIVE: Coronary artery disease is the leading cause of mortality among women in the industrial countries. Unfortunately, the routinely available noninvasive tests used to screen the presence of coronary artery disease have been relatively insensitive and nonspecific for women. The aim of this study was to evaluate the importance of pretest coronary artery disease probability and to determine whether the evaluation of left ventricular diastolic parameters is a relevant diagnostic tool in women with suspected coronary artery disease. PATIENTS AND METHODS: Electrocardiography at rest and during exercise, echocardiography at rest with evaluation of systolic and diastolic functional parameters, dobutamine stress echocardiography, exercise thallium myocardial scintigraphy, and coronary angiography were performed in 180 consecutive patients with suspected coronary artery disease. RESULTS: Coronary angiography revealed significant coronary artery disease in 104 patients. Angina pectoris, resting and exercise electrocardiography had a very low pretest probability in women. Dobutamine stress echocardiography, myocardial scintigraphy and the evaluation of left ventricular diastolic function showed less relevant gender-related differences and had a significantly better pretest probability. CONCLUSION: Dobutamine stress echocardiography and exercise thallium myocardial scintigraphy are reliable methods of diagnosing coronary artery disease in women. Echocardiographic assessment of diastolic left ventricular function represents another screening test for the evaluation of suspected coronary artery disease in women. All three methods, however, are not able to discriminate between coronary macro- or microangiopathy. PMID- 12371079 TI - [Randomized, double-blind crossover study of bioavailability of levothyroxine]. AB - OBJECTIVE: The synthetic thyroid hormone levothyroxine-sodium (LT4) is still the treatment of choice to replace thyroid hormone deficiency in hypothyroidism, and for adjuvant treatment of euthyroid goiter. A change of LT4 preparations during treatment may lead to major changes of thyroid hormone levels. In this study, we compared the bioavailability of two LT4 preparations, L-Thyroxin Henning 100 and Eferox 100. PATIENTS AND METHODS: In a double-blind trial, 60 euthyroid volunteers were randomly assigned to two treatment groups. Over a period of 2 weeks, each group received 0.1 mg/d of the different preparations according to a "crossover design". To monitor the efficacy of the different drugs, baseline serum thyrotropin (TSH) and free thyroxine (fT4) levels were measured with the help of immunoenzyme tests. RESULTS: Compared to Eferox, L-Thyroxin Henning led to continuously higher fT4 levels (p = 0.0004). The area under the concentration time curve (AUC) of fT4 also confirmed this highly significant difference. With respect to the influencing factors, a higher bioavailability in men compared to women (p = 0.004) was noted. Also, the increase of body weight was related to a lower bioavailability (p = 0.002). Regarding the baseline TSH serum levels, a reduction of 70% in the L-Thyroxin Henning group versus only 56% in the Eferox group was noted after a period of 14 days. Clinical symptoms of hyperthyroidism were not observed in the volunteers under both substances. CONCLUSION: In this study, L-Thyroxin Henning 100 showed a significantly higher bioavailability than Eferox 100 (p = 0.001). According to these findings, we do recommend regular measurements of serum TSH and fT4 levels when changing LT4 preparations of different brands, to cope with metabolic decompensation by using a new LT4 dosage. PMID- 12371080 TI - [Gilbert disease and type I and II Crigler-Najjar syndrome due to mutations in the same UGT1A1 gene locus]. AB - BACKGROUND: Gilbert syndrome and the Crigler-Najjar syndromes Type I and II are disorders of bilirubin conjugation with consecutive indirect hyperbilirubinemia of different severity. Morbus Gilbert is a mild hyperbilirubinemia, which is only of significance in case of drug therapy or differential diagnosis. Crigler-Najjar syndrome II leads to a more serious kind of hyperbilirubinemia. In case of Crigler-Najjar syndrome I patients are suffering from a very severe hyperbilirubinemia, which often causes death during the first months of life. MOLECULAR GENETICS: The molecular defects of these three syndromes have been characterized during the last decade. They are caused by mutations in the UGT1A1 gene locus. This locus codes for the enzyme bilirubin uridine 5'-diphosphate-(UDP ) glucuronosyltransferase (UGT1A1). In the case of Gilbert syndrome two bases are inserted into the promoter of the gene. In Crigler-Najjar syndrome type I and II mutations lead to the exchange of amino acids, changes of the reading frame or to stop codons. CONCLUSION: All three forms of indirect hyperbilirubinemia are caused by mutations in the UGT1A1 gene locus, which codes for the enzyme UDP glucuronosyltransferase. PMID- 12371081 TI - [Ethics in clinical routine care: example of prognosis information]. AB - OBJECTIVES: The article discusses from an ethical point of view the question whether a physician should tell the patient the whole truth about a poor prognosis. From a legal viewpoint, the therapeutic privilege gives physicians in most countries the right to limit information, if they are concerned that this information will severely harm the patient. METHODS AND RESULTS: An overview about empirical studies, especially surveys of physicians and patients, shows that most patients always wish to know their prognosis, while physicians would less often tell the whole truth. Physicians explain their attitudes by referring to the ethical principles of nonmaleficience and beneficience. These principles are apparently in conflict with the principles of veracity and respect of patient autonomy. However, it can be shown that this conflict does not persist when empirical data about consequences of truthful information are considered: telling the truth seems not to have negative, but rather positive consequences on the overall well-being of the patient. After having summarized the empirical studies that have examined the consequences of truthful information about severe and incurable diseases, the article argues for always telling patients the truth if they want to know it. CONCLUSION: Many conflicts in medical ethics are between prima facie principles. In cases where the principles of beneficience and nonmaleficience are used in the argumentation, some of the conflicts can be eliminated when the ethical judgment is made on a thorough empirical basis, as shown by the example of truth-telling about prognosis. PMID- 12371082 TI - [Nephrology update. Kidney protection with antihypertensive drugs: I]. AB - Diabetes has become the most common single cause of end-stage renal disease in many countries. The coexistence of diabetes mellitus and hypertension dramatically increases the risk of developing target organ complications including renal disease. There are good arguments that ESRD in the patient with diabetes is largely preventable with the interventions currently available. For type 2 diabetes the UK Prospective Diabetes Study Group Trial clearly documented that the frequency of microangiopathic sequelae can be diminished by glycaemic control and even more impressively by intensified antihypertensive treatment. An analysis of recent randomized long-term clinical trials that evaluated the rate of decline in renal function demonstrated that the lower the blood pressure within the range of normotensive values, the greater the preservation of renal function. Since the 1994 Working Group Report on Hypertension and Diabetes suggested a goal blood pressure of 130/80 mmHg should be achieved in patients with diabetes and/or renal insufficiency; lower blood pressure levels, i.e. less than 125/75 mmHg are recommended for patients with proteinuria > 1 g/d and renal insufficiency regardless of etiology. Antihypertensive regimens should include an ACE inhibitor or an AT1-receptor blocker in order to provide maximum renal benefits in diabetic and non-diabetic renal diseases. Such low blood pressure are virtually impossible to achieve with monotherapy. In most cases the combination of two and more antihypertensive drugs is necessary. The purpose of this report is to update the previous recommendations with a focus on level of blood pressure control, proteinuria reduction and retarding the progression of renal disease. PMID- 12371083 TI - [Pregnancy-associated thrombotic microangiopathy--a diagnostic and therapeutic challenge]. AB - BACKGROUND: Thrombotic microangiopathies are diseases rarely associated with pregnancy. The pathogenesis might be related to severe preeclampsia and HELLP syndrome. CASE REPORT: In May 2000, we saw a 26-year-old primigravida in the 39th gestational week with worsening anemia, thrombocytopenia, and increasing liver enzymes. The diagnosis of HELLP syndrome was made and delivery initiated. Postpartum liver function stabilized, but anemia, thrombocytopenia, and preexisting hypertension worsened. Additionally, renal function deteriorated, and she had to be dialyzed 12 days after delivery. Renal biopsies were performed on day 12, 34, and 60 after delivery. The material showed a thrombotic microangiopathy, initially in an active stage. Later, fibrosis of the preglomerular arterioles and the glomeruli as well as progressive tubulointerstitial damage could be shown. Plasmapheresis was started; substitution was performed with fresh frozen plasma (FFP). Simultaneously, the patient was treated with corticosteroids. After 24 days, we began with cyclophosphamide pulses. Overall, 28 plasmapheresis sessions and three cyclophosphamide pulses were given. In spite of this aggressive regimen, renal function did not recompensate, and renal replacement therapy with continuous ambulatory peritoneal dialysis (CAPD) was initiated. CONCLUSION: This course shows that mortality could be decreased using plasmapheresis therapy, but further research is needed to introduce more specific, kidney-protective regimens. PMID- 12371084 TI - [Severe electrolyte imbalance and edema in therapy with rosiglitazone]. AB - CASE REPORT: A case of a 49-year-old male with preexisting liver damage is reported. The patient was admitted to hospital with severe electrolyte disorder and face edema after therapy first with 4 mg for 2 months and later for 5 months with 8 mg rosiglitazone. The initial electrolyte values were: sodium 110 mmol/l, potassium 3.3 mmol/l, calcium 2.0 mmol/l, chloride 81 mmol/l. An already known hypercholesterolemia worsened substantially to values up to 28.5 mmol/l. Under substitution therapy with sodium chloride infusion and potassium, the electrolyte level normalized rapidly. The hypercholesterolemia improved over several weeks after stopping the drug, and the general condition of the patient improved clearly. CONCLUSION: Rosiglitazone has been certified in Germany since July 2000. Although a liver toxicity with rosiglitazone has been denied, the administration of this drug in patients with liver damage is contraindicated. Especially when prescribing new drugs one has to pay special attention to contraindications and comedication since often not all therapeutic mechanisms and side effects are fully known/understood. Interaction between different drugs and their influences on existing diseases are only noticed after a widespread application of the drug. PMID- 12371085 TI - [Chronic cough in muscular dystrophy]. PMID- 12371086 TI - [Parenteral nutrition: at what price? An ethical orientation to "percutaneous endoscopic gastrostomy" (PEG catheter) nutrition]. PMID- 12371087 TI - [Quality management in health care: error prevention and managing errors in medicine]. AB - BACKGROUND: About 3.7% of in-house-treated patients in Switzerland, the USA and Australia are victims of treatment-related health problems which probably are related to avoidable "adverse events" in more than 50% of the occurrences. Reasons are primarily systematic incidents, e.g., organizational deficiencies in the health system and only secondarly individual mistakes. As there are no systematic studies available, it is not proven if those figures can be transferred to the German Health Care System. Here, experts anticipate up to 12.000 proven treatment errors per year. PREVENTION OF AND DEALING WITH ADVERSE EVENTS: Dedicated programs for identification and prevention of adverse events should be implemented--besides systematic quality improvement--to improve professional handling and prevention of adverse events. This consists of a) assessment of the existing problem using existing data bases and/or implementation of mandatory documentation and information routines as well as reporting systems, b) development of sanction-free reporting routines within the legal framework, c) dissemination of behavior-oriented training systems for recognition and prevention of adverse events as well as incentives for the participation in such training systems, d) implementation of automatic routines for prevention of adverse events (e.g., computer-based monitoring of ADE or computer-based reminder systems based on clinical guidelines). PMID- 12371088 TI - [Future scale and market capacity of urban water environmental infrastructure in China: a system dynamic model]. AB - With the application of system dynamics, a dynamic, nonlinear model (SDMUWEIC) was developed in this paper in order to reflect the relationships of population, economic, resources and environment. Through a systematic procedure of model validation and uncertainty analysis, the model was applied for predicting and analyzing the future market capacity and constituents of urban water infrastructure. It illustrated the volumes and trends of potential capital market in construction, general mechanical equipments and water treatment instruments as well as their relevant influencing factors including water pricing and urbanization rate. Several different scenarios were further under test to reveal the sensitivity of different uncertain components. PMID- 12371089 TI - [Mercury in the peat bog ecosystem in Xiaoxing'an mountain in China]. AB - The mercury content in Tangwang River forested catchment of Xiaoxingan Mountain in Northeast of China was studied. The average total mercury (THg) in peat profile ranged from 65.8 ng/g to 186.6 ng/g with the highest in the depth of 5-10 m. THg in the peat surface was higher than the background in Heilongjiang province, and higher than the Florida Evergrade in America and Birkeness in Sweden. MeHg ranged from 0.16 ng/g to 1.86 ng/g with the highest in the depth of 10-15 cm. MeHg was 0.2-1.2% of THg. They all decreased with the depth. There was no strong significant correlation between the THg and MeHg (p = 0.05, r = 0.28). THg in upland mor layer of soil (0-20 cm) was comparable to the peat surface (0 20 cm), but in deeper layer THg in peat was much higher than the forested mineral soil. THg in the peat bog increased, but MeHg decreased after it was drained 30 years ago. THg in plant was different, THg in the moss (119 ng/g, n = 12) was much larger than the herbage, the arbor and the shrub. The peat bog was contaminated by mercury coming from the atmosphere to some degree. PMID- 12371090 TI - [A comparison of efficiency of different auxiliaries used in phosphorus determination by ignition method]. AB - Researches on phosphorus recoveries from NaH2PO4 and (NaPO3)n (n > or = 2) after ignition at temperature from 150 degrees C to 550 degrees C with auxiliary, such as MgSO4, Mg(NO3)2, MgCl2, MgAc2, CaCl2 and so on, were finished. It was found that the phosphorus can not be completely recovered when NaH2PO4 or (NaPO3)n (n > or = 2) was ignited together with MgSO4 at routine ashing temperature (500 degrees C) with the final mixture extracted with 0.2 mol/L HCl at 80 degrees C for 0.5 h. In contrast, MgCl2, MgAc2, Mg(NO3)2 and CaCl2 can all make the phosphorus recoveries completely. MgCl2 (or MgAc2) rather than MgSO4 in the research was suggested. To be utilized as ashing auxiliary in the phosphorus determination with ignition method. Although Mg(NO3)2 is a highly effective auxiliary, yet danger of explosion, toxicity of nitrogen dioxide and more manipulation steps may impede its widespread utilization. PMID- 12371091 TI - [MSW incineration fly ash melting by DSC-DTA]. AB - Melting characteristics of two kinds of municipal solid waste incineration(MSWI) fly ash were studied in this paper by high temperature differential scanning calorimetry and differential temperature analysis. MSWI fly ash was considered as hazardous waste because it contains heavy metals and dioxins. The experiments were performed in either N2 or O2 atmosphere in temperature range of 20 degrees C 1450 degrees C at various heating rates. Two different MSW incineration fly ashes used in the experiments were collected from our country and France respectively. The process of fly ash melting exhibits two reactions occurring at temperature ranges of about 480 degrees C-670 degrees C and 1136 degrees C-1231 degrees C, respectively. The latent heat of polymorphic transformation and fusion were approximately 20 kJ/kg and 700 kJ/kg, while the total heat required for melting process was about 1800 kJ/kg. The paper also studied effect of CaO to melting. A heat flux thermodynamic model for fly ash melting was put forward and it agrees well with experimental data. PMID- 12371092 TI - [Safety threshold of fluorine in endemic fluorosis regions in China]. AB - Four endemic fluorosis regions in China and their environmental epidemiological characteristics were summarized in this paper. It shows that the epidemiology of endemic fluorosis is closely related to geochemical parameters of local environment. The food-web and dose-effect relationship of fluoride from environment to human body in different types of endemic fluorosis regions were studied. And the safety threshold of fluoride in different regions was determined. The results have provided a scientific basis for environmental risk assessment of fluoride in China. PMID- 12371093 TI - [Micronuclei induced by chronic inhalation of SO2 in mouse bone marrow cells]. AB - In the chronic inhalation experiment of sulfur dioxide(SO2), micronuclei(MN) frequencies in the polychromatophilic erythroblasts(PCE) of mouse bone marrow and the frequencies of cells with MN were significantly increased in dose-dependent manner. There was a significant difference between the male and the female animals. The results also showed that SO2 inhibited urethone-induced MN formation. These results furtherly confirm that SO2 inhalation was a clastogenic and genotoxic agent to mammalian cells, and the combined effects of SO2 and other mutagens are complex. PMID- 12371094 TI - [Effect of covering with new soil on reducing the accumulation of radio-strontium in plant]. AB - Effects of covering with new soil on reducing the accumulation of 89Sr by soybean and Chinese cabbage (especially in the edible part of crop) were studied on simulated pollutants by using the isotope-tracer techniques. The results showed that the absorption and accumulation of 89Sr in the soybean and Chinese cabbage could be decreased significantly by covering soil polluted 89Sr with new soil. The specific activity of 89Sr in bean hull, bean straw, bean root and Chinese cabbage were reduced by 82.8%, 56.4%, 38.7%, 66.5% and 68.8% respectively when the depth of covering with new soil reached 9 cm. The specific activity of absorption and accumulation 89Sr in the crop decreased with depth of the new soil profile. The specific activity of 89Sr in the crop follows a negative linear relation with depth of the new soil profile by analyzing the experiment data with linear regression method. PMID- 12371095 TI - [Effects of SO2 on NO reduction with methanol over Ag/Al2O3 catalyst]. AB - Ag/Al2O3 catalyst with 5% Ag loading was prepared by the single step sol-gel mixture method and the effects of adding SO2 to reaction feed on NO reduction by CH3OH over the catalyst was investigated in the presence of oxygen. The results showed that in the absence of SO2 and H2O the catalyst displayed lower activity temperature and higher N2O selectivity, which was attributed to the partial reduction of oxidized Ag into metallic Ag under reaction conditions. Selective catalytic reduction activity was not decreased but significantly increased, N2O formation was suppressed and most effective NO reduction temperature shifted to higher temperature by pre-sulfated Ag/Al2O3 or addition of SO2 to reaction mixture. XPS analysis showed that the sulfate-like species were formed by the effect of SO2. PMID- 12371096 TI - [Calculating critical load of acid deposition with dynamic model]. AB - Any natural ecosystem is a steady system which will ultimately approach a new steady state when it receives a fixed quantity of acid deposition. Dynamic models can be used to predict the changing potential of the chemical state of an ecosystem under different acid depositions. According to the theory of signal and system, this potential can be modeled by the first-order exponential attenuation function to get the value of chemical index when the ecosystem achieves steady state. A dose-response curve can be obtained according to the different quantity of acid deposition and the corresponding steady-state values of chemical index. Consequently, the critical load for an ecosystem can be evaluated from the dose response curve, i.e., the quantity of acid deposition under which the steady state value of the chemical index will achieve the critical chemical value. Taking MAGIC as an example, this method was applied to estimate critical loads of S deposition for a water body on Emei Mountain in Sichuan province and a lake on Nanshan Mountain in Chongqing city, the values being 1.54 and 6.51 keq.(hm2.a)-1, respectively. Through comparing the critical loads calculated using the method proposed in this paper with the target loads calculated with MAGIC and the critical loads estimated using SSWC method, it can be concluded that the method proposed in this paper are reasonable in estimating critical loads of acid deposition. PMID- 12371097 TI - [Determination and characteristics of OH radical in urban atmosphere in Beijing]. AB - The measurement of atmospheric hydroxyl radicals (OH) in Beijing was carried out using two methods based on high performance liquid chromatography (HPLC). The diurnal variation of OH concentration was obtained. The maximum concentration during sunny daytime was about 8 x 10(7) cm-3 in summer, while it decreased to about 2 x 10(7)-4 x 10(7) cm-3 in autumn. The relationships between OH and other pollutants were studied and discussed. Positive linear correlation between OH and UV-B intensity, O3, HNO2 was observed, while negative correlation between OH and NOx was found. PMID- 12371098 TI - [Physical and chemical characters of materials from several mineral aerosol sources in China]. AB - Loess, sand and coal-ash samples were collected at several mineral aerosol sources to analyze the particle size as well as the major and trace elements. The results show that the physical and chemical characters were quite different between these samples. The loess was mainly made up of small particle size that 91% of the sample could be transported as mineral aerosol to a long distance. The sand consists of relative large-size particles, with only 15% sample being formed into mineral aerosol. The coal-ash samples were quite different in particle sizes in the two regions, with 39% of particles < 74 microns in Fushun samples while only 7% in Huhehaote samples. Chemical compositions varied significantly in different particle sizes, with trace elements obviously enriched in the least size. The elements to Al ratios (E/Al) were quite alike between the Loess and the sand, but very different from the coal ash. The similarity of the E/Al ratios between the Loess, the sand and the deep-sea clay suggest that the deserts and the Loess in the northwest China may be important sources for sediment of the northwest Pacific Ocean. PMID- 12371099 TI - [Effects on fine particles by the continued high temperature weather in Beijing]. AB - The continued high temperature weather (CHTW) appeared in Beijing since June 23 1999. The measurements showed the PM2.5 mass concentrations were 2 to 3 times of that in non-CHTW period. However the diffusion conditions were still fine in these days. Both the highest hourly concentrations of O3 and the SO4(2-) composition in PM2.5 can identify that the photochemical action was very active. It is the very reason to produce more fine particles in atmosphere. PMID- 12371100 TI - [Oxidative degradation of chlorinated hydrocarbons under anaerobic conditions]. AB - Based on column experiments, the oxidative degradations of some chlorinated hydrocarbons under three less-reduced redox conditions were investigated. The results showed that in the presence of nitrate and manganese oxide, 1,2 dechloroethane (1,2-DCA) and vinyl chloride (VC) could be oxidized. The transformation rates of 1,2-DCA under denitrification and manganese reduction were 1.18/h and 0.54/h, respectively, while those of VC were 0.29/h and 0.15/h, respectively. In the presence of iron, degradation of VC was not clear. In addition, the degradation of 1,2-DCA was inhibited. For other chlorinated hydrocarbons, such as 1,1,1-trichloroethane, trichloroethene, cis-dichloroethene and trans-dichloroethene, no degradation occurred under the three studied redox conditions. Monochlorobenzene exhibited relative high removal in the columns, however, due to its high soil adsorption potential, it was not known yet whether microbial activities were involved. PMID- 12371101 TI - [Treatment of a 2,4-dichlorophenol contaminated wastewater in an air-lift inner loop bioreactor]. AB - An air lift inner-loop bioreactor packed with honeycomb-like ceramic carrier was immobilized with a 2,4-Dichlorophenol-degrading pure culture and was investigated to degrade 2,4-Dichlorophenol and phenol. In fed-batch operation mode, 2,4-DCP biodegradation rate increased with run numbers and followed zero-order kinetics model when it existed alone, but when 2,4-DCP was present in the mixture with phenol, phenol degradation rate had an apparent trend to increase whereas 2,4-DCP removal rate became slower and slower. In continuous operation, 2,4-DCP at the concentration ranged from 6.9 to 102.4 mg/L could be degraded well at the dilution rate of 0.16 h-1 and the average removal percentage was 96.5%. Carbon sources changed from 2,4-DCP to acetate sodium and peptone in the course of operation for 12 days did not cause the bacteria loss the DCP-degrading ability. PMID- 12371102 TI - [Photoelectrocatalytic degradation of Rhodamine B using mesh Ti/TiO2 electrode]. AB - An innovative mesh titanium dioxide (TiO2) electrode was prepared by anodisation. The morphology and the crystalline texture of the TiO2 film on electrode were examined by SEM and Raman spectroscopy respectively. The results indicated that the structure and properties of the film depended on anodisation rate, and the anatase was the dominant component under the controlled experimental conditions. Biasing could increase the efficiency of photocatalytic degradation of organic matters. Mineralization Rhodamine B (Rh B) was complete relatively in photoelectro-catalytic (PEC) oxidation. Both chromogen destruction of Rh B and de ehtylation in PEC degradation took place simultaneously. PMID- 12371103 TI - [Effect of COD/SO4(2-) ratio on the ecological characteristic in acidogenic sulfate-reducing reactor]. AB - Continuous-flow experiment was conducted in acidogenic sulfate-reducing reactor, which was fed with high strength sulfate wastewater, to study the change law of pH value, ORP (oxidation reduction potential), VFAs (volatile fat acids), ALK (alkalinity) and the distribution of preponderant populations subjected by decreasing COD/SO4(2-) ratio from 4.2 to 2.0. It was demonstrated that during this course of change, ORP and ALK increased, while pH value and the proportion of acetic acid accounting for VFAs decreased significantly, and the type of climax community was changed from stable-type with high COD/SO4(2-) ratio to sub stable-type with low COD/SO4(2-) ratio, but it was still belonged to typical acetic-acid type climax community. PMID- 12371104 TI - [Flow field test on the tangential section of polypropylene tubular membrane module annular gap in rotating linear tangential flow]. AB - A new type of polypropylene tubular membrane apparatus of rotating cross flow was designed to study experimentally the flow field characteristics of the tangential section of the membrane annular gap. The authors designed rotary linear tangential flow tubular membrane separator and its test system for the first time. Through the system, the flow field of rotary linear tangential flow with the advanced Particle Image Velocimetry (PIV) was tested for the first time. A lot of streamlines and vorticity maps of the tangential section of separator in different operation conditions were obtained. The velocity distribution characteristics were analyzed quantitatively: 1. At non-vortex area, no matter how the operation parameters change, the velocity near to rotary tangential flow entrance was higher than the velocity far from entrance at the same radial coordinates. At vortex area, generally the flow velocity of inner vortex was lower than the outer vortex. At the vortex center, the velocity was lowest, the tangential velocity were equal to zero generally. At the vortex center zone, the tangential velocity was less than the axial velocity. 2. Under test operations, the tangential velocity and axial velocity of vortices borders are 1-2 times of average axial velocity of membrane module annular gap. The maximum tangential velocity and axial velocity of ellipse vortices were 2-6 times of average axial velocity of membrane module annular gap. 3. The vortices that are formed on the tangential section, there existed mass transfer between inner and outer parts of fluid. Much fluid of outer vortices got into the inner ones, which was able to prevent membrane tube from particles blocking up very soon. PMID- 12371105 TI - [Laboratory-scale continuous treatment of monosodium glutamate manufacturing wastewater using yeast]. AB - Dehydrogenation activity(DHA), test indicated that mixed yeast strains isolated from high strength monosodium glutamate wastewater could endure the high concentrations of COD, SO4(2-), and NH4+ containing in glutamate wastewater. The mixed yeast strains were inoculated to a biological contact oxidation reactor, and the reactor was used to treat the glutamate wastewater. Under a COD load ranging from 2.0 to 14.3 kg/(m3.d), the COD removal rate was over 80%. On the other hand, supplementation of phosphorus was necessary to maintain a stable COD removal performance. Variation of effluent pH seemed to have no apparent influence on COD removal rate. The optimum pH for the growth of yeast, however, was found to be in the range of 3.5-5.0. The effluent wastewater contained high concentration of yeast bodies, which could be utilized as a forage additive because of its high protein content (57.9%) and well-balanced amino acid distribution. PMID- 12371106 TI - [Studies on ceramic dual function membrane bioreactor for wastewater treatment]. AB - A new bioreactor including ceramic dual function membrane, filtration and aeration, was developed and used for wastewater treatment. The turbidity of effluent by the ceramic membrane was comparable to that of 4-24 hours by sedimentation. Aeration and filtration could be switched constantly to the ceramic membrane in the bioreactor and the clogging of ceramic membrane could be well prevented. The wastewater was treated in batch and continues operation modes respectively and the experimental results showed that the COD loading was 1.1 kg/(m3.d) and 1.5 kg/(m3.d) respectively. PMID- 12371107 TI - [Dynamic properties of Al-humic flocs]. AB - Using a microscopic technique, the dynamic properties of Al-humic flocs were studied and the regularity of the variation of floc structure, i.e. fractal dimension, was discussed. The results show that during coagulation floc size increased with agitation time and finally reached an equilibrium state. As flocs grow their structure gradually become looser with an increase of void ratio, and results in a substantial decrease of their fractal dimension from the initial value of 1.8 to about 1.4. There exist an exponential relation between the fractal dimension Df and the floc size df as Df = Adf-n. It was explained by theoretical analysis that the decrease of Df with increase of df as was the result of random collision and agglomeration of primary particles. The alum dosage directly affected the rate of floc growth and its equilibrium size. Overdosing may bring about an apparent decrease of the fractal dimension of the flocs. PMID- 12371108 TI - [Application of the reaction theory to flow pattern on the integrated vertical flow constructed wetland]. AB - This study was conducted to determine the flow pattern of the integrated vertical flow constructed wetland (IVCW) by using the reaction theory. The retention time distribution (RTD), obtained by tracer experiment, showed the retention time was between 19 and 35 h under 200-800 mm/d hydraulic loading rate, and the actual flow pattern was between the ideal plug flow and continuous stirred flow based on the characteristic value of the RTD. Furthermore, the dispersed flow model was found adequate in simulating the actual flow pattern of the IVCW and the Peclect number was determined between 11 and 19. Comparison the planted and unplanted IVCW, the plant root was found favorable for the flow pattern to approximate the ideal plug flow. PMID- 12371109 TI - [Purification of dichloromethane waste gas in a biotrickling filter]. AB - An experimental investigation on purification of dichloromethane waste gas was conducted in a 50 mm diameter biotrickling filter packed with randomly-stacked polypropylene. A heterotrophic population was acclimated from facility's secondary sludge, and was further inoculated over the surface of the packing. The inoculation process lasted about 30 days, and then a biofilm was developed. The counter-current operation was carried out in the filter. The pH and temperature of the circulating liquid were controlled as 7.0 +/- 0.5 and 28.5 +/- 2 degrees C respectively. The biofilm system was well accommodative to the fluctuation of operating conditions. When the concentration of dichloromethane was in the range of 0.7 to 3.12 g/m3, and the gas empty-bed residence time was 15.7 s, the removal efficiency of 72.0 to 99.1% could be achieved. It was also showed that the acid environment in the filter could refrain the degradation of dichloromethane. PMID- 12371110 TI - [Accumulation and cycle of heavy metal in Sonneratia apetala and S. caseolaris Mangrove Community at Futian of Shenzhen, China]. AB - The absorption, accumulation, distribution and cycle of Cu, Pb, Zn, Cr and Ni in S. apetala and S. caseolaris Mangrove Community at Futian Mangrove Nature Reserve of Shenzhen were studied. The results showed that the Cu, Pb, Zn, Cr and Ni contents in forest soil were increased from bottom to surface layer, and the storage of the five heavy metals in the surface layer(depth 0-30 cm) was Zn > Pb > Ni > Cr > Cu. The concentration ability was S. caseolaris > S. apetala > K. candel. The existing accumulation of Cu, Pb, Zn, Cr and Ni in the community were respectively 23,019.61 micrograms/m2, 23,429.65 micrograms/m2, 117,870.41 micrograms/m2, 6835.79 micrograms/m2, 12,995.08 micrograms/m2. The annual absorption were 6592.20 micrograms/m2, 2664.77 micrograms/m2, 24,123.56 micrograms/m2, 853.25 micrograms/m2, 1990.86 micrograms/m2, respectively. The annual return were 3179.50 micrograms/m2, 1300.64 micrograms/m2, 8401.31 micrograms/m2, 398.99 micrograms/m2, 646.20 micrograms/m2, respectively. The annual net retention accumulation of Cu, Pb, Zn, Cr and Ni in the community were 3413.07 micrograms/m2, 1364.13 micrograms/m2, 15,722.25 micrograms/m2, 454.25 micrograms/m2, 1344.66 micrograms/m2. The turn over period of Cu, Pb, Zn, Cr and Ni were 8, 19, 15, 18 and 21 years. PMID- 12371111 TI - [The dynamics rules of soil organic matter of turnover ecosystems traced by stable carbon isotopes]. AB - On the basis of different photosynthetic pathway, there's obvious difference in delta 13 C values between C3 plants and C4 plants. Use this characteristic, the delta 13C values in different size and density fractions of two profile soil samples either in farm land and forest lands near Maolan Karst virgin forest was analyzed, there plant C3 plants previously and plant C4 plants now. Results showed that the delta 13C values of different size fractions in forest soil aere delta 13Ccoarse sand < delta 13Cfine sand < delta 13Ccoarse silt < delta 13Cclay < delta 13Cfine silt, and the delta 13C values of different size fractions in farmland soil were delta 13Ccoarse sand > delta 13Cfine sand > delta 13Ccoarse silt > delta 13Cclay > delta 13Cfine silt, it indicated that the soil organic matter was fresh in coarse sand and oldest in fine silt. The delta 13C values of different density fractions in forest soil were delta 13Clight < delta 13Cheavy, and the delta 13C values of different density fractions in farmland soil were delta 13Clight > delta 13Cheavy, it also indicated that the soil organic matter was fresh in light fractions and old in heavy fractions. PMID- 12371112 TI - [Effect of selenium on the response of the active oxygen scavenging system in the leaves of paddy rice under the stress of herbicide]. AB - The effects of selenium on the protection system in the leaves of paddy rice were studied under the stress of herbicide mefenacet. The results showed that selenium could cause increase of the height and root length, enhanced the contents of protein, glutathione(GSH) and the activities of antioxidant enzyme, leading to decreased level of activated oxygen and malondialdehyde(MDA), slowering the autoxidation rate. All these could alleviate the harmful effect of mefenacet on paddy rice. The paired t-test results showed that the effect of selenium on the response of the active oxygen scavenging system were significant. PMID- 12371113 TI - [Distribution of tributyltin between water and liposome]. AB - Distribution of the tributyltin (TBT) between egg-phosphatidylcholine membranes (liposome) and water under different pH and a comparison between the partition of TBT into lipid membranes and into octanol were studied. The distribution of this compound into both lipid membranes and octanol increased with pH. The major difference was that at lower pH, where ionized TBT dominated distribution was greater into lipid membranes compared to that of octanol. At high pH, where TBT predominates as neutral species, the distribution ratios were very similar in the octanol and the liposome-water system. These differences can be attributed to the properties of the biomembrane. The results indicated that the liposome-water partition model appears to be a better molecular descriptor for ionogenic organic compounds. PMID- 12371114 TI - Injection drug use in North America. AB - Injection drug use of psychotropic agents for nontherapeutic purposes is associated with some of the most pernicious infectious diseases seen in the United States. There is an inextricable link between infection, injection drug use (IDU) and other risk behaviors, especially those related to sexual activity. A number of national surveys now provide excellent databases to track the breadth, scope and impact of IDU across the United States. The prevalence of heroin use has increased over the past decade with larger numbers of users presenting for treatment of drug use disorders. A host of serious infections can result from IDU. Prevention and early intervention with evidence-based harm reduction strategies are crucial to reducing and eliminating these odious consequences. PMID- 12371115 TI - Opioids, immunology, and host defenses of intravenous drug abusers. AB - Overall, it is apparent that opioids do affect host defense mechanisms. Heroin users present with an altered and functionally impaired immune system and have a higher prevalence of infectious diseases than do nonaddicts. Individuals exposed to opioid treatment for pain management during surgical procedures or maintained on oral methadone for treatment of drug addiction show either no effect or a suppressed immune system, depending on dosage and, in the case of methadone maintained patients, duration of drug treatment. Confounding factors in these studies undermine definitive conclusions about the mechanisms by which opioids induce their immunomodulatory effects. Animal models have provided the means by which investigators can study the effects of opioids in a complex, biologic system that is easily manipulated and controlled. Findings from these studies have confirmed human data associating a pathogenic susceptibility with opioid use. Animal models have shown the complexity of this association. Interaction of the CNS, the autonomic nervous system, and the HPA axis is required for the varied effects of opioids on the immune system. By implication, exogenous opioids may be mimicking pathways by which endogenous opioids are involved in regulating immune defenses. To minimize the increased incidence of infectious diseases in heroin users and individuals clinically exposed to opioids, it will be important to determine the individual and collective effects of the opioid-induced activation of these pathways and the consequences of that activation to the immune system. PMID- 12371116 TI - Management of the hospitalized injection drug user. AB - Patients with a history of injected drug use are often distrustful of the medical system. This attitude is generally based on problems in the management of pain or withdrawal or on hostility from medical providers in the past. When treated with respect and appropriate medical concern, hospitalized injection drug users will often actively seek to begin recovery from drug use. Identifying injection drug use is the first step in providing appropriate care. Assessing and systematically addressing key management issues allows the provider to care for the hospitalized injection drug user effectively. The well-trained provider can be a significant catalyst for behavior change in the addicted population. PMID- 12371117 TI - Central nervous system infections in injection drug users. AB - Central nervous system infections in injection drug users are often devastating in terms of excess morbidity and mortality. In injection drug users with infective endocarditis, embolization from infected valvular vegetations may cause cerebral infarction, intracranial hemorrhage, and the formation of brain abscess. Focal intracranial infections (i.e., brain abscess and spinal epidural abscess) may occur in the absence of infective endocarditis, resulting from bacteremia that seeds the brain or epidural space. Antimicrobial therapy, combined with surgical intervention, may be essential to improve outcome from these neurologic complications. Toxin-mediated diseases (especially tetanus and wound botulism) are also seen in injection drug users. Inoculation of Clostridium spp at injection sites may lead to toxin generation and disease. Clinicians must maintain a high level of suspicion for these diagnoses in injection drug users. PMID- 12371118 TI - Ocular manifestations of injection drug use. AB - Injection drug use can result in a variety of severe ocular conditions. Hematogenous dissemination of various fungi and bacteria may produce endophthalmitis with resultant severe visual loss. Retinal arterial occlusive disease may result from talc and other particulate emboli. Most commonly, life threatening systemic diseases such as endocarditis and HIV infection secondarily affect the eye. Because many of these conditions may result in blindness if untreated, accurate diagnosis and prompt initiation of therapy are essential. PMID- 12371119 TI - Sinopulmonary complications of illicit drug use. AB - Illicit drug use is frequently complicated by sinopulmonary illnesses. These complications fall into two major categories: (1) direct effects of drug exposure, and (2) indirect effects caused by HIV-induced immunosuppression. This article reviews the more common sinopulmonary syndromes associated with illicit drug use. PMID- 12371120 TI - Infective endocarditis in the injection drug user. AB - Although infective endocarditis is certainly not the most common infection seen in injecting drug users, it is the infection that clinicians most commonly think of when they consider infectious complications of injected drug use. The microbiology of infective endocarditis in injection drug users has remained relatively stable over the last several decades. Tricuspid valve endocarditis has been associated most frequently with injection drug use, but recent reports have suggested that involvement of left-sided valves is seen more often now than in the past. The use of transesophageal echocardiography has greatly advanced the ability to diagnose infective endocarditis and the cardiac complications of valvular infection. PMID- 12371121 TI - Hepatic disease in injection drug users. AB - A wide variety of infectious diseases affect injection drug users. One of the most common is viral hepatitis. In the United States, hepatitis B affects 1.5 million people, and hepatitis C affects more than 4 million people, many of whom are past or current users of injected drugs. Although the treatment for chronic hepatitis B and hepatitis C has improved, protocols specifically designed for injection drug users, especially given their lifestyle and reportedly low compliance rates, are seriously lacking. These disorders can lead to cirrhosis, hepatocellular carcinoma, and the need for liver transplantation in a sizeable proportion of cases. Therefore, early intervention should be a top priority. PMID- 12371122 TI - Bone and joint infections in injection drug users. AB - Skeletal infections in injection drug users have an insidious onset, present with indolent symptoms, and often occur in unusual locations. Unless physicians are familiar with the disease entities unique to the injection drug user, the diagnosis is frequently delayed. Systemic signs of infection are often lacking. The organisms causing the infection represent a wide spectrum; hence, empiric therapy is not generally recommended. Plain-film radiographs are of little help for early diagnosis. Imaging studies, especially radionucleotide studies and CT or MR imaging scans, can help localize the site of infection. For etiologic diagnosis of these infections, bone biopsy or needle aspiration of the involved bone or joint is required. The choice of antibiotic agent should be based on culture results and the antimicrobial susceptibility of the causative organism. Treatment may also involve surgical drainage or debridement of affected structures. Failure to manage acute bone and joint infection aggressively inevitably leads to chronic, often incurable, infection. Successful therapy requires a team approach including the internist and consultants from orthopedic surgery, infectious diseases, and substance abuse counselors. PMID- 12371123 TI - Skin and soft tissue infections in injection drug users. AB - Skin and soft tissue infections are the most common cause for hospital admission of injection drug users. Cutaneous and subcutaneous abscesses are the most frequent type of SSTI and occur most commonly when drug users are no longer able to inject intravenously and resort to injection directly into skin or muscle. Abscesses may be difficult to differentiate from uncomplicated cellulitis or may be confused with pseudoaneurysms, hematoma, phlegmon, or thrombosed vein. Special studies, including ultrasonography; CT scans, and MR imaging; or careful incision and inspection may be necessary to clarify the extent of infection and the presence of abscess. These procedures may also help differentiate a subcutaneous abscess from a vascular structure. Uncomplicated cellulitis most commonly responds to antibiotic therapy directed toward Staphylococcus aureus and Streptococcus spp. In several recent studies, cutaneous and subcutaneous abscesses have been found to be caused by polymicrobial infections and to include anaerobic organisms as well as aerobic gram-positive cocci in a little more than 50% of cases. Complete, often repeated, incision and drainage is a prerequisite for successful outcome in these cases. Complications of SSTI are many and are potentially life threatening. They include direct extension of subcutaneous abscess into vital areas or structures, necrotizing fasciitis and myositis, bacteremia, and sepsis. An outbreak of a highly lethal SSTI that recently occurred in Scotland, Ireland, and England seems to have resulted from infection with Clostridia spp, including C. novyi and C. perfringens. A rare but well documented SSTI in injection drug users is pyomyositis, an abscess-forming infection of skeletal muscle. More than 20 cases have been reported in temperate climates to date. Although not life-threatening, chronic cutaneous venous ulcers of the lower extremities are common and debilitating, requiring long-term multidisciplinary care for successful healing. PMID- 12371124 TI - Radiologic study of injection drug use complications. AB - The complications of injection drug use (IDU) can lead to a wide variety of clinical problems that range from complications localized to the injection site to more disseminated disease. The radiologic workup of these problems uses multiple modalities, depending on the location and type of problem to be investigated. This article discusses and illustrates some of the local and disseminated complications that can occur after IDU. PMID- 12371125 TI - HIV-1 infection in injection drug users. AB - Injection drug use is an efficient and ongoing means of HIV transmission and is the principal mode of transmission in some parts of the world. In the United States, approximately 10,000 injection drug users are believed to acquire HIV each year. The US Public Health Service hopes to decrease all HIV transmission in the United States by 50% in the next 5 years, by promoting care and prevention services to infected persons. Subtle differences in the virology and immunopathogenesis of HIV between injection drug users and other groups at risk are still being investigated. So far such differences have no practical implication. Comparison of progression rates and survival with HIV across risk groups has been difficult because of the many competing causes of death unrelated to HIV among injection drug users, but overall HIV disease progression rates are similar across risk groups, after adjusting for age. Some AIDS-related opportunistic infections are more common (such as tuberculosis) or less common (such as Kaposi's sarcoma) among injection drug users, based on rates of exposure and latent infection. Other comorbidities, including chronic psychiatric disorders and hepatitis C disease, are more common among injection drug users than among others with HIV infection. Highly active antiretroviral treatment seems to be as effective in persons with a history of injected drug use as in others. Increasing the numbers of HIV-infected injection drug users who know their diagnosis, increasing their access to care and prevention services, and increasing their adherence to a therapeutic regimen are the current challenges in confronting the HIV-epidemic among injection drug users. To overcome these obstacles, clinicians must have both technical knowledge and skill in assisting patients with behavior change. PMID- 12371126 TI - Sexually transmitted diseases in injection drug users. AB - The recommended work up for diagnosis of STDs in injection drug users is presented in the box. Diagnostic work up for sexually transmitted disease in injection drug users Asymptomatic patients-screening work up Serology VDRL, HIV antibody, hepatitis B surface antigen, hepatitis C antibodies AND Endocervical specimen Gonococcal culture, gonococcal DNA detection (probe) OR Amplification (PCR), chlamydial DNA detection or amplification OR Urine specimen-gonococcal and chlamydial DNA amplification (PCR) AND Vaginal specimen pH, clue cells, Trichomonas Endourethral specimen Gonococcal DNA amplification, chlamydial DNA amplification OR Urine specimen-gonococcal and chlamydial amplification Symptomatic patients-diagnostic work up All the above AND Genital ulcers Dark field microscopy, Herpes simplex virus-DNA detection or culture, and, depending on geographic risk factors, Gram's stain for Hemophilus duceryl Exophytic lesions Clinical diagnosis of genital warts, skin biopsy if treatment fails VDRL, Venereal Disease Research Laboratory; PCR, polymerase chain reaction. PMID- 12371127 TI - Clinicopathological study of involvement of granulocyte colony stimulating factor and granulocyte-macrophage colony stimulating factor in non-lymphohematopoietic malignant tumors accompanied by leukocytosis. AB - Involvement of granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) in non-lymphohematopoietic malignant tumors accompanied by leukocytosis was clinicopathologically investigated. Among 1,778 autopsy cases in the last 20 years, 485 lesions of 439 cases with non-lymphohematopoietic malignant tumors accompanied by leukocytosis with a white blood cell count of 10,000/mm3 or greater during the course were immunohistologically examined for G-CSF and GM-CSF. Three (0.7%) and two cases (0.5%) were G-CSF- and GM-CSF-positive, respectively. GM-CSF mRNA was confirmed by using non-fixed cryopreserved tumor tissues in one case positive for GM-CSF. G CSF-positive cases were large cell carcinoma of the lung, adenocarcinoma of the colon, and adenocarcinoma of the stomach, and GM-CSF-positive cases were spindle cell carcinoma of the lung and malignant thymoma. In the case with stomach carcinoma, the primary lesion showing moderately differentiated adenocarcinoma was negative, but the lung metastatic lesion showing less differentiated adenocarcinoma was G-CSF-positive. The survival period was six months or less in four out of five positive cases. The highest white blood cell count in five CSF positive cases was markedly elevated: 29,400-103,500/mm3 (mean: 59,700/mm3). In four cases, excluding one case which may have been markedly affected by chemotherapy, the bone marrow showed hyperplasia, and the number of the granulocyte series cells significantly increased. There were three cases (0.7%) negative for both G-CSF and GM-CSF, although they showed marked leukocytosis (60,000/mm3 or higher) which were higher than the mean count of CSF-positive cases and was not observed in autopsy cases with non-tumorous diseases. Other stimulating factors may be involved in the development of leukocytosis in such cases. PMID- 12371128 TI - Lectin-histochemical and -cytochemical study of periodic acid Schiff-positive lysosome granules as a histological feature of the female mouse kidney. AB - Renal proximal straight tubules (PST) of the female mouse contain periodic acid Schiff-positive lysosome granules. An excellent example of this is found in the kidneys of female DBA/2Cr mice. In the present study, lectin-histochemistry showed that lectin-positive granules occur in the PST of DBA/2Cr mice. Out of twenty-one lectins studied, the granules bound WGA, s-WGA, LEL, STL, DSL, GSL-II, VVL, RCA-I, ECL, PSA, LCA and PHA-E. Such granules were also observed in the proximal convoluted tubules (PCT). In addition, heterogeneous binding to the SBA or DBA was observed in the PST. Lectin-cytochemistry for s-WGA, STL, VVL, RCA-I, ECL and PSA, showed that: 1) lysosomes bind a higher level of s-WGA or STL than VVL, RCA-I, ECL or PSA; 2) PSA binding is similar in PST and PCT; 3) there are many PCT lysosomes that are negative for s-WGA, STL, VVL, RCA-I, and ECL lectin binding; and 4) s-WGA binding is highly specific to the lysosomes of the PST. Based on the binding specificities of each lectin, it was suggested that the mannose content of PST and PCT lysosomes is similar, and that PST lysosomes have a high level of N-acetylglucosamine, N-acetylgalactosamine, galactose or galactosyl (beta 1, 4) N-acetylglucosamine. PMID- 12371129 TI - Influence of metal ion solutions on rabbit osteoclast activities in vitro. AB - The purpose of the present study was to compare the effects of various metal ions (aluminium, chromium, cobalt, gold, iron, strontium, titanium and vanadium) on rabbit osteoclast activities, with respect to their number, size, resorptive capacity and their capacity to release proteinases. Marked heterogeneous osteoclastic behaviour was observed early in culture with metal ions (24 h) in term of resorption parameters. In contrast, protease activities (cysteine proteinase and metalloproteinase activities) were not modulated in our culture conditions. Aluminium, iron, gold and titanium reduced the number of osteoclasts significantly. Aluminium and gold had no effect on osteoclast-mediated resorption on dentin-slices, although aluminium induced a greater number of very small lacunae. Titanium reduced only the mean surface area per lacunae, cobalt reduced the mean surface area of lacunae and increased their number, and iron reduced both parameters. Strontium had no effect on osteoclast formation and on total dentin slice surface resorbed. However, strontium increased the number of small lacunae formed on dentin-slices by osteoclasts. Chromium had no effect on osteoclast activities. These findings indicate that metal ions induce very early effects on osteoclasts, which can contribute to periprosthetic pathologies via different cellular mechanisms. PMID- 12371130 TI - Morphological changes in the rat exocrine pancreas after pancreatic duct ligation. AB - In the present study, morphological changes of the exocrine pancreas in rats after pancreatic duct ligation were examined with light microscopy (hematoxylin eosin, TUNEL, and PCNA staining) and scanning electron microscopy in order to elucidate the effects of increased pancreatic duct pressure. On the fifth day after pancreatic duct ligation, ductular proliferation, periductal fibrosis, and disappearance of acini were observed. TUNEL and PCNA staining demonstrated many apoptotic acinar cells and proliferating ductal cells immediately after ligation, which reached a maximal number on the 2nd or 3rd day. Tortuous or helical interlobular pancreatic ducts with inner surfaces containing many crater-like depressions and long cilia were found after ligation. These changes were almost identical to those observed in the pancreatic tissue of model chronic pancreatitis rats, WBN/Kob rats, and stroke-prone spontaneously hypertensive (SHRSP) rats. In summary, the morphological changes observed after pancreatic duct ligation were similar to those of chronic pancreatitis, therefore, the characteristic changes of pancreatic ducts observed in chronic pancreatitis may be caused by increased pancreatic duct pressure. PMID- 12371131 TI - An electron microscopic study of neuronal degeneration and glial cell reaction in the retina of glaucomatous rats. AB - The present investigation was focused on the ultrastructural changes in the neurons and glial cells in the retina of rats with experimentally-induced glaucoma. An experimental glaucoma model was created by limbal-derived vein cauterization. Animals were sacrificed at 1, 3 weeks and 3 months post-operation. Retinae were dissected and processed for electron microscopy. Neuronal degeneration was observed in all the different layers of the retina at both 1 and 3 weeks post-operation. Some degenerating neurons were found in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL). And the dying neurons presented apoptotic-like more than necrotic neurons. Many degenerating axons and axon terminals were observed between neurons in the GCL, inner plexiform layer (IPL), INL, and outer plexiform layer (OPL). Activated astrocytes and microglial cells were present in close association with degenerating neurons and axons. The Muller cells in the INL also presented longer and darker processes with more microfilaments than in normal cells. Degenerating neuronal debris, degenerating axonal profiles and electron-dense bodies were often found in the cytoplasm of macrophages. The results suggest that both microglial cells and astrocytes are activated in the process of neuronal degeneration in the retina of experimentally-induced glaucomatous rats. It is hypothesized that they may play a protective role in removing degenerating neuronal elements in the retina after the onset of glaucoma. PMID- 12371132 TI - Enhanced accumulation of the isoprostane, 8-epi-PGF2 alpha, in human aortic and pulmonary valves of patients with coronary heart disease. AB - The aim of the present study was to investigate whether the isoprostane 8-epi PGF2 alpha differently accumulates in semilunar valves of patients suffering from coronary heart disease (CHD, n = 19) as compared to valves from healthy heart donors (controls, n = 6). Sections from isolated aortic and pulmonary valves were analyzed by semiquantitative immunohistochemistry. The 8-epi-PGF2 alpha-content was determined by using a specific radioimmunoassay. The accumulation of 8-epi PGF2 alpha in both valves was higher in CHD-patients in comparison to controls (Aortic valves: 36.49 +/- 11.26% vs. 15.78 +/- 3.04%; pulmonary valves: 46.79 +/- 9.80% vs. 14.99 +/- 3.57%). The results from the radioimmunoassay revealed comparable findings in both groups (CHD vs. controls: 395.95 +/- 86.09 vs. 139.50 +/- 47.46 pg/mg protein in the aortic valves and 430.47 +/- 76.30 vs. 147.33 +/- 53.84 pg/mg protein in pulmonary valves). Pulmonary valves seem to be more susceptible to oxidative stress than aortic valves as evidenced by a higher accumulation of 8-epi-PGF2 alpha in CHD patients. Considering the data presented in this study, we suggest that 8-epi-PGF2 alpha is a valuable indicator of oxidative injury in human semilunar valves. PMID- 12371133 TI - Effects of phthalate esters on actin cytoskeleton of Py1a rat osteoblasts. AB - We evaluated, by confocal laser scanning microscopy, the actin cytoskeleton of immortalized rat Py1a osteoblasts treated with phthalate esters (butyl benzyl phthalate, BBP and dibutyl phthalate, DBP), endocrine disruptors with estrogenic activity. We observed some peculiar modifications of actin cytoskeleton and cells changing from a spindle shape to a rounded form. In particular, F-actin formed thick bundles around the cell membrane but only a weak labeling was observed in rounded cells. Also influence on apoptosis and short-term effects on FGF-2 were studied. It was found that BBP and DBP exert their action in a similar way, act in a transient manner and do not induce apoptosis. PMID- 12371134 TI - Microscopic changes induced by the intratracheal inoculation of amniotic fluid and meconium in the lung of neonatal rats. AB - Meconium aspiration syndrome is a major contributor to neonatal respiratory distress in infants and it has been sporadically recognized in neonatal animals. This investigation was designed to study the short and long term effects of meconium and amniotic fluid in the lungs of neonatal rats. Seven-day-old rats (n = 123) divided in three groups were intratracheally inoculated with saline solution, amniotic fluid or meconium. Rats were euthanatized on 1, 3, 7, 14, 28, 56 and 112 postinoculation days (PID) and the lungs were examined by light microscopy. Saline solution did not induce any change while amniotic fluid elicited only a mild foreign body response which disappeared by PID 14. In contrast, meconium induced an exudative alveolitis characterized by recruitment of neutrophilsn in the bronchoalveolar spaces. Meconium also induced atelectasis, hyperinflation and thickening of alveolar septa all of which had disappeared by PID 14. Starting at PID 7, neutrophils were progressively replaced by macrophages, giant cells, and some fibroblasts. There were sporadic foci of mineralization starting at PID 14 and lasting up to PID 112. Some mineralized foci became lined with cuboidal epithelial cells at PID 28. Meconium was slowly degraded but still evident by PID 112. It was concluded that inoculation of meconium in neonatal rats induces acute microscopic changes typical of meconium aspiration syndrome. The long term lesions induced by meconium consisted of persistent multifocal histiocytic alveolitis and bronchiolitis reaction with occasional foci of calcification. PMID- 12371135 TI - Immunohistochemical evidence of lactoferrin in hepatic biopsies of patients with viral or cryptogenetic chronic liver disease. AB - Lactoferrin (Lf) expression has been immunohistochemically investigated in 117 formalin-fixed paraffin-embedded liver bioptic samples obtained from an equal number of patients affected by chronic hepatitis (HCV = 76; HBV = 17; HBV + HDV = 14; cryptogenetic = 10); in addition, 10 autoptic specimens of normal liver were studied as control. The Lf immunoreactivity was evaluated by an intensity distribution (ID) score. The Lf immunoexpression was observed in 88 out of 117 (75%) cases of chronic hepatitis; interestingly, all liver specimens from HBV hepatitis showed a constant Lf reactivity with the highest ID-score, whereas the evidence of Lf was encountered in 54/76 (71.1%) HCV as well as in 11/14 (78.6%) HDV chronic hepatitis, thus documenting a variable degree of Lf immunostaining in relation to different viruses. Moreover, in 6/10 (60%) cases of cryptogenetic hepatitis Lf immunoexpression was documented, whereas all normal liver controls were unreactive. In HCV specimens, the Lf nuclear immunoreactivity appeared to increase with the progression of the disease, with a greater expression in genotype 1. In contrast, no relationship among Lf ID-scores and different stages or grades of HBV, HDV or cryptogenetic hepatitis was encountered. This fact may suggest a role for Lf as an unspecific defensive agent in chronic inflammatory liver diseases, similarly to that elsewhere reported in other inflammatory tissue injuries. PMID- 12371136 TI - Protective role of the complement regulatory protein human CD-55 in cardiac xenograft: a descriptive study and a revision of the literature. AB - The limited and inadequate availability of organs from human donors has resulted in the utilisation of xenografts as an alternative tool. Nevertheless, hyperacute rejection (HAR) following xenograft determines the loss of the transplanted organ. The "primum movens" is the activation of the complement pathway mediated by the binding of natural xenogenic antibodies to the endothelium of the graft, followed by the lysis of the endothelial cells with subsequent oedema, thrombosis and necrosis of the transplanted organ. In this work we describe morphological and biomolecular observations of isolated human-decay accelerating factor (h-DAF, CD55) transgenic pig hearts, after perfusion for four hours with human blood. H DAF is a membrane glycoprotein inhibiting the complement activation in humans. We describe considerably reduced damages in transgenic hearts, compared to controls. The cardiac function resulted preserved. Our data are in agreement with what was already shown by other groups using different experimental models. In conclusion, we encourage the use of new sources of transgenic animals, pointing out the importance of morphological analysis in evaluation of xenograft. PMID- 12371137 TI - Aspergillus fumigatus causes in vitro electrophysiological and morphological modifications in human nasal epithelial cells. AB - The role of the airway epithelium in the development of invasive aspergillosis in immunocompromised hosts has rarely been studied although patients at risk for this infection frequently have epithelial damage. We developed an in vitro model of primary culture of human nasal epithelial cells (HNEC) in air-liquid interface, which allows epithelial cell differentiation and mimics in vivo airway epithelium. We subsequently tested 7-day and 24-hour Aspergillus fumigatus filtrates on the apical side of HNEC to know whether A. fumigatus, the main species responsible for invasive aspergillosis, produces specific damage to the epithelial cells. The results were compared with those obtained with non pathogenic filamentous fungi. Seven-day culture filtrates of A. fumigatus and Penicillium chrysogenum induced electrophysiological modifications whatever the fungus tested. In contrast, only 24-hour A. fumigatus filtrates induced a specific decrease in transepithelial resistance, hyperpolarization of the epithelium, and cytoplasmic vacuolization of HNEC compared with both A. niger and Penicillium chrysogenum. The inhibition of the A. fumigatus effects with amiloride suggests that the 24-hour fungal filtrate acts through sodium channels of HNEC. These early modifications of the epithelial cells could facilitate colonization of the airways by A. fumigatus. To know whether the molecules involved are specific to A. fumigatus or simply produced more rapidly than by other filamentous fungi warrants further investigation. In this perspective, the primary culture of HNEC represents a suitable model to study the interactions between airway epithelial cells and A. fumigatus. PMID- 12371138 TI - Bone remodelling and tumour grade modifications induced by interactions between bone and swarm rat chondrosarcoma. AB - Chondrosarcoma is currently defined as a malignant cartilage tumour arising de novo or within a pre-existing benign cartilage tumour. Chondrosarcoma can be surgically resected, but all grades have significant rates of local recurrence. The purpose of the present study was to develop an animal intraosseous chondrosarcoma model simulating the progression of human chondrosarcoma and elucidating its behaviour and biology. An intraosseous Swarm rat model was designed to assess interactions between bone and chondrosarcoma. A comparison of tumour grading was carried out according to transplantation site. The effects of chondrosarcoma cells (SRC cells) on the mineralisation capacities of osteoblasts and on osteoclast differentiation were studied in relation to modifications observed in vivo at the cellular level. Transplantation of Swarm rat chondrosarcoma within bone marrow or contiguous to induced periosteal lesions led to extensive bone remodelling with trabecular bone rarefaction and periosteal apposition. Transplantation in close contact to bone but without any periosteal lesion had no effect on bone, suggesting that bone healing factors interact with tumour development. With the intramedullary model, the development of tumours of different grade confirms that bone environment is an important factor in malignancy. A decrease of bone nodule formation was noted after cocultures of SRC cells with rat bone marrow, but there was no modification of osteoclast differentiation after cultures of total rabbit bone cells with SRC cells. These data reveal the importance of interactions between bone environment and tumour in inducing bone remodelling and variations in tumour malignancy. PMID- 12371139 TI - Androgen receptor mRNA under-expression in poorly differentiated human hepatocellular carcinoma. AB - Many studies suggest that hepatocellular carcinoma (HCC) is an androgen-dependent tumor with an incidence five times higher in males, but few data are available on the androgen receptor (AR) mRNA levels in different physiological classes of human liver specimens. In this study 108 human hepatic samples have been analyzed for AR mRNA expression by a comparative RT-PCR assay. These consisted of 35 non tumoral hepatic samples (3 normal parenchymas, 4 steatosis, 10 hepatitis, 18 cirrhosis), 38 tumoral specimens derived from uninodular and multinodular HCCs and 35 peritumoral hepatic tissues. Normalized AR mRNA levels in tumoral and peritumoral liver tissues spanned from 0 to 146% and from 7 to 125% respectively. Only in a relatively small percentage of HCCs, the levels of expression of AR mRNA were higher than in the corresponding peritumoral tissues (16% of total HCCs). Although extremely variable, the AR mRNA levels were related to histological tumoral differentiation and proved to be lower in the highly dedifferentiated HCCs as compared to the well differentiated ones. Therefore, the evaluation of AR expression in HCC patients might be relevant for the planning of clinical studies on anti-androgen therapies, which might be useful only in the cases in which a high level of AR mRNA is detected, considering the high heterogeneity of AR mRNA levels which characterizes HCC samples. It is likely that the HCCs, expressing low or undetectable levels of AR mRNA, would not benefit by the anti-androgen therapy. PMID- 12371140 TI - Modulation of pulmonary neuroendocrine cells in idiopathic interstitial pneumonia. AB - In order to reveal modulation of the number of pulmonary neuroendocrine cells (PNEC) in interstitial lung diseases and to clarify significance of cell proliferation activity in occurrence of PNEC, we counted airway PNEC of the patients of idiopathic interstitial pneumonia, secondary interstitial pneumonia and control lungs, and compared the number of PNEC with airway Ki-67 labeling. The lung tissue samples were obtained by video-assisted thoracoscopic surgery from 22 patients with usual interstitial pneumonia (UIP), 7 with non-specific interstitial pneumonia (NSIP), 8 with chronic hypersensitivity pneumonia (CHP), 13 with collagen vascular disease (CVD), and were compared with age-matched control lungs. The tissues were immunostained for chromogranin A and for Ki-67. Average incidence of bronchiolar PNEC in normal, UIP, NSIP, CHP, CVD lungs was 0.169%, 0.348%, 0.326%, 0.175% and 0.201%, respectively, and average Ki-67 labeling index in them was 0.241%, 1.186%, 1.605%, 1.058%, and 2.353%, respectively. And, in UIP lungs, PNEC incidence or Ki-67 labeling index was different according to pathological lesions. Thus, PNEC increase in the bronchiole of UIP, and the incidence of PNEC varies according to degree of activity of epithelial cell proliferation probably related to epithelial cell injury. Moreover, enhanced expression of human homolog of achaete-scute complex (hASH1) mRNA in UIP lungs suggests that hASH1 could play roles in the regulation of PNEC. PMID- 12371141 TI - Effect of low or high dietary calcium on the morphology of the rat femur. AB - The present study compared the effect of a calcium deficit or surfeit on femurs. Young female rats were fed with the normal (1.18%), low (0.05%), or high (2.00%) calcium diet for 3, 7, 15 or 30 days. Two groups received the low calcium diet for the first 15 days and then were followed by the normal (L-N) or high calcium diets (L-H) for the sequential 15 days. The morphology of the femur was studied together with serum calcium, parathyroid hormone (PTH), calcitonin and bone mineral density (BMD). We did not find any significant changes in the serum PTH level and bone morphology in the high calcium group. In the low calcium group, the serum PTH level increased, BMD of the whole body, the femoral weight and the femoral trabecular bone decreased as compared with the normal calcium group. There was a greater proportion of resorbing surface, less resting surface and larger vascular canal openings in the femoral endosteal surfaces in the low calcium group. In the L-N or L-H group, the femoral trabecular bone increased and the femoral resorbing surface decreased as compared with those of the low calcium group. These findings suggest that high calcium intakes do not affect the bone mass, and low calcium intakes have a deleterious effect on bone status, which may be related to vascular alternations of the bone. Reversing the low income calcium intake by a higher calcium diet can partially improve the bone alternations induced by low calcium intake. PMID- 12371142 TI - Molecular mechanisms in the pathogenesis of traumatic brain injury. AB - Traumatic brain injury (TBI) is a serious neurodisorder commonly caused by car accidents, sports related events or violence. Preventive measures are highly recommended to reduce the risk and number of TBI cases. The primary injury to the brain initiates a secondary injury process that spreads via multiple molecular mechanisms in the pathogenesis of TBI. The events leading to both neurodegeneration and functional recovery after TBI are generalized into four categories: (i) primary injury that disrupts brain tissues; (ii) secondary injury that causes pathophysiology in the brain; (iii) inflammatory response that adds to neurodegeneration; and (iv) repair-regeneration that may contribute to neuronal repair and regeneration to some extent following TBI. Destructive multiple mediators of the secondary injury process ultimately dominate over a few intrinsic protective measures, leading to activation of cysteine proteases such as calpain and caspase-3 that cleave key cellular substrates and cause cell death. Experimental studies in rodent models of TBI suggest that treatment with calpain inhibitors (e.g., AK295, SJA6017) and neurotrophic factors (e.g., NGF, BDNF) can prevent neuronal death and dysfunction in TBI. Currently, there is still no precise therapeutic strategy for the prevention of pathogenesis and neurodegeneration following TBI in humans. The search continues to explore new therapeutic targets and development of promising drugs for the treatment of TBI. PMID- 12371143 TI - The possible role of the gut neuroendocrine system in diabetes gastroenteropathy. AB - Gastrointestinal symptoms such as nausea and vomiting, heartburn, abdominal pain, diarrhoea, constipation and faecal incontinence are common in patients with diabetes. Diabetes gastroenteropathy is a clinically relevant problem. In addition to the increased morbidity it causes, it results in severely impaired metabolic control, which in turn increases the risk of hyper-/hypoglycaemia. Moreover, the poorly controlled blood glucose level increases the risk of secondary diabetes complications, namely, retinopathy, nephropathy, neuropathy and cardiovascular affection. Gastrointestinal symptoms may also cause malnutrition in patients with diabetes, which, together with the disturbed immune defence in diabetes, may cause intercurrent infections. Gastrointestinal symptoms in patients with diabetes are attributed to disturbed gastrointestinal motility. Gastrointestinal dysmotility in diabetes is believed to be caused by autonomic neuropathy and/or hyperglycaemia. The neuroendocrine system of the gut secretes peptides/amines that play an important role in regulating gastrointestinal motility. It is conceivable, therefore, to assume that a disturbance in this regulatory system may contribute to the pathogenesis of gastrointestinal complications in diabetes. The present review gives an updated overview of the abnormalities in the gastrointestinal neuroendocrine system in diabetes, speculates upon the possible role of these abnormalities in the pathogenesis of diabetes gastroenteropathy and, finally, predicts the possible clinical implications of these findings. PMID- 12371144 TI - Focal adhesion kinase: protein interactions and cellular functions. AB - Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review. PMID- 12371145 TI - Mortalin: a potential candidate for biotechnology and biomedicine. AB - Mortalin is a novel member of the hsp70 family of proteins that exhibits a different staining pattern in normal and immortal cells. It was also cloned as glucose regulated protein, GRP75 and peptidebinding protein, PBP74. It has been assigned multiple functions ranging from stress response, intracellular trafficking, antigen processing, control of cell proliferation, differentiation and tumorigenesis. The present article compiles and reviews information on multiple sites and functions of mortalin. In view of its upregulation in many tumors and transcriptional inactivation function of p53, its potential use in biotechnology and biomedicine is discussed. PMID- 12371146 TI - Desmosomes and disease: an update. AB - Desmosomes play a critical role in the maintenance of normal tissue architecture. Skin blistering can occur when desmosomal adhesion is compromised by antibodies in autoimmune diseases such as pemphigus. Inherited mutations in genes encoding desmosomal constituents can adversely affect the skin, and result in heart abnormalities. Desmosomes may have a tumour suppressor function: expression of desmosomal components is reduced in some human cancers, and desmosomal cadherins have the capacity to suppress the invasiveness of cells in culture. Transgenic animal research has provided important insights into the role of these junctions in normal epithelial morphogenesis and disease. PMID- 12371147 TI - The controversial nuclear matrix: a balanced point of view. AB - The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus. PMID- 12371148 TI - Tumour morphology--interplay between chromosome aberrations and founder cell differentiation. AB - Studies of haematological neoplasms have shown that alterations in structure and/or expression of transcription factor genes may play a crucial role for transforming stem cells or progenitor cells into malignant cells. These mutations typically arise through balanced translocations and appear to induce a block in cellular differentiation. The impact of the transforming mutation is highly dependent on the lineage of the founder cell and each specific translocation is limited to one or a few morphological subtypes. Originating from immature cells, these neoplasms have a high self-replicative capacity and are already before transformation protected from senescence by constitutive telomerase expression. Most solid tumours, on the other hand, probably originate from cells at higher levels of differentiation and require multiple mutations in oncogenes and tumour suppressor genes for neoplastic transformation. Absence of telomerase activity in the tumour-founding cell line predisposes to abnormal shortening of telomeric repeats in these cells during early clonal expansion. In turn, this triggers chromosomal breakage-fusion-bridge events through which the tumour genome is constantly reorganised, resulting in a complex and heterogeneous pattern of chromosome aberrations in the tumour cell population; the abnormal mitotic processes also give rise to cellular pleomorphism and nuclear atypia. Tumour morphology thus appears to be determined not only by the lineage of the transformed cell but also by its propensity for chromosomal instability. PMID- 12371149 TI - CD26: an expanding role in immune regulation and cancer. AB - In this review, we highlight major aspects of the biology of CD26, a dipeptidyl peptidase IV (DPPIV)-containing surface glycoprotein with multiple functions. In particular, we discuss findings demonstrating that CD26/DPPIV has an essential role in immune regulation as a T cell activation molecule and a regulator of chemokine function. We also review recent studies that identify key cellular molecules that physically associate with CD26 and the potential consequences of their interaction, including those with clinically-related implications. Furthermore, we present work suggesting a role for CD26 in the pathogenesis and behavior of selected human cancers, both solid tumors and hematological malignancies. We present recent studies that investigate the potential role of CD26 as a molecular target for novel treatment modalities for T cell lymphoid malignancies and possibly other hematological malignancies, with work involving the use of anti-CD26 monoclonal antibody, CD26-transfected cells as well as soluble CD26 molecules. PMID- 12371150 TI - Control of the cell cycle by neurotrophins: lessons from the p75 neurotrophin receptor. AB - Although traditionally little attention has been paid to the interplay between neurotrophins and the cell cycle, a number of recent findings suggest an important role for these growth factors in the regulation of this aspect of the cellular physiology. In this article, we review the evidence from a number of studies that neurotrophins can influence cell cycle progression or mitotic cycle arrest both in the nervous system as well as in other cell types. The contrary response of different cells to neurotrophins in terms of cell cycle regulation derives in part from the fact that these factors use two different receptor types to transmit their signals: members of the Trk family and the p75 neurotrophin receptor (p75NTR). With this in mind, we outline the current state of our knowledge regarding the molecular basis underlying the control of cell cycle progression by neurotrophins. We focus our interest on the receptors that transduce these signals and, in particular, the striking finding that p75NTR interacts with proteins that can promote mitotic cycle arrest. Finally, we discuss the mechanisms of cell death mediated by p75NTR in the context of cell cycle regulation. PMID- 12371151 TI - Glucose transport and metabolism in chondrocytes: a key to understanding chondrogenesis, skeletal development and cartilage degradation in osteoarthritis. AB - Despite the recognition that degenerative cartilage disorders like osteoarthritis (OA) and osteochondritis dissecans (OCD) may have nutritional abnormalities at the root of their pathogenesis, balanced dietary supplementation programs have played a secondary role in their management. This review emphasizes the importance and role of nutritional factors such as glucose and glucose-derived sugars (i.e. glucosamine sulfate and vitamin C) in the development, maintenance, repair, and remodeling of cartilage. Chondrocytes, the cells of cartilage, consume glucose as a primary substrate for ATP production in glycolysis and utilize glucosamine sulfate and other sulfated sugars as structural components for extracellular matrix synthesis and are dependent on hexose uptake and delivery to metabolic and biosynthetic pools. Data from several laboratories suggests that chondrocytes express multiple isoforms of the GLUT/SLC2A family of glucose/polyol transporters. These facilitative glucose transporter proteins are expressed in a tissue and cell-specific manner, exhibit distinct kinetic properties, and are developmentally regulated. They may also be regulated by endocrine factors like insulin and insulin-like growth factor I (IGF-I) and cytokines such as interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha). Recent studies suggest that degeneration of cartilage may be triggered by metabolic disorders of glucose balance and that OA occurs coincident with metabolic disease, endocrine dysfunction and diabetes mellitus. Based on these metabolic, endocrine and developmental considerations we present a novel hypothesis regarding the role of glucose transport and metabolism in cartilage physiology and pathophysiology and speculate that supplementation with sugar derived vitamins and nutraceuticals may benefit patients with degenerative joint disorders. PMID- 12371152 TI - The role of CD44 in the development and prognosis of head and neck squamous cell carcinomas. AB - CD44, the product of a single gene, exists as several isoforms generated by alternative exon splicing and posttranslational modifications, and is widely distributed in different cells and tissues including those of squamocellular origin. CD44 is a cell surface glycoprotein involved in many cellular processes acting as a receptor for cell to cell or cell to matrix adhesion, as a signal transmitter and as a growth factor-presenting molecule. Numerous studies based on immunohistochemical analyses of paraffin-embedded or frozen tissue sections using different monoclonal antibodies to CD44 isoforms and molecular biological techniques have provided evidence that in many types of tumours there is overexpression of CD44 isoforms and aberrant processing of immature CD44 transcripts relative to non-neoplastic control tissues, suggesting a role of CD44 in tumour development and progression. In contrast to these malignancies, one or more of the CD44 splice-variant isoforms are down-regulated in squamous cell carcinomas of the head and neck. CD44-deficient mice develop normally without giving rise to spontaneous tumours, but CD44-negative cells appear to be more susceptible to oncogenic transformation. Reduction in the expression of CD44 may confer growth advantage and malignant properties to tumour cells. The clinical significance of CD44 in squamous cell carcinomas of the head and neck as a tumour marker for cancer diagnosis and prognosis is discussed. PMID- 12371153 TI - On the mechanism of homocysteine pathophysiology and pathogenesis: a unifying hypothesis. AB - Studies have shown that hyperhomocysteinemia is an important and independent risk factor for a variety of human cardiovascular diseases. In this paper, a unifying hypothesis is proposed which suggests that hyperhomocysteinemia may exert its pathogenic effects largely through metabolic accumulation of S-adenosyl-L homocysteine, a strong noncompetitive inhibitor of the catechol-O methyltransferase (COMT)-mediated methylation metabolism of various catechol substrates (such as catecholamines and catechol estrogens). In the case of endogenous catecholamines in peripheral tissues, inhibition of their methylation by S-adenosyl-L-homocysteine will result in elevation of blood or tissue levels of catecholamines, and consequently, over-stimulation of the cardiovascular system's functions. Moreover, because the vasculature is constantly exposed to high levels of endogenous catecholamines (due to high levels of circulating neurohormone epinephrine plus rich innervation with sympathetic nerve terminals), vascular endothelial cells would incur chronic cumulative damage caused by the large amounts of the oxidative products (catechol quinones/semiquinones and oxyradicals) generated from endogenous catecholamines. This mechanistic explanation for the vascular toxicity of hyperhomocysteinemia is supported by many experimental findings, and it also fully agrees with the known protective effects of folate, vitamins B6 and B12 in hyperhomocysteinemic patients. In addition, based on the predictable effects of hyperhomocysteinemia on the methylation of catecholamines in the central nervous system as well as on the methylation of catechol estrogens in estrogen target organs, it is also suggested that hyperhomocysteinemia is an important risk factor for the development of neurodegerative disorders (Parkinson's and Alzheimer's diseases) and estrogen induced hormonal cancers. More studies are warranted to test these intriguing ideas. PMID- 12371154 TI - Animal in vivo models of EBV-associated lymphoproliferative diseases: special references to rabbit models. AB - Animal models of human EBV-associated diseases are essential to elucidate the pathogenesis of EBV-associated diseases. Here we review those previous models using EBV or EBV-like herpesviruses and describe the details on our two newly developed rabbit models of lymphoproliferative diseases (LPD) induced by simian EBV-like viruses. The first is Cynomolgus-EBV-induced T-cell lymphomas in rabbits inoculated intravenously (77-90%) and orally (82-89%) during 2-5 months. EBV-DNA was detected in peripheral blood by PCR from 2 days after oral inoculation, while anti-EBV-VCA IgG was raised 3 weeks later. Rabbit lymphomas and their cell lines contained EBV-DNA and expressed EBV-encoded RNA-1 (EBER-1). Rabbit lymphoma cell lines, most of which have specific chromosomal abnormality, showed tumorigenicity in nude mice. The second is the first animal model for EBV-infected T-cell LPD with virus-associated hemophagocytic syndrome (VAHS), using rabbits infected with an EBV-like herpesvirus, Herpesvirus papio (HVP). Rabbits inoculated intravenously with HVP-producing cells showed increased anti-EBV-VCA-IgG titers, and most (85%) subsequently died of fatal LPD and VAHS, with bleeding and hepatosplenomegaly, during 22-105 days. Peroral spray of cell-free HVP induced viral infection with seroconversion in 3 out of 5 rabbits, with 2 of the 3 infected rabbits dying of LPD with VAHS. Atypical T lymphocytes containing HVP DNA and expressing EBER-1 were observed in many organs. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. These rabbit models are also useful and inexpensive alternative experimental model systems for studying the biology and pathogenesis of EBV, and prophylactic and therapeutic regimens. PMID- 12371155 TI - Protein kinase C isoforms and lipid second messengers: a critical nuclear partnership? AB - A growing body of evidence, accumulated over the past 15 years, has highlighted that the protein kinase C family of isozymes is capable of translocating to the nucleus or is resident within the nucleus. The comprehension of protein kinase C isoform regulation within this organelle is under development. At present, it is emerging that lipid second messengers may play at least two roles in the control of nuclear protein kinase C: on one side they serve as chemical attractants, on the other they directly modulate the activity of specific isoforms. One of the best characterized lipid second messenger that could be involved in the regulation of nuclear PKC activity is DAG. The existence of two separate pools of nuclear DAG suggests that this lipid second messenger might be involved in distinct pathways that lead to different cell responses. Nuclear phosphatidylglycerol, D-3 phosphorylated inositol lipids and nuclear fatty acids are involved in a striking variety of critical biological functions which may act by specific PKC activation. The fine tuning of PKC regulation in cells subjected to proliferating or differentiating stimuli, might prove to be of great interest also for cancer therapy, given the fact that PKC-dependent signaling pathways are increasingly being seen as possible pharmacological target in some forms of neoplastic diseases. In this article, we review the current knowledge about lipid second messengers that are involved in regulating the translocation and/or the activity of different protein kinase C isoforms identified at the nuclear level. PMID- 12371156 TI - Regulation of smooth muscle cell accumulation in diabetes-accelerated atherosclerosis. AB - Diabetes leads to accelerated formation/progression of lesions of atherosclerosis. Cardiovascular disease thus develops earlier in people with type 1 or type 2 diabetes compared to people without diabetes, and cardiovascular (macrovascular) disease is the major cause of death in adults with diabetes. The molecular and cellular mechanisms leading to diabetes-accelerated atherosclerosis are not well understood. The arterial smooth muscle cell (SMC), one of the three or four principal cell types in atherosclerosis, has been extensively studied over the years. Proliferation and accumulation of SMCs are believed to play important roles in the progression of macrophage-rich lesions to fibroatheromas. Further progression of these atheromas into complicated vulnerable lesions that are likely to cause the acute clinical symptoms of atherosclerosis (myocardial infarction and stroke) may involve cell death and loss of SMCs from the fibrous cap of the lesion. Recent animal studies have shown that diabetes causes a marked increase in SMC accumulation and proliferation in atheromas. Hyperglycemia, advanced glycation end-products, insulin and lipid abnormalities associated with the diabetic environment have been suggested to increase SMC accumulation. Indeed, it is becoming increasingly clear that macrovascular disease associated with diabetes is a multifactorial disease. We review the factors and mechanisms that may regulate SMC proliferation and accumulation in different stages of lesion progression in diabetes. We propose that lipid abnormalities associated with diabetes can act in combination with growth factors present in the diabetic environment to increase SMC accumulation and accelerate lesion progression. PMID- 12371157 TI - Review of collecting duct carcinoma with focus on clinical and pathobiological aspects. AB - In recent years, the concept of collecting duct carcinoma (CDC) has been established. CDCs constitute about 0.4 to 2% of RCCs. Macroscopically, CDCs occur in the renal medulla. On the cut surface, they are generally firm, white or grey and poorly circumscribed. Histologically, CDCs are characterized by various cytological and histological appearances. Furthermore, desmoplastic stromal reaction around the tumor and atypical hyperplastic changes or carcinoma in situ in the adjacent medullary collecting duct are frequently observed. Histological distinction from papillary RCCs is most important, because both tumors share some structural and histochemical features, and it seems that some investigators have confused diagnostic criteria for CDCs. On the other hand, the concept of medullary carcinoma, which preferentially occurs in a black race and shows histological features similar to those of CDC, has also recently been established. Although there have been few studies on chromosomal abnormalities of CDCs and consistent abnormalities have not been identified, a recent study using microsatellite analysis has shown a high frequency (60%) of LOH in 1q32.1-32.2. Further studies are needed to elucidate the genetic characteristics of CDCs and to determine the relationship or difference between CDCs and medullary carcinomas. PMID- 12371158 TI - Optical properties of intact leaves for estimating chlorophyll concentration. AB - Changes in leaf chlorophyll content can serve as relative indicators of plant vigor and environmental quality. This study identified reflectance, transmittance, and absorptance wavebands and band ratios within the 400- to 850 nm range for intact leaves that could be used to estimate extracted leaf chlorophyll per unit leaf area (areal concentration) with minimal error. Leaf optical properties along with chlorophyll a, b, and a + b concentrations were measured for the planar-leaved sweetgum (Liquidambar styraciflua L.), red maple (Acer rubrum L.), wild grape (Vitis rotundifolia Michx.), and switchcane [Arundinaria gigantea (Walter) Muhl.], and for needles of longleaf pine (Pinus palustris Miller). Generally, reflectance, transmittance, and absorptance corresponded most precisely with chlorophyll concentrations at wavelengths near 700 nm, although regressions were also strong in the 550- to 625-nm range. A power function was superior to a simple linear function in yielding low standard deviations of the estimate (s). When data were combined among the planar-leaved species, s values were low at approximately 50 mumol/m2 out of a 940 mumol/m2 range in chlorophyll a + b at best-fit wavelengths of 707 to 709 nm. Minimal s values for chlorophyll a + b ranged from 32 to 62 mumol/m2 across species when band ratios having numerator wavelengths of 693 to 720 nm were used with the application of a power function. Optimal denominator wavelengths for the band ratios were 850 nm for reflectance and transmittance and 400 nm for absorptance. This information can be applied in designing field portable chlorophyll meters and in the landscape-scale remote sensing of plant responses to the environment. PMID- 12371159 TI - Vegetation stress detection through chlorophyll a + b estimation and fluorescence effects on hyperspectral imagery. AB - Physical principles applied to remote sensing data are key to successfully quantifying vegetation physiological condition from the study of the light interaction with the canopy under observation. We used the fluorescence reflectance-transmittance (FRT) and PROSPECT leaf models to simulate reflectance as a function of leaf biochemical and fluorescence variables. A series of laboratory measurements of spectral reflectance at leaf and canopy levels and a modeling study were conducted, demonstrating that effects of chlorophyll fluorescence (CF) can be detected by remote sensing. The coupled FRT and PROSPECT model enabled CF and chlorophyll a + b (Ca + b) content to be estimated by inversion. Laboratory measurements of leaf reflectance (r) and transmittance (t) from leaves with constant Ca + b allowed the study of CF effects on specific fluorescence-sensitive indices calculated in the Photosystem I (PS-I) and Photosystem II (PS-II) optical region, such as the curvature index [CUR; (R675.R690)/R2(683)]. Dark-adapted and steady-state fluorescence measurements, such as the ratio of variable to maximal fluorescence (Fv/Fm), steady state maximal fluorescence (F'm), steady state fluorescence (Ft), and the effective quantum yield (delta F/F'm) are accurately estimated by inverting the FRT PROSPECT model. A double peak in the derivative reflectance (DR) was related to increased CF and Ca + b concentration. These results were consistent with imagery collected with a compact airborne spectrographic imager (CASI) sensor from sites of sugar maple (Acer saccharum Marshall) of high and low stress conditions, showing a double peak on canopy derivative reflectance in the red-edge spectral region. We developed a derivative chlorophyll index (DCI; calculated as D705/D722), a function of the combined effects of CF and Ca + b content, and used it to detect vegetation stress. PMID- 12371160 TI - Narrow-waveband reflectance ratios for remote estimation of nitrogen status in cotton. AB - Tailoring nitrogen (N) fertilizer applications to cotton (Gossypium hirsutum L.) in response to leaf N status may optimize N use efficiency and reduce off-site effects of excessive fertilizer use. This study compared leaf and canopy reflectance within the 350 to 950 nm range in order to identify reflectance ratios sensitive to leaf chlorophyll (Chl), and hence N status, in cotton. Plants were grown outdoors in large pots using half-strength Hoagland's (control) solution until some three-row plots received a restricted supply of N. Treatments comprised control, 20% of control N at first flower bud (square) onward; 0 and 20% of control N at first flower onward; and 0% of control N at fruit-filling onward. Despite leaf N values ranging from 51 to 19 g kg-1 across treatments and sampling dates, a weak correlation was obtained between Chl and N (r2 = 0.32, df = 70). In general, N stress led to increased reflectance at 695 +/- 2.5 nm (R695) and decreased reflectance at R410, and changes in leaf N were best correlated with either R695 or R755 in leaves and either R410 or R700 in canopies. The strongest associations between leaf constituent and canopy reflectance ratio were Chl vs. R415/R695 (r2 = 0.72), carotenoids vs. R415/R685 (r2 = 0.79), and N vs. R415/R710 (r2 = 0.70). The R415 measure appears to be a more stable spectral feature under N stress, as compared with more pronounced changes along the reflectance red edge (690-730 nm). Multiple regression identified a three waveband canopy reflectance model that explained 80% of the variability in leaf N. Results indicate that remote sensing of N status in cotton is feasible using narrow-waveband reflectance ratios that involve the violet or blue region of the spectrum (400 to 450 nm) and the more commonly featured red-edge region. PMID- 12371161 TI - Spectral properties of salt crusts formed on saline soils. AB - Rapid identification and large-scale mapping of salt-affected lands will help improve salinity management in watersheds and ecosystems. This study was conducted to examine spectral reflectance of soils treated with saline solutions containing NaCl, NaHCO3, Na2SO4, and CaSO4.2H2O. Spectral reflectance was measured upon salt crusts formed on two soils (Torrifluvents) subirrigated with saline solutions of 500, 1000, and 1500 mmolc L-1 with a spectroradiometer in the visible and near-infrared region (400-2500 nm). Spectral analyses revealed that samples of gypsum crusts have diagnostic absorption features near 1023, 1225, 1457, 1757, 1800, and 2336 nm, whereas halite crusts have diagnostic absorption features near 1442, 1851, 1958, and 2226 nm. Several broad absorption features were seen in the spectra of the crusts of sodium bicarbonate at 1243, 1498, 1790, 1988, and 2356 nm. The spectrum of soils treated with sodium sulfate exhibited absorption features at 1243, 1472, 1677, 1774, 1851, 1968, and 2245 nm. Crystal size or salt concentrations did not affect the positions of the absorption bands of the salt crusts. However, reflectance increased as particle sizes decreased or with increasing presence of salt crusts. Spectroscopy can be used under certain conditions to identify the presence of primary diagnostic spectral features of gypsum, nahcolite, thenardite, and halite crusts. PMID- 12371162 TI - Environmental mapping based on spatial variability. AB - Environmental maps show the probable environmental states of different types of land use or development of landscape in a geographic context. Remotely sensed data are particularly efficient for environmental mapping in order to outline major environmental types. Multiple schemes of image classification used in environmental mapping are either traditionally statistical or heuristic. While the former methods do not take account of spatial variability in space and aerial data, the latter ones does not lend themselves to optimal solutions we present. Novel probabilistic models of piecewise-homogeneous images are used in environmental mapping to segment real images. The models consider both an image and a land cover map. Such a pair constitutes an example of a Markov random field specified by a joint Gibbs probability distribution of images and maps. Parameters of the model are estimated by using a stochastic approximation technique. Its convergence to the desired values is studied experimentally. Addition of spatial attributes appears to be necessary in most areas where the differences in spatial data between regions in the image occur. Experiments in generating the pairs of images and environmental maps and in segmenting the simulated as well as real images are discussed. PMID- 12371163 TI - Accelerated deployment of an agricultural nutrient management tool: the Maryland Phosphorus Site Index. AB - In 1998, the Maryland legislature mandated nitrogen (N) and phosphorus (P) nutrient management planning for nearly all of Maryland's commercial agricultural operations. State regulations required that a phosphorus indexing tool (P Index) be used for determining the potential for P losses from agricultural land, even though a reliable P Index did not exist. The development and assessment of the P Index as a dependable tool for the evaluation of the potential for P losses was constrained by a very aggressive implementation schedule imposed by state regulations. The Maryland Phosphorus Site Index (PSI) was evaluated on 646 state representative field sites beginning in the spring of 1999 and continuing through the spring of 2000. Of the representative fields, 69% were determined to have a "low" P loss rating, 19% were in the "medium" P loss rating category, 8% were determined to be a "high" risk for P loss, and 4% rated as "very high" P loss potential. Fifty-five percent of the fields evaluated had soil test phosphorus (STP) levels less than the 75 mg kg-1 Mehlich-1 P environmental threshold established by state regulations. The frequency distribution of PSI performance was evaluated for several subcategories of the statewide data set. The Maryland PSI will be deployed for use in constructing farm nutrient management plans well before its predictive capabilities can be objectively and rigorously validated. Field validation is essential. In the meantime, the Maryland PSI should function adequately as a tool to assist in the prioritization of field P loss risk potential. PMID- 12371164 TI - Cottongrass effects on trace elements in submersed mine tailings. AB - Phytostabilization may limit the leakage of metals and As from submersed mine tailings, thus treatment of acid mine drainage with lime could be reduced. Tall cottongrass (Eriophorum angustifolium Honckeny) and white cottongrass (E. scheuchzeri Hoppe) were planted in pots with unlimed (pH 5.0) and limed (pH 10.9) tailings (containing sulfides) amended with sewage sludge (SS) or a bioashsewage sludge mixture (ASM). Effects of the amendments on plant growth and plant element uptake were studied. Also, effects of plant growth on elements (Cd, Cu, Pb, Zn, and As), pH, electrical conductivity (EC), and concentrations of SO4(2-), in the drainage water as well as dissolved oxygen in tailings, were measured. Both plant species grew better and the shoot element concentrations of white cottongrass were lower in SS than in ASM. Metal concentrations were lowest in drainage water from limed tailings, and plant establishment had little effect on metal release, except for an increase in Zn levels, even though SO4(2-) levels were increased. In unlimed tailings, plant growth increased SO4(2-) levels slightly; however, pH was increased and metal concentrations were low. Thus, metals were stabilized by plant uptake and high pH. Amendments or plants did not affect As levels in the drainage water from unlimed tailings. Thus, to reduce the use of lime for stabilizing metals, phytostabilization with tall cottongrass and white cottongrass on tailings is a sound possibility. PMID- 12371165 TI - Gaseous contaminant emissions as affected by burning scrap tires in cement manufacturing. AB - We studied the environmental impact (gaseous emissions) of using scrap tires as a fuel substitute at a cement plant that produces one million tons of cement per year. Using a combination of tires and coal as opposed to only coal caused variations in the pollutant emission rate. The study recorded a 37% increase in the rate of emission for CO, a 24% increase for SO2, an 11% decrease for NOx, and a 48% increase for HCl when tires were included. The rate of emission for metals increased 61% for Fe, 33% for Al, 487% for Zn, 127% for Pb, 339% for Cr, 100% for Mn, and 74% for Cu, and decreased 22% for Hg. On the other hand, the emission rate of organic compounds dropped by 14% for polycyclic aromatic hydrocarbons, 8% in naphthalene, 37% in chlorobenzene, and 45% in dioxins and furans. We used a Gaussian model of atmospheric dispersion to calculate the average pollutant concentration (1-h, 24-h, and annual concentrations) in the ambient air at ground level with the help of the ISC-ST2 software program developed by the USEPA. When tires were used, we observed (i) a 12 to 24% increase in particulate matter, this range considering the concentration variation depending on the average used (1-h, 24-h, and annual basis), 31 to 52% in CO, 22 to 34% in SO2, 39 to 52% in HCl, 12 to 27% in Fe, -3 to 8% in Al, 30 to 37% in Zn, and 270 to 885% in Pb; (ii) a decrease of 8 to 13% in NOx, 9 to 13% in polycyclic aromatic hydrocarbons, 6 to 7% in naphthalene, 32 to 39% in chlorobenzene, and 32 to 45% in dioxins and furans. The results obtained showed that the maximum ground-level concentrations were well within the environmental standards (for operation with only coal as well as for operation with a combination of coal and tires). PMID- 12371166 TI - Ammonia, methane, and nitrous oxide emission from pig slurry applied to a pasture in New Zealand. AB - Much animal manure is being applied to small land areas close to animal confinements, resulting in environmental degradation. This paper reports a study on the emissions of ammonia (NH3), methane (CH4), and nitrous oxide (N2O) from a pasture during a 90-d period after pig slurry application (60 m3 ha-1) to the soil surface. The pig slurry contained 6.1 kg total N m-3, 4.2 kg of total ammoniacal nitrogen (TAN = NH3 + NH4) m-3, and 22.1 kg C m-3, and had a pH of 8.14. Ammonia was lost at a fast rate immediately after slurry application (4.7 kg N ha-1 h-1), when the pH and TAN concentration of the surface soil were high, but the loss rate declined quickly thereafter. Total NH3 losses from the treated pasture were 57 kg N ha-1 (22.5% of the TAN applied). Methane emission was highest (39.6 g C ha-1 h-1) immediately after application, as dissolved CH4 was released from the slurry. Emissions then continued at a low rate for approximately 7 d, presumably due to metabolism of volatile fatty acids in the anaerobic slurry-treated soil. The net CH4 emission was 1052 g C ha-1 (0.08% of the carbon applied). Nitrous oxide emission was low for the first 14 d after slurry application, then showed emission peaks of 7.5 g N ha-1 h-1 on Day 25 and 15.8 g N ha-1 h-1 on Day 67, and decline depending on rainfall and nitrate (NO3) concentrations. Emission finally reached background levels after approximately 90 d. Nitrous oxide emission was 7.6 kg N ha-1 (2.1% of the N applied). It is apparent that of the two major greenhouse gases measured in this study, N2O is by far the more important tropospheric pollutant. PMID- 12371167 TI - Evaluation of leachate recirculation on nitrous oxide production in the Likang Landfill, China. AB - Landfill leachate recirculation is efficient in reducing the leachate quantity handled by a leachate treatment plant. However, after land application of leachate, nitrification and denitrification of the ammoniacal N becomes possible and the greenhouse gas nitrous oxide (N2O) is produced. Lack of information on the effects of leachate recirculation on N2O production led to a field study being conducted in the Likang Landfill (Guangzhou, China) where leachate recirculation had been practiced for 8 yr. Monthly productions and fluxes of N2O from leachate and soil were studied from June to November 2000. Environmental and chemical factors regulating N2O production were also accessed. An impermeable top liner was not used at this site; municipal solid waste was simply covered by inert soil and compacted by bulldozers. A high N2O emission rate (113 mg m-2 h-1) was detected from a leachate pond purposely formed on topsoil within the landfill boundary after leachate irrigation. A high N2O level (1.09 micrograms L-1) was detected in a gas sample emitted from topsoil 1 m from the leachate pond. Nitrous oxide production from denitrification in leachate-contaminated soil was at least 20 times higher than that from nitrification based on laboratory incubation studies. The N2O levels emitted from leachate ponds were compared with figures reported for different ecosystems and showed that the results of the present study were 68.7 to 88.6 times higher. Leachate recirculation can be a cost effective operation in reducing the volume of leachate to be treated in landfill. However, to reduce N2O flux, leachate should be applied to underground soil rather than being irrigated and allowed to flow on topsoil. PMID- 12371168 TI - Treatment of 2,4-dichlorophenol polluted soil with free and immobilized laccase. AB - Enzyme treatment is currently considered for remediation of terrestrial systems polluted with organic compounds. In this study, two soils from Pennsylvania with 2.8 or 7.4% organic matter contents (Soils 1 and 2, respectively) were amended with 14C-labeled 2,4-dichlorophenol (2,4-DCP) and incubated with a laccase from Trametes villosa (free or immobilized on montmorillonite). 2,4-DCP was either transformed to methanol-soluble polymeric products (11-32%) or covalently bound to soil organic matter (53-85%); unaltered 2,4-DCP could be recovered from soil by methanol extraction (0-38%) at the completion of a 14-d incubation period. In Soil 1, both free and immobilized laccase removed 100% of 2,4-DCP without regard for moisture conditions. In Soil 2, immobilized laccase removed more 2,4-DCP (about 95%, regardless of moisture conditions) than free enzyme (55, 75, and 90% at 30, 55, and 100% of maximum water-holding capacity, respectively). Binding of 2,4-DCP in the humin fraction was nearly the same for free and immobilized laccase. More 2,4-DCP, however, was bound to humic and fulvic acids in the presence of immobilized laccase than in the presence of free laccase. In general, immobilized laccase performed better than free laccase. However, for practical applications, the higher activity of immobilized laccase is offset by a 23% loss in enzyme activity during immobilization, which approximates the 30% increase in free laccase needed to achieve the same level of remediation. Furthermore, immobilized laccase is more costly than free T. villosa laccase. PMID- 12371169 TI - Soil and plant selenium at a reclaimed uranium mine. AB - Selenium (Se) associated with reclaimed uranium (U) mine lands may result in increased food chain transfer and water contamination. To assess post-reclamation bioavailability of Se at a U mine site in southeastern Wyoming, we studied soil Se distribution, dissolution, speciation, and sorption characteristics and plant Se accumulation. Phosphate-extractable soil Se exceeded the critical limit of 0.5 mg/kg in all the samples, whereas total soil Se ranged from a low (0.6 mg/kg) to an extremely high (26 mg/kg) value. Selenite was the dominant species in phosphate and ammonium bicarbonate-diethylenetriamine pentaacetic acid (AB-DTPA) extracts, whereas selenate was the major Se species in hot water extracts. Extractable soil Se concentrations were in the order of KH2PO4 > AB-DTPA > hot water > saturated paste. The soils were undersaturated with respect to various Se solid phases, albeit with high levels of extractable Se surpassing the critical limit. Calcium and Mg minerals were the potential primary solids controlling Se dissolution, with dissolved organic carbon in the equilibrium solutions resulting in enhanced Se availability. Adsorption was a significant (r2 = 0.76-0.99 at P < 0.05) mechanism governing Se availability and was best described by the initial mass isotherm model, which predicted a maximum reserve Se pool corresponding to 87% of the phosphate-extractable Se concentrations. Grasses, forbs, and shrubs accumulated 11 to 1800 mg Se/kg dry weight. While elevated levels of bioavailable Se may be potentially toxic, the plants accumulating high Se may be used for phytoremediation, or the palatable forage species may be used as animal feed supplements in Se-deficient areas. PMID- 12371170 TI - Reclamation of high-elevation, acidic mine waste with organic amendments and topsoil. AB - The Summitville Mine was a high-elevation (3500 m) gold mine in southwestern Colorado. The mine was abandoned in 1992, leaving approximately 200 ha of disturbance comprised partially of an open pit, a cyanide heap leach pad, and two large waste rock piles. Reclamation of these mine facilities is challenging due to extreme climatic conditions in conjunction with high acid-production potential and low organic matter content of waste materials on site. In addition, stockpiled topsoil at the site is acidic and biologically inactive due to long term storage, and may not be suitable for plant growth. The purpose of this study was to evaluate the effects of organic amendments (mushroom compost vs. biosolids) and topsoil (stockpiled vs. nonstockpiled) on aboveground biomass and plant trace element uptake. An on-site field study was established in 1995 to identify the most effective combination of treatments for successful reclamation of on-site waste rock materials. Incorporation of organic matter significantly increased aboveground biomass, with mushroom compost being more effective than biosolids, but did not significantly influence trace element uptake. Conversely, the use of topsoil did not affect aboveground biomass, but did influence trace element uptake. Treatments that received topsoil supported plant growth with significantly higher trace element tissue concentrations than treatments that did not receive topsoil. In general, it was found that waste rock could be directly revegetated when properly neutralized, fertilized, and amended with organic matter. Additionally, stockpiled topsoil, when neutralized with lime, supported plant growth equivalent to that on nonstockpiled topsoil. PMID- 12371171 TI - Application of classification-tree methods to identify nitrate sources in ground water. AB - A study was conducted to determine if nitrate sources in ground water (fertilizer on crops, fertilizer on golf courses, irrigation spray from hog (Sus scrofa) wastes, and leachate from poultry litter and septic systems) could be classified with 80% or greater success. Two statistical classification-tree models were devised from 48 water samples containing nitrate from five source categories. Model 1 was constructed by evaluating 32 variables and selecting four primary predictor variables (delta 15N, nitrate to ammonia ratio, sodium to potassium ratio, and zinc) to identify nitrate sources. A delta 15N value of nitrate plus potassium > 18.2 indicated animal sources; a value < 18.2 indicated inorganic or soil organic N. A nitrate to ammonia ratio > 575 indicated inorganic fertilizer on agricultural crops; a ratio < 575 indicated nitrate from golf courses. A sodium to potassium ratio > 3.2 indicated septic-system wastes; a ratio < 3.2 indicated spray or poultry wastes. A value for zinc > 2.8 indicated spray wastes from hog lagoons; a value < 2.8 indicated poultry wastes. Model 2 was devised by using all variables except delta 15N. This model also included four variables (sodium plus potassium, nitrate to ammonia ratio, calcium to magnesium ratio, and sodium to potassium ratio) to distinguish categories. Both models were able to distinguish all five source categories with better than 80% overall success and with 71 to 100% success in individual categories using the learning samples. Seventeen water samples that were not used in model development were tested using Model 2 for three categories, and all were correctly classified. Classification tree models show great potential in identifying sources of contamination and variables important in the source-identification process. PMID- 12371172 TI - Trace metal leaching through a soil-grassland system after sewage sludge application. AB - To determine whether sludge applications to soil would lead in the short term to toxicity to plants and trace metal leaching to ground water, we studied the fate of some trace and major elements in a brown soil-meadow system just after repeated sewage sludge applications. The main pathways were quantified over a 37 mo period with undisturbed monolith lysimeters including two controls, four lysimeters treated with 3 x 100 m3 ha-1, and four with 3 x 400 m3 ha-1 of sewage sludge. In drainage waters the effect was limited in time and, in the case of NO3 N and Cl, delayed by 1 to 4 mo and lasted several months before returning to background conditions. Nickel and Cu concentrations in solution increased also after sludge application and had not return to background conditions after 20 mo. Trace metal concentrations did not reach toxic levels in herbage and N, Cu, Cd, and Zn concentrations were correlated with the first sludge input only. Calculated over a 37-mo period, total element output was significantly increased for Ca, NO3-N, and Ni only, because of the time-dependent response to sludge application and high variability between replicates. Output was maximal for Cd, with 1.5% of total input for the 100 m3 ha-1 treatment. Particulate matter in drainage water accounted for an average of 20% of trace metal leaching. The main long-term risk was the rapid increase in trace metal concentrations in the topsoil, which may eventually lead to toxic levels in herbage. PMID- 12371173 TI - Changes in the rhizosphere of metal-accumulating plants evidenced by chemical extractants. AB - The plants Salix viminalis L. (common osier) and Thlaspi caerulescens J. Presl & C. Presl have been studied often because of their high potential to extract heavy metals from soils. The soil properties favoring this phytoextraction are not yet fully known. In this study we compared three frequently used single-extracting agents (NaNO3, diethylenetriaminepentaacetic acid [DTPA], and ethylenediaminetetra-acetic acid [EDTA]) with a sequential extraction procedure to describe changes in the different Cd, Cu, and Zn pools in the rhizosphere of S. viminalis and T. caerulescens grown on calcareous and acidic Swiss soils in a pot experiment. The sequential extraction was used to assess the chemical affinities of these heavy metals (HM) in the soil whereas the single extractants were used for estimating the bioavailable HM pools in the soils. Cadmium depletion in several pools was most apparent in the acidic soil, with a significant decrease observed in the NaNO3-, DTPA-, and EDTA-extractable fractions following T. caerulescens growth compared with control pots. The sequential extraction showed that most Cd extracted by the plant from the acidic soil originated from the organic pool, which implies that heavy metals bound to organic matter may constitute a significant part of the bioavailable Cd pool in soils. In the calcareous soil only a small amount of Cd was taken up by T. caerulescens, and this came mainly from the carbonate-bound fraction. This study shows that T. caerulescens, and to a lesser extent S. viminalis, can alter the heavy metal distribution in different soil pools within 90 d. PMID- 12371174 TI - Relationships between bacterial tolerance levels and forms of copper and zinc in soils. AB - The effects of various fractions of copper (Cu) and zinc (Zn) on soil bacteria were evaluated by the heavy metal tolerance level of the bacterial community (IC50) in soil samples collected near a mine. The IC50 values had no relationship with the total concentrations of Zn and Cu in the soils, but were weakly correlated with the 0.05 M CaCl2-extractable form of each metal in the soils (Cu: R2 = 0.670, p < 0.01; Zn: R2 = 0.453, p < 0.05). It was found that the IC50 correlated strongly with the total concentration of each metal in the extracts from water-saturated soil samples, described below as "soil solution" (Cu: R2 = 0.789, p < 0.01; Zn: R2 = 0.617, p < 0.01). The speciation of these metals in the soil solutions was estimated using an equilibrium thermodynamic computer model, SOILCHEM. Simulated free Cu ion ranged from 18 to 98% of total Cu, and organic complexes of Cu ranged from < 1 to 56%. In all samples, Zn existing as the free ion was estimated to be more than 80% of total Zn in the soil solutions. The IC50 values were also correlated with the estimated free metal ion activities, but with slightly lower correlation coefficients than found for total concentration in the soil solutions (Cu: R2 = 0.735, p < 0.01; Zn: R2 = 0.610, p < 0.01). The results suggest that not only high metal ion activities, but also total dissolved metal concentrations in soil solutions may affect the bacterial community. PMID- 12371175 TI - Kriging method evaluation for assessing the spatial distribution of urban soil lead contamination. AB - Describing contaminant spatial distribution is an integral component of risk assessment. Application of geostatistical techniques for this purpose has been demonstrated previously. These techniques may provide both an estimate of the concentration at a given unsampled location, as well as the probability that the concentration at that location will exceed a critical threshold concentration. This research is a comparative study between multiple indicator kriging and kriging with the cumulative distribution function of order statistics, with both local and global variograms. The aim was to determine which of the four methods is best able to delineate between "contaminated" and "clean" soil. The four methods were validated with a subset of data values that were not used in the prediction. Method performance was assessed by calculating the root mean square error (RMSE), analysis of variance, the proportion of sites misclassified by each method as either "clean" when they were actually "contaminated" or vice versa, and the expected loss for each misclassification type. The data used for the comparison were 807 topsoil Pb concentrations from the inner-Sydney suburbs of Glebe and Camperdown, Australia. While there was very little difference between the four methods, multiple indicator kriging was found to produce the most accurate predictions for delineating "clean" from "contaminated" soil. PMID- 12371176 TI - Relationships between stream size, suspended particles, and filter-feeding macroinvertebrates in a great plains drainage network. AB - Suspended fine particles (seston) are an important component of energy and nutrient cycling in streams, but they can also be pollutants. We examined seston dynamics and filter-feeding macroinvertebrate communities in sites representing headwaters to large rivers in the Kansas River drainage, northeastern KS. Seston samples were collected at least seasonally during low to moderate flows for one year beginning in the summer of 1999, and quality was assessed by determining organic content and C to N ratio. A rapid bioassessment approach was used to examine filter-feeders. Relationships between stream size and seston concentrations were markedly influenced by anthropogenic activities. There was no relationship between total seston concentration and stream size across all sites (r = 0.14, p > 0.05), but a significant, positive relationship was evident when impounded and suburban sites were excluded (r = 0.73, p < 0.01); this same trend was evident for organic and inorganic components. Seasonal patterns of C to N ratio were evident, with generally lower values during winter and highest values in summer. However, seasonal patterns were dampened in suburban sites and virtually absent below impoundments. Filter-feeder richness was correlated with average organic seston concentrations (r = 0.8, p < 0.01), but this relationship was also obscured by impoundments and suburban development. In particular, impoundments had a dramatic, negative effect on richness. Abundance of most hydropsychid caddisfly taxa was positively correlated with organic seston concentration. Results indicate there are significant patterns regarding seston, filter-feeders, and stream size in this Great Plains river system, but patterns are strongly influenced by human activities. These relationships are relevant to management issues regarding suspended particles and the potential development of bioassessment techniques. PMID- 12371177 TI - Soil testing to predict phosphorus leaching. AB - Subsurface pathways can play an important role in agricultural phosphorus (P) losses that can decrease surface water quality. This study evaluated agronomic and environmental soil tests for predicting P losses in water leaching from undisturbed soils. Intact soil columns were collected for five soil types that a wide range in soil test P. The columns were leached with deionized water, the leachate analyzed for dissolved reactive phosphorus (DRP), and the soils analyzed for water-soluble phosphorus (WSP), 0.01 M CaCl2 P (CaCl2-P), iron-strip phosphorus (FeO-P), and Mehlich-1 and Mehlich-3 extractable P, Al, and Fe. The Mehlich-3 P saturation ratio (M3-PSR) was calculated as the molar ratio of Mehlich-3 extractable P/[Al + Fe]. Leachate DRP was frequently above concentrations associated with eutrophication. For the relationship between DRP in leachate and all of the soil tests used, a change point was determined, below which leachate DRP increased slowly per unit increase in soil test P, and above which leachate DRP increased rapidly. Environmental soil tests (WSP, CaCl2-P, and FeO-P) were slightly better at predicting leachate DRP than agronomic soil tests (Mehlich-1 P, Mehlich-3 P, and the M3-PSR), although the M3-PSR was as good as the environmental soil tests if two outliers were omitted. Our results support the development of Mehlich-3 P and M3-PSR categories for profitable agriculture and environmental protection; however, to most accurately characterize the risk of P loss from soil to water by leaching, soil P testing must be fully integrated with other site properties and P management practices. PMID- 12371178 TI - Relating net nitrogen input in the Mississippi River basin to nitrate flux in the lower Mississippi River: a comparison of approaches. AB - A quantitative understanding of the relationship between terrestrial N inputs and riverine N flux can help guide conservation, policy, and adaptive management efforts aimed at preserving or restoring water quality. The objective of this study was to compare recently published approaches for relating terrestrial N inputs to the Mississippi River basin (MRB) with measured nitrate flux in the lower Mississippi River. Nitrogen inputs to and outputs from the MRB (1951 to 1996) were estimated from state-level annual agricultural production statistics and NOy (inorganic oxides of N) deposition estimates for 20 states that comprise 90% of the MRB. A model with water yield and gross N inputs accounted for 85% of the variation in observed annual nitrate flux in the lower Mississippi River, from 1960 to 1998, but tended to underestimate high nitrate flux and overestimate low nitrate flux. A model that used water yield and net anthropogenic nitrogen inputs (NANI) accounted for 95% of the variation in riverine N flux. The NANI approach accounted for N harvested in crops and assumed that crop harvest in excess of the nutritional needs of the humans and livestock in the basin would be exported from the basin. The U.S. White House Committee on Natural Resources and Environment (CENR) developed a more comprehensive N budget that included estimates of ammonia volatilization, denitrification, and exchanges with soil organic matter. The residual N in the CENR budget was weakly and negatively correlated with observed riverine nitrate flux. The CENR estimates of soil N mineralization and immobilization suggested that there were large (2000 kg N ha 1) net losses of soil organic N between 1951 and 1996. When the CENR N budget was modified by assuming that soil organic N levels have been relatively constant after 1950, and ammonia volatilization losses are redeposited within the basin, the trend of residual N closely matched temporal variation in NANI and was positively correlated with riverine nitrate flux in the lower Mississippi River. Based on results from applying these three modeling approaches, we conclude that although the NANI approach does not address several processes that influence the N cycle, it appears to focus on the terms that can be estimated with reasonable certainty and that are correlated with riverine N flux. PMID- 12371179 TI - Ion cycling in hemlock-northern hardwood forests of the southern Lake Superior region: a preliminary study. AB - Upland forests of the southern Lake Superior region are diverse and contain a shifting mosaic of eastern hemlock [Tsuga canadensis (L.) Carr.] and northern hardwood forests dominated by sugar maple (Acer saccharum Marsh.). In this study, we survey the relative effects of management practice (old growth vs. managed), forest cover type (hemlock vs. northern hardwood), and soil great group (Entic Haplorthod vs. Alfic Oxyaquic Fragiorthod) on ion cycling as a precursor to a longer-term, more detailed study. Bulk precipitation, throughfall, and soil leachates at three depths were collected for two growing seasons in eight stands on the Ottawa National Forest in the Upper Peninsula of Michigan. A total of 1210 solutions were analyzed for pH, Na, K, Mg, Ca, Cl, NO3, and SO4. Losses of base cations (Ca, Mg, K) and SO4 from the bottom of the rooting zone generally were greater in old-growth than in managed northern hardwoods on both fragic and nonfragic soils. Leaching losses of base cations and NO3 usually were greater beneath old-growth northern hardwoods than beneath old-growth hemlock on both soil types and for both forest cover types and management practices on fragic than nonfragic soils. Management practice, forest cover type, and soil type all appear to affect ion cycling within these forests. All of the stands featured striking losses of base cations that probably are influenced strongly by NO3 and SO4 in atmospheric deposition. PMID- 12371180 TI - Sorption and degradation characteristics of phosmet in two contrasting Australian soils. AB - The organophosphate insecticide phosmet [phosphorodithioic acid, s-((1,3-dihydro 1,3-dioxo-2H-isoindol-2yl)methyl), o,o-dimethyl ester] is used to control red legged earth mites (Halotydeus destructor), lucerne flea (Sminthurus viridis), and Oriental fruit moth (Cydia molesta) in horticulture and vegetable growing. This study was undertaken with two soils of contrasting properties to determine the extent to which sorption and degradation of the insecticide might influence its potential to leach from soil into receiving waters. Two soils were used: a highly organic, oxidic clay soil (Ferrosol) and a sandy soil low in organic matter (Podosol), sampled to 0.3 m depth. The extent of sorption and decomposition rate of a phosmet commercial formulation were measured in laboratory experiments. Sorption followed a Freundlich isotherm at all depths. The Freundlich coefficient K was significantly correlated (p = 0.005) with organic C content in the Podosol, and significantly correlated (p = 0.005) with organic C and clay content in the Ferrosol. K was highest (48.8 L kg-1) in the 0- to 0.05-m depth of the Ferrosol, but lowest (1.0 L kg-1) at this depth in the Podosol. Degradation followed first-order kinetics, with the phosmet half-life ranging from 14 h (0-0.05 m depth) to 187 h (0.2-0.3 m depth) in the Ferrosol. The half-life was much longer in the sandy Podosol, ranging from 462 to 866 h, and did not change significantly with depth. Soil organic C and to a lesser degree clay content influenced phosmet sorption and degradation, but the interaction was complex and possibly affected by co-solvents present in the commercial formulation. PMID- 12371181 TI - Pesticides in surface water, sediment, and rainfall of the northeastern Pantanal basin, Brazil. AB - Within the last 25 years an intensive agriculture has developed in the highland regions of Mato Grosso state (Brazil), which involves frequent pesticide use in highly mechanized cash-crop cultures. To provide information on pesticide distribution and dynamics in the northeastern Pantanal basin (located in southern Mato Grosso), we monitored 29 pesticides and 3 metabolites in surface water, sediment, and rainwater of the study area during the main application season. In environmental samples, 19 pesticides and 3 metabolites were detected in measurable quantities, resulting in at least one pesticide detection in 68% of surface water samples (n = 139), 62% of sediment samples (n = 26), and 87% of rainwater samples (n = 91). Surface water samples were most frequently contaminated by endosulfan compounds (alpha-, beta-, -sulfate), ametryn, metolachlor, and metribuzin, although in low (< 0.1 microgram L-1) concentrations. Sediment samples exhibited concentrations up to 4.5 micrograms kg 1 of p,p'-DDT, p,p'-DDE, endosulfan-sulfate, beta-endosulfan, and ametryn. In contrast, rainwater was polluted with substantial amounts of endosulfan, alachlor, metolachlor, trifluralin, monocrotofos, and profenofos (maximum concentrations = 0.3 to 2.3 micrograms L-1) in the highlands. Lowland rainwater samples taken 75 km from the next application area contained 5- to 10-fold lower mean pesticide concentration than in the highlands. Cumulative deposition rates of the pesticide sum within the study period ranged from 423 micrograms m-2 in the highlands to 14 micrograms m-2 in the lowlands. The atmospheric input of pesticides to ecosystems seemed to be of higher relevance in the tropical study area than known from temperate regions. PMID- 12371183 TI - Imazaquin adsorbed on pillared clay and crystal violet-montmorillonite complexes for reduced leaching in soil. AB - Ground water pollution due to herbicide leaching has become a serious environmental problem. Imazaquin [2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2 yl)quinoline-3-carboxylic acid] is an herbicide used to control broadleaf weeds in legume crops. Imazaquin is negatively charged at the basic pH of calcareous soils and exhibits high leaching potential in soils. Our aim was to design formulation of imazaquin to reduce herbicide leaching. Imazaquin sorption on pillared clay (PC) and crystal violet (CV)-montmorillonite complexes was studied. The CV-montmorillonite complexes become positively charged with adsorption of CV above the cation exchange capacity (CEC) of montmorillonite, and thus can sorb imazaquin. The Langmuir equation provides a good fit to isotherms of imazaquin sorption on PC and CV-montmorillonite complexes, but for charged complexes an equation that combines electrostatics with specific binding was preferred. Maximal imazaquin sorption was 17.3 mmol kg-1 for PC and 22.2 mmol kg-1 for CV montmorillonite complexes. The extents of imazaquin desorption into water were 21% for PC and 5% for CV-clay complexes. The presence of anions decreased imazaquin sorption on both sorbents in the sequence phosphate > acetate > sulfate. Reduction of imazaquin sorption by the anions and the extent of its desorption in electrolyte solutions were higher for PC than for CV-clay complexes. Leaching of imazaquin from CV-montmorillonite formulations through soil (Rhodoxeralf) columns was two times less than from PC formulations and four times less than that of technical imazaquin. The CV-montmorillonite complexes at a loading above the CEC appear to be suitable for preparation of organo-clay imazaquin formulations that may reduce herbicide leaching significantly. PMID- 12371182 TI - Soil-to-root transfer and translocation of polycyclic aromatic hydrocarbons by vegetables grown on industrial contaminated soils. AB - Polycyclic aromatic hydrocarbons (PAHs) are possible contaminants in some former industrial sites, representing a potential risk to human health if these sites are converted to residential areas. This work was conducted to determine whether PAHs present in contaminated soils are transferred to edible parts of selected vegetables. Soils were sampled from a former gasworks and a private garden, exhibiting a range of PAH concentrations (4 to 53 to 172 to 1263 and 2526 mg PAHs kg-1 of dry soil), and pot experiments were conducted in a greenhouse with lettuce (Lactuca sativa L. var. Reine de Mai), potato (Solanum tuberosum L. var. Belle de Fontenay), and carrot (Daucus carota L. var. Nantaise). At harvest, above- and below ground biomass were determined and the PAH concentrations in soil were measured. In parallel, plates were placed in the greenhouse to estimate the average PAH-dust deposition. Results showed that the presence of PAHs in soils had no detrimental effect on plant growth. Polycyclic aromatic hydrocarbons were detected in all plants grown in contaminated soils. However, their concentration was low compared with the initial soil concentration, and the bioconcentration factors were low (i.e., ranging from 13.4 x 10(-4) in potato and carrot pulp to 2 x 10(-2) in potato and carrot leaves). Except in peeled potatoes, the PAH concentration in vegetables increased with the PAH concentration in soils. The PAH distribution profiles in plant tissues and in soils suggested that root uptake was the main pathway for high molecular weight PAHs. On the opposite, lower molecular weight PAHs were probably taken up from the atmosphere through the leaves as well as by roots. PMID- 12371184 TI - Sorption interactions between imazaquin and a humic acid extracted from a typical Brazilian oxisol. AB - Soil sorption of most hydrophobic organic compounds (e.g., nonpolar pesticides) is directly related to soil organic matter (SOM) content. Humic substances are the major SOM components, containing carboxylic, phenolic, amine, quinone, and other functional groups, and specific structural configurations. In this paper, sorption interactions between imazaquin (2-[4,5-dydro-4-methyl-4-(1-methylethyl) 5-oxo-1H- imidazol-2-yl]-3-quinoline-carboxylic acid) herbicide (IM) and a humic acid (HA) extracted from a typical Brazilian Oxisol were studied with electron paramagnetic resonance (EPR) and Fourier-transform infrared (FTIR) spectroscopic techniques. A polarographic technique was used to quantify sorption. The IM amount sorbed on the HA was much higher than that on the whole soil within the pH range studied, emphasizing the prominent role played by SOM on IM sorption. Moreover, IM sorption increased as the soil-solution pH decreased. This enhancement in sorption was attributed to the hydrophobic affinity of the herbicide by the HA and to the electrostatic interaction between the protonated quinoline group of IM and the negative sites of the HA. Hydrophobic regions in the HA's interior at low pH (< 5.0) were recently demonstrated by an EPR detectable spin-label molecule. The FTIR and EPR spectroscopy and polarography data indicated weak interaction between IM and the soil and its HA, involving hydrogen bonding, proton transfer, and cation exchange (at low pH), and mainly hydrophobic interactions. However, no strong reaction mechanism, such as charge transfer, was involved. In addition, this research suggested that soil amendment with organic material might increase magnitude of IM sorption, consequently avoiding leaching and carryover problems usually found for mobile and persistent herbicides such as imazaquin. PMID- 12371185 TI - Arsenic accumulation in the hyperaccumulator Chinese brake and its utilization potential for phytoremediation. AB - The unique property of arsenic hyperaccumulation by the newly discovered Chinese brake (Pteris vittata L.) fern is of great significance in the phytoremediation of arsenic-contaminated soils. The objectives of this study were to (i) examine arsenic accumulation characterized by its distribution pattern in Chinese brake, and (ii) assess the phytoextraction potential of the plant. Young ferns with five or six fronds were transferred to an arsenic-contaminated soil containing 98 mg As kg-1 and grown for 20 wk in a greenhouse. At harvest, the Chinese brake produced a total dry biomass of 18 g plant-1. Arsenic concentration in the fronds was 6000 mg kg-1 dry mass after 8 wk of transplanting, and it increased to 7230 mg kg-1 after 20 wk with a bioconcentration factor (ratio of plant arsenic concentration to water-soluble arsenic in soil) of 1450 and a translocation factor (ratio of arsenic concentration in shoot to that in root) of 24. The arsenic concentrations increased as the fronds aged, with the old fronds accumulating as much as 13,800 mg As kg-1. Most (approximately 90%) of the arsenic taken up by the Chinese brake was transported to the fronds, with the lowest arsenic concentrations in roots. About 26% of the initial soil arsenic was removed by the plant after 20 wk of transplanting. Our data suggest that the arsenic hyperaccumulating property of the Chinese brake could be exploited on a large scale to remediate arsenic contaminated soils. PMID- 12371186 TI - Basal area growth of sugar maple in relation to acid deposition, stand health, and soil nutrients. AB - Previous studies have shown in noncalcareous soils that acid deposition may have increased soil leaching of basic cations above the input rate from soil weathering and atmospheric depositions. This phenomenon may have increased soil acidity levels, and, as a consequence, may have reduced the availability of these essential nutrients for forest growth. Fourteen plots of the Forest Ecosystem Research and Monitoring Network in Quebec were used to examine the relation between post-industrial growth trends of sugar maple (Acer saccharum Marsh.) and acid deposition (N and S), stand decline rate, and soil exchangeable nutrient concentrations. Atmospheric N and S deposition and soil exchangeable acidity were positively associated with stand decline rate, and negatively with the average tree basal area increment trend. The growth rate reduction reached on average 17% in declining stands compared with healthy ones. The results showed a significant sugar maple growth rate reduction since 1960 on acid soils. The appearance of the forest decline phenomenon in Quebec can be attributed, at least partially, to soil acidification and acid deposition levels. PMID- 12371187 TI - Ion partitioning among soil and plant components under drip, furrow, and sprinkler irrigation regimes: field and modeling assessments. AB - Soil and water resources can be severely degraded by salinity when total salt input exceeds output in irrigated agriculture. This study was conducted to examine partitioning of Ca2+, Na+, and Cl- between soil and soybean [Glycine max (L.) Merr.] plants under different irrigation regimes with both field and modeling assessments. In drip and sprinkler treatments, the irrigation water was salinized with NaCl and CaCl2 salts to simulate a Cl- and Na+ dominant saline drainage water. In the furrow irrigation treatment, the soil was salinized, prior to planting, with NaCl and CaCl2 salts to simulate a Cl- and Na+ dominant saline soil. A total of 756 soil and 864 plant samples were collected and analyzed for the salt ions to obtain ion partitioning and mass balance assessments. Modeling of salt ion uptake by plants and distribution in the soil profile was performed with a two-dimensional solute transport model for the three irrigation regimes. Results indicated that about 20% of the applied Ca2+ was recovered in harvested soybean biomass in all treatments. Plant uptake of either Na+ or Cl- was less than 0.5% in the drip and furrow, and about 2% in the sprinkler irrigation treatment. Significant increases in soil salinity were found in the sprinkler plot that received the highest cumulative amount of salts. Simulated ion distributions in the soil were comparable with the measurements. Compared with the total seasonal salt input, mass balances between 65 and 108% were obtained. Most salt inputs accumulate in the soil, and need to be removed periodically to prevent soil salinization. PMID- 12371189 TI - Salmonella typhimurium survival and viability is unaltered by suspended particles in freshwater. AB - Rolling microcosm experiments were conducted to determine whether suspended particles affect the survival and viability of a model pathogen, Salmonella choleraesuis, serotype typhimurium (American Type Culture Collection no. 23567), in a freshwater microbial community. Water from the Duluth, MN harbor of Lake Superior (including native microorganisms) was inoculated with clay, silt, or flocculent organic particles in a range of concentrations and a streptomycin resistant strain of S. typhimurium. Microcosms (incubated at 20 degrees C) were rolled horizontally (3 rpm) and sampled periodically for total bacteria and total, viable, and culturable S. typhimurium. Total S. typhimurium abundance decreased rapidly in all experiments (8.5-73.1% d-1). Total bacteria did not decrease as rapidly as the S. typhimurium population in any experiment, suggesting that a microcosm effect was not responsible for the decline in S. typhimurium populations. Loss rates of attached and free cells were similar, indicating that attachment to particles did not enhance the persistence of Salmonella cells beyond our minimum detectable differences. After eight days, only 0.1 to 11.9% of the initial S. typhimurium inocula were detected by direct counts. Suspended particles had a minimal effect on the survival and viability of S. typhimurium; the losses of total, viable, or culturable Salmonella were generally the same across particle treatments and concentrations. Silt and flocculent particles affected loss rates of total and viable S. typhimurium similarly to inorganic particles (clay). It appears unlikely that suspended particles would provide a means for S. typhimurium to persist at hazardous levels in freshwater. PMID- 12371188 TI - Secondary metabolite concentrations and terpene emissions of Scots pine xylem after long-term forest fertilization. AB - Secondary compounds are known to be associated with the resistance of conifer xylem against insects and fungi. The effects of long-term forest fertilization with nitrogen (N) or with N, calcium (Ca), and phosphorus (P) on secondary compounds in the xylem of 50-yr-old Scots pine (Pinus sylvestris L.) trees were examined. Xylem samples were collected from trees growing in three locations in southern Finland: Vilppula, Padasjoki, and Punkaharju. Forests were fertilized every fifth (Vilppula and Padasjoki) or tenth (Punkaharju) year since the 1950s. We compared concentrations of individual and total monoterpenes and resin acids in the heartwood and sapwood of Scots pine. Terpene emissions were analyzed from the sapwood and total phenolics from the heartwood. Fertilization did not have any significant effect on the concentrations and emissions of xylem monoterpenes. Concentrations of several individual terpenes in sapwood were positively correlated with the corresponding terpene emission. The concentrations of individual resin acids (i.e., abietic and dehydroabietic) decreased significantly in Punkaharju, but increased in the sapwood of N-fertilized trees compared with control ones at Padasjoki and Vilppula. The concentrations of resin acids in the heartwood were not significantly affected by fertilization. Both fertilization treatments decreased the total phenolic concentrations in the heartwood of trees growing in Padasjoki. There was a significant positive correlation between the total phenolics and total resin acid concentration. Overall, resin acids and phenolics seemed be more responsive than monoterpenes to N treatment. These results suggest that forest fertilization might cause slight changes in secondary compound concentrations of xylem, and thus might have significance in the decay resistance of wood. PMID- 12371190 TI - Phosphorus and nitrate nitrogen in runoff following fertilizer application to turfgrass. AB - Intensively managed golf courses are perceived by the public as possibly adding nutrients to surface waters via surface transport. An experiment was designed to determine the transport of nitrate N and phosphate P from simulated golf course fairways of 'Tifway' bermudagrass [Cynodon dactylon (L.) Pers.]. Fertilizer treatments were 10-10-10 granular at three rates and rainfall events were simulated at four intervals after treatment (hours after treatment, HAT). Runoff volume was directly related to simulated rainfall amounts and soil moisture at the time of the event and varied from 24.3 to 43.5% of that added for the 50-mm events and 3.1 to 27.4% for the 25-mm events. The highest concentration and mass of phosphorus in runoff was during the first simulated rainfall event at 4 HAT with a dramatic decrease at 24 HAT and subsequent events. Nitrate N concentrations were low in the runoff water (approximately 0.5 mg L-1) for the first three runoff events and highest (approximately 1-1.5 mg L-1) at 168 HAT due to the time elapsed for conversion of ammonia to nitrate. Nitrate N mass was highest at the 4 and 24 HAT events and stepwise increases with rate were evident at 24 HAT. Total P transported for all events was 15.6 and 13.8% of that added for the two non-zero rates, respectively. Total nitrate N transported was 1.5 and 0.9% of that added for the two rates, respectively. Results indicate that turfgrass management should include applying minimum amounts of irrigation after fertilizer application and avoiding application before intense rain or when soil is very moist. PMID- 12371191 TI - Sorption and transport behavior of naphthalene in an aggregated soil. AB - Soil solution chemistry influences the sorption and transport behavior of hydrophobic organic compounds (HOCs) in soil. We used both batch and column studies to investigate the influence of ionic strengths (0.03 and 1.5 M) and flow velocities (12 and 24 cm h-1) on sorption and transport of naphthalene (NAP) in aggregated soil. Sorption parameters such as the Freundlich coefficient (Kf) and exponent (n) calculated from batch studies and column experiments were also compared. Retardation of NAP transport was greater at higher solution ionic strength, which may be attributed to greater sorption affinity due to enhanced aggregation of the sorbent. The effect of ionic strength on sorption of NAP observed in the batch study was consistent with the results from the column study. The Kf and n values obtained from the batch study for the two ionic strengths ranged from 7.8 to 13.7 and 0.68 to 0.80, respectively, whereas the Kf and n values obtained from the column study ranged from 7.9 to 9.9 and 0.73 to 0.85, respectively. The effluent breakthrough curve (BTC) of NAP at a flow rate of 24 cm h-1 showed significant chemical and physical nonequilibrium behavior, implying that a considerable amount of sorption in aggregated soil was time dependent when flow was relatively fast. The BTCs calculated with the parameters determined from batch studies compared poorly with the measured BTCs. The potential for nonequilibrium transport should be incorporated in models used for predicting the fate and transport of HOCs. Furthermore, caution is required when extrapolating the results from batch studies, especially for aggregated soils. PMID- 12371192 TI - Solute transport modeling under cultivated sandy soils and transient water regime. AB - Drainable lysimeters offer the possibility to integrate heterogeneous solute leaching conditions caused by row crops and transient water regime, and to conveniently measure water and solute fluxes at the drainage outlet. To compare solute leaching behavior in and around drainable lysimeters operating under a transient water regime in potato (Solanum tuberosum L.) fields, parameters of the convective lognormal transfer (CLT) function model were fitted using bromide (Br ) flux concentrations (Cf) measured in lysimeters and from Br- resident concentrations (Cr) measured in adjacent soil cores. Expected mean values Ez(I) obtained from Cr and Cf CLT parameters were equivalent and well correlated (R2 = 0.78). However, estimated median values mu of the CLT function were smaller when derived from Cr (1.05 to 1.28) compared with Cf (1.23 to 2.14). Most mu values were also smaller than previously reported values for a 30-cm reference depth, indicating that 50% of solute mass would leach more readily in these coarse sandy soils. Higher variance and dispersion of Cr compared with those of Cf could be related to a smaller sampling support (sample size/sampling area) in the case of Cr measured by soil coring, or to disruption of solute transport mechanisms in the repacked lysimeter. Retained Br- in the top soil layer after 12 to 17 cm of cumulative drainage was indicated by measured Cr. Neither CLT function simulated well residual topsoil Cr values, indicating that Br- plant cycling or preferential flow probably interfered even though tuber Br- uptake was relatively small. PMID- 12371193 TI - Export of manure phosphorus and nitrogen in turfgrass sod. AB - Regulatory mandates have increased demand for best management practices (BMPs) that will reduce nutrient loading on watersheds impaired by excess manure P and N. Export of manure P and N in turfgrass sod harvests is one BMP under consideration. This study quantified amounts and percentages of P and N removed in a sod harvest for different rates of manure and inorganic P and N. Six treatments comprised an unfertilized control, two manure rates with and without supplemental inorganic N, and inorganic P and N only. The treatments were applied to 'Tifway' bermudagrass (Cynodon dactylon L. x C. transvaalensis Burtt-Davey), '609' buffalograss [Buchloe dactyloides (Nutt.) Engelm.], and 'Reveille' bluegrass (Poa arachnifera Torr. x P. pratensis L.) under field conditions. Comparisons among treatments revealed small variations of P and N content in clippings and the plant component of sod, but large variations in the soil component of sod for each turf species. In addition, 2 to 10 times more P and 1.3 to 5 times more N was removed in soil than in plant components of sod for the two manure rates with and without added inorganic N. Percentages of applied P and N in harvested sod were similar for the two manure rates with and without added N for each species, but differed among turf species for each P (46 to 77%) and N (36 to 47%). The large amounts and percentages of manure P and N removed by sod harvest support the feasibility of this BMP in efforts to reduce nutrient loads on watersheds. PMID- 12371194 TI - Nutrient processing capacity of a constructed wetland in western Ireland. AB - In Ireland, constructed wetland systems are increasingly being used to perform tertiary treatment on municipal waste effluent from small towns and villages located in areas whose receiving waters are deemed sensitive. The bedrock formation in the west of Ireland is primarily karst limestone and where the overburden-soil cover is very shallow, such waters are highly sensitive to pollution sources, as little or no natural attenuation and/or treatment will occur. Constructed wetland technology has been seen to offer a relatively low cost alternative to the more conventional tertiary treatment technologies, particularly when dealing with low population numbers in small rural communities. This paper examines the waste treatment performance, in terms of nutrient (P and N) reduction, of a recently constructed surface-flow wetland system at Williamstown, County Galway, Ireland. Performance evaluation is based on more than two years of water quality and hydrological monitoring data. The N and P mass balances for the wetland indicate that the average percentage reduction over the two-year study period is 51% for total N and 13% for total P. The primary treatment process in the wetland system for suspended solids (between 84 and 90% reduction), biological oxygen demand (BOD) (on average, 49% reduction), N, and P is the physical settlement of the particulates. However, the formation of algal bloom during the growing season reduces the efficiency of the total P removal. PMID- 12371195 TI - Bioavailability of organic phosphorus in a submerged aquatic vegetation-dominated treatment wetland. AB - Enzymatic hydrolysis and mineralization of organic phosphorus (P) were determined in surface water samples collected from inflow and outflow of a submerged aquatic vegetation (SAV)-dominated treatment wetland of the Florida Everglades. Water samples were fractionated into three size fractions (> 0.4 micron, < 0.4 to > 0.05 micron, and < 0.05 micron) with a sequential flow filtration technique. The fractionated water samples were incubated to hydrolyze with alkaline phosphatase (APase) and phosphodiesterase (PDEase), and to mineralize at different redox and pH. Unlike APase, which hydrolyzed < or = 10% of organic P, PDEase hydrolyzed > or = 71% of organic P in unfiltered water from both inflow and outflow waters, suggesting the domination of bioavailable diester P in the water. Phosphodiesterase completely hydrolyzed organic P in the < 0.4- to > 0.05-micron and < 0.05-micron fractions, as compared with < or = 35% in the > 0.4-micron fraction. However, the P mineralization in inflow and outflow waters at different redox and pH showed that P associated with particulate > 0.4 micron had been mineralized the most. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy showed that surficial sediments from the inflow region contained a high proportion of polynucleotides, nucleoside monophosphates, and previously unreported glycerophosphoethanolamine and phosphoenolpyruvates. However, at the outflow, the relative proportion of polynucleotides and nucleoside monophosphates was reduced substantially. This suggests that the SAV wetland may sequester P via accretion of organic matter. PMID- 12371196 TI - Nursing practice issues in assisted living facilities. PMID- 12371197 TI - Retaining nurses by releasing the power to care. PMID- 12371198 TI - Enhancement of ultrasonic cavitation yield by multi-frequency sonication. AB - The paper reports the enhanced effect of multi-frequency ultrasonic irradiation on cavitation yield. The cavitation yield is characterized by electrical conductivity determination, fluorescence intensity determination and iodine release method. Two-frequency (28 kHz/0.87 MHz) orthogonal continuous ultrasound, two-frequency (28 kHz/0.87 MHz) orthogonal pulse ultrasound and three-frequency (28 kHz/1.0 MHz/1.87 MHz) orthogonal continuous ultrasound have been used. It has been found that the combined irradiation of two or more frequencies of ultrasound can produce a significant increase in cavitation yield compared with single frequency irradiation. The possible mechanisms of the enhanced effect are briefly discussed. PMID- 12371199 TI - Improved synthesis of chalcones under ultrasound irradiation. AB - Claisen-Schmidt condensation of acetophenone with aromatic aldehydes catalyzed by pulverized KOH and KF-Al2O3 results chalcones in 52-97% and 83-98% yields respectively in alcoholic solvent under ultrasound irradiation. PMID- 12371200 TI - Fluorescence enhancement of the aqueous solution of terephthalate ion after bi frequency sonication. AB - The fluorescence enhancement of the aqueous solution of terephthalate ion (TA) under orthogonal sonication of 28 kHz and 1.7 MHz ultrasonic wave has been studied. It has been found that the fluorescence intensity of TA solution after bi-frequency ultrasonic irradiation is obviously higher than the sum of those under two individual ultrasonic irradiations. PMID- 12371201 TI - The ultrasonically induced reaction of benzoyl chloride with nitrobenzene: an unexpected sonochemical effect and a possible mechanism. AB - A mixture of benzoyl chloride and nitrobenzene is not known to be chemically reactive, indeed the mixture is chemically inert when subjected individually to either ultrasonic irradiation or heating. However, if this system is initially subjected to ultrasound and then heated for several hours at 200 degrees C, a reaction does occur and the products are benzoic anhydride, hydrochloric acid, nitrous and nitric acid together with some minor products. To the best of our knowledge, this is the first example of a reaction where the effect of ultrasound does not appear to be the consequence of the direct action of acoustic cavitation bubbles. A possible explanation of this behaviour is advanced which involves an electron transfer reaction in which nitrobenzene is first activated by ultrasound and then acts as oxidant in the thermal stage of reaction. PMID- 12371203 TI - Sonodynamic effect of erythrosin B on sarcoma 180 cells in vitro. AB - The ultrasonically induced cytotoxic effect of erythrosin B (EB) on isolated sarcoma 180 cells was investigated. The tumor cells were suspended in an air saturated phosphate buffered saline and exposed to ultrasound at 1.93 MHz in a standing-wave mode for up to 60 s in the presence and absence of EB. The rate of cell damage induction by ultrasound was enhanced by 4-5 times with 160-microM EB, while no cell damage was observed with EB alone. This enhancement was significantly inhibited by histidine. Sonochemical generation of active oxygen species in the presence of EB, measured by ESR spectroscopy, was also inhibited by histidine. These results indicate the involvement of a sonochemical mechanism. PMID- 12371204 TI - The effect of ultrasound upon the oxidation of thiosulphate on stainless steel and platinum electrodes. AB - Ultrasound was found to increase the oxidation peak current and hence the decomposition rate of thiosulphate 50-fold compared to silent conditions. The effects of the ultrasonic frequency (20 and 38 kHz) and power upon the electrochemical oxidation of thiosulphate in aqueous KCl (1 mol dm-3) at stationary stainless steel and platinum electrodes were studied chronoamperometrically and potentiostatically (at various scan rates). No sigmoidal-shaped voltammograms were observed for the redox couple S4O6(2-)/S2O3(2 ) in the presence of ultrasound. However, application of ultrasound to this redox couple provided an increase in the oxidation peak current at the frequencies employed, the magnitude of which varied with concentration, scan rate and ultrasonic power. Under sonication at 20 and 38 kHz, the oxidation peak potential shifted anodically with increasing ultrasonic power. This anodic shift in potential may be due to the formation of hydroxyl radicals, changes in electrode surface composition and complex adsorption phenomena. The large increase in oxidation peak currents and the rates of decomposition of thiosulphate, in the presence of ultrasound, are explained in terms of enhanced mass transfer at the electrode due to cavitation and acoustic streaming together with microstreaming coupled with adsorption phenomena. It is also shown that changes in macroscopic temperature throughout the experiment are insufficient to cause the observed enhanced diffusion. PMID- 12371202 TI - Frequency effect on the sonochemical remediation of alachlor. AB - The effect of ultrasonic frequency on the sonodegradation of alachlor is described. The rate observed for the destruction of alachlor is approximately 25 times faster at 300 kHz under argon saturation than at 20 kHz under comparable acoustic input energy. The effect of variation of a number of extrinsic parameters such as dissolved gases, radical scavengers and hydroxyl radical promoters is also explored. Argon-saturated solutions display an enhancement in rate by a factor of two compared to either oxygen- or air-saturated solutions upon sonication at 300 kHz. The principal ultrasonic degradation products have been determined in air, argon, and oxygen. The products results primarily from cleavage of the N-methoxymethyl unit when sonication occurs in argon and air. Oxygen addition has been observed when the saturating gas is oxygen. The nature of the active site for reactivity of alachlor is discussed. PMID- 12371205 TI - Microelectrode study of single cavitational bubbles induced by 500 kHz ultrasound. AB - Insight is gained into about the processes governing cavitational activity and acoustic streaming induced by high frequency (500 kHz) ultrasound by the use of microelectrodes with short time resolution electrochemical equipment to allow monitoring of the activity of single cavitating bubbles. Current transients are interpreted as showing the flux of solution towards the electrode surface due to microstreaming. In order to explain the current amplitude, a simplified model is produced. Important parameters such as bubble size and shape on the surface as well as the boundary layer thickness for microstreaming are taken into account. This model leads to the amplitude of the oscillations of the cavitating bubble. Introducing realistic bubble sizes, this amplitude is found to be in the order of 1 micron. The conclusions arising from this work allow a further interpretation of previous observations at millimeter scale electrodes. PMID- 12371206 TI - Needless tests. PMID- 12371207 TI - The barber-surgeons of yesteryear. PMID- 12371208 TI - Psychiatry: current directions. PMID- 12371209 TI - Treating a high risk population: a collaboration of child psychiatry and pediatrics. PMID- 12371210 TI - Effectiveness of a multidisciplinary outpatient headache management program. PMID- 12371211 TI - Depression in the medically ill. PMID- 12371212 TI - Clinical and research issues in the evaluation and treatment of families. AB - Despite an influx of new pharmacological agents in the eighties and nineties, clinicians and researchers recognize that family intervention offers one area of adjunctive therapy that may improve the odds of recovery, increase quality of life, or help patients and family members deal with a chronic or recurring illness. PMID- 12371213 TI - Management of behavioral complications of dementia. AB - The behavioral and psychotic complications of dementia are common clinical problems, requiring a review of past psychiatric diagnosis, as well as medical differential diagnosis to uncover potentially treatable causes. Behavioral factors can play an important contributing role and behavioral interventions can sometimes significantly improve or reduce symptoms' emergence. Psychotropic medications when properly selected and monitored can significantly reduce agitated behavioral and psychotic symptoms associated with dementia and may have very positive impact on a patient's quality of life, a family's ability to manage patients in the community, and a resident's nursing home management. PMID- 12371214 TI - Images in medicine. Type I Gaucher's disease. PMID- 12371215 TI - Immunization update, 2002. PMID- 12371216 TI - Trends and patterns in place of death, 1989-2000. PMID- 12371217 TI - Progress in the control of prostate cancer in Rhode Island, 1987-2000. PMID- 12371218 TI - Principles of tissue transfer techniques in urethral reconstruction. AB - Many reconstructive surgical techniques require tissue transfer, or movement of tissue from a donor to a recipient site. The techniques of tissue transfer in reconstructive urologic surgery assume knowledge of skin anatomy, the basic principles of tissue transfer, and the composition and characteristics of the donor and recipient tissues. These topics will be addressed in this article. PMID- 12371219 TI - Management of chordee in children and young adults. AB - Penile curvature is a spectrum of disease affecting boys with and without hypospadias. The etiology of chordee includes skin tethering, fibrotic bucks or dartos fascia, corporeal body disproportion and rarely a fibrotic urethra. Several surgical techniques (plication, excision, and graft insertion) are currently employed to repair penile curvature. Recent neuroanatomical studies of the developing fetal penis have shown that the dorsal nerve branches from the 11 and 1 o'clock positions to the 5 and 7 o'clock positions, being absent in the midline. Since the neuroanatomy is similar in both the hypospadiac and normal penis, we now recommend performing penile straightening in both hypospadiac and non hypospadiac patients with significant curvature by the placement of plication sutures at the 12 o'clock position. Placement of dorsal midline plication sutures corrects curvature without risk to the underlying nerve structures. PMID- 12371220 TI - Tubularized incised plate (TIP) hypospadias repair. AB - In the past 6 years TIP urethroplasty has become a preferred technique for distal hypospadias repair, and has also been used for proximal lesions and reoperations. In this article technical aspects of the procedure are described and published outcomes reviewed. TIP hypospadias repair has gained widespread acceptance because of its versatility, low complication rate and reliable creation of a vertically-oriented meatus. PMID- 12371221 TI - Glandular hypospadias repair. AB - Glandular hypospadias represents approximately 15% of the hypospadias variants seen. This article will examine common surgical approaches applicable to the child with glandular hypospadias. Hypospadias repairs discussed in this article will include urethromeatoplasty, MAGPI, the GAP procedure, MIV glans plasty, urethral advancement procedure, and parameatal based flap variants, including the Mathieu and Barcat procedures. Because these anomalies are cosmetically less aberrant than more proximal variants, only those surgical techniques which assure a normal-appearing penis should be undertaken. PMID- 12371222 TI - Midshaft hypospadias. AB - The key to successful hypospadias surgery is minimal tissue handling, tension free reconstruction, the use of well-vascularized tissue, and knowledge of which repairs are indicated. Preservation of the urethral plate along with an onlay graft has a low complication rate and should be attempted for midshaft hypospadias repair. Although tubularized pedicle flaps increase the risk of complications such as urethral strictures, urethral diverticula, and fistulas, they provide a good alternative in the presence of a poorly developed urethral plate. For correction of the penile curvature, we recommend ventral lengthening procedures in cases where simple dorsal plication will result in shortening of an already compromised penile length. Using these principles, excellent cosmetic and functional results can be expected for treatment of midshaft hypospadias. PMID- 12371223 TI - Proximal hypospadias. AB - Approximately 20% of hypospadiac urethras are located proximally, anywhere from the penoscrotal to the perineal region. Repair of proximal defects is still one of the most challenging surgical procedures facing the hypospadiologist. Modifications of current techniques for the correction of proximal hypospadias as well as new innovative techniques continue to be proposed. PMID- 12371224 TI - Complications of hypospadias repair. AB - Hypospadias is one of the most frequently encountered congenital malformations of the genitourinary system. The incidence in studies of large populations has been reported to be from 1 to 8 per 1000 male births. For centuries the imagination and creativity of surgeons has been challenged to create a phallus that is both functional and cosmetic. Over the last 30 years numerous new operations and techniques have been developed with the objective of achieving improved cosmetic results with minimal complications. PMID- 12371225 TI - Megalourethra and urethral diverticula. AB - Megalourethra and urethral diverticula encompass a diverse group of congenital and acquired urethral defects. The appropriate management of these anomalies relies on a keen appreciation of phallic anatomy and an understanding of urethral embryology. A thorough history and physical examination--including a careful evaluation of the urinary tract--is necessary to identify associated congenital anomalies. Finally, satisfactory surgical management demands meticulous attention to surgical technique. PMID- 12371226 TI - Management of epispadias. AB - Although epispadias is considered to be the least severe defect of the exstrophy epispadias complex, the treatment of this anomaly is far from trivial. Epispadias does not involve the body of the bladder or the hindgut but does affect the urethra and can affect the bladder neck. As a consequence, it presents with a spectrum of severity that can affect urinary continence if the epispadias anomaly is proximal enough to affect the urinary sphincter mechanism. PMID- 12371227 TI - Imaging of the male urethra for stricture disease. AB - Imaging of the urethra for suspected stricture disease should initially consist of conventional imaging with a dynamic RUG. It is easy to perform and detects clinically relevant strictures involving the anterior urethra and those with extension into the membranous urethra. Additional studies, including antegrade imaging, sonographic urethrography, and MRI are best used in conjunction with RUG as clinically indicated to better define the extent of disease and assist in guiding reconstruction. Post-operatively, VCUG is appropriate to evaluate complete healing and adequacy of repair. Sonourethrography is a simple technique that provides a dynamic, precise assessment of anterior urethral strictures. It is best employed as a staging study in men with known symptomatic strictures in whom the need for operative therapy is clear. For short bulbar strictures ultrasound is more accurate in measuring stricture length than conventional radiographic RUG and is therefore helpful in determining whether to excise or graft. For long or complex strictures assessment of the stricture's diameter may be helpful in determining flap width or in identifying the focal urethral segments to be excised. The simplicity, precision, and availability of sonography along with the absence of radiation exposure make sonourethrography a valuable staging tool for the reconstructive urologist. MRI is valuable for defining the distorted pelvic anatomy that is frequently associated with posterior urethral strictures resulting from trauma. By determining the location of the prostate and the length of the prostatomembranous defect, MRI may help determine whether a transperineal or transpubic approach for reconstruction is necessary. PMID- 12371228 TI - Excision and primary anastomosis for anterior urethral stricture. AB - Excision with spatulated primary anastomosis (EPA) is an excellent reconstructive option for short bulbar urethral strictures with success rates between 90 and 95% in appropriately selected patients. Patient selection requires a careful history, physical examination, and radiographic staging. Failure with this reliable method is caused by inadequate excision of urethral stricture and incomplete mobilization of the urethra with excessive anastomotic tension. Complications that include wound and urinary tract infections, chordee, and erectile dysfunction, are uncommon. EPA warrants strong consideration as a first line treatment due to its excellent and durable long-term results. PMID- 12371229 TI - Ventral onlay graft techniques for urethroplasty. AB - Techniques of anterior urethroplasty have evolved over the last 40 years due to advances in tissue transfer techniques and the changing etiology of urethral stricture. The use of free grafts has come full circle from full thickness skin grafts to the current popularity of buccal mucosa grafts. Grafts fell out of favor due to poor results with full thickness skin grafts and the more widespread use of pedicle flaps. Although controversy still exists as to the appropriate use of flaps versus grafts, the evidence does not demonstrate an advantage in outcomes: overall success rates for grafts and flaps are similar (84.3 and 85.9% respectively). Nevertheless, grafting techniques are now considered first line therapy for the majority of strictures that cannot be excised and reanastomosed. PMID- 12371230 TI - Dorsal onlay techniques for urethroplasty. AB - Dorsal onlay graft urethroplasty is a versatile procedure which may be combined with various substitute materials such as preputial skin, buccal mucosa grafts or pedicled flaps. Others substitute materials such as human urethral mucosa from corpses or collagen matrix will be possible in future. The long-term results of a wide series of patients showed a final success rate from 92% to 97%. Any kind of substitution urethroplasty deteriorate over time, and in our series of patients with an extended follow-up from 21.5 to 43 months the success rate of dorsal onlay graft urethroplasty decreased from 92% to 85%. With regard to substitute material concerns (buccal mucosa versus preputial skin) a long-term follow-up is mandatory to establish if buccal mucosa is superior to foreskin as urethral substitute material. At present, the authors currently use both according to patient preference, status of the genital tissues or strictures characteristics. PMID- 12371231 TI - Penile circular fasciocutaneous flaps to reconstruct complex anterior urethral strictures. AB - The circular fasciocutaneous penile flap meets all criteria for tissue transfer and urethral reconstruction. It reliably provides ample hairless tissue, usually 13 to 15 cm long, without compromising cosmesis or function. We find it ideal for long strictures in the distal or pendulous urethra, where the decreased substance of the corpus spongiosum may jeopardize graft viability. A second major advantage is its versatility: it can be used throughout the entire anterior urethra, from the membranous area to the meatus. In addition, the circular fasciocutaneous penile flap is easily combined with other tissue-transfer techniques when necessary, enabling one-stage reconstruction in the majority of cases. The flap may be tubularized for replacement urethroplasty or divided and used in two separate stenotic areas. Onlay reconstruction is preferable to flap tubularization and has provided a better initial and long-term outcome. The circular fasciocutaneous penile flap provides superior results even in patients with complex refractory strictures in whom previous attempts at anterior urethroplasty have failed. We believe its superiority resides in the transfer of well-vascularized tissue to the compromised area. Complications can be minimized by avoiding prolonged placement in the exaggerated lithotomy position and by meticulous attention to principle of reconstructive surgery. PMID- 12371232 TI - Scrotal and perineal flaps for anterior urethral reconstruction. AB - The use of scrotal and perineal flaps for anterior and posterior urethral reconstruction has been unfairly maligned with claims of poor waterproofing qualities, formation of diverticula, and the potential to import hair into the urethra. Actually, scrotal and perineal skin do not appear to differ from other genital skin with regards to permeability to surface liquids, and although these islands are difficult to tailor, when properly prepared they are no more likely to create diverticula than other genital skin islands. Similarly, if the flap is prepared from hirsute skin, hair will be imported into the urethra; however, hairless scrotal areas can be mobilized as skin islands, and the skin overlying the perineal artery is non-hirsute or nearly non-hirsute in many individuals. PMID- 12371233 TI - Urethral reconstruction of strictures resulting from treatment of benign prostatic hypertrophy and prostate cancer. AB - Urethral strictures commonly result from treatments for prostate disease, such as transurethral resection, radical prostatectomy, and radiotherapy. Treating these strictures can be difficult: it may be complicated by previous irradiation, and endoscopy often fails. We review the risk factors for development of strictures resulting from the treatment of prostate disease and discuss the success rates of both endoscopic and open therapies. PMID- 12371234 TI - Reconstruction of posterior urethral disruption. AB - Posterior urethral disruption may be a devastating complication of pelvic trauma. The acute management of these injuries is reviewed as well as the controversy surrounding early versus delayed repair. The various approaches to delayed repair of pelvic fracture urethral distraction defects are presented and the technique of perineal repair is discussed in detail. PMID- 12371235 TI - Muscular, myocutaneous, and fasciocutaneous flaps in complex urethral reconstruction. AB - The ability to achieve a long-term, stable, stricture-free, hairless urethral lumen in patients with complex anterior stricture and compromised genital skin is one of the ongoing challenges of reconstructive urologic surgery. The conservative approach by endoscopic urethrotomy or dilatation with a self catheterization schedule rarely affects a definitive cure except in the short filmy superficial strictures of the bulbous portion of the urethra. Genital fasciocutaneous island flaps are currently the golden standard for definitive, reliable resolution of anterior urethral strictures in patients who have not undergone a prior surgical procedure that may alter the penile or scrotal circulation, or those with skin loss from trauma, decubiti, radiation, or balanitis xerotica obliterans. PMID- 12371236 TI - Staged urethroplasty: indications and techniques. AB - There is still a place for staged urethroplasty. There are some indications for staged urethral reconstruction such as strictures associated with chronic inflammation, fistula, false passage, urethral stones, urethral diverticula, abscess, failed prior repair, complicated hypospadias, severe trauma, neurologic diseases, extensive BXO strictures and long strictures. Staging a urethroplasty should not be considered a step backwards rather instead we should learn from experience and realize there are some patients who are too complex to reconstruct in a single stage. PMID- 12371237 TI - Management of fossa navicularis strictures. AB - The correction of strictures involving the fossa navicularis poses a distinct reconstructive challenge. Unlike surgical repair of strictures involving other urethral segments where the primary concern is restoration of urethral patency, management of fossa navicularis strictures also requires particular attention to cosmesis. Paramount to the success of any of the described procedures is the careful selection of nondiseased tissue for substitution. If the penile skin is healthy, the preferred urethral substitute is the fasciocutaneous ventral transverse island flap. The inherent characteristics of this versatile flap (i.e., well-vascularized predictable pedicle, nonhair bearing, negligible contraction) provide for an excellent time-tested glandular urethral substitute. In rare cases in which there is a suggestion of penile skin inflammation or scarring, extragenital tissue transfer techniques should be considered. Equally important is the need to substitute the entire length of diseased urethra, preferably as an onlay, preserving the dorsal urethral wall. Persistent proximal urethral disease will eventually result in further stricture formation. Finally, the choice of glanduloplasty is particularly important in achieving a cosmetically appealing outcome. A glans-cap repair is preferred because of the limited dissection required with this relatively simple and bloodless technique. Careful selection of the most appropriate combined urethral substitution and glans reconstruction techniques, as well as meticulous attention to surgical details, are mandatory for achieving a satisfactory functional and cosmetic outcome with fossa navicularis strictures. PMID- 12371238 TI - Experimental and clinical experience with tissue engineering techniques for urethral reconstruction. AB - Tissue engineering has been proposed as a strategy for urethral reconstruction. This may involve matrices alone, wherein the body's natural ability to regenerate is used to orient or direct new tissue growth, or the use of matrices with cells. Acellular collagen matrices derived from donor bladder submucosa have been used both experimentally and clinically for onlay urethral replacement with good success at our center. If a tubularized urethral repair is needed, the use of cells on the collagen matrix is essential for adequate tissue formation. Tissue engineering techniques are useful for urethral reconstruction. PMID- 12371239 TI - Effective hospital safety orientation. AB - How healthcare safety trainers can gain the attention of employees at all levels so that they truly understand their role in meeting problem situations which arise in seven Environment of Care areas. PMID- 12371240 TI - Preventing construction fires. AB - Causes usually are simple: careless smoking, no house-keeping, sloppy maintenance on electrical tools, portable heating, lack of adequate fire watch, or faulty wiring. PMID- 12371241 TI - Documentation of training: developing essential records. AB - In response to the need for proper documentation of training given to security officers to insure that training had actually been given and to better achieve training standardization, two types of forms have been developed by a security department at a medical center. PMID- 12371242 TI - Hospital security over the years: keeping up with the changes. AB - Entering his twenty-fifth year in hospital security, the author looks back on his experiences in an everchanging field including: the Three Mile Island "melt-down" panic; his first injury; the '80s AIDS "epidemic;" handling the victims of a prison riot; changes brought about by a major hospital merger; and the new things he continues to learn. PMID- 12371243 TI - Forming a partnership with law enforcement. AB - Despite budgetary limitations, security can work with local law enforcement and other departments to enhance its professional status. PMID- 12371244 TI - Employee screening today: the information is there, but be careful how you use it. AB - Doing a good job of conducting thorough reference checks and background investigations of job applicants has become increasingly important in healthcare in preventing personnel-related crime and the legal consequences of negligence in hiring people who commit such crimes. However, there are many obstacles to obtaining information on applicants. In this report, we'll explore in depth the current state of background checking as described by an official of a company which provides background checking services, as well as the latest in legal practices from a leading attorney in the field. PMID- 12371245 TI - Trends in infant abduction. AB - Progress has been made in reducing the number of infant abductions in the last five years, but there are still some serious problems for security and nursing managers, not the least of which is complacency. The article analyzes statistics on infant kidnapping which have been maintained by the National Center for Missing & Exploited Children (NCMEC) since 1983. PMID- 12371246 TI - Delivering security in today's new threat environments. AB - New security issues that have arisen since 9/11 have put facility security managers, especially those in hospitals, in the forefront of responsibility should a serious act of terrorism occur. Upgrading the skills of hospital security staff personnel is essential, the author contends. PMID- 12371247 TI - The critical role of hospitals involved in national bioterrorism preparedness. AB - This article is excerpted from a speech by Homeland Security Director Ridge on April 8, 2002 at the annual meeting of the American Hospital Association. In it he spells out what is expected from hospitals in preparing for the next terror attack, with the emphasis on bioterrorism and what assistance is being and will be provided. PMID- 12371248 TI - Under surveillance: is it time to reexamine your facility's security model. AB - The many different security models in place in health facilities across the country--including 'campus police,' off-duty officers, proprietary programs, contract security, and combined proprietary and contract security--will come under increased scrutiny by JCAHO in the event of an internal or external disaster situation. In this article, the author addresses the need for reevaluating current security models so that they can be defended as reasonable and appropriate should a future incident or litigation call them into question. PMID- 12371249 TI - Anger management in healthcare. AB - Anger is not necessarily bad, according to the author. There are times when it is appropriate. At other times it can escalate unpleasantly. The article describes the different stages of anger and the author's advice on the proper response at each stage. He also lists anger reducing and anger enhancing response behaviors to be aware of. PMID- 12371250 TI - Workplace assault management training: an outcome evaluation. AB - In this comprehensive study of health workers taking care of patients who are prone to challenging and aggressive behavior, injury rates for workers who had received PART (Professional Assault Response Training) as recommended by OSHA and other agencies were compared with those who received no training. Surprisingly, those with training had a higher number of reported injuries and a per capita worker's compensation payout double that received by those without training. The possible reasons for these results are analyzed, along with their implications for those responsible for supporting healthcare workers at risk for violence. The author concludes that training in itself may not be an adequate prevention strategy, but may need to be implemented by other approaches. PMID- 12371251 TI - Security planning for the hospital library: protecting your information assets. AB - The hospital library has become a multi-service information center with a variety of costly resources. This article presents an overview of two important areas of hospital library security: access control and premises protection. It discusses access control policies, auditing of premises security, and implementing premises security measures. PMID- 12371252 TI - A food services security survey. AB - Following a recent increase in reported theft, embezzlement, and pilferage in the storage and production areas of the hospital's Nutrition & Food Services, department, the protective service department, in cooperation with the NFS director, conducted a survey of overall security systems and staff awareness as well as specific vulnerabilities to theft which required correction. PMID- 12371253 TI - Achieve lifelong success: a matter of a few simple conventions. PMID- 12371254 TI - Nursing care of the pediatric patient following strabismus repair surgery. AB - This paper presents the holistic approach to the care of the pediatric patient with strabismus. It discusses preoperative management, contemporary surgical procedures including postoperative suture adjustments, complications and the care and management of the patient and family on the pediatric unit. It reviews discharge instructions including pain management, postoperative safety and the follow-up appointment with the surgeon. Methods of determining the level of knowledge of the child's primary caregiver are also a major focus. PMID- 12371255 TI - Making a difference through education in the life of the patient with pseudotumor cerebri. PMID- 12371256 TI - Hearing screening. PMID- 12371257 TI - Strabismus surgery. PMID- 12371258 TI - Danger: ultraviolet light! PMID- 12371259 TI - Eye on research: retinal microchip implants. PMID- 12371260 TI - (New) public management of mentally disordered offenders. Part II: A vision with promise. PMID- 12371261 TI - Clarifying automatism. PMID- 12371262 TI - Heinousness in capital crimes. Myths of proportionality and social protection. PMID- 12371263 TI - Measures of perceived coercion in prison treatment settings. PMID- 12371264 TI - Human rights vs. public protection. English mental health law in crisis? PMID- 12371265 TI - Perceived coercion and procedural justice in the Broward mental health court. PMID- 12371266 TI - The genetics of human obesity: recent progress. AB - The risk of becoming obese is higher in some families than in others. The risk (the lambda coefficient) is two to three fold for moderate obesity, but up to five to eight fold for severe obesity. Several genes exhibit mutations that can cause early onset severe obesity. These mutations are rare and account for only a small fraction of the cases of obesity. At this time, more than fifty genes have been shown in various studies to influence the energy balance, nutrient partitioning, or the age of onset of obesity. The results of these studies are generally disappointing and often contradictory. One approach is to scan the genome with a high number of polymorphic markers to identify chromosomal regions harboring genes implicated in the development of obesity. Such studies can be helpful in defining new targets to explore. PMID- 12371267 TI - [Physiopathology of obesity]. AB - Obesity results from an imbalance between energy intake and energy expenditure. Human studies indicate that obese individuals have an increased basal metabolic rate secondary to an increased fat-free mass. A blunted dietary thermogenesis is observed but is not of sufficient magnitude to lead to a major weight gain. Indirect evidence suggests that physical activity may be low in obese individuals. In healthy lean subjects, overfeeding leads to a stimulation of spontaneous physical activity. The ensuing increase in energy expenditure may play a role in the prevention of weight gain. This response, however, shows a high interindividual variability. There is overall little evidence that major alterations of energy expenditure are present in obese individuals. It is likely that an alteration of mechanisms of food intake control plays a prominent role in the pathogenesis of this disease. PMID- 12371268 TI - [Obesity: therapeutic aspects]. AB - Obesity is now recognized as a chronic disease. Its treatment implies a prolonged negative energy balance, by reducing caloric intake and/or increasing energy expenditure. In practice, three therapeutic approaches can be considered: 1) life style modifications, combining well-balanced hypocaloric diet and regular physical exercise, the key-issue in obesity management; 2) in case of failure and as adjunct treatment, anti-obesity drugs, especially orlistat, an intestinal lipase inhibitor, and sibutramine, a central appetite regulator; and 3) in patients with extreme refractory obesity, surgical procedures consisting of gastric restriction (gastroplasty) or intestinal bypass. Anti-obesity treatments must be evaluated in the long run, in terms of efficacy/safety ratio, upon criteria of weight loss, reduction in associated risk factors, improvement of quality of life and, if possible, reduction of morbidity and mortality. PMID- 12371269 TI - [Clinical genetics at the crossroads: the example of hereditary kidney diseases]. AB - Clinical genetics has reached a crossroads. Indeed recent progress in molecular genetics has generated great hope. This progress has consequences beyond genetic diseases, leading to the understanding of much more general mechanisms of disease. This progress has also permitted a better classification of genetic entities, such as Alport's syndrome, based on the molecular mechanisms involved. Lastly this progress allows us to design pharmacologic therapeutic approaches. Enzyme replacement therapy in Fabry's disease is a recent example of this approach. Difficulties are much greater in diseases such as autosomal dominant polycystic kidney disease. To reach these goals, height collaboration between molecular genetics and cell biology in the post-gene era, and between clinical genetics and other medical specialties, is required not only in the field of pediatric diseases, but also in late-onset genetic diseases. PMID- 12371270 TI - [Is it possible to prevent prostate cancer?]. AB - The present paper gives a comprehensive overview of recent data, especially prospective randomized trials, which support an important role for nutrition in the development of prostate cancer. Prostate cancer seems to be an ideal candidate for chemoprevention, in order to interfere by modification of nutritional habits with its onset, its incidence and ultimately with its progression, especially in high risk groups. PMID- 12371271 TI - [The neural crest and evolution of vertebrates]. PMID- 12371272 TI - [Genes: movers of life and disease]. AB - Genes are elements of the genetic material (deoxyribonucleic acid, dna). They code for a protein product. The human genome contains 35,000 genes. Half of our genetic information is inherited from our father, the other half from our mother. Mitrochondria and their DNA are entirely of maternal origin, which allowed Bryan Sykes to establish that modern human subjects can be classified into seven different lineages. The seven daughters of Eve, Bryan Sykes, 2001). Some genes code for structural proteins, some others code for regulatory ones. The efficient control of the cell cycle is carried out by numerous "normal" proteins. Some proteins, mutated on "abnormally" regulated are inducers of diseases, such as cancers or degenerative diseases. PMID- 12371273 TI - [Identification of cancer antigens of relevance for specific cancer immunotherapy]. AB - Cytolytic T lymphocytes (CTL) play a major role in the recognition and destruction of tumor cells by the immune system. In the last ten years, our team has identified at the molecular level a number of markers, called antigens, whose presence at the surface of tumor cells allow CTL to recognize such cells. Some of these antigens, including those encoded by the MAGE genes, are absent on all normal cells, and therefore constitute ideal targets for cancer vaccines aimed at increasing the activity of anti-tumor lymphocytes. Such vaccines are currently tested in clinical trials with melanoma patients. These antigens consist of small peptides that are presented by HLA molecules and that result from the degradation of intracellular proteins. This degradation is performed by an intracellular proteolytic complex called the proteasome. We recently observed that dendritic cells, which in the lymph node are responsible for antigen presentation to the lymphocytes in order to initiate the immune response, are inefficient to produce some peptides because they contain a different proteasome called "immunoproteasome". This unexpected observation may have important implications for the choice of vaccination strategies. PMID- 12371275 TI - [Gigantism: a mystery explained]. AB - Acromegaly was first described by Pierre Marie in 1886. Some years later, it became clear that acromegaly and gigantism share the same etiology: GH hypersecretion due to a pituitary adenoma, induces gigantism if already present during puberty, or an acromegalic if it appears only during the adulthood. During the XXth century, the disease has been well described and is now well controlled with several treatments. Recently, genetic alterations responsible for the disease have been elucidated. PMID- 12371274 TI - [Medically assisted reproduction in couples carrying the human immunodeficiency virus]. AB - Today, in developed countries, many HIV-infected people remain in good health thanks to antiviral medication, and a growing number of them want to have children. The benefit of resorting to assisted procreation and the contamination prevention strategies, throughout pregnancy, are summarized as well as the changes in ethical considerations. The balance between the importance of the message of prevention and the benefit for patients of being assisted in their desire for a child, has evolved towards a growing interest for medical intervention in order to avoid the risks of spontaneous conception outside health care structures. We are presenting the medical structure adapted at Erasme hospital and the 38 first requests taken into account by our pluridisciplinary team. This approach, which is coherent from a scientific point of view, respects both the autonomy of people, carrying HIV as well as the essential interest of the child, in being born uninfected, and also has the enormous advantage of allowing access to parenthood without destroying the consistency and coherence of the message of prevention of sexual contamination. PMID- 12371276 TI - [Progress, checks and errors in arterial surgery during the last two decades]. AB - The Advancement of Vascular Surgery since the eighties. During the last two decades, Vascular Surgery became a specialty, vascular technics and materials were greatly improving and surgical indications have been rationalized. Results greatly improved. However they currently came up against an incompressible rate of complications. It explains the increasing place of endovascular therapies and sporadic experiments of laparoscopic surgery. As all procedures remain associated with early and late risks, vascular surgeons should systematically monitor their results in order to maintain them at the best level. PMID- 12371278 TI - [From I to we]. PMID- 12371277 TI - [Hematopoietic stem cells: source, indications and perspectives]. AB - The haematopoietic stem cell (HSC) has been first described in the mouse and now identify in human as well. Exposed to a cocktail of growth factor, this HSC can self re-new and/or differentiate into the three lineages we have in the peripheral blood. These HSC are of major importance in the clinics since they can be used for some marrow (or stem cell) transplantation, and lead to the cure of a number of malignant and non malignant hemopathies. We have today three sources of HSC: the bone marrow, the mobilized peripheral blood stem cell and the cord blood. Bone marrow used to be the classical source of HSC after harvesting by aspirations in the iliac crest. However, this approach is now supplanted by the recovery of HSC in peripheral blood using a cell separation after four days of G CSF administration. These are several advantages of this technique, but the most important one is the more rapid hematopoietic recovery after transplantation, reducing the risk of infection and transfusion. A recent source of HSC is the umbilical cord blood. At the moment of delivery, the cord blood is extremely enriched in HSC due to the migration of these cells from the liver to the bone marrow stroma, where they will persist after birth. We have learned that the marrow stroma display a major role in the regulation of hematopoiesis and the pathogenesis of several malignant hemopathies can be explained by disturbance in the function of stromal cell. We have particularly studied the patho-genesis of chronic lymphocytic leukaemia. We have also observed that a subpopulation of stromal cells, the mesenchymal cells are of major importance in the microenvironment. In addition, the plasticity of these cells is demonstrated in vitro and we have currently a research program investigating its differentiation in neural cells. All these observations bring new promises in the treatment of hemopathies but also in some other neurological degenerative diseases. PMID- 12371279 TI - [Quo vadis, nursing care? Critique of common sense]. PMID- 12371280 TI - [Outline of a regulation for nursing specialties: least common denominator or fragile compromise?]. PMID- 12371281 TI - [Outlines of regulations for changing nursing education--position: a demand for action]. PMID- 12371282 TI - [Presentation of student dissertations: shared knowledge is better practice]. PMID- 12371283 TI - [EasySoft. user meeting in Berlin: progress often needs an impetus]. PMID- 12371284 TI - [Metabolic syndrome: the trap of wealth]. PMID- 12371285 TI - [Diet therapy in hyperuricemia and gout: warnings about meat and alcohol]. PMID- 12371286 TI - [Thyroid diseases in the elderly: masked symptoms]. PMID- 12371287 TI - [Nutrition in the hospital: taking eating seriously again]. PMID- 12371288 TI - [Positions on the election: politics throws the ball to nursing]. PMID- 12371289 TI - [Learning in nursing: a sweat-producing story]. PMID- 12371290 TI - [Systemic organization counseling in the hospital: communicating--but properly]. PMID- 12371291 TI - [Basics in work relations: personnel planning--what to observe?]. PMID- 12371292 TI - [Artificial nutrition by PEG catheters--an ethical orientation: respecting human dignity]. PMID- 12371293 TI - [How do homosexual men experience nursing care?]. PMID- 12371294 TI - What is a truly innovative drug? New definition from the International Society of Drug Bulletins. PMID- 12371295 TI - Collaborating with pharmaceutical research. Family physicians beware! PMID- 12371296 TI - Conscientious family physicians and polypharmacy. PMID- 12371297 TI - Up-to-date information omitted. PMID- 12371298 TI - Funding support for primary care research. PMID- 12371299 TI - Cochrane reviews not so useful. PMID- 12371300 TI - Taking ginger for nausea and vomiting during pregnancy. AB - QUESTION: Many of my patients prefer to use natural or herbal medicines, such as ginger, before taking drugs to treat nausea and vomiting of pregnancy. Is there evidence that ginger is safe to use during pregnancy? Is it effective? ANSWER: Although ginger is used in many cultures to treat the symptoms of nausea and vomiting, no trials have established its safety for use during pregnancy. On the other hand, its efficacy has been documented in two randomized, blinded controlled trials. PMID- 12371301 TI - Grapefruit. PMID- 12371302 TI - Lengthy prescription refills. PMID- 12371303 TI - Do cyclooxygenase inhibitors increase risk of cardiovascular thrombotic events? PMID- 12371304 TI - Cyclooxygenase-2 inhibitor update. PMID- 12371305 TI - Dealing with office emergencies. Stepwise approach for family physicians. AB - OBJECTIVE: To develop a simple stepwise approach to initial management of emergencies in family physicians' offices; to review how to prepare health care teams and equipment; and to illustrate a general approach to three of the most common office emergencies. QUALITY OF EVIDENCE: MEDLINE was searched from January 1980 to December 2001. Articles were selected based on their clinical relevance, quality of evidence, and date of publication. We reviewed American family medicine, pediatric, dental, and dermatologic articles, but found that the area has not been well studied from a Canadian family medicine perspective. Consensus statements by specialty professional groups were used to identify accepted emergency medical treatments. MAIN MESSAGE: Family medicine offices are frequently poorly equipped and inadequately prepared to deal with emergencies. Straightforward emergency response plans can be designed and tailored to an office's risk profile. A systematic team approach and effective use of skills, support staff, and equipment is important. The general approach can be modified for specific patients or conditions. CONCLUSION: Family physicians can plan ahead and use a team approach to develop a simple stepwise response to emergency situations in the office. PMID- 12371306 TI - Research electives in rural health care. AB - PROBLEM BEING ADDRESSED: As academic medical institutions being to address the education and service needs of rural Canadians, research will make its way to the foreground. Rural physicians are well positioned to lead in this venture, but often have little time or energy to take on extra duties. Rural populations differ in essential ways from urban populations. Certainly, the limitations of geography, funding, and population density alter medical surveillance, treatment, and research in ways that are largely undocumented. OBJECTIVE OF PROGRAM: To undertake research projects of interest to our group of rural clinicians and to expose medical students to both research and rural practice. MAIN COMPONENTS OF PROGRAM: Seven rural family physicians welcomed medical students into their group practice for summer research electives. Topics were chosen in advance by the medical group, and one member was designated as supervisor for each student. A local nurse educator also provided support to students and to clinicians after the students' departure. Several projects were undertaken simultaneously each summer; the result was several published peer-reviewed articles and good teaching and learning experiences. CONCLUSION: Rural research electives provide a valuable experience for students and preceptors. Such initiatives deserve broad promotion and support. PMID- 12371307 TI - New drugs with novel therapeutic characteristics. Have they been subject to randomized controlled trials? AB - OBJECTIVE: To determine how many randomized controlled trials on the safety or efficacy of new drugs are published when these drugs are first marketed in Canada, and to determine the quality of the information in those trials. DESIGN: A MEDLINE search was conducted on each drug identified as having novel therapeutic characteristics and first marketed between 1990 and 2000. MAIN OUTCOME MEASURES: Number of trials dealing with the safety or efficacy of each drug published at the time the drug was marketed. Number of patients taking the study drug, length of the trial, and type of control. RESULTS: The number of trials varied substantially. For some drugs, there were more than 20 studies; for others only a single study. Many trials were small and short-term, and used placebo controls. CONCLUSION: Too few trials or inadequate trials on the safety and efficacy of new drugs are published when these drugs are first marketed in Canada. The lack of published trials means that physicians do not know whether results are generalizable to their patients, how to position the drug in relation to other treatments, or whether the drugs have long-term safety and efficacy. PMID- 12371309 TI - Short report: parental knowledge of rectal acetaminophen. PMID- 12371308 TI - Preventive screening. What factors influence testing? AB - OBJECTIVE: To determine factors associated with having preventive screening tests in a population-based sample of Ontario women. DESIGN: Secondary analysis of data from Statistics Canada's National Population Health Survey linked to data from the Ontario Health Insurance Plan to ascertain whether women aged 20 or older had Pap smears, mammography, bone densitometry, or cholesterol testing. Factors associated with having testing were subjected to logistic regression analysis. SETTING: Ontario. PARTICIPANTS: Women aged 20 or older; from 19,600 Canadian households, 2232 Ontario women gave consent to linkage of administrative databases. MAIN OUTCOME MEASURES: Age-specific population screening rates. Odds ratios and probabilities of having screening in relation to socioeconomic, geographic, and physician-associated factors. RESULTS: Having screening was associated with age, income, education, and place of residence. Women with regular physicians were more likely to have Pap smears (odds ratio [OR] 4.4, range 1.7 to 12), densitometry (OR 22, range 3.6 to 140), and cholesterol testing (OR 8.0, range 2.3 to 29). Women who had periodic health examinations were more likely to have Pap smears (OR 6.7, range 4.6 to 9.8), mammograms (OR 3.7, range 2.3 to 5.9), densitometry (OR 3.7, range 1.3 to 10.5), and cholesterol testing (OR 3.0, range 2.0 to 4.5). The probability of having testing increased with number of visits a year to a doctor, but ceased to increase after three visits. CONCLUSION: Having screening tests was associated with socioeconomic factors including income, education, and place of residence. Patients who went to doctors for episodic care only were less likely to have preventive screening than patients who went for periodic health examinations. PMID- 12371310 TI - Infectious disease guides. For users of hand-held computers. AB - The Sanford Guide is an independent, comprehensive authoritative reference on treatment of infectious disease . It can be difficult to navigate, and it costs $25 (US). The ePocrates ID program is smaller but is quicker to use. It also interfaces with a drug database. The ABX POC-IT Guide is well designed and includes antibiotic monographs but currently has the least extensive disease database of the three programs, and furthermore, it lacks pediatric data. All three programs appear to use evidence-based recommendations. PMID- 12371311 TI - Residents' page. PMID- 12371312 TI - [Characteristics of the clinical picture of combined injuries of the organ of vision and eye appendages under conditions of peace-time and war injuries]. AB - A total of 100 patients with combined injuries of the organ of vision and eye appendages, inflicted in peacetime and under war conditions, were examined. The clinical picture of combined injuries in the wounds inflicted at the battlefield (gunshot and resultant from mine explosions) and of traffic, communal, criminal, and occupational injuries was studied using traditional ophthalmological and common clinical instrumental methods of examination. Common regularities of the traumatic process were revealed in combined injuries: mutual aggravation of injuries to the eye and its appendages, a considerable incidence of bilateral injuries, and simultaneous impacts of several damaging factors. The most unfavorable prognosis as regards visual functions in the group observed was for patients with war injuries, particularly those caused by mine explosions. PMID- 12371313 TI - [Laser instrumental surgery in prevention and treatment of retinal detachment. I]. AB - Detachment of the retina, developing in the presence of aggravating factors, and its elimination by means of an original laser surgical method became the object of this study. Visulas YAG laser (Karl Zeiss) fitted with an endolaser output was used. Coagulation energy was 0.2-0.9 Wt, YAG laser energy 0.1-9.2 mJ, up to 170 pulses. Analysis was carried out in 38 patients with subretinal adhesions, incarceration of the retina in the scleral wound, and rupture of the retina with its edge folded and elevated. All laser interventions were operations of choice and became the first stage in laser surgical treatment of detachment of the retina. Relapses occurred in 10 (27.7%) cases. PMID- 12371314 TI - [Possibilities of immunosuppressive therapy in reconstructive keratoplasty (experience gained in the use of cyclosporin)]. AB - Cyclosporin was used for preventing the graft rejection reaction after reconstructive operations on the anterior segment of the eye. Analysis of the results of surgical treatment of patients with vascularized corneal leukomas of different etiology in 23 patients (25 eyes) who received postoperative oral cyclosporin therapy and in 25 patients to whom this drug was instilled showed that cyclosporin promoted transparent healing of the keratotransplant and its use was not associated with serious side effects. PMID- 12371315 TI - [Correction of high astigmatism and astigmatic anisometropia by intrastromal photokerato-ablation in children and adolescents]. AB - A total of 151 photorefraction operations for correction of medium and high astigmatism in 38 children (9-15 years) and 47 adolescents (16-17 years) carried out by LASIK method are analyzed. The operations were performed under local anesthesia with EC-5000 eximer laser (Nidek) and Hansatome microkeratome (B & L). Complicated and common myopic astigmatism (2.07 +/- 0.89 to 2.91 +/- 0.63 diopters) was decreased by 61.2% in children and by 62.2% in adolescents. Visual acuity without correction increased by 0.58 +/- 01.2. Correction of astigmatic anisometropia resulted in its decrease to 0.75-1.5 diopters in 77.9% children and 62.8% adolescents. Hypermetropic astigmatism decreased by 64.9% of its initial value in children and by 56.9% in adolescents. Mixed astigmatism decreased by 62.7% of its initial value. The resultant visual acuity without correction was 0.39 +/- 0.13 in children and 0.47 +/- 0.17 in adolescents. According to questionnaires distributed among parents, 13.5% patients studied better at school after the operation and 19.2% went in for sports in athletic sections. The main causes for the intervention were limitations in choice of profession (for 49.6%) and desire to practice modern athletics (in 13.8%). Photorefraction correction of astigmatism in children and adolescents brings about an effective stable result decreasing refraction amblyopia and improving the quality of life. PMID- 12371316 TI - [Morphological changes in failure of the lenticular ligamentous-capsular system]. AB - Morphological changes in case of failure of the lenticular ligamentous-capsular system are described. Histological changes in this complex led the authors to the conclusion that morphological substrata of the "traumatic" and "surgical" lenticular subluxation are similar and are characterized by coarse mechanical changes in the lens capsule, with the relatively intact structure of the ligamentous system at a considerable length. Subluxation of the lens resultant from oral drug therapy is characterized by pronounced changes in the lens capsule and in the short cinnous ligament fibers. Age-associated or involution subluxation is caused primarily by pathological changes in the lens capsule, which seems to experience high strain from the remaining zonular fibers. PMID- 12371317 TI - [Neuro-ophthalmological symptoms in patients with para-clinoid aneurysms]. AB - Neuroophthalmological symptoms in patients with large and giant paraclinoid aneurysms is presented, based on analysis of case histories, examination of visual functions, and evaluation of ophthalmoscopic picture. Variants of paraclinoid aneurysm location with respect to optic tract structures and the visual function are discussed. Methods of modulating the peripheral and central neurons of the optic tract are described. PMID- 12371318 TI - [Clinical informative value of new methods of ultrasonic examination in the differential diagnosis of various forms of congenital diseases of the eyes in children]. AB - Clinical informative value of computer echography for the differential diagnosis of severe intrauterine uveitis (68 children, 96 eyes), retinopathy neonatorum (37 children, 74 eyes), Coats' retinitis (35 children, 35 eyes), and retinoblastoma (17 children, 26 eyes) was studied in a group of 157 children (231 eyes) with opaque refracting media. Ultrasonic diagnostic equipment with a high level of computer support essentially improved the resolving capacity of echography and detected accessory acoustic signs of some congenital ocular diseases. PMID- 12371319 TI - [Study of the nervous-vascular reactivity of the bulbar conjunctiva in patients with primary open-angle glaucoma with normalized intraocular pressure]. AB - Clinical evaluation of adrenoreception was carried out in 96 patients with various clinical patterns of primary open-angle glaucoma with normalized intraocular pressure by changes in vascular reactivity in the bulbar conjunctiva capillaries in response to epinephrine and norepinephrine. Neuro-vascular reactions of the bulbar conjunctiva in response to norepinephrine and epinephrine were different in patients with different patterns of primary open-angle glaucoma with normalized intraocular pressure. A significant correlation between the rate of progress of optic disk glaucomatous atrophy and the parameters of ocular adrenoreceptor structures of the eye was detected. PMID- 12371320 TI - [Significance of serotonin in the pathogenesis of diabetic retinopathy and central chorioretinal dystrophy]. AB - A total of 147 patients with preproliferative and proliferative diabetic retinopathy (DR), with the exudative hemorrhagic and nonexudative stages (93 females and 54 males), were consulted at Helmholtz Institute of Ophthalmic Diseases in 1997-2001. Control group consisted of 39 healthy subjects aged 25-76 years. The group of patients with preproliferative DR consisted of 27 patients with compensated diabetes mellitus. The course of diabetes in 39 patients with proliferative DR was evaluated as medium severe during the subcompensation stage. The clinical picture of the fundus oculi was characterized by pronounced hemorrhagic activity. Slight retinal hemorrhages were seen in the patients with preproliferative DR. Nodules, defects of pigmented epithelium, atrophic foci were seen in the central zone of the fundus oculi in patients with nonexudative stage of central chorioretinal dystrophy; edema in the central zone, polymorphic hemorrhages, solid exudate were observed in the patients with the exudative hemorrhagic stage; subretinal neovascular membrane was detected in some patients. Erythrocyte deformability coefficient, platelet aggregation coefficient to ADP, and platelet factor were evaluated by common methods. Serotonin was measured by the fluorometric method (B. M. Kogan's method) at clinical biochemical laboratory of urgent methods of examination of N. V. Sklifosovsky Institute of Emergency. The erythrocyte deformability coefficient was notably increased in the patients with proliferative DR and central chorioretinal dystrophy in comparison with the normal value. Plasma serotonin concentration was increased significantly only in the patients with proliferative DR, while in the rest groups this concentration was notably decreased. Study of platelet aggregation gave contradictory results. The values of platelet factor 4 differed from the control negligibly. Serotonin insufficiency in patients with proliferative DR was paralleled by increased plasma serotonin concentration and thrombocytopathy. In patients with preproliferative DR and central chorioretinal dystrophy serotonin insufficiency was associated with decreased concentration of serotonin and thrombocytopathy. Erythrocyte rigidity was similarly increased in patients with proliferative DR and central chorioretinal dystrophy. PMID- 12371321 TI - [Study of the effectiveness of ultraviolet irradiation of blood in the treatment of traumatic uveitis]. AB - Sixty-five patients (65 eyes) with traumatic uveitis were treated. Ultraviolet irradiation of autoblood was included in therapeutic complexes of 28 patients. 37 patients received traditional therapy (corticosteroids, nonsteroid inflammatory agents, etc.). Addition of UV exposure of autoblood to combined therapy for traumatic uveitis more effectively (92.9 vs. 75.7%) and sooner liquidated posttraumatic inflammatory reaction (8.10 +/- 1.5 vs. 12.7 +/- 1.7 days), decreased the hospital stay (11.0 +/- 2.0 vs. 15.8 +/- 1.3 days), and eventually more often improved the visual acuity (in 42.9 vs. 24.3% patients). Hence, UV exposure of autoblood is an effective, safe, and virtually atraumatic method of treatment. PMID- 12371322 TI - [Keratocyte apoptosis in keratoconus]. AB - Analysis of 37 corneal disks removed in perforating keratoplasty for stages II-IV keratocone and of 15 corneal disks (control) from subjects dead from mechanical injuries at the age of 17-35 years was carried out. The expression of CD95 receptors on keratocyte membranes was evaluated by indirect immunofluorescent label using rabbit antibodies. Keratocyte apoptosis was evaluated by indirect immunofluorescent labeling of nuclear DNA nucleotides. Estimation of the volumic share of keratocytes expressing CD95 on cell membrane showed it to be almost 8 fold higher and apoptosis index almost 5-fold higher in the keratocone corneal disks than in the control. The results indicate the presence of a Fas-mediated mechanism of keratocyte apoptosis and its manifold predominance in the cornea in keratocone. These results are clinically significant for the choice of pathogenetic therapy of patients with keratocone. PMID- 12371323 TI - [Video technology of qualitative control of eyeball movements in nystagmus]. AB - A method for controlling voluntary movements of the eye is suggested, based on video-image processing and computer analysis. The program can be used for analysis of eye movements by its trajectory and spectrum. Results of measurements of voluntary movements of the eye in patients with nystagmus are presented. The results of measurements, characterizing the changes in nystagmus before and after operation for strabismus correction are discussed. PMID- 12371324 TI - [A rare clinical case of Sipple disease]. AB - Multiple endocrine neoplasia (MEN) unites a group of syndromes caused by tumors or hyperplasia of several endocrine glands. We examined and treated patients (father and daughter) with Sipple disease, or type II MEN; the diagnosis was confirmed at Institute of Clinical Endocrinology (Moscow). The aim of our report was to supplement the clinical picture of the disease. Clinical examinations showed thickened corneal nerves, the dry eye syndrome, and abnormal refraction. PMID- 12371325 TI - [A case of long-lasting keratitis caused by a fragment of the eyelash grown through the upper eyelid cartilage]. PMID- 12371326 TI - [Autoregulation of ocular vessels in health and in primary open-angle glaucoma]. PMID- 12371327 TI - [Use of cytokines and their complexes in ophthalmology]. PMID- 12371328 TI - [Present-day automated perimetry]. PMID- 12371329 TI - [Tikhon Ivanovich Eroshevskii (the 100th anniversary of his birth)]. PMID- 12371330 TI - [Triplex scanning of orbital vessels in patients with normal pressure glaucoma]. AB - Bloodflow in the orbital artery and central retinal artery was studied by triplex scanning in 35 patients with normal pressure glaucoma, and 14 subjects without signs of glaucoma. Significant differences in the studied parameters were detected in the patients in comparison with the controls: decreased linear bloodflow velocity in the orbital (p < 0.01) and central retinal arteries (p < 0.01), increased peripheral resistance indexes in both arteries (p < 0.05) in patients with normal pressure glaucoma, and decreased bloodflow velocity in the central retinal artery in primary open-angle glaucoma (p < 0.05). The time course of hemodynamic parameters was different in normal pressure and primary open-angle glaucoma. These data confirm the important role of vascular disorders in the pathogenesis of normal pressure glaucoma. PMID- 12371331 TI - [Surgical strategy in the treatment of severe and extremely severe eye burns. 2]. AB - The author presents the results of using his own surgical strategy in multiple modality treatment of 76 patients with severe and extremely severe burns of the eyes. He proves that active surgical strategy during the early period of burn process notably decreased the incidence of complications, preserved the eyeball for further reconstructive operations, and thus improved the functional outcomes of severe burns of the eyes. PMID- 12371332 TI - ICD-9-CM committee discusses new diagnosis proposals. PMID- 12371333 TI - Taking a closer look at physician-based coding. PMID- 12371334 TI - Facing new challenges every day. PMID- 12371335 TI - Laboratory services regulations impact national coverage decisions, HIM. AB - This is Part 2 of a two-part article on laboratory services regulations. Part 1, which appeared in the September Journal of AHIMA, dealt with the administrative policies contained in these regulations. Part 2 addresses the specific national coverage decisions that were developed as part of this regulatory process. For more information on each coverage policy, review Addendum B of the regulations. PMID- 12371336 TI - The first line of defense against privacy complaints. PMID- 12371337 TI - Keeping current on legislation vital to HIM professionals. PMID- 12371338 TI - Moving forward with document imaging and never looking back to paper. AB - Many healthcare facilities are exploring document imaging solutions and enjoying the benefits of increased accessibility and faster workflows. The transition requires significant planning, however. In this article, learn how several healthcare organizations implemented document imaging systems and how their facilities have changed as a result. PMID- 12371339 TI - Implementing a document imaging system. AB - The benefits of a document imaging system are myriad once it's installed--it's the implementation that you need to prepare for. In this article, get a bird's eye view of one facility's implementation and the many lessons learned along the way. PMID- 12371340 TI - Turning production data into management tools. AB - Staring down another backlog in your HIM department? Use routine data collection activities to strengthen your case for additional staff, increased budget dollars, or an incentive program. PMID- 12371341 TI - Tackling tough management issues: advice from the top. AB - Wondering how your peers across the country are managing the myriad challenges that confront HIM professionals? We assembled a panel of experts to address common problems and concerns. PMID- 12371342 TI - Professional practice solutions. When are history and physical (H&P) requirements for outpatients necessary? PMID- 12371343 TI - A closer look at clinical pertinence standards. PMID- 12371344 TI - Mind your business associate access: six steps. PMID- 12371345 TI - Practice brief. Implementing the minimum necessary standard. PMID- 12371346 TI - Creating privacy rule implementation efficiency. PMID- 12371347 TI - The effect of fast speech rate on stuttering frequency during delayed auditory feedback. AB - Delayed auditory feedback (DAF) has been documented to improve fluency in those who stutter. The increased fluency has been attributed to the slowed speech rate induced by DAF, but recent experiments have suggested that increasing the speech rate may also decrease stuttering under DAF. This investigation described the effect of combining a fast speech rate and DAF on the fluency of four people who stutter. Fluency of the two mildly dysfluent subjects was the same for both no DAF and DAF conditions at normal and at fast oral reading rates. In contrast, the two severely dysfluent subjects improved in fluency from the no DAF to the DAF conditions. They were found to be dysfluent at both normal and fast oral reading rates without DAF. The results of the study point to the need for further research on the relationship between speech rate and stuttering frequency under conditions of DAF and no DAF. EDUCATIONAL OBJECTIVES: Readers will learn about and be able to describe how the frequency of stuttering is affected by: (1) speech rates; (2) DAF; and (3) how stuttering severity influences such effects. PMID- 12371348 TI - An experimental investigation of the impact of the Lidcombe Program on early stuttering. AB - Preliminary Phase I and II trials for the Lidcombe Program of early stuttering intervention have found favorable outcomes and that the treatment is safe. Although speech-language pathologists (SLPs) often need to intervene with pre schoolers' early stuttering, many of these children will recover at some time in the future without such intervention. Consequently, they need to know whether the Lidcombe Program's effect on stuttering is greater than that of natural recovery. Participants were 23 pre-school children who were randomly assigned to either a control group or a treatment group that received the Lidcombe Program for 12 weeks. A repeated measures ANOVA showed no main effect on stuttering for the group (control/treatment), a significant main effect for the measurement occasion (at the start and at the end of the treatment period), and a significant interaction between group and measurement occasion. Stuttering in the treatment group reduced twice as much as in the control group. These results are interpreted to mean that the introduction of the Lidcombe Program has a positive impact on stuttering rate, which exceeds that attributable to natural recovery. PMID- 12371349 TI - Laryngeal anesthetization for the treatment of acquired disfluency: a case study. AB - The subject of this case study is an adult who became severely disfluent after a motor vehicle accident in which he did not suffer significant injuries. His disfluency persisted for 4 months notwithstanding a short trial of speech therapy. Hyperfunctional phonation subsystem disturbances were identified on follow-up evaluations. Laryngeal anesthetization was achieved via a transcutaneous lidocaine injection. The patient improved dramatically within 15 min of the procedure. More than 18 months later, he has retained normal speech fluency without any additional post-injection intervention. Theoretical discussions are rendered to help interpret this treatment outcome. EDUCATIONAL OBJECTIVES: The reader will be able to describe (1) the laryngeal movements and adjustments commonly associated with stuttered speech; (2) the speech dysfunctions a patient evidenced following a motor vehicle accident; and (3) the author's treatment rationale for resolving the patient's speech disorder. PMID- 12371350 TI - National Stuttering Association members' opinions about stuttering treatment. AB - As stuttering support groups, such as the National Stuttering Association (NSA), have gained prominence and visibility, it has become increasingly important for speech-language pathologists (SLPs) to learn about the people who participate in such groups. This article presents results of a brief survey completed by 200 members of the NSA to examine the opinions of support group members regarding the field of speech-language pathology and stuttering treatment options. Results indicate that NSA members hold a variety of opinions, both positive and negative, about the resources available to them. Findings highlight a number of ways in which SLPs can work with stuttering support groups, both to learn more about the needs of people who stutter and also to provide needed information about treatment options that are available for people who stutter. EDUCATIONAL OBJECTIVES: The reader will learn about and be able to describe (1) the role of support groups in stuttering treatment; (2) the people who are members of the NSA; and (3) their opinions about various issues related to stuttering and its treatment. PMID- 12371351 TI - Recasts in parents' language to their school-age children who stutter: a preliminary study. AB - Language samples collected from 13 school-age children diagnosed as stutterers (CWS) in conversation with a parent and a similar corpus of data collected from a matched control group of 13 normally-fluent children (CWNS) were analyzed for the quality and quantity of recast usage in the parents' language. Recasts, a frequently studied type of child-directed language (CDL) pattern hypothesized to increase the length and complexity of young children's expressive language, have not been studied typically in older children's conversations with parents nor have they been studied in the CDL of parents of CWS. Comparisons of these samples revealed that both groups of parents were frequent users of imitations, as well as simple and complex type recasts. Indeed, the rank order of recast types used was identical for the two groups of caregivers. When parents' conversations with the CWS were analyzed further, it was noted that they were not more likely to focus on stuttered utterances as the utterances on which to base their recasts (i.e., "platform utterances") than on the child's fluently-produced utterances. These results suggest there is one additional conversation parameter for which there are evident similarities in the CDL used by parents of CWS and CWNS. Clinical implications gleaned from this preliminary study are discussed, as are suggestions for future study. EDUCATIONAL OBJECTIVES: The reader will learn about and be able to describe (1) the role of recasts in young children's language development; (2) the types of recasts that were observed in the conversations of parents of CWS and CWNS; and (3) the similarities observed in the recasts produced by parents of CWS and CWNS. PMID- 12371352 TI - [Adverse events following immunization with BCG vaccine in Poland 1994-2000]. AB - BACKGROUND: In Poland, since 1955 BCG mass vaccinations have been compulsory. During the last two decades the vaccination program has been developed and includes: 1) primary vaccination of the newborn, 2) vaccination of children aged 7 and 12, and 3) revaccination of 18 year old adolescents. More than 95% of newborns and about 80% of older children of this population have been vaccinated. BCG vaccine being in use in Poland bases on the Brazilian/Moreau sub strain and has been producing by the Polish Laboratory. Since 1994 the central register of adverse events of BCG has been introduced by National Tuberculosis and Lung Diseases Institute (NTLDRI). OBJECTIVES: The aim of this study was to evaluate the frequency, trends and clinical type of adverse events after BCG vaccination. In addition, correctness of case-finding and treatment of BCG complications was evaluated. METHODS: From January 1, 1994 to December 31, 2000 individual data on adverse events, on special card was reported to NTLDRI. Categories of BCG complications was based on classifications proposed by A. Lotte. RESULTS: Data of 7,354,780 BCG vaccinated persons was analyzed. Of them 1,559 cases reported AEFI. From the further analysis 98 cases with insufficient data were excluded. The frequency of these cases has been estimated to be 0.2@1000 (2 per 100,000 vaccinated) and was stabile. Among AEFI cases, 773 reported local complication (ulcer, abscess, K.F.) and 647 (44.3%) lymphadenitis including 353 cases with enlargement of lymph nodes and 287 cases of suppurative lymphadenitis (4 per 100,000 vaccinated). Majority of AEFI cases were infants (76%). Only 1 case (infant) with disseminated BCG disease, probably due to the partial interferon gamma receptor deficiency, was reported. Main errors in the diagnosis of complications and the management of adverse events after BCG vaccination were lack of data on the diameter of local changes and size of lymph nodes, as well as attempts to remove the lymph nodes. CONCLUSION: The results of the study indicate that BCG vaccinations in Poland rarely produce adverse reactions and therefore may be considered as safe method of tuberculosis prevention. Adverse reactions to BCG appears to be underreported. Surveillance of adverse reactions to vaccines should be included in the national immunization program. PMID- 12371353 TI - [Infectious diseases in Poland in 2000]. AB - The decreasing tendency in incidence of infectious diseases observed in Poland in previous years as compared with 2000 has weakened or stopped. Increase in the incidence of selected infectious diseases can be linked with the improvement of surveillance resulting from the better diagnostics and greater attention paid to these diseases (including borreliosis, salmonella, and Haemophilus influenzae meningitis). Between 1999 and 2000, the most intense decrease in the number of mumps, measles, and scarlet fever cases as an effect of the end of epidemics was observed. At the same time increase in the number of pertussis, rubella, chickenpox, and meningitis cases was noticed. In 2000, the first case of human rabies since 1986 has been reported. In 2000, compared with 1999, among all notified deaths percentage of deaths attributed to infectious diseases (0.83%) and infectious diseases death rate (0.79 per 10,000) were slightly higher and were the highest in the last decade. As in 1999 the observed increase was effect of the influenza deaths increase (358 deaths, mortality 0.022%). The main disease causing the largest number of deaths, as in previous years, was tuberculosis (36.5% of total infectious diseases deaths). PMID- 12371354 TI - [Measles in Poland in 2000]. AB - The decreasing tendency in the number of measles cases has been observed (incidence 0.2 per 100,000). No cases were noted in two voivodeships, in 6 voivodeship from one to two cases were noted. In the remaining 8 voivodeships the highest incidence was noted in Slaskie and Wielkopolskie voivodeships (0.4 per 100,000). Comparing to 1999 the incidence among children aged 0-4 decreased nearly three times from 1.4 to 0.5 per 100,000 whereas the incidence among children aged 8 and 9 and adults aged 20-24 increased. Serological confirmation of cases suspected for measles improved too slowly (assay for IgM)--out of 107 suspected cases only 49 (47.6%) were serologically tested. PMID- 12371355 TI - [Pertussis in year 2000]. AB - Reemergence of pertussis in a form of epidemic was observed in Poland in 1997/1998. After some decrease of incidence in 1999, year 2000 brought another increase in pertussis morbidity (2,269 cases; incidence 5.9/100,000), most of them in the older age group of 10-14. It indicates rather sustained trend of increased incidence and shift in the age of infected. It may be due to decrease of immunity with age. According to the vaccination calendar in Poland, last pertussis vaccination is given before the age of two. More cases occurred among females (6.3/100,000) than among males (5.4/100,000) and in urban areas (7.8/100,000) than in the rural ones (2.8/100,000). Big differences in numbers of cases reported between different districts and between urban and rural populations bring strong possibility of insufficient sensitivity of the surveillance in many regions of Poland. It is concluded that increase of pertussis incidence in Poland, causes urgent need of additional vaccination in the age of 5 to secure protection for the older age groups. PMID- 12371356 TI - [Scarlet fever in Poland in 2000]. AB - In 2000, as compared with 1999, a 14% decrease in the number of scarlet fever cases was noted. The incidence was 21.6 per 100,000 population and was one of the lowest since World War II. In particular voivodeships incidence ranged from 9.4 to 34.3 per 100,000 population. In urban areas the incidence was 71.3% higher than in rural ones. Of all registered cases 95.3% were children under 15 years of age. The age distribution of scarlet fever cases in 2000 was similar to the distribution observed in the last decade. The highest incidence was noted among children aged 6 and 5, respectively 257.0 and 224.4 per 100,000. About 2.1% of all cases were hospitalized. No scarlet fever deaths were noted. PMID- 12371357 TI - [Mumps in Poland in 2000]. AB - A total of 17,548 mumps cases were reported in Poland in 2000. A 5-fold decrease in incidence of the disease (from 233.4 to 45.4 per 100,000 population) was noted, when compared with 1999. Approximately 4.8% of mumps cases were hospitalized (849 cases). The majority of the reported cases were children aged 5 9 (51.4% of all cases). In all voivodships the incidence was lower than in previous years. The probable cause of the decline of mumps cases was a marked amelioration of MMR vaccination coverage among 3-year old children. The MMR vaccine is not included into the national program of immunization, the vaccination is recommended for 2-year and 7-year old children. PMID- 12371358 TI - [Influenza in Poland in 2000]. AB - In 2000 the number of cases of influenza and influenza-like illness (ILI) registered in Poland amounted to 1,596,920 (68.1% of 1999 cases). The highest influenza incidence was reported in Lodzkie voivodship (9,388.8 cases per 100,000). Among children aged 0 to 14 years the number of influenza and ILI cases amounted to 408,495 (incidence 5,518.8 cases per 100,000) and was 25.5% of the total number of cases recorded in 2000. The number of patients referred to hospital was 7,028 and 358 persons died due to influenza and its complications. No influenza strains were isolated in 2000. Immunofluorescence test confirmed infection with influenza type A in 39 patients, while with type B--in 2 patients. Sero-surveys carried out in the epidemic season 2000/2001 showed that the highest antihemagglutinin antibody levels were produced for influenza strain A(H3N2) and B. The highest antibody titers were recorded in the age group 15-25, while the lowest levels--in the age group 0-3 years. PMID- 12371359 TI - [Rubella in Poland in 2000]. AB - In 2000, as compared with 1999, a 49% increase in the number of rubella cases was noted. It corresponded to the development a new compensatory epidemic (the peak of the last epidemic was observed in 1997). A total of 49,181 cases were registered, including 1 case of congenital rubella. Incidence on the country level was 119.5 per 100,000 population. In particular voivodeships it ranged from 24.0 to 314.8. Incidence in urban areas was 35% higher than in rural areas. Incidence among women (109.0) was 16% lower than among men (130.6). One of the probable reasons of this difference was intensive immunization of 13-year old girls, which have been providing for several years. Among all registered rubella cases 94.2% were children under 15 years of age. The highest incidence was noted among 7-year old (1,216.0) and 6-year old (1,148.9) children. About 0.3% of rubella cases were hospitalized. No rubella deaths were noted. PMID- 12371360 TI - [Meningitis and encephalitis in Poland in 2000]. AB - A total of 2,033 cases of meningitis and 570 of encephalitis were reported in Poland in 2000. Among cases of meningitis 1,051 (51.7%) were classified as viral and 982 (48.3%) as bacterial. Etiological factors were determined in 36.7% (360/982) cases of bacterial meningitis. Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae were found in 10.3% (101/982), 8.7% (85/982), and 7.5% (74/982) cases, respectively. As in previous years, N. meningitidis typed B was strongly predominating. Out of 570 cases of encephalitis, 170 (29.8%) were tick borne, of which most were reported from endemic areas of north-eastern part of the country. PMID- 12371361 TI - [Salmonellosis in Poland in 2000]. AB - In 2000, 22,799 cases of salmonellosis were reported to the sanitary epidemiological stations, incidence rate = 59.0 per 100,000 population. Above 65% of patients were hospitalized. The seasonal peak was noted in May and June. Most of cases (70%) were laboratory confirmed by isolation Salmonella strains types. Salmonella Enteriditis was the most frequent type: 91% of cases and 70% of infected healthy persons. Other serotypes--Typhimurium, Infantis, Hadar and Virchow, caused 5% Salmonella cases only. Seven types not registered in the country up to 2000 were identified (S. Bargny, Kimuenza, Kisii, Limete, Nitra, Rissen, Winterthur). The most affected age group were children under five (337.3/100,000). The most serious clinical syndromes and extraintestinal manifestations like septicaemia, arthritis, meningitis, osteomyelitis pneumonia and other, were observed in 87 patients with at least one non-fecal specimen culture-positive for non-typhoidal Salmonella. In older patients, other diseases like carcinoma, leukaemia, lupus erythematosus, contributed to Salmonella infection. Twelve of those patients had died. PMID- 12371362 TI - [Dysentery in Poland in 2000]. AB - The notified number of dysentery cases is still falling down since the year 1995 when bacteriological examination of feces for diagnostic purposes lost free of charge status obtained in 1928 under the legislation act of infectious disease investigation and obligatory registration. Only 121 dysentery cases were notified in the year 2000 (incidence 0.31/100,000 population), while 292 cases were notified in 1999 (incidence 0.76/100,000). No one death case was notified. Only 2 outbreaks (one due to S. flexneri 4a and one due to S. sonnei), both involving 26 patients, were notified in 2000, less than in 1999 when four outbreaks involving 146 patients were registered. In the year 2000, S. sonnei was the dominant etiological agent in 80% of notified dysentery cases and S. flexneri only in 20% of cases was the next. S. dysenteriae 3 was found only in one patient S. boydii in no one. All Shigella strains were susceptible to nitrofurans (ex.nifuroxaside), gentamicin, nalidixic acid, cefotaxime, and imipenem. Only two strains, including the one important from India, were susceptible to co trimoxasole, but not to doxycycline. PMID- 12371364 TI - [Botulism in Poland in 2000]. AB - A total of 72 cases of botulism were registered in Poland in 2000, with corresponding incidence 0.19 per 100,000 population. In the rural areas 56 (incidence 0.38), and in the urban areas 16 (incidence 0.07) cases were registered. In 2000, there were 46 outbreaks of one person, 7 outbreaks of two people, and 4 of three people noted. Meat dishes were the main vehicle of the botulinum toxin (41 cases; 56.9%). Of them, prevailed homemade conserves (bottling jars) prepared from pork meat (18.1%). Home made sausages were associated with 13.9%, canned fish with 12.5%, sausages of commercial production- with 12.5%, and dishes from poultry with 11.1% cases. Two deaths from botulism were registered in Poland in 2000. PMID- 12371363 TI - [Foodborne infections and intoxications in Poland in 2000]. AB - The total number of 26,701 cases of bacterial foodborne infections and intoxications were registered in 2000. The incidence was 96.9/100,000 population. S. Enteritidis was found in 96.9% of cases in outbreaks caused by Salmonella sp. The main vehicle of foodborne infections and intoxications in outbreaks (in Poland defined as 4 and more sick people) was food prepared from eggs (44.4% cases in outbreaks, 54.4% cases caused by Salmonella). Private homes prevailed (56.1% of outbreaks) among the places of the ready made food production. Six epidemics with 100 and more cases each were registered. Four deaths were noted in outbreaks in 2000. PMID- 12371365 TI - [Intoxications caused by chemicals for plant protection in Poland in 2000]. AB - A total number of 107 cases of intoxications caused by chemicals for plant protection were registered in Poland in 2000 (incidence 0.28 per 100,000 population). The majority of intoxications (74.8%) occurred after an intake of substances. In 28.0% cases it was suicidal intake, in 43.0% cases accidental intake, and in 3.7% with food. In 15.9% cases exposure took place at agricultural labor. Insecticides caused 64.5% of the total number of cases. In rural regions 83.2% of the sick people were subject of intoxication by chemicals for plant protection and in the urban regions 16.8%. The incidence among men was higher than incidence among women (0.37 and 0.19 respectively). In 2000, no group intoxications were noted. In the result of intoxication by chemicals for plant protection seven people died. PMID- 12371366 TI - [Hepatitis B in Poland in 2000]. AB - A total of 2,825 (7.3 per 100,000 population) cases of hepatitis B (including 130 co-infections with both HBV and HCV) were reported in 2000 in Poland. Comparing to 1999 the total number of cases decreased by 683 and incidence by 1.8. This decline resulted mainly from vaccination of children and high risk patients though the improvement of sterilization procedures could be also of significance, especially in persons below 60 years of age. The number of hospitalizations in 2000 increased by 15%. Regional differences in both incidence and percentage of hospitalizations were observed. The incidence of hepatitis B per 100,000 was higher in urban (8.3) than in rural (7.4) population. The lowest incidence (0.6) was observed in children aged 0-4 years; in older children (5-9 years of age) the incidence rates were 8.6 in boys and 3.4 in girls. The highest incidence, ranging from 4.5 to 9.1 was found in the age group 14-24 years, especially in boys and young men living in urban areas. PMID- 12371367 TI - [Hepatitis C in Poland in 2000]. AB - In 2000, a total of 2,086 (5.4 per 100,000 population) acute and chronic hepatitis C cases were noted. The differences in incidence between groups were observed: incidence in urban areas (7.2 per 100,000) was 190% those in rural areas (2.5 per 100,000) and among men (6.4/100,000) was 40% those among women (4.5/100,000). Hepatitis C cases aged 40-49 constituted the most predominant group (475 cases, 22.8%). Because of development of diagnosis and reporting presented data can be a subject to the information bias and thus should be taken cautiously. PMID- 12371368 TI - [Hepatitis A in Poland in 2000]. AB - A total of 262 cases of hepatitis A were reported in 1999. The incidence was estimated to be 0.7 per 100,000 and represented 74% decrease compared to the preceding year. The incidence rates within rural and urban populations were similar. The highest incidence was reported among persons 20-24 years old. Patients in this age group constituted 20.2% of the total number of cases. The above data shows that since 1997 epidemiological situation of hepatitis A is making for low endemicity pattern. PMID- 12371369 TI - [Tetanus in Poland in 2000]. AB - In last decade gradual decrease in numbers of reported cases of tetanus has been observed. In 2000, 14 cases of tetanus (10 women and 4 men) were reported in Poland. All those cases occurred among people of age 40 or more, 35.7% of cases died. Case fatality increased with age. All cases were unvaccinated or vaccination status was unknown. The data show effectiveness of vaccination program in younger age groups, but they also show need for promotion of post exposure prophylaxis, especially among older people both in rural and in urban areas. No case of neonatal tetanus was noted in Poland since 1984. PMID- 12371370 TI - [Rabies in Poland in 2000]. AB - In Poland in 2000, one case of human rabies was reported. It has been the first case of human rabies since 1986. The person with rabies was bitten by cat and has not received post-exposure treatment. Mass oral vaccination of wild animals against rabies which was introduced in 1993, showed a positive impact on the epizootic situation of rabies in Poland. However, we observed two-fold increase of animal rabies cases this year. Sources of wild and domestic animal rabies are present on the north-east, east, and south-east parts of the country, uncovered by oral vaccination. Out of 9,210 persons vaccinated in Poland against rabies, 2,587 (19%) were bitten by or were in contact with a rabid animals. Main reason for vaccination against rabies were contacts with animals category C (rabies not excluded, 5,741 cases; 62%) or category D (animals healthy during the exposition, 882 cases; 10%). PMID- 12371371 TI - [Human brucellosis in Poland in 2000]. AB - Most of the cases of human brucellosis registered in 2000 in Poland constitute chronically ill professionals (17 cases) and of them--15 with a long time (more than 16 years) of professional activity. Six cases of acute brucellosis were registered in 2000. Among them four were imported from Spain, and two were acquired in Poland. PMID- 12371372 TI - [Trichinellosis in Poland in 2000]. AB - In the year 2000 thirty six cases of trichinellosis were reported and registered in Poland. Most of them (26 cases) were noted in western region of the country (wielkopolskie voivodeship). Consumption of meat from four pigs and six boars caused 32 infections. In four sporadic cases--sources of the infection were not discovered. A total of 19 men and 17 women were infected (including three children up to 14 years of age). Twenty-six persons were hospitalized. No deaths from trichinellosis were reported in the year 2000. PMID- 12371373 TI - [Cestode infections in Poland in 2000]. AB - In 2000, 439 intestinal cestode infections were registered in Poland. Among them 359 were caused by Taenia saginata, three by T. solium, 52 by Taenia sp., two by Hymenolepis nana, two by Diphyllobothrium latum, and one by Dipylidium caninum. Moreover, 29 cases of echinococcosis were also registered. The obtained results confirmed the low frequency of intestinal cestodoses in Poland. PMID- 12371374 TI - [AIDS and HIV infections in Poland in 2000]. AB - By the end of 2000 cumulative numbers of 1,024 AIDS cases and 524 AIDS deaths were diagnosed and registered in Poland. Until 1999 (134 new AIDS cases) number of annually diagnosed AIDS cases remained relatively low, but continuous increasing of new diagnoses has been observed. Regardless of possible influence of highly active antiretroviral treatment, AIDS figures--especially registered for 2000--can be incomplete, like numbers of AIDS deaths decreasing since 1995. The biggest amount of newly discovered HIV infections (809) was reported in 1990. Next years these numbers ranged from 384 in 1993 to 637 in 1998. Majority of AIDS cases and HIV-infected persons in Poland were drug users, respectively 49.3% and 63.7%. Persons affected heterosexually and without information about the way of HIV transmission account for an increasing proportion of new infections. Quality of collected epidemiological data will depend on easy access to HIV testing and quality of the data provided: data completeness, punctual notification and consistency with obligatory definitions. PMID- 12371375 TI - [Malaria in Poland in 2000]. AB - In 2000, 24 malaria cases were registered in Poland. All of them were imported, mainly from Africa (15 cases). Plasmodium falciparum infection was confirmed in 15 cases, P. vivax--in seven. Among 24 malaria cases 22 were men and 20 in 20-49 years old group. Eleven persons travelled abroad in the connection with their job, six--as tourists, three--came from malaria endemic or epidemic countries. In 2000, 25-year old man with malaria falciparum died. PMID- 12371376 TI - Balancing values and imperatives: a study of nursing service in an ICU. AB - There has been significant attention from the managers and purchasers of health services regarding the economic advantages that result from changes to the patterns of health care delivery in the acute hospital setting. The impact of these changes, whilst often rendering advantage at the economic management level of health care, can have different consequences for the people who deliver and the people who receive health service. This paper reports on a study that was conducted with a group of nurses to investigate the practice milieu of a critical care unit in the context of changes to health service management. Interpretive methods were used to capture the perspective of the nurses and the way they interpret the multiple factors that influence their practice and their practice environment. The findings indicate that the nurses in the study setting interpret these factors according to the influences they have on the structure, the geography and the value of their work. Explication of these findings provides a research base to inform recommendations relating to improving the practice milieu of the critical care environment. PMID- 12371377 TI - Chest x-ray quiz. Ebstein's anomaly. PMID- 12371378 TI - Registered nurses' lived experience of advocacy within a critical care unit: a phenomenological study. AB - Anecdotally, it has often been expressed by registered nurses (RNs) working within critical care environments that they are patient advocates. However, to date, little systematic research has been undertaken to validate this assertion. Thus this project, which explored the lived experience of RNs working within a critical care unit in a country area of Australia, was conceived. The five participants of this study were all Division 1 RNs possessing a critical care certificate and a minimum of 4 years' nursing experience. Through their participation in an in-depth audiotaped interview they revealed a wealth of experiences and ideas about their involvement as patient advocates. The results of this research indicate that the phenomenon of nurse advocacy is a multi faceted process and embraces many kinds of activities that nurses engage in on behalf of their clients. The findings of this study indicate that some of the participants' experiences are congruent with elements of advocacy contained within the nursing literature and statements of professional nursing bodies. However, there are some findings in this study that are not consistent with available literature. For instance, these participants markedly reject the notion that advocacy is an inappropriate concept for nurses, despite suggestions in the literature that this is an inappropriate role. Instead they wholeheartedly embrace this role, asserting it as central to their practice. Further, although the literature identifies potential controversies regarding enactment of the role of advocacy, the participants of this study are silent on these matters. It is not known what this silence implies and, in light of the study findings, it is recommended that nursing organisations, theorists and clinicians consider whether it is worthwhile to more clearly confirm the nature and role of advocacy within Australian nursing. PMID- 12371379 TI - Nurse-initiated x-ray of isolated limb fractures in the emergency department: research outcomes and future directions. AB - Patients presenting at emergency departments with isolated limb trauma face chronic delays due to over crowding and over utilisation of these departments for primary health care. Excessive waiting periods for assessment, definitive diagnostic procedures and subsequent treatment plans compound the current access block for emergency care, with these factors contributing to patient dissatisfaction. To improve patient satisfaction and decrease waiting times for some patients, the literature suggests that nurse initiated x-rays at the point of triage may be of value. Nurses can appropriately determine the need for radiological assessment in patients with isolated limb trauma--a high correlation has been found between doctors' and nurses' ordering of x-rays. To ensure x-rays are accurately and appropriately ordered, it is suggested that strict guidelines and structured educational programmes for nurses be implemented. The findings from this literature review suggest that extending the triage role to include nurse initiated x-rays has the potential to decrease waiting times and to increase patient satisfaction in the emergency department. PMID- 12371380 TI - Central line management--progress! PMID- 12371382 TI - The initiation and administration of drugs for advanced life support by critical care nurses in the absence of a medical practitioner. AB - Current legislation does not permit the administration of first line resuscitation medications by suitably qualified Division 1 registered nurses (RNs) in the absence of a medical officer. This omission by the Drugs, Poisons and Controlled Substances Act 1981 (Vic) and the Drugs, Poisons and The Controlled Substances Regulations 1995 (Vic) leaves many critical care nurses in a vulnerable legal position. The primary aim of this study was to gauge the view of critical care nurses with respect to lobbying for change to the current legislation. In addition, the study aimed to explore and describe the educational preparation, practice perceptions and experiences of RNs working in critical care regarding cardiopulmonary resuscitation and the administration of first line advanced life support (ALS) medications in the absence of a medical officer. It was anticipated that data collected would demonstrate some of the dilemmas associated with the initiation and administration of ALS medications for practising critical care nurses and could be used to inform controlling bodies in order for them to gain an appreciation of the issues facing critical care nurses during resuscitation. A mailout survey was sent to all members of the Victorian Branch of the Australian College of Critical Care Nurses (ACCCN). The results showed that the majority of nurses underwent an annual ALS assessment and had current ALS accreditation. Nurses indicated that they felt educationally prepared and were confident to manage cardiopulmonary resuscitation without a medical officer; indeed, the majority had done so. The differences in practice issues for metropolitan, regional and rural nurses were highlighted. There is therefore clear evidence to suggest that legislative amendments are appropriate and necessary, given the time critical nature of cardiopulmonary arrest. There was overwhelming support for ACCCN Vic. Ltd to lobby the Victorian government for changes to the law. PMID- 12371383 TI - [Comparative evaluation of the clinical effectiveness and the adverse effects of three different forms of nitrates in high oral doses in patients with stable angina pectoris]. AB - The aim of this study was the comparative evaluation of antianginal efficacy and the adverse effects of 3 nitrates in oral doses: isosorbide dinitrate 80 mg in slow release form (ISDN-80), nitroglycerin 15 mg--slow release (NITRO-15) and pentaerythritol tetranitrate 100 mg in normal tablets (PENTA-100) in patients (pts) with stable angina pectoris. In a randomized, double-blind, cross-over and placebo (P) controlled study 15 men, with mean age 54.8 +/- 8.0 years, with stable angina, received single doses of: ISDN-80, NITRO-15, PENTA-100 or P. Clinical efficacy of the drugs was evaluated by analysis of the walking times: total (TT), to angina (TA), and to ischemia (TI) on treadmill during stress tests performed 2 and 6 hours (h) after drug ingestion; the adverse effects were registered during 6 h follow up. RESULTS: 2 h after ingestion all 3 study drugs improved significantly: TT, TA and TI in comparison to P. After 6 h the same parameters were improved by: ISDN-80 and NITRO-15, but PENTA-100 improved only TT and TA. After 6 h ISDN-80 significantly improved in comparison to NITRO-15: TT by 19.7% (p < 0.01), TA by 21.2% (p < 0.01) ant TI by 25.0% (p < 0.05), and in comparison to PENTA-100: TT by 32.1% (p < 0.001), TA by 33.4% (p < 0.001) and TI by 41.1% (p < 0.01). After 6 h NITRO-15 significantly improved TI in comparison to PENTA-100 by 13.1% (p < 0.05). The headaches, the most frequent adverse effects, occurred after ingestion of ISDN-80 in 6 pts, NITRO-15 in 4 pts, PENTA 100 in 3 pts and P in 1 pt. Among three evaluated nitrates ISDN-80 significantly improved the effort tolerance and the coronary reserve in the strongest way, NITRO-15 was intermediate in the clinical efficacy, but PENTA-100, the drug with the weakest antianginal efficacy, was the reason of the least number of the adverse effects. PMID- 12371385 TI - [The utility of high density lipoprotein cholesterol subclasses measurement]. AB - The aim of our study was to estimate the cholesterol concentrations in high density lipoprotein subfractions (HDL2 i HDL3) in plasma of patients with normocholesterolemia and atherosclerosis of coronary arteries and in patients with myeloproliferative diseases. Blood samples from patients with normal plasma cholesterol levels were analyzed in spite of the fact that atherosclerosis of coronary arteries usually exist with hypercholesterolemia and myeloproliferative diseases usually coexist with hypocholesterolemia. In this way we wanted to determine changes in HDL2 i HDL3 metabolism which occur independently from changes of cholesterol levels in other lipoproteins. Our results showed that in patients suffering from atherosclerosis of coronary arteries with normolipemia the lowering of plasma cholesterol level in subfractions HDL2 and HDL3 was observed. In patients with myeloproliferative disease with normocholesterolemia the slight lowering of cholesterol level in subfraction HDL3 may be connected with moderate hypertriglyceridemia. In both groups of patients the lowering of HDL cholesterol in plasma was accompanied with the increased lipid peroxidation. PMID- 12371384 TI - [Simvastatin and extrinsic coagulation pathway in peritoneally dialyzed patients]. AB - Peritoneally dialyzed subjects (CAPD) are prone to dyslipidemia and have a high risk of cardiovascular death. Statins (hydroxy-methylglutaryloCoA reductase inhibitors) show beneficial effects on serum lipids and hemostasis in kidney diseases. The purpose of this study was to assess platelet functions, some hemostatic parameters-extrinsic coagulation pathway-total, truncated, free TFPI (tissue factor pathway inhibitor), TF (tissue factor), TFPI/Xa and TF/VIIa complexes, as well as a marker of endothelial cell injury--von Willebrand factor- vWF and serum lipids in 10 hyperlipidemic CAPD patients treated with simvastatin (Zocor, MSD, at a dose of 10 mg at bedtime) for 3 months. Cholesterol and LDL fell significantly as early as after 1 month and remained lowered during further months of the therapy. No significant changes in von Willebrand factor, free TFPI, TF, TFPI/Xa and TF/VIIa complexes were found during therapy with simvastatin. Truncated TFPI decreased significantly as early as after 1 month and total TFPI decreased after 3 months of the therapy with simvastatin. Simvastatin is an effective hypolipemic agent. It seems that simvastatin have no or only little effect on endothelial function and extrinsic coagulation pathway in peritoneally dialyzed patients. PMID- 12371386 TI - [Sodium-proton exchanges and platelet procoagulant activity in type 1 diabetic patients]. AB - Platelet sodium-proton exchange rate and phospholipid dependent procoagulant activity were measured in 31 type 1 diabetics (mean age 32.3 +/- 10.1 years) and 35 healthy subjects (mean age 35.4 +/- 9.4 years). The activity of platelet Na+/H+ exchanger was measured in platelet rich plasma, using an optical swelling assay, according to Rosskopf et al. Platelet procoagulant activity was measured in platelet rich plasma, platelet poor plasma and platelet/microparticles filtrated plasma, using Russell's viper venom (according to Jy and Horstman) and calibrated with ship L-alpha-phosphatidylethanolamine. We found that Na+/H+ exchange rate was significantly higher in diabetic patients in comparison to the controls (p = 0.0009). There was also a positive correlation between the activity of Na+/H+ exchanger and phospholipid dependent procoagulant activity in all plasma fractions. We did not find a significant association between Na+/H+ exchanger activity and metabolic parameters studied, however in patients with HbA1c level > 7.5% higher Na+/H+ exchange rates were noted. Total procoagulant activity did not rise significantly in diabetic patients, but was markedly higher in platelet poor and platelet filtrated plasma. It was supposed that it originated from platelet derived microparticles, enriched in phospholipids. Our results suggest that an increased platelet Na+/H+ exchange rate and raised procoagulant activity connected with platelet microparticles may enhance the risk of vascular damage in type 1 diabetic patients. PMID- 12371387 TI - [Histamine receptors expression on lymphocytes and neutrophils in glucocorticosteroid sensitive and resistant asthma patients]. AB - Histamine is a physiological mediator which exerts both, effector and regulatory influence through its receptors on various cells. In the study we evaluated the expression of histamine receptors (HR) on inflammatory cells (lymphocytes and neutrophils) in glucocorticosteroid sensitive (n. = 17) and resistant (n. = 19) asthmatic patients and healthy subjects (n = 14). Moreover, we examined in vitro the influence of cortisol on HR expression in both studied groups with asthma and in a control group. We concluded that HR expression on lymphocytes was similar in all studied subjects. However, HR expression on neutrophils in both groups of asthmatic patients was significantly decreased compared to the control group. After the in vitro incubation with cortisol, we observed a significant decrease in HR expression on neutrophils (10(-8)-10(-5) M) and lymphocytes (10(-8)-10(-4) M) only in healthy subjects. Taking into consideration that histamine proinflammatory action is mediated by H1 receptor, and that in patients treated with long-time systemic GCS therapy we did not observe any decrease in HR expression in vitro, we suggest additional therapy with the second-generation antihistamines. These drugs not only block H1 receptors, but also have an anti inflammatory effect. PMID- 12371388 TI - [Is hyperthyroidism often present in patients with thyroid differentiated carcinoma?]. AB - The coexistence of thyroid cancer and hyperthyroidism is confirmed by many authors. It appears that the frequency of both disease can be greater as a result of lack of proper and penetrating biopsy diagnosis and of qualification to surgery treatment patients with hyperfunctional goiter and Graves disease, especially coexisting with nodes. The aim of our study was estimation of occurrence of hyperthyroidism in the patients with thyroid differentiated cancer. We examined group of 217 patients with diagnosed thyroid differentiated cancer, 20 patients (9.1%) of them were earlier hyperthyroidism diagnosed. 17 of them were hyperfunctional nodular goiter diagnosed and three as Graves disease, confirmed by presence of anti-TSH receptor antibodies (TRAK). Before thyroidectomy ultrasonography showed nodular goiter in 17 patients and hypoechogenic goiter with nodules in 3 patients. After thyroidectomy at the hyperthyroid patients in 16 papillary thyroid cancer and in 4 follicular thyroid cancers were diagnosed. The frequency of coexistence of cancer and hyperthyroidism in our material amounted for 9.1%. The results of our observation do not diverge from facts given in world literature and is point out the need for precise analysis of patients with hyperthyroidism and of proper qualification to surgery treatments changes suspected to be malignant process. PMID- 12371390 TI - [Autoimmune hepatitis in the sight of contemporary immunology]. PMID- 12371389 TI - [Is there any difference in quality of life after 12 months surgical treatment of acquired heart valve disease in comparison to results after three months?]. AB - The quality of life in eighty patients three and twelve months after surgical treatment of acquired heart valve disease was assessed. The significant extension of distance in 6 minute walk test and clinical improvement measured in change of NYHA functional classes after three and twelve months was noticed. However twelve months after valve replacement in comparison to results after three months no further improvement of quality of life in the matter of physical, psychical and social factors was seen. PMID- 12371391 TI - [Anemia in hypothyroidism]. PMID- 12371392 TI - [Microalbuminuria as a sign of genetically determined renal injury]. PMID- 12371393 TI - [Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV)]. PMID- 12371394 TI - [Role of growth factors in pancreas carcinogenesis]. PMID- 12371395 TI - Progress in Polio Eradication: Vaccine Strategies for the End Game. Paris, France, 28-30 June 2000. Proceedings. PMID- 12371396 TI - [XI Congress of the Catalan Society of Gastroenterology. Reus, 2002. Abstracts]. PMID- 12371397 TI - Index of Suspicion. PMID- 12371398 TI - Special focus on thyroid-associated ophthalmopathy. PMID- 12371399 TI - What is an "at-will" employer? PMID- 12371400 TI - [Non-Hodgkin lymphoma]. PMID- 12371401 TI - [Proceedings of the 2nd Scientific Conference on Thyroid Carcinoma. Szczyrk, December 10-13, 2000]. PMID- 12371402 TI - Bibliography. Current world literature. Pharmacology and therapeutics. PMID- 12371403 TI - Bibliography. Current world literature. Molecular cell biology and physiology of solute transport. PMID- 12371404 TI - Guidelines for prenatal diagnosis. PMID- 12371405 TI - [Primary care and alcohol use disorders: evaluation of a faculty-development program in Venezuela]. AB - OBJECTIVE: Primary care offers an opportunity to identify and treat persons who drink alcohol above permissible levels. In order to prepare primary care practitioners around the world to prevent and treat alcohol-related problems, the National Institute on Alcohol Abuse and Alcoholism of the United States of America has developed and tested a model international program for educating physicians about such problems. The model was designed to increase the clinical, teaching, and research skills of medical school faculty who work with medical students, residents, and primary care physicians. Venezuela was one of the countries selected for the initiative. METHODS: During September 1999 a five-day faculty-development course consisting of 19 workshops was conducted at the University of Zulia, which is located in the city of Maracaibo, Zulia, Venezuela. Teaching strategies included class presentations, role plays, case presentations, skills-building workshops, and having each participant develop a teaching plan that he or she would use. RESULTS: Thirty-three faculty members from 9 of Venezuela's 10 medical schools participated in the project. The 18 female and 15 male participants had an average age of 44 years. The areas of specialization of the 33 participants were: family medicine (9 participants), psychiatry (7), pediatrics (6), obstetrics (4), internal medicine (3), and unspecified (4). Of the 33 participants, 25 of them (76%) completed a six-month follow-up interview. This group said they had significantly increased their competence in 14 clinical areas and that they had successfully implemented new teaching activities within their respective medical schools and residency programs. CONCLUSIONS: This model proved to be an effective strategy for increasing training for physicians in the prevention and treatment of alcohol-related problems in Venezuela. The evaluation confirms similar findings in other countries where the program has been implemented. PMID- 12371406 TI - Medicare fraud-busters target patient transfer reporting. PMID- 12371407 TI - Abstracts of the Sardinia Group of the Italian Biochemical Society, June 2001, the Workgroup on Neurochemistry of the Italian Society of Biochemistry and Molecular Biology, May 2002, and the SIB Group of Marine and Environmental Biochemistry and the Italian Society of Marine Biology, June 2002. PMID- 12371409 TI - 25th European Conference on Visual Perception. Glasgow, Scotland, 25-29 August 2002. Abstracts. PMID- 12371408 TI - Current awareness on yeast. PMID- 12371410 TI - 16th International Symposium Epidemiology in Occupational Health, Jack Pepys Symposium on Prevention of Occupational Asthma, 3rd International Congress on Women's Health: Occupation, Cancer and Reproduction. Abstracts. PMID- 12371411 TI - Abstracts of the XVIII International Pigment Cell Conference. The Netherlands, 9 13 September 2002. PMID- 12371412 TI - ACTRIMS-ECTRIMS 2002. Abstracts of the 7th Annual Meeting of the Americas Committee for Treatment and Research in Multiple Sclerosis and 18th Congress of the European Committee for Treatment and Research in Multiple Sclerosis. September 18-21, 2002. Baltimore, Maryland, USA. PMID- 12371413 TI - Proceedings of the XXVII Congress of the Society of Biomechanics. Valenciennes, 12-13 September 2002. PMID- 12371414 TI - Abstracts of the 14th Annual Transcatheter Cardiovascular Therapeutics Symposium. September 24-28, 2002. Washington, DC, USA. PMID- 12371415 TI - Abstracts of the 67th Annual Meeting of the American College of Gastroenterology. October 20-23, 2002. Seattle, Washington, USA. PMID- 12371416 TI - ALSODatabase: database of SOD1 (and other) gene mutations in ALS on the Internet. European FALS Group and ALSOD Consortium. AB - Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for 20-30% of all familial cases of ALS and for 1-2% of all ALS cases. The exact number of these mutations is, however, unknown, as is the number of individuals worldwide with them who have ALS or are at risk of developing the disease. Information on the genetic and clinical data of ALS patients with the SOD1 gene mutations is, also, only available for one-third of all families and is incomplete in some of those families. Some attempts have been made to correlate the genotype with the phenotype of the patients with SOD1 gene mutations and the available evidence suggests that only a few mutations could be linked to a consistent age of onset or pattern of survival. The ALS scientific community has recognized an urgent need for collecting the genetic and clinical information on all SOD1 individuals and the ALSOD Consortium was set up with the aim of pooling all available data into a uniform centralized database. Recent advances in information technology and the wide accessibility of the Internet have provided an ideal vehicle through which data can not only be recorded but also searched, amended and updated in real time. Here, we describe the work done so far by the ALSOD Consortium and plans for the future. PMID- 12371417 TI - Measurement of selectin tether bond lifetimes. PMID- 12371418 TI - Abstracts of the 8th Central European Lung Cancer Conference. September 1-4, 2002. Vienna, Austria. PMID- 12371419 TI - Abstracts from the 5th Congress of the European Association for Neuro-Oncology. Florence, Italy, 7-10 September 2002. PMID- 12371421 TI - American Dental Education Association 79th Annual Meeting. March 2-6, 2002. San Diego, California, USA. Abstracts. PMID- 12371420 TI - Abstracts of the 10th International Congress on Antiphospholipid Antibodies. Taormina, Sicily, Italy, September 29-October 3, 2002. PMID- 12371422 TI - American College of Emergency Physicians Research Forum. October 7-8, 2002. Seattle, Washington, USA. Abstracts. PMID- 12371424 TI - American Society for Reproductive Medicine 58th Annual Meeting. October 12-17, 2002. Seattle, Washington, USA. Abstracts. PMID- 12371423 TI - Abstracts of the IXth International Congress of the Metastasis Research Society. Chicago, Illinois, USA. 20-22 September 2002. PMID- 12371425 TI - European Helicobacter Study Group. XVth International Workshop on Gastrointestinal Pathology and Helicobacter. Athens, Greece, 11-14 September 2002. Abstracts. PMID- 12371426 TI - Abstracts of the 29th Scandinavian Congress of Rheumatology. Tromso, Norway, 15 18 August 2002. PMID- 12371427 TI - National Drug Abuse Survey shows drug use increasing, covers mental illness for the first time. PMID- 12371428 TI - IOM report reviews factors related to suicide, outlines plans for national network of research labs. PMID- 12371429 TI - The J-Delay scale: a measure of the likelihood of patient delay in breast cancer. AB - Patient delay in seeking treatment for breast cancer is a major contributing factor to morbidity and mortality. No instruments have previously been developed to predict the likelihood of patient delay. This report describes the development and testing of the J-Delay scale, designed to estimate a woman's risk of making the judgment to delay versus to seek immediate evaluation of self-discovered breast symptoms that might signal breast cancer. The J-Delay scale items were developed in four qualitative studies (28 focus groups, combined N = 147 women). The J-Delay scale was tested in 2 large, community-based samples varying across age, income, and ethnicity (combined N = 1,290). Content validity was supported by narrative analysis. Criterion validity was supported by the correct prediction of patient delay in 69%-86% of surveyed and interviewed women with symptoms of 3 months' or more duration. Internal consistency reliability was .83 in English samples (n = 596 and 352) and .81 in a Spanish sample (n = 222). Test-retest reliability after 3-4 months (n = 251) was supported by consistent assignment in 88.8% of cases. The J-Delay is a valid and reliable tool to identify women at risk for patient delay. Women identified as likely to delay medical attention must be targeted for early detection interventions focused on patient delay. PMID- 12371430 TI - Health promotion with adolescents: examining theoretical perspectives to guide research. AB - A guiding theoretical framework in research serves not only to guide a single research study, but also to link previous and future research that is guided by the same framework. Existing theoretical perspectives appropriate for use with adolescent health promotion research were reviewed. Instead of randomly selecting several theories for comparison, an intensive review of the literature was conducted to identify which theories were most commonly used with adolescent health promotion research. The results of this review revealed some interesting and noteworthy information regarding the state of theory use in adolescent health research for the last decade. Information is provided on theoretical perspectives by journal and year of publication. Trends are analyzed so that nurses can evaluate the current state of the science. Social cognitive theory (Bandura, 1986), the health belief model (Becker, 1978), and the health promotion model (Pender, 1996) emerged as the most significant theories for adolescent health promotion research and thus are discussed at the end of the article. PMID- 12371431 TI - Nursing information. PMID- 12371432 TI - A glimpse over the horizon. PMID- 12371433 TI - Nursing shortage. PMID- 12371434 TI - Exploring the dimensions of access to health services: implications for nursing research and practice. AB - Access to health services is a major concern across North America and abroad, with particular salience for the residents of rural and remote areas and the health professionals committed to providing services to them. Intrinsic to this discussion is clarification of the phenomenon of access to health services, a concept that remains nebulous and obscure to consumers, health care providers, and policymakers alike. Multiple understandings of access to health services impedes progress in the development of policy, the creation of programs, and the transformation of health services. Considerable discussion of theory concerning access to health services is articulated in public or community health literature and that of other disciplines; however, limited attention to this topic is apparent in nursing literature. This report articulates definitions, dimensions, and frameworks of access to health services from available literature and existing theory. Further, key points are identified and discussed for consideration in nursing research on the term access and implications for practice. PMID- 12371435 TI - El cambio de vida: conceptualizations of menopause and midlife among urban Latina women. AB - The experience of menopause among Latina women has seldom been described. The purpose of this study was to conceptualize and contextualize the experience of menopause from the perspective of Latina women. A series of focus group sessions were conducted with postmenopausal Latina women living in a large midwestern city. Themes derived from content analysis included: (a) The primacy of health and the importance of harmony and balance; (b) El cambio de vida--something you have to go through; and (c) This time is for me: reorientation and restructuring. Rediscovery and redefinition as opposed to being defined by physical symptoms marked this life phase. Implications of study findings are discussed within the context of an emerging biopsychosocial perspective of midlife and menopause transition. PMID- 12371436 TI - Effects of side cooling on temperature, humidity and water recycling efficiency in a culture vessel for a space experiment--results of ground experiment. AB - Seed-to-seed experiments using dwarf plants will be conducted in Cell Biology Experiment Facility (CBEF) set up in the International Space Station (ISS). Development of a simple system to recycle water transpired by plants is necessary to save space and electrical power. A cooling system using a cooling plate that cools one side of the ventilated culture vessel to enhance water vapor condensation was developed. Effects of side cooling on air temperature, relative humidity and water recycling efficiency in the culture vessel were investigated on the ground. Decreasing the cooling plate temperature lowered temperatures of cooled side and inside air. Cooling treatment also decreased relative humidity inside the vessel less than 90% and lowered the amount of water vapor lost from the vessel through ventilation filters. This seemed to be due to the increased water vapor condensation onto the cooled side. To investigate the effect of increases in outside humidity on water recycling efficiency in the culture vessel, a water vapor transfer model was established. The calculation results indicate that an increase in relative humidity around the vessel can decrease water vapor loss from the vessel, increase water condensation onto the cooled side, and therefore, enhance water recycling efficiency in the vessel. The side cooling system seems to be useful for the CBEF in ISS because relative humidity in the CBEF is controllable. PMID- 12371437 TI - Successful human cadaveric renal homograft with major blood-group incompatibility. PMID- 12371438 TI - Clinical guidelines for the treatment of depressive disorders. PMID- 12371439 TI - Engineered yeasts simulating P450-dependent metabolisms: tricks, myths and reality. PMID- 12371440 TI - The toxicity of phenothiazine. AB - Phenothiazine, the parent compound of a multitude of present-day drugs, has been employed on an extensive scale for its insecticidal, fungicidal, antibacterial and anthelmintic properties. Almost a catholicon, its widespread use in animals and man has led to the uncovering of many adverse reactions encompassing effects on blood elements, neuromuscular problems and photosensitization. The high lipophilicity of phenothiazine and the formation of two redox systems amongst its many metabolites can facilitate the occurrence of generalised macromolecular disruption. Information from the literature has been garnered and appraised in this review to enable an insight into the possible mode(s) of interaction of phenothiazine with living systems. PMID- 12371441 TI - The functional state of the xenobiotic metabolizing system in rat liver following chronic administration of diethylnitrosamine or its precursors. AB - The effect of chronic administration of 0.002% N-nitrosodiethylamine (DENA), 0.002% diethylamine (DEA) and 0.0005% sodium nitrite (SN) on the functional state of the xenobiotic metabolizing system in rat liver was investigated. Administration of DEA and DENA increased concentration of cytochromes P-450 and b5. SN did not affect the enzymes of the monooxygenase system. Coadministration of DEA and SN maximally increased the concentration of cytochrome P-450. It is not possible to explain the phenomenon of combined administration of SN and DEA by simple summation of the effects caused by them separately. The activity of microsomal glutathione S-transferase did not change when DEA and SN were given together, yet increased when they were administered separately. The maximum increase of the total activity of cytosol glutathione S-transferases was observed following DENA. In all four experimental groups a decrease of isoenzyme 5-5 activity was observed. Investigation of Se-independent glutathione peroxidase activity showed the multivariance of response of the glutathione S-transferase family to the compounds studied. The concentration of hepatic free SH-groups increased following administration of DENA and decreased dramatically when SN and DEA were coadministered. When they were given separately the concentration remained at control level. PMID- 12371442 TI - Metabolic and chemical studies on alpha,N-diarylnitrones. AB - The metabolism of a number of alpha,N-diarylnitrones has been studied in vitro using male hamster microsomes. All substrates produced benzaldehyde and an uncharacterised substance as metabolites. If the metabolic reaction was carried out in the light, or if the metabolic extracts were exposed to light, the chemical formation of two new compounds was observed. One compound had chromatographic and spectroscopic properties identical to the corresponding amide; the other compound had properties which suggested it was an oxaziridine. The results are discussed in relation to the formation of amides as metabolites of N-benzyl anilines. It is concluded that nitrones are not on the above metabolic pathway, but that they may form amides by chemical rearrangement. PMID- 12371443 TI - The case for cases: publishing high-quality case reports in Homeopathy. PMID- 12371444 TI - A study of the inter-observer reliability of paper case analysis. AB - Two hundred homeopathic practitioners were each sent a questionnaire. The main body of the questionnaire requested the analysis of a paper case, with an optional second case. The response rate was disappointing at about 15%. There was consensus of 33% for the selection of the correct medicine in the analysis of the first case. The study was small but poses further questions about case analysis and the diverse interpretations of the law of similars. PMID- 12371445 TI - Homeopathy and health related Quality of Life: a survey in six European countries. AB - A pilot survey of 1025 patients receiving homeopathic treatment in six European countries is reported. An initial questionnaire included demographic information and questions from health-related Quality of Life (QoL) scales. A follow-up questionnaire collected data on changes in QoL. The demographic characteristics of respondents were similar to previous studies. 82.8% of respondents had previously tried conventional treatments. Satisfaction with consultations was high. The changes of QoL status are positive but weak. 2.7% of patients experienced side-effects which they attribute to homeopathic treatment. 7.8% of patients reported significant aggravation at the beginning of the homeopathic treatment, 25.4% slight aggravation of symptoms. A new survey should be performed with a better motivation of doctors and patients. Further description of treatment would be helpful. PMID- 12371446 TI - The starting point: pathography. AB - The issues which have dominated discussion of homeopathic medicine hitherto are the efficacy and effectiveness of the medicines themselves and the problem of their mechanism of action. The resolution of these is of profound clinical and scientific importance. But there is another aspect of homeopathic methodology that is of equal, and perhaps even more fundamental importance, and that does not depend on whether or how the medicines work. This is the detailed study, almost unique now in western medicine, of the disease process and the healing process; the evolution, manifestation and resolution of the illness in the individual patient. This paper reviews the epidemiology and the 'pathography' that are inherent in the homeopathic method, and discusses their implications for medical science and clinical practice, and their value to medical education; their importance to the identity of the medicine of the future and the doctor of the future. PMID- 12371447 TI - Vitalism, complexity and the concept of spin. AB - Vital Force is a concept that has suffered at the hands of the current medical model. An attempt is made to show how it might be possible to explain Vital Force in terms of complexity theory. A metaphor is introduced for the operation of Vital Force in terms of gyroscopic motion. PMID- 12371448 TI - Homeopathy in survivors of childhood sexual abuse. AB - The objective of this review was to ascertain the incidence of childhood sexual abuse: to ascertain the long-term effects of childhood sexual abuse and to collate the experiences of homeopaths in caring for survivors. Childhood sexual abuse (CSA) is common with a high percentage of homeopathic patients giving a positive history. It is associated with many common clinical conditions, particularly pelvic pain. Survivors are more likely to present for medical help and to be hospitalised. Homeopaths have found work with such patients to be difficult, with cases being complex and multi-layered. New models for case analysis have been developed. PMID- 12371449 TI - Homeopathic treatment of chronic headache: a critique. AB - The author critically reviews a randomised controlled trial by homeopathy for chronic headache and an observational follow-up study of the same patient cohort. The results showed no difference between homeopathy and placebo. The author believes that these results were a 'false negative' due to inadequate homeopathic treatment, particularly relating to the duration of symptoms and handling of homeopathic aggravations. Guidelines for future studies are proposed. PMID- 12371450 TI - Response to Vithoulkas: homeopathic fantasies about science, a meta-critique. PMID- 12371451 TI - The need for the correct sequence of remedies. AB - In chronic disease, often several medicines are required to complete a cure. These should follow a correct sequence to have an optimal effect. Giving the second or the third indicated medicine first may result in temporary amelioration but will not start the process of cure. The following case exemplifies this rule. PMID- 12371452 TI - Two cases of pulmonary TB treated with homeopathy. PMID- 12371453 TI - Nux vomica and animal experiments. PMID- 12371454 TI - Who's that talking? AB - The case of a man aged 37, with symptoms suggesting paranoid schizophrenia is discussed. There was a rapid and sustained response to Kalium bromatum. PMID- 12371455 TI - Homeopathy in dementia and agitation. AB - Three cases of elderly, institutionalised patients with problems relating to dementia and agitation and good response to homeopathic treatment are presented. PMID- 12371456 TI - Provings and magic. PMID- 12371457 TI - Homeopathic pathogenetic trials and the constitutional type questionnaire. PMID- 12371458 TI - Reinventing vitalism. PMID- 12371459 TI - Homeopathic constitutional type questionnaire correlates of conventional psychological and physical health scales: individual difference characteristics of young adults. AB - This study examined associations between scores for 19 different remedies on the constitutional type questionnaire (CTQ) and scores on standardized psychological and medical trait and state scales from health psychology research. Subjects were 104 young adult American college students (mean age 20 years; 67% female). Scales included the chemical intolerance index (CII) for environmental sensitivity, the NEO personality inventory, Marlowe-Crowne social desirability (MCSD) Scale for defensiveness, Harvard parental caring scale (HPCS) for perceived mother and father traits, Profile of Mood State (POMS) scale, Pennebaker symptom checklist (PSC), and a 3-item global health rating scale. The majority of CTQ constitutional type scores correlated significantly with greater NEO neuroticism, lower MCSD defensiveness, and greater psychological distress on the POMS subscales. NEO Extraversion and Openness subscales correlated with specific CTQ scores in directions consistent with clinical remedy pictures. CTQ Carcinosin differed from other remedies, showing no significant correlations with other scales. As hypothesized (a) persons high on CTQ scores for Carcinosin and low in parental caring (HPCS) had the highest symptom score; (b) those high on CTQ scores for Sulphur and low on HPCS had the poorest global health ratings; (c) individuals high on four different CTQ type scores (Carcinosin, Lachesis, Nux vomica, Sulphur) and high on environmental sensitivity (CII) exhibited the highest symptom scores. Taken together, the data offer additional validation of the CTQ and provide a foundation for studying interactions of constitutional type with both psychosocial and physicochemical environmental factors in homeopathic provers and patients. PMID- 12371460 TI - Homeopathic treatment of hot flushes: a pilot study. AB - Hot flushes are a common problem, especially for menopausal women for whom hormone replacement therapy (HRT) is contra-indicated or who prefer not to take it and patients receiving Tamoxifen. Some seek homeopathic treatment. We report an uncontrolled, pilot outcome study, conducted at the Tunbridge Wells Homeopathic Hospital (TWHH) in 1998-1999. The study was conducted in out-patient consultations booked in the usual way. Thirty-one patients referred to the Department for menopausal flushes and seen for an initial consultation and at least one follow-up review, were assessed in three groups: Hot flushes: No history of carcinoma of the breast. Hot flushes: Treatment for breast carcinoma, not receiving Tamoxifen. Hot flushes: Treatment for breast cancer including Tamoxifen. For all patients, the initial and follow-up assessments included review of hot flush frequency and severity. Patients also completed their own self-assessment rating after follow-up consultations. The results indicate useful symptomatic benefit for all three groups of patients. PMID- 12371461 TI - Comparative effect of Coffea cruda potencies on rats. AB - The effects of Coffea cruda 30 and 200c and caffeine on the sleep pattern of rats were investigated. Treatments were administered orally at the beginning of the sleeping period. EEG from the parietal region was recorded. Delta (0.5-2.5 Hz) and slow (< 1 Hz) waves are two of the major oscillation types that characterize neocortical electrical activity. The spectral power in these bands and the power ratio between 0.32-0.48 Hz and the delta band (slow/delta power ratio) for control and treatment groups were analyzed blind. Power in the delta band was significantly higher than baseline for Coffea 30c and caffeine (15.5mg/kg). An increase in the slow/delta power ratio between control and treatment was detected for Coffea cruda 30 and 200c. Coffea 30c and caffeine have similar effects on sleep pattern, enhancing delta power; Coffea cruda 200c appears to affect only the synchronization. PMID- 12371462 TI - Alcohol concentration in the preparation of mother tinctures of vegetable origin. The example of Holarrhena antidysenterica. AB - The strength of solvent affects the therapeutic efficacy of homeopathic tinctures of vegetable origin. On the basis of physical, chemical and biological assay of different tinctures made with different concentrations, in the case of Holarrhena antidysenterica Wall, the best solvent was 70% v/v ethanol. This is different from the standard strong alcohol recommended in homeopathic pharmacopoeia. It may be necessary to review the procedure followed for the preparation of homeopathic mother tinctures of vegetable origin. PMID- 12371463 TI - Homeopathic effect: a network perspective. AB - There are two aspects to the problem of describing the homeopathic effect in physical terms: the nature of the therapeutic agent, and the system on which it acts. The latter can be considered as a network, which provides a conceptual framework that throws new light on long-standing questions, based on generic results such as the enhanced susceptibility of networks near critical states. It suggests a characterisation of health and disease in terms of distance from a critical state. The Internet provides a concrete analogy. This predicts a limiting condition on the acceptable loss of highly connected nodes in the system, and suggests a procedure to measure its connectivity and related parameters. PMID- 12371464 TI - Learning experiences--what works for postgraduates. AB - Supervision and postgraduate education for health professionals studying homeopathy was investigated using feedback and comparative questioning of participants who have taken part in group supervision. Objectives set by supervisors, influenced by previous feedback, are scored and ranked. The key areas of postgraduate learning of homeopathy and how well the supervision model met these are analysed and discussed. Learning experiences are ranked in order of preference. Aspects of case presentation are compared for interest and relevance. PMID- 12371465 TI - Homeopathic treatment of Chronic Fatigue Syndrome: three case studies using Jan Scholten's methodology. AB - This paper explores the treatment of Chronic Fatigue Syndrome following a viral infection in young people. The methodology is based on that of Dr Jan Scholten, Holland, who has systematically described the homeopathic themes of all elements in the periodic table. Three case studies are presented, Cobaltum Phosphoricum, Calcium Phosphoricum and Cadmium Phosphoricum were prescribed. The common themes and the differentiating features of these Phosphate salts are described in detail to show how the homeopathic similimum is found and cure achieved. PMID- 12371466 TI - The experience of living with fibromyalgia: confronting an invisible disability. AB - Fibromyalgia (FM) is a complex, chronic, painful musculoskeletal syndrome which is characterized by extreme fatigue, disordered sleep, and other associated physical and cognitive problems. Because its etiology is unknown, and because no specific pathophysiological mechanisms have been found to underlie the syndrome, making a diagnosis is very difficult. FM adversely affects the quality of life, and the societal costs based on medical expenses, lost wages, lost tax revenue and compensation expenditures are very significant. The purpose of this phenomenological study was to describe and enhance the understanding of the experience of living with FM. The participants included nine women ranging in age from 30 years to 56 years who had been diagnosed with the condition for more than a year. Data were collected by means of unstructured interviews. Thematic analysis, using van Manen's (1990) methodology, identified eight themes: (a) pain the constant presence, (b) fatigue-the invisible foe, (c) sleep-the impossible dream, (d) thinking of a frog (e) dealing with a flare-up, (f) longing for a normal life, (g) the power of naming-seeking a diagnosis, and (h) living within the boundaries. These themes were integral parts of the whole story, and through their interrelationships, the essence confronting an invisible disability was captured. The findings of this qualitative study have implications for nursing practice, education and research. It has become an increasing challenge for our health care system to adequately cope with the large numbers of persons diagnosed with chronic illnesses. Administrators of these systems can benefit from the information learned during this study. PMID- 12371467 TI - The nurse clinical trial coordinator: benefits and drawbacks of the role. AB - It has become common for nurses to be recruited into and/or seek careers outside of the traditional domain of hospital-based work. This article draws on interview data to consider a position nurses are occupying within biomedicine, that of the nurse clinical trial coordinator. It examines and analyzes the value attributed to such positions by nurse trial coordinators. The analysis reveals that nurses identify three aspects of the position-social relations, the acquisition of skills and knowledge, and work and control-as having both advantages and disadvantages over other work roles within nursing. It concludes with suggestions for further research on the role and involvement of nurses in clinical research. PMID- 12371468 TI - Adequacy of time per visit in community nursing. AB - This article is a study of the experiences of community based nurses; specifically, their ratings of the adequacy of time they had to complete treatment and prevention activities. Perception of adequacy of time to complete job functions is important because of its links to job satisfaction and job stress. The largest predictor of a sense of inadequate time was visit characteristics. Specifically, it was the mental health speciality team which was most likely to experience inadequate time to deliver treatment and prevention activities. Possible explanations include the time required to deliver care to this patient population, and/or the greater travelling distances and coordination activities linked to provision of services to this patient population. Nurse characteristics were also important in the analysis. Nurses with an RN designation were less likely to report stress with the time they had to complete their activities. Years of community nursing experience was also an important predictor; individuals with greater community experience were less likely to report inadequate time for their duties. PMID- 12371469 TI - Integrating concepts for the development of qualitatively-derived theory. AB - The development of qualitatively-derived theory (QDT) remains a challenge for researchers wishing to retain the complexity of reality. The techniques of concept integration provide a means to link concepts according to their shared attributes and logically according to their mutual interactions, reactions, and responses. While retaining all of the advantages of qualitative induction, integrating concepts in this manner places QDT theory at the upper end of mid range theory, or disclosive theory, to produce a theory of higher abstraction and broader scope. PMID- 12371470 TI - Transcending the limits of method: cultivating creativity in nursing. AB - In this article the author suggests that by nurturing the development of creativity in individual nurses and cultivating a culture of creativity in the profession, our knowledge and practice as a discipline could be strongly enhanced. This cultivation process, however, requires that we move beyond "using" theory to develop methods of practice. Such an approach to theory serves to solidify and constrain practice and inhibits the full contribution of nursing theory and knowledge. Drawing on the work of philosophers in the pragmatist tradition, the author considers how we might redescribe form to better support the creative process and ultimately enhance the development and contribution of nursing knowledge and theory. PMID- 12371471 TI - Knowledge Impact Conference 2001: action plans linking nursing knowledge to practice outcomes. AB - The work of the Knowledge Conferences in Northeastern USA over 2 decades has been significant. A major contribution is a Consensus Statement that provides the basis for dialogue about nursing knowledge worldwide. In addition to proceedings that have been circulated, the best of the most recent series of conferences-from 1996 through the recent conference in October 2001-will be included in the forthcoming book on knowledge and practice. PMID- 12371472 TI - Nursing information. PMID- 12371473 TI - A glimpse over the horizon.... PMID- 12371474 TI - Photoreduction of the chloropropionic acid of carfentrazone-ethyl in sodium sulfide [corrected]. AB - The purpose of this study was to determine whether the conversion of carfentrazone-chloropropionic acid to carfentrazone-propionic acid in sunlit rice paddies is attributed to photoreduction. Model solutions (Na2S with quinoids) irradiated by laboratory ultraviolet light dechlorinated carfentrazone chloropropionic acid (1.6-28.4% yield of carfentrazone-propionic acid), though Na2S alone was also reactive. Minor conversion (0-2.5%) occurred in the dark, along with dehydrochlorination to carfentrazone-cinnamic acid. Carfentrazone propionic acid formed proportionally to the Na2S concentration (0-50.7% at 1.3-91 mM), whereas acidic pH inhibited reactivity. Photoreduction with Na2S was verified with 2-chloropropionic acid conversion to propionic acid (53.5%) and by minor 4-chlorophenoxyacetic acid dechlorination. Dissolved Na2S was the primary photoreductant, whereas reduced quinones degraded carfentrazone-chloropropionic acid by an alternate reaction. A survey of ambient rice field conditions indicates that carfentrazone-chloropropionic acid photoreduction is not directly attributed to H2S/HS- in this environment, though reduced quinones may be involved to an unknown extent. PMID- 12371475 TI - Sponge halogenated natural products found at parts-per-million levels in marine mammals. AB - Several unknown, abundant brominated compounds (BCs) were recently detected in the blubber of dolphins and other marine mammals from Queensland (northeast Australia). The BCs were interpreted as potential natural products due to the lack of anthropogenic sources for these compounds. This study investigated whether some of the BCs accumulated by diverse marine mammal species are identical with natural BCs previously isolated from sponges (Dysidea sp.) living in the same habitat. Isolates from sponges and mollusks (Asteronotus cespitosus) were compared with the signals detected in the mammals' tissue. Mass spectra and gas chromatography retention times on four different capillary columns of the isolates from sponges and mammals were identical in all respects. This proves that the chemical name of the compound previously labeled BC-2 is 4,6-dibromo-2 (2',4'-dibromo)phenoxyanisole and that the chemical name of BC-11 is 3,5-dibromo 2-(3',5'-dibromo,2'-methoxy)phenoxyanisole. Using a quantitative reference solution of BC-2, we established that the concentrations of the brominated metabolites found in the marine mammals are frequently >1 mg/kg. The highest concentration (3.8 mg/kg), found in a sample of pygmy sperm whale (Kogia breviceps), indicates that BC-2 is a bioaccumulative, natural organohalogen compound. This is supported by the concentrations of the BCs in our samples being equal to the highest concentrations of anthropogenic BCs in any environmental sample. The quantitative determination of BC-2 in blubber of marine mammals from Africa and the Antarctic suggests that BC-2 is widespread. These results are direct proof that marine biota can produce persistent organic chemicals that accumulate to substantial concentrations in higher trophic organisms. PMID- 12371476 TI - Vapor phase transport of unexploded ordnance compounds through soils. AB - Unexploded ordnance (UXO) is a source of concern at several U.S. Department of Defense (DOD) sites. Localization of munitions and fate and transport of the explosive compounds from these munitions are a major issue of concern. A set of laboratory experiments were conducted in specially designed flux chambers to measure the evaporative flux of three explosive compounds (2,4-dinitrotoluene, 2,6-dinitrotoluene, and 1,3-dinitrobenzene) from three different soils. The effect of different soil moisture contents, the relative humidity of air contacting the soil surface, and soil temperature on the chemical fluxes were evaluated. A diffusion model was used to describe the chemical transport mechanism in the soil pore air. The soil-air partition constant was treated as a fit parameter in the model because of the uncertainty in the a priori estimation. The model predicts the qualitative trends of the experimental fluxes satisfactorily. Under extremely dry conditions, the flux decreased more rapidly than that predicted by the model. The fluxes from soils at 24 degrees C were higher than those at 14 degrees C, indicating a larger volatilization driving force at the higher temperature. PMID- 12371477 TI - Application of the luciferase recombinant cell culture bioassay system for the analysis of polycyclic aromatic hydrocarbons. AB - An aryl hydrocarbon (Ah) receptor-based luciferase cell culture bioassay developed to detect 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other halogenated aromatics was modified and optimized to detect and quantitate polycyclic aromatics (PAHs). Twenty-four PAHs were analyzed, and subsequent EC50 and EC20 concentrations (based on the median and 20% TCDD maximal response, respectively) and appropriate induction equivalency factors (calculated by comparison to the response obtained with TCDD) were determined from dose-response experiments. Six compounds were shown to be active in the system, with benzo[k]fluoranthene > benz[a,h]anthracene indeno[1,2,3-cd]pyrene > benzo[a]pyrene > benzo[b]fluoranthene > chrysene. A complex mixture of 16 PAHs was also analyzed using this system, and overall induction equivalency (or 1-EQ) of the mixture was shown to be very similar to that predicted from the sum of the activity estimated for each individual PAH. Overall, our results strongly support the use of this system for the detection and relative quantitation of Ah receptor active PAHs. PMID- 12371478 TI - Products of aqueous chlorination of 4-nonylphenol and their estrogenic activity. AB - Seven reaction products (2-chloro-4-nonylphenol [NP], 2,6-dichloro-4-NP, trichlorophenol, 4-propyl-2'-hydroxyphenol, 4-isobutyl-2'-hydroxyphenol, 4 isoamyl-2'-hydroxyphenol, and 4-isopentyl-2'-hydroxyphenol) were identified by gas chromatography/mass spectrometry (GC/MS) in order to assess the estrogenic activity originated from 4-nonylphenol (4-NP) in drinking water. The estrogenic activities of the aqueous chlorinated 4-NP solution at 10, 60, and 120 min chlorination time were assessed by a yeast two-hybrid system based on the ligand dependent interaction of two proteins, a human estrogen receptor (ER), and a coactivator. It was found that all three solutions inhibited transcriptional activation induction by 4-NP. Further experiments showed that these solutions also inhibited beta-galactosidase induction by 17beta-estradiol. For the solution at 10 min, the inhibition was found to be due to its toxicity, with an inhibition concentration (IC50) of about 10-fold of concentration of chlorinated 4-NP solution, suggesting the existence of some products with higher yeast toxicity than that of the parent 4-NP. Similar inhibition trends were also found in the dose response of the two solutions at 60 and 120 min, with an IC50 of 10-fold concentration. In these cases, the effects were considered to result from their antagonist action because the two solutions show lower yeast toxicity of which IC50 is 45-fold concentration. This suggests that some products in the chlorinated 4-NP solution elicit the antiestrogenic activities. PMID- 12371479 TI - Silver concentrations in Colorado, USA, watersheds using improved methodology. AB - River water samples were collected at five sites in the state of Colorado, USA, to assess the impact of municipal and industrial discharges on Ag concentrations and speciation in surface waters. Samples were collected and analyzed for total (unfiltered collections), filtered (0.1 and 0.4 microm), particulate (> or = 0.45 microm), and colloidal Ag (3 kDa-0.1 m) using ultraclean protocols. A series of laboratory experiments were conducted to assess bias from sample storage, digestion, and preconcentration protocols. In general, upstream unfiltered and particulate Ag concentrations fell within a fairly narrow range, 3.1 to 21 ng/L and 0.2 to 1.7 microg/g, respectively. Downstream unfiltered and particulate Ag concentrations showed a more broad range, 2.8 to 1,110 ng/L and 0.5 to 104 microg/g, respectively, and reflected attenuated impacts of Ag-laden discharge effluents. However, Ag concentrations in the 0.1-microm filter-passing fraction 0.8 to 1.2 km downstream from major treatment plant effluents were all below the chronic silver criteria. On average, more than 60% of the 0.1-microm filter passing Ag was associated with colloidal macromolecular organic matter. Silver concentrations in colloids (microg/g) were, on average, the same as those in suspended particulate matter. The percentage abundance of colloidal Ag was similar to that of dissolved organic carbon, suggesting that strong Ag binding ligands exist in both the colloidal and the particle size fractions, as these macromolecular ligands likely play a major role in Ag speciation. PMID- 12371480 TI - Aerobic degradation of ethyl-tert-butyl ether by a microbial consortium: selection and evaluation of biodegradation ability. AB - A microbial consortium that degrades ethyl-tert-butyl ether (ETBE) as the sole source of carbon and energy under aerobic conditions was selected from a gasoline polluted soil. This consortium consists of a variety of microorganisms with a predominance of filamentous morphology. Degradation of ETBE was found to be solely related to bacterial activity. After prolonged cultivation followed by successive transfers, the consortium's degradation ability was improved and reached a specific degradation rate of 95 mg/g(protein)/h (about 146 mg/g(dry wt)/h). This exceeds the previously reported rates in the literature for ETBE degrading microorganisms as pure or mixed cultures. Furthermore, a stoichiometric balance of chemical oxygen demand (COD) removal and oxygen uptake with ETBE removal provides indirect evidence of complete degradation. The consortium's activity was not inhibited by high ETBE concentrations (< or = 1,600 mg/L), and large inoculum sizes (> or = 120 mg(protein)/L) were desirable for a faster and complete degradation of ETBE. The enriched consortium was also able to completely degrade methyl-tert-butyl ether (MTBE), tert-amyl methyl ether (TAME), and tert butyl alcohol (TBA). both alone and in mixture with ETBE, without any measurable release of major degradation intermediates. In each case, MTBE and TAME exhibited the most significant resistance to degradation while TBA was rapidly degraded. PMID- 12371481 TI - Monitoring of aromatic monosulfonic acids in coastal waters by ion-pair liquid chromatography followed by electrospray-mass spectrometric detection. AB - Spain is one of the European countries that still discharges untreated wastewaters and sewage sludge into the sea. Aromatic monosulfonated compounds were detected in 36 seawater samples collected bimonthly on the Catalonian coast (Spain) over a period of 18 months. These compounds are of environmental concern because of their limited biodegradability and high mobility within the aquatic system. A method based on a sequential solid-phase extraction procedure (SSPE) followed by ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IPC-ESI-MS) in negative ionization mode was used to monitor aromatic monosulfonated compounds in the Catalonian coastal waters. Triethylamine (TEA) and acetic acid (HOAc) were used as volatile mobile-phase additives. A comparison of two polymeric solid-phase extraction (SPE) materials for the extraction of 15 aromatic monosulfonated compounds of environmental concern was performed. The investigated adsorbents were the polystyrene-divinylbenzene materials Lichrolut EN, from Merck, and Isolute ENV+, from International Sorbent Technology (IST). The influence of the matrix on the analysis of seawater samples was also studied. Applications of the developed analytical procedure to seawater samples gave limits of detection (n = 3) ranging from 0.6 to 45 pg on column for the target analytes. Barcelona and Tarragona locations were chosen along the Catalonian coast as sampling sites because of their large sewage discharges and intensive industrial activities. Samples were acquired from submarine outfalls of two rivers, Besos and Llobregat, located in the north and south of Barcelona city, respectively. Samples from petrochemical industry submarine outfalls located near the commercial port of Tarragona were also studied. Concentrations of benzenesulfonates (BS) and naphthalenesulfonates (NS) were of the order of ng/ml. Most of the collected samples were found to contain both isomers of mononaphthalenesulfonate. The study showed that these compounds can contaminate marine coastal waters. PMID- 12371482 TI - Sorption kinetics of selected volatile organic compounds in humin. AB - Each component of the chemically heterogeneous soil exhibits a unique sorption behavior toward organic sorbates. The sorption kinetics of some volatile organic compounds (VOCs) in pressed humin disks was investigated by tracking the weight change of the disks with a microbalance. Higher sorbing capacity for more polar VOCs as well as C13 nuclear magnetic resonance data indicates that humin was more hydrophilic than Aldrich humic acid (Milwaukee, WI, USA). The apparent diffusivity of acetone, toluene, and hexane in the disks ranged from 10(-8) to 10(-10) cm2/s. The sorbed toluene in humin does not seem persistent to desorption; however, acetone and hexane, either as polar or linear compounds in humin, show persistence against desorption. On completion of the desorption experiments, there were approximately 35 and 20% sorbate residue for acetone and hexane, respectively. PMID- 12371483 TI - Effect of pH on cadmium toxicity, speciation, and accumulation during naphthalene biodegradation. AB - Lowering pH of a microbiological medium from 7 to 4 decreased cadmium toxicity during naphthalene biodegradation by a Burkholderia sp. Cadmium speciation and cadmium accumulation in the system were studied to explain this effect. Cadmium speciation was determined by direct measurement and by geochemical modeling. Previous studies have implicated the monovalent hydroxylated cadmium (CdOH+) species in the effect of pH on cadmium toxicity. Modeling analysis predicted CdOH+ formation only at very low concentrations (< or = 0.0128 microM), while the measured concentration of divalent ionic cadmium (Cd2+) was at least three orders of magnitude greater, suggesting that Cd2+ is the more significant metal form. With respect to cadmium accumulation, cells contained in media adjusted to pH 4 accumulated only 2.76 +/- 0.76 mg Cd/g cells, whereas cells in media adjusted to pH 7 accumulated 8.52 +/- 0.71 mg Cd/g cells. These data suggest that cadmium toxicity is correlated with increased cadmium accumulation rather than the formation of CdOH as pH is increased. At low pH, the decrease in cadmium accumulation may be caused by increased competition between hydrogen and cadmium ions for binding sites on the cell surface or by an increase in metal efflux pump activity due to an increase in the proton gradient that drives the efflux pump. PMID- 12371484 TI - Development and application of an oil toxicity and exposure model, OilToxEx. AB - An oil toxicity and exposure model (OilToxEx) was developed and validated for estimation of impacts to aquatic organisms resulting from acute exposure to spilled oil. Because oil exposure is shorter than the time required for equilibrium between the organism and the water to be reached, the time and temperature dependence of toxicity is addressed. Oil toxicity is a function of aromatic composition and the toxicity of individual aromatics in the mixture. Lethal concentration to 50% of exposed organisms (LC50), as a function of octanol water partition coefficient (Kow), and an additive model are used to estimate the toxicity of monoaromatic and polycyclic aromatic hydrocarbon mixtures in water soluble fractions (WSF) and oil-in-water dispersions (OWD) of oil. The toxicity model was verified by comparison with oil bioassay data where the exposure concentrations of aromatics were measured. The observed toxicity in the bioassays could be accounted for by the additive narcotic effects of the dissolved aromatics in the exposure media. Predicted LC50s were compared to those calculated from measured concentrations after spills to verify the exposure model for field conditions. These results indicate that the additive toxicity and exposure model may be used to estimate toxicity of untested oils and spill conditions. PMID- 12371485 TI - Minimum cross-sectional diameter: calculating when molecules may not fit through a biological membrane. AB - Some compounds that are predicted to be toxic to aquatic organisms instead show no toxicity. In some cases, this phenomenon occurs because the molecules are too large physically to pass through biological membranes, at least by passive transport. The size of the smallest circle through which a molecule can pass is called its minimum effective cross-sectional diameter. Until now, no method has been generally available for determining this parameter. Here, we present such a method, based on vector analysis, that produces results in seconds or minutes, even for relatively large molecules, on a desktop computer. The only input required is the Cartesian coordinates and van der Waals radii of the constituent atoms. The gas-phase, energy-minimized structure is used as an approximation, because to our knowledge, no practical means of experimentally measuring an effective diameter in solution is currently available. PMID- 12371486 TI - Prediction of uptake dynamics of persistent organic pollutants by bacteria and phytoplankton. AB - Phytoplankton and bacteria play an important role on the biogeochemical cycles of persistent organic pollutants (POPs). However, experimental data and quantitative knowledge of the kinetics of uptake and depuration of most POPs by bacteria and phytoplankton are scarce. In the present paper, a procedure to predict the sorption kinetics to bacteria and phytoplankton is developed. The prediction method is the combination of a mechanistic model for sorption and quantitative structure-activity relationships relating bioconcentration factors and membrane permeability to the chemical physical-chemical properties. The model consists of two compartments where the first compartment is the cellular surface and the second compartment is the cell biomass or matrix. Equations for estimating uptake and depuration rate constants into the matrix and adsorption and desorption rate constants onto the surface are obtained. These expressions depend on the physical chemical properties of the chemical, the environmental temperature, the microorganism size, and species-specific quality of organic matter. While microorganism shape has a secondary influence on uptake dynamics, microorganism size and chemical hydrophobicity arise as the key factors controlling the kinetics of POP incorporation into bacteria and plankton. Uptake, depuration, adsorption, and desorption rate constants are reported for POPs such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), polychlorinated dioxins and furans (PCDD/Fs), and POPs of emerging concern, such as polybrominated diphenyl ethers (PBDEs). Finally, implications of uptake and depuration dynamics on the biogeochemical cycling of POPs are discussed. PMID- 12371487 TI - Persistent organochlorine residues and their bioaccumulation profiles in resident and migratory birds from North Vietnam. AB - Concentrations of persistent organochlorines (OCs), such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), hexachlorobenzene (HCB), and chlordane compounds (CHLs), were determined in whole-body homogenates of resident and migratory birds collected from the Red River estuary, North Vietnam, during March and October 1997. Contamination pattern was in the order of DDTs > PCBs > HCHs > CHLs > HCB in both resident and migratory birds. Residue concentrations, according to the feeding habit, showed little variability, which may reflect relatively similar trophic levels of the bird species analyzed. Resident birds accumulated greater concentrations of DDTs as compared to migrants. In contrast, HCH residues were greater in migratory species. Higher proportions of p,p'-DDT to total DDT concentrations were found in many species of residents and migrants, indicating recent exposure to technical DDT in northern Vietnam. Congener-specific PCB analysis showed the predominance of penta- and hexachlorobiphenyls in all the species analyzed. Estimation of hepatic microsomal enzyme activities suggested higher metabolic capacity for PCB congeners in shore birds from Vietnam as compared to higher-trophic predator birds and marine mammals. Comparison of OC residues in avian species in Asia-Pacific revealed that DDT residues in resident birds in North Vietnam are among the highest values reported for the countries surveyed, suggesting recent usage of DDT in Vietnam. Available data for birds, fish, and bivalves from the recent Asia-Pacific Mussel Watch Program suggested that Vietnam might be a potential source of DDT contamination in Asian developing countries. To our knowledge, this is the first study of the OC accumulation in avian species from Vietnam. PMID- 12371488 TI - Asia-Pacific mussel watch: monitoring of butyltin contamination in coastal waters of Asian developing countries. AB - Butyltin compounds (BTs) including mono-, di-, and tributyltin and total tin (sigmaSn), were determined in green mussels (Perna viridis) from various Asian developing countries, such as Cambodia, China (Hong Kong and southern China), Malaysia, India, Indonesia, the Philippines, and Vietnam, to elucidate the contamination status, distribution, and possible sources and to assess the risks on aquatic organisms and humans. Butyltin compounds were detected in green mussels collected from all the sampling location investigated, suggesting widespread contamination of BTs along the coastal waters of Asian developing countries. Among butyltin derivatives, tributyltin (TBT) was the predominant compound, indicating its ongoing usage and recent exposures in Asian coastal waters. Higher concentrations of BTs were found in mussels collected at locations with intensive maritime activities, implying that the usage of TBT as a biocide in antifouling paints was a major source of BTs. In addition, relatively high concentrations of BTs were observed in mussels from aquaculture areas in Hong Kong and Malaysia, as it has been reported in Thailand. With the recent improvement in economic status in Asia, it is probable that an increase in TBT usage will occur in aquaculture. Although contamination levels were generally low in mussel samples from most of the Asian developing countries, some of those from polluted areas in Hong Kong, India, Malaysia, the Philippines, and Thailand revealed levels comparable to those in developed nations. Furthermore, the concentrations of TBT in some mussels from polluted areas exceeded the threshold for toxic effects on organisms and estimated tolerable average residue levels as seafoods for human consumption. A significant correlation was observed between the concentrations of sigmaBTs and sigmaSn in mussels, and sigmaBTs were made up mostly 100% of sigmaSn in mussels taken from locations having intensive maritime/human activities. This suggests that anthropogenic BTs represent the major source of tin accumulation in mussels. To our knowledge, this is a first comprehensive report on butyltin pollution monitoring in developing countries in the Asia-Pacific region. PMID- 12371489 TI - Development and validation of models predicting the toxicity of major seawater ions to the mysid shrimp, Americamysis bahia. AB - The concentration and balance of major ions that comprise total dissolved solids (TDS) can influence the toxicity of effluents discharged to freshwater and marine environments. An additional complicating factor in waters released to saltwater systems is the effluent salinity since the toxicity of major ions changes with the salinity of the test solution. A study was conducted to evaluate the toxicity of six major seawater ions (bicarbonate, borate, calcium, magnesium, potassium, and sulfate) to the mysid shrimp, Americamysis bahia, at salinities of 10 and 20/1000. Logistic regression models were developed to predict organism survival at deficient and excess concentrations of the ions. Calcium and potassium caused significant mortality to mysid shrimp in both excess and deficient (relative to artificial seawater) solutions. Bicarbonate, borate, and magnesium displayed significant toxicity only in excess concentrations, while sulfate had no adverse impacts at any of the concentrations tested. As the salinity of the test solutions decreased, mysid shrimp tolerated increasingly lower calcium and potassium concentrations. Similarly, as salinity increased, the upper tolerance levels of calcium, potassium, and magnesium also increased. The models developed during these studies, and similar models developed by other researchers, were used to evaluate 11 actual effluents with unexplained toxicity that might be associated with TDS ions. The models correctly identified calcium as the primary toxicant in 9 of the 11 effluents. These results indicate the models can be used as an important tool to identify toxicity associated with major seawater ions. PMID- 12371490 TI - Predicting chemical contaminants in freshwater sediments through the use of historical biochemical endpoints in resident fish species. AB - Previous studies in Bayou Bartholomew, Arkansas, USA, indicated significant relationships between the individual health of fish sampled from 13 sites and specific biochemical responses. Evaluation of several biochemical endpoints in 1994 indicated the bioavailable occurrence of either polychlorinated biphenyl (PCB) congeners or metals. To evaluate this possibility, in December 2000, sediments were collected at four sites on Bayou Bartholomew, where fish, collected in July 1994, had previously demonstrated the highest hepatic cytochrome P4501A (CYP1A) and heme oxygenase (HO) expression. Samples were analyzed for 89 PCB congeners, 18 organochlorine pesticides, and 25 metals. Total PCB concentrations ranged from 6.5 to 704 ng/g dry weight. Although several PCB congeners were observed, 81, 87, 99, 114, and 153 represented up to 33, 22, 29, 92, and 55% of the sum of PCBs from the four sites, respectively. Total organochlorine pesticide concentrations ranged from undetectable to 53.2 ng/g dry weight. Lindane, heptachlor, dachtal, dieldrin, hexachlorobenzene, dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyl ethane (DDE), and dichlorodiphenyl dichloroethane (DDD) isomers were detected in most samples. Detected organophosphate pesticides included malathion, chlorpyrifos, and dimethoate. Both p,p'-DDE and p,p'-DDD were the most predominant of the pesticides measured (0.5-14.1 and 0.7-58.5 ng/g dry wt). All metal concentrations analyzed were below sediment quality guideline values. Metals exceeding national average concentrations were cobalt (2 of 4 [2/4] sites), copper (1/4), molybdenum (4/4), antimony (3/4), selenium (4/4), tin (4/4), and zinc (1/4). These results were consistent with PCBs being causative agents for the biochemical and adverse individual responses observed in fish sampled from this waterway. PMID- 12371491 TI - Endocrine disruptors in sewage treatment plants, receiving river waters, and sediments: integration of chemical analysis and biological effects on feral carp. AB - Occurrence of alkylphenol ethoxylates or their metabolites (alkylphenols and carboxylated derivatives), as well as natural and synthetic steroids in sewage treatment plant (STP) effluents and in their receiving waters, has been related to biological effects, measured as alterations of plasma vitellogenin (VTG) concentration in natural fish populations. Water composites of STP influents, effluents, sludge, river water, sediment, and feral carps (Cyprinus carpio) were analyzed over a seven-month period in two tributaries of the Llobregat River (NE Spain). Solid-phase extraction/liquid chromatography/mass spectrometry (SPE-LC MS) analysis revealed concentrations of up to 31 microg/L for nonylphenol ethoxylates (NPEOs), 15 microg/L for nonylphenol (NP), and 35 microg/L for nonylphenoxy carboxylate (NPE1C) in river water downstream of STPs. These compounds were also found to accumulate in river sediment with concentrations ranging from 10 to 820 microg/kg of NPEOs and from 22 to 645 microg/kg for NP. Natural and synthetic estrogens and progestogens also occurred in the water and sediments analyzed but in the ng/L and microg/kg range, respectively. Vitellogenin fluctuated among sites and sampling periods, but it was found to be increased in male carp collected downstream of the main STP. A correlation between endocrine-disrupting compounds (EDCs) in water and sediment and plasma VTG concentration in male carp was observed, especially for alkylphenolic compounds in water and sediment samples (r = 0.83-0.84 for n = 24) and for estriol and estrone in water (r = 0.78 and 0.94 for n = 9 and 8, respectively). PMID- 12371492 TI - Level and extent of mercury contamination in Oregon, USA, lotic fish. AB - We conducted a probability survey of 154 Oregon, USA, stream and river sites to assess the spatial extent of mercury (Hg) contamination in fish tissue. Samples consisted of whole-fish analyses of both small (<120 mm) and large (>120 mm) fish at each site, when both were present. Overall, Hg levels (microgram/g) in small fish (mean = 0.031; standard deviation [SD] = 0.029), large piscivores (mean = 0.284; SD = 0.175), and large invertivores (mean = 0.055; SD = 0.047) were found within fairly narrow ranges; always above detection (0.0025 microgram/g) and almost always below 0.4 microgram/g. Given the great ecoregion diversity across Oregon, the narrow range in fish tissue Hg levels suggests that atmospheric transport is an important vehicle for Hg distribution. In small fish, Hg levels were almost always low and showed little meaningful difference among fish taxa. In large fish, Hg levels were significantly related to fish length. Piscivores (pikeminnow and bass) had significantly higher Hg levels, and the slope of their Hg level/length relationship was much steeper than for invertivores. Salmonids, the most commonly occurring fish taxon in Oregon, exceeded 0.1 microgram Hg/g (deemed protective for fish-eating mammals) in an inferred 15% of stream lengths where they occurred. Pikeminnows and bass were found at fewer sites, but they exceeded 0.1 microgram Hg/g in an inferred 96 and 70%, respectively, of stream lengths where they occurred. PMID- 12371493 TI - Responses of benthic invertebrates to combined toxicant and food input in floodplain lake sediments. AB - Benthic communities in floodplain lake ecosystems are often exposed to varying levels of both food and toxicants. Inhibition through toxicants of sensitive species and stimulation through increased amounts of food of opportunistic species have been observed in separate studies. The aim of this study was therefore to assess the responses of benthic invertebrates to combined food and contamination input. Hence, seven floodplain lakes located along the River Waal, The Netherlands, with different levels of food (being either phytoplankton or macrophyte dominated) and toxicants were selected. The responses of the sensitive mayfly Ephoron virgo and the opportunistic midge Chironomus riparius to these sediments were assessed in 10-d growth bioassays with both species and a 28-d emergence experiment with C. riparius. A decrease in both survival and growth of E. virgo was observed with increasing contaminant levels. In contrast, C. riparius responded to the food quantity and quality in the sediments in spite of the toxicants present. Therefore, we conclude that the midge C. riparius is not a suitable test organism for the assessment of sediment toxicity. Alternatively, it proved to be an appropriate test organism to determine the nutritional value of sediments. The mayfly E. virgo turned out to be a much more appropriate test organism for sediment toxicity bioassays because it responds to the toxicant levels in the sediments rather than to the nutritional value. Our results demonstrate that the trophic state of an ecosystem (macrophyte or plankton dominated) influences the ecological risk of toxicants to benthic invertebrates in a species-specific way. It is concluded that not the toxicant load but the combination of food and contaminants determines the persistence of benthic invertebrates and therewith the benthic invertebrate composition in complexly polluted ecosystems. PMID- 12371494 TI - A combined microcosm and field approach to evaluate the aquatic toxicity of azinphosmethyl to stream communities. AB - We evaluated the potential effects of the organophosphate insecticide azinphosmethyl (AZP) in a combined microcosm and field approach. The upper regions of the Lourens River, South Africa, are free of contamination (control site), whereas the subsequent stretches flowing through a 400-ha orchard area receive transient insecticide pollution (e.g.. 0.82 microg/L AZP, 344 microg/kg chlorpyrifos) following spray drift and runoff (contaminated site). Stones taken from the control site were transferred to outdoor microcosms (1.5 x 0.2 x 0.2 m), providing 12 core species and approximately 350 individuals per microcosm. Microcosms were contaminated for 1 h with AZP (control, 0.2, 1, 5, and 20 microg/L; three replicates each), and acute effects on survival were evaluated 6 d following exposure. The two strongest treatments (measured concentrations: 19.2 +/- 1.0 and 4.9 +/- 0.3 microg/L, respectively) resulted in a significantly (analysis of variance) reduced invertebrate density, attributed mainly to various insect taxa, such as Demoreptus sp., Castanophlebia sp., Simuliidae, and Chironomidae. In contrast, Aeshna sp., Dugesia sp., Ceratopogonidae, and Cheumatopsyche sp. were unaffected. In parallel, we conducted a quantitative macroinvertebrate survey at the control site and the contaminated site of the Lourens River after the seasonal pesticide application period. The two sites contained a similar number of species but differed considerably in their species composition and abundances. Five of the eight species that were affected by AZP in the microcosm study occurred in the field at significantly lower densities at the contaminated than at the control site or were absent at the contaminated site. All of the four species that were unaffected in the microcosm occurred at significantly higher densities at the contaminated field site. Only 3 of the 12 species reacted differently in the microcosm and the field study. We conclude that microcosm studies employing a field-relevant design could be linked successfully to field studies and our results suggest that transient pesticide contamination affects the aquatic communities of the Lourens River. PMID- 12371495 TI - Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese. AB - In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feather-molting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species. PMID- 12371496 TI - Ten-week exposure to treated sewage discharge has relatively minor, variable effects on reproductive behavior and sperm production in goldfish. AB - It is well established that effluent from many sewage treatment plants (STPs) possesses estrogenic properties; however, the biological significance of this estrogenicity to fish reproductive behavior and sperm production is unknown. This study tested the hypothesis that 10-week exposure to a STP effluent with well established estrogenic properties compromises the reproductive vigor of mature goldfish by decreasing the intensity of their reproductive drive and sperm production. Male goldfish were exposed for 10 weeks to one of four treatments: well water, a solvent control, 50 ng/L estradiol (E2), or undiluted effluent from a major metropolitan STP. Two trials were conducted, one in the winter and one in the summer when the effluent was chlorinated. Exposure to effluent in both the summer and winter trials induced small, but nonsignificant, decreases in the frequency of male spawning behaviors but had no effect on sperm production. Exposure to E2 caused a clear reduction in spawning behavior in one trial while causing reduced sperm production in both trials. We conclude that the effect of effluent exposure from this STP on adult fish is likely to be relatively minor and that the effluent's estrogenic properties may vary over the course of the year. PMID- 12371497 TI - Genetic and demographic responses of mercury-exposed mosquitofish (Gambusia holbrooki) populations: temporal stability and reproductive components of fitness. AB - Two previous mesocosm studies showed changes in glucosephosphate isomerase-2 (Gpi 2) allele frequencies in mosquitofish populations exposed to mercury for 111 d or two years. A previous selection component analysis of single-generation populations exposed for 111 d to 18 microg/L Hg suggested that female sexual selection and fecundity selection could contribute to changes in Gpi-2 allele frequencies. The present multigeneration study was conducted to determine the stability of Gpi-2 allele frequencies over four years of mercury exposure, measure the reproductive fitness of Gpi-2 genotypes inhabiting control and mercury-contaminated mesocosms to determine a mechanism explaining changes in Gpi 2 allele frequencies, investigate differences in the demographic characteristics of mercury-exposed and control populations, and investigate the water quality of the mesocosms to determine if variables other than mercury show concordant patterns among mesocosms. Differences in Gpi-2 allele frequencies between control and mercury-exposed populations were stable over four years (approximately eight generations) of mercury exposure. Mercury-exposed female mosquitofish had a lower probability of being gravid than control females (p = 0.001). Mercury-exposed females also had lower fecundity (total number of eggs and embryos) than control females (p = 0.036). Unlike the results of the more intense mercury exposures in the single generation study, no strong evidence was found that Gpi-2 genotype influenced fecundity or the probability of being gravid in both control and mercury-exposed females. The quantification of fitness components is difficult but has the potential to enhance our understanding of how toxicants alter allele frequencies in exposed populations. PMID- 12371498 TI - Effects of depleted uranium on the health and survival of Ceriodaphnia dubia and Hyalella azteca. AB - Depleted uranium (DU) has been used as a substitute for the fissionable enriched uranium component of atomic weapons tested at Los Alamos National Laboratory (LANL) (Los Alamos, NM, USA) since the early 1950s, resulting in considerable concentrations of DU in the soils within the test sites. Although the movement of DU into major aquatic systems has been shown to be minimal, there are many small order ephemeral streams and areas of standing water in canyons throughout LANL that may be affected by inputs of DU via runoff, erosion, and leaching. Ninety six-hour acute and 7-d chronic toxicity assays were conducted to measure the toxicity of DU on survival and reproduction of Ceriodaphnia dubia. A 14-d water only assay was conducted to measure survival and growth of Hyalella azteca. The estimated median lethal concentration (LC50) to produce 50% mortality of the test population for the 96-h Ceriodaphnia dubia assay was 10.50 mg/L. Reproductive effects occurred at a lowest-observable-effect concentration > or = 3.91 mg/L with a no-observable-effect concentration of 1.97 mg/L. The estimated 14-d LC50 for the Hyalella azteca assay was 1.52 mg/L. No significant relationship was detected between growth and DU concentrations. Concentrations at which toxicity effects were observed in this study for both invertebrates exceeded concentrations of total uranium observed in runoff from LANL lands. Thus, it is likely that current runoff levels of uranium do not pose a threat to these types of aquatic invertebrates. PMID- 12371499 TI - Effects of ligand-bound silver on Ceriodaphnia dubia. AB - In aqueous media, ionic silver concentrations are low and transport occurs in the colloidal phase. In the aquatic environment, silver forms 1:1 complexes with thiol-containing compounds such as cysteine and glutathione. In order to quantitatively characterize the risk associated with silver in aquatic ecosystems, the bioavailabilities and toxicities of silver cysteinate and silver glutathionate were characterized. Static renewal bioassays were conducted with Ceriodaphnia dubia to estimate chronic toxicity, using mortality and reproduction as endpoints. Silver nitrate was the most lethal compound, with a median lethal concentration (8-d LC50) of 0.32 microg Ag/L (95% confidence interval [CI] = 0.19 0.54). The 48-h LC50 for AgNO3 was 0.5 microg/L and did not change significantly through 8 d. The presence of food in the bioassay did not change the 48-h LC50 for AgNO3. Silver glutathionate (AgGSH) and silver cysteinate (AgCys) induced less mortality during the 8-d bioassay. Silver cysteinate appeared to have the greatest effect on fecundity, with a no-observable-effect concentration (NOEC) less than 0.001 microg/L. Silver nitrate and AgGSH had lowest-observable-effect concentration (LOEC) values (nominal concentrations) of 0.01 and 0.6 microg/L, respectively. Results indicate that the ligand-bound silver in these laboratory studies is bioavailable and impairs reproduction of C. dubia at low aqueous concentrations. PMID- 12371500 TI - Polychlorinated biphenyls and toxaphene in Pacific tree frog tadpoles (Hyla regilla) from the California Sierra Nevada, USA. AB - Pacific tree frog (Hyla regilla) tadpoles were collected throughout the Sierra Nevada mountain range, California, USA, in 1996 and 1997 and analyzed for the presence of polychlorinated biphenyls (PCBs) and toxaphene. Whole-tadpole sigma PCB levels ranged from 244 ng/g (wet wt) at lower elevations on the western slope to 1.6 ng/g high on the eastern slope, whereas sigma toxaphene levels ranged from 15.6 to 1.5 ng/g. Linear regression of PCB and toxaphene residue levels versus elevation indicated a significant relationship, with an r2 value of 0.33 for PCB and 0.45 for toxaphene indicating a significant elevation effect on PCB and toxaphene bioaccumulation in Sierra Nevada H. regilla. Tadpole samples from sites in east-facing versus west-facing drainage basins showed significant differences in PCB and toxaphene residue levels, suggesting the possibility of a rain-shadow effect in the long-range atmospheric transport of these contaminants to the Sierra Nevada Mountains. PMID- 12371501 TI - An evaluation of the success of dredging as remediation at a DDT-contaminated site in San Francisco Bay, California, USA. AB - Lauritzen Canal, a portion of San Francisco Bay near Richmond, California, USA, was heavily contaminated with dichlorodiphenyltrichloroethane (DDT) and dieldrin as a result of releases from a pesticide-formulating firm. In 1996 and 1997, 82,000 m3 of contaminated sediment was removed from the canal by dredging. This study evaluated the success of the dredging based largely on body burdens of DDT and its metabolites (sigmaDDT) in resident biota, with some data on sediment- and water-contaminant levels and sediment toxicity testing. Sediment disturbance during dredging introduced a pulse of sigmaDDT into the Lauritzen Canal ecosystem, and body burdens of fish and invertebrates increased 2- to 76-fold, depending on the species. Approximately 1 1/2 years after remediation, 11 of 14 indicators showed contamination comparable with or worse than the contamination that existed prior to dredging. Monitoring of mussels up to four years postdredging suggests some modest improvement, although the sigmaDDT body burden of canal mussels remained far above the norm for San Francisco Bay. The elevated sigmaDDT body burdens in biota that persisted for years after remediation reflect recent exposure and are not merely a result of slow metabolic elimination of the sigmaDDT pulse associated with dredging. Sediment sigmaDDT concentrations were low immediately after dredging, but within months, the canal bottom became covered with a veneer of fine sediment as contaminated as that that had been removed. The source of this material has not been conclusively established, but we suspect it came from slumping and erosion from the flanks of the canal beneath docks and around pilings where dredging was not done. In retrospect, either capping in place or more thorough dredging may have been more successful in reducing pesticide exposure of the biota, although there were difficulties associated with both alternatives. PMID- 12371502 TI - Quantitative structure-activity relationship for the photoinduced toxicity of polycyclic aromatic hydrocarbons to the luminescent bacteria Vibrio fischeri. AB - Sunlight can greatly enhance the toxicity of polycyclic aromatic hydrocarbons (PAHs). Photosensitization reactions (e.g., generation of singlet-state oxygen) and photomodification reactions (e.g., photooxidation of PAHs to more toxic species) are both pathways of photoinduced toxicity of PAHs. Previously, a quantitative structure-activity relationship (QSAR) was developed for PAHs showing that a photosensitization factor (PSF) and photomodification factor (PMF) can be additively combined to describe photoinduced toxicity. That QSAR model was developed for the photoinduced toxicity of 16 PAHs to the higher plant Lemna gibba. The objective of this study was to apply the QSAR model developed for L. gibba to another organism. The organism chosen was the luminescent marine bacteria Vibriofischeri. Toxicity data used for the QSAR model were inhibition of luminescence and inhibition of growth of V. fischeri. Both short-term (15 min) and long-term (18 h) assays of toxicity were used. Light did not impact on PAH toxicity in the short-term assay, and thus the QSAR model did not correlate well with these data. Conversely, light greatly enhanced toxicity when the long-term assay was employed. The PMFs for the PAHs from the L. gibba QSAR showed a moderate correlation to bacterial toxicity in the long-term assay, whereas the PSFs showed only a weak correlation to toxicity. As was the case for L gibba, summing the PMF and the PSF resulted in a strong correlation to toxicity that had predictive value. Thus, a QSAR model derived for plants accurately described the toxicity of PAHs to a bacterial species. This indicates that the bipartite mechanism of PAH-photoinduced toxicity may be applicable to other organisms. PMID- 12371503 TI - Integrative assessment of benthic macroinvertebrate community impairment from metal-contaminated waters in tributaries of the Upper Powell River, Virginia, USA. AB - Benthic macroinvertebrate communities of the North Fork Powell River (NFP), southwest Virginia, USA, appear to be impacted by aluminum (Al) and iron (Fe) from acid mine drainage (AMD) beyond the zone of pH depression. As part of a watershed restoration project, we used integrative techniques, including water column, sediment, and in situ toxicity tests; sediment and water column chemistry; and habitat assessments, to detect AMD impacts. An analysis of variance, least significant difference post hoc test, and Spearman correlations were used to test the sensitivity of these integrative techniques to detect various (acidic or neutralized) levels of AMD input and to determine the mode of impairment (metal-contaminated sediments or water) to the benthic macroinvertebrate community. Benthic macroinvertebrate indices were the most sensitive endpoint to AMD inputs and were significantly correlated (p < or = 0.05) with water column metal concentrations in in situ and water column toxicity tests. Sediment chemistry and toxicity did not detect AMD impacts and were not significantly correlated with benthic macroinvertebrate indices. These results suggest that the primary mode of impairment to the benthic macroinvertebrate communities beyond the zone of pH depression were waterborne Al and Fe. PMID- 12371504 TI - Oil effect in freshly spiked marine sediment on Vibrio fischeri, Corophium volutator, and Echinocardium cordatum. AB - The purpose of this study was to provide data to be used in The Netherlands for development of ecotoxicologically based quality criteria for oil-contaminated sediments and dredged material. In addition, the relation of toxicity to specific oil boiling-point fraction ranges was explored. Natural marine sediment, with a moisture, organic carbon, and silt content of approximately 80, 1.8, and 33% of the dry weight, respectively, was artificially spiked using a spiking method developed in this project. Aliquots of one part of the sediment were spiked to several concentrations of Gulf distillate marine grade A (DMA) gasoil (containing 64% C10-19) and aliquots of the other part to several concentrations of Gulf high viscosity grade 46 (HV46) hydraulic oil (containing 99.2% C19-40). Thus, for each individual oil type, a concentration series was created. Vibrio fischeri (endpoint: bioluminescence inhibition), Corophium volutator (endpoint:mortality), and Echinocardium cordatum (endpoint:mortality) were exposed to these spiked sediments for 10 min, 10 d and 14 d, respectively. Based on the test results, the effective concentration on 50% of the test animals was statistically estimated. For DMA gasoil and HV46 hydraulic oil, respectively, the effective concentrations were 43.7 and 2,682 mg/kg dry weight for V. fischeri, 100 and 9,138 mg/kg dry weight for C. volutator, 190, and 1064 mg/kg dry weight for E. cordatum. This study shows that the toxicity is strongly correlated with the lower boiling-point fractions and especially to those within the C10-C19 range. PMID- 12371505 TI - Modeling explicitly and mechanistically median lethal concentration as a function of time for risk assessment. AB - A mechanistic model that explains how toxic effects depend on the duration of exposure has been developed. Derived from the dynamic energy budget (DEB)tox model, it expresses the hazard rate as a function of the toxic concentration in the organism. Using linear approximations in accordance with the general simplifications made in DEBtox, the concentration that induces x% of lethality (LCx) and in particular the lethal concentration 50% (LC50) are expressed explicitly as functions of time. Only three parameters are required: an asymptotic effect concentration, a time constant, and an effect velocity. More sophisticated (but still analytic) models are possible, describing more complex toxicity patterns such as an increase of sensitivity with time or, conversely, an adaptation. These models can be fitted to the common and widespread LC50 endpoints available from the literature for various aquatic species and chemicals. The interpretation of the values assigned to the parameters will help explain the toxicity processes and standardize toxicity values from different sources for comparisons. PMID- 12371506 TI - Cellular, synaptic and network effects of neuromodulation. AB - All network dynamics emerge from the complex interaction between the intrinsic membrane properties of network neurons and their synaptic connections. Nervous systems contain numerous amines and neuropeptides that function to both modulate the strength of synaptic connections and the intrinsic properties of network neurons. Consequently network dynamics can be tuned and configured in different ways, as a function of the actions of neuromodulators. General principles of the organization of modulatory systems in nervous systems include: (a) many neurons and networks are multiply modulated, (b) there is extensive convergence and divergence in modulator action, and (c) some modulators may be released extrinsically to the modulated circuit, while others may be released by some of the circuit neurons themselves, and act intrinsically. Some of the computational consequences of these features of modulator action are discussed. PMID- 12371507 TI - Metalearning and neuromodulation. AB - This paper presents a computational theory on the roles of the ascending neuromodulatory systems from the viewpoint that they mediate the global signals that regulate the distributed learning mechanisms in the brain. Based on the review of experimental data and theoretical models, it is proposed that dopamine signals the error in reward prediction, serotonin controls the time scale of reward prediction, noradrenaline controls the randomness in action selection, and acetylcholine controls the speed of memory update. The possible interactions between those neuromodulators and the environment are predicted on the basis of computational theory of metalearning. PMID- 12371508 TI - Dopamine-dependent plasticity of corticostriatal synapses. AB - Knowledge of the effect of dopamine on corticostriatal synaptic plasticity has advanced rapidly over the last 5 years. We consider this new knowledge in relation to three factors proposed earlier to describe the rules for synaptic plasticity in the corticostriatal pathway. These factors are a phasic increase in dopamine release, presynaptic activity and postsynaptic depolarisation. A function is proposed which relates the amount of dopamine release in the striatum to the modulation of corticostriatal synaptic efficacy. It is argued that this function, and the experimental data from which it arises, are compatible with existing models which associate the reward-related firing of dopamine neurons with changes in corticostriatal synaptic efficacy. PMID- 12371509 TI - TD models of reward predictive responses in dopamine neurons. AB - This article focuses on recent modeling studies of dopamine neuron activity and their influence on behavior. Activity of midbrain dopamine neurons is phasically increased by stimuli that increase the animal's reward expectation and is decreased below baseline levels when the reward fails to occur. These characteristics resemble the reward prediction error signal of the temporal difference (TD) model, which is a model of reinforcement learning. Computational modeling studies show that such a dopamine-like reward prediction error can serve as a powerful teaching signal for learning with delayed reinforcement, in particular for learning of motor sequences. Several lines of evidence suggest that dopamine is also involved in 'cognitive' processes that are not addressed by standard TD models. I propose the hypothesis that dopamine neuron activity is crucial for planning processes, also referred to as 'goal-directed behavior', which select actions by evaluating predictions about their motivational outcomes. PMID- 12371510 TI - Actor-critic models of the basal ganglia: new anatomical and computational perspectives. AB - A large number of computational models of information processing in the basal ganglia have been developed in recent years. Prominent in these are actor-critic models of basal ganglia functioning, which build on the strong resemblance between dopamine neuron activity and the temporal difference prediction error signal in the critic, and between dopamine-dependent long-term synaptic plasticity in the striatum and learning guided by a prediction error signal in the actor. We selectively review several actor-critic models of the basal ganglia with an emphasis on two important aspects: the way in which models of the critic reproduce the temporal dynamics of dopamine firing, and the extent to which models of the actor take into account known basal ganglia anatomy and physiology. To complement the efforts to relate basal ganglia mechanisms to reinforcement learning (RL), we introduce an alternative approach to modeling a critic network, which uses Evolutionary Computation techniques to 'evolve' an optimal RL mechanism, and relate the evolved mechanism to the basic model of the critic. We conclude our discussion of models of the critic by a critical discussion of the anatomical plausibility of implementations of a critic in basal ganglia circuitry, and conclude that such implementations build on assumptions that are inconsistent with the known anatomy of the basal ganglia. We return to the actor component of the actor-critic model, which is usually modeled at the striatal level with very little detail. We describe an alternative model of the basal ganglia which takes into account several important, and previously neglected, anatomical and physiological characteristics of basal ganglia-thalamocortical connectivity and suggests that the basal ganglia performs reinforcement-biased dimensionality reduction of cortical inputs. We further suggest that since such selective encoding may bias the representation at the level of the frontal cortex towards the selection of rewarded plans and actions, the reinforcement-driven dimensionality reduction framework may serve as a basis for basal ganglia actor models. We conclude with a short discussion of the dual role of the dopamine signal in RL and in behavioral switching. PMID- 12371511 TI - Dopamine: generalization and bonuses. AB - In the temporal difference model of primate dopamine neurons, their phasic activity reports a prediction error for future reward. This model is supported by a wealth of experimental data. However, in certain circumstances, the activity of the dopamine cells seems anomalous under the model, as they respond in particular ways to stimuli that are not obviously related to predictions of reward. In this paper, we address two important sets of anomalies, those having to do with generalization and novelty. Generalization responses are treated as the natural consequence of partial information; novelty responses are treated by the suggestion that dopamine cells multiplex information about reward bonuses, including exploration bonuses and shaping bonuses. We interpret this additional role for dopamine in terms of the mechanistic attentional and psychomotor effects of dopamine, having the computational role of guiding exploration. PMID- 12371512 TI - The computational role of dopamine D1 receptors in working memory. AB - The prefrontal cortex (PFC) is essential for working memory, which is the ability to transiently hold and manipulate information necessary for generating forthcoming action. PFC neurons actively encode working memory information via sustained firing patterns. Dopamine via D1 receptors potently modulates sustained activity of PFC neurons and performance in working memory tasks. In vitro patch clamp data have revealed many different cellular actions of dopamine on PFC neurons and synapses. These effects were simulated using realistic networks of recurrently connected assemblies of PFC neurons. Simulated D1-mediated modulation led to a deepening and widening of the basins of attraction of high (working memory) activity states of the network, while at the same time background activity was depressed. As a result, self-sustained activity was more robust to distracting stimuli and noise. In this manner, D1 receptor stimulation might regulate the extent to which PFC network activity is focused on a particular goal state versus being open to new goals or information unrelated to the current goal. PMID- 12371513 TI - Dopamine controls fundamental cognitive operations of multi-target spatial working memory. AB - This study addresses computationally how the prefrontal cortical circuit performs operations of multiple items in spatial working memory. The basic idea is that dopamine controls the circuit dynamics for the operations by changing the ratio of the NMDA-channel transmission to the AMPA-channel transmission. There is evidence that this ratio is a function of dopamine D1 receptor activation. The simulation shows that the model circuit performs several different operations of multi-target spatial working memory depending on this ratio. When the ratio is low, 'replacement' occurs from the previously loaded target to a new one. In intermediate levels of the ratio, a new target is 'added' to the previously loaded target, resulting in the coexistence of more than one target. For higher ratios, the circuit 'rejects' other succeedingly received target stimuli. This study suggests four important issues: First, the cortical circuit can perform operations of multi-target spatial working memory. Second, the circuit can switch the modes of the operations by changing the NMDA-to-AMPA ratio. Third, dopamine would have major roles in the operations of multi-target spatial working memory. Fourth, the intracortical inhibition (especially of the cross-directional) plays an important role in regulating the competition between targets. PMID- 12371514 TI - An integrative theory of the phasic and tonic modes of dopamine modulation in the prefrontal cortex. AB - This paper presents a model of both tonic and phasic dopamine (DA) effects on maintenance of working memory representations in the prefrontal cortex (PFC). The central hypothesis is that DA modulates the efficacy of inputs to prefrontal pyramidal neurons to prevent interferences for active maintenance. Phasic DA release, due to DA neurons discharges, acts at a short time-scale (a few seconds), while the tonic mode of DA release, independent of DA neurons firing, acts at a long time-scale (a few minutes). The overall effect of DA modulation is modeled as a threshold restricting incoming inputs arriving on PFC neurons. Phasic DA release temporary increases this threshold while tonic DA release progressively increases the basal level of this threshold. Thus, unlike the previous gating theory of phasic DA release, proposing that it facilitates incoming inputs at the time of their arrival, the effect of phasic DA release is supposed to restrict incoming inputs during a period of time after DA neuron discharges. The model links the cellular and behavioral levels during performance of a working memory task. It allows us to understand why a critical range of DA D1 receptors stimulation is required for optimal working memory performance and how D1 receptor agonists (respectively antagonists) increase perseverations (respectively distractability). Finally, the model leads to several testable predictions, including that the PFC regulates DA neurons firing rate to adapt to the delay of the task and that increase in tonic DA release may either improve or decrease performance, depending on the level of DA receptors stimulation at the beginning of the task. PMID- 12371515 TI - Opponent interactions between serotonin and dopamine. AB - Anatomical and pharmacological evidence suggests that the dorsal raphe serotonin system and the ventral tegmental and substantia nigra dopamine system may act as mutual opponents. In the light of the temporal difference model of the involvement of the dopamine system in reward learning, we consider three aspects of motivational opponency involving dopamine and serotonin. We suggest that a tonic serotonergic signal reports the long-run average reward rate as part of an average-case reinforcement learning model; that a tonic dopaminergic signal reports the long-run average punishment rate in a similar context; and finally speculate that a phasic serotonin signal might report an ongoing prediction error for future punishment. PMID- 12371516 TI - Local analysis of behaviour in the adjusting-delay task for assessing choice of delayed reinforcement. AB - The adjusting-delay task introduced by Mazur (Quantitative analyses of behavior: V. The effect of delay and of intervening events on reinforcement value, 1987, pp. 55-73) has been widely used to study choice of delayed reinforcers. This paradigm involves repeated choice between one reinforcer delivered after a fixed delay and another, typically larger, reinforcer delivered after a variable delay; the variable delay is adjusted depending on the subject's choice until an equilibrium point is reached at which the subject is indifferent between the two alternatives. Rats were trained on a version of this task and their behaviour was examined to determine the nature of their sensitivity to the adjusting delay; these analyses included the use of a cross-correlational technique. No clear evidence of sensitivity to the adjusting delay was found. A number of decision rules, some sensitive to the adjusting delay and some not, were simulated and it was observed that some effects usually supposed to be a consequence of delay sensitivity could be generated by delay-independent processes, such as a consistent, unchanging relative preference between the alternatives. Consequently, the use of explicit analysis of delay sensitivity is advocated in future research on delayed reinforcement. PMID- 12371517 TI - Neuromodulation of decision and response selection. AB - We present a model for the attentional neuromodulation of decision and selection processes. The model assumes that phasic responses in the brain nucleus Locus Coeruleus modulate, via the transmission of norepinephrine, the synaptic efficiency of neural circuits, at specific (stimulus and task dependent) time intervals. The model is applied first, to a task of perceptual choice, simulating attentional fluctuations and accounting for a series of behavioral and neurophysiological data. Second, the flexibility of information processing, whereby the parameters of the local circuits are modified online, is illustrated in the application of the model to a task of selection from short-term memory. PMID- 12371518 TI - Simplified dynamics in a model of noradrenergic modulation of cognitive performance. AB - Neurophysiological work in monkeys has shown that changes in tonic and stimulus induced activity in the noradrenergic brainstem nucleus locus coeruleus (LC) are tightly correlated with fluctuations in behavioral performance in a visual discrimination task. Simulation work suggests transitions between observed modes of LC activity may be mediated by changes in the level of coherent firing among individual LC neurons. We have simplified this simulation by abstracting the LC to a simple, excitable system in two variables modeled after the FitzHugh-Nagumo relaxation oscillator. This abstracted LC simulates population-level dynamics of the nucleus relevant to its influence on behavior. Coherence within the nucleus was simulated as a single parameter which amplified external input to the abstracted LC while attenuating tonic, uncorrelated activity. Simulated results captured LC dynamics and their relationship with behavior observed in monkeys. Behavior was further simulated over a range of potential LC states not, as yet, directly observed. These results suggest a reverse sigmoidal relationship between false alarm rate and coherence, and a more complex, non-monotonic relationship between response time and coherence. PMID- 12371519 TI - Control of exploitation-exploration meta-parameter in reinforcement learning. AB - In reinforcement learning (RL), the duality between exploitation and exploration has long been an important issue. This paper presents a new method that controls the balance between exploitation and exploration. Our learning scheme is based on model-based RL, in which the Bayes inference with forgetting effect estimates the state-transition probability of the environment. The balance parameter, which corresponds to the randomness in action selection, is controlled based on variation of action results and perception of environmental change. When applied to maze tasks, our method successfully obtains good controls by adapting to environmental changes. Recently, Usher et al. [Science 283 (1999) 549] has suggested that noradrenergic neurons in the locus coeruleus may control the exploitation-exploration balance in a real brain and that the balance may correspond to the level of animal's selective attention. According to this scenario, we also discuss a possible implementation in the brain. PMID- 12371520 TI - Neuromodulation, theta rhythm and rat spatial navigation. AB - Cholinergic and GABAergic innervation of the hippocampus plays an important role in human memory function and rat spatial navigation. Drugs which block acetylcholine receptors or enhance GABA receptor activation cause striking impairments in the encoding of new information. Lesions of the cholinergic innervation of the hippocampus reduce the amplitude of hippocampal theta rhythm and cause impairments in spatial navigation tasks, including the Morris water maze, eight-arm radial maze, spatial reversal and delayed alternation. Here, we review previous work on the role of cholinergic modulation in memory function, and we present a new model of the hippocampus and entorhinal cortex describing the interaction of these regions for goal-directed spatial navigation in behavioral tasks. These mechanisms require separate functional phases for: (1) encoding of pathways without interference from retrieval, and (2) retrieval of pathways for guiding selection of the next movement. We present analysis exploring how phasic changes in physiological variables during hippocampal theta rhythm could provide these different phases and enhance spatial navigation function. PMID- 12371521 TI - Cholinergic modulation of sensory representations in the olfactory bulb. AB - We present a computational model of the mammalian olfactory bulb (OB) designed to investigate how cholinergic inputs modulate olfactory sensory representations. The model integrates experimental data derived from diverse physiological studies of cholinergic modulation of OB circuitry into a simulation of bulbar responses to realistic odorants. Experimentally-observed responses to a homologous series of odorants (unbranched aliphatic aldehydes) were simulated; realistic cholinergic inputs to the OB model served to increase the discriminability of the bulbar responses generated to very similar odorants. This simulation predicted, correctly, that missing cholinergic inputs to the OB would result in greater generalization between similar aliphatic aldehydes. Based on the assumption that the overlap between the neural representations of two sensory stimuli is predictive of their perceptual similarity, we tested this prediction in a behavioral experiments with rats. We show that, indeed, rats with selective lesions of cholinergic neurons that project to the OB and cortex discriminate less well between aliphatic aldehydes with similar carbon chain lengths than do rats that received sham lesions. PMID- 12371522 TI - Acetylcholine in cortical inference. AB - Acetylcholine (ACh) plays an important role in a wide variety of cognitive tasks, such as perception, selective attention, associative learning, and memory. Extensive experimental and theoretical work in tasks involving learning and memory has suggested that ACh reports on unfamiliarity and controls plasticity and effective network connectivity. Based on these computational and implementational insights, we develop a theory of cholinergic modulation in perceptual inference. We propose that ACh levels reflect the uncertainty associated with top-down information, and have the effect of modulating the interaction between top-down and bottom-up processing in determining the appropriate neural representations for inputs. We illustrate our proposal by means of an hierarchical hidden Markov model, showing that cholinergic modulation of contextual information leads to appropriate perceptual inference. PMID- 12371523 TI - Sensory-motor gating and cognitive control by the brainstem cholinergic system. AB - As an essential component of ascending activating systems, cholinergic neurons with diffuse projections are supposed to be involved in the regulation of cognitive processes such as attention, consciousness, learning, and memory. As for the role of cholinergic projections from the basal forebrain nuclei to cerebral cortical regions including hippocampus, a couple of models have been proposed that acetylcholine facilitates extrinsic inputs to the cortex and inhibits intracortical processing. In this review, to explore the possibility that there exists a generalized principle on the role of cholinergic systems in the brain, we summarized the knowledge so far obtained on the action of a brainstem cholinergic nucleus, the pedunculopontine tegmental nucleus (PPTN) at their target regions. By in vitro experiments we clarified that cholinergic inputs to the intermediate layer of the superior colliculus, presumably originating from the PPTN, facilitate generation of its motor outputs for the initiation of saccades. Furthermore, cholinergic inputs may enhance excitatory responses of mesopontine dopaminergic cells, for instance to reward-related signals. In addition, we observed that PPTN neurons showed multi-modal activities in behaving monkeys; their activities were related to execution and preparation of saccades, the level of task performance, and reward. The multi-modal activities encoded in the PPTN may suggest that PPTN associates movement-related activities with those related to task performance and reward. Together with the already reported facilitatory action on the sensory processing at the visual thalamus, these observations suggest that the brainstem cholinergic system facilitates the central processes for motor command generation and extrinsic sensory processing. For our final goal of exploring the general working principle of the cholinergic systems, further studies are needed to clarify the effects of the brainstem cholinergic system on the intrinsic processing in the brain. PMID- 12371524 TI - On-line learning in changing environments with applications in supervised and unsupervised learning. AB - An adaptive on-line algorithm extending the learning of learning idea is proposed and theoretically motivated. Relying only on gradient flow information it can be applied to learning continuous functions or distributions, even when no explicit loss function is given and the Hessian is not available. The framework is applied for unsupervised and supervised learning. Its efficiency is demonstrated for drifting and switching non-stationary blind separation tasks of acoustic signals. Furthermore applications to classification (US postal service data set) and time series prediction in changing environments are presented. PMID- 12371525 TI - Neuromodulation and plasticity in an autonomous robot. AB - In this paper we implement a computational model of a neuromodulatory system in an autonomous robot. The output of the neuromodulatory system acts as a value signal, modulating widely distributed synaptic changes. The model is based on anatomical and physiological properties of midbrain diffuse ascending systems, in particular parts of the dopamine and noradrenaline systems. During reward conditioning, the model learns to generate tonic and phasic signals that represent predictions and prediction errors, including precisely timed negative signals if expected rewards are omitted or delayed. We test the robot's learning and behavior in different environmental contexts and observe changes in the development of the neuromodulatory system that depend upon environmental factors. Simulation of a computational model incorporating both reward-related and aversive stimuli leads to the emergence of conditioned reward and aversive behaviors. These studies represent a step towards investigating computational aspects of neuromodulatory systems in autonomous robots. PMID- 12371526 TI - Equivalent efficacy and safety of a new HFA-134a formulation of BDP compared with the conventional CFC in adult asthmatics. AB - The present study demonstrates the equivalent efficacy for BDP 500 microg bid given via MDI with the new HFA-134a propellant (Chiesi Farmaceutici S.p.A., Parma) compared to a conventional CFC propellant (Becotide, Allen & Hanburys, UK). One hundred and sixteen adult patients with stable mild to moderate asthma (FEV1 > or = 60% of predicted normal) entered a 2-week run-in period where they maintained their own inhaled corticosteroids and were then assigned to a 12-week treatment with the test drug in a randomized, multicentre, double-blind, double dummy, parallel-group design. Ninety-one patients completed the study period. Morning and evening peak expiratory flow rate (PEFR), use of rescue salbutamol, number of daytime and nighttime asthma attacks, number of nighttime awakenings, and clinical symptoms were recorded daily by patients on a diary card. Pulmonary function tests (FEV1, FVC, PEFR, MEF50 and FEF25) were completed at study entry, at the start of treatment and every 2 weeks thereafter. Morning (08.00-10.00 AM) serum cortisol was measured at the start and at the end of treatment. Adverse events were collected for the total study period. Equivalence between groups was demonstrated for the primary end-point morning PEFR, as well as for evening PEFR and FEV1 (the 95% CI of the treatments' difference was within the 5% of the LSM of BDP CFC). The other secondary pulmonary function tests measured at the clinic visit showed a satisfactory asthma control, albeit without statistically significant differences between groups. Decreases in the use of rescue salbutamol and in clinical symptoms were also reported in both groups, with no differences between them. Adverse events were reported in 81.4% of patients in the BDP HFA group and in 82.5% in the CFC group. There were 73 and 59 adverse drug reactions in the two groups, respectively; the difference was mainly due to differences in taste. No drug-related serious adverse events were reported in either group. No difference was seen for morning serum cortisol between baseline and end of treatment, or between groups. In conclusion, the BDP-HFA 134a formulation proved to be statistically equivalent to the standard BDP CFC product over 12 weeks in adult patients with mild to moderate asthma. PMID- 12371527 TI - Prevalence of latex sensitization in health care workers of a general hospital in Palermo, Sicily. AB - STUDY OBJECTIVE: To assess the prevalence of latex sensitization in a group of hospital employees in a general hospital. DESIGN: Cross-sectional study on hypersensitivity to latex gloves among health-care workers. SETTING: A general hospital in Palermo, Sicily. PATIENTS: 196 health-care workers answered a questionnaire about their case history of allergic diseases (i. e., rhinitis and/or asthma) and about symptoms after wearing latex gloves. All subjects were tested by skin prick test (SPT) with commercial latex extract and aeroallergens and had blood draw for total serum IgE and latex-specific IgE testing and glove use test. MAIN RESULTS: 42% of the subjects who answered the questionnaire reported at least one symptom after wearing latex gloves. All symptoms were local, and none of the subjects reported systemic reactions. The most common symptom was itching, but none of subjects with only itching presented a positive SPT or specific serum IgE to latex. The SPT to latex was positive in 19 of 196 subjects (9.7%). Specific IgE to latex were found in 15/196 subjects (7.6%). Glove-use test was positive in 14/196 (7.1%). CONCLUSIONS: The overall prevalence of latex sensitivity in health-care workers in our epidemiological setting is 7.1%. An accurate diagnosis must take in account the integration of in vivo and in vitro tests with previous history of allergic disease. PMID- 12371528 TI - Nitric oxide sera levels as an inflammatory marker in asthma. AB - BACKGROUND: Nitric oxide is an important regulator of various biological activities in human physiology, and it has been studied in the physiopathology of asthma, especially in exhaled air. OBJECTIVE: We studied the presence of NO in sera of patients with distinct degrees of asthma severity in order to determine a parameter of diagnosis and control of efficacious treatment. METHODS: We determined the presence of NO based upon the Griess reaction in the sera of 124 donors--34 controls and 90 asthmatic subjects. RESULTS: Asthmatic patients presented higher levels of nitric oxide in peripheral blood (56.54 +/- 33.37) compared to the control group (3.06 +/- 4.48). A statistically significant difference (p < 0.0001) between the nitric oxide sera levels from the two groups was demonstrated. CONCLUSION: This study demonstrates that nitric oxide sera levels can be used as an additional inflammatory marker in asthma, especially as an auxiliary diagnostic method in children where NO exhaled air analysis is difficult. PMID- 12371529 TI - Incidence of pollinosis in the city of A Coruna: correlation with aerobiological data. AB - An atmospheric pollen count was carried out in the city of A Coruna during 1999 using two pollen traps located at two different points in the city. A total number of 6979 and 3536 pollen grains, respectively, were identified, the majority during the Spring and Summer. Further, patients living near the pollen traps were selected from among those diagnosed as suffering from respiratory allergies by the Allergy Department of A Coruna's Juan Canalejo Hospital. The patients had at least one positive skin test for some pollen type, had not received immunotherapy in the last year, and were willing to fill in a symptoms booklet during the study period. The results obtained reveal the pollen types that produce the greatest number of skin sensitization cases (Poaceae, Plantago, Chenopodium and Parietaria), with a positive correlation between the atmospheric pollen concentration of such taxa and the frequency of allergy symptoms. This has enabled the setting of pollen values, above which A Coruna's inhabitants are considered to be at risk. PMID- 12371530 TI - IgE-mediated urticaria from formaldehyde in a dental root canal compound. AB - A patient had suffered twice from an acute urticaria after treatment with two different dental root canal compounds (RCC). Skin prick tests with the single components and formaldehyde 1%aq. were positive to formaldehyde 1%aq., but irritant with formaldehydeRCC. Specific IgE to formaldehyde were positive. Patch tests were positive to formaldehydeRCC only. In the literature 28 patients with immediate symptoms to formaldehyde containing RCC have been described. PMID- 12371531 TI - Desensitization to latex by percutaneous route. AB - Latex allergy is a newly emerging problem. In the last decades its prevalence has increased progressively, especially among health care personnel and patients. Preventive measures have been suggested to reduce the risk of sensitization, but this is very difficult because of the ubiquity of latex products. Since only two clinical reports are available in the literature, suggesting that subcutaneous desensitizing treatments resulted in important side effects, we decided to attempt a desensitization through alternative routes. After having succeeded in carrying out sublingual desensitization, we report the case of a latex-allergic patient who successfully underwent percutaneous desensitization. PMID- 12371532 TI - Urticaria to cetirizine. AB - A patient with recurrent idiopathic urticaria reported exacerbations after treatment with cetirizine. Prick test to cetirizine was negative. Double-blind challenge tests with mizolastine, loratadine, fexofenadine, dexchlorpheniramine, ebastine, ketotifen, and placebo were negative, whereas hydroxyzine and its active metabolite, cetirizine, reproduced the urticaria. Identification of uncommon adverse reactions to H1 antihistamines is important, particularly because they may mimic the underlying disease. PMID- 12371533 TI - Chlamydia pneumoniae infection and its role in asthma and chronic obstructive pulmonary disease. AB - Chlamydia pneumoniae (CP) is a common cause of respiratory tract infections, and several studies have asked whether it may play a pathogenic role in connection with bronchial asthma and chronic obstructive pulmonary disease (COPD). Evidence that CP infection is associated with these diseases is a cardinal item. However, evaluation of CP infection is hampered by difficulties in obtaining agreement on the definition of a gold standard. In the literature, serology is based on different cutoff points of antibody titres, which complicates the definition of CP seropositive findings and the classification of acute infection, chronic and past infection. In connection with acute and chronic infection, it is important to demonstrate the presence of CP by culture or polymerase chain reaction (PCR) in the respiratory tract, especially in the lower airways. Often, the results of serology is not associated with the findings by culture or PCR testing, which may involve the risk of inconclusive evidence. Evaluation of a possible presence of CP by clinical improvement after treatment with antibiotics is difficult since uncontrolled studies have been used and other microorganisms are also affected by antibiotics. Furthermore, many patients improve without antibiotics, and improvement has also been observed in patients remaining culture positive after treatment with antibiotics. It should also be noted that the antiinflammatory effects of antibiotics may improve the clinical status of patients. Despite these obstacles, studies point to the possibility that in some patients acute CP infections may lead to acute exacerbations of bronchial asthma. Whether a persistent CP infection contributes to chronic asthma or severe COPD, or whether it incites the diseases in previously healthy individuals is a question for further studies. Whether a causal relationship exists between CP infection and obstructive pulmonary disease or whether these patients are more susceptible to CP infection is unknown. Nevertheless, a cooperative role of CP in the proinflammatory mechanisms involved in these diseases remains to be examined since cellular studies show that CP stimulates the production and expression of cytokines, chemokines and adhesion molecules, actions that may amplify and prolong the inflammation. PMID- 12371534 TI - Elevated IgM anti-IgE in sera from allergic patients. AB - The role of anti-IgE autoantibodies in IgE-related allergic diseases has not been elucidated sufficiently. For example, anti-IgE antibodies have been reported to cause both proallergic and antiallergic blocking reactions. Contrary to other authors, some authors revealed a positive correlation between total IgE and the amount of IgE/IgG complexes detected. By comparing the IgE levels of allergic patients with those of control persons and rheumatoid arthritis patients the present study contributes to our understanding of the role of anti-IgE autoantibodies. The sera were tested by means of ELISA concerning their content of free and complexed IgG and IgM anti-IgE. In addition, the amounts of IgE/IgG and IgE/IgM complexes were determined in the sera of allergic and control persons after Superose 6 column separation. We found that the allergic patients revealed significantly higher values of free IgM anti-IgE than patients with rheumatoid arthritis and than control persons (mean 0.470, p = 0.0164 and p = 0.0061, respectively). The corresponding values for IgE/IgM complexes also tend to be higher (mean 0.431, p = 0.0784 and p = 0.0601, respectively). However, the corresponding contents of IgG anti-IgE autoantibodies and IgE/IgG complexes did not differ significantly. After Superose 6 column separation, we detected IgE/IgG in fractions corresponding to MW of 150 to 180 kDa for the sera of both allergic and control persons. In contrast, the IgE/IgM complexes were found in fractions corresponding to MW 330 kDa. We conclude that the increased IgM anti-IgE autoantibody titer in the sera of allergic patients is not correlated with the high total IgE level. Moreover, we suggest that the IgE/IgG complexes form de nova during ELISA. Unlike the IgE/IgG complexes, the IgE/IgM complexes are assumed to occur already in circulating blood. PMID- 12371535 TI - Detection of the genes induced in activated lymphocytes by modified differential display. AB - Activated lymphocytes induced by mitogens or antigens express various sets of genes, including those involved in the expression of cytokines, surface molecules, and nuclear proteins. To detect inducible genes in activated lymphocytes, we used the RNA arbitrarily primed polymerase chain reaction (RAP PCR) method, which is modified by original differential display. By this method we identified eight clones; four contained sequences almost identical to that of the genes for heat shock 90Kd protein1 alpha, STAT2, Ig kappa constant region and interferon receptor 1, two had 70% homology to mitochondrial ATP synthase and bromodomain-containing 2 genes, and two had less than 40% homology to known DNA sequences. By RT-PCR, the heat shock 90 Kd protein1 alpha, STAT2 and interferon receptor 1 genes showed increased expression in phytohemagglutinin (PHA) stimulated or antigen stimulated T cell line. Differential display is a useful method for detection of inducible genes from a small amount of material and at various time points, although the homology of PCR primer influences the displayed genes. PMID- 12371536 TI - Airway obstruction induced by inhaled acetaldehyde in asthma: repeatability relationship to adenosine 5'-monophosphate responsiveness. AB - Inhaled acetaldehyde and adenosine 5'-monophosphate (AMP) cause bronchoconstriction in asthmatics by a mechanism believed to involve histamine release from airway mast cells. This study investigates the repeatability of the acetaldehyde challenge and the relationship between airway responsiveness to acetaldehyde and AMP. To this end, we examined the effect of inhaled acetaldehyde on airway tone in comparison with either methacholine or AMP in 16 asthmatics. Furthermore, the repeatability of the acetaldehyde challenge was assessed in 14 subjects with mild asthma. The response to each bronchoconstrictor agent was measured by the PC20 (provocative concentration required to produce a 20% fall in FEV1). The geometric mean (range) PC20 values were 3.1 mmol/l (0.5-46.0 mmol/l) for methacholine, 883.1 mmol/l (190.7-1816.1 mmol/l) for acetaldehyde, and 50.1 mmol/l (3.2-1152.1 mmol/l) for AMP. Thus, acetaldehyde was 18-fold less potent than AMP in causing bronchoconstriction. A similar correlation was observed between PC20 acetaldehyde and either PC20 AMP (r = 0.58, p = 0.02) or PC20 methacholine (r = 0.56, p = 0.02). The challenge procedure with acetaldehyde was moderately repeatable (coefficient of repeatability = +/- 1.4 doubling concentrations, intraclass correlation coefficient = 0.64). We conclude that inhaled acetaldehyde is less potent than AMP in causing bronchoconstriction in asthma, and that the response to inhaled acetaldehyde is repeatable. Furthermore, the present data lends indirect support to the suggestion that acetaldehyde responsiveness and AMP responsiveness are not identifying the same alterations in the airways. PMID- 12371538 TI - Influence of the antenna diagram on a stellar interferometer that is suffering from telescope-pointing errors. AB - We report our experimental investigations of the influence of differential telescope-pointing errors on data corruption in an optical stellar interferometer. This effect was investigated theoretically as a function of the telescope antenna diagram, which depends on the aperture diameter. Using a laboratory breadboard consisting of a three-telescope array, we carried out the experiments with various aperture diameters and complex objects. The results matched the simulation and demonstrate that, when there is no error in pointing, a large aperture size induces correctible error but that, with a pointing error, data corruption becomes critical. In both cases, the larger the apertures, the more corrupt the data. PMID- 12371539 TI - Action from tunable periodic structures. II. Experimental observation of electric field-induced diffraction peaks. AB - As previously predicted [Appl. Opt. 40, 5583 (2001)], we have now observed electric field-induced diffraction peaks in transmission and reflection experiments by use of a LiNbO3 sample with interdigital planar electrodes that serve as a diffraction grating. The magnitudes of the new peaks in the reflection experiments are ten times larger than those in the transmission experiments. We interpret these effects in terms of a field-induced refractive-index change produced by the linear electro-optic effect. The positive and negative changes in the refractive index produce two diffraction gratings that are period doubled with respect to the original grating and that have a phase difference between them. The superposition of the diffracted light from these gratings is shown to account for the new peaks. From the relative magnitude of the new peak to that of the central peak, we estimate the refractive-index change to be 0.004. PMID- 12371537 TI - IL-4 supplemented B-cell cultures of allergic children show reduced IgA and IgG production in response to additional stimulation with IL-10. AB - BACKGROUND: Cytokines play an important role in mediating immunoglobulin switch, the secretion of protective mucosal immunoglobulins, and the development of allergic diseases. This study investigates whether B cells from allergic and healthy children have different capacities to secrete immunoglobulins after stimulation with IL-4, IL-6, IL-10, IL-11, and IL13. METHODS: We analyzed the peripheral venous blood of 44 healthy probands and of 109 allergic patients with a mean age of 13 years, allergic to grass pollen, birch pollen, and house dust mites. Lymphocytes were isolated by a density gradient and B cells were enriched by using a Magnetic Activated Cell Separator (MACS) and anti-CD19 microbeads. B Cells were co-cultured with human CDw32 (Fc gammaRII) expressing mouse Ltk fibroblasts and mouse anti-human CD40 monoclonal antibodies (CD40 system). The interleukins IL-4, IL-6, IL-10, IL-11, and IL-13 were supplemented in various combinations. After 14 days, concentrations of IgE, IgG, IgA, and IgM were measured in the supernatants with ELISA. RESULTS: Suppression of IgA-, IgG, and IgM- synthesis was induced by stimulation of B cells with IL-4. After additional application of IL-10, IgA, IgG, and IgM synthesis was significantly increased. When cultures stimulated with IL-4 were additionally supplemented with IL-10, IgA, and IgG synthesis of B cells obtained from allergic individuals was significantly decreased compared to nonallergic individuals. IgE-secretion of B cells from allergic individuals was significantly increased compared to nonallergic individuals after stimulation with IL-4. CONCLUSION: Our results implicate that IL-4 is essential for the regulation of immunoglobulin class switch to IgE and that IL-4 is an important cytokine for the development of allergic diseases. The capacity of B cells in allergic children to produce less IgA and IgG in response to additional stimulation with IL-10 of cultures supplemented with IL-4 could play an important role in mediating a mucosal immune system vulnerable to allergens. This phenomenon could contribute to the pathogenesis of allergic diseases. PMID- 12371540 TI - Formation of a planar coarse wavelength-division multiplexer and demultiplexer with reflection volume phase gratings. AB - A compact configuration for a coarse wavelength-division multiplexer (CWDM) and a coarse wavelength-division demultiplexer (CWDDM) that are based on reflection volume phase gratings is formed. The design and calculated results for four channel CWDM and CWDDM configurations in the region near 800 nm are presented. Theoretical predictions are experimentally verified with a four-channel CWDDM whose channels are centered at 775, 800, 825, and 850 nm. PMID- 12371541 TI - Long-trace profiler with cyclic optical configuration. AB - The results of the development of a much simpler optical configuration for the long-trace profiler (LTP) are presented. The current technique employs a cyclic optical configuration to achieve zero optical path difference for the closely spaced, laterally separated laser beams. The accuracy of measurement is found to remain as good as that in the case of the widely used LTP, although the geometrical alignment problem is simplified significantly. PMID- 12371542 TI - Holographic parabolic mirror as a receiver optical front end for wireless infrared communications: experimental study. AB - The inherent multifunctionality of holographic optical elements and their light physical weight make them an attractive solution for the receiver optics of portable terminals in indoor infrared wireless communication systems. A parabolic holographic mirror has been recorded in silver halide at a visible wavelength, and its replay wavelength has been shifted to the near infrared. Employment of proprietary swelling technology resulted in a permanent replay wavelength shift without the need for hologram sealing. Despite the relatively low diffraction efficiency of holograms recorded in silver halide in principle, an improvement in the receiver signal-to-noise ratio of more than 20 dB has been measured. The results of the conducted experiments proved undoubtedly the great potential of curved holographic mirrors as a key element of the receiver optical front end in IR wireless communication systems. PMID- 12371543 TI - Differential wavelength-scanning heterodyne interferometer for measuring large step height. AB - An interferometer based on the differential heterodyne configuration and wavelength-scanning interferometry for measuring large step heights is presented. The proposed interferometer is less sensitive to environmental disturbances than other interferometers and can accurately measure interference phases. A tunable diode laser is utilized to illuminate the interferometer and thus solve the phase ambiguity problem. Counting the interference fringes as the wavelength is scanned through a known change in wavelength directly determines the step height. Three gauge blocks of different lengths, 5, 10, and 50 mm, are individually wrung on a steel plate to simulate large step heights. Comparing the results measured by the proposed interferometer with those by the gauge block interferometer reveals that the accuracy is approximately 100 nm. PMID- 12371544 TI - Long-term temporal stability of the National Institute of Standards and Technology spectral irradiance scale determined with absolute filter radiometers. AB - The temporal stability of the National Institute of Standards and Technology (NIST) spectral irradiance scale as measured with broadband filter radiometers calibrated for absolute spectral irradiance responsivity is described. The working standard free-electron laser (FEL) lamps and the check standard FEL lamps have been monitored with radiometers in the ultraviolet and the visible wavelength regions. The measurements made with these two radiometers reveal that the NIST spectral irradiance scale as compared with an absolute thermodynamic scale has not changed by more than 1.5% in the visible from 1993 to 1999. Similar measurements in the ultraviolet reveal that the corresponding change is less than 1.5% from 1995 to 1999. Furthermore, a check of the spectral irradiance scale by six different filter radiometers calibrated for absolute spectral irradiance responsivity based on the high-accuracy cryogenic radiometer shows that the agreement between the present scale and the detector-based scale is better than 1.3% throughout the visible to the near-infrared wavelength region. These results validate the assigned spectral irradiance of the widely disseminated NIST or NIST traceable standard sources. PMID- 12371545 TI - Realization of the National Institute of Standards and Technology detector-based spectral irradiance scale. AB - A detector-based spectral irradiance scale has been realized at the National Institute of Standards and Technology (NIST). Unlike the previous NIST spectral irradiance scales, the new scale is generated with filter radiometers calibrated for absolute spectral power responsivity traceable to the NIST high-accuracy cryogenic radiometer instead of with the gold freezing-point blackbody. The calibrated filter radiometers are then used to establish the radiance temperature of a high-temperature blackbody (HTBB) operating near 3,000 K The spectral irradiance of the HTBB is then determined with knowledge of the geometric factors and is used to assign the spectral irradiances of a group of 1,000-W free electron laser lamps. The detector-based spectral irradiance scale results in the reduction of the uncertainties from the previous source-based spectral irradiance scale by at least a factor of 2 in the ultraviolet and visible wavelength regions. The new detector-based spectral irradiance scale also leads to a reduction in the uncertainties in the shortwave infrared wavelength region by at least a factor of 2-10, depending on the wavelength. Following the establishment of the spectral irradiance scale in the early 1960s, the detector-based spectral irradiance scale represents a fundamental change in the way that the NIST spectral irradiance scale is realized. PMID- 12371546 TI - Comparative study with double-exposure digital holographic interferometry and a shack-hartmann sensor to characterize transparent materials. AB - We compare wave-front measurements using double-exposure digital holography and a Shack-Hartmann sensor. A voltage-driven liquid-crystal wedge modulates the optical wave front and provides a refractive-index gradient typical of interesting transparent materials. Measurement accuracy and reliability are similar for both methods. In our opinion, digital holographic interferometry has several advantages for both laboratory and field environments. When compared with Shack-Hartmann methods, these advantages include hardware simplicity and robustness, relative insensitivity to sample dynamic range, and less computational demanding and more straightforward data evaluation algorithms. We believe that digital holography provides the methodology of choice for field studies of transparent materials such as microgravity protein crystal growth experiments. PMID- 12371547 TI - Microscopic phase-shifting profilometry based on digital micromirror device technology. AB - A microscopic three-dimensional (3-D) shape measurement system based on digital fringe projection has been developed and experimentally investigated. A Digital Micromirror Device along with its illumination optics is integrated into a stereomicroscope, which projects computer-generated fringe patterns with a sinusoidal intensity profile through the microscope objective onto the object surface being measured. The fringe patterns deformed by the object surface are recorded by a CCD camera. The microscopic 3-D shape of the object surface is measured and reconstructed by use of a phase-shifting technique. We discuss design considerations and error analysis of the system. Experimental results successfully demonstrate the capability of this technique for surface profile measurement of rough surfaces at the micrometer level. PMID- 12371548 TI - X-ray-ultraviolet beam splitters for the Michelson interferometer. AB - With the aim of realizing a Michelson interferometer working at 13.9 nm, we have developed a symmetrical beam splitter with multilayers deposited on the front and back sides of a silicon nitride membrane. On the basis of the experimental optical properties of the membrane, simulations have been performed to define the multilayer structure that provides the highest reflectivity-transmission product. Optimized Mo-Si multilayers have been successfully deposited on both sides of t he membrane by use of the ion-beam sputtering technique, with a thickness-period reproducibility of 0.1 nm. Measurements by means of synchrotron radiation at 13.9 nm and at an angle of 45 degrees provide a reflectivity of 14.2% and a transmission of 15.2% for a 60% s-polarized light, close to the simulated values. Such a beam splitter has been used for x-ray laser Michelson interferometry at 13.9 nm. The first interferogram is discussed. PMID- 12371550 TI - Absolute distance measurement by two-point-diffraction interferometry. AB - We present a point-diffraction interferometer that has been specially devised to perform absolute distance measurements in three dimensions. It is composed of two main parts: One is a target that moves in three dimensions, and the other is a stationary two-dimensional array of photodetectors. The target is made of point diffraction sources that emit two spherical wave fronts, whose interference is monitored by the photodetectors. Application of a phase-shifting technique allows the phase values of the photodetectors to be precisely measured, which are then fitted to a geometric model of multilateration so as to determine the xyz location of the target by minimization of least-squares errors. Experimental results show that the proposed diffraction interferometer is capable of measuring the xyz coordinates of the target with a volumetric uncertainty of less than 1.0 microm over a working volume of a 100-mm side. PMID- 12371549 TI - Evaluation of the performance of polished mirror surfaces for the TAMA gravitational wave detector by use of a wave-front tracing simulation. AB - We evaluated the performance of polished mirror surfaces for the TAMA interferometric gravitational wave detector by comparing the experimental results with a wave-front tracing simulation. The TAMA mirror surfaces were polished to a roughness of a few nanometer rms. We confirmed that these polished mirrors do not limit the present TAMA sensitivity and that the target shot-noise sensitivity will be achieved with these mirrors, even if a power-recycling technique is introduced in the next stage of the TAMA. PMID- 12371551 TI - Improvement of the interferometric method for measuring electro-optic coefficients of poled polymer thin films. AB - The interferometric method for measuring the linear electro-optic coefficients of polymer films has been improved. This improved method is based on using the antipiezoelectric effect of a quartz crystal to compensate for the change in the optical path length that is due to the electro-optic effect of a polymer film. The electro-optic coefficients of six kinds of new poled polymer film have been determined at a wavelength of 633 nm by this new method. This technique offers a simple, rapid, and highly sensitive method for measuring the electro-optic coefficients of polymer films. PMID- 12371552 TI - Dual-wavelength interferometric technique with subnanometric resolution. AB - A new fringe subdivision method that employs a large synthetic wavelength to subdivide fringes formed by a small single wavelength is proposed. Based on this subdivision method, we demonstrate a novel dual-wavelength interferometric technique with subnanometric resolution, whose potential fringe subdivision factor derived from an evaluation of the interferometer can reach up to 1/440,000. Theoretical analysis and experimental results with a resolution of 0.05 nm are presented to show the feasibility of the interferometric technique. PMID- 12371553 TI - Two-longitudinal-mode He-Ne laser for heterodyne interferometers to measure displacement. AB - We propose a new configuration for a high-resolution heterodyne interferometer that employs a two-longitudinal-mode He-Ne laser with an intermode beat frequency of 600-1000 MHz. The high beat frequency is downconverted to 5 MHz such that the phase change of the interferometer output is precisely measured with a displacement resolution of 0.1 nm. A thermal control scheme is adopted to stabilize the cavity length of the He-Ne plasma tube such that a frequency stability of 2 parts in 10(9) is obtained by suppression of frequency drifts caused by t he phenomena of frequency pulling and polarization anisotropy. This two-longitudinal-mode He-Ne laser yields a high output power of 2.0 mW, which permits multiple measurements of as many as 10 machine axes simultaneously. PMID- 12371554 TI - High-precision shape measurement by white-light interferometry with real-time scanner error correction. AB - White-light interferometric techniques allow high-precision shape measurement of objects with discontinuous structures by detecting the peak of the coherence envelope. These techniques assume a specific change in the optical path difference (OPD) between the interfering beams; however, the scanning device effecting that change often introduces OPD errors that are carried over to the measurements. We present a technique for measuring OPD changes from the collected interference fringes during each measurement. Information about the scan is directly fed into the algorithm, which compensates for the errors, resulting in improved measurement accuracy. The method corrects not only the scanner errors but also slowly varying vibrations. In addition, this technique can be easily adapted to any existing low-coherence interferometer because no large data storage or postprocessing is required. PMID- 12371555 TI - Three-dimensional phase imaging with the intensity transport equation. AB - Phase can be retrieved from intensity measurements with the intensity transport equation. Three-dimensional image formation of weak phase objects based on this method is investigated. It is shown that, although the refractive index of a thin object can be measured, the three-dimensional variation of refractive index of an arbitrary object cannot, in general, be reconstructed, as spatial frequencies with a zero-axial component are not detected. However, this may not be a problem if regions with known refractive index are present in the sample. PMID- 12371556 TI - Tilting tolerance analysis of a broadband polarization-preserving beam displacer. AB - We numerically investigated the performance of an achromatic polarization preserving beam displacer that was proposed by Galvez [Opt. Lett.26,971(2001)]. First, the four-prism configuration of the Galvez scheme is verified by simulation. We examined the extension of wavelength beyond visible light by considering the corresponding power loss. The degree of polarization is also considered. The tolerance of tilts of reflecting surfaces is comprehensively discussed. It is shown that the second reflector of a four-prism system is the most critical component. Performance of various materials, including plastics, is evaluated. We explain a simplified implementation based on two prisms (the classic Porro configuration), which also improves material performance. PMID- 12371557 TI - Fiber-linked Faraday effect current sensor by use of a flint glass cell with dielectric-coated retardation-compensated total reflection surfaces. AB - A new type of Faraday effect optical current transformer has been developed that uses a single block of square flint glass with dielectric-coated total reflection surfaces as the sensing element. Numerical calculation has shown that the coating of two dielectric layers of 45.59-nm-thick Ta2O5 and 448.35-nm-thick SiO2 films on a flint glass surface produces zero retardation total reflection for the 45 degrees incident angle of light at lambda = 840 nm with great incident angle, wavelength, and film thickness tolerances. A fiber-linked current transformer has been constructed and experimentally demonstrated to exhibit high isolation from surrounding currents as well as high stability against mechanical disturbances. PMID- 12371558 TI - Visible spectral dependence of the scattering and absorption coefficients of pigmented coatings from inversion of diffuse reflectance spectra. AB - A spectral-projected gradient method and an extension of the Kubelka-Munk theory are applied to obtain the relevant parameters of the theory from measured diffuse reflectance spectra of pigmented samples illuminated with visible diffuse radiation. The initial estimate of the spectral dependence of the parameters, required by a recursive spectral-projected gradient method, was obtained by use of direct measurements and up-to-date theoretical estimates. We then tested the consistency of the Kubelka-Munk theory by repeating the procedure with samples of different thicknesses. PMID- 12371559 TI - Extreme-ultraviolet thin-film interference in an Al-Mg-Al multiple-layer transmission filter. AB - Thin-film interference has been observed in the transmittance of a filter consisting of 263.5-nm-thick magnesium with a 32.2-nm-thick aluminum layer on each side. The transmittance, measured by synchrotron radiation, has an oscillatory behavior in the 25-70-nm wavelength range. On the basis of the calculation of the transmittance, the oscillatory behavior results from interference associated with the relatively transmissive magnesium and aluminum layers and the reflection from the oxidized aluminum surface layers. The bandpass performance of magnesium and aluminum layers deposited on a silicon photodiode detector is presented. PMID- 12371560 TI - Simple computations involving two-component symmetric trilayers. AB - The equivalent properties of a symmetric three-layer structure are derived by a nontraditional method that provides useful insights and a simplified application to some thin-film design problems. The equations derived from this method may be used to design a three-layer replacement for a single layer in an interference coating. The equations are especially helpful for cases that involve complex numbers, such as metal layers or above-critical angle propagation in dielectric layers. Several multilayer design problems are solved to demonstrate the application of this approach. PMID- 12371561 TI - Speckle-contrast monitoring of tissue thermal modification. AB - Measurements of the contrast value of time-averaged speckle-modulated images of cartilage tissue are used to study tissue thermal modification in the case of laser-light treatment. This modification is related to thermally induced internal stress relaxation in the matrix of the treated tissue. The specific feature of the evolution of time-averaged speckle contrast with a change in the current temperature of modified collagen tissue is the typical looplike form of the contrast-temperature dependencies associated with irreversible changes in tissue structure and correlated with changes in the tissue diffuse transmittance and the tissue internal stress mentioned by other researchers. PMID- 12371562 TI - Large-aperture automatic focimeter for the measurement of optical power and other optical characteristics of ophthalmic lenses. AB - We propose an optical apparatus enabling the measurement of spherical power, cylindrical power, and optical center coordinates of ophthalmic lenses. The main advantage of this new focimeter is to provide a full bidimensional mapping of the characteristics of ophthalmic glasses. This is made possible thanks to the use of a large-area and high-resolution position-sensitive detector. We describe the measurement principle and present some typical mappings, particularly for progressive lenses. We then discuss the advantages in terms of speed and versatility of such a focimeter for the measurement of complex lens mappings. PMID- 12371563 TI - Direct Raman imaging techniques for study of the subcellular distribution of a drug. AB - Direct Raman imaging techniques are demonstrated to study the drug distribution in living cells. The advantage of Raman imaging is that no external markers are required, which simplifies the sample preparation and minimally disturbs the drug mechanism during imaging. The major challenge in Raman imaging is the weak Raman signal. In this study, we present a Raman image model to describe the degradation of Raman signals by imaging processes. Using this model, we demonstrate special purpose image-processing algorithms to restore the Raman images. The processing techniques are then applied to visualize the anticancer agent paclitaxel in living MDA-435 breast cancer cells. Raman images were obtained from a cancer cell before, during, and after drug treatment. The paclitaxel distribution illustrated in these images is explained by means of the binding characteristics of the paclitaxel and its molecular target-the microtubules. This result demonstrates that direct Raman imaging is a promising tool to study the distribution of a drug in living cells. PMID- 12371564 TI - Nitric oxide breath testing by tunable-diode laser absorption spectroscopy: application in monitoring respiratory inflammation. AB - We used a high-resolution mid-IR tunable-laser absorption spectroscopy (TLAS) system with a single IV-VI laser operating near 5.2 microm to measure the level of exhaled nitric oxide (eNO) in human breath. A method of internal calibration using simultaneous eNO and exhaled CO2 measurements eliminated the need for system calibration with gas standards. The results observed from internally calibrating the instrument for eNO measurements were compared with measurements of eNO calibrated to gas standards and were found to be similar. Various parameters of the TLAS system for eNO breath testing were examined and include gas cell pressure, exhalation time, and ambient NO concentrations. A reduction in eNO from elevated concentrations (approximately 44 parts in 10(9)) to near-normal levels (<20 parts in 10(9)) from an asthmatic patient was observed after the patient had received treatment with an inhaled glucocorticoid anti-inflammatory medication. Such measurements can help in evaluating airway inflammation and in monitoring the effectiveness of anti-inflammatory therapies. PMID- 12371565 TI - Ultrasound-modulated optical tomography of biological tissue by use of contrast of laser speckles. AB - Ultrasound-modulated optical tomography based on the measurement of laser-speckle contrast was investigated. An ultrasonic beam was focused into a biological tissue sample to modulate the laser light passing through the ultrasonic column inside the tissue. The contrast of the speckle pattern formed by the transmitted light was found to depend on the ultrasonic modulation and could be used for imaging. Variation in the speckle contrast reflected optical inhomogeneity in the tissue. With this technique, two-dimensional images of biological-tissue samples of as much as 25 mm thick were successfully obtained with a low-power laser. The technique was experimentally compared with speckle-contrast-based, purely optical imaging and with parallel-detection imaging techniques, and the advantages over each were demonstrated. PMID- 12371566 TI - Insurance product design and its effects: trade-offs along the managed care continuum. AB - This paper uses 1996-97 Community Tracking Study data to analyze the effects of different insurance product designs on service use, access, and consumer assessments of care for nonelderly people with employer-sponsored insurance. Product types are defined by features including use of networks, gatekeeping, capitation, and group/staff model delivery systems. We found no evidence of differences across product types in unmet need or delayed care or use of hospitals, surgery, or emergency rooms. At the same time, different product designs present purchasers with a clear trade-off between paying more out of pocket and encountering more administrative barriers to care. In addition, an increasing proportion of consumers report dissatisfaction with choice of physicians and low trust in physicians as one moves along the managed care continuum from unmanaged to heavily managed products. Our findings have implications for efforts to regulate managed care. The existence of a trade-off between out-of-pocket costs and administrative barriers to care means that some forms of regulation run the risk of reducing choices available to consumers. This is particularly true of regulations that would change the nature of managed care products by prohibiting the use of specific care management tools. To the extent that the backlash against managed care targets restrictions on choice and administrative hassles among consumers who nonetheless choose more heavily managed products because of their lower cost, eliminating heavily managed products would leave those consumers worse off. PMID- 12371567 TI - Small firms' demand for health insurance: the decision to offer insurance. AB - This paper explores the decisions by small business establishments (< 100 workers) to offer health insurance. We estimate a theoretically derived model of establishments' demand for insurance using nationally representative data from the 1997 Robert Wood Johnson Foundation Employer Health Insurance Survey and other sources. Findings show that offer decisions reflect worker demand, labor market conditions, and establishments' costs of providing coverage. Premiums have a moderate effect on offer decisions (elasticity = -.54), though very small establishments and those employing low-wage workers are more responsive. This suggests that premium subsidies to employers would be an inefficient means of increasing insurance coverage. Greater availability of public insurance and safety net care has a small negative effect on offer decisions. PMID- 12371568 TI - Using survey measures to assess risk selection among Medicare managed care plans. AB - This paper quantifies risk selection among competing Medicare managed care plans, using beneficiary survey data from the Consumer Assessments of Health Plans Survey. Selection, measured by variation in plan-level prevalence of health conditions and predicted costs, was substantial. A plan with moderate (one standard deviation) adverse selection would have predicted costs 11.6% above an average plan. Only a small part of this variation was explained by the geographical differences in the prevalence of health conditions among or within Metropolitan Statistical Areas, indicating that the selection was driven by plan attributes. Plans serving members with greater health needs have the potential to establish programs to serve these sick members well, yet this places plans at financial risk. Hence, improved risk adjustment for chronic conditions may be warranted. Moreover, survey measures have the potential to measure the prevalence of such conditions reliably and consistently across plans. PMID- 12371569 TI - Risk selection among SSI enrollees in TennCare. AB - The issue of risk selection is especially important for states that enroll blind and disabled beneficiaries of Supplemental Security Income (SSI) in Medicaid managed care. SSI beneficiaries have persistent needs for care, have a wide variety of chronic conditions, and often need atypical and complex services. Risk selection occurs when the health care needs of beneficiaries enrolled in a specific plan differ systematically from the needs of the overall beneficiary population and payments do not reflect those needs. We assess the extent of risk selection among managed care plans for SSI beneficiaries over the first three years of Tennessee's Medicaid managed care program, TennCare. Using claims data containing fee-for-service expenditures prior to enrollment in managed care, we find substantial evidence of persistent risk selection among plans. Results are robust to most alternative measures of risk selection for most plans. PMID- 12371570 TI - Predicting the use of outpatient mental health services: do modeling approaches make a difference? AB - Studies attempting to project the impact of providing health coverage to the uninsured population have demonstrated considerable variation in the estimated costs of mental health care. Different modeling approaches to project health care use and costs have been shown to address some data characteristics well, but not all of them. Using data from Health Care for Communities, a recent national household survey, this paper attempts to estimate and predict the use of mental health outpatient services if insurance coverage were extended to the uninsured. The study employs two-part models, with the second part based on an ordinary least squares (OLS) approach and a generalized linear model (GLM), and a zero inflated negative binomial model (ZINB). Estimates and predictions are not sensitive to the modeling approaches chosen, although the ZINB model out performs the two-part models in terms of out-of-sample prediction. PMID- 12371571 TI - Physician fees and managed care plans. AB - One of the objectives of managed care organizations (MCOs) has been to reduce the rate of growth of health care expenditures, including that of physician fees. Yet, due to a lack of data, no one has been able to determine whether MCOs have been successful in encouraging the growth of price competition in the market for physician services in order to slow the growth in physician fees. This study uses a unique, national-level data set to determine what factors influenced the physician fees that MCOs negotiated during the 1990-92 period. The most influential characteristics were physician supply and managed care penetration, which suggest that the introduction of competition into the health care market was an effective force in reducing physician fees. PMID- 12371572 TI - Effects of rising costs on health insurance coverage: private and public choices are not independent of one another. PMID- 12371573 TI - Advancing the role of nonprofit health care. AB - This article comes out of a series of discussions among a diverse group of chief executive officers (CEOs) and other leaders of nonprofit hospitals, long-term care facilities, health maintenance organizations, and other insurance providers, including several nonprofit Blue Cross Blue Shield (BCBS) plans. The group was convened as part of Howard Berman's Walter J. McNerney Fellowship project. (Berman is CEO of Excellus, Inc., a nonprofit Blue Cross Blue Shield affiliate that insures the health of more than 2.15 million people in upstate New York. He was awarded the McNerney Fellowship in April 2001 by the Health Research and Educational Trust, an American Hospital Association affiliate. The Fellowship goes annually to at least one fellow to highlight or pursue work that will provide new insights into how different sectors of the health care system can better work together for improved outcomes.) The group has met several times over the past year around a shared concern: the current challenges to nonprofit health care organizations and the future role for nonprofits in the re-visioning and creation of an American health care system that is characterized by universal access, patient-centered quality, and national affordability. Members have supported the public relations campaign of the "Alliance for Advancing Nonprofit Health Care," an effort initiated by the Caucus, an independent group of nonprofit BCBS plans. The group continues to explore the need for a broad-based coalition of providers, insurers, and other organizations to effectively protect and enhance the role of nonprofit health care. PMID- 12371574 TI - Bridge to services: drug injectors' awareness and utilization of drug user treatment and social service referrals, medical care, and HIV testing provided by needle exchange programs. AB - Using qualitative interviews conducted in 1999, we examine awareness and use of drug user treatment and social service referrals, medical care, and HIV testing provided by needle exchange programs (NEPs) among injectors who use NEPs (N=26) and injectors who get their syringes from other sources (N=20). A four-category typology of NEP service knowledge and use emerges from these interviews: "Active involvement--use of services; "Stepping stone"--no use of services but knowledge that specific services are available; "Vague awareness"--nonspecific knowledge of service availability: and "Unaware:--no awareness of the service provision function of NEPs. We describe patterns of distribution of respondents among these categories and suggest policy implications. PMID- 12371575 TI - Substance use in the construction industry: a comparison of assessment methods. AB - Most users of illicit drugs are employed adults, with substance use rates especially high in the construction industry. In an effort to shed light on the nature and extent of drug use among construction industry workers, and to compare drug use assessment methods, substance use among construction workers, 60% of whom were apprentices, across six sites was assessed by questionnaire, urinalysis, and hair analysis. Nearly 17% of the participants reported current drug use, although drug use differed dramatically by site. Drug use rates also differed by respondent characteristics, participation rates, and assessment method. The strengths and weaknesses of each assessment method are discussed, along with the rationale for combining methods. PMID- 12371576 TI - The impact of a family empowerment intervention on juvenile offender heavy drinking: a latent growth model analysis. AB - We report the results of a growth model analysis of the impact of a Family Empowerment Intervention (FEI) on the heavy drinking over a 36-month follow-up period among youths processed at the Hillsborough County Juvenile Assessment Center. Families involved in the project were randomly assigned to either receive an Extended Services Intervention (ESI) or the FEI. Families in the ESI group received monthly phone contacts and, if indicated, referral information; FEI families received three one-hour, home-based meetings per week for approximately 10 weeks from a clinician-trained paraprofessional. By seeking to improve family functioning by empowering parents, it was hypothesized that target youths' behavior and psychosocial functioning would improve. Although the difference between FEI and ESI was not significant, the reported frequency of getting very high or drunk on alcohol declined more over time for FEI completers than FEI noncompleters. The results provide support for the impact of the FEI services. PMID- 12371577 TI - A survey of attitudes among drug user treatment providers toward the treatment of inhalant users. AB - This study assessed the attitudes of drug user treatment program directors towards the problem of inhalant "abuse." In 2000, surveys were mailed to directors asking about treatment success and prognosis for inhalant users, level of neurological damage incurred by users, availability of treatment resources, their program's policies toward admission of users, and staff training needs for inhalant use. Two open-ended questions queried their assessment of barriers to treatment and subjective feelings about the topic of inhalant use. Five hundred and fifty responses were received. Findings show that program directors perceive a great deal of neurological damage incurred through inhalant use and have a general pessimism about treatment effectiveness and recovery. The respondents also felt that there were insufficient resources for inhalant user treatment and that special staff training in the area was needed. The majority of the directors indicated that they have or would treat inhalant users. Implications for future research and policy change are discussed. PMID- 12371578 TI - Patient compliance and maternal/infant outcomes in pregnant drug-using women. AB - Treatment compliance is an important variable in drug use intervention. For pregnant drug-misusing women, compliance with treatment has been particularly problematic, even in specialized and more intensive treatment programs. The present study, conducted from March 1999 to June 2000, compared maternal/infant outcomes in pregnant drug-using women who were either compliant or noncompliant with drug use interventions offered through a prenatal care clinic. Compliant women (N = 11) completed four therapy sessions (behavioral reinforcement of drug abstinence + brief motivational therapy), while noncompliant women (N = 20) participated in zero to three therapy sessions. The two groups were similar on demographic and drug use severity measures. Compliant mothers, however, gave birth to infants with higher birthweights than noncompliant mothers. Over half of compliant mothers were also drug-free at delivery, compared to one-fourth of noncompliant mothers. These data support an association between treatment compliance and birth outcomes, and highlight the need to develop strategies for improving compliance with such interventions. PMID- 12371579 TI - Nurses' use of alcohol and other drugs: findings from a national probability sample. AB - This study examined the prevalence of alcohol and other drug (AOD) use among nurses in the 1984 National Longitudinal Survey of Youth (NLSY) using methods similar to those employed in a study comparing nurses and nonnurses from the 1980 1984 Epidemiological Catchment Area program (ECA). Conditional logistic regression was used to estimate the degree to which AOD use was associated with occupation. Results indicating that substance use is unrelated to occupation lend support to earlier findings from the ECA. PMID- 12371580 TI - Diagnosis and management of early lung cancer. AB - Bronchogenic carcinoma remains the leading cause of cancer deaths in the United States. Approximately 80% of newly diagnosed cases are non-small cell lung cancer (NSCLC); 80% of these present with disseminated or locally advanced disease. Unfortunately, only 10% are potentially surgically curable patients with early stage disease (T1N0/T2N0). Most patients with early-stage disease are asymptomatic, with their lung cancer detected as a result of non-cancer related procedures. Studies have shown that chest radiography as a screening modality resulted in a higher discovery of early disease, but did not translate to a significant reduction in lung cancer mortality. Recent work on low-dose helical CT, however, has renewed interest in the challenge of detecting early-stage lung cancer. PMID- 12371581 TI - Positron emission tomography (PET) and combined imaging modalities for staging lung cancer. AB - FDG PET in its current form supplements but does not yet replace other noninvasive imaging modalities for the evaluation and staging of the patient with NSCLC. Clinicians await further data from well-designed clinical trials to help integrate FDG PET into current clinical practice. Looking forward, sophisticated radiolabeling techniques promise to improve both the diagnostic accuracy of PET and our ability to deliver targeted cancer therapy to patients. PMID- 12371582 TI - Molecular staging of lung and esophageal cancer. AB - In both esophageal and NSCLC, the TNM stage at diagnosis remains the most important determinant of survival. Significant research to investigate the biology of NSCLC and esophageal carcinoma is ongoing, and the roles of proto oncogenes, tumor suppressor genes, angiogenic factors, extracellular matrix proteases, and adhesion molecules are being elucidated. While evidence is accumulating that various markers are involved in NSCLC and esophageal tumor virulence, the current studies are compromised by small sample sizes, heterogeneous populations, and variations in techniques. Large prospective studies with homogenous groups designed to evaluate the role of these various markers should clarify their potential involvement in NSCLC and esophageal cancer. Identification of occult micrometastases in lymph nodes and bone marrow using immunohistochemical techniques and rt-PCR is intriguing. These techniques are promising as a method to more accurately stage patients, and therefore to predict outcomes and to determine therapies. Perhaps the most promising area of research is the development of novel drugs whose mechanism of action targets the pathways of various molecular markers. Molecular biologic substaging offers an opportunity to individualize a chemotherapeutic regimen based on the molecular profile of the tumor, thus providing the potential for improved outcomes with less morbidity in patients with both NSCLC and esophageal cancer. PMID- 12371583 TI - Neuroendocrine tumors of the bronchopulmonary tract: a reappraisal of their classification after 20 years. AB - This article is an overview of the classification of pulmonary neuroendocrine neoplasms, their presentation, their pathologic appearance, and their clinical management. In addition, the original classification, based on histologic features, is reassessed in the light of newer areas in study, including neurosecretory products, neuroendocrine markers, ultrastructural studies, ploidy analysis, cell adhesion markers, apoptosis, oncogene mutation analysis, and genetic alterations. The histologic classification proposed in 1983 remains the single most valuable factor in establishing the diagnosis and, together with the TNM status, the prognosis of this group of interesting neoplasms. PMID- 12371584 TI - Video-assisted thoracic surgery (VATS) resection for lung cancer. AB - VATS is a relatively new technology that has become the standard of care for basic procedures such as drainage of pleural effusion and blebectomy. VATS anatomic lung resection is more controversial. Published studies demonstrate several advantages of VATS over a standard posterolateral thoracotomy. A minimally invasive approach causes less inflammatory reaction. Acute and chronic pain are diminished. As a result, the length of hospitalization is shorter. Early and late shoulder dysfunction is less and return to work time is shorter. Taken together, these factors suggest a better overall outcome using a VATS approach. From an oncologic standpoint, lymph node dissection can be accomplished and locoregional recurrence is low. The validity of VATS for lung cancer will be determined by long-term data. A phase III national (intergroup) protocol is being drafted and will help to answer these questions. PMID- 12371585 TI - Sentinel node biopsy for staging lung cancer. AB - The importance and value of accurate clinical and pathological staging of lung cancer is emphasized. The philosophy, techniques, and general results of the sentinel node technique for lymphatic mapping are reviewed. The experience with this technique in patients with non-small cell lung cancer is discussed and the future use of lymphatic mapping is pondered. PMID- 12371586 TI - Radical resections for T4 lung cancer. AB - T4 lung cancers are a heterogeneous group of locally advanced lung cancers. Treatment is palliative for the majority of patients, ranging from supportive care to chemoradiotherapy. In certain patients, however, surgery is beneficial and may be curative. Patients with T4N0M0 cancers invading the distal trachea, carina, left atrium, aorta, superior vena cava, or vertebral bodies may be surgical candidates. Radical resections of these T4 lung cancers have potential for cure if no mediastinal lymph node metastases (N2 or N3) occur and if resection is complete. Increased postoperative mortality exists and extends beyond 30 days, as evidenced by a 30-day mortality of 8% and a 90-day mortality of 18%. Improved palliation (median survival of 19 months) and cure (31% five year survival) are possible in patients who meet the criteria, who undergo radical resection, and who are followed by physicians in facilities with special interests in extended resections. The use of induction therapy and surgery in T4 patients may further increase survival and the number of T4 patients in whom radical resection is possible. Radical resections are contraindicated in patients with T4 lung cancers associated with malignant pleural effusions. Unfortunately, these patients have the worst prognosis. If surgical palliation is an option, only pulmonary resection with pleurectomy and not pleuropneumonectomy should be considered. In contrast, lung cancers with the best prognosis are those T4 tumors diagnosed because of a satellite tumor nodule within the same lobe. Because radical resections are usually not required, operative mortality is not increased. Five-year survival in patients with satellite intralobar tumor nodules without mediastinal nodal metastases is comparable to survival of highly selected T4N0M0 patients who undergo radical resection. These two extremes of T4 lung cancers, malignant pleural effusion and satellite intralobar tumor nodules, generally are not considered for or do not require radical resections. It is debatable that the definition of T4 should include these entities. PMID- 12371587 TI - Parenchymal sparing operations for bronchogenic carcinoma. AB - By the end of the 1950s, the principles of tracheobronchial and pulmonary artery (PA) reconstruction had been established, and their successful clinical application had taken place. It was not until very recently, however, that these techniques aroused widespread interest among thoracic surgeons as a means to achieve complete cancer resection while preserving functioning lung parenchyma. At the present time, sleeve resection of the bronchus and/or PA has a definite role in the surgical management of lung cancer. Growing interest in this field is evidenced by an increasing number of technical variations intended to adapt the basic technique to the different anatomical settings. Also pitfalls, complications, and their prevention and treatment are being extensively described. Last but not least, functional and oncological long-term results, comparing favorably with those of more extended resections, are being reported by many groups. This demonstrates that sleeve lobectomy is no longer reserved only for particularly skillful surgeons. Sleeve lobectomy has achieved its rightful position among the techniques commonly used in thoracic surgery after 40 years of improving understanding and alternating enthusiasm and legitimate doubts. PMID- 12371588 TI - Multi-modality treatment of non-small cell lung cancer. AB - Despite significant advances in radiation therapy techniques and a variety of newer chemotherapeutic agents, when multimodality treatment for stage I and II tumors has been tested by Phase III randomized prospective trials of adequate size, no significant survival advantage over surgery alone has been found in most instances. Modalities tested include preoperative radiation therapy, and postoperative chemotherapy and radiation therapy. Trials are presently underway to test preoperative chemotherapy for stages Ib, II, and T3NI (S9900) and to test adding surgery for patients with N2 disease who have been treated by chemotherapy and radiation therapy (INT 0139). Results of a recently completed trial (JBR10) will answer the question of whether postoperative chemotherapy is of benefit for patients with stages T2N0 or T1-2N1. Until these trials are completed, surgeons should resist the temptation to use newer but unproven therapies except within established approved protocols. PMID- 12371589 TI - Management of malignant tracheobronchial obstruction. AB - Malignant airway obstruction produces symptoms of dyspnea, cough, and stridor with a significant impact on quality of life. With progressive airway narrowing, a critical stenosis is life threatening, due to impending suffocation. Bronchoscopy provides the mainstay of establishing the diagnosis as well as defining the extent and degree of airway obstruction. Therapeutic bronchoscopy provides the ability to achieve prompt airway stabilization while completing a patient evaluation and workup. Patients may have malignant airway obstruction from primary tracheobronchial tumors, adjacent primary tumors with airway invasion, or metastatic disease to the airway. Surgical resection is the preferred definitive therapy for primary airway tumors. A subset of lung or thyroid malignancies that invade the airway may also be amenable to primary surgical resection, as long as a complete resection of tumor can be obtained along with primary airway reconstruction. The majority of patients with malignant airway obstruction will be unresectable due to locally advanced disease, metastatic disease, or contraindicating comorbidity. In these patients, therapeutic bronchoscopy provides a prompt and reliable palliation of the airway obstruction. The simplest and most immediately reliable strategy for endoluminal tumor is mechanical core-out of the tumor. Laser vaporization, photodynamic therapy, cryotherapy, and endobronchial brachytherapy all are adjuncts to the coring out of endoluminal tumor that may prolong the period of palliation achieved. Airway stenting is the only endoluminal therapy available for the management of malignant obstruction from extrinsic disease, and is also a useful adjunct to providing coverage of endoluminal tumor. Flexible and creative application of these techniques provides the best chance for successful airway palliation. Although the long-term outlook in these patients is often dismal, relief of airway obstruction results in a marked improvement in quality and length of life. PMID- 12371590 TI - Thoracic empyema. AB - This article summarizes the pathogenesis, clinical presentation, radiological manifestations, and treatment options of patients with parapneumonic effusions or empyema. Emphasis is placed on an expeditious workup and appropriate selection of the multiple therapeutic options. PMID- 12371591 TI - Review of long-term results of stereotactic psychosurgery. AB - Stereotactic psychosurgery is an effective method for treating some medically intractable psychiatric illnesses. However, it is unfamiliar and the long-term clinical results have not been reported in Asia. The long-term results of psychosurgery are evaluated and the neuroanatomical basis is discussed. Twenty one patients underwent stereotactic psychosurgery for medically intractable psychiatric illnesses since 1993. All were referred from psychiatrists for these disorders. Two patients showed aggressive behavior, 12 had obsessive-compulsive disorder (OCD), and seven had depression with anxiety disorders. Bilateral amygdalotomy and subcaudate tractotomy were performed for aggressive behavior, limbic leucotomy was performed for OCD, and subcaudate tractotomy with or without cingulotomy was performed for depression with anxiety. OCD was evaluated with the Yale-Brown Obsessive Compulsive Scale (YBOCS), the visual analogue scale, the Clinical Global Impression Scale, and the Overt Aggression Scale (OAS). The Mini Mental State Examination and the Wechsler Adult Intelligence Scale-Revised were used for the evaluation of aggressive behavior. The 17-item Hamilton Depression Rating Scale (HAMD) was used for evaluation of depression. Ventriculography was used in the first seven patients and magnetic resonance imaging-guided stereotaxy was used in the recent 14 cases for localization of the target. The lesions were made with a radiofrequency lesion generator. OAS scores in the two patients with aggressive behavior during follow up declined from 8 to 2 with clinical improvement. All 12 patients with OCD returned to their previous life and showed the mean YBOCS scores decreased from 34 to 3. Ten patients with OCD could be followed up (mean 45 months). All patients returned to their previous social life. In seven patients with depression with anxiety, HAMD scores declined from 28.5 to 16.5. There was no operative mortality and no significant morbidity except for one case of mild transient urinary incontinence. These long-term results indicate that stereotactic psychosurgery is a safe and effective method of treating some medically intractable psychiatric illnesses. PMID- 12371592 TI - Combined embolization and microsurgery for cerebral arteriovenous malformation. AB - Thirty-seven of 396 patients with arteriovenous malformations (AVMs) were treated by preoperative embolization and surgical excision in our medical center between 1991 and 1997. The AVMs were Spetzler-Martin grade I in six, grade II in 11, grade III in 12, grade IV in four, and Grade V in four. A total of 69 embolization procedures were performed using estrogen alcohol and polyvinyl acetate as liquid embolization materials. AVM grades improved following embolization in 10 patients. Three hemorrhages occurred several days after the final embolization, probably due to hemodynamic stress on the unoccluded perforating feeders or drainer occlusion. Surgical resection was performed under guidance of intraoperative digital subtraction angiography. Although the AVMs were excised totally in one stage with no major neurological deficits, normal perfusion pressure breakthrough occurred in a patient with prominent collateral circulation of the AVM which developed after the main feeder embolization. Preoperative staged embolization with estrogen alcohol and polyvinyl acetate is effective to expand the surgical indication and to achieve complete resection of difficult AVMs. Direct surgery should be performed as soon as possible if hemodynamic changes are observed following embolization. PMID- 12371593 TI - Huge chronic subdural hematoma mimicking cerebral infarction on computed tomography--case report. AB - A 70-year-old male presented with progressive consciousness disturbance. He had a history of cerebrovascular accident in the left cerebral hemisphere. The initial diagnosis was newly developed extensive left cerebral hemispheric infarction based on computed tomography. However, magnetic resonance imaging and surgical findings were consistent with chronic subdural hematoma (CSDH). The preexisting neurological disease may have allowed such unexpected CSDH expansion. PMID- 12371594 TI - Complete superior and inferior sagittal sinus thromboses with multiple cranial nerve pareses and transient ischemic attack--case report. AB - A 27-year-old woman with headache and right peripheral facial nerve paresis persisting for over 25 days, and left hemiparesis for 2 days, which had all been gradually improving, was admitted to our hospital as she suddenly developed horizontal and vertical diplopia. She had right fourth and sixth cranial nerve pareses, papilledema, and right orbital venous congestion, and also experienced a seizure on the day of admission. Magnetic resonance (MR) imaging and MR venography revealed complete superior and inferior sagittal sinus thromboses and significant collateral venous channels, but no parenchymal lesion. Fourth and seventh cranial nerve pareses and the left hemiparesis resolved completely within 2 days, but she concurrently developed an episode of right hemiparesis, which lasted for 30 minutes. The patient recovered with medical therapy. MR venography showed recanalization of both sinuses. She was neurologically intact except for minimal right abducens nerve paresis at discharge, 40 days after admission. Multiple cranial nerve pareses with transient ischemic attack is an extremely rare manifestation of superior sagittal sinus thrombosis. Transient functional disturbance due to temporary reduction of tissue perfusion caused by overload of the collateral channels is more likely to be responsible for the transient ischemic attack and reversible ischemic neurological deficit. PMID- 12371595 TI - Ruptured dissecting aneurysm of the vertebral artery associated with occlusive internal carotid artery dissection--case report. AB - A 64-year-old male presented with subarachnoid hemorrhage. Angiography showed a dissecting aneurysm of the right vertebral artery (VA), and severe stenosis of the right internal carotid artery (ICA). He was treated conservatively in the early stage. Repeat angiography showed enlargement of the dissecting aneurysm of the VA and partial resolution of the stenosis of the right ICA. Intraaneurysmal coil embolization with proximal coil occlusion was performed following a balloon occlusion test. The postoperative course was uneventful. Based on the neuroradiological findings, the stenotic lesion of the right ICA was considered to be due to dissection. Analysis of serial changes in dissecting lesions in the craniocervical arteries is important for the correct choice of treatment, especially in patients with multi-vessel dissections. The surgical options should be determined on an individual basis. PMID- 12371596 TI - De novo cerebral aneurysms manifesting as repeated subarachnoid hemorrhage and cerebral ischemic stroke--case report. AB - A 29-year-old man suffered repeated subarachnoid hemorrhage and cerebral ischemic stroke over a period of 6 years. Cerebral angiography at each episode disclosed development of multiple de novo aneurysms at the bilateral middle cerebral arteries (MCAs), internal carotid arteries, right anterior cerebral artery, and right vertebral artery. Two of the ruptured aneurysms were treated by surgical and endovascular treatment, but he died of the effects of rupture of a de novo right MCA aneurysm. Histological examination at autopsy disclosed marked degenerative changes in all layers of the cerebral vessels, which were probably congenital in origin. PMID- 12371597 TI - Aberrant right subclavian artery--three case reports. AB - Conventional angiography detected three cases of aberrant right subclavian artery. A 51-year-old female presented with a small infarction in the left medulla oblongata and severe stenosis of the left subclavian artery. A 59-year old female presented with multiple cerebral infarctions and severe atherosclerotic changes in the intracranial arteries. A 58-year-old female presented with aneurysmal subarachnoid hemorrhage. The aberrant right subclavian artery was asymptomatic in all patients. Knowledge of this anatomical variation is important in diagnostic neuroangiography and interventional neuroradiology. PMID- 12371598 TI - Distinctive pleomorphic xanthoastrocytoma-like tumor with exclusive abortive or aberrant neuronal differentiation and repeated recurrence--case report. AB - A 9-year-old girl with a 1-month history of generalized seizure presented with a distinctive tumor resembling pleomorphic xanthoastrocytoma. Neuroimagings showed a right frontotemporal lobe tumor. Histological examination of the resected tumor indicated similarity to pleomorphic xanthoastrocytoma without staining for glial fibrillary acidic protein. The neuronal immunoreactivity and ultrastructural features showed two discrepancies: Numerous cytoplasmic processes containing rich structures suspected to be microtubules and neurofilaments were present, but neurofilament protein 70 kd/200 kd staining was negative; and many tumor cells showed synaptophysin staining, but no synaptic structures or vesicles were observed. She suffered recurrence 14 months after the first surgery. The specimen from the second operation revealed no malignant transformation with a MIB-1 labeling index of 1.9%. Only 2 months after the second operation, there was a second recurrence. Irradiation was administered (60.2 Gy). Twenty-eight months later, no tumor progression was seen. This tumor was an unconventional type with "abortive" or "aberrant" neuronal differentiation or an extreme variant of pleomorphic xanthoastrocytoma. PMID- 12371599 TI - Neuro-Behcet's disease manifesting as a neoplasm-like lesion--case report. AB - A 50-year-old man presented with neuro-Behcet's disease (NBD) manifesting as a large neoplasm-like lesion affecting the brainstem, basal ganglia, and white matter of the cerebral hemisphere. He had no history of disease except for psychoneurosis. On admission, neurological examination found left hemiparesis and dysarthria. Magnetic resonance (MR) imaging showed multiple small ring-like enhancement in the basal ganglia, brainstem, and deep white matter. Biopsy of the mass was performed. Histological examination revealed invasion of inflammatory cells in the white matter, especially around the blood vessels. After the brain biopsy, the patient developed oral aphthae, genital ulcers, and skin eruptions, which are indicative of Behcet's disease. MR imaging after three courses of steroid pulse therapy revealed that the edematous lesion had become smaller with minimum midline shift. NBD should be considered in the differential diagnosis of lesions with multiple ring-like enhancement extending from the basal ganglia to the brainstem, because dermatological manifestations are sometimes obscured during periods of remission. PMID- 12371600 TI - Spinal cord edema preceding syringomyelia associated with Chiari I malformation- case report. AB - A 38-year-old woman with Chiari I malformation presented with spinal cord edema preceding syringomyelia manifesting as a 5-month history of nuchal pain and numbness of the upper extremities. Magnetic resonance imaging showed spinal cord edema, a poorly defined syrinx at the C-2 to T-2 levels, and distorted cerebellar tonsils. Computed tomography revealed cerebrospinal fluid (CSF) density in the center of spinal cord edema, and positron emission tomography revealed no uptake of L-[methyl-11C]methionine, indicating a non-neoplastic lesion. Craniocervical decompression achieved excellent clinical and neuroradiological outcomes. The success of surgical treatment supports the theory that patients with Chiari I malformation have increased transmural flow of CSF, causing spinal cord edema that progresses to syringomyelia. Early treatment of patients with spinal cord edema is indicated to prevent permanent spinal cord injury due to progressive syringomyelia. PMID- 12371601 TI - Fulminant subdural empyema treated with a wide decompressive craniectomy and continuous irrigation--case report. AB - A 56-year-old male presented with fulminant subdural empyema manifesting as rhinorrhea, periorbital cellulitis, fever, convulsions, and consciousness disturbance. Neuroimaging showed pansinusitis with skull destruction and extensive subdural empyema. Decompressive craniectomy, irrigation of the empyema, and subdural drainage were performed. Endoscopic sinus surgery was performed to remove the source of infection at the same time. Streptococcus milleri was cultured from the pus. Continuous irrigation of the subdural space with saline containing gentamicin for 7 days resulted in prompt elimination of pus and debris. The patient was discharged with only a slight neurological deficit. PMID- 12371604 TI - Concentration- and time-dependent effect of aminooxyacetic acid on cortical epileptogenicity. AB - In the present electrophysiological study the effect of aminooxyacetic acid (AOAA) on the cortical epileptogenicity, and on the basic electro-cortical activity was investigated in anesthetized rats. AOAA did not induce spontaneous epileptiform discharges but modified the somato-sensory evoked responses and the cortical epileptogenicity (induced by 4-aminopyridine) in the same manner depending on its concentration. AOAA at low concentrations increased the amplitude of evoked responses and the ipsilateral manifestation of epileptiform activity, however, at high concentrations significantly suppressed both the evoked responses and the induction and expression of seizures discharges. The anticonvulsive effect of AOAA was time-dependent (reached its maximum after 2h AOAA pre-treatment) and reversible. AOAA at low concentrations probably increases the efficacy of the NMDA excitatory system and decreases GABA-synthesis, resulting neuronal hyperexcitation. However, AOAA at high concentrations can lead to an effective cortical inhibition through intra- and extracellular accumulation of GABA. The gradual GABA accumulation - up to a certain level - at the synapses could also explain the time-dependency of the anticonvulsive effect of AOAA. PMID- 12371602 TI - Neuropsychological sequelae of subthalamic nucleus deep brain stimulation in Parkinson's disease: a critical review. AB - Neuropsychologists are increasingly involved in surgical candidacy evaluations and postoperative neurobehavioral assessments of patients with movement disorders, most notably those with Parkinson's disease (PD). We review here the initial studies regarding neuropsychological outcomes of deep brain stimulation (DBS) within the subthalamic nucleus (STN) for treatment of PD. Overall, these initial investigations provide preliminary support for the cognitive and neurobehavioral safety of STN DBS. Improvements in self-reported symptoms of depression and diminished verbal fluency were the most common findings, whereas changes in global cognitive abilities, memory, attention, and frontal/executive functions were inconsistent and most often described as nominal and/or transient. The generalizability of this literature is hindered by several methodological limitations, including small samples and the absence of appropriate control participants. The clinical and theoretical implications of these initial studies are highlighted and recommendations are offered to guide future research. PMID- 12371605 TI - Amyloid-beta1-42 treatment does not have a specific effect on cholinergic neurons in in vitro basal forebrain neuronal cultures of rat. AB - The neurotoxic effect of amyloid-beta peptide (1-42) was investigated in cultures of neuronal tissue derived from the basal forebrain of embryonic rat. The axonal varicosities of the cholinergic cells were revealed by vesicular acetylcholine transporter staining, and the axonal varicosities in general by synaptophysin immunohistochemistry. The results demonstrate that the treatment of in vitro neuronal cultures with 20 microM amyloid-beta peptide (1-42) for 2 days on day 5, 12 or 15 exerted a neurotoxic effect on both the cholinergic and the non cholinergic neurons. In the same cultures, the absolute number of synaptophysin positive axon varicosities was reduced to greater extent (control: 203 +/- 37/field vs treated: 101 +/- 16/field) than the number of vesicular acetylcholine transporter-immunoreactive (control: 48 +/- 4/field vs treated: 0/field) structures. It is concluded that amyloid-beta peptide (1-42) does not have a specific effect only on the cholinergic neurons, but affects non-cholinergic neurons as well. PMID- 12371606 TI - Seasonal changes in thyroid activity in the female sheath-tailed bat, Taphozous longimanus (Chiroptera: Emballonuridae). AB - The present study was designed to investigate changes in thyroid activity during the reproductive cycle in Taphozous longimanus. Thyroid gland showed marked seasonal variation in weight and secretory activity. It was inactive in quiescence and early to mid-winter dormancy and active during recrudescence and breeding period during late winter dormancy. The serum 3,5,3'-triiodothyronine (T3) and thyroxine (T4) concentrations showed significant variation and closely coincided with thyroid activity. The T3 and T4 concentrations were higher in recrudescence, late winter dormancy and minimum in quiescence and initial stages of first pregnancy. The body weight (r = 0.56), ovary weight (r = 0.73), and thyroid weight (r = 0.70) showed correlation with each other and with T3 and T4 concentrations. The correlation between body weight, thyroid weight and T3 and T4 concentrations in non-pregnant bats was higher when compared with pregnant bats. The T3 and T4 levels remained low during the initial stages of development in first pregnancy when compared with the initial stages of second pregnancy. The scant food supply and low levels of T3 and T4 and low temperature during initial stages of first pregnancy might be responsible for differential rate of fetal development in two successive pregnancies in T. longimanus. PMID- 12371603 TI - The effects of estrogen replacement therapy on neuropsychological functioning in postmenopausal women with and without dementia: a critical and theoretical review. AB - We review 42 studies examining the effects of estrogen replacement therapy (ERT) on memory and cognition in nondemented postmenopausal women. Although there are an appreciable number of nonsignificant findings, the number of significant findings favoring ERT users considerably outnumbers the rare findings of better performance in controls. Experimental studies demonstrate a consistent beneficial effect on verbal memory, but these are short-term studies of the more acute effects of ERT. The observational studies suggest that there may be a long lasting effect of continued ERT on cognitive functioning, but these studies need to be interpreted with caution because of the lack of random assignment and a possible "healthy user bias." We also summarize findings from studies on the effects of ERT on Alzheimer's disease (AD). ERT is associated with a decreased risk for dementia, but there is little evidence for a positive effect on cognition in women with AD. Definitive answers to questions about the long-term effects of ERT on cognitive aging and risk of developing AD should be provided by 3 ongoing clinical trials. PMID- 12371607 TI - Relationship between corticosterone and body weight, androstenedione and insulin during the period of delayed ovulation in a vespertilionid bat, Scotophilus heathi. AB - The aim of the present study was to evaluate the relationship between corticosterone, body weight, insulin and androstenedione in order to understand the role of adrenal in contributing hyperandrogenism during delayed ovulation in S. heathi. The circulating corticosterone concentration in female S. heathi showed significant seasonal variation. The peak corticosterone concentration observed during August-September coincides with increased feeding activities in S. heathi. The present study noted a seasonal variation in relationship of corticosterone with insulin and androstenedione in S. heathi. An inverse relationship of corticosterone with insulin and androstenedione was found during August to December, but not during January to May. A seasonal variation in the effect of adrenocorticotropic hormone (ACTH) on adrenal corticosterone production in vitro was observed during reproductive cycle. Corticosterone production in vitro by adrenal declined significantly as compared to the control during quiescence in September. The finding suggests that adrenal attained the peak responsiveness to ACTH during September. ACTH significantly enhanced the androstenedione production by the adrenal in vitro during December, when the circulating androstenedione was also high in S. heathi. This suggests that the adrenal may also contribute to hyperandrogenism during the period of delayed ovulation in S. heathi. Further studies are required to reveal the unique pattern of seasonal relationship between corticosterone, insulin and androstenedione in S. heathi. PMID- 12371608 TI - N-acetil-l-cysteine and 2-amino-2-thiiazoline N-acetyl-l-cysteinate as a possible cancer chemopreventive agents in murine models. AB - The aim of this study was to investigate N-acetyl-cysteine (NAC) and its 2-amino 2-thiazoline salt (NACAT) as potential chemopreventive agents on experimentally induced lung tumours by urethane (U) in mice. Female BALB/c mice were used. U was given by intraperitoneal injections during 2 weeks (single dose - 10 mg/mouse, total - 50 mg/mouse). Mice were treated daily per os with NAC 1/10 LD50, NACAT 1/10 or 1/100 LD50 starting 2 weeks prior U administration, then during U treatment and thereafter for 2 months. The duration of experiment was 4 months. The results showed that NAC (1000 mg/kg) reduced the lung tumour incidence to 30% that of controls, P < or = 0.05. Most effective of NACAT was 100 mg/kg dose; it reduced an average of lung adenomas per mouse by 26%, P < or = 0.05, but lower dose (10 mg/kg) was less effective. In order to achieve similar chemopreventive effect (approximately 30%) on mice, it is necessary to use 0.38 mM/kg of NACAT or 6.13 mM/kg of NAC. It means that 16 times less of NACAT is required, if calculated by molar concentration. In general, NAC and NACAT have a moderate chemopreventive effect on lung tumorigenesis induced by urethane in mice. PMID- 12371609 TI - Murine and human hematopoietic colony formation: a possible regulatory role for intracellular histamine. AB - Increasing number of data suggests that locally produced histamine is involved in regulation of hematopoiesis. In this study the granulocyte/macrophage (CFU-GM) colony formation by normal murine or human bone marrow cells, leukaemic colony formation (CFU-L) by a murine leukemia cell line (WEHI 3B), and colony formation by bone marrow cells from patients with chronic myeloid leukemia (CML) have been examined. We detected mRNA and protein expression of histidine decarboxylase (HDC), the only enzyme responsible for histamine synthesis both in normal bone marrow progenitor cells and in leukaemic progenitors. The significance of in situ generated histamine was shown on colony formation by inhibitory action of alphaFMH (blocking HDC activity, i.e. de novo histamine formation) and by N,N diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE) disturbing the interference of histamine with intracellular binding sites. These data provide further confirmation of the role of histamine in development and colony formation of bone marrow derived cells. PMID- 12371610 TI - A synthetic corticosteroid, dexamethasone regulates generation of soluble form of interleukin-6 receptor of human lymphocytes, in vitro. AB - In contrast to most of the soluble cytokine receptor antagonists properties, the soluble IL-6 receptor (sIL-6R) occurring in various body fluids of healthy persons and patients with various diseases is an agonist. The enhancing effect is due to its ability to form complex with IL-6 and to bind to gp130 making constitutively IL-6 receptor negative cells responsive for IL-6. The generation as well as the functional role of soluble IL-6 receptor is poorly understood. Earlier, we found that the sIL-6R levels in sera of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were higher than those of the control group measured by ELISA sandwich technology. In the present study we detected different levels of sIL-6R in the supernatants of lymphocyte cultures of healthy persons and patients with RA as well as SLE. Moreover, we found, that in vitro dexamethasone treatment stimulated generation of sIL-6R in both healthy persons and in active SLE, while it strongly suppressed production of sIL-6R in both RA groups. At mRNA level, we found that in SLE both the IL-6R mRNA encoding the membrane spanning and alternatively spliced (soluble) variants increased. Surprisingly, the strong decrease of sIL6R protein in RA was not found at mRNA level. PMID- 12371611 TI - Intra- and interspecific molecular polymorphism of thrips species. AB - Molecular polymorphism of six species of Thysanoptera of both sexes, collected from different locations and host plants in Hungary was studied by using RAPD-PCR technique. The specimens were classified according to sampling sites (Godollo, Nagykovacsi and Valko), host plants (Lathyrus tuberosus, Medicago sativa, Taraxacum officinale, Trifolium pratense), sexes, and larvae in case of Aeolothrips intermedius. On the basis of the total of 103 fragments generated by 15 RAPD primers the genetic distances were calculated by cluster analysis using simple matching method. The dendrogram resulted in two main groups: Aeolothripidae (Aeolothrips intermedius) and Thripidae (Frankliniella intonsa, Kakothrips robustus, Odontothrips confusus, Thrips dilatatus and T. tabaci). Within the family Thripidae two subgroups were observed including (i) F. intonsa, T. dilatatus and T. tabaci, and (ii) K. robustus and O. confusus. Two population specific and one sex-linked fragments were identified by the RAPD primers, OPQ 14, NO11 and OPA08, respectively. PMID- 12371612 TI - Nuclear translocation of p90Rsk and phosphorylation of CREB is induced by ionomycin in a Ras-independent manner in PC12 cells. AB - In the present study we examined the possible role of p90Rsk in pathways leading to neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) and the calcium ionophore ionomycin. PC12-M17 cells, expressing a dominant inhibitory Ras protein, do not undergo neuronal differentiation in response to NGF like wild-type PC12 cells, but exhibit neurite outgrowth when treated with NGF in combination with ionomycin. However, the blockade of Ras in these cells results in failure of activation of mitogen-activated protein kinase (MAPK)/extracellular signal regulation kinase (ERK) (MEK) and ERK activation as well, therefore kinases other than those of the ERK pathway might play a role in the induction of neuronal differentiation in this case. Here we show that p90Rsk translocates to the nucleus in response to ionomycin in both wild-type PC12 and PC12-M17 cells, and this spatial distribution is followed by increased phosphorylation of the cAMP response element binding protein (CREB). Since CREB is believed to be the transcription factor that can integrate Ca2+, growth factor and cAMP-induced signals, we suggest that p90Rsk may be one of the kinases which is able to replace ERKs under certain circumstances, thereby participating in Ras independent neuronal differentiation induced by NGF plus ionomycin. PMID- 12371613 TI - Distribution and pattern of water-soluble proteins of lens as revealed by gel filtration chromatography in fish (Catla catla) of different age groups. AB - The water soluble proteins of the lens collected from different age group of the fish Catla catla were subjected to Sephadex G 200 gel filtration to fractionate and to characterize the protein and further to determine the elution volume (Ve) of different fractions. The relative proportion of each fraction was also calculated. Total number of fractions F1 to F4 was discernible through gel filtration. The molecular weight of these varied from 35,000 Da to 640,000 Da. Three subfractions were also noted in F1 fraction. Interesting observations were, presence of alpha, betaH betaL crystallins in all age groups and LMW proteins in older age group. a-crystallin present in major amounts in comparison to betaH & betaL crystallins. Decrease of alpha-crystallins (except in age group II) with the increase of age, counter balanced by the increase of betaH crystallins. Increase of betaH crystallins was followed by the decrease of betaL crystallins. Results suggest that proportionate distribution of different classes of proteins determined by column chromatography is widely variable. PMID- 12371614 TI - Differential regulation of the two metallothionein genes in common carp. AB - The expression of two metallothionein (MT) genes was followed in carp (Cyprimus carpio) in vivo during exposure to As and Cu. Changes in the levels of MT-1 and MT-2 mRNA in the liver and kidney were detected by semi-quantitative RT-PCR. The inducibility of the two MT isoforms was tissue- and metal-specific. Regardless of whether As or Cu was applied, the liver was more responsive than the kidney. Copper influenced the expression of both isoforms: the MT-1 and MT-2 mRNA levels increased in both the liver and the kidney in a time- and dose-dependent manner. Arsenic affected mostly the MT-2 expression, while the level of the MT-1 transcript did not change significantly in either organ. PMID- 12371615 TI - Influence of the polyphenolic tannic acid on the toxicity of the insecticide deltametihrin to fish. A comparative study examining both biochemical and cytopathological parameters. AB - Humics and pesticides are present in aquatic environment and the toxicological consequences of their chemical interaction is well studied. However, data concerning the mechanism of the biochemical action of humic-pesticide combinations are scarce, especially in vertebrates. Thus we have chosen to study the in vivo effects of the plant polyphenolic tannic acid and the pyrethroid insecticide deltamethrin [Decis] alone or in combination on hepatic xenobiotic metabolizing enzyme activities and the associated redox-parameters in carp, as the complex assessment of these systems are regarded to serve as a relevant biomarker of environmental pollution. Stress effects and tissue damage were followed by determination of the plasma glucose level, the activities of plasma transaminases, and by electron microscopy. Tannic acid alone exerted weak prooxidant effect due to its marked antioxidant enzyme inhibitory activity. Deltamethrin, applied in a very low dose, induced oxyradical production in fish via activation of cytochrome P450 isozymes. This effect was promoted by the antioxidant enzyme inhibitory action of tannic acid, when the two chemicals were combined; however, the ultrastructural damage of the hepatocytes was reduced by the common cytoprotective capacity of the phenolic. Numerous humics are known to alter the toxicity of pesticides and their influence depends on their type and concentration. Therefore, our work taken together with other comparative studies may contribute to the assessment of the impact of humics in nature, especially in case of environmental pollution. PMID- 12371616 TI - Response of Anabaena species to different nitrogen sources. AB - Nitrogenase activity, ammonia excretion and glutamine synthetase (GS) activity were examined in five strains of Anabaena (A. anomala ARM 314, A. fertilissima ARM 742, A. variabilis ARM 310, A. oryzae ARM 313 and A. oryzae ARM 570) in the presence of 2.5 mM NO3-N (KNO3), 2.5 mM NH-4-N [(NH4)2SO4] and diatomic nitrogen (N2). Ammonium-N was more inhibitory to nitrogenase activity as compared to NO3-N in all the strains. Maximum GS activity was exhibited in NO3-N medium, irrespective of the cyanobacterial strains studied. Uninduced release of ammonia was observed in all the species, with A. oryzae ARM 313 and Anabaena variabilis ARM 310 exhibiting maximum excretion of 0.25-0.31 and 0.27-1.23 mu moles NH4 mg Chl(-1) respectively on the 15th day of incubation. The glutamine synthetase activity of A. oryzae ARM 313 was relatively very high as compared to Anabaena variabilis ARM 310. There was no nitrate reductase activity in any of the Anabaena sp. grown on NH3-N or N2-N on the 15th day of incubation. PMID- 12371617 TI - Kinetics of ochratoxin A production in different Aspergillus species. AB - Kinetics of ochratoxin A production was examined in a number of ochratoxin producing isolates representing different sections of the Aspergillus genus. Both weak and high ochratoxin producers were tested using immunochemical or high performance liquid chromatograhic methods. All isolates were found to produce the highest amounts of ochratoxin A after 7-10 days of incubation. Ochratoxin production varied between 30 - 5 x l0(5) ng ml(-1) among the Aspergillus isolates tested. The A. albertensis and A. melleus isolates examined were found to produce ochratoxin A constitutively. A. albertensis produced the highest amounts of ochratoxin A at 30 degrees C after 7 days' incubation in YES liquid medium. Ergosterol content and ochratoxin production of A. albertensis cultures were in good correlation. PMID- 12371618 TI - Selection of strain and optimization of mutanase production in submerged cultures of Trichoderma harzianum. AB - Nineteen fungal strains belonging to different genera were tested for extracellular mutanase production in shaken flasks. The optimal enzymatic activity was achieved by Trichoderma harzianum F-470, a strain for which the mutanase productivity has not yet been published. Some of factors affecting the enzyme production in shaken flasks and aerated fermenter cultures have been standardized. Mandels mineral medium with initial pH 5.3, containing 0.25% mutan and inoculated with 10% of the 48-h mycelium, was the best for enzyme production. A slight mutanolytic activity was also found when sucrose, raffinose, lactose and melibiose were carbon sources. Application of optimized medium and cultural conditions, as well as use of a fermenter with automatic pH control set at pH 6.0 enabled to obtain a high mutanase yield (0.33 U/ml, 2.5 U/mg protein) in a short time (2-3 days). The enzyme in crude state was stable over a pH range of 4.5-6.0, and at temperatures up to 35 degrees C; its maximum activity was at 40 degrees C and at pH 5.5. PMID- 12371619 TI - Allergen-specific T lymphocytes as targets for specific immunotherapy: striking at the roots of type I allergy. AB - In the past decades allergic diseases have tremendously increased and hypersensitivity reactions represent a growing health concern in industrialized countries. Despite various effective therapeutic options for the treatment of allergic diseases, only specific immunotherapy (SIT) has been shown to have effects on the underlying immunological mechanisms, namely functional changes at the level of T helper (Th) lymphocytes. It was found that allergen-specific CD4+ Th2 lymphocytes play a key role in the pathophysiology of atopic diseases. During successful SIT, the Th2-dominated immune response is modified towards a Th1 response, leading to a decline in allergen-specific IgE levels in the long term. In order to improve the efficacy and safety of SIT, novel approaches were developed targeting allergen-specific Th2 lymphocytes since specific inactivation or modulation towards Th1 cells could interfere with the disease process. In view of this aspect, this review will basically focus on two new, promising approaches to improve SIT: (1) the use of hypoallergenic proteins characterized by reduced IgE-binding capacities but retained T lymphocyte-activating properties and (2) oligodeoxynucleotides containing CpG motifs as an example of adjuvants which foster Th1 immune responses. Both approaches promise to be capable of adjusting the pathological Th2 immune response. PMID- 12371620 TI - Type I interferons as immunoregulatory molecules; implications for therapy in experimental autoimmune uveoretinitis. AB - Clinical trials have shown that the type I interferon (IFN)-alpha/beta have some beneficial effects on organ-specific autoimmune diseases, such as Behcet's diseases and multiple sclerosis, although the precise mechanisms remain largely unresolved. T helper cells 1 (Th1)-mediated autoimmune responses are involved in the initiation and/or progression of human uveitis, such as Behcet's disease. The animal model of experimental autoimmune uveoretinitis (EAU), characterized by a monophasic clinical course, has contributed to the understanding of the pathogenesis of human uveitis. Th1 producing IFN-gamma induce EAU development, while Th2 producing IL-4/IL-10 prevent the disease. However, depending on the cytokine milieu, the pro-inflammatory cytokine IFN-gamma may attenuate the autoimmune responses and anti-inflammatory cytokine IL-4 exacerbates it. Chemokines also play a crucial role in EAU development, which might be resolved by Th2-mediated immune responses. The administration of IFN-alpha/beta prevents EAU development, accompanied by a diminished production of IFN-gamma/IL-10. Interestingly, however, IFN-alpha/beta also have some beneficial effects on patients with Th2-like phenotype in addition to Th1-like phenotypes. Thus, the immuno-modulatory action of IFN-alpha/beta may be dependent on the context of cytokine combination and/or their concentrations. PMID- 12371621 TI - V gene replacement in T and B lymphocytes: illicit or regimented rearrangement? AB - Lymphocytes can undergo novel types of secondary genetic rearrangements that alter the specificity of a pre-existing B cell receptor (BCR) or T cell receptor (TCR). V gene replacement is one of them and it replaces an already rearranged V gene segment with an upstream germline V gene segment. This review focuses on the molecular aspect of rearrangement. The role of this mechanism in the immune system is debated. PMID- 12371622 TI - The TCR/CD3 complex: molecular interactions in a changing structure. AB - The T cell receptor-CD3 (TCR/CD3) complex is a multichain structure in charge of antigen recognition in T cells. Despite many genetic, structural, and functional data obtained in recent years, essential questions concerning the TCR/CD3 complex still remain open, including: 1) the precise number of polypeptides in each TCR/CD3 complex, their interactions and spatial arrangement, 2) the role(s) of each polypeptide in antigen recognition and/or in receptor signal transmission, and 3) the relationship between the TCR/CD3 complex and other membrane or cytoplasmic molecules involved in downstream signaling. In this work we shall review data concerning some of these issues, proposing a model of the overall structure of the TCR/CD3 complex to explain its known features. PMID- 12371623 TI - DNA viruses and genetic modification of dendritic cells. AB - An alternative approach to the use of tumor-peptide-loaded dendritic cells (DC) in immunotherapy would be the use of genetically modified DC using viral vectors expressing tumor-associated antigens (TAA). However, viruses have developed several immune escape mechanisms and, thus, one has to study the interaction between viruses and DC before these viruses can be used as an alternative strategy. Here we report that vaccinia virus (VV) as well as herpes simplex virus type 1 (HSV-1) are able to potently infect monocyte-derived DC, however, this infection leads to the inhibition of the DC-mediated T cell stimulation in vitro. PMID- 12371624 TI - The emerging distinct role of TNF-receptor 2 (p80) signaling in chronic inflammatory disorders. AB - Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine with strong proinflammatory and immunomodulatory properties. TNF-alpha plays a critical role in many acute or chronic inflammatory diseases and anti-TNF strategies have proven to be clinically effective. Two TNF-specific cell surface receptors, TNF R1 (p60) and TNF-R2 (p80), have been identified and the function of these receptors and the downstream intracellular signal-transduction pathways have been extensively studied in vitro. For a long time p60 was considered to be the predominant mediator of TNF signaling, whereas p80 was ascribed only an auxilliary function. However, there is increasing clinical and experimental evidence for an important independent role of p80 signaling in chronic inflammatory conditions. To date, most data exist for Crohn's disease. Upregulation of p80 and increased p80 signaling aggravates experimental colitis and is likely to contribute to the chronicity of inflammation in vivo. Further studies are required to elucidate critically important steps in TNF signaling that might be dysregulated. This will lead to a better understanding of the pathogenesis of these diseases and potentially reveal new, more specific therapeutic targets. PMID- 12371625 TI - The cGMP synthesis and PKG1 expression in murine lymphoid organs. AB - Numerous reports indicate that cyclic 3',5' guanosine monophosphate (cGMP) is involved in the regulation of immune processes. However, the mechanisms responsible for the synthesis of this nucleotide and its signaling pathways in immune cells are still not well recognized. The aim of our studies was to establish: 1) which form of guanylyl cyclase (GC) synthesizes cGMP in murine lymphoid organs and 2) whether the same organs express the isoforms PKG1alpha and/or PKG1beta of protein kinase G, known as possible target for synthesized cGMP. Cells isolated from thymus, lymph nodes, and spleen were treated with activators (SNP, ANP, CNP, STa) of soluble or particulate cyclases. Sodium nitroprusside (SNP) elevated intracellular cGMP 2-fold in thymic and lymph node cells and about 10-fold in spleen cells. Atrial natriuretic peptide (ANP) caused modest but statistically significant increases of cGMP in cells of all three organs. Additionally, spleen cells elevated their cGMP content about 2-fold in response to C-type natriuretic protein (CNP). In cellular homogenates of the all analyzed organs, the antibody anti-PKG1beta stained the 78 kDa band corresponding to the molecular mass of PKG1. Only homogenates of spleen cells were stained by the antibody recognizing PKG1alpha. Our results indicate that in the investigated organs cGMP may be synthesized mainly by soluble GC in response to nitric oxide. The modest increase of cGMP upon stimulation by ANP suggests that in all these organs either exists only a small subpopulation of cells that express particulate cyclase GC-A or GC-A is expressed at very low level. In spleen cells, however, cyclase GC-B appears to be the more active enzyme. Elevated cGMP concentration may in turn activate PKG1beta in thymus, lymph node, and spleen cells and also PKG1alpha in spleen cells. PMID- 12371626 TI - Systemic isotretinoin in the treatment of a Behcet's patient with arthritic symptoms and acne lesions. PMID- 12371627 TI - Thoughts on the proposed links between Behcet's disease and familial Mediterranean fever. PMID- 12371628 TI - Colchicine neuromyopathy: a report of six cases. AB - Colchicine has been in use for therapeutic purposes for many years. It can, however, cause subacute onset muscle and peripheral nerve toxicity in patients with chronic renal failure. In this report we describe 6 patients who developed neuromyopathy after the administration of colchicine. All patients presented with proximal muscle weakness, elevated serum creatine kinase (CK) levels, and neuropathy and/or myopathy on electromyography (EMG). The diagnosis of colchicine toxicity was confirmed in all cases by the normalization of CK levels and EMG after discontinuation of the drug. Toxicity developed in 4 renal failure patients on therapeutic doses of the drug, while one patient took a massive dose for suicidal reasons, and the other was on high-dose therapy. Patients using colchicine--especially those with renal failure--should be warned about the side effects of the drug and physicians should be careful in the administration of the drug. PMID- 12371629 TI - The effects of nitric oxide donors and inhibitors on neutrophil functions in Behcet's disease. AB - OBJECTIVE: The effects of nitric oxide donor SNAP and nitric oxide inhibitors L NMMA and AG on the functions of neutrophils in patients with Behcet's Disease (BD) were investigated in vitro. METHODS: Oxidative burst and phagocytosis of neutrophils were evaluated by flow cytometry in patients with Behcet's disease (n = 32), inflammatory (n = 17) and healthy controls (n = 14), in the presence of L NMMA, AG and SNAP. RESULTS: The stimulation index of oxidative burst was found to be significantly decreased following PMA stimulation in patients with active BD compared to inflammatory and healthy controls. Oxidative function of neutrophils were inhibited in all 3 groups in the presence of L-NMMA, AG and SNAP L-NMMA inhibited the oxidative burst of neutrophils obtained from healthy controls more than inflammatory controls and BD (80% vs 52% and 53% respectively, p = 0.001). No significant difference of phagocytosis inhibition was found with L-NMMA, AG and SNAP and there were also no differences between the groups (% 9-39). CONCLUSION: Nitric oxide donors and inhibitors may have a therapeutic role in Behcet's disease by suppresing neutrophil activity. PMID- 12371630 TI - Anti-Saccharomyces cerevisiae antibodies--a novel serologic marker for Behcet's disease. AB - OBJECTIVE: To evaluate the prevalence and clinical correlations of antibodies against Saccharomyces cerevisiae (ASCA) among patients with BD. METHODS: Twenty seven BD patients were studied. Data from medical files and from patients' interviews was collected, regarding the entire spectrum of disease manifestations, and a severity score was calculated for each patient. IgA- and IgG-ASCA levels, determined by ELISA, were studied in all BD patients and in three control groups: patients with recurrent aphthous stomatitis (RAS), systemic lupus erythematosus (SLE) and healthy volunteers. RESULTS: Thirteen BD patients (48.1%) were ASCA-positive, compared to one patient in each control group (10%, p = 0.01). The mean value of IgG-ASCA in the BD patients was 20.7 +/- 12.3 units, significantly higher than in patients with RAS (10.0 +/- 5.5, p < 0.001), SLE (11.8 +/- 9.3, p < 0.03) or healthy volunteers (10.8 +/- 9.8, p < 0.02). Mean IgA ASCA level was 16.8 +/- 8.8 units in the BD patients, significantly higher compared to healthy volunteers (11.0 +/- 5.0, p = 0.02) but similar to patients with RAS (17.0 +/- 5.3). No correlation was found between ASCA and any BD associated clinical manifestation nor the presence of HLA-B5. No difference was found in the rate of major oral ulcers nor in the systemic disease severity score between positive- and negative-ASCA patients (27.3% vs. 30.8%, and 7.31 +/- 1.80 vs. 7.28 +/- 2.27 respectively, NS). CONCLUSION: The results of our study associate, for the first time, the presence of a distinct antibody, i.e. ASCA, with BD. ASCA were not linked to a specific clinical manifestation of the disease and probably do not pose an increased risk for a more severe disease course. PMID- 12371631 TI - Molecular diagnosis of FMF: lessons from a study of 446 unrelated individuals. AB - BACKGROUND: Traditionally, the diagnosis of familial Mediterranean fever (FMF) has been based on clinical manifestations and the physician's experience. Following the cloning of the gene associated with this disease (MEFV), genetic analysis of its mutations has become available, providing a new tool for the establishment or confirmation of the diagnosis of FMF. OBJECTIVES: We analyzed the results of molecular testing for MEFV mutations in 600 individuals. We wished to determine how many of them bore mutations and what percentage had clinically active FMF. We also compared the rate of genetic confirmation of the FMF diagnosis in referrals with suspected FMF seen by general practitioners with that of persons sent for genetic analysis by FMF experts. METHODS: Of 600 individuals tested for FMF mutations, we analyzed separately 446 unrelated persons for the combination of their mutations, epidemiological data, and clinical manifestations. The five most common mutations in the present cohort were analyzed using the amplification refractory mutation system (ARMS). RESULTS: Of the 446 subjects analyzed, 249 (55%) bore mutations: 147 of these were homozygotes or compound heterozygotes, all of whom had FMF according to clinical criteria. Of the remaining 102 heterozygotes, 72 had FMF according to clinical criteria. Two patients with none of the five mutations also had FMF: North African Jews bore mainly mutations M694V and E148Q. The M6941 mutation was found exclusively in Palestinian Arabs. The rate of confirmation of FMF diagnosis by mutation analysis in subjects sent by FMF experts was significantly higher than that of persons referred by general practitioners. Analysis of the molecular testing of the multicase families (154 individuals) revealed that 141 of them bore MEFV mutations and that 4 persons homozygous for E148Q were asymptomatic. CONCLUSIONS: Molecular analysis of FMF mutations confirmed the diagnosis in about 60% of the referrals with suspected FMF. Some (33%) of the patients were heterozygotes, and there were also FMF patients with none of the 5 mutations analyzed. A second opinion by an FMF expert may decrease the need for mutation analysis in subjects suspected of having FMF. PMID- 12371632 TI - Is there an association of plasma homocysteine levels with vascular involvement in patients with Behcet's syndrome? AB - OBJECTIVE: To assess whether or not plasma homocysteine levels play a part in vascular involvement in Behcet's syndrome (BS). METHODS: 74 consecutive BS patients fulfilling the criteria of the International Study Group for BS, 35 healthy control (HC) and 14 rheumatoid arthritis (RA) patients on methotrexate (MTX) were studied. BS patients were then classified as those with and without vascular involvement. Fasting plasma homocysteine, folate, and vitamin B12 concentrations were measured by enzyme immunoassay and chemiluminescent immunoassay methods respectively. RESULTS: Plasma homocysteine levels were found to be higher in the BS patients than in the healthy control (16.08 +/- 7.5 vs. 12.9 +/- 6.3 micromol/L, p < 0.03). The homocysteine levels in the RA group on MTX were higher compared with both the BS and HC groups (28.7 +/- 9.9; p < 0.0001). No remarkable difference pertaining to homocysteine levels was found between BS patients with or without thrombosis (p < 0.86). Hyperhomocysteinemia was also detected in 11 out of 22 (50%) of the patients with vascular involvement, which proved to be of no significant difference in comparison with those without vascular involvement (20/52, 38%; chi2 = 0.26, p > 0.05). Active BS smokers exhibited a higher concentration of homocysteine in contrast to non smoker BS sufferers (20 +/- 8.4 vs 14.1 +/- 6.1 micromol/l; p < 0.004). Smoking was determined to have a positive correlation with vascular involvement (r = 0.26, p < 0.046), as well as with homocysteine levels (r = 0.31, p < 0.012) in BS. Upon logistic regression analysis, smoking was found to have a significant relationship with vascular involvement (odds ratio 3.12 [95% CI 2.02-4.22] p = 0.04). There was no significant difference between the study groups with respect to their B12 vitamin and folate levels. We were unable to make any correlation between homocysteine and vitamin B12 or folate in any of the groups (p > 0.05). CONCLUSIONS: No association was found between homocysteine levels and vascular involvement in our BS patients. We determined that smoking seems to pose a risk for vascular involvement in BS patients. PMID- 12371633 TI - Synovial histology in three Behcet's disease patients with orthopedic surgery. AB - Specimens of synovial tissues from 5 affected joints of 3 patients with Behcet's disease were available for histopathological examination. All specimens were infiltrated by lymphocytes and neutrophils, and exhibited marked vascularity and infiltration of lymphoid cells among the vessels. Marked plasma cell infiltration and lymphoid follicle formation were found in one synovial tissue sample. There was no evidence of infection or vasculitis. These findings suggest that the histopathological characteristics of synovial tissue in Behcet's disease may have a wide range, some of which may even resemble the synovial tissue of rheumatoid arthritis. PMID- 12371634 TI - Adamantiades-Behcet's disease with inner ear involvement. AB - Adamantiades-Behcet's disease is a chronic recurrent inflammatory disorder involving the small and large vessels. Typical loci of manifestations are the mucous membranes, skin and eyes, as well as the joints and central nervous system. Other organs are not commonly involved. We present two patients, one with ocular and the other with mucocutaneous manifestation of Adamantiades-Behcet's disease. In addition, the first patient reported three episodes of sudden hearing loss while under immunosuppressive therapy for his eye involvement. The second, therapy-naive patient complained of tinnitus in his left ear. Careful examination revealed vestibular involvement in the first patient and retrocochlear involvement in the second. Inner ear involvement is an uncommon manifestation of Adamantiades-Behcet's disease. In case of relevant signs or history, such as hearing disturbance, tinnitus and/or vertigo, patients should be examined for inner ear involvement. PMID- 12371635 TI - Successful treatment of familial Mediterranean fever attacks with thalidomide in a colchicine resistant patient. AB - Colchicine is the treatment of choice in familial Mediterranean fever (FMF) both for attacks and for prevention of secondary amyloidosis. The overall non responder rate varies from 5-10 to 40%. Thalidomide is known to blunt the acute phase response. We report the efficacy of the addition of thalidomide to colchicine in controlling the febrile attacks and acute phase response in a patient with FMF resistant to 2 mg colchicine per day. PMID- 12371636 TI - Fire and ICE: the role of pyrin domain-containing proteins in inflammation and apoptosis. AB - The genetic bases for several human autoinflammatory syndromes have recently been identified, and the mutated proteins responsible for these diseases are rapidly being characterized. Here, we examine two of these newly identified proteins, pyrin (also called marenostrin, product of the familial Mediterranean fever locus, MEFV) and cryopyrin (product of the CAIS1 locus, and mutated in familial cold urticaria, Muckle Wells syndrome and chronic infantile neurological cutaneous and articular syndrome). Both pyrin and cryopyrin contain an N-terminal domain that encodes a death domain-related structure, now known as the pyrin domain, or PyD. We trace the molecular interactions mediated by these PyDs, examine the evolution of the family of molecules containing this domain, and discuss the function of PyD-containing proteins and their homologues. Synthesis of the available data indicates that both pyrin and cryopyrin interact via their PyDs with a common adaptor protein, ASC. ASC itself participates in at least three important cellular processes: apoptosis, recruitment and activation of pro caspase-1 (with associated processing and secretion of IL-1beta), and activation of NF-kappaB (a transcription factor involved in both initiation and resolution of the inflammatory response). Through PyD:PyD interactions, pyrin and cryopyrin, as well as several related, but still uncharacterized PyD containing proteins, appear to modulate the activity of all three of these processes, each of which plays a crucial role in the inflammatory pathways that characterize the innate immune system. PMID- 12371637 TI - Aphtous stomatitis in a patient with Behcet's disease and HIV was associated with an increased HIV load. PMID- 12371638 TI - The disappearance of pulmonary artery aneurysms and intracardiac thrombus with immunosupressive treatment in a patient with Behcet's disease. PMID- 12371639 TI - Is there a heterozygote advantage for familial Mediterranean fever carriers against tuberculosis infections: speculations remain? PMID- 12371640 TI - Highlights of the 10th International Congress on Behcet's Disease. PMID- 12371642 TI - Neural stem cells: progression of basic research and perspective for clinical application. AB - It has long been thought that functional regeneration of the injured central nervous system (CNS) is impossible, as Santiago Ramony Cajal described in the early 20th century, "once the development was ended, the fonts of growth and regeneration ... dried up irrevocably". In mammalian neural development, most neuronal production (neurogenesis) occurs in the embryonic stage. However, recent findings indicate that neurogenesis continues in the olfactory bulb, hippocampus, and dentate gyrus of adult mammalian animals, from the neural stem cells (NSCs). Recently developed techniques have made it possible to isolate, culture, and grow pluripotent self-renewing NSCs from both embryonic and adult brains. This basic research is attracting a lot of attention because of the hope that it will lead to regeneration and reconstruction therapy for the damaged CNS. In this review, recent findings on the stem cell biology of the CNS and strategies for its potential therapeutic application will be discussed. PMID- 12371641 TI - Early blunted cortisol response to insulin induced hypoglycaemia in familial Mediterranian fever. AB - OBJECTIVE: To study cortisol, adrenocorticotropic hormone and C-reactive protein responses to specific stimuli in familial Mediterranean fever (FMF). METHODS: For the purpose of measuring cortisol, ACTH, and CRP responses to insulin induced hypoglycaemia during attack-free periods, 14 FMF patients, 11 patients with ankylosing spondylitis or Behcet's disease as disease controls (DC), and a further 10 healthy control subjects (HC) were involved in this study. None of the subjects had ever received corticosteroids before this study. Cortisol and ACTH levels were measured by chemiluminescence enzyme immunoassay. RESULTS: No attack was observed among FMF patients during the test. No significant difference in the mean cortisol values after insulin induced hypoglycaemia was observed between the groups involved at any stage of the test. The integral cortisol response to hypoglycaemia expressed as the AUC (0-90 min) was found not to differ among the study groups (1827 +/- 115.6 in FMF; 2196 +/- 205.4 in DC, p = 0.12; 1771 +/- 98.4 in HC, p = 0.9). The delta response of cortisol to insulin induced hypoglycaemia wasfound to be statistically lower (-4 +/- 0.8 mg/dl vs. -1.9 +/- 0.7 microg/dl; p<0.03) only for the 0 to 30 min interval in patients with FMF compared to HC respectively. Similar results, though of no statistical significance, were also found for the 0 to 45 min interval (1.17 +/- 2.2 microg/dl in FMF patients vs. 3.3 +/- 2 microg/dl in HC; p = 0.6). The mean basal CRP level of patients with FMF was remarkably higher than that in HC. Although the mean CRP level at 90 min for FMF cases with cortisol levels under 12 microg/dl at 30 min was found to be higher than those with cortisol levels over 12 microg/dl at 30 min, no significant difference was observed. CONCLUSION: An early blunted cortisol response observed in a stressful situation in FMF patients may well account for the curious relationship between stress and an inflammatory reaction and/or attack. Furthermore, the fact that the CRP level was relatively higher in FMF patients with lower cortisol levels might also highlight the importance of endogen cortisol in the inflammatory feature of this disease. PMID- 12371643 TI - Diagnosis and treatment of obturator hernia. AB - Obturator hernia is a rare type of hernia, but it is a significant cause of intestinal obstruction due to the associated anatomy. Correct diagnosis and treatment of obturator hernia is important, because delay can lead to high mortality. Twelve patients with obturator hernia were managed during a 11-year period, including 11 women and 1 man with a mean age of 82 years. We compared our experience with the previously published data to establish standards for the diagnosis and treatment of this hernia. All 12 patients presented with intestinal obstruction. The median interval from admission to operation was 2 days. The Howship-Romberg sign was positive in 5 patients. A correct diagnosis was made in all 8 patients who underwent pelvic CT scanning. Surgery was performed via an abdominal approach (n = 7) or an inguinal approach (n = 5). The hernial orifice was closed using the uterine fundus (n = 6), a patch (n = 5), and direct suture (n = 1). Mean follow-up time was 33 months, and no recurrence has been detected. The poor physical condition of patients might have led to a delay in diagnosis and treatment. In troubled patients with nonspecific intestinal obstruction, CT scanning is useful for the early diagnosis of obturator hernia. Correct CT diagnosis of obturator hernia allows us to select the inguinal approach combined with patch repair, which is minimally invasive surgery. PMID- 12371644 TI - Pemphigus as a paradigm of autoimmunity and cell adhesion. AB - Pemphigus is a group of autoimmune blistering diseases of the skin and mucous membranes that are characterized histologically by intraepidermal blisters due to the loss of cell-cell adhesion of keratinocytes and immunopathologically by the finding of pathogenic IgG autoantibodies directed against the cell surface of keratinocytes. Identification of the target antigens has redefined pemphigus as an autoimmune disease against desmosomal cadherin or desmoglein. The IgG autoantibody-mediated functional inhibition of desmoglein which plays an important role in the cell-cell adhesion of keratinocytes results in blister formation. Patients with pemphigus vulgaris and pemphigus foliaceus have IgG autoantibodies against desmoglein3 and desmoglein1, respectively. Even complex clinical variations of pemphigus vulgaris and foliaceus are now logically explained by the desmoglein compensation theory. As an extension of this theory, the exfoliative toxin produced by Staphylococcus aureus, which causes staphylococcal scalded skin syndrome and bullous impetigo, was found to specifically cleave desmoglein1 and induce the identical histology to pemphigus foliaceus. Another recent innovation has been the development of an active disease mouse-model for pemphigus using autoantigen knockout mice, in which self tolerance of the defective gene product is not acquired. When splenocytes from desmoglein3 knockout mice are adoptively transferred into mice expressing desmoglein3, anti-desmoglein3 IgG is stably produced in the recipient mice that develop the phenotype of pemphigus vulgaris. This model will be valuable not only for dissecting the cellular and molecular mechanisms in pathogenic antibody production but also for developing novel therapeutic strategies. PMID- 12371645 TI - Dynamic carpal stability. AB - The term carpal instability is commonly used, but what carpal stability actually is has not been defined. Much of the mechanically complex wrist's versatility is due to the intercalated three bone proximal carpal row. Landsmeer described the collapse tendency associated with intercalated segments. The factors which provide static stability are the oblique alignment of the scaphoid, the obliquely aligned dorsal and palmar ligamentous complexs, the intrinsic perilunate ligaments, the transiting transcarpal tendons and the negative intraarticular pressure. The proximal carpal row adjusts its posture on the counterbalancing flexion/pronation torque exerted by the scaphoid and the extension/supination torque exerted by the triquetrum. The dynamic factors are the compressive force exerted across the joint acting on the joint surfaces and the effect of the bowstringing force provided by the flexor carpi radialis acting at the scaphoid tuberosity. The proximal carpal row has a tendency to translate ulnarly along the ulnarly sloped radial articular surface while the distal row has a tendency to slide radially on the radially sloped lunatatotriqueteral distal articular surface. This activity produces differential tension in the ligaments attaching to the triquetrum which effects an extension/supination stance of the triquetrum. The force couple acting on the scaphoid effects the flexion tendency of the scaphoid. The bowstringing of the flexor carpi radialis also counteracts scaphoid flexion. Alterations in any of these factors may upset the delicate mechanical balance of the joint. PMID- 12371646 TI - The use of foetal human brain tissue as brain implants: phenotype manipulation by genetic manipulation and biochemical induction. AB - The use of dopaminergic mesencephalic (VM) human foetal brain tissue as implants to neurosurgically treat Parkinson's disease has been in progress since the 1980's. A major limitation in the use of VM tissue is the amount of tissue available from each human embryo. Usually tissue from about 7 embryos is required to treat each patient unilaterally. To overcome this we have developed various strategies. One is to convert embryonic cerebral cortex in human embryos into dopaminergic tissue which is stable, and which will secrete dopamine in vivo once implanted. The cerebral cortex is about 500 times larger than the VM and can therefore provide a lot more tissue for transplantation. This can be achieved by genetic manipulation of the embryonic cerebral cortex tissue, involving the lipo transfection of multiple copies of the human tyrosine hydroxylase gene into both neurones and glial cells. In another approach we have biochemically manipulated the development of the cerebral cortex to direct the neurotransmitter phenotype towards the dopaminergic type, and away from other phenotypes. This tissue, too, is stable and will synthesise and secrete dopamine when transplanted. Our third approach has been to manipulate pluripotential neural cells which are yet to develop into neurones and glial cells. These cells can be expanded in number many fold before treatment to direct their development into stable dopaminergic neurones in large numbers (70%), which synthesise and release dopamine. When used as transplants in animal models of Parkinson's disease, these various types of artificially induced dopaminergic tissue are very effective at reducing the Parkinsonian syndrome. PMID- 12371647 TI - Neurochemical organization of the first visual synapse. AB - The retina employs two main synaptic relays in which information converges to higher order cells, and at the same time is modified by lateral inhibitory interneurons. At the first synaptic layer, rod and cone terminals contact second order neurons (horizontal and bipolar cells), and in turn, horizontal cells contact cones and bipolar cells. In this talk/review we describe the structures and the neurochemicals involved in transmitting the visual signal at this synaptic complex. PMID- 12371648 TI - A case of dilated cardiomyopathy with end-stage heart failure treated by prolonged continuous hemodiafiltration. AB - A 55-year-old Asian man first visited to our hospital with complaining of exertional dyspnea eight years ago, and was diagnosed as having idiopathic dilated cardiomyopathy. One of his siblings also suffered from idiopathic dilated cardiomyopathy. His symptoms became worse gradually, and he was hospitalized again because of disturbance of consciousness on February 21, 2001. Hemodynamic monitoring with a Swan-Ganz catheter was started, which revealed that the cardiac index was 1.1 L/ min/BSA, cardiac output 1.8 L/min, and pulmonary artery pressure 43/33 mmHg. The echocardiographic observation showed that the left ventricular ejection fraction was 32%, and serum BNP was elevated to 5,411 pg/mL. Multi-organ failure including renal and hepatic dysfunction developed because of the low cardiac output status. Continuous hemodiafiltration (CHDF) was introduced to reduce the volume overload, improve renal failure, and eliminate adverse cytokines. Although his hemodynamic status was temporarily improved after starting CHDF, weaning from CHDF was difficult and he finally died from cardiogenic shock after two month of intensive therapy. The autopsy showed thinning of the left ventricular wall, and histological examination revealed diffuse fibrous hyperplasia and myocardial fiber deficit in the ventricular myocardium. CHDF was effective in reducing the volume overload and improving renal function; however, heart transplantation is inevitable for the patients with severe heart failure due to dilated cardiomyopathy. PMID- 12371649 TI - Vancomycin-resistant enterococci in shellfish, unchlorinated waters, and chicken. AB - Vancomycin-resistant enterococci (VRE) have been a cause of increasing concern chiefly regarding the infection of hospital patients. There is suspicion, but limited evidence, that food and environmental spread may be important. Biomonitoring by examination of bivalve shellfish was used to assess the occurrence of VRE entering the environment. Using pre-enrichment and Lewisham and Slanetz and Bartley agars, 2/125 (1.6%) of shellfish were found to contain enterococci resistant to high levels of vancomycin. Lewisham agar allows relatively rapid identification of VRE. In a second phase of the work using pre enrichment and Slanetz and Bartley agar, 4/151 (2.7%) shellfish and 5/27 (18.5%) raw chickens contained VRE. Using filtration and pre-enrichment, no VRE were found in 54 unchlorinated water samples. The study shows that environmental prevalence of VRE is low, and that raw chickens are frequently contaminated. PMID- 12371650 TI - Prediction of food thermal process evaluation parameters using neural networks. AB - Two neural networks (ANN) were developed to predict thermal process evaluation parameters g and f(h)/U (the ratio of heating rate index to the sterilizing value), respectively. The temperature change required for the thermal destruction curve to traverse one log cycle (z), cooling lag factor (j(c)) andf(h)/U were input variables for predicting g and z, while j(c) and g were inputs for predicting f(h)/U. The data used to train and verify the ANN were obtained from reported values. Shrinking of input and output variables using natural logarithm function improved the prediction accuracy. The use of "Wardnets" with three slabs of 14 nodes in each slab, with a learning rate of 0.7 and momentum of 0.9 provided the best predictions. The g (unshrunk values) was predicted with a mean relative error of 1.25 +/- 1.77%, and a mean absolute error of 0.11 +/- 0.16 degrees F. The f(h)/U was predicted with a mean relative error of 1.41 +/- 3.40%, and a mean absolute error of 2.43 +/- 15.97, using 10 nodes in each slab. The process time calculated using the g from the ANN models closely followed the time calculated from the tabulated gvalues (RMS=0.612 min, average absolute error=0.466 min with an S.D. of 0.400 min). PMID- 12371651 TI - Effect of medium composition and temperature and pH changes on exopolysaccharide yields and stability during Streptococcus thermophilus LY03 fermentations. AB - To increase the exopolysaccharide (EPS) yields from Streptococcus thermophilus LY03 and to unravel the nature of the EPS degradation process, fermentation experiments were carried out with this strain in a customized MRS medium, using different additional carbohydrates or amino acids possibly related to growth and EPS production. No significant increase of the EPS yields or activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase that are correlated with EPS production, or of the activity of dTDP glucose pyrophosphorylase involved in the rhamnose synthetic branch of EPS biosynthesis, was observed. The EPS monomer composition remained unchanged for all experiments. Fermentations with a sudden temperature increase or lowered pH were carried out as well to try to avoid EPS degradation upon prolonged fermentation. It was demonstrated that EPS degradation took place enzymatically. Incubations of purified high-molecular-mass EPS with cell-free culture supernatant or cell extracts showed its degradation by enzymes with an endo activity. This glycohydrolytic activity probably encompasses several enzymes having a molecular mass lower than 50,000 and 10,000 Da, and seems to be rather stable at high temperature and low pH. These results contribute to a better understanding of the physiological and chemical factors influencing EPS production and degradation. PMID- 12371652 TI - Analysing collaborative trials for qualitative microbiological methods: accordance and concordance. AB - In qualitative (detection) food microbiology, the usual measures of repeatability and reproducibility are inapplicable. For such studies, we introduce two new measures: accordance for within laboratory agreement and concordance for between laboratory agreement, and discuss their properties. These measures are based on the probability of finding the same test results for identical test materials within and between laboratories, respectively. The concordance odds ratio is introduced to present their relationship. A method to test whether accordance differs from concordance is discussed. PMID- 12371653 TI - The effect of commercial production and product formulation stresses on the heat resistance of Escherichia coli O157:H7 (NCTC 12900) in beef burgers. AB - The effects of commercial beef burger production and product formulation on the heat resistance of Escherichia coli O157:H7 (NCTC 12900) in beef burgers were investigated. Fresh beef trimmings were inoculated with E. coli O157:H7 to approximately log10 7.0 cfu g(-1) and subjected to standard beef burger production processes, including freezing, frozen storage and tempering. The tempered trimmings were processed in line with commercial practice to produce burgers of two formulations, a 'Quality' burger containing 100% beef and an 'Economy' burger containing 70% beef and 30% other ingredients (salt, seasoning, soya, onion and water). The burgers were then frozen and stored. Control 'unprocessed' burgers were produced to each of the above formulations using fresh beef trimmings. All burger types were heat-treated at 55, 60 or 65 degrees C. Samples were examined by plating on Tryptone Soya Agar (TSA), incubated at 37 degrees C for 2 h, before overlaying with SMAC (TSA/SMAC) and incubation at 37 degrees C. The resultant counts were used to derive D-values for E. coli O1 57:H7. At each treatment temperature, the D-values from each burger formulation using frozen tempered trimmings were significantly lower (P < 0.001) than the D values from that formulation using fresh trimmings. At each treatment temperature, the D-values from Economy burgers using processed trimmings were significantly higher (P < 0.001) than the D-values from Quality burgers using processed trimmings. A similar trend of significantly higher (P<0.001) D-values for Economy burgers was observed using fresh trimmings. This study found that commercial processing and product formulation have profound effects on the heat resistance of E. coli O157:H7 in beef burgers. PMID- 12371654 TI - Antifungal activity of octyl gallate. AB - Antifungal activities of propyl (C3), octyl (C8) and dodecyl (C12) gallates (3,4,5-trihydroxybenzoate) were tested against Saccharomyces cerevisiae ATCC7754 and Zygosaccharomyces bailii ATCC 60483. Octyl gallate was found to be the only active compound with the minimum fungicidal concentration of 25 microg/ml (89 microM) against S. cerevisiae and of 50 microg/ml (177 microM) against Z. bailii, respectively. The inactivation study showed that octyl gallate was fungicidal against both S. cerevisiae and Z. bailii at any stage of growth. These fungicidal activities were not influenced by pH values. Octyl gallate at 100 microg /ml reduced plasma membrane fluidity to 48% of control. On the other hand, dodecyl gallate at the same concentration reduced it to 76% of control. Only octyl gallate inhibited glucose-induced medium acidification, indicating direct or indirect inhibition of plasma membrane H +-ATPase. The primary fungicidal activity of octyl gallate comes from its ability to act as a nonionic surface active agent (surfactant), though it can not be inferred that membrane damage, such as a decrease in the membrane fluidity, is the only cause of the lethal effect. PMID- 12371655 TI - Risk assessment of the use of sub-optimal levels of weak-acid preservatives in the control of mould growth on bakery products. AB - The hurdle technology approach was used to prevent fungal growth of common contaminants of bakery products including isolates belonging to the genera Eurotium, Aspergillus and Penicillium. Several levels (0.003%, 0.03% and 0.3%) of calcium propionate, potassium sorbate and sodium benzoate were assayed on a model agar system in a full-factorial experimental design in which the other factors assayed were pH (4.5, 6 and 7.5) and a(w) (0.80, 085, 0.90 and 0.95). Potassium sorbate was found to be the more suitable preservative to be used in combination with the common levels of pH and a(w) in Spanish bakery products. Sub-optimal concentrations (0.003% and sometimes 0.03%) led to an enhancement of fungal growth. None of the preservatives had a significant inhibitory effect at neutral pH. PMID- 12371656 TI - What is the source of ochratoxin A in wine? AB - During a microvinification trial using natural mouldy grapes from a research experimental vineyard, ochratoxin A (OTA) contaminated white wine was obtained. Potential OTA-producing mycobiota of grape samples used in this microvinification process was assessed. Only Aspergillus carbonarius isolates were detected as producers of OTA. Our report is a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine. PMID- 12371657 TI - Handedness effects on transient evoked otoacoustic emissions in schoolchildren. AB - Handedness, as a potentially influencing, nonpathologic factor, has not been investigated in relation to transient evoked otoacoustic emissions (TEOAEs). The present study aimed to examine the effects of handedness on the TEOAE spectrum in entry-level schoolchildren, with attention also to possible ear asymmetry. A total of 228 subjects (114 males, 114 females, mean age = 6.3 years) were tested using the ILO292 Otodynamics Analyzer (Quickscreen mode) in quiet rooms in 22 schools. For statistical analysis, subjects were matched for factors such as handedness, gender, age, and history of recent ear infection. The results from subjects with passing TEOAE, pure-tone screening, and tympanometry revealed no significant handedness effect overall, although a significant ear asymmetry effect on the measurement parameters of AB difference, noise level, response level, whole-wave reproducibility, band reproducibility, and signal-to-noise ratios was found. PMID- 12371658 TI - Real-ear-to-coupler difference predictions as a function of age for two coupling procedures. AB - The predicted real-ear-to-coupler difference (RECD) values currently used in pediatric hearing instrument prescription methods are based on 12-month age range categories and were derived from measures using standard acoustic immittance probe tips. Consequently, the purpose of this study was to develop normative RECD predicted values for foam/acoustic immittance tips and custom earmolds across the age continuum. To this end, RECD data were collected on 392 infants and children (141 with acoustic immittance tips, 251 with earmolds) to develop normative regression equations for use in deriving continuous age predictions of RECDs for foam/acoustic immittance tips and earmolds. Owing to the substantial between subject variability observed in the data, the predictive equations of RECDs by age (in months) resulted in only gross estimates of RECD values (i.e., within +/- 4.4 dB for 95% of acoustic immittance tip measures; within +/- 5.4 dB in 95% of measures with custom earmolds) across frequency. Thus, it is concluded that the estimates derived from this study should not be used to replace the more precise individual RECD measurements. Relative to previously available normative RECD values for infants and young children, however, the estimates derived through this study provide somewhat more accurate predicted values for use under those circumstances for which individual RECD measurements cannot be made. PMID- 12371659 TI - Comparison of electrically evoked whole-nerve action potential and electrically evoked auditory brainstem response thresholds in nucleus CI24R cochlear implant recipients. AB - In this study, differences between electrically evoked whole-nerve action potential (EAP) and electrically evoked auditory brainstem response (EABR) measurements within Nucleus CI24R cochlear implant recipients were evaluated. Precurved modiolus-hugging internal electrode arrays, such as the CI24R, are designed to provide more direct stimulation of neural elements of the modiolus. If the electrode array is closer to the modiolus, electrically evoked and behavioral levels might be lower than were previously recorded for the straight electrode array, the CI24M. EAP and EABR growth functions and behavioral levels were obtained for 10 postlingually deafened adults. Results revealed no significant differences between EAP and EABR threshold levels, and these levels were not significantly lower than those obtained using the CI24M. PMID- 12371660 TI - Interaural asymmetries revealed by dichotic listening tests in normal and dyslexic children. AB - Normal and dyslexic right-handed children were assessed with three dichotic listening tests, the Dichotic Digits test, the Competing Words subtest of the SCAN, and the Dichotic Consonant-Vowel test. Performance was measured as both number and percentage of correct responses in the right and left ears. Laterality was defined as a simple difference in percentage between the two ears. Differences across the tests were revealed for all children, with the greatest differences occurring for left-ear responses. Only one dichotic listening test, Competing Words from the SCAN, produced a consistent right-ear advantage across all of the children tested. Between groups of children, differences in performance and in laterality were demonstrated. Using a criterion of poorer than 76 percent correct for the left ear, the Competing Words subtest of the SCAN identified 7 of the 10 dyslexic children as abnormal, with no false alarms in the control group. PMID- 12371661 TI - Behavioral and electrophysiologic evidence of auditory processing disorder: a twin study. AB - We administered a battery of both behavioral and electrophysiologic measures to a pair of fraternal twin girls, one of whom exhibited symptoms consistent with an auditory processing disorder. Both twins were within normal limits on standardized tests of cognitive and language skills. Basic audiometric measures, as well as behavioral tests of simultaneous masking, backward masking, gap detection, and frequency-sweep discrimination, showed little difference between the twins. Significant differences, however, were evident on event-related potentials (ERPs) in response to both within-channel and across-channel gap detection tasks. Substantial differences were also noted for ERPs to both linguistic and nonlinguistic targets in dichotic listening paradigms. The pattern of electrophysiologic results was consistent with a deficit in the efficiency of interhemispheric transfer of auditory information. A possible reason for the greater effectiveness of electrophysiologic over behavioral measures is discussed. PMID- 12371662 TI - Cell volume control and signal transduction in apoptosis. AB - Apoptosis is a physiological form of death in which cells turn-on an intrinsic genetic program that eventually leads to their destruction in a highly regulated manner. This process renders elimination of "unwanted cells" in the body, and accounts for cellular turnover and homeostasis of tissues in multicellular organisms. Consequently, an imbalance in the apoptotic rate in a particular tissue can lead to profound effects in the whole organism. Exposure of cells to apoptotic stimuli induces a rapid loss of cell volume (apoptotic volume decrease) that plays a pivotal role in the decision of a cell to undergo apoptosis. Interestingly, the apoptotic volume decrease is driven by changes in ionic fluxes across the plasma membrane that promote a decrease in the intracellular ions that ultimately also leads to a reduction in intracellular ionic strength. Despite an intensive research effort however, the cellular and molecular mechanisms that trigger changes in cell volume during apoptosis remain poorly understood. Nevertheless, this apoptotic volume decrease has been shown to be a necessary component of the apoptotic cascade and an important point of modulation for the entire cell death process. In this review, we will focus on the importance of the apoptotic volume decrease in the context of signaling and modulation of programmed cell death. PMID- 12371663 TI - Protective effect of topical iodine preparations upon heat-induced and hydrofluoric acid-induced skin lesions. AB - In this study, the protective prophylactic and post-exposure effects of novel topical iodine preparations were demonstrated upon heat- and hydrofluoric acid induced skin lesions in the haired guinea pig. Prophylactic treatment of thermal bums with a liquid iodine preparation resulted in statistically significant reductions of 39% and 30%, respectively, in acute inflammation and hemorrhage microscopic dermal parameters indicative of acute tissue damage. A clear trend of iodine-induced reduction in dermal necrosis occurred, and the epidermal healing markers, acanthosis and hyperkeratosis, were increased. Postexposure treatment of thermal burns with an iodine ointment preparation immediately after occurrence also conferred significant therapeutic reduction in parameters of tissue damage such as epidermal ulceration (87%), acute inflammation (58%), and hemorrhage (30%). Gross pathological evaluation showed that prophylactic and postexposure treatments with the liquid iodine preparation significantly reduced the heat induced ulceration area by 97% and 65%, respectively. In addition, immediate treatment with an ointment iodine formulation significantly decreased the ulceration area by 98%; its tetraglycol vehicle also had a beneficial effect. Postexposure treatment with the iodine ointment proved efficacious upon hydrofluoric acid-induced skin burns. We observed statistically significant reductions of 76% and 68% in ulceration areas at intervals of 5 and 10 minutes between exposure and treatment, whereas a weaker effect was observed at a longer time interval of 15 minutes. Our findings suggest the therapeutic usage of these newly developed iodine preparations for thermally induced and hydrofluoric acid induced skin burns. PMID- 12371664 TI - Evaluation of carcinogenic responses in the Eker rat following short-term exposure to selected nephrotoxins and carcinogens. AB - This study examined the response of the Eker rat to nephrotoxic compounds and to genotoxic nonrenal carcinogens. Groups of male Eker rats received either no treatment; a vehicle treatment; treatment with a noncarcinogenic nephrotoxin (aluminum nitrilotriacetate, 2 mg/kg/day of aluminum, intraperitoneally, 3 days per week or cyclosporine A, 30 mg/kg/day, orally by gavage, 7 days/week); or treatment with a genotoxic nonrenal carcinogen (furan, 8 mg/kg/day, orally by gavage, 5 days/week or 2,4-diaminotoluene, 6.5 mg/kg/day, orally by gavage, 7 days/week or 2-nitropropane, 89 mg/kg/day, orally by gavage, 3 days/week). Duration of treatment was 4 and/or 6 months. Tissues from the Eker rats were evaluated microscopically and numbers of proliferative renal lesions were counted. Administration of nephrotoxic compounds (Al-NTA and cyclosporine) significantly increased the number of preneoplastic and neoplastic renal lesions in the Eker rat compared to concurrent vehicle controls. The genotoxic nonrenal carcinogens had no consistent effect on numbers of preneoplastic or neoplastic renal lesions and did not produce neoplasms in the expected target organ (liver). PMID- 12371665 TI - 4-Vinylphenol-induced pneumotoxicity and hepatotoxicity in mice. AB - 4-Vinylphenol (4-hydroxystyrene, 4-ethenylphenol, 4-VP) occurs naturally in some foods and has been used as a flavoring agent in food products. It is used synthetically in the production of polymers and resins. It has also been reported to be a minor metabolite of styrene in rats and humans. Varying doses of 4 vinylphenol were administered ip to mice. Hepatotoxicity was assessed by measuring serum sorbitol dehydrogenase (SDH) and by light microscopy. Pneumotoxicity was assessed by measuring proteins, cells, and lactate dehydrogenase activity in bronchoalveolar lavage fluid (BALF) and by light microscopy. 4-VP caused a dose-dependent increase in serum SDH and mild hepatocellular swelling. It caused an increase in cell number and lactate dehydrogenase activity in BALF. Microscopically, there was widespread and severe necrosis of the bronchioles by 12 hours. Re-epithelialzation of the bronchioles was evident by 48 hours. These studies indicate that 4-vinylphenol is both hepatotoxic and pneumotoxic. PMID- 12371666 TI - Propylene glycol monomethyl ether (PGME): inhalation toxicity and carcinogenicity in Fischer 344 rats and B6C3F1 mice. AB - A series of inhalation studies with propylene glycol monomethyl ether (PGME) vapor were undertaken to characterize its subchronic toxicity in mice and chronic toxicity/oncogenicity in rats and mice. Groups of male and female Fischer 344 rats and B6C3F1 mice were exposed to 0, 300, 1,000, or 3,000 ppm vapor from 1 week to 2 years. Primary treatment-related effects included: initial sedation of animals exposed to 3,000 ppm and its subsequent resolution correlating with induction of hepatic mixed function oxidase activity and S-phase DNA synthesis; elevated mortality in high-exposure male rats and mice (chronic study); elevated deposition of alpha2u-globulin (alpha2U-G) and associated nephropathy and S-phase DNA synthesis in male rat kidneys; accelerated atrophy of the adrenal gland X zone in female mice (subchronic study only); and increased occurrence and/or severity of eosinophilic foci of altered hepatocytes in male rats. No toxicologically relevant statistically significant increases in neoplasia occurred in either species. A numerical increase in the incidence of kidney adenomas occurred in intermediate-exposure male rats; however, the association with alpha2U-G nephropathy, a male rat specific effect, indicated a lack of relevance for human risk assessment. PMID- 12371667 TI - Hepatoblastomas in mice in the US National Toxicology Program (NTP) studies. AB - Over the last 8 years, a 5-fold increase in the incidence of mice with spontaneous hepatoblastomas and a moderate increase in the incidence of chemically induced hepatoblastomas in B6C3F1 mice occurred in 2-year NTP studies compared to the previous 7 years. There was a positive association between an increased incidence of mice with hepatoblastoma and an increased incidence of mice with hepatocellular tumors in the treated mice. The rate of pulmonary metastases for hepatoblastoma was similar to that of pulmonary metastasis for hepatocellular carcinomas. Although a variety of chemicals caused an increased incidence of mice with hepatoblastoma, there was no apparent association between a specific chemical structure or a biological class of compounds and their capacity to induce hepatoblastomas. Hepatoblastomas frequently arose within hepatocellular carcinomas or adenomas and were induced by the same compounds that induced hepatocellular neoplasms. Therefore, it seems reasonable to combine the incidence of mice with hepatoblastomas and the incidence of mice with hepatocellular carcinomas in hazard identification studies. PMID- 12371668 TI - Apparent mesonephric duct (rete anlage) origin for cysts and proliferative epithelial lesions in the mouse ovary. AB - Ovaries and adjacent parovarian (mesovarial) tissues of CD-1 mice of various ages were examined to characterize cystic and proliferative epithelial lesions of the ovary and parovarian tissues. Ovaries and adjacent tissues from 6 mice each at approximately 3 and 8 months of age were processed for light microscopy and step sections (50-micron intervals) of the entire tissue were examined. Tissues were collected from 40 mice each at 16 through 24 months of age and 3 step sections per mouse were examined. Mesonephric duct remnants were found in all mice at 3 and 8 months. Ducts were usually in the mesovarial adipose tissue and near or within the ovarian hilus and were often loosely associated with smooth muscle of the mesovarial ligament. The epithelium of the ducts varied from low cuboidal to columnar, occasional individual cells were ciliated, and small papillary configurations of epithelium were occasionally present. Ducts were dilated (> 1 mm) more often in mice at 8 months compared to 3 months, and some were continuous with cystic spaces within the ovaries. As mice aged (16-24 months), dilation and cystic change in ducts and associated compression of ovarian tissue away from the ovarian hilus became common. The epithelium of dilated ducts was generally flattened, but foci of cuboidal to columnar epithelium and/or occasional ciliated cells were present. Many ducts contained foci of hyperplastic and/or hypertrophic epithelium, and papillary projections of epithelium were occasionally found. Some of the latter lesions were consistent with a diagnosis of papillary cystadenoma. Hyperplasia of associated fibromuscular stroma was limited to a few apparently extraovarian ducts. The results of this study indicate that remnant mesonephric ductular structures are common in and adjacent to the ovaries of CD-1 mice. As mice age, these ducts become dilated and, in some, the epithelium becomes hyperplastic and/or hyperptrophic. These mesonephric duct remnants appear to be a common source of ovarian and parovarian cyts cysts and epithelial neoplasms of the ovary in mice. PMID- 12371669 TI - Comparative prevalence, multiplicity, and progression of spontaneous and vinyl carbamate-induced liver lesions in five strains of male mice. AB - The overall and age-specific prevalences and multiplicities of spontaneous and chemically induced hepatocellular neoplasia were compared among male B6D2F1, B6C3F1, C3H (C3H/HeNCr1 MTV-), B6CF1, and C57BL/6 (C57BL/6NCr1) mice following a single intraperitoneal injection of 0.03 microM vinyl carbamate (VC)/g body weight or vehicle alone at 15 days of age. Additional groups of B6C3F1, C3H, and C57BL/6 males received 0.15 microM VC/g body weight at 15 days of age. For male B6D2F1, B6C3F1, C3H, B6CF1, and C57BL/6 mice, the estimated overall prevalences (and multiplicities) of hepatocellular adenomas or carcinomas in vehicle controls were 14.1% (0.19), 12.3% (0.15), 8.2% (0.10), 7.2% (0.09), and 2.4% (0.02), respectively. The analogous estimates in the low-dose group were 59.2% (1.19), 72.9% (4.07), 48.6% (1.99), 22.8% (0.29), and 43.9% (0.82). Analogous estimates for B6C3F1, C3H, and C57BL/6 mice in the high-dose group were 45.3% (4.29), 59.7% (6.63), and 46.8% (1.74), respectively. Age-specific multiplicity estimates suggested a progression from altered hepatocellular foci (AHF) to hepatocellular neoplasms. Further evidence of progression was provided by the temporal occurrence of hepatocellular adenomas before carcinomas, and the apparent origination of carcinomas within adenomas. Pulmonary metastases were observed in many of the mice with hepatocellular carcinomas. These findings confirm previous observations of strain differences in liver neoplasm response, suggest a progressive development from AHF to adenomas, and ultimately to carcinomas, and show sensitivity to VC-induced hepatocarcinogenesis in all 5 strains. PMID- 12371670 TI - Longitudinal magnetic resonance imaging quantitation of rat liver regeneration after partial hepatectomy. AB - This report demonstrates the advantages of using a noninvasive soft tissue imaging technique--magnetic resonance imaging (MRI)--to monitor liver regeneration after 70% partial hepatectomy in the rat in a longitudinal manner. Six animals were scanned prior to and on 6 subsequent occasions up to 9 days after surgical removal of the median and left lateral lobes. Within the observed time frame liver volumes were restored to approximately 88% of presurgery values. Final liver volumes correlated well with postmortem liver weights (R = 0.93). Regeneration is well-quantified empirically by a 4 parameter logistic equation: % Regeneration = 84 - (84/(1 + (Days/2.31)(2.34))) The rate of regeneration was maximal at 1.5 days, which coincided with the maximum increase of Mitotic Index- a measure of cell proliferation, determined in a subsequent study. Pre- and postpartial hepatectomy measurements remove two potentially confounding unknowns- the presurgery liver volume, and the amount of liver actually excised. 3D reconstructions of the liver effectively illustrate the morphological changes associated with the procedure, and the regrowth of liver tissue. PMID- 12371671 TI - Characterization of uterine leiomyomas in CD-1 mice following developmental exposure to diethylstilbestrol (DES). AB - Experimental animal and clinical studies have well established the association of prenatal exposure to diethylstilbestrol (DES) and the subsequent development of reproductive tract abnormalities, including poor reproductive outcome and neoplasia. Overwhelmingly, the focus has been on DES-induced epithelial lesions, particularly vaginal adenosis and adenocarcinoma; however, uterine smooth muscle cells are also recognized as cellular targets of DES. This descriptive report characterizes uterine leiomyomas that occur in outbred CD-1 mice following exposure to DES prenatally on days 9 to 16 of gestation or on neonatal days 1 to 5. These DES-induced uterine leiomyomas have typical histomorphologic, and some immunohistochemical characteristics of spontaneously occurring uterine smooth muscle tumors of B6C3F1 mice previously described in our laboratory, and they are also similar to uterine leiomyomas (fibroids) commonly observed in premenopausal women. PMID- 12371672 TI - Mismatch negativity results from bilateral asymmetric dipole sources in the frontal and temporal lobes. AB - The event-related potential (ERP) reflecting auditory change detection (mismatch negativity, MMN) registers automatic selective processing of a deviant sound with respect to a working memory template resulting from a series of standard sounds. Controversy remains whether MMN can be generated in the frontal as well as the temporal cortex. Our aim was to see if frontal as well as temporal lobe dipoles could explain MMN recorded after pitch-deviants (Pd-MMN) and duration deviants (Dd-MMN). EEG recordings were taken from 32 sites in 14 healthy subjects during a passive 3-tone oddball presented during a simple visual discrimination and an active auditory discrimination condition. Both conditions were repeated after one month. The Pd-MMN was larger, peaked earlier and correlated better between sessions than the Dd-MMN. Two dipoles in the auditory cortex and two in the frontal lobe (left cingulate and right inferior frontal cortex) were found to be similarly placed for Pd- and Dd-MMN, and were well replicated on retest. This study confirms interactions between activity generated in the frontal and auditory temporal cortices in automatic attention-like processes that resemble initial brain imaging reports of unconscious visual change detection. The lack of interference between sessions shows that the situation is likely to be sensitive to treatment or illness effects on fronto-temporal interactions involving repeated measures. PMID- 12371673 TI - Cortical potential imaging of episodic memory encoding. AB - Previous findings suggest that episodic memory encoding is subserved by different brain structures that interplay and transform experience into memories. The present study aims to identify topographic location of the underlying neural generators, and correlate their activities with the subsequent memory effect using electrophysiological neuroimaging of the event-related potentials (ERPs) recorded from 11 healthy subjects participating in word encoding tasks. Cortical potentials were imaged noninvasively from scalp ERPs. Different levels of brain activation were found in the left inferior prefrontal, left temporal and left parietal lobes with different latencies after onset of event. It is concluded that these regions work jointly across both spatial and temporal domains to promote verbal memory formation. PMID- 12371674 TI - Brain activity during syntactic and semantic processing--a magnetoencephalographic study. AB - Drawings of objects were presented in series of 54 each to 14 German speaking subjects with the tasks to indicate by button presses a) whether the grammatical gender of an object name was masculine ("der") or feminine ("die") and b) whether the depicted object was man-made or nature-made. The magnetoencephalogram (MEG) was recorded with a whole-head neuromagnetometer and task-specific patterns of brain activity were determined in the source space (Minimum Norm Estimates, MNE). A left-temporal focus of activity 150-275 ms after stimulus onset in the gender decision compared to the semantic classification task was discussed as indicating the retrieval of syntactic information, while a more expanded left hemispheric activity in the gender relative to the semantic task 300-625 ms after stimulus onset was discussed as indicating phonological encoding. A predominance of activity in the semantic task was observed over right fronto-central region 150 225 ms after stimulus-onset, suggesting that semantic and syntactic processes are prominent in this stage of lexical selection. PMID- 12371675 TI - Stimulus duration influences the dipole location shift within the auditory evoked field component N100m. AB - The equivalent source of the neuromagnetic auditory evoked field (AEF) component N100m shifts systematically within its latency range. In the current study, possible effects of stimulus duration on this shift were analysed. 15 subjects were stimulated monaurally with tones of different duration (50, 100, 200 ms) and AEFs were recorded successively over both hemispheres. Dipoles were calculated in 5-ms-steps from 15 ms before to 15 ms after the N100m peak maximum. A dipole location shift within the N100m latency from posterior to anterior and from superior to inferior was observed. The shift in anterior-posterior direction was found to be larger in the right compared to the left hemisphere. Stimulus duration significantly affected the degree of dipole shift in this direction. It was found to be shorter the shorter the stimulus. PMID- 12371676 TI - Scalp topography of the spontaneous K-complex and of delta-waves in human sleep. AB - OBJECTIVE: Together with spindles, K-complexes are well known hallmarks of stage 2 sleep (S2). However, little is known about their topographical distribution in comparison to delta-waves and to K-complexes superimposed by spindles. PATIENTS AND METHODS: In this study, the topographical distribution of spontaneous K complexes and delta-waves in S2 and delta-waves in stage 4 sleep (S4) in 10 healthy young adults (aged 20 to 35 years, 7 female) was investigated. K complexes with and without spindles in S2, delta-waves with and without spindles in S2, and delta-waves in S4 distributed all over the night were visually selected. EEG power maps and statistical parametric maps were calculated. RESULTS: Absolute delta power of S2 K-complexes appeared to be significantly higher than of S2 delta-waves and delta power of S4 delta-waves was higher than of S2 delta-waves. In K-complexes and delta-waves, power was found to be highest over medio-frontal regions in the delta frequency band (0.5-4.0 Hz) with a second maximum occipitally in delta-waves, no matter whether superimposed by a spindle or not. CONCLUSION: K-complexes and delta-waves in S2 differ in topographical distribution. Even though in S2 delta-waves have less power, they have a similar topographical distribution in S2 and S4, supporting the hypothesis that delta waves in S2 further develop towards delta-waves in slow wave sleep. The delta frequency components of K-complexes and delta-waves are unaffected by spindles. PMID- 12371677 TI - Generators of visual evoked potentials for faces and eyes in the human brain as determined by dipole localization. AB - Human visual evoked potentials were recorded during presentation of photos of human and animal faces and various face features. Negative waves with approximate peak latencies of 165 msec (N170) were bilaterally recorded from the occipito temporal regions. Mean peak latencies of the N170 were shorter for faces than eyes only. Analyses of amplitudes of evoked potentials indicated that the N170 elicited by faces reflected activity of a specific neural system which was insensitive to detailed differences among individual faces regardless of species, and consequently suggest that this system might function to detect existence of faces in general. On the other hand, the mean amplitude of the N170 elicited by human eyes was significantly larger than those by animal eyes. These differences in response latencies and amplitudes of the N170 suggest existence of at least 2 different visual evoked potentials with similar latencies (i.e., N170) which are sensitive to faces in general and human eyes, respectively. Dipole source localization analysis indicated that dipoles for the N170 elicited by eyes were located in the posterior inferior temporal gyrus, and those for faces, located initially in the same region, but moved toward the fusiform and lingual gyri at the late phase of the N170. The results indicated that information processing of faces and eyes separated at least as early as the latency of the N170 at the posterior inferior temporal gyrus as well as the fusiform and lingual gyri, and might provide neurophysiological and anatomical bases to an initial structural encoding stage of human faces. PMID- 12371678 TI - Genetic structure of the Ards Peninsula, Northern Ireland: evidence from civil registers of marriage 1840-1911. AB - This paper uses marital migration data transcribed from the Civil Registers of Marriage 1840-1911 to estimate kinship from migration matrices and isonymy in the Ards Peninsula, Northern Ireland. The distribution of religious denominations (Presbyterian, Episcopalian, and Roman Catholic) varies systematically throughout the region, with up to 77% Roman Catholic in the south and 81% Presbyterian in the north. Portavogie, a fishing village on the east coast, is exclusively Protestant, with a population 93% Presbyterian. Comparison of migration and isonymy with geographical distance by multidimensional scaling and the MATFIT procedure show Portavogie to be an outlier, more distantly related to other areas than its geographical position would predict. We suggest that this discrepancy is due to settlement history and occupational and religious isolation. Mantel tests show that marital migration is significantly related to geographical distance (rMG = 0.4257), as is the distribution of religious denominations (rRG = 0.5548) through settlement history. Migration is dependent on religion (rMR = 0.3674), and isonymy is dependent on migration (rIM= 0.2531) but not on geography or religion. With Portavogie omitted from the analysis, the dependence of migration on geography and on religion increases (rMG = 0.5583, rMR = 0.5646), as does the correlation between religion and geography (rRG = 0.7213). The dependence of isonymy on migration increases (rIM = 0.5103), and significant correlations between isonymy and religion (rIR = 0.4135) and isonymy and geography (rIG = 0.4660) appear. We argue that a full explanation of population structure requires geographical distance, settlement history, and the influence of religion and occupation to be taken into account. PMID- 12371679 TI - Absence of selection on birth weight in modern Italy. AB - The effect of stabilizing and directional selection on birth weight has been analyzed for Italian births from 1954 to 1994, a period of rapid improvement in environmental conditions. The population of newborns was subdivided according to gestational age, one of the main covariates of birth weight. In the last cohort of births, no selection at all (neither stabilizing nor directional) was found in full-term babies, which represent more than 95% of total deliveries. Preterm babies are still selected against, even if at lower rates than in the past. It can therefore be claimed that improved and widely available prenatal and neonatal care has dramatically changed the selection patterns previously associated with birth weight in the majority of the Italian population. The mortality rates associated with birth-weight variations lying in a wide interval (2.5 kg-4.5 kg) are nowadays very similar, and both stabilizing and directional selection have practically disappeared. PMID- 12371680 TI - Ethnicity and type 2 diabetes in Rio de Janeiro, Brazil, with a review of the prevalence of the disease in Amerindians. AB - To what extent can ethnic factors contribute to the prevalence of type 2 diabetes and impaired glucose tolerance (IGT) in an urban Brazilian population? Conversely, how can environmental factors such as diet change these prevalences in a given ethnic group, in this case Brazilian Indians? To answer these questions estimates of ethnic admixture in Afro- and Euro-Brazilians from Rio de Janeiro, Brazil, were established using eight genetic systems and compared with the prevalences of these conditions obtained previously. This information was integrated with results obtained inside and outside of Brazil. The similarity of prevalences for type 2 diabetes and IGT in Afro- and Euro-Brazilians may be related to the extensive gene flow that occurred between them and to similar socioeconomic levels in the samples investigated. On the other hand, changes in the traditional diet are probably conditioning the appearance of diabetes among Brazilian and other South American Indians. PMID- 12371681 TI - Apolipoprotein H genetic variability in the population of Krk Island, Croatia. AB - Apolipoprotein polymorphisms are emerging as suitable markers for the study of the formation of human populations. In contrast to the data available for apolipoprotein E, the data regarding apolipoprotein H (protein, apoH; gene, APOH) variations are only beginning to accumulate. By blood plasma isoelectric focusing and immunoblotting, we analyzed the distribution of apoH phenotypes in 397 individuals (192 males; 205 females) from seven villages of an autochthonous population of the eastern Adriatic island of Krk. APOH allele frequencies were: APOH*2 = 0.877, APOH*3 = 0.098, APOH*1 = 0.025, with the majority of the sample being homozygous. No significant differences between villages were observed. When these data were compared to those of other populations studied so far, a significant association between APOH allele frequencies and latitude was observed. We hypothesize that this association reflects differences in diet composition across different climatic zones. PMID- 12371682 TI - Diversity at eight polymorphic Alu insertion loci in Chinese populations shows evidence for European admixture in an ethnic minority population from northwest China. AB - We have analyzed eight human-specific Alu insertion polymorphisms in four Chinese populations belonging to three ethnic groups (98 Hans from Shanghai, 80 Hans from Guangzhou, 85 Uyghurs, and 60 Sibos). All populations exhibited high levels of average heterozygosity, and those in Uyghur and Sibo were higher than predicted by the island model of population structure. The degree of genetic differentiation among these populations is statistically significant, and lower than those observed in most parts of the world except for Europe and Sahul (Australia and New Guinea). Phylogenetic analysis of these data with published data from 29 worldwide populations shows that there is a close genetic affinity among all the East Asian populations except for the Uyghur, and that the Uyghur population was found to lie between the East Asian and the West Asian populations on the population tree. The greater heterozygosity and the significant genotype associations between unlinked loci observed for the Uyghurs support the scenario that the Uyghurs might have originated from an admixture between Europeans and East Asians. This study also provides further support for the "out-of-Africa" hypothesis of modern human evolution in East Asia. PMID- 12371683 TI - Functional and postural lateral preferences in humans: interrelations and life span age differences. AB - This study aimed to provide data on lateral preferences among older subjects, to analyze age differences, and to determine interrelations between lateral preferences. Four functional preferences (handedness, footedness, eyedness, earedness) and three postural lateral preferences (hand-clasping, arm-folding, leg-crossing) were assessed in 628 Germans (252 men, 376 women) aged between 19 and 90 years. Sex differences, age differences, and associations between lateralities were analyzed applying chi-square tests. Logistic regression analyses considering age, sex, and interactions between variables were applied to analyze combined effects on laterality measures. Right-sided preference for handedness, footedness, eyedness, earedness, and leg-crossing characterized 86.8%, 77.1%, 70.9%, 67.8%, and 56.6%, respectively, of subjects, while a left sided preference for hand-clasping and arm-folding characterized 56.4% and 60.2%, respectively, of all participants. Results are within the range of other populations. Only footedness differed between the sexes: there were more left footed men. Older cohorts showed a rightward shift in handedness, eyedness, earedness, and leg-crossing, the opposite for arm-folding. No age-related differences exist in footedness or hand-clasping. Logistic regression models indicate no interaction between age and sex for each laterality measure. The four functional lateralities are significantly interrelated. All also are positively associated with leg-crossing. Conversely, the postural lateralities generally are not correlated, although leg-crossing and arm-folding are, inversely. The observed relationships among lateralities support the hypothesis that handedness, footedness, leg-crossing, and earedness might be aspects of a larger phenotype that is independent of hand-clasping and arm-folding. PMID- 12371684 TI - Inbreeding, isonymy, and kin-structured migration in the principality of Andorra. AB - Andorra, one of the smallest countries in Europe, is geographically very isolated. Located in the Central Pyrenees, it is surrounded by high mountains. This paper investigates the endogamous levels and relationships between demographic or geographical variation and inbreeding coefficients calculated through isonymic methods. Our results suggest that political and geographical frontiers are not significant enough to pose effective genetic barriers. The overall inbreeding coefficient average (0.0031) is moderate with respect to other populations in the same region. Temporal and geographical variations of total inbreeding and their components are explained in relation to changes in population size and intensity, and to origin and destination of migrant collectives. Although the spatial distribution of the population of Andorra is the main contributing factor to inbreeding, the kin-structured composition of immigrant collectives is another fundamental factor that helps to explain the levels and variation of inbreeding. PMID- 12371685 TI - Short tandem repeat polymorphism of the D2S1242 locus in a Japanese population. AB - Allele frequencies and sequence characteristics of the D2S1242 short tandem repeat (STR) locus were studied in a Japanese population sample. A total of 10 D2S1242 alleles and 34 genotypes were identified in 273 unrelated Japanese individuals. The five most common alleles detected had frequencies of over 10%. No deviations from Hardy-Weinberg equilibrium were found when the expected allele values were compared with the observed values. Sequence analysis of each allele showed a tetranucleotide polymorphism. Alleles 9 to 14 had different sequence structures than alleles 15 to 19. Allele 18 had a different sequence in the Japanese sample compared to an Austrian sample. The power of discrimination was 0.95. The present results demonstrate that the D2S1242 STR locus is a useful genetic marker in the Japanese population. PMID- 12371686 TI - Ethnic admixture composition of two western Amazonian populations. AB - A small riverine community, Portuchuelo (8 degrees 37'S, 63 degrees 49'W), and a rural county, Monte Negro (10 degrees 15'S, 63 degrees 18'W), both in the state of Rondjnia, Brazil, were studied for the purposes of ascertaining health conditions and the causes of the variability of some infectious diseases. The sample included 181 inhabitants of Portuchuelo and 924 of Monte Negro. Data on 11 blood polymorphisms (ABO, Rh, MNSs, Kell, Fy, haptoglobin, hemoglobin, ACP1, PGM1, GLO1, and CA2) were used to determine the ethnic composition of the inhabitants of Portuchuelo and Monte Negro. The contributions of Africans, Amerindians, and Europeans to the ethnic composition of the studied populations were, respectively, 0.21 +/- 0.046, 0.44 +/- 0.064, and 0.35 +/- 0.069 in Portuchuelo; and 0.25 +/- 0.032,0.12 +/- 0.046, and 0.63 +/- 0.054 in Monte Negro. PMID- 12371687 TI - Birth order and its association with the onset of chronic fatigue syndrome. AB - Chronic fatigue syndrome (CFS) is a medically unexplained illness that is diagnosed on the basis of a clinical case definition; so it probably is an illness with multiple causes producing the same clinical picture. One way of dealing with this heterogeneity is to stratify patients based on illness onset. We hypothesized that either the whole group of CFS patients or that group which developed CFS gradually would show a relation with birth order, while patients who developed CFS suddenly, probably due to a viral illness, would not show such a relation. We hypothesized the birth order effect in the gradual onset group because those patients have more psychological problems, and birth order effects have been shown for psychological characteristics. We compared birth order in our CFS patients to that in a comparison group derived from U.S. demographic data. We found a tendency that did not reach formal statistical significance for a birth order effect in the gradual onset group, but not in either the sudden onset or combined total group. However, the birth order effect we found was due to relatively increased rates of CFS in second-born children; prior birth order studies of personality characteristics have found such effects to be skewed toward first-born children. Thus, our data do support a birth order effect in a subset of patients with CFS. The results of this study should encourage a larger multicenter study to further explore and understand this relation. PMID- 12371688 TI - Competition Commission points to complex monopolies in the supply of POMs. PMID- 12371689 TI - Response of pheasants to live attenuated turkey rhinotracheitis vaccine. AB - The entire crop of 18,120 pheasants for the 2000 rearing season (May 8 to August 7) of one estate in the south of England was vaccinated at one day and five weeks of age with a turkey rhinotracheitis (TRT) vaccine. Blood samples and oropharyngeal swabs were taken from the second week's hatching every three weeks throughout the growing season to assess the response of the birds. There was evidence of seroconversion in samples collected three weeks after vaccination, with positive titres being maintained in 33 per cent or more of the population up to at least 22 weeks of age. Positive titres were also recorded in samples taken on December 6 from shot birds between 22 and 30 weeks of age. Positive titres to infectious bronchitis virus (IBV) were identified in a high proportion of the poults as early as one day of age. Reverse-transcriptase PCR detected IBV-like virus and TRT of the same subtype as the TRT vaccine administered three weeks previously. PMID- 12371690 TI - Effects of different us isolates of porcine reproductive and respiratory syndrome virus (PRRSV) on blood and bone marrow parameters of experimentally infected pigs. AB - Seventy five-week-old, crossbred, caesarean-derived, colostrum-deprived pigs were randomly divided into five groups of 14 pigs and assigned one of five treatments: the intranasal inoculation of 1 (5.7) TCID50 of one of four plaque-purified isolates of porcine reproductive and respiratory syndrome virus (PRRSV) (VR2385, VR2431, ISU-984 and ISU-22), or uninfected cell culture and media. Haematological variables were measured for 21 days and bone marrow was analysed when the pigs were killed three, seven, 10, 21 or 28 days after the inoculation. The PRRSV infected pigs had non-regenerative anaemia and markedly increased myeloid:erythroid ratios from three to 21 days after inoculation. There was a significant (P < 0.05) difference in the severity of the anaemia induced by the four PRRSV isolates; the most highly pneumovirulent strains (VR2385, ISU-984 and ISU-22) induced more severe anaemia than the least virulent isolate (VR2431). The anaemia induced by PRRSV was probably due to a direct or indirect effect on erythroid precursor cells in the bone marrow. PMID- 12371691 TI - Effects of grazing undrenched weaner deer on chicory or perennial ryegrass/white clover pasture on the viability of gastrointestinal nematodes and lungworms. AB - This study determined the in vitro effects on the viability of internal parasites of grazing undrenched weaner deer on either chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. One experiment investigated the hatching and development of gastrointestinal nematode eggs and larvae, and the development and motility of L1 lungworm (Dictyocaulus eckerti) larvae, and a second experiment used larval migration inhibition assays to test the viability of L1 lungworm larvae extracted from the faeces of weaner deer grazed on either chicory or pasture when they were incubated with rumen and abomasal fluids from fistulated deer also grazing on chicory or pasture. The incubations were undertaken with and without added condensed tannins purified from chicory and with or without polyethylene glycol (PEG) to bind the tannins. Chicory had no effect on the hatching and development of gastrointestinal nematode eggs and larvae. Grazing chicory reduced the number of lungworm larvae developing to the L3 stage, and L1 lungworm larvae from the faeces of chicory grazed deer were less viable in rumen and abomasal fluid than larvae from pasture grazed animals. Abomasal fluid was significantly (P < 0.001) less inhibitory to the migration of L1 lungworms than rumen fluid. When the larvae were incubated in rumen and abomasal fluids from chicory-grazed deer, their passage through sieves was significantly (P < 0.001) reduced in comparison with when they were incubated in the fluids from pasture-grazed deer Adding condensed tannins to rumen fluid increased the inhibition of the migration of L1 lungworm larvae but PEG removed this inhibition; this effect was not observed with abomasal fluid. PMID- 12371692 TI - Teladorsagiosis in young lambs and extended postparturient susceptibility in moxidectin-treated ewes grazing heavily contaminated pastures. PMID- 12371693 TI - Incidence and antibiotic resistance of pathogenic Escherichia coli among poultry in Belgium. PMID- 12371694 TI - Closed reduction of an atlanto-occipital and atlantoaxial dislocation in a foal. PMID- 12371695 TI - Abattoir study of maedi-visna virus infection in Turkey. PMID- 12371696 TI - RCVS disciplinary hearings. PMID- 12371697 TI - RCVS disciplinary hearings. PMID- 12371698 TI - BVA governance. PMID- 12371699 TI - Future of the RCVS library. PMID- 12371700 TI - Future of the RCVS library. PMID- 12371701 TI - Hospital standards. PMID- 12371702 TI - English for peptide science. PMID- 12371703 TI - Solution structure of nociceptin peptides. AB - Peptides embedded in the sequence of pre-pro-nociceptin, i.e. nociceptin, nocistatin and orphanin FQ2, have shed light on the complexity of the mechanisms involving the peptide hormones related to pain and have opened up new perspectives for the clinical treatment of pain. The design of new ligands with high selectivity and bioavailability, in particular for ORL1, is important both for the elucidation and control of the physiological role of the receptor and for their therapeutic importance. The failure to obtain agonists and antagonists when using, for nociceptin, the same substitutions that are successful for opioids, and the conformational flexibility of them all, justify systematic efforts to study the solution conformation under conditions as close as possible to their natural environment. Structural studies of linear peptides in solution are hampered by their high flexibility. A direct structural study of the complex between a peptide and its receptor would overcome this difficulty, but such a study is not easy since opioid receptors are membrane proteins. Thus, conformational studies of lead peptides in solution are still important for drug design. This review deals with conformational studies of natural pre-nociceptin peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yielded a completely reliable bioactive conformation, but the global conformation of the peptides in biomimetic environments can shed light on their interaction with receptors. PMID- 12371704 TI - Synthesis and study of a gramicidin B mutant possessing a ditryptophan crosslink. AB - Recent studies of peptide dimers linked by Trp-Trp (ditryptophan) crosslinks suggest that the crosslinks can reinforce antiparallel beta-structure. Depending on environment, gramicidins A, B and C form either helical ion channels with parallel beta-structure or non-functional pores with antiparallel beta-structure. In the channel conformation of the gramicidins Trp9 and Trp15 are close in space, but in the pore conformation Trp9 and Trp15 are far apart. We hypothesized that a ditryptophan crosslink between Trp9 and Trp15 could pre-organize gramicidin in an active conformation. To test the potential for preorganization, an intramolecular ditryptophan crosslink was formed between Trp9 and Trp15 in a W13F mutant of gramicidin B. Photooxidative conditions were shown to generate ditryptophan crosslinks in low yields. While not preparatively useful, photooxidative tryptophan crosslinking may have implications for protein aging processes like cataract formation. The ditryptophan crosslink in the gramicidin B mutant substantially lowered the antibiotic activity of the gramicidin B mutant, unlike the ditryptophan crosslink in the antibiotic X-indolicidin. The biaryl chromophore generated diagnostic Cotton effects in the CD spectrum that revealed the absolute stereochemistry of the biaryl chromophore, but the biaryl chromophore obscured diagnostic features below 220 nm. However, changes in peptide conformation were reflected in changes in the biaryl region of the CD spectrum above 240 nm. PMID- 12371705 TI - Trypsin-catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, Streptomyces griseus and chum salmon). AB - A procedure has been developed for the synthesis of oligopeptide amide using inverse substrates as acyl donors with amino acid amide instead of p-nitroanilide as acyl acceptor and trypsins of different origin (bovine, Streptomyces griseus and chum salmon trypsins) as the catalyst. The effectiveness of this procedure was demonstrated by the synthesis of a pentapeptide, Boc-[Leu5]-enkephalin amide, as a model compound. The method was the first enzymatic method shown to be successful at each successive coupling step for the synthesis of the oligopeptide. Bovine and chum salmon trypsins were superior to Streptomyces griseus trypsin as the catalyst. PMID- 12371706 TI - Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays. AB - Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol bound peptides in biochemical binding and functional assay systems is demonstrated. PMID- 12371707 TI - On lung nerves and neurogenic injury. AB - Information accumulated in recent years has begun to unveil a previously unsuspected complexity in the innervation of the lungs. We know now that the conducting airways receive a highly redundant supply of vagal motor and sensory fibers; that many of these fibers cross over from the contralateral side of the brain to reach distant portions of the lung, thereby assuring the symmetry and simultaneity of the bronchomotor responses; and that, perhaps in recognition of the different functions and properties of proximal and distal airways, vagal motor fibers have a distinctive segmental distribution. Both sensory and motor neurons serve as the input and output elements of a complex brain stem neuronal network, which integrates the regulation of airway smooth muscle tone into the control of ventilation. This network has a local counterpart in the airway walls, where a heterogeneous population of intrinsic neurons may act not only as a relay for cholinergic stimuli, but also as a local mechanism of inflammatory modulation. The interruption of the nerve supply to the lungs (for instance after lung transplantation) abolishes the integration of bronchomotor and ventilatory activities, and, by increasing airway deformation, may initiate fibroproliferative responses in the airway walls. In addition, the destruction of vagal motor and sensory fibers leaves behind a surviving population of denervated intrinsic neurons, which may act as a disregulated mechanism of inflammatory amplification. PMID- 12371708 TI - Telemedical devices in diabetes management. AB - This paper reviews telemedicine and its recent expansions within diabetes management. Diabetes mellitus continues to be one of the major chronic diseases with up to 11% of national health care expenditure, when all late complications are taken into account. Over and above this, the incidence of diabetes is increasing in pandemic fashion. Diabetes has been the focus of telemedicine and information technology over the past two decades. Useful applications supporting high quality treatment exist in clinical management, education, decision support and modelling. Development of high-speed networks enables transmission of good quality photographs making consultations from distant locations possible. Databases and data analysis are fundamental to diabetes outcome research. Less successful has been the development of electronic medical records although this is a dream of many. Evidence of improved clinical outcome using telemedical applications still awaits the breakthrough. PMID- 12371709 TI - Molecular actions of drugs that sensitize cardiac myofilaments to Ca2+. AB - Ca(2+)-sensitizers are inotropic agents that modify the response of myofilaments to Ca2+, and are potentially valuable drugs in the treatment of heart failure. These agents have diverse chemical structures, and in some cases also have effects as inhibitors of phosphodiesterase activity. Advantages of their actions include vasodilation combined with inotropic effects. Reduction in the amounts of Ca2+ required to activate the myofilaments also lowers the oxygen consumption required for Ca2+ transport, lowers the threat of arrhythmias, and may blunt Ca(2+)-dependent transcriptional and translational mechanisms leading to hypertrophy and failure. Although diastolic abnormalities and impaired relaxation were thought to be potential undesirable effects of Ca(2+)-sensitizers, studies of hearts beating in situ indicate that this may not be a major problem. We focus here on Ca(2+)-sensitizers that act on cardiac troponin C, the Ca2+ receptor that triggers activation of the actin-myosin interaction. Structural studies have identified a unique mode of Ca2+ signaling in cardiac troponin C that should aid in targeting drugs to the heart. Moreover, identification of docking sites of Ca(2+)-sensitizers on troponin C suggest new directions for rational drug design. PMID- 12371711 TI - Effects of preventive group education on the resistance of LDL against oxidation and risk factors for coronary heart disease in bypass surgery patients. AB - BACKGROUND: Comprehensive preventive education for heart patients is effective in reducing cardiac events. However, very demanding counselling protocols cannot easily be implemented as an integral part of clinical practice in hospitals. AIM: To evaluate whether recurrent preventive group education for coronary artery bypass grafting patients affects the resistance of LDL against oxidation and the classical risk factors for coronary heart disease. METHODS: A prospective, controlled study with one-year follow-up was carried out in Southern Finland. Coronary artery bypass patients were allocated late (> or = 18 months) after the operation in the intervention and control groups. RESULTS: Seventy two patients (65 men and 7 women) completed the study. Twelve-hour group education on healthy life-style had a significant (25%, P = 0.01) but transient positive effect on the resistance of LDL against oxidation. There was a trend towards increased physical activity in the intervention group. The impact of education on patients' weight was also more consistent (1.2 kg at 12 months, P < 0.05), whereas long-term effects on fibrinogen and serum lipids were small. CONCLUSIONS: Repeated group education applied as an economically feasible part of specialist care had only small positive effects on patients' risk factors. There was a significant, but transient, increase in the resistance of LDL against oxidation. However, effective lipid lowering drug treatment is indicated for most coronary artery bypass patients rather than repeated health education alone. PMID- 12371710 TI - Disordered proteins in dementia. AB - Aggregates of dysfunctional proteins and peptides in or between brain neurons are key neuropathological features of dementia and are believed to directly cause or substantially contribute to the development of these diseases. Fundamental parts of the mechanisms underlying the dysregulation of proteins in Alzheimer's disease, frontotemporal dementia, prion diseases and other dementing disorders are now well characterized, mainly due to the discovery of genes causing dominantly inherited disease forms (Table 1). As of today, no efficient pharmacotherapies are available, but new insights into the underlying molecular mechanisms are providing strategies to prevent or even cure these devastating disorders. PMID- 12371712 TI - Mathematics-assisted mapping in analysis of medical disease. AB - Genetic mapping in analysis of medical disease is performed under several assumptions and (experimental) conditions, which are made about the data in general and the disease in particular. Here we discuss these conditions, what they mean, and what kind of deleterious effects they might have on the analysis. We also illustrate how to proceed and what kind of possibilities the statistical analysis may provide to medical scientists. PMID- 12371713 TI - In silico approaches to microarray-based disease classification and gene function discovery. AB - The automated analysis of transcriptional profiling data promises a wealth of information that may be used to develop a more complete understanding of gene function and interactions. Moreover, it may improve the effectiveness of complex diagnostic tasks. This article discusses important data mining and management techniques to analyse genome-wide expression data. It reviews some of the major discovery goals, methods and applications in a number of biomedical domains. Finally, this paper highlights key problems that need to be approached by a new generation of computational solutions. PMID- 12371714 TI - Bioinformatics: from genome to drug targets. AB - The complete sequence determination of the human genome marks the start of a new era in biological science, with focus shifting from sequencing to functional mechanisms of gene products. In addition to effects on gene expression, most of the currently used therapeutic drugs either have enzymes or membrane proteins as their molecular targets of action. These membrane proteins include ion channels and transporters of small molecules, and receptors that convey signals from one side of a membrane to the other. Membrane proteins are thus involved in a variety of cellular processes and have a large potential as targets for new drug discovery. However, detailed structural information is still lacking for the majority of membrane proteins since their association with membrane constituents make NMR (nuclear magnetic resonance) spectroscopic and X-ray diffraction determinations difficult. Molecular modelling by biocomputing is a methodological alternative for structural studies of membrane proteins, but has to be based on experimental structural information in addition to computational techniques. A combination of bioinformatics and experimental techniques was used to model membrane proteins from two different classes, secondary transporters of the sodium:neurotransmitter symporter family (SNF transporters), and G-protein coupled receptors (GPCRs). The protein models were used to examine ligand-protein interactions and signalling/transport mechanisms, and to design experimental site directed mutagenesis studies. Such studies have provided new insight into the detailed molecular mechanisms of two important classes of membrane proteins, which may be of value in the discovery and development of new pharmaceuticals. PMID- 12371715 TI - Importance of increased ultrafiltration volume and impact on mortality: sepsis and cytokine story and the role for CVVH. AB - There is growing interest in extracorporeal blood purification therapies (EBPT) as adjuvants in the complex therapy of sepsis and multiple organ dysfunction syndrome (MODS). Nowadays the only routinely used purification technique is 'renal replacement therapy' (RRT) during acute renal failure (ARF), one of the almost inevitable and deadly components of MODS. RRT has been the first and still is the most utilised and effective type of EBPT. Evidence is growing about its ability to maintain homeostatic balance in critically ill patients, and specifically in septic patients with MODS. Clinical trials have been recently designed to modify or improve these therapies. In detail, the following issues have been currently addressed: effects on blood purification provided by different therapies, adequacy of prescription and delivery of therapy, toxins and solutes to be removed with these techniques. Based on these speculations we will briefly review the current understanding of these issues and the rationale for application of RRT in the intensive care unit (ICU). In particular, we will focus on the importance of increased ultrafiltration volume and its impact on mortality in the general ICU population and in septic patients. PMID- 12371716 TI - Continuous renal replacement therapies. AB - Continuous Renal Replacement Therapy (CRRT) is frequently used in patients admitted to intensive care units with multiple organ failure and acute renal failure. These patients are prone to developing hypotension making it very difficult to use conventional haemodialysis for their treatment. When compared to conventional haemodialysis CRRT has obvious clinical advantages. These advantages are mostly due to slow volume and uraemic toxin removal leading to better haemodynamic tolerability for such patients. In our unit during the year 2000, 58 patients were submitted to CRRT: 14 of the patients underwent treatment with continuous veno-venous haemofiltration and 44 were submitted to continuous veno venous haemodiafiltration. The mean patient age was 61.7 years (range: 20-87), 36 male and 22 females. Twenty patients (43.1%) had sepsis, 18 (31%) were post open heart surgery, 7 (12%) had multiple organ failure, 4 (6.9%) were polytraumatised, 3 (5.2%) were post neurosurgery and 1 (1.8%) was a liver transplant patient. Despite the grave prognosis of these patients, 22 (37.8%) survived and 36 (62.2%) died. Of the patients that survived, 10 (17.2%) recovered renal function and 12 (20.6%) remained on a regular haemodialysis programme. The authors conclude that CRRT seems to be an alternative to conventional haemodialysis for the treatment of those patients with acute renal failure. PMID- 12371717 TI - Vascular access in acute renal failure. AB - Today, central venous access catheters play an important role in the treatment and management of many dialysis patients. Their use and care may influence the patient's overall outcome. Therefore, it is critical to have a thorough knowledge of the vascular anatomy, types of catheters, placement techniques and maintenance and management of complications. The incidence nowadays of acute renal failure in intensive care patients is reported as high as 25%. Acute renal failure is one of the few causes of organ failure in which complete recovery is possible, provided the patient survives the associated comorbid conditions. There are various extracorporeal dialysis techniques available to treat this category of patients using mostly a central dialysis catheter as vascular access. It is important to select carefully the type of catheter and to create a specific vascular access system in order to be able to perform the selected dialysis technique in the most optimal efficient conditions. Any inadequacies of access will create dialysis insufficiency leading to more comorbid conditions and even higher mortality. In this article, we will describe the different possibilities as well as the nursing management of that type of vascular access in patients with acute renal failure. PMID- 12371718 TI - Acute renal failure in childhood. AB - The definition of acute renal failure is a sudden reduction in renal function of at least 50 percent and is characterised by rising serum levels of waste products such as urea and creatinine, by disturbed water and electrolyte metabolism and changes in the amount or composition of the urine. The clinical manifestations are extremely variable, there are very many causes and the outcome depends mainly on the underlying condition, which is either in the kidneys or in the body as a whole. This paper will discuss the pathophysiology of acute renal failure and the aetiology including prerenal, renal and postrenal causes. Clinical findings, diagnosis and treatment will be discussed and complications of this life threatening condition highlighted. The final part of the paper deals with the prognosis of acute renal failure. PMID- 12371720 TI - Early management and prevention of acute renal failure. AB - Despite major advances in nutritional support, membrane technology and dialytic techniques, the mortality of patients with acute renal failure (ARF) who require dialysis is still almost 50% (1). Increased patient age and co-morbidity confer a poorer prognosis, and the condition is certainly commoner in this patient group. Hence, one study showed that the age-related annual incidence of ARF increased from 17 per million in adults under 50 years to 949 per million in the 80-89 age group (2). Over 60% of cases of ARF ultimately result from renal hypoperfusion and consequent intra-renal ischaemic damage, which leads to acute tubular necrosis (ATN) (3). Ischaemic ARF may thus result from a diversity of systemic and intra-renal circulatory stresses including acute losses of blood and extra cellular fluids, from low cardiac output states such as following ischaemic or toxic myocardial damage, and even from drug-induced renal perfusion shutdown (ACE inhibitors, non-steroidal anti-inflammatory agents). Many cases of ARF have a multi-factorial aetiology (e.g. post-surgical sepsis with hypovolaemia, hypotension and injudicious antibiotic use), and these patients, who often have other organ failure, fit into the poorer prognostic category. A large number of patients with ischaemic ARF pass through a phase of potentially reversible pre renal oliguria; early recognition and prompt, appropriate treatment of these pre renal factors can prevent progression to established ARF, with the genuine prospect of improved patient morbidity and mortality, and this is the main scope of this article. Early diagnosis in other patients with ARF, such as those with acute inflammatory renal disease (e.g. vasculitis) or urinary tract obstruction, will allow appropriate prompt treatment and the possibility for reversal of the ARF. The following account, which is composed of personal experience, that of colleagues, and the literature (1,4), is not intended to provide a comprehensive guide to the management of ARF, but seeks to highlight important common pitfalls and fundamental principles in the recognition and subsequent preventive treatment of these patients. PMID- 12371719 TI - Anticoagulation therapy in acute renal failure extracorporeal treated patients. AB - In extracorporeal techniques, as used for Acute Renal Failure (ARF) treatments, blood is constantly exposed to foreign surfaces. These foreign surfaces include the catheter(s), blood tubings, the dialyser membrane and all artificial materials used for such techniques in general. Blood begins to clot as soon as it strikes a foreign surface. The foreign surface initiates the clotting process. The extracorporeal circuit is prone to clotting during acute treatments unless some form of anticoagulation is employed. On the other hand, this specific group of patients is often at increased risk of bleeding (cfr. Post-operative patients and post-renal-transplant patients, patients with Multiple Organ Failure (MOF) often including liver disturbances). It should be kept in mind that patients with renal failure have a defect in platelet function resulting in altered platelet vessel wall interaction. Over time, as the extracorporeal treatment progresses, the platelets become more adhesive or sticky. The blood becomes more likely to clot. Performing effective therapy with low bleeding risk in ARF patients is a challenge requiring knowledge, skills and experience. PMID- 12371721 TI - Ethical aspects of withdrawing/withholding renal replacement therapies on patients in acute renal failure in an intensive care unit. AB - The majority of patients being treated for acute renal failure in intensive care units have multiple medical problems. Accordingly, the withdrawal of renal replacement therapies should be considered as part of a general decision about whether to initiate or continue with treatment per se. Several guidelines on withdrawing and withholding therapy have been produced and some common themes emerge: concerns to avoid euthanasia, potential for benefit, patient consent (shared decision-making), team consensus/decision-making, and the provision of appropriate palliative care and resource implications. Each of these is considered in turn, although the word limit for this paper does not permit detailed exposition. PMID- 12371722 TI - Cardiovascular outcome in critically ill patients treated with continuous haemofiltration--beneficial effects of bicarbonate-buffered replacement fluids. PMID- 12371723 TI - Who should manage continuous renal replacement in the intensive care setting? A nursing viewpoint. AB - Since its inception, continuous renal replacement therapy (CRRT) has been performed in critical care units with or without the involvement of nephrology nursing support (1,2). It is apparent that the issue of providing care to patients requiring this therapy is not so much a debate on the nursing control of CRRT, but a focused discussion on the nursing management and delivery of care to the patient receiving CRRT in the intensive care setting. Although the choice of a nursing care model for CRRT is dependent on many clinical and organisational factors, the use of one nursing specialty to deliver CRRT care can leave gaps in practice. The Joint or Collaborative Nephrology/critical care nursing model brings the highest level of nursing expertise to the bedside. The joint model tends to promote collaboration between two distinct nursing specialties, with opportunities for setting joint standards and promoting research. With this in mind, this discussion will examine some of the factors affecting structuring of nursing care, describe nursing models currently in use, compare the attributes of each, and conclude which model is preferred for the delivery of nursing care for CRRT. PMID- 12371724 TI - Nursing care plan for renal patients following the Krohwinkel model. PMID- 12371725 TI - Nutritional aspects of acute renal failure. AB - The patient with acute renal failure is a very ill patient suffering from high urea levels causing poor appetite, nausea and vomiting. These patients are usually treated with a low sodium, low protein and, if the potassium in the blood is high, with a low potassium diet (1). This paper discusses whether or not this is the correct treatment. The symptoms of high urea levels in the blood together with increased needs for energy and protein makes it very hard to prevent the patient becoming malnourished. Often energy-enriched drinks are necessary to achieve recommendations and it is prudent to let the patient eat and drink what they desire. By calculating the energy and protein needs and comparing these with the intake and the state of illness and by following the patient's body weight over time we can obtain information about the state of nourishment. When we alter the food that's offered we achieve better intake and reduce the risk of malnourishment. PMID- 12371726 TI - The effects of acute renal failure on the family. AB - This paper will explore the effects of Acute Renal Failure (ARF) on the family of the patient. The patient's needs are the concern of the health care team and are more medically orientated than those of the family, whose needs are prevailingly social ones. However, the family's reactions do affect the patient and his/her response to their illness. The diagnosis of any major illness is devastating for the patient and his/her family, and in discussing ARF and its effects, it is possible to realise the common needs of the relatives of all acutely sick people. There is little written about the family's needs when ARF is diagnosed because of the commonalities with the diagnosis of any sudden critical illness. However it is in the relationships between the families and the specialist renal nurse where the differences lie. The partnership which develops in chronic renal care between the renal nurse and his/her patient translates onto the acute unit and makes the care that the specialist acute nurse provides different to other wards in a hospital. On diagnosis, the patient will be treated either on an Intensive Care Unit or in the acute haemodialysis unit depending on the causes of the renal failure and the co-morbidities. The personnel treating the patient will differ depending where the treatment is delivered; but wherever the patient finds themselves the needs of the family are generally the same (1). There is much debate about where is the best place to treat people with ARF (2) but most patients are moved to the acute haemodialysis ward as soon as possible, where the specialist nursing staff have great influence over the treatment and care of the patient. It is here, where the expert nurse and his/her skills are most in evidence and the skills of the nurse are vital, as s/he will spend the most time with the patient (3). It is the time spent during treatment when the family can relate to one caregiver and relationships develop. PMID- 12371727 TI - Effects of different doses in continuous veno-venous haemofiltration on outcomes of acute renal failure: a prospective randomised trial. AB - BACKGROUND: Continuous veno-venous haemofiltration is increasingly used to treat acute renal failure in critically ill patients, but a clear definition of an adequate treatment dose has not been established. We undertook a prospective randomised study of the impact of different ultrafiltration doses in continuous renal replacement therapy on survival. METHODS: We enrolled 425 patients, with a mean age of 61 years, in intensive care who had acute renal failure. Patients were randomly assigned ultrafiltration at 20 ml/h-1/kg(-1) (group 1, n = 146), 35 ml/h(-1)/kg(-1) (group 2, n = 139), or 45 ml/h(-1)/ kg(-1) (group 3, n = 140). The primary endpoint was survival at 15 days after stopping haemofiltration. We also assessed recovery of renal function and frequency of complications during treatment. Analysis was by intention to treat. RESULTS: Survival in group 1 was significantly lower than in groups 2 (p = 0.0007) and 3 (p = 0.0013). Survival in groups 2 and 3 did not differ significantly (p = 0.87). Adjustment for possible confounding factors did not change the pattern of differences among the groups. Survivors in all groups had lower concentrations of blood urea nitrogen before continuous haemofiltration was started than non-survivors. 95%, 92% and 90% of survivors in groups 1, 2 and 3, respectively, had full recovery of renal function. The frequency of complications was similarly low in all groups. INTERPRETATION: Mortality among these critically ill patients was high, but increase in the rate of ultrafiltration improved survival significantly We recommend that ultrafiltration should be prescribed according to patient's bodyweight and should reach at least 35 ml/h(-1)/kg(-1). PMID- 12371728 TI - Renal replacement therapy in the intensive care unit with a single-pass batch system. PMID- 12371729 TI - Deaths due to dialyser repair: a new clinical syndrome. PMID- 12371730 TI - Enjoying another European experience and meeting EDTNA/ERCA colleagues. PMID- 12371731 TI - EDTNA/ERCA guidelines: technical section. PMID- 12371732 TI - Chemical additives in new RO systems. AB - Recent analysis of water and dialysate samples shows that due to new RO systems chemical substances can migrate into the osmotic water. A large number of tests have shown that there are no additives in drinking and softened water, but that the problem arises after the RO system. For the tests, samples were taken in the drinking water, after the softener and after the RO system. When the problem was located there were also samples taken from a similar water treatment system, and further samples were taken from two identical RO systems from another supplier. Instead of an RO system that removes all kind of substances, we're now confronted with systems that add volatile aromatic carbon hydrogens. Further tests have shown that dialysate contains the same level of these carbon hydrogens. It seems that even after having primed the artificial kidney in the blood compartment carbon hydrogens are present. The effects of these substances can be alarming because levels have been found equal to limits that are being proposed for drinking water under new European legislation. Is this the moment to look at our RO water quality level, and state new limits for new additives, should we work out new prescriptions for water quality or should we let the rules remain as they are? PMID- 12371733 TI - The influence of bacteria in dialysis water on its endotoxin level. AB - The influence of bacteria in dialysis water and its endotoxin level is one of the main important issues of purified water for haemodialysis. The current methods, assessing the number of water bacteria, are insufficient; due to their large diversity, only a small fraction of them grow on our used culture agars. Bacterial cells are classified into three categories: the A cells represent cultivable cells, B cells are living though not cultivable whereas the C cells are dead cells. Methods assessing these different cell categories are discussed. Endotoxin is only released from the C-type cells and is removed by the Reverse Osmosis membranes. It is our task to ensurethat we keep the A-cell level low after the Reverse Osmosis. The purified water must not remain very long in the pipe system and the use of Permeate tanks should be avoided. PMID- 12371734 TI - Safe use of water treatment units in dialysis: manufacturers or users liability? PMID- 12371735 TI - Endotoxin and 'on line' production of substitution fluid in haemodiafiltration and haemofiltration. AB - In dialysis, machines intended for use in the treatment modes haemodiafiltration (HDF) and haemofiltration (HF) have been on the market about 10 years. These machines are equipped to be able to produce the substitution fluid to be used for direct infusion. In principle, this is done by using the reverse osmosis water of the clinic and mixing in concentrates as usual to form dialysis fluid. The dialysis fluid is then filtered in order to prepare it for use as substitution fluid. The volumes used when the substitution fluid is prepared on-line are usually 20-50 litres in HDF mode and 70-150 litres in HF mode. This means that a patient treated 3 times a week is exposed to a total volume of substitution fluid ranging from 3,000 litres to a maximum 23,000 litres a year. Where else in medicine do we see something coming close to this? Because of these large infusion volumes, the issue of endotoxin levels becomes as important as the issue of sterility. PMID- 12371736 TI - Routine disinfection of the total dialysis fluid system. AB - The importance of bacteria and endotoxin free, sterile dialysis fluid for long term, high quality haemodialysis treatment is obvious and very much demanded (1,2). Dead spaces and connections between units (segments) of fluid production and delivery in elder systems are a continuous source for bacteria growth, biofilm generation and endotoxin release (3). After varying success with routine disinfection of system components showing partly fast recovery and growth of bacteria (i.e. < 48 hours) we changed to routine disinfection of the entire fluid production and distribution system. We call this'system disinfection'. We report the methods and results from observation of practice over 28 months of disinfection. The fluid system is composed of a soft water tank, reverse osmosis (double RO), RO fluid loop, central bicarbonate production and delivery system and dialysis stations with and without ultrafilter and citric-thermal disinfection before and after each haemodialysis. The system disinfection is carried out bimonthly with peracetic acid 3.5% in > 0.1% solution at a mean temperature of > 15 degrees C and at a minimum of 60 minutes of disinfection time. Samples for microbiological testing and endotoxin measurement were assessed 3-4 monthly at 7 measurement points. The tests were carried out 7 times on the 11th day (mean value [MV]) after routine system disinfection. The result was in 0.2 CFU/ml (MV) in 40 tests. The endotoxin levels (IU/L) were all < 0.25 except one at 0.325 in RO water. Endotoxin was assessed 5 times in 26 tests over 28 months. Samples were taken at 10.5 (MV) days after system disinfection. The Gel Clot or turbometric method was used. Efficient and preventive routine system disinfection of an entire dialysis fluid production and distribution system as standard in modern equipment - can support sufficient quality in dialysis fluid produced and distributed by elder and composed systems. PMID- 12371737 TI - Hand decontamination: what interventions improve compliance? AB - BACKGROUND: Haemodialysis units have traditionally been classified as high-risk areas in relation to infection control. Hand decontamination is the fundamental infection control practice, although this is not always performed optimally Deficiencies in the technique, frequency and appropriateness of this vital practice are frequently reported in health care journals. A literature search was carried out to determine what interventions improve compliance with hand decontamination practice. METHOD: A literature search was carried out of electronic databases from 1988-2000. Forty articles matched the key search words. When inclusion/exclusion criteria had been applied, seven primary research articles were found to be suitable. Appropriate tools were used to critically analyse the research. The findings were then synthesised into similar themes. FINDINGS: The interventions used in the seven articles utilised a combination of staff and patient education, feedback and motivational interventions to improve compliance. The evidence showed that these interventions met with different degrees of success. Multiple interventions were generally more successful than individual efforts. The improvement in hand decontamination practice often fell back to base line measurements after the intervention had ceased. IMPLICATIONS FOR PRACTICE: The findings can cautiously be used to improve practice. A combination of continuous, innovative education and motivation should improve and maintain this important skill. PMID- 12371738 TI - The role of the renal specialist nurse in prevention of renal failure. AB - This article will investigate the care required for those with reduced renal function before renal replacement therapy (RRT) commences. Renal nurses are often involved with the technical, monitoring and evaluative aspects of RRT for those with end stage renal failure. However, many patients may experience reduced renal function many years before reaching the stage of needing RRT. Renal nurses are already involved in the preparation of patients for RRT, but are not presently exercising their specialist skills in the period before this time by contributing to the prevention of end stage renal failure (ESRF). Screening programmes carried out in various parts of the world demonstrate that many members of the population have undetected renal insufficiency, and may benefit from intervention from the nephrology team to prevent further renal dysfunction. It is for this group of patients that this article will consider the potential for the renal nurse to expand their scope of practice. PMID- 12371739 TI - Dialysis--a family matter. A personal tribute to the relatives of kidney patients. AB - The patient and his or her treatment is the main focus of the multidisciplinary team in the Renal Unit, yet the most important part of the patient's life is that spent at home, outside the hospital. It is important to recognise the contribution to care of the patient's family, especially the spouse. It is also important to understand the carer's needs. Carers experience many of the stresses associated with End Stage Renal Failure, while being relatively unsupported and unappreciated by hospital staff. This paper focuses on the need for imagination and understanding of the carer's predicament, and suggests ways in which carer's needs can be met. PMID- 12371740 TI - Improving the nurse-patient relationship: a multi-faceted approach. AB - AIM: The aim of the symposium was to provide strategies to help overcome different types of nurse-patient communication problems, with particular reference to the renal disease setting. In this context, the importance of effective patient education to increase patient compliance played a vital role. The case studies presented and the subsequent discussions helped increase the awareness of important medical aspects, such as the increased risk of anaemia related complications in diabetic patients and the importance of early referral and anaemia treatment in patients with renal disease. Strategies proposed were supported by best practice guidelines and the latest scientific data. METHODOLOGY: A moderator led an interactive session that included two video case studies, scientific presentations and panel discussions. The audience played an active role by expressing their opinions via keypads. The case studies and the views of the audience were discussed by a panel of experts, who related their experiences and advice. CONCLUSIONS: A good nurse-patient relationship is essential to meet the clinical, psychological and social needs of the patient in order to optimise treatment. Effective patient education is an important tool to meet patient's needs and increase treatment compliance. Moreover, optimal treatment of patients requires a multidisciplinary approach to allow early identification and treatment of associated co-morbidities. Anaemia-related co morbidities are particularly important in diabetic patients with renal disease, since this patient group develops anaemia at an early stage and has a significantly increased risk of cardiovascular complications. In renal disease, there are well-defined guidelines and treatment options for anaemia-related co morbidities, which can easily be adapted to a patient's needs in order to offer flexible, individualised treatment with the aim of improving treatment outcomes. PMID- 12371741 TI - The use of clinical reasoning to promote patient choice. PMID- 12371742 TI - Distribution of study observations. PMID- 12371743 TI - Identification and expression of mammalian long-chain PUFA elongation enzymes. AB - In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a delta6 desaturation step followed by retroconversion (Sprecher, H., Biochim. Biophys. Acta 1486, 219-231, 2000). We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA. The database search led to the isolation of two cDNA clones from human and mouse. These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5. The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide. Expression of these open reading frames in baker's yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20- and 22-carbon long chain PUFA, as determined by the conversion of 20:4n-6 to 22:4n-6, 22:4n-6 to 24:4n-6, 20:5n-3 to 22:5n-3, and 22:5n-3 to 24:5n-3. The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20- and C22-carbon) n-6 and n-3 PUFA substrates by the significant increase in the levels of 24:4n-6 and 24:5n-3, respectively. This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis. PMID- 12371744 TI - Effect of dietary conjugated linoleic acid (CLA) on metabolism of isotope-labeled oleic, linoleic, and CLA isomers in women. AB - The purpose of this study was to investigate the effect of dietary CLA on accretion of 9c-18:1, 9c,12c-18:2, 10t,12c-18:2, and 9c,11t-18:2 and conversion of these FA to their desaturated, elongated, and chain-shortened metabolites. The subjects were six healthy adult women who had consumed normal diets supplemented with 6 g/d of sunflower oil or 3.9 g/d of CLA for 63 d. A mixture of 10t,2c-18:2 d4, 9c,11t-18:2-d6, 9c-18:1-d8, and 9c,12c-18:2-d2, as their ethyl esters, was fed to each subject, and nine blood samples were drawn over a 48-h period. The results show that dietary CLA supplementation had no effect on the metabolism of the deuterium-labeled FA. These metabolic results were consistent with the general lack of a CLA diet effect on a variety of physiological responses previously reported for these women. The 2H-CLA isomers were metabolically different. The relative percent differences between the accumulation of 9c,11t 18:2-d6 and 10t,12c-18:2-d4 in plasma lipid classes ranged from 9 to 73%. The largest differences were a fourfold higher incorporation of 10t,12c-18:2-d4 than 9c,11t-18:2-d6 in 1-acyl PC and a two- to threefold higher incorporation of 9c,11t-18:2-d6 than 10t,12c-18:2-d4 in cholesterol esters. Compared to 9c-18:1-d8 and 9c,12c-18:2-d2, the 10t,12c-18:2-d4 and 9c,11t-18:2-d6 isomers were 20-25% less well absorbed. Relative to 9c-18:1, incorporation of the CLA isomers into 2 acyl PC and cholesterol ester was 39-84% lower and incorporation of 10t,12c-18:2 was 50% higher in 1-acyl PC. This pattern of selective incorporation and discrimination is similar to the pattern generally observed for trans and cis 18:1 positional isomers. Elongated and desaturated CLA metabolites were detected. The concentration of 6c,10t,12c-18:3-d4 in plasma TG was equal to 6.8% of the 10t,12c-18:2-d4 present, and TG was the only lipid fraction that contained a CLA metabolite present at concentrations sufficient for reliable quantification. In conclusion, no effect of dietary CLA was observed, absorption of CLA was less than that of 9c-18:1, CLA positional isomers were metabolically different, and conversion of CLA isomers to desaturated and elongated metabolites was low. PMID- 12371745 TI - Dietary CLA alters yolk and tissue FA composition and hepatic histopathology of laying hens. AB - The effect of dietary CLA along with n-3 PUFA on yolk FA profile and hepatic lipid accumulation was investigated. Laying hens (n = 40) were randomly assigned to four experimental diets containing 0, 0.5, 1.0, or 2.0% CLA. Menhaden oil was used as the source of n-3 PUFA. Dietary CLA did not affect the total lipid content of egg yolk (P > 0.05). The amounts of CLA isomers (cis-9 trans-11, trans 10 cis-12) in the egg yolk were proportional to the levels of CLA in the diet (P < 0.05). The total CLA content in the egg yolk was 0, 0.97, 2.4, and 5.3 wt%, respectively (P < 0.05). Addition of CLA resulted in an increase in saturated FA (P < 0.05) with a concomitant reduction in monounsaturated FA (P < 0.05) in the yolk, liver, abdominal fat, breast, and thigh muscle. No difference in saturated and monounsaturated FA content in heart and spleen tissue was noted. Dietary CLA at all concentrations resulted in an increase (P < 0.05) in the total number of fat vacuoles and lipid infiltration in hepatocytes. The number of cells with 75% or higher lipid vacuolation in the cytoplasm was also increased (P < 0.05) by 2.0% CLA. Dietary CLA at 0.5% levels resulted in an increase (P < 0.05) in the total lipid content of hepatic tissue. The total lipid content in leg muscle was lower (P < 0.05) in CLA-fed birds. However, no effect of CLA on lipid content of breast muscle, heart, spleen and adipose tissue was observed (P > 0.05). The current study used CLA in a FFA form. The effects of using CLA in other form such as TG on avian hepatic tissue need to be investigated. PMID- 12371746 TI - Retinal sensitivity loss in third-generation n-3 PUFA-deficient rats. AB - A previous study conducted in guinea pigs suggested that ingestion of diets high in EPA and DHA may result in suboptimal retinal function. The aim of the present study was to evaluate retinal function in pigmented (Long-Evans) rats, raised to a third generation on diets that were either deficient in n-3 PUFA or adequate (with the addition of DHA). Electroretinographic assessment employed full-field white flash stimulation. Photoreceptor responses were evaluated in terms of peak amplitudes and implicit times (a-wave, b-wave), intensity-response functions (Naka-Rushton), and the parameters of a model of transduction (P3). Retinal phospholipid FA composition was measured by capillary GLC. DHA levels were reduced by 55% in n-3-deficient animals compared with the n-3-adequate group, whereas the levels of docosapentaenoic acid n-6 were 44 times higher in n-3 deficient animals. The level of arachidonic acid was marginally higher (12.8%) in n-6-adequate animals. The n-3-deficient animals exhibited significantly reduced retinal sensitivity (sigma and S values were both affected by 0.29 log units) and increased b-wave implicit times compared with those fed the n-3-adequate diet. These data suggest that n-3 PUFA are required for development of retinal sensitivity, more so than other indices of retinal function assessed by current methods, such as maximal response amplitude. However, the benefit for retinal function of adding preformed DHA to diets already replete in n-3 PUFA remains unclear. PMID- 12371747 TI - n-3 FA increase liver uptake of HDL-cholesterol in mice. AB - In humans, diets rich in fish oil (containing n-3 FA) decrease the incidence of coronary artery diseases. This is thought to be caused by the induction in liver and skeletal muscle of genes involved in lipid oxidation, and to the repression in liver and adipose tissue of genes responsible for lipogenesis. n-3 FA are known to reduce the synthesis of FA and TG in the liver, resulting in a decrease of plasma concentrations of TG-rich lipoproteins. On the other hand, little is known of a possible effect of n-3 FA on HDL metabolism. To investigate this question, female C57Bl/6J mice were fed an n-3 FA-enriched diet for 16 wk. As expected from previous studies, we found that total cholesterol, TG, and phospholipids were reduced in the plasma of treated mice. We also found that HDL cholesterol decreased after this treatment and that the in vivo fractional catabolic rate of HDL-cholesteryl ester was significantly higher in treated mice than in control mice fed a standard diet. Consistent with these results, treated mice exhibited increased uptake of HDL-cholesteryl ester in the liver. Moreover, quantitative reverse transcriptase-PCR analysis showed a two- to threefold increase in scavenger receptor B-1 gene expression. Taken together, these results suggest that an n-3 FA-enriched diet stimulates one step in the reverse cholesterol transport in mice, probably by increasing the amount of the scavenger receptor class B-1. These effects of n-3 FA on HDL metabolism may contribute to their beneficial effects on the vasculature. PMID- 12371748 TI - Impaired lipoprotein metabolism in obese offspring of streptozotocin-induced diabetic rats. AB - The time course of changes in lipoprotein metabolism of obese offspring of mildly diabetic rats was studied with respect to serum lipoprotein composition as well as LCAT and tissue lipoprotein lipase (LPL) activities. Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on day 5 of gestation. Control pregnant rats were injected with citrate buffer. At birth, obese pups had higher serum glucose, insulin, and lipoprotein (VLDL, LDL-HDL1, HDL(2-3)) levels than control pups. After 1 mon of life, all of these parameters in obese rats became similar to those of controls. However, LCAT, adipose tissue LPL, and hepatic triacylglycerol lipase activities were high. At 2 mon of age, VLDL-TAG levels were higher in obese females than in controls. By the age of 3 mon, obese offspring had developed insulin resistance with hyperglycemia, hyperinsulinemia, and higher serum lipoprotein concentrations. Indeed, qualitative abnormalities of lipoproteins were seen and were typical of obese and diabetic human beings. Fetal hyperinsulinemia should be considered as a risk factor for later metabolic diseases, including dyslipoproteinemia. PMID- 12371750 TI - Enrichment of LDL with EPA and DHA decreased oxidized LDL-induced apoptosis in U937 cells. AB - Oxidized LDL (oxLDL) may contribute to the accumulation of apoptotic cells in atherosclerotic plaques. Although it is well established in monophasic chemical systems that the highly unsaturated EPA and DHA will oxidize more readily than FA that contain fewer double bonds, our previous studies showed that enrichment of LDL, which has discrete polar and nonpolar phases, with these FA did not increase oxidation. The objective of this study was to compare the extent of apoptosis induced by EPA/DHA-rich oxLDL to that induced by EPA/DHA-non-rich oxLDL in U937 cells. LDL was obtained from one healthy subject three times before and after supplementation for 5 wk with 15 g/d of fish oil (FO), an amount easily obtainable from a diet that contains fatty fish. After supplementation, an EPA/DHA-rich LDL was obtained. Oxidative susceptibility of LDL, as determined by measuring the formation of conjugated dienes and the accumulation of cholesteryl ester hydroperoxides, was not higher in EPA/DHA-rich LDL. The oxLDL-induced cell apoptosis was detected by the activation of caspase-3, the translocation of PS to the outer surface of the plasma membrane using the Annexin V-fluorescein isothiocyanate binding assay, and the presence of chromatin condensation and nuclear fragmentation using the 4,6-diamidino-2-phenylindole staining assay. All three measures showed that after FO supplementation, EPA/DHA-rich oxLDL-induced cell apoptosis decreased. The decrease was not related to the concentration of lipid hydroperoxides. This study suggests that a possible protective effect of EPA/DHA-rich diets on atherosclerosis may be through lessening cell apoptosis in the arterial wall. PMID- 12371749 TI - High oleic acid oil suppresses lung tumorigenesis in mice through the modulation of extracellular signal-regulated kinase cascade. AB - This study was undertaken to estimate the effect of dietary high oleic acid oil (OA) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in mice. Diet containing 10% oil was fed to mice through experimental periods. On day 30 after NNK injection (100 mg/kg body weight, i.p.), the treatment increased the level of prostaglandin E2 (PGE2) as well as proliferating cell nuclear antigen, a marker of cell proliferation in a high linoleic acid oil (LA)-fed group but not in an OA-fed group. The NNK treatment also induced the activation of an extracellular signal-regulated kinase (Erk) cascade (Erk, Mek and Raf-1) in an LA-fed group. On the other hand, OA feeding abolished the NNK-induced activation of the Erk cascade. In conjugation with these events, OA feeding reduced lung tumor incidence and tumor multiplicity (percentage of mice with tumors) in mice compared with LA feeding at the 20th experimental week. These results suggest that OA suppresses lung tumorigenesis and that this suppression is correlated with the inhibition of PGE2 production and inactivation of the Erk cascade. PMID- 12371751 TI - Eicosapentaenoic acid promotes apoptosis in Ramos cells via activation of caspase 3 and -9. AB - Eicosapentaenoic acid (EPA; 20:5n-3) may reduce the cell number in cultured leukemia/lymphoma cells owing to reduced cell proliferation, induction of cell death, or a combination of these processes. EPA has been shown to promote apoptosis in Ramos cells, and our present study was focused on a possible cell cycle arrest and the pathways by which the apoptotic process is induced. Apoptosis may proceed along the intrinsic (mitochondrial) or the extrinsic (death receptor) pathway, which are mediated via different caspases. Caspases are a class of homologous cysteine proteases recognized as pivotal mediators of apoptosis. We investigated whether EPA affects progression of the cell cycle or promotes apoptosis directly. By incorporation of [3H]thymidine and [3H]valine, we showed that DNA, as well as protein synthesis, was reduced after incubation of Ramos cells with EPA for 6 h. We monitored cell cycle distribution by 5-bromo-2' deoxyuridine staining and observed no cell cycle arrest in the EPA-incubated cells. Incubation of cells with EPA caused PS-flipping, as demonstrated by annexin V-binding (flow cytometry), and cleavage of poly(ADP-ribose) polymerase measured by Western blot analysis. Furthermore, we observed increased activity of caspase-3 and -9, but not of caspase-8. Whereas inhibitors of caspase-3 and -9 reduced EPA-induced apoptosis, inhibition of caspase-8 did not. This suggests that EPA may promote apoptosis via the intrinsic pathway in Ramos cells. Thus, the reduction in cell number can be explained by a direct apoptotic effect of EPA rather than via cell cycle arrest. PMID- 12371752 TI - Positional distribution of FA in TAG of enzymatically modified borage and evening primrose oils. AB - Stereospecific analysis was carried out to establish positional distribution of FA in the TAG of DHA, EPA, and (EPA + DHA)-enriched oils. In this study, TAG of enzymatically modified oils were purified using a silicic acid column. The TAG were then subjected to positional distribution analysis using a modified procedure involving reductive cleavage with Grignard reagent. The results showed that in DHA-enriched borage oil (BO), DHA was randomly distributed over the three positions of TAG, whereas gamma-linolenic acid (GLA) was mainly esterified at the sn-2 and -3 positions. In DHA-enriched evening primrose oil (EPO), however, DHA and GLA were concentrated in the sn-2 position. In EPA-enriched BO, EPA was randomly distributed over the three positions of TAG, similar to that observed for DHA. In EPA-enriched EPO, however, this FA was mainly located at the primary positions (sn-1 and sn-3) of TAG. In both oils, GLA was preferentially esterified at the sn-2 position. In (EPA + DHA)-enriched BO, EPA and DHA were mainly esterified at the sn-1 and -3 positions of TAG, whereas GLA was mainly located at the sn-2 position. In (EPA + DHA)-enriched EPO, GLA was mainly located at the sn 2 and -3 positions; EPA was preferentially esterified at the sn-1 and -3 positions, and DHA was found mainly at the sn-3 position. PMID- 12371753 TI - Characterization of trans-monounsaturated alkenyl chains in total plasmalogens (1 O-alk-1'-enyl-2-acyl glycerophospholipids) from sheep heart. AB - In the present study, we investigated the alkenyl chains from sheep heart plasmalogens (1-O-alk-1'-enyl-2-acyl glycerophospholipids) after their conversion into trimethylene dioxyalkanyl (TMDOA) derivatives. Particular attention was given to monounsaturated alkenyl chains (C18 mainly). For this purpose, a combination of silver ion TLC and GLC on highly polar, very long capillary columns was applied to TMDOA derivatives. Approximately 30 different alkenyl chains could be separated, and the main observation was that the component previously reported as a cis-9 18:1 alkenyl chain in plasmalogens embraces in fact a wide range of trans and cis isomers, in amounts equal to 7.9 and 5.6%, respectively, of total alkenyl chains. Concerning the trans-monoenoate fraction, isomers with their ethylenic bond spanning from delta6-delta8 to delta16 were tentatively identified on the basis of their distribution profile, which was similar to that of trans-18:1 acids prepared and isolated from sheep adipose tissue. The main trans-monoenoic C18 alkenyl chain in sheep heart plasmalogens would thus have its double bond in position 11, which seems logical, as alkenyl chains are derived from the corresponding alcohols, themselves issued from the corresponding FA, and in this particular case, vaccenic (trans-11 18:1) acid. cis Monoenoic C18 alkenyl chains also appear more complex than realized earlier, showing in particular isomers with their ethylenic bond farther than the delta9 position, in addition to the main isomer derived from oleic acid. Several trans 16:1 alkenyl chains could be observed (totaling ca. 1%), but cis-16:1 isomers were present in trace amounts only. PMID- 12371754 TI - A direct method for regiospecific analysis of TAG using alpha-MAG. AB - An analytical procedure was developed for regiodistribution analysis of TAG using alpha-MAG prepared by an ethyl magnesium bromide deacylation. In the present communication, the deacylation procedure is shown to lead to representative alpha MAG, allowing the composition of the native TAG in the alpha-position to be determined directly. The composition in the beta-position can then be estimated from the composition of the alpha-MAG and TAG according to the formula 3 x TAG - 2 x alpha-MAG. The estimates are superior to those obtained using the alpha,beta DAG and Brockerhoff calculations as they come closer to the theoretical value and have smaller SD. The present procedure, first demonstrated on a synthetic TAG, was then successfully applied to the analysis of borage oil, milkfat, and tuna oil. PMID- 12371756 TI - New companion animal tumor registry in the works. PMID- 12371757 TI - Terrorism law includes penalties for extremists targeting animal-related businesses. PMID- 12371755 TI - Evaluation of two GC columns (60-m SUPELCOWAX 10 and 100-m CP Sil 88) for analysis of milkfat with emphasis on CLA, 18:1, 18:2 and 18:3 isomers, and short- and long-chain FA. AB - Milkfat is a complex mixture of many diverse FA, some of which have demonstrated health benefits including anticancer properties. Attempts are under way to enrich milkfats with long-chain n-3 PUFA and CLA. It has been recommended that the analysis of these milkfats requires gas chromatography (GC) equipped with long, highly polar capillary columns. However, many analyses have been reported using CARBOWAX type (polyethylene glycol) capillary columns, such as SUPELCOWAX 10, even though the separation characteristics of many of the FA and their isomers present in milkfats have not been described in detail. This includes the isomers of CLA, cis- and trans-octadecenoic acid (18:1), linoleic acid (18:2n-6), and linolenic acid (18:3n-3), and the long-chain PUFA. On the other hand, the resolution of these FA and their isomers has been more fully described using the highly polar capillary columns, such as CP Sil 88 and SP2560 because of the improved resolution obtained using these polar columns. The present study was undertaken to characterize the separation of these FA present in milkfats using a 60-m SUPELCOWAX 10 column, to compare the results to those from a 100-m CP Sil 88 column, and to determine if these two columns could possibly serve to complement each other for the analysis of total milkfat. The advantages of the SUPELCOWAX 10 column were a better resolution of the short-chain saturated from their monounsaturated FA (MUFA) analogs, and a complete separation of the alpha linolenic (18:3n-3) and eicosadecenoic acid (20:1) isomers. It also provided an alternative elution order of the linoleic (18:2n-6), 18:3n-3 and gamma-linolenic (18:3n-6) acid isomers. On the other hand, the CP Sil 88 column provided a better resolution of the CLA isomers, MUFA, the isolated cis and trans MUFA fractions, the PUFA, and many the 18:2n-6 and 18:3n-3 isomers. A complete analysis of milk lipids using the CP Sil 88 column required the prior separation of total FAME using silver ion-TLC. The results of the present study confirm that the 100-m highly polar capillary GC columns are mandatory for the analysis of milk lipids, and at best, the 60 m SUPELCOWAX 10 capillary column serves as a complementary GC column to provide different separations in certain regions based on its intermediate polarity. PMID- 12371758 TI - Use of noninvasive dental dolorimetry to evaluate analgesic effects of intravenous and intrathecal administration of morphine in anesthetized dogs. AB - OBJECTIVE: To determine whether changes in amplitude of the reflex-evoked muscle action potential (REMP) elicited by noninvasive dental dolorimetry (electrical stimulation of the tooth pulp) in anesthetized dogs may be used to objectively evaluate the effectiveness of IV and intrathecal (IT) administration of morphine. ANIMALS: 6 male Beagles that were 2 to 6 years old. PROCEDURE: Dogs were used in a crossover design with at least a 5-day washout period between treatments. Each dog received morphine, saline (0.9% NaCl) solution, and oxytocin via the IV and IT routes of administration; however, only results for morphine and saline treatments were reported here. Dogs were anesthetized and prepared for noninvasive dental dolorimetry. After IV or IT administration, electrical stimulation was applied to a tooth, and REMPs of the digastricus muscle were recorded at 5-minute intervals for 60 minutes. To determine differences in REMP amplitude between treatments, a linear regression line was fitted for each dog treatment combination. RESULTS: The IV administration of morphine significantly inhibited REMP amplitude, compared with IV administration of saline solution. Intrathecal administration of morphine significantly inhibited REMP amplitude, compared with IT administration of saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: Noninvasive dental dolorimetry in anesthetized dogs has promise as a technique for use in evaluating the analgesic potential of drugs administered IV and IT through evaluation of their effect on REMP amplitude recorded for the digastricus muscle. PMID- 12371759 TI - Effect of intrathecal and intravenous administration of oxytocin on amplitude of the reflex-evoked muscle action potential after electrical stimulation of the tooth pulp in anesthetized dogs. AB - OBJECTIVE: To determine whether intrathecal (IT) or IV administration of oxytocin will diminish amplitude of the reflex-evoked muscle action potential (REMP) in the digastricus muscle during electrical stimulation of the tooth pulp in anesthetized dogs, thus suggesting an analgesic effect for oxytocin. ANIMALS: 6 male Beagles that were 2 to 6 years old. PROCEDURE: Dogs were used in a crossover design with at least a 5-day washout period between treatments. Each dog received morphine, saline (0.9% NaCl) solution, and oxytocin by both the IT and IV routes of administration. Noninvasive dental dolorimetry was used to assess changes in pain threshold following administration of treatments. Effectiveness of analgesia was determined on the basis of change in REMP amplitude in the digastricus muscle. RESULTS: Morphine administered IV significantly inhibited REMP amplitude, compared with IV administration of saline solution or oxytocin. There was not a significant change in REMP amplitude between saline solution and oxytocin administered IV. Intrathecal administration of morphine significantly inhibited REMP amplitude, compared with IT administration of saline solution or oxytocin. Intrathecal administration of oxytocin significantly increased REMP amplitude, compared with IT administration of saline solution or morphine. CONCLUSIONS AND CLINICAL RELEVANCE: Although IV administration of oxytocin did not have an effect on REMP amplitude, compared with IV administration of saline solution, IT administration of oxytocin had the opposite effect of morphine and increased REMP amplitude of the digastricus muscle. These data do not support the use of oxytocin as an analgesic agent in dogs. PMID- 12371760 TI - Characterization of matrix metalloproteinase-2 and -9 in cerebrospinal fluid of clinically normal dogs. AB - OBJECTIVE: To characterize matrix metalloproteinase (MMP)-2 and -9 in CSF of clinically normal dogs. SAMPLE POPULATION: Samples of CSF collected from 23 dogs. PROCEDURE: Dogs were anesthetized, CSF samples were collected, and dogs were then euthanatized. Each CSF sample was evaluated immediately for RBC count, WBC count, and protein and glucose concentrations, and cytologic examination also was performed. Samples were considered normal when protein concentration was < 25 mg/dL and CSF contained < 6WBCs/microL and < 25 RBCs/microL. Samples were stored at -70 degrees C. Sections of brain tissue were collected and processed for histologic examination. The MMPs were evaluated by use of gelatin zymography and a polyclonal antibody-based sandwich ELISA. RESULTS: Mean WBC count for CSF samples was < 1 WBC/microL (range, 0 to 3 WBCs/mL). Mean protein concentration was 12 mg/dL (range, 8 to 17 mg/dL). Mean RBC count was 3.65 RBCs/microL (range, 0 to 21 RBCs/microL). All CSF samples generated a clear band on zymography gels that corresponded to the human commercial standard of proenzyme MMP-2. Other major clear bands were not detected on zymography gels. Bands correlating to MMP 9 were not detected in any samples. The ELISA results revealed a mean +/- SD proenzyme MMP-2 concentration of 5.61 +/- 1.92 ng/mL (range, 3.36 to 10.83 ng/mL). CONCLUSIONS AND CLINICAL RELEVANCE: The proenzyme form of MMP-2 is detectable in CSF of clinically normal dogs, whereas MMP-9 is not detectable. Additional investigation of MMPs in CSF from dogs with various diseases of the nervous system is indicated. PMID- 12371761 TI - Effects of carprofen and dexamethasone on canine chondrocytes in a three dimensional culture model of osteoarthritis. AB - OBJECTIVE: To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. PROCEDURE: Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP-13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. RESULTS: Addition of IL-1beta caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1beta. CONCLUSIONS AND CLINICAL RELEVANCE: Human recombinant IL-1beta resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis. PMID- 12371762 TI - Effect of administration of a phospholipid emulsion on the initial response of horses administered endotoxin. AB - OBJECTIVE: To evaluate the effect of a phospholipid emulsion (PLE) on the initial response of horses to administration of endotoxin. ANIMALS: 12 healthy adult horses. PROCEDURES: Horses were assigned to 2 treatment groups (6 horses/group). The control group was administered 1 L of saline (0.9% NaCl) solution, and the treated group was administered PLE (200 mg/kg, IV); treatments were administered during a period of 120 minutes. An infusion of endotoxin was initiated in both groups starting 1 hour after initiation of the saline or PLE solutions. Physical examination and hemodynamic variables were recorded, and blood samples were analyzed for concentrations of tumor necrosis factor (TNF)-alpha, interleukin-6, thromboxane B2 (TxB2), 6 keto-prostaglandin F (PGF)1alpha, total leukocyte count, and PLE concentrations. An ANOVA was used to detect significant differences. RESULTS: Administration of PLE resulted in significantly lower rectal temperature, heart rate, cardiac output, right atrial pressure, and pulmonary artery pressure and higher total leukocyte counts in treated horses, compared with values for control horses. The TNF-alpha concentration was significantly less in treated horses than in control horses. The TxB2 and 6 keto-PGFF1alpha concentrations were significantly different between treated and control horses at 30 minutes (TxB2) and at 30 and 60 minutes (6 keto-PGF1alpha). CONCLUSIONS AND CLINICAL RELEVANCE: Prior infusion of PLE in horses administered a low dose of endotoxin decreased rectal temperature, heart rate, pulmonary artery pressure, and TNF-alpha concentrations. Results of this study support further evaluation of PLE for use in the treatment of horses with endotoxemia. PMID- 12371763 TI - Comparative virulence of isolates of bovine viral diarrhea virus type II in experimentally inoculated six- to nine-month-old calves. AB - OBJECTIVE: To determine the comparative virulence of 5 isolates of bovine viral diarrhea virus (BVDV) type II by inoculating 6- to 9-month-old beef calves with isolates originating from the tissues of cattle affected with naturally occurring, transient, acute, nonfatal infections or naturally occurring, peracute, fatal infections. ANIMALS: 22 calves that were 6 to 9 months old. PROCEDURE: The study used BVDV isolates 17011, 713, and 5521 that originated from fetuses aborted from cows with transient, nonfatal, acute BVDV infections and isolates 23025 and 17583 that originated from the tissues of cattle with peracute, fatal BVDV infections. Calves were allotted to 6 groups (1, mock infected control calves [n = 2]; 2, inoculated with BVDV 17011 [4]; 3, inoculated with BVDV 713 [4]; 4, inoculated with BVDV 5521 [4]; 5, inoculated with BVDV 23025 [4]; and 6, inoculated with BVDV 17583 [41]. Rectal temperatures and clinical signs of disease were recorded daily. Total and differential WBC and platelet counts were performed. Histologic examination and immunohistochemical analysis were conducted to detect lesions and distribution of viral antigens, respectively. RESULTS: Calves inoculated with BVDV 23025 or 17583 developed more severe clinical signs of disease (fever and diarrhea), more severe lymphopenia, and more severe lesions (alimentary epithelial necrosis, lymphoid depletion, and BVDV antigen deposition in lymphatic tissues), compared with calves inoculated with BVDV 713, 5521, or 17011. CONCLUSIONS AND CLINICAL RELEVANCE: Relative severity of experimentally induced infections corresponded to severity of clinical signs of naturally occurring infections with respective BVDV isolates. PMID- 12371764 TI - Differentiation of Haemobartonella canis and Mycoplasma haemofelis on the basis of comparative analysis of gene sequences. AB - OBJECTIVE: To determine whether Haemobartonella canis and Mycoplasma haemofelis (formerly known as H felis [large form]) can be differentiated by use of comparative analysis of gene sequences. SAMPLE POPULATION: Blood samples obtained from 3 dogs infected with H canis and 2 cats infected with M haemofelis. PROCEDURE: The partial 16S rDNA and ribonuclease P RNA (RNase P) genes were amplified, cloned, and sequenced in blood samples obtained from H canis-infected dogs and M haemofelis-infected cats. The DNA sequences were subjected to comparative analysis. RESULTS: The 16S rDNA sequences of H canis and M haemofelis were nearly identical (homology of 99.3 to 99.7%). In contrast, RNase P gene sequences had a lower degree of sequence homology between the 2 organisms (94.3 to 95.5%). CONCLUSIONS AND CLINICAL RELEVANCE: Haemobartonella canis and M haemofelis are not identical organisms. Molecular differentiation of H canis and M haemofelis is more clearly evident by use of comparative analysis of RNase P gene sequences than by comparative analysis of 16S rDNA gene sequences. PMID- 12371765 TI - In vitro evaluation of an intraluminal solution to attenuate effects of ischemia and reperfusion in the small intestine of horses. AB - OBJECTIVE: To evaluate the efficacy of intraluminal administration of a customized solution during low-flow ischemia and reperfusion in the jejunum of horses. SAMPLE POPULATION: Segments of jejunum obtained from 13 healthy adult horses. PROCEDURE: In isolated segments of jejunum maintained in an extracorporeal circuit, arterial flow was reduced to 20% of baseline for 40 minutes (ischemia) followed by 60 minutes of reperfusion. In 2 groups, a customized solution (concentrations, 12.5 and 25%, respectively) was placed in the lumen prior to low-flow ischemia and maintained during reperfusion. The control group received intraluminal lactated Ringer's solution for the same duration. Various metabolic, hemodynamic, histologic, and permeability variables were recorded. RESULTS: The 12.5% solution resulted in less histomorphologic injury and reduced mucosal permeability to albumin, compared with the 25% solution and the lactated Ringer's solution. Morphologic injury and permeability were reduced in tissues that received the 25% solution, compared with the control group, but this difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Use of a 12.5% customized solution appeared to minimize injury in the isolated extracoporeal jejunal loop, which provides some indication that it might be useful in clinical situations. PMID- 12371766 TI - I plasma concentrations of adrenocorticotrophic hormone and alpha-melanocyte stimulating hormone in ferrets (Mustela putorius furo) with hyperadrenocorticism. AB - OBJECTIVE: To determine plasma concentrations of adrenocorticotrophic hormone (ACTH) and alpha-melanocyte stimulating-hormone (alpha-MSH) in healthy ferrets and ferrets with hyperadrenocorticism. ANIMALS: 16 healthy, neutered, privately owned ferrets, 28 healthy laboratory ferrets (21 sexually intact and 7 neutered), and 28 ferrets with hyperadrenocorticism. PROCEDURES: Healthy ferrets were used for determination of reference plasma concentrations of ACTH and a-MSH. Diagnosis of hyperadrenocorticism was made on the basis of history, clinical signs, urinary corticoid-to-creatinine ratios, ultrasonography of the adrenal glands, and macroscopic or microscopic evaluation of the adrenal glands. Blood samples were collected during isoflurane anesthesia. Plasma concentrations of ACTH and alpha MSH were measured by radioimmunoassay. RESULTS: Plasma concentrations of ACTH in 23 healthy neutered ferrets during the breeding season ranged from 4 to 145 ng/L (median, 50 ng/L). Plasma concentrations of alpha-MSH in 44 healthy neutered or sexually intact ferrets during the breeding season ranged from < 5 to 617 ng/L (median, 37 ng/L). Reference values (the central 95% of the values) for ACTH and alpha-MSH were 13 to 100 ng/L and 8 to 180 ng/L, respectively. Plasma concentrations of ACTH and alpha-MSH in ferrets with hyperadrenocorticism ranged from 1 to 265 ng/L (median, 45 ng/L) and 10 to 148 ng/L (median, 46 ng/L), respectively. These values were not significantly different from those of healthy ferrets. Plasma ACTH concentrations of sexually intact female ferrets in estrus were significantly higher than those of neutered females. CONCLUSIONS AND CLINICAL RELEVANCE: Ferrets with hyperadrenocorticism did not have detectable abnormalities in plasma concentrations of ACTH or alpha-MSH. The findings suggest that hyperadrenocorticism in ferrets is an ACTH and alpha-MSH-independent condition. PMID- 12371767 TI - Computed tomography of the elbow joint in clinically normal dogs. AB - OBJECTIVE: To use computed tomography (CT) to provide a detailed description of elbow joint structures in clinically normal dogs. ANIMALS: 6 clinically normal adult mixed-breed dogs weighing 24 to 37 kg and one 12-month-old Labrador Retriever weighing 27 kg. PROCEDURE: To perform CT of both elbow regions, dogs were anesthetized and placed in lateral recumbency. One- and 2-mm contiguous slices were obtained by use of a third generation computed tomographic scanner. Good resolution and anatomic detail were acquired from the computed tomographic images by use of a bone (window width, 3,500 Hounsfield units; window level, 500 Hounsfield units) and soft-tissue setting (window width, 400 Hounsfield units; window level, 66 Hounsfield units). After euthanasia, the forelimbs from the Labrador Retriever were removed and frozen in water at -18 degrees C. Elbow joints were sectioned into approximately 1-mm-thick slab sections by use of an electric planer. Anatomic sections were photographed and compared with the corresponding computed tomographic images. Computed tomographic reconstructions of the elbow joint were created in sagittal and dorsal planes. RESULTS: Structures on the computed tomographic images were matched with structures in the corresponding anatomic sections. The entire humeroradioulnar joint surface could be evaluated on the reconstructed images in the sagittal and dorsal plane. CONCLUSIONS AND CLINICAL RELEVANCE: Computed tomographic images provide full anatomic detail of the bony structures of the elbow joint in dogs. Muscles, large blood vessels, and nerves can also be evaluated. These results could be used as a basis for evaluation of computed tomographic images of the forelimbs of dogs with elbow joint injuries. PMID- 12371768 TI - Effect of growth and training on muscle adaptation in Thoroughbred horses. AB - OBJECTIVE: To determine the effect of growth and training on metabolic properties in muscle fibers of the gluteus medius muscle in adolescent Thoroughbred horses. ANIMALS: Twenty 2-year-old Thoroughbreds. PROCEDURE: Horses were randomly assigned to 2 groups. Horses in the training group were trained for 16 weeks, and control horses were kept on pasture without training. Samples were obtained by use of a needle-biopsy technique from the middle gluteus muscle of each horse before and after the training period. Composition and oxidative enzyme (succinic dehydrogenase [SDHI) activity of each fiber type were determined by use of quantitative histochemical staining procedures. Whole-muscle activity of SDH and glycolytic enzyme (phosphofructokinase) as well as myosin heavy-chain isoforms were analyzed biochemically and electrophoretically, respectively. RESULTS: The SDH activity of type-I and -IIA fibers increased during growth, whereas whole muscle activity was unchanged. Percentage of type-IIX/B muscle fibers decreased during training, whereas that of myosin heavy-chain IIa increased. The SDH activity of each fiber type as well as whole-muscle SDH activity increased during training. An especially noticeable increase in SDH activity was found in type IIX/B fibers. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in muscle fibers of adolescent Thoroughbreds are caused by training and not by growth. The most noticeable change was for the SDH activity of type-IIX/B fibers. These changes in the gluteus medius muscle of adolescent Thoroughbreds were considered to be appropriate adaptations to running middle distances at high speeds. PMID- 12371769 TI - Changes in concentrations of neuroendocrine hormones and catecholamines in dogs with myocardial failure induced by rapid ventricular pacing. AB - OBJECTIVE: To describe neuroendocrine responses that develop in dogs subjected to prolonged periods of ventricular pacing. ANIMALS: 14 adult male hound-type dogs. PROCEDURE: Samples were obtained and neuroendocrine responses measured before (baseline) and after 3 periods of ventricular pacing. A pacemaker was used to induce heart rates of 180, 200, and 220 beats/min (BPM). Each heart rate was maintained for 3 weeks before increasing to the next rate. Atrial natriuretic peptide, antidiuretic hormone, aldosterone, norepinephrine, epinephrine, and dopamine concentrations and plasma renin activity were measured. Severity of left ventricular compromise was estimated. RESULTS: Shortening fraction decreased significantly with increasing heart rates (mean +/- SE, 35.5 +/- 1.4, 25.0 +/- 1.4, 19.5 +/- 1.9, and 12.2 +/- 2.3 for baseline, 180 BPM, 200 BPM, and 220 BPM, respectively). Atrial natriuretic peptide concentrations increased significantly at 180 BPM (44.1 +/- 3.0 pg/mL) and 200 BPM (54.8 +/- 5.5 pg/mL), compared with baseline concentration (36.8 +/- 2.6 pg/mL). Dopamine concentration increased significantly at 200 BPM (70.4 +/- 10.4 pg/mL), compared with baseline concentration (44.2 73 pg/mL). Norepinephrine concentrations increased significantly from baseline concentration (451 +/- 46.2 pg/mL) to 678 +/- 69.8, 856 +/- 99.6, and 1,003 +/- 2676 pg/mL at 180, 200, and 220 BPM, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs subjected to ventricular pacing for 9 weeks developed neuroendocrine responses similar to those that develop in humans with more chronic heart failure and, except for epinephrine concentrations, similar to those for dogs subjected to ventricular pacing for < 6 weeks. PMID- 12371770 TI - Effect of caprine arthritis-encephalitis virus infection on expression of interleukin-16 in goats. AB - OBJECTIVE: To determine the effect of caprine arthritis-encephalitis virus (CAEV) infection on expression of interleukin-16 (IL-16). ANIMALS: 6 goats experimentally infected with CAEV and 6 age-matched healthy uninfected control goats. PROCEDURE: Peripheral blood mononuclear cells (PBMCs) and synovial membrane cells from infected and control goats cultured with or without phytohemagglutinin were analyzed for IL-16 mRNA by use of a reverse transcriptase polymerase chain reaction assay with goat-specific primers, after cloning and sequencing of a 384-bp fragment of the goat IL-16 gene. Synovial fluid, serum, and culture supernatants of PBMCs and synovial cells of control and CAEV-infected goats were analyzed for IL-16 by use of an ELISA. RESULTS: The 384-bp product was 86% homologous to the corresponding human IL-16 nucleotide sequence. Higher expression of IL-16 mRNA in PBMCs (unstimulated or stimulated with phytohemagglutinin) was detected in samples from CAEV-infected goats, compared with control goats, but the difference was not significant. Synovial membrane cells infected in vitro had higher expression than uninfected control cells. Higher IL-16 concentration was detected in synovial fluid, serum, and culture supernatants of PBMCs of infected goats than in samples from control goats. CONCLUSIONS AND CLINICAL RELEVANCE: infection with CAEV increases expression of IL-16, a proinflammatory and chemotactic cytokine. This cytokine appears to be constitutively expressed at low concentrations in normal uninfected PBMCs and synovial membrane cells. Increased production of IL-16 in CAEV infection may partly be responsible for increased lymphoid cell infiltrations observed in arthritic joints and other tissues of CAEV-infected goats. PMID- 12371771 TI - Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs. AB - OBJECTIVE: To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION: Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE: The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS: All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE: Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP. PMID- 12371772 TI - Inheritance of pancreatic acinar atrophy in German Shepherd Dogs. AB - OBJECTIVE: To assess the heritability of pancreatic acinar atrophy (PAA) in German Shepherd Dogs (GSDs) in the United States. ANIMALS: 135 GSDs belonging to 2 multigenerational pedigrees. PROCEDURE: Two multigenerational pedigrees of GSDs with family members with PAA were identified. The clinical history of each GSD enrolled in the study was recorded, and serum samples for canine trypsin-like immunoreactivity (cTLI) analysis were collected from 102 dogs. Dogs with a serum cTLI concentration < or = 2.0 microg/L were considered to have exocrine pancreatic insufficiency (EPI) and were assumed to have PAA. RESULTS: Pedigree I consisted of 59 dogs and pedigree II of 76 dogs. Serum cTLI concentrations were measured in 48 dogs from pedigree I and 54 dogs from pedigree II. A total of 19 dogs (14.1%) were determined to have EPI, 9 in pedigree I (15.3%) and 10 in pedigree II (13.6%). Of the 19 dogs with EPI, 8 were male and 11 were female. CONCLUSIONS AND CLINICAL RELEVANCE: Evaluation of data by complex segregation analysis is strongly suggestive of an autosomal recessive mode of inheritance for EPI in GSDs in the United States. PMID- 12371773 TI - Cutaneous analgesia, hemodynamic and respiratory effects, and beta-endorphin concentration in spinal fluid and plasma of horses after acupuncture and electroacupuncture. AB - OBJECTIVE: To determine cutaneous analgesia, hemodynamic and respiratory effects, and beta-endorphin concentration in spinal fluid and plasma of horses after acupuncture and electroacupuncture (EA). ANIMALS: 8 healthy 10- to 20-year-old mares that weighed between 470 and 600 kg. PROCEDURE: Each horse received 2 hours of acupuncture and 2 hours of PAES at acupoints Bladder 18, 23, 25, and 28 on both sides of the vertebral column as well as sham needle placement (control treatment). Each treatment was administered in a random order. At least 7 days elapsed between treatments. Nociceptive cutaneous pain threshold was measured by use of skin twitch reflex latency (STRL) and avoidance to radiant heat (< or = 50 degrees C) in the lumbar area. Skin temperature, cardiovascular and respiratory variables, and beta-endorphin concentration in spinal fluid (CSF-EN) and plasma (plasma-EN) were measured. RESULTS: Acupuncture and PAES significantly increased STRL and skin temperature. The CSF-EN was significantly increased from baseline values 30 to 120 minutes after onset of PAES, but it did not change after acupuncture and control treatments. Heart and respiratory rates, rectal temperature, arterial blood pressure, Hct, total solids and bicarbonate concentrations, base excess, plasma-EN, and results of blood gas analyses were not significantly different from baseline values after acupuncture, PAES, and control treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of PAES was more effective than acupuncture for activating the spinal cord to release beta-endorphins into the CSF of horses. Acupuncture and PAES provided cutaneous analgesia in horses without adverse cardiovascular and respiratory effects. PMID- 12371774 TI - Detection of a genetic mutation for myotonia congenita among Miniature Schnauzers and identification of a common carrier ancestor. AB - OBJECTIVE: To develop a molecular genetic test to detect the mutant skeletal muscle chloride channel (CIC-1) allele that causes myotonia congenita in Miniature Schnauzers and to analyze the relationship of affected and carrier dogs. ANIMALS: 372 Miniature Schnauzers from the United States, Canada, Australia, and Europe that were tested between March 2000 and October 2001. PROCEDURE: The sequence surrounding the mutation in the CIC-1 allele was amplified by use of a unique pair of primers. Polymerase chain reaction (PCR) products were digested with the restriction enzyme Hpy CH4 III and separated on a 6% polyacrylamide gel. Pedigrees from all available carrier and affected dogs were analyzed, and a composite pedigree was established. RESULTS: Enzyme digestion of PCR products of the normal CIC-1 allele resulted in 3 fragments of 175, 135, and 30 bp, whereas PCR products of the mutant allele resulted in fragments of only 175 and 165 bp. Of the 372 Miniature Schnauzers, 292 (78.5%) were normal, 76 (20.4%) were carriers, and 4 (1.1%) were affected (myotonic) dogs. Frequency of the mutant allele was 0.113. Pedigree analysis revealed that a popular sire, documented to be a carrier, was a common ancestor of all carriers and affected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR-based enzyme digestion DNA test was developed. The mutant allele for this disease is frequent in Miniature Schnauzers that are related to a common carrier ancestor. Breeding dogs should be tested by this specific DNA test to help limit the spread of this deleterious mutation. PMID- 12371775 TI - Use of a high-molecular-weight carboxymethylcellulose in a tissue protective solution for prevention of postoperative abdominal adhesions in ponies. AB - OBJECTIVE: To evaluate efficacy and safety of IP administration of high-molecular weight carboxymethylcellulose (HMW CMC) for the prevention of postoperative intra abdominal adhesions in ponies. ANIMALS: 10 ponies. PROCEDURE: A 1% solution of HMW CMC was instilled intra-abdominally prior to surgery in 5 ponies, whereas 5 control ponies did not receive HMW CMC. Postoperative adhesions were induced by use of a bowel-abrasion method comprising laparotomy, typhlotomy, and abrasion of jejunal serosa at multiple sites with placement of 3 sutures at each site. Day of surgery was day 0. After surgery, ponies were monitored, and hematologic, serum biochemical, and peritoneal fluid analyses were performed on days 1, 2, 3, 5, 7, and 10. On day 10, ponies were euthanatized. Intra-abdominal adhesions were recorded, and tissue samples were collected for histologic examination. RESULTS: A significantly greater number of adhesions, number of multiple adhesions, and mean incidence of adhesions were identified in control ponies, compared with CMC treated ponies. Mean peritoneal fluid WBC count on day 7 and serum fibrinogen concentrations on days 5 and 7 were significantly higher in control ponies, compared with CMC-treated ponies. Results of serum biochemical analyses did not differ significantly between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: Intra-abdominal use of 1% HMW CMC during surgery was effective for preventing postoperative adhesions in ponies. Use of HMW CMC did not have detrimental effects on wound healing, intra-abdominal defenses, or patient health. A 1% solution of HMW CMC may be used routinely during abdominal surgery of horses for prevention of postoperative adhesions. PMID- 12371776 TI - Use of multisite quantitative ultrasonography for noninvasive assessment of bone in horses. AB - OBJECTIVE: To evaluate the usefulness of multisite quantitative ultrasonography for noninvasive assessment of bone in horses. SAMPLE POPULATION: 12 healthy horses and both forelimbs from 8 clinically normal horses. PROCEDURE: For in vivo measurements, various regions of interest (ROI) were examined on the third metacarpal bone, radius, and tibia. Precision error for speed of sound (SOS) measurements was obtained by measuring each ROI of 4 horses 10 times with probe repositioning. Additionally, 3 operators measured each aspect of the third metacarpal bone of 6 horses 5 times each. For ex vivo measurements, third metacarpal bones were examined at 9 ROI, and SOS measurements were performed before and after soft tissue removal. One ROI of a single forelimb was subjected to 96 ex vivo measurements with 3 different contact media. RESULTS: The lateral aspect of the third metacarpal bone had significantly higher SOS values than the dorsal and medial aspect of the third metacarpal bone. No difference was obtained between SOS values of the lateral and medial aspect of the radius. The tibia had significantly higher SOS values than the lateral aspect of the radius and the dorsal and medial aspect of the third metacarpal bone. Intraoperator coefficients of variation ranged from 0.62 to 3.15%, and interoperator coefficients of variation ranged from 0.78 to 2.70%. Values of SOS were highest when silicone oil was used as the contact medium. CONCLUSIONS AND CLINICAL RELEVANCE: Speed of sound measurements obtained by quantitative ultrasonography in axial transmission mode can be used to precisely measure superficial cortical bone properties of third metacarpal bone, radius, and tibia in horses. PMID- 12371777 TI - The independent contribution of driver, crash, and vehicle characteristics to driver fatalities. AB - Several driver, crash, and vehicle characteristics may affect the fatality risk of drivers involved in crashes. To determine the independent contribution of these variables to drivers' fatality risk, we used data from single-vehicle crashes with fixed objects contained in the US Fatal Accident Reporting System. A multivariate logistic regression revealed that the odds ratio (OR) of a fatal injury increased with age, reaching 4.98 (99% confidence interval (CI) = 2.01 12.37) for drivers aged 80 + compared with drivers aged 40-49 years. Female gender (OR = 1.54, 99% CI = 1.35-1.76) and blood alcohol concentration greater than 0.30 (OR = 3.16. 99% CI = 1.96-5.09) were also associated with higher fatality odds. In comparison with front impacts, driver-side impacts doubled the odds of a fatality (OR = 2.26, 99% CI = 1.92-2.65), and speeds in excess of 111 kilometers per hour (kph: 69 mph) prior to or at impact were related to higher fatality odds (OR = 2.64, 99% CI = 1.82-3.83) compared with speeds of less than 56 kph (35 mph). Three-point seatbelts were protective against fatal injuries (OR = 0.46, 99% CI = 0.39-0.53 compared with no belt). These data suggest that increasing seatbelt use, reducing speed, and reducing the number and severity of driver-side impacts may prevent fatalities. The importance of age and gender suggests that the specific safety needs of older drivers and female drivers may need to be addressed separately from those of men and younger drivers. PMID- 12371778 TI - Using logistic regression to estimate the influence of accident factors on accident severity. AB - Logistic regression was applied to accident-related data collected from traffic police records in order to examine the contribution of several variables to accident severity. A total of 560 subjects involved in serious accidents were sampled. Accident severity (the dependent variable) in this study is a dichotomous variable with two categories, fatal and non-fatal. Therefore, each of the subjects sampled was classified as being in either a fatal or non-fatal accident. Because of the binary nature of this dependent variable, a logistic regression approach was found suitable. Of nine independent variables obtained from police accident reports, two were found most significantly associated with accident severity, namely, location and cause of accident. A statistical interpretation is given of the model-developed estimates in terms of the odds ratio concept. The findings show that logistic regression as used in this research is a promising tool in providing meaningful interpretations that can be used for future safety improvements in Riyadh. PMID- 12371779 TI - Traffic safety and the switch to a primary seat belt law: the California experience. AB - This study explores whether the change of an existing seat belt law from secondary to primary enforcement enhances traffic safety. In particular, we examine traffic fatalities and injuries in California from 1988 to 1997. During the first half of this period, California law provided for secondary enforcement of its mandatory seat belt law, but in 1993 it upgraded the law to primary enforcement. Controlling for the number of motor vehicle collisions, a Box-Tiao intervention analysis of the time series is used to compare the monthly fatalities and injuries before and after the change in the enforcement provision. The results show that California experienced an improvement in traffic safety in terms of a significant reduction in injuries, but the change in enforcement provision had no statistically significant impact on fatalities. PMID- 12371780 TI - The effect on accidents of technical inspections of heavy vehicles in Norway. AB - This paper presents a study of the effects on accidents of technical inspections of heavy vehicles (trucks and buses) in Norway. Multiple regression analysis is applied in order to estimate the effects of technical inspections, controlling for annual trend in accident rate, the number of new drivers and annual economic growth. It is found that abolishing inspections may result in an increase of 5 10% in the number of heavy vehicles involved in injury accidents; increasing the number of inspections by 100% is associated with a similar reduction in the number of accidents. These results are not statistically significant and highly uncertain. The study clearly illustrates many of the difficulties often encountered in non-experimental accident research. PMID- 12371781 TI - Signal detection in conditions of everyday life traffic dilemmas. AB - This paper shows how the paradigm of signal detection could serve as a viable means for the analysis of drivers' choices in conditions of everyday life traffic dilemmas. The participants were 28 drivers, most of them professional, who spend at least 6 h a day on the road. All agreed to have a note-taking silent passenger for the entire journey, every day during a period of 3-4 weeks. All completed the sensation-seeking questionnaire. Their 'to do or not to do' choices in conditions of four (out of a total of six) traffic dilemmas (amber light, distance keeping, stopping in road-crossing and merging in routes) were analyzable in terms of a modification of the paradigm of signal detection. In accord with the basics of the paradigm of signal detection, the rate of success of the drivers to detect signals of danger on the road (perceptual sensitivity) fell into the range of partial uncertainty (more than 50% and not too much above this level)! The choices made by thrill-and-adventure-seeking drivers were more lenient than the choices of the drivers who scored lower on this dimension. PMID- 12371782 TI - Further results from a trial comparing a hidden speed camera programme with visible camera operation. AB - As described in a previous paper [Accident Anal. Prev., 33 (2001) 277], the hidden camera programme was found to be associated with significant net falls in speeds, crashes and casualties both in 'speed camera areas' (specific signed sites to which camera operation is restricted) and on 100 km/h speed limit roads generally. These changes in speeds, crashes and casualties were identified in the trial area in comparison with a control area where generally highly visible speed camera enforcement continued to be used (and was used in the trial area prior to the commencement of the trial). There were initial changes in public attitudes associated with the trial that later largely reverted to pre-trial levels. Analysis of 2 years' data of the trial showed that falls in crash and casualty rates and speeds associated with the hidden camera programme were being sustained. It is not possible to separate out the effects of the concealment of the cameras from other aspects of the hidden speed camera programme, such as the four-fold increase in ticketing. This increase in speed camera tickets issued was an expected consequence of hiding the cameras and as such, an integral part of the hidden camera programme being evaluated. PMID- 12371783 TI - Factors influencing the probability of an incident at a junction: results from an interactive driving simulator. AB - Using data generated from a fixed-base interactive driving simulator, which was used to evaluate a driver decision aid, a model is built to predict the probability of an incident (i.e. an accident or a 'near miss') occurring as a result of a right-turn across left-hand traffic at an unsignalised junction. This can be considered to be the product of two separate probabilities, the first being the probability that the gap between a pair of vehicles in the traffic stream is accepted, and the second the probability that the time needed to cross the on-coming stream of traffic causes the time-to-collision with the nearest vehicle in this traffic stream to be less than a second. The model is developed from the results of experimental trials involving a sample of drivers, the majority of whom were aged 60 years or older, in order to demonstrate the effect of various parameters on these probabilities. The parameters considered include the size of the gap between successive vehicles, vehicle characteristics such as size, colour and velocity, driver characteristics such as age and sex, and both daytime and night-time conditions. PMID- 12371784 TI - Young drivers' decision making and safety belt use. AB - Past research in safety belt use has primarily focused on describing the relationship between drivers' demographic characteristics and safety belt use. This study compared the impact of situational factors (the direction of collision, the type of road, and the presence of an airbag system), demographic factors, and constructs (criteria) elicited from subjects regarding safety belt use. Based on the results obtained, a conceptual model was developed. The model indicated that drivers' decision-making process when judging the level of accident risk and usefulness of safety belts differs from those that determine actual behavior. Perceived risk was related to road type, perceived consequences of an accident, perceived usefulness of safety belts, self responsibility, the time available for the driver to warn the other driver, dangerous behavior, and gender. These variables showed that people were able to rationally judge the risk. Despite the fact that people judge behavior in what appeared to be a rational manner, risk perception was not a good predictor of belt use. Belt use was mainly influenced by individual factors such as gender, grade point average (GPA), and age. Other factors impacting safety belt use included the perceived frequency of an accident and the S.D. of perceived usefulness of safety belts. PMID- 12371785 TI - 'Road rage' in Arizona: armed and dangerous. AB - Little is known about the relationship between firearm carrying and hostile behavior on the roadway. To explore a possible association between firearm carrying by drivers and hostile driving behavior we conducted a random-digit-dial survey of 790 licensed drivers in Arizona. In addition to demographic questions, we asked whether respondents had carried a gun while driving in the 12 months prior to the survey. Respondents were also asked if they, in anger, had personally made obscene gestures, cursed or shouted at other drivers, impeded another drivers progress with their vehicle, aggressively 'followed another driver too closely', or brandished a gun at another driver. We used multivariable logistic regression to explore correlates of hostile driving behavior while taking into account several demographic and behavioral characteristics. Overall 11% of drivers always (4%) or sometimes (7%) carried a gun with them in their vehicle; 34% report having made obscene gestures/cursed/shouted angrily; 28% report aggressively following or blocking other drivers with their vehicle. In both crude and multivariate adjusted analyses, self-report of engaging in hostile behavior while driving was significantly more common among men, young adults, and individuals who carried a firearm in their car. Our findings suggest that, at least among Arizona motorists, having a gun in the car is a strong marker for aggressive and illegal behavior behind the wheel. PMID- 12371786 TI - The effects of standard enforcement on Michigan safety belt use. AB - The purpose of this study was to assess the effects of standard enforcement legislation on safety belt use in Michigan through a series of seven statewide direct observation surveys. A secondary purpose of the study was to compare the results in Michigan to the results in other states that have changed the provision of their mandatory safety belt use law from secondary to standard enforcement. The study found that standard enforcement has been effective in increasing safety belt use in Michigan. Immediately following the implementation of standard enforcement, Michigan's belt use rate increased to 83.5%, 13.4 percentage points higher than the highest rate previously observed. One year after the change, safety belt use in Michigan was still nearly 10 percentage points greater than the highest observed rate before standard enforcement legislation was enacted. Results indicated that safety belt use decreased slightly in the year following the implementation of standard enforcement. This appears to be an overall trend across all observed groups, and not due to any single demographic category. The results also suggest that standard enforcement legislation appears to have a greater effect on groups with historically low belt use, such as young people, males, passengers, and Black/African-Americans. When compared with other states that have made the change from secondary to standard enforcement, the increase in the safety belt use rate in Michigan was comparable to the increase seen in states with relatively high safety belt use prior to standard enforcement. However, states that had low safety belt use rates prior to adopting standard enforcement legislation observed a larger percentage point increase in the year following their change to standard enforcement. PMID- 12371787 TI - Effects of an afternoon nap on nighttime alertness and performance in long-haul drivers. AB - The effects of an afternoon nap on alertness and psychomotor performance were assessed during a simulated night shift. After a night of partial sleep restriction, eight professional long-haul drivers either slept (nap condition) or engaged in sedentary activities (no-nap condition) from 14:00 to 17:00 h. Alertness and performance testing sessions were conducted at 12:00 (pre-nap baseline), 24:00, 02:30, 05:00 and 07:30 h, and followed 2-h runs in a driving simulator. In the nap condition, the subjects showed lower subjective sleepiness and fatigue, as measured by visual analog scales, and faster reaction times and less variability on psychomotor performance tasks. Electrophysiological indices of arousal during the driving runs also reflected the beneficial effects of the afternoon nap, with lower spectral activity in the theta (4-7.75 Hz), alpha (8 11.75 Hz) and fast theta-slow alpha (6-9.75 Hz) frequency bands of the electroencephalogram, indicating higher arousal levels. Thus, a 3-h napping opportunity ending at 17:00 h improved significantly several indices of alertness and performance measured 7-14 h later. PMID- 12371788 TI - Responses of teenagers and their parents to California's graduated licensing system. AB - In 1998, California adopted a strong graduated licensing system that lengthened the learner's permit stage from 1 month to a mandatory 6 months and introduced passenger and nighttime restrictions for initial license holders. The passenger restriction (no passengers younger than 20 for the first 6 months) is stronger than such restrictions in any other state; the nighttime ban is relatively weak, not beginning until midnight. Surveys were undertaken to learn what teenagers and their parents thought about the new requirements and how they responded to them. Two groups of beginning California license holders were surveyed three times during the first year of licensure; their parents were interviewed twice. One group (n = 543) was subject to the graduated licensing requirements, the other (n = 814) was not. Parents strongly endorsed the new system. The vast majority approved of the new permit requirements and the nighttime and passenger restrictions. Among parents whose children were subject to the new requirements, 79% were strongly in favor of the new system and only 4% were neutral or opposed. Teenagers were less favorable toward the new requirements. Most approved of the new learner's permit rules, and the majority of teenagers favored the night restriction, but only about one-third endorsed the passenger restriction. Compliance with the new rules was not close to universal, but the new licensing system resulted in young people holding their learner's permits longer, accumulating more practice driving prior to licensure and decreased the amount of reported driving after midnight and transportation of teenagers when initially licensed. Most teenagers subject to the new rules said they were able to do the activities they wanted despite the changes; almost three-quarters said they were not affected much by either the nighttime or passenger restriction. Overall the results indicate that the new licensing system is accepted favorably by teenagers and their parents and has substantially increased the types of behaviors that collectively should lead to crash and injury reductions. PMID- 12371789 TI - Ethical conflicts over access to services: patient effects and worker influence in home health. AB - A survey of home health social workers (N = 51) explored the effects on patients of ethical conflicts over access to services. The findings suggest that patients were as likely to be discharged or not receive services as they were to receive the services without paying a fee. Social workers rated themselves as moderately influential in the resolution of the conflict. Their influence was significantly correlated with patients more often receiving services and less often being discharged. Social work influence was enhanced by recognition of professional expertise and/or through informal networking within the agency. Implications for practice and education are discussed. PMID- 12371791 TI - Translating psychosocial insight into ethical discussions supportive of families in end-of-life decision-making. AB - A large number of Americans would rather rely on family and friends more than their physicians about end-of-life care and decisions. Moving beyond traditional clinical ethics and its dyadic focus on the physician-patient relationship, this article presents an approach to ethical decision-making at the end of life that is more inclusive of the patient's family and has the potential to advance social work practice in end-of-life care. Initial attention is given to how psychosocial and bioethical perspectives and practices interact to shape understanding of moral issues in end-of-life decisions. Morally relevant principles are then adapted from contextual therapy as being useful for including more of a family focus and viewing ethical decision-making at the end of life as a family process. Specifically, focus is on exploring the ethical dynamics of family systems that impact the decision-making process and translating psychosocial insight into ethical discussions that are supportive of families. The case of a patient with sudden and unexpected brain death and without advance directives demonstrates one family's unresolved grief and illustrates how its members were helped to reason morally about end-of-life choices. Contributions of a social worker and bioethicist are illustrated. PMID- 12371790 TI - Social work services in home health care: challenges for the new prospective payment system era. AB - The implementation of the Prospective Payment System (PPS) provides a unique opportunity for social workers to be better integrated into home health care. To do so, it is important for social workers to define their roles and eliminate any barriers to providing social workers services, which may improve patient outcomes. Two focus groups with home health nurses (n = 10) and social workers (n = 8) were conducted in a large urban home health agency to define social work roles and identify barriers to providing social work services. This paper categorizes the barriers to providing social work services into informational, systems/organizational, and inter-professional barriers and presents possible solutions to these barriers as home health agencies strive to provide care under PPS. PMID- 12371792 TI - Take some time to look inside their hearts: hospice social workers contemplate physician assisted suicide. AB - This article presents a subset of data from a larger study that explored the impact of the legal choice of physician assisted suicide (PAS) on hospice providers. Eight social workers shared their personal and professional voices about a very controversial and difficult issue. Oregon is the only place in the country where PAS is legal and these social workers practice in an environment where the choice of PAS has been an option for two years. Three overarching themes emerged from the data: (1) the dilemmas that arise from the hospice philosophy; (2) the conflicts that emerge between the choice of PAS and social works' cardinal values and practice principles; and (3) the struggles with personal values and PAS. PMID- 12371793 TI - The development of programs for pregnant and parenting teens. AB - The impact of early pregnancy and parenting on adolescents is well documented, with its negative effects on achieving adequate education, job skills training, gainful employment and economic independence duly recognized. While current interest and resources focus on abstinence only programs as major pregnancy prevention initiatives, we are increasingly failing those young women who have chosen early parenthood as a life option. This article will describe the efforts of a large urban hospital to provide hospital-based comprehensive services to pregnant and parenting teens to reduce the negative consequences to them and their children, and to help them to become effective parents and lead healthy, productive lives. PMID- 12371794 TI - Barriers and influences in seeking health care among lower income minority women. AB - The purpose of this study was to identify and describe perceived barriers to seeking health care, determine perceptions of confidence in health care practitioners, and explore strategies to enhance, promote, and improve early health care intervention among low income minority women. Focus group methodology was used to collect data and content analysis was used to analyze the data. Results revealed four broad categories for discussion: (a) confidence in the physician, (b) frequency of engaging in screening procedures, (c) barriers and influences in seeking health care, and (d) a wish list for covered health care services. The study underscored the importance of both spirituality and family in the lives of aging minority women. The paper includes implications for public policy and suggests an agenda for public policy advocates in the new millennium. PMID- 12371796 TI - Oncologic emergencies: superior vena cava syndrome. PMID- 12371797 TI - A 52-year-old man with sudden onset of a facial rash. PMID- 12371798 TI - So you're being sued: do's and don'ts for the defendant. AB - Many physicians find themselves involved in malpractice litigation during their careers, either as defendants or as expert witnesses. Knowing a few do's and don'ts can help make the process easier, prevent professional and financial disaster for you or other medical professionals, and even prevent a lawsuit altogether. PMID- 12371799 TI - The newer antimuscarinic drugs: bladder control with less dry mouth. AB - Two newer antimuscarinic anticholinergic drugs--tolterodine and extended-release oxybutynin--are approximately as effective in treating overactive bladder as immediate-release oxybutynin, but are more tolerable. I review clinical trial data on the newer agents. PMID- 12371800 TI - Acute ischemic stroke: is there a role for hypothermia? AB - Preliminary clinical trials suggest that therapeutic hypothermia might improve the outcomes of patients with acute stroke. Definitive trials are under way. PMID- 12371801 TI - A 74-year-old woman with hemodynamic complications of acute MI. PMID- 12371802 TI - Poor hypertension control: let's stop blaming the patients. AB - Physician behavior--not patient noncompliance--is the major cause of poor hypertension control in the United States, many studies show. Hypertension control is unlikely to improve unless physicians become more aggressive in treating mildly elevated systolic blood pressure. PMID- 12371803 TI - Elder abuse and neglect: what physicians can and should do. AB - Although reports of elder abuse to official agencies have been steadily increasing, physicians report only 2% of reported cases. Multiple barriers to reporting exist. This article reviews the terminology, epidemiology, and clinical signs associated with elder abuse in the community and offers practical strategies for intervention. PMID- 12371804 TI - Treating hyperglycemia in type 2 diabetes: new goals and strategies. AB - To achieve glycemic goals in type 2 diabetes, one must usually use combinations of oral agents or oral agents plus insulin. This paper discusses the metabolic derangements of type 2 diabetes, the different classes of antihyperglycemic drugs, and strategies for using these drugs rationally. PMID- 12371805 TI - Should exercise electrocardiography be a routine part of the periodic health physical? PMID- 12371806 TI - Hot extraction and characterization of a ligninlike fraction involved in larvicidal effects of decomposed leaf litter against mosquito. AB - Hot water-extraction was performed on decomposed leaf litter in order to solubilize the toxic fraction involved in the dietary interaction against mosquito larvae in subalpine breeding sites. The toxic fraction was partially extracted by water with an optimum temperature of 60 degrees C and recovered in an insoluble form. Phytochemical characterization was achieved through differential enzymatic hydrolyses, using the laccase mediator delignifying system, and aluminum chloride chelation monitored by standard bioassays; comparative spectrophotometric analyses in ultraviolet light after solubilization in acetyl bromide; and comparative reversed-phase high-performance liquid chromatography of the phenolic aldehydes after alkaline nitrobenzene oxidation. The results suggested the involvement of ligninlike compounds in the toxicity of the isolated fraction. Toxicity of this fraction appeared far stronger than that of the crude leaf litter. The involvement of this ligninlike fraction in the dietary toxicity of leaf litter against larval mosquito was then investigated. PMID- 12371807 TI - Peramine alkaloid variation in Neotyphodium-infected Arizona fescue: effects of endophyte and host genotype and environment. AB - We determined concentrations of peramine, the only alkaloid produced by Neotyphodium-infected (E+) Arizona fescue plants (of the four major types typically assayed in infected grasses), in a long-term field experiment. Four plant genotypes with (E+) and without (experimentally removed, E-) their respective haplotypes (two haplotypes in two plant genotypes) of Neotyphodium were grown in the field under manipulated soil moisture and nutrients. Peramine production required the presence of the endophyte; plants without their endophytes did not contain peramine. Whereas the endophyte was necessary for peramine production, levels of peramine did not vary by Neotyphodium haplotype within plant genotypes. Furthermore, peramine levels did not differ among soil moisture and nutrient treatments, and growth and reproductive measures of the host grass explained little of variation in peramine levels. Instead, peramine levels differed significantly between plant genotypes harboring the same endophyte haplotype, suggesting that plant genotype, rather than endophyte haplotype or environment, largely determines levels of peramine in Arizona fescue. The results suggest that whereas the endophyte is required for peramine production, the plant genotypic background in which the endophyte is embedded, rather than endophyte haplotype or environmental factors, mostly influences peramine levels within this population of Arizona fescue. PMID- 12371808 TI - Semiochemical-mediated flight responses of sap beetle vectors of oak wilt, Ceratocystis fagacearum. AB - The sap beetle, Colopterus truncatus (Coleoptera: Nitidulidae), is one of the primary vectors of the oak wilt pathogen, Ceratocystis fagacearum, in the north central United States. Field behavioral assays utilizing various release rates and blends of three methyl-branched hydrocarbon aggregation pheromone components showed that flight responses of this beetle were similar in Illinois and Minnesota populations. In both locations, both sexes of the beetle responded synergistically to a combination of the three-component pheromone and fermenting whole-wheat bread dough. Further, Colopterus truncatus preferred a high release rate over a low release rate of the three-component blend. In both locations, the response of C. truncatus to a simplified version of the pheromone consisting of (2E,4E,6E)-3,5-dimethyl-2,4,6-octatriene (1) and (2E,4E,6E,8E)-3,5,7-trimethyl 2,4,6,8-decatetraene (3) was not significantly different from the response to the three-component blend. An experiment in Illinois with all possible combinations of the components demonstrated that the decatetraene (3) was the crucial component in the blend; of all treatments, the maximal response was elicited by 3 + dough. Chipped bark, phloem, and xylem from northern pin oak, Quercus ellipsoidalis, was not attractive to C. truncatus in Minnesota. During a weekly survey over two seasons in Minnesota, C. truncatus flew in response to the three component pheromone between early April and early July, with the maximum responses coming on May 4, 2000 and April 20, 2001. During both years, more than 98% of the beetles were trapped between April 14 and June 1. During the same survey, Glischrochilus spp. (Nitiduildae) flew during longer periods of the summer, particularly in 2001. The sex ratio of C. truncatus responding during all experiments was female-biased (1.8:1, female-male), which is characteristic of other male-produced coleopteran aggregation pheromones. Other sap beetles that play a minor role in the pathobiology of C. fagacearum also responded in experiments conducted in Minnesota. Carpophilus brachypterus Say was cross attracted to the two- and three-component blends of the C. truncatus pheromone and dough, whereas two Glischrochilus spp. were attracted to all treatments that contained dough. PMID- 12371809 TI - Purine metabolizing capability of Enterobacter agglomerans affects volatiles production and attractiveness to Mexican fruit fly. AB - We investigated two strains of Enterobacter agglomerans that differ in their ability to metabolize uric acid for (1) attractiveness to sugar-fed Mexican fruit flies, and (2) production of volatile chemicals that may be responsible for the attractiveness. The two strains were cultured on a medium that contained uric acid as the primary nitrogen source to simulate bird feces, a natural substrate for this bacterium. Active cultures of both strains were more attractive than uninoculated uric acid medium to both sexes of sugar-fed flies in wind-tunnel bioassays. The uricase(+) strain was more attractive than the uricase(-) strain to males and to females <9 days old, but not to older females. Volatiles found by solid-phase microextraction in greater amounts in headspace above active cultures of both strains than above uninoculated medium were ammonia, dimethyldisulfide, 3 methylbutanol, 2-phenylethanol, 2,5-dimethylpyrazine, and trimethylpyrazine. The uricase(+) strain produced more ammonia, dimethyldisulfide, and trimethylpyrazine than the uricase(-) strain. An additional chemical, 3-hydroxybutanone, appears to be produced exclusively by the uricase(+) strain. The uricase(-) strain produced more 2-phenylethanol than the uricase(+) strain. Differences in volatiles are consistent with the generally greater attractiveness of the uricase(+) strain compared with the uricase(-) strain as ammonia, 3-hydroxybutanone, and trimethylpyrazine have been demonstrated attractive to sugar-fed Mexican fruit flies. PMID- 12371810 TI - Kairomone strains of Euclytiaflava (Townsend), a parasitoid of stink bugs. AB - Tachinid flies commonly use the pheromones and allomones of stink bugs (Pentatomidae) as host-finding kairomones. Pheromone-baited traps for predaceous (Podisus spp.) and phytophagous (Euschistus spp.) pentatomids were used to obtain tachinid parasitoids in order to study the semiochemical relationships between these parasitic flies and their stink bug hosts. Gas chromatography electroantennogram detector (GC-EAD) experiments and field tests were conducted to determine if pheromone strains of the tachinids, Euclytia flava and Hemyda aurata, occur in nature and to determine if the EAD-active compound, (E)-2 octenal (a common allomone compound of Heteroptera), affects attraction of tachinid parasitoids to synthetic Podisus pheromones. Addition of (E)-2-octenal to Podisus spp. synthetic pheromones in field traps tended to suppress attraction of the bugs, whereas (E)-2-octenal decreased, did not affect, or increased pheromonal attraction of tachinid parasitoids depending on the host species pheromone being tested and the habitat type in which the traps were deployed. Evidence from GC-EAD experiments of E. flava associated with different stink bug hosts suggested that kairomone-strains of this tachinid parasitoid coexist naturally. The significance of cryptic kairomone strains of parasitoids for classical biological control is discussed, and the mechanisms whereby parasitoids evolve kairomonally mediated host-shifts is considered. PMID- 12371811 TI - Field response of Ips paraconfusus, Dendroctonus brevicomis, and their predators to 2-methyl-3-buten-2-ol, a novel alcohol emitted by ponderosa pine. AB - Methylbutenol (MBO) is a major component of the aggregation pheromone of the European spruce beetle Ips typographus and also has been found to be emitted in large amounts by several species of pine native to western North America. This study investigates the influence this signal may have on the behavior of North American bark beetles and examines whether MBO functions as a defensive compound for emitting pines. The response of two North American bark beetles (Ips paraconfusus and Dendroctonus brevicomis) and their predaceous beetles (Trogositidae and Cleridae) to MBO, pheromone, and monoterpenes in varying release rates was investigated in the field using Lindgren funnel traps. MBO exhibited no repellent properties when tested alone, nor did MBO appear to have any effect on the aggregation response of these bark beetles and their predators to their pheromones. These results provide no support for a defensive function of MBO. PMID- 12371812 TI - Toxic and aversive diterpenes of Euphorbia esula. AB - Leafy spurge (Euphorbia esula L.), a plant introduced into the Great Plains of North America from Europe, has become a serious economic and ecological threat to the productivity of agricultural and natural areas. Cattle, the predominant livestock species on the Great Plains, as well as common wild ruminant species in this region appear to consume little if any leafy spurge. This is likely because they experience a toxic response after consuming small amounts of this plant, and they consequently learn to avoid it. Domestic sheep and goats can consume considerable amounts of leafy spurge and are used to help control it, but even these species may suffer a toxic response at high levels of intake. Toxic diterpene ingenols have been isolated from leafyspurge tissues, but compounds in this plant have not been evaluated with respect to their capacity to induce food aversion learning in ruminants. We conducted bioassay-guided fractionations of leafy spurge in an attempt to isolate toxic and aversive compounds. These bioassay-guided fractionations led to identification of ingenol and one of its diesters as two toxic compounds in leafy spurge that are potentially aversive to cattle. PMID- 12371813 TI - Geophagy as a correlate of folivory in red-handed howler monkeys (Alouatta belzebul) from eastern Brazilian Amazonia. AB - Howler monkeys, Alouatta spp., are the most folivorous of neotropical primates (Platyrrhini), although Amazonian red-handed howlers (Alouatta belzebul) are relatively frugivorous. The feeding ecology of a free-ranging group of A. belzebul was monitored at a site in eastern Brazilian Amazonia over a tenmonth period (November 1997 to August 1998). The first half of the study period encompassed the peak of the wet season, during which the study group's diet was predominantly frugivorous (scan sample records: 53.5% fruit, 40.8% leaves), and the second half, the transition from wet to dry season, characterized by a marked shift to folivory (18.9% fruit, 77.9% leaves). This shift was accompanied by a marked increase in mature, as opposed to flush leaves, which are relatively rich in secondary compounds. Ingestion of soil from termitaria was recorded on a total of 26 occasions, all of which occurred during the second half of the study period. Soil from termitaria was relatively rich in elements such as Ca and Na and in organic carbon, in comparison with that from the forest floor. The extent to which the monkeys ingested soil for their mineral supplements, or as an aid for the digestion of leaves, in particular the absorption of secondary compounds, remains unclear. The marked correlation with the observed patterns of folivory suggests that the latter function may have been the primary motive for geophagy in this species. PMID- 12371814 TI - Occurrence of p-nonylphenol isomers in wild species of Cichorium endivia subsp. divaricatum. AB - p-Nonylphenol isomers and other aromatic compounds were found in roots and stalks of the plant Cichorium endivia subsp. divaricatum. These compounds were characterized by high-resolution capillary GC-MS. PMID- 12371815 TI - Defensive secretion of Therea petiveriana: chemical identification and evidence of an alarm function. AB - The volatile constituents of the supposed defensive secretions of the glandular pouches of the adults of both sexes of the cockroach Therea petiveriana have been shown to contain N-3-methylbutylacetamide (MBA) and N-3-methylbutylpropanamide (MBP), which represented 60% of the volatile fraction. The other 40% included acidic, aromatic, and aldehydic compounds. Behavioral experiments demonstrated that the secretion acts as an alarm pheromone for adults. PMID- 12371816 TI - Identification of sex pheromone composition of click beetle Agriotes brevis candeze. AB - Geranyl butyrate (GB) and (E, E)-farnesyl butyrate (FB) were identified in the pheromone gland extract of females of the click beetle, Agriotes brevis (Candeze) (Coleoptera: Elateridae) as the major sex pheromone components. Polyethylene vial dispensers containing 20-200 mg of a 1:1 mixture caught high numbers of beetles. Captures did not decrease even after 73 days of field exposure of dispensers. At sites where both Agriotes sputator L. and A. brevis were present, the above baits were selectively catching only A. brevis, despite the fact that GB is also the main pheromone component of A. sputator, suggesting that FB has a role in reproductive isolation. In the early part of the season, traps into which the insects could both crawl and fly captured more A. brevis than designs where the insects could only fly in. Trap design was not important later in the season. This indicates the need for future development of a trap suitable for use throughout the whole season. PMID- 12371817 TI - Pheromone-based trapping of West Indian sugarcane weevil in a sugarcane plantation. AB - Attraction of Metamasius hemipterus (Oliver) to gallon and bamboo traps baited with insecticide-treated sugarcane, the male-produced pheromone, 4-methyl-5 nonanol, and 2-methyl-4-heptanol is more efficient if ethyl acetate is added. The optimal traps are ground-level gallon traps baited with insecticide-laced sugarcane, pheromone, and ethyl acetate. Capture rates of ground-level gallon traps are doubled by placing an insecticide-laced pad under the trap, but significantly decreased by placing the trap on a stick above ground. The efficiency of ground-level gallon traps is the same as ground level ramp traps. Mass-trapping M. hemipterus in newly planted sugarcane using ground level bamboo traps baited with insecticide-laced sugarcane and pheromone over six months revealed populations were low for the first two months, became maximum at five months, and declined thereafter. Capture rates of traps bordering newly planted and mature sugarcane were not significantly different from capture rates of traps in the interior of the plots. Capture rates of bamboo traps containing only insecticide-laced sugarcane and deployed at 30 traps/ha averaged 6 weevils/trap/week compared with 66 weevils/trap/week for traps additionally containing pheromone lures and deployed at 5 traps/ha. Capture rates for bamboo traps baited with insecticide-laced sugarcane and pheromone and deployed at 10 and 15 traps/ha were 43 and 38 weevils/trap/week, respectively. Total captures were higher in those plots with a higher density of insecticide-laden sugarcane and pheromone baited traps, and the differences were approximately proportional to trap density in the range of 5-15 traps/ha. Capture rates of traps containing insecticide-laced sugarcane and pheromone were always higher than of traps containing only insecticide-laced sugarcane, but in the first two months after planting the differences were much greater than in months 3-6 after planting. PMID- 12371818 TI - Intermale variation in aggregation pheromone release in Prostephanus truncatus. AB - Intermale variation in pheromone signaling has been confirmed and quantified by measurements of pheromone produced by single adult male Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae). Males varied in both the amounts of the two components of their aggregation pheromone and the ratio of one component to the other. The mean rates of production of the pheromone components T1 and T2 were 1.9 and 0.5 microg/day, respectively. There were repeatable differences among males in the amounts of T2 produced and the proportion of T1 in the pheromone blend over two weeks. Of the 15 males studied, one released a large burst of pheromone in a short period, while the remainder, if they did release, did so over an extended period. This suggested that there may be two alternative release strategies and the significance of this is discussed. PMID- 12371819 TI - Trans-bergamotenes-male pheromone of the ectoparasitoid Melittobia digitata. AB - The first male-produced sex attractant pheromone in the parasitic Hymenoptera has been identified. The elaborate courtship behavior of Melittobia digitata, an idiobiont that parasitizes the last larval instar or the pupal stage of solitary bees and wasps, involves a series of coordinated movements of legs, wings, and antennae, initiated after the female is attracted to the blind, flightless male. We identified alpha- and beta-trans-bergamotene as the active compounds of the male M. digitata sex attractant. Variation in the release of the sex pheromone by males and the pheromone load during aging is also described. PMID- 12371820 TI - Evidence that ribonuclease activity present in beetle regurgitant is found to stimulate virus resistance in plants. AB - Phaseolus vulgaris L. cv. 'Pinto' bean is a local lesion host for the plant pathogen Southern bean mosaic virus (SBMV) and its vector is the Mexican bean beetle, Epilachna varivestis Mulsant. The objective of this study was to determine if prior feeding by the beetle would affect 'Pinto' bean's resistance to SBMV and determine if ribonuclease (RNase), a major constituent of beetle regurgitant, mediated the plant's response to the virus. 'Pinto' bean plants fed upon by beetles had increased resistance to plant viruses compared to non-wounded or mechanically wounded and buffer-treated plants. Plants that were mechanically wounded and treated with RNase had increased resistance to plant viruses that was equal to plants fed upon by adult beetles. The induction of plant pathogen defenses could be a good adaptation for the plant in the presence of a beetle and pathogen threat. This evidence suggests that RNase activity in the beetle regurgitant could function as an insect-derived elicitor of plant resistance to viruses. PMID- 12371821 TI - Do Eurasian beavers smear their pelage with castoreum and anal gland secretion? AB - The scent-matching hypothesis postulates that scent marks provide an olfactory link between a resident owner and his territory, and that this enables intruding animals to recognize the chance of escalated conflicts. However, it is unclear if Eurasian beavers (Castor fiber) mark their own pelage with castoreum from their castor sacs (i.e., the same material used in territorial marking); and/or if beavers mark their pelage with anal gland secretion (AGS) from the anal glands to waterproof the pelage and to act as a "living-scent mark." Chemical analysis (gas chromatography and mass spectrometry) of hair samples from 22 live-trapped beavers revealed that castoreum compounds were not present in any samples, AGS compounds were found from 3 animals (13.6%) around the cloaca, and the compound squalene was found in all the samples. Beavers may release castoreum directly into the water when it meets an intruder. Thereby, the "scent mark" in the water can provide an olfactory link between a resident owner and his territory. Squalene, in contrast to AGS, may be essential for keeping beaver pelts water repellant. PMID- 12371822 TI - Ni(0)/ZnCl(2)-promoted coupling of enones and enynes. Domino process via formation of three C-C bonds and cleavage of one C-C bond. AB - Domino coupling of enones and enynes, which proceeds via the formation of three carbon-carbon bonds and the cleavage of one carbon-carbon bond, was developed using a Ni(0) and ZnCl2 system. PMID- 12371823 TI - Heavy-atom kinetic isotope effects, cocatalysts, and the propagation transition state for polymerization of 1-hexene using the rac-(C(2)H(4)(1 indenyl)(2))ZrMe(2) catalyst precursor. AB - Using 13C NMR techniques, the 12C/13C kinetic isotope effects (KIEs) for the polymerization of 1-hexene catalyzed by rac-(C2H4(1-indenyl)2)ZrMe2 in the presence of four different cocatalysts (tris(pentfluorophenyl)borane, tris(pentafluorophenyl)alane, anilinium tetrakis(pentafluorophenyl)borate, and methylalumoxane) have been determined. All cocatalysts yield similar KIEs within experimental uncertainty, with values of 1.009(1) and 1.017(2) at C1 and C2, respectively. Ab initio DFT computational modeling of the polymerization KIE indicates that alkene binding to the catalyst must be reversible, with the majority of the KIE developing in the subsequent migratory insertion reaction. PMID- 12371824 TI - Characterization of endoperoxide and hydroperoxide intermediates in the reaction of pyridoxine with singlet oxygen. AB - The photosensitized oxidation of vitamin B6, pyridoxine, is investigated by product and kinetic analysis. Singlet oxygen quenching rates, measured by time resolved laser flash generation of singlet oxygen followed by monitoring singlet oxygen phosphorescence decay, confirm previous observations that pyridoxine is a moderate quencher. The quenching rate for 3-methoxypyridine is 100 times slower than that for 3-hydroxypyridine, indicating the hydroxy moiety is required for efficient quenching. The chemical quenching rate constant, kr, was estimated by comparison with a known singlet oxygen reaction. Results indicate that the chemical quenching rate of pyridoxine dominates the total quenching. The major reaction product in methanol was isolated and characterized by NMR and MS. The data are consistent with a solvent adduct of the substituted 2,5-pyridinedione. At low temperature, two semistable intermediates were characterized by NMR. The data are consistent with a hydroperoxide and endoperoxide. These intermediates suggest initial attack of singlet oxygen para to the hydroxy group followed by either proton transfer to form the hydroperoxide or addition of the peroxide to the imine to form the endoperoxide. In the presence of protic solvents, the solvent adducts to the imine and elimination of water yield the observed 2,5 pyridinedione product. PMID- 12371825 TI - Chemical properties of a para-benzyne. AB - 5,8-Didehydroisoquinolinium ion, a para benzyne analogue, was generated in a Fourier transform ion cyclotron resonance mass spectrometer, and its reactivity toward various neutral reagents was examined. A direct comparison of the reaction kinetics of the para benzyne, a meta isomer, and analogous monoradicals, indicates that the para benzyne is a poorer electrophile but a more reactive radical than its meta isomer. PMID- 12371826 TI - A new route to coordination complexes of nitroxyl (HN=O) via insertion reactions of nitrosonium triflate with transition-metal hydrides. AB - Nitrosonium triflate reacts with cold methylene chloride solutions of mer,trans ReH(CO)3(PPh3)2 (1) with 1,1-insertion of NO+ into the Re-H bond to give the orange nitroxyl complex [mer,trans-Re(NH=O)(CO)3(PPh3)2][SO3CF3] (3) in 86% isolated yield. Use of [NO][PF6] or [NO][BF4] gives analogous insertion products at low temperature, which decompose on warning to ambient temperature to the fluoride complex mer,trans-ReF(CO)3(PPh3)2 (4). A related 1,1-insertion is observed in the reaction of 1 with [PhN2][PF6] in acetone that affords the yellow orange phenyldiazene salt [mer,trans-Re(NH=NPh)(CO)3(PPh3)2][PF6] (2), which has been characterized by X-ray crystallographic methods. The methyl derivative mer,trans-Re(CH3)(CO)3(PPh3)2 (5) also undergoes a 1,1-insertion reaction with [NO][SO3CF3] to give the nitrosomethane adduct [mer,trans Re{N(CH3)=O}(CO)3(PPh3)2][SO3CF3] (6) as red crystals in 75% yield. The nitroxyl complex [cis,trans-OsBr(NH=O)(CO)2(PPh3)2][SO3CF3] (8) can be similarly prepared as orange crystals in 52% yield by reaction of cis,trans-OsHBr(CO)2(PPh3)2 (7) with [NO][SO3CF3] in cold methylene chloride solution. PMID- 12371828 TI - New liquid crystalline phases with layerlike organization. AB - Novel lamellar mesophases which are quite distinct from conventional smectic mesophases were obtained with a bolaamphiphilic triblock molecule composed of a rigid biphenyl core, two polar 2,3-dihydroxypropoxy groups in the terminal 4- and 4'-positions, and a semiperfluorinated chain [O(CH2)6C10F21] in the lateral 3 position. The competitive combination of microsegregation and rigidity in this molecule leads to layer structures in which the bolaamphiphilic cores segregate from the lateral chains into distinct sublayers. In these sublayers the biphenyl cores are aligned parallel to the layer planes. Decreasing the temperature leads to a subsequent inset of orientational and positional order of the biphenyl unit, which leads to a transition from an uniaxial SmA phase to a biaxial SmAb phase and finally to a mesophase with an additional periodicity within the aromatic sublayers. Here, microsegregation occurs on two distinct levels: The segregation of the nonpolar chains from the aromatic cores leads to the "bulk" layer structure and segregation of polar and aromatic subunits within the aromatic sublayers gives rise to an additional periodicity within the aromatic sublayers. These phases can be regarded as smectic phases built up by quasi-2D layers with nematic, respectively SmA-like order, separated by isotropic layers of the lateral chains. PMID- 12371827 TI - Characterization of the relevant excited states in the photodissociation of CO ligated hemoglobin and myoglobin. AB - The relevant excited states in the rapid photodissociation process of hemoglobin and myoglobin are examined by means of time-dependent density functional theory. Our calculations clearly show that the photodissociation is mediated by two repulsive states (5 A' ' and 3 A') which cross the lowest excited states (1 A' and 1 A' ') at an internuclear Fe-C distance of about 2 A. Electron detachment/attachment density plots nicely explain the repulsive nature of the 5 A' ' and 3 A' states. PMID- 12371829 TI - Social isomers in encapsulation complexes. AB - We introduce here a new form of isomerism-social isomerism-that arises when two different guests are confined to a cylindrical host capsule. The isomerism deals with the orientationof one guest with respect to the other when they are in a container. Specifically, para-substituted toluenes coencapsulated with typical solvent molecules such as benzene, chloroform, and cyclohexane show two different isomeric arrangements that interconvert slowly on the NMR time scale. The dimensions of the capsule prevent the guests from squeezing past one another or tumbling freely. PMID- 12371830 TI - How electron flow controls the thermochemistry of the addition of olefins to nickel dithiolenes: predictions by density functional theory. AB - The reaction of a nickel dithiolene complex (1) and ethylene is a two-step process, in which the trans-product (2) forms first in the direct addition of the olefin to 1, while the more thermodynamically stable cis-product (3) involves isomerization of 2. The introduction of electron-withdrawing groups (cyano or trifluoromethyl) not only significantly lowers the activation energy (TS1) for the formation of trans-product, but it also strongly stabilizes the products (2, 3) such that they are favored by the free energy. However, these substituents leave the barrier for the conformational transformation step (TS2) nearly unchanged. On reduction, the previously favored adduct is now strongly disfavored. PMID- 12371831 TI - Asymmetric total synthesis of (-)-azaspirene, a novel angiogenesis inhibitor. AB - The asymmetric total synthesis of (-)-azaspirene, an angiogenesis inhibitor, has been accomplished, establishing its absolute stereochemistry. The key steps are a MgBr2.OEt2-mediated, diastereoselective Mukaiyama aldol reaction, a NaH-promoted, intramolecular cyclization of an alkynylamide, and the aldol reaction of a ketone containing functionalized gamma-lactam moiety without protection of tert-alcohol and amide functionalities. PMID- 12371832 TI - Diametrically opposite trends in alkene insertion in late and early transition metal compounds: relevance to transition-metal-catalyzed polymerization of polar vinyl monomers. AB - Variable-temperature 1H NMR studies of the reaction of cationic (alpha-diimine)Pd alkyl complexes with alkenes are presented. The studies reveal that vinyl bromide coordinates to the Pd(II)-Me complex followed by migratory insertion and beta bromo elimination, to generate free propene. Propene further reacts to give beta agostic Pd(II)-tert-butyl species. From the reactions with vinyl bromide, stable chloro-bridged dicationic Pd complex was isolated and characterized. For a series of alkenes (CH2=CHX), the rate for migratory insertion decreases as follows: X = CO2Me > Br > H > Me. PMID- 12371833 TI - Formation of a new crystalline silicate structure by grafting dialkoxysilyl groups on layered octosilicate. AB - Dialkoxydichlorosilanes ((RO)2SiCl2, R = alkyl) react almost completely with interlayer silanol groups in a layered silicate octosilicate to create a new crystalline silicate structure consisting of new five-membered rings arranged regularly on both sides of the silicate layers. The introduction of dialkoxysilyl groups to the interlamellar region of layered silicates with regular reaction sites provides a new methodology for the design and construction of novel crystalline silicate frameworks by a soft chemical route. PMID- 12371834 TI - Development of a donor-acceptor concept for enzymatic cross-coupling reactions of aldehydes: the first asymmetric cross-benzoin condensation. AB - Highly enantioenriched mixed benzoins are obtained selectively through a biocatalytical cross-coupling reaction of aromatic aldehydes using ThDP-dependent enzymes. PMID- 12371835 TI - A supramolecular oscillator composed of carbon nanocluster C(120) and a rhodium(III) porphyrin cyclic dimer. AB - A rhodium(III) porphyrin cyclic dimer (1) included carbon nanocluster C120 (2) to form a pi-electronic supramolecular complex (1 superset2), in which 2 oscillated back and forth within its cavity. Spectroscopic titration of 1 with 2 and line shape analysis on variable-temperature 1H NMR spectral profiles of 1 superset2 and reference inclusion complexes in toluene-d8, chlorobenzene-d5, and dichlorobenzene-d4 indicated that the association constant (Kassoc) of 1 superset2 and oscillation activity of included 2 are both larger as the affinity of the solvent toward fullerenes is smaller. The DeltaS values for the oscillation were all positive, indicating that the oscillation of 2 involves desolvation of a protruding C60 moiety of included 2 from the host cavity. PMID- 12371836 TI - New partially oxidized 1-D platinum chain complexes consisting of carboxylate bridged cis-diammineplatinum dimer cations. AB - The first/second examples of partially oxidized 1-D platinum chain compounds consisting of cationic dimer units have been obtained from electro-oxidation of an aqueous solution containing cis-[Pt(NH3)2(OH2)2]2+ and acetate/propionate. The analytical and crystallographic studies reveal the mixed-valency of Pt(2.2+)infinity. The XPS confirms the presence of both Pt(II) and Pt(III). The solid-state physical measurements reveal that they are diamagnetic semiconductors and display a fairly broad, low-energy absorption band in the range of 500-3200 nm. PMID- 12371837 TI - Direct synthesis of mesostructured lamellar molybdenum disulfides using a molten neutral n-alkylamine as the solvent and template. AB - A series of novel mesostructured lamellar molybdenum disulfides with the d spacings from 17 to 30 A can be prepared by the reaction of Mo(CO)6 with elemental sulfur using a molten n-alkylamine as the solvent as well as the template at 140 degrees C. Such intercalated phases can be transformed into mesoporous molybdenum disulfides by slow thermal treatments at 200 degrees C. PMID- 12371838 TI - DNA-binding of semirigid binuclear ruthenium complex delta,delta-[mu-(11,11' bidppz)(phen)(4)ru(2)](4+): extremely slow intercalation kinetics. AB - We here report a remarkably slow rearrangement of binding modes for a binuclear ruthenium(II) complex upon interaction with DNA. It has been previously shown that Delta,Delta-[mu-(11,11'-bidppz)(phen)4Ru2]4+ binds to DNA in one of the grooves. However, we find that this is only an initial, metastable, binding mode, which is extremely slowly reorganized into an intercalative binding geometry. The slow rearrangement and dissociation, revealed by flow linear dichroism and fluorescence spectroscopy, are concluded to be a result from the complex being threaded through the DNA, with one of the bridging aromatic dppz ligands intercalated between the base pairs of the DNA, placing one metal center in the minor groove and one in the major groove. A negative LD, a high luminescence quantum yield, and long luminescence lifetimes, similar to the intercalating complex Delta-[Ru(phen)2dppz]2+, indicate intercalation of the bidppz moiety. The unique slow dissociation of the complex in its final DNA-binding mode suggests that this class of threading, partially intercalated binuclear complexes may be interesting in the context of cancer therapy. Also, their unique optical and photophysical properties could make such complexes, either alone or scaffolded by DNA structures, of interest for the development of nanometer-sized molecular optoelectronic devices. PMID- 12371839 TI - Anomalous magnetic properties of MnO nanoclusters. AB - In this communication, we experimentally report, for the first time, that the MnO nanoclusters with cluster diameters of 5-10 nm show a ferromagnetic behavior with a phase transition from ferromagnetic to paramagnetic phases at 27 K even though their bulk phase is antiferromagnetic. We observed large coercivities up to 9500 Oe and a large remanence of 1.72 emu/g at 2 K, which are typically observed values for ferromagnetic materials. Although it is not clear, this abnormal ferromagnetic behavior of MnO nanoclusters may arise from cluster size effects. PMID- 12371840 TI - Co(3)[Co(CN)(5)](2): a microporous magnet with an ordering temperature of 38 K. AB - The reaction between [Co(H2O)6]2+ and [Co(CN)5]3- in deoxygenated water yields a dark blue solid of composition Co3[Co(CN)5]2.8H2O (1). X-ray powder diffraction data indicate a cubic Prussian blue-type framework, wherein the hexacyanometalate positions have only a two-thirds occupancy of square-pyramidal [Co(CN)5]3- units. Upon dehydration, the compound retains crystallinity and exhibits a Type I dinitrogen sorption isotherm, characteristic of a microporous solid. Magnetic measurements show 1 to behave as a ferrimagnet with an ordering temperature of 48 K, which is reduced to 38 K in the dehydrated solid. At 5 K, both 1 and dehydrated 1 exhibit magnetic hysteresis with a coercive field of 1160 G (the highest value yet reported for a cubic Prussian blue analogue) and remnant magnetizations of 1540 and 745 cm3G/mol, respectively. Thus, dehydrated 1 represents the first compound for which microporosity and long-range magnetic ordering have been demonstrated to coexist. PMID- 12371841 TI - Reversal of enantioselectivity in the asymmetric rhodium- versus iridium catalyzed hydroboration of meso substrates. AB - The Ir-catalyzed asymmetric hydroboration of bicyclic hydrazines with ee and chemical yields up to 64% is reported. The switch from rhodium to iridium leads systematically to opposite enantiomers in this desymmetrization reaction. PMID- 12371842 TI - Direct assessment of the reduction potential of the [4Fe-4S](1+/0) couple of the Fe protein from Azotobacter vinelandii. AB - Recently, it has been demonstrated that the [4Fe-4S] cluster of the Fe protein of nitrogenase from Azotobacter vinelandii can be reduced to an unprecedented all ferrous state. In this work, the reduction potential for the formation of the all ferrous state is measured by the reactions of the reduced and oxidized Fe protein with a variety of chemical redox active agents, and by mediated spectroelectrochemical titration. Redox titrations obtain a potential ca. -790 mV/NHE for the formation of the all-ferrous state, a value consistent with the chemical reactivity experiments and with recent theoretical calculations. At present, no known redox protein in A. vinelandii is capable of generating the all ferrous Fe protein. PMID- 12371843 TI - Rhodium-catalyzed asymmetric 1,4-addition of aryltitanium reagents generating chiral titanium enolates: isolation as silyl enol ethers. AB - The addition of aryltitanium triisopropoxide (ArTi(OPr-i )3) to alpha,beta unsaturated ketones proceeded with high enantioselectivity (94-99.8% ee) in the presence of 3 mol % of [Rh(OH)((S )-binap)]2 in THF at 20 degrees C to give high yields of the titanium enolates as 1,4-addition products. The titanium enolates were converted into silyl enol ethers by treatment with chlorotrimethylsilane and lithium isopropoxide. PMID- 12371844 TI - Remarkable activity of a novel cyclic seleninate ester as a glutathione peroxidase mimetic and its facile in situ generation from allyl 3-hydroxypropyl selenide. AB - 1,2-Oxaselenolane Se-oxide is a novel cyclic seleninate ester that functions as a remarkably efficient glutathione peroxidase mimetic by catalyzing the reduction of tert-butyl hydroperoxide to tert-butyl alcohol in the presence of benzyl thiol. The seleninate ester can be conveniently generated in situ by oxidation of allyl 3-hydroxypropyl selenide with tert-butyl hydroperoxide. Its catalytic activity surpasses that of several other known GPx mimetics containing cyclic selenenamide structures, which were also tested for comparison. PMID- 12371846 TI - Enzyme mediated extracellular synthesis of CdS nanoparticles by the fungus, Fusarium oxysporum. AB - The biosynthesis of Q-state CdS nanoparticles by reaction of aqueous CdSO4 solution with the fungus, Fusarium oxysporum, is demonstrated. Nanoparticle formation proceeds by release of sulfate reductase enzymes by the fungus, conversion of sulfate ions to sulfide ions that subsequently react with aqueous Cd2+ ions to yield highly stable CdS nanoparticles. Elucidation of an enzymatic pathway using fungi opens up the exciting possibility of developing a rational, biosynthesis strategy for nanomaterials over a range of chemical compositions which is currently not possible. PMID- 12371845 TI - Cyclization by intramolecular carbolithiation of alkyl- and vinyllithiums prepared by reductive lithiation: surprising stereochemistry in the lithium oxyanion accelerated cyclization. AB - The versatility of intramolecular carbolithiation of simple alkenes to yield cyclopentylmethyllithiums by unconjugated organolithiums is greatly increased (1) by generating the organolithiums by reductive lithiation of phenyl thioethers with aromatic radical anions and (2) by using allylic or homoallylic alcohol groups on the receiving alkene. This type of reductive lithiation allows virtually any kind of organolithium to be generated, usually in a connective manner. Furthermore, the allylic or homoallylic lithium oxyanionic groups on the alkene greatly accelerate the reactions and lead in most cases to completely stereoselective cyclization at -78 degrees . Most significantly, the trans stereoselectivity is the opposite from that observed when the organometallic is allylic. A four-membered ring has also been generated by this method. PMID- 12371847 TI - Time-resolved optical Kerr-effect spectroscopy of low-frequency dynamics in Di-L alanine, poly-L-alanine, and lysozyme in solution. AB - The low-frequency spectra of peptides and proteins in solution have been investigated with optical heterodyne-detected Raman-induced Kerr-effect spectroscopy. Spectra were obtained for di-l-alanine ALA(2) and poly-l-alanine (PLA) in dichloroacetic acid solution. The conformational dependence of those spectra at low frequency has been analyzed. ALA(2) displays a band centered at 50 cm-1, whereas the alpha-helical PLA shows two shoulders at 60 and 140 cm-1. The similarity of the spectral features observed in PLA to those in water can be explained by analogous acoustic translational modes in the hydrogen network of the PLA alpha-helix. The mostly alpha-helical protein lysozyme in aqueous solution has also been investigated and showed significantly more structure with modes at 10, 35, 73, 106, and 164 cm-1. PMID- 12371848 TI - Generation of 30-50 nm structures using easily fabricated, composite PDMS masks. AB - This communication demonstrates an approach to generate simple nanostructures with critical dimensions down to 30 nm over cm2-sized areas using composite PDMS masks. These masks were patterned with feature sizes down to 100 nm. When used in phase-shifting lithography, these masks generated arrays of structures in photoresist with line widths as small as 30 nm, slots in metal with features down to 40 nm, and wells in epoxy with diameters as small as 100 nm. The wells were used to prepare arrays of uniformly sized nanocrystals of salts. PMID- 12371849 TI - Immobilized enzymes as catalytically-active tools for nanofabrication. AB - One efficient strategy for creating nanostructures on surfaces is to use the catalytic properties of a surface molecule. This strategy benefits from the amplification and chemical specificity inherent in catalysis. We describe a demonstration of the key step of such a strategy: the surface trapping of a product generated by a nanometer-scale patch of surface-bound enzyme. Nanografting was used to create a approximately 70-nm patch of carboxylic acid groups surrounded by antibiofouling oligio(ethyleneoxide) groups on the surface of a gold ball. A catalytic site was prepared by immobilization of acetylcholine esterase to the carboxylic acid patch, and a product trap was prepared by scratching a small hole in the antibiofouling surface to reveal the gold surface. Two hours after addition of acetylthiocholine, the trap was filled. This demonstrated that the enzyme had catalyzed a reaction and that the product had been used to modify the surface film. PMID- 12371850 TI - The metal is the kinetic site of protonation of (diimine)Pt dimethyl complexes. AB - Protonolysis of (diimine)PtMe2 (1) complexes in CD2Cl2 containing CD3CN at -78 degrees C yields (diimine)PtMe2(H)(NCCD3)+ (4), (diimine)PtMe(NCCD3)+ (5), and methane. The relative yields of 5 and methane decrease with increasing concentrations of CD3CN. This is consistent with protonation of 1 occurring directly at the metal, rather than at a methyl group. The principle of microscopic reversibility then implies that the deprotonation in "Shilov-type C-H activation" occurs from a Pt(IV) hydridomethyl intermediate, rather than from a Pt sigma-methane complex. PMID- 12371851 TI - Inhibitor scaffolds as new allele specific kinase substrates. AB - The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N(6)-(benzyl) ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38 T106G/A157L/L167A). 3-Benzyl-PPTP was preferred by v-Src (T338G) (k(cat)/K(M) = 3.2 x 10(6) min(-)(1) M(-)(1)) over ATP or the previously described ATP analogue, N(6) (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (k(cat)/K(M) = 2.6 x 10(4) min(-)(1) M(-)(1)) comparable to ATP (k(cat)/K(M) = 5.0 x 10(4) min(-)(1) M(-)(1)) largely due to a significantly better K(M) (6.4 microM vs 530 microM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N(6)-(benzyl)-ATP with respect to the wild-type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma-(32)P]-3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, even while competing with cellular levels (4 mM) of unlabeled ATP. The fact that this new more highly orthogonal nucleotide is accepted by three widely divergent kinases studied here suggests that it is likely to be generalizable across the entire kinase superfamily. PMID- 12371852 TI - Observation of topical catalysis by sphingomyelinase coupled to microspheres. AB - Sphingomyelinase, SMase (EC 3.1.4.12), was coupled onto amino-derivatized acrylate microspheres and was shown to retain its catalytic activity. The immobilized enzyme allows one to carry out topical enzymatic reaction in a controlled manner. Accordingly, these spheres were held with a micropipet and using micromanipulator brought into contact with a giant liposome membrane composed of phosphatidylcholine and sphingomyelin (SOPC/C16:0-SM, 0.75:0.25, molar ratio), representing the substrate for the immobilized enzyme. The macroscopic consequences of the enzyme reaction were visualized using fluorescence microscopy as well as differential interference contrast microscopy. The surface contact of the giant vesicle and immobilized enzyme causes membrane microdomain formation and domain clustering (capping) in the membrane and subsequent shedding of small vesicles from the membrane into the interior of the giant liposome. The method described represents a novel approach to study enzymatic reactions and allows manipulating giant vesicles as well as cultured cells in a spatially controlled manner. PMID- 12371853 TI - 3-pyrrolines are mechanism-based inactivators of the quinone-dependent amine oxidases but only substrates of the flavin-dependent amine oxidases. AB - We previously reported that 3-pyrroline and 3-phenyl-3-pyrroline effect a time dependent inactivation of the copper-containing quinone-dependent amine oxidase from bovine plasma (BPAO) (Lee et al. J. Am. Chem. Soc. 1996, 118, 7241-7242). Quinone cofactor model studies suggested a mechanism involving stoichiometric turnover to a stable pyrrolylated cofactor. Full details of the model studies are now reported along with data on the inhibition of BPAO by a family of 3-aryl-3 pyrrolines (aryl = substituted phenyl, 1-naphthyl, 2-naphthyl), with the 4 methoxy-3-nitrophenyl analogue being the most potent. At the same time, the parent 3-phenyl analogue is a pure substrate for the flavin-dependent mitochondrial monoamine oxidase B from bovine liver. Spectroscopic studies (including resonance Raman) on BPAO inactivated by the 4-methoxy-3-nitrophenyl analogue are consistent with covalent derivatization of the 2,4,5 trihydroxyphenylalanine quinone (TPQ) cofactor. The distinction of a class of compounds acting as an inactivator of one amine oxidase family and a pure substrate of another amine oxidase family represents a unique lead to the development of selective inhibitors of the mammalian copper-containing amine oxidases. PMID- 12371854 TI - Solution NMR techniques for large molecular and supramolecular structures. AB - Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa. With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H. This paper describes the experimental schemes used and details of their setup for individual measurements. The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines. Optimal transfer times for 2D [(15)N,(1)H]-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa. These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure. The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s. Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually. PMID- 12371855 TI - Conformational heterogeneity observed in simulations of a pyrene-substituted DNA. AB - NMR studies previously carried out for a DNA system with a pyrene-substituted base did not observe NOEs involving the adenine located 5' to the pyrene, and thus the conformation of the adenine was poorly defined in the resulting family of refined structures. However, chemical shift data suggested that an AT base pair may be present. We have carried out fully unrestrained molecular dynamics simulations starting from several members of the family of structures, and these simulations support the existence of an AT base pair for this region. Simulations in both explicit and implicit solvent were carried out, with each converging to either anti or syn conformation for adenine and base pairing in all cases. During these simulations, large and dramatic conformational changes are observed that suggest pathways for complex conformational changes in the highly packed DNA interior. Our analysis reveals little difference in the energies of these syn and anti conformations, in contrast to control calculations carried out for standard DNA (in the absence of a neighboring pyrene). While no interconversion between the conformations was observed in standard simulations, reversible anti/syn exchange was directly simulated using the locally enhanced sampling approach. No exchange was seen in the non-pyrene control sequence. Together, these results suggest that an increased flexibility is introduced as a consequence of the pyrene substitution, offering an explanation that is consistent with the available experimental data. These results increase our optimism that simulations in atomic detail may provide accurate models for experimental observations in complex systems. PMID- 12371856 TI - Synthesis, structure, and photochemistry of exceptionally stable synthetic DNA hairpins with stilbene diether linkers. AB - The structure and properties of 18 hairpin-forming bis(oligonucleotide) conjugates possessing stilbene diether linkers are reported. Conjugates possessing bis(2-hydroxyethyl)stilbene 4,4'-diether linkers form the most stable DNA hairpins reported to date. Hairpins with as few as two T:A base pairs or four noncanonical G:G base pairs are stable at room temperature. Increasing the length of the hydroxyalkyl groups results in a decrease in hairpin thermal stability. On the basis of the investigation of their circular dichroism spectra, all of the hairpins investigated adopt B-DNA structures, except for a hairpin with a short poly(G:C) stem which forms a Z-DNA structure. Both the strong fluorescence of the stilbene diether linkers and their trans-cis photoisomerization are totally quenched in hairpins possessing neighboring T:A and G:C base pairs. Quenching is attributed to an electron-transfer mechanism in which the singlet stilbene serves as an electron donor and T or C serves as an electron acceptor. In contrast, in denatured hairpins and hairpins possessing neighboring G:G base pairs the stilbene diether linkers undergo efficient photoisomerization. PMID- 12371857 TI - Chemistry of the aromatic 9-germafluorenyl dianion and some related silicon and carbon species. AB - Dipotassio-9-germafluorenyl dianion (3b) was synthesized by reduction of 9,9 dichloro-9-germafluorene (4b) with sodium/potassium alloy in tetrahydrofuran. The X-ray crystal structure of 3b, like that for the analogous silicon compound 3a, shows C-C bond length equalization in the five-membered metallole rings and C-C bond length alternation in the six-membered benzenoid rings, indicating aromatic delocalization of electrons into the germole ring of 3b. Calculated nucleus independent chemical shift (NICS) values indicate that the five-membered ring is more aromatic than the six-membered rings in 3a and 3b. Derivatization of 3b with Me(3)SiCl gave 9,9-bis(trimethylsilyl)-9-germafluorene (5). Controlled oxidation of 3b yielded dipotassio-9,9'-digerma-9,9'-bifluorenyl dianion (6). Reaction of 6 with MeOH yielded 9,9'-digerma-9,9'-bifluorene (7). The X-ray structure of 6 indicates C-C bond length alternation in the five-membered rings. Thus dianion 6, like its silicon analogue 8, has the negative charges localized at metal atoms and no aromatic character. Dipotassio-9,9'-bifluorenyl dianion (9), the carbon analogue of 6, exhibits aromaticity with its X-ray crystal structure showing the C-C bond length equalization in both the five- and six-membered rings. Derivatization of 9 with MeI gave 9,9'-dimethyl-9,9'-bifluorene (10). The structure of 10 shows that the two fluorenyl rings are cis to each other with a torsional angle of 59 degrees and a long C-C single bond (1.60 A) connecting them. PMID- 12371858 TI - Synthesis and characterization of polymethacrylate-based nitric oxide donors. AB - A synthetic path for the preparation of methacrylic homo- and copolymers containing secondary amine groups that can be converted into nitric oxide (NO) releasing N-diazeniumdiolates is described. The polymers are obtained by a multistep procedure involving synthesis of methacrylate monomers containing boc protected secondary amine sites, free radical benzoyl peroxide initiated polymerization, deprotection of the amine sites, and subsequent reaction of the polymers with NO in the presence of sodium methoxide. Monomers with both linear and cyclic pendant secondary amines are examined as polymer building blocks. In most cases, polymers are obtained for both types with compositions that agree well with initial monomer ratios and with number average molecular weights (M(n)) ranging from 1.69 to 2.58 x 10(6) Da. The final N-diazeniumdiolated methacrylic amine polymers are shown to release NO for extended periods of time with "apparent" t(1/2) values ranging from 30 to 60 min when suspended in phosphate buffer, pH 7.4. Total NO loading and release for these materials can reach 1.99 micromol per mg of polymer and is proportional to the amine content of the polymer. It is further shown that by using a dimethacrylate cross-linking agent in conjunction with the various methacrylate amines, suspension polymerization methods can be employed to create small (100-200 microm) polymeric methacrylate microbeads. Such microbeads that can be sequentially deprotected and converted to NO release particles via in-situ diazeniumdiolate formation as carried out for the non-crosslinked polymers. PMID- 12371859 TI - Stereochemical memory in the temperature-dependent photodenitrogenation of bridgehead-substituted DBH-type azoalkanes: inhibition of inverted-housane formation in the diazenyl diradical through the mass effect (inertia) and steric hindrance. AB - The photochemical denitrogenation of the cyclopentene-annelated DBH-type azoalkanes 1 has been examined in solution as a function of bridgehead substitution and temperature. For all derivatives, namely, the unsubstituted 1a(H/H), monomethyl 1b(Me/H) dimethyl 1c(Me/Me), monophenyl 1d(Ph/H), and diphenyl 1e(Ph/Ph), the temperature-dependent ratio of syn and anti housanes 2 provides experimental support for a competition between the singlet (high temperature) and triplet (low temperature) reaction channels in the direct photolysis. The syn/anti ratio of the housanes 2 depends on the extent and type of bridgehead substitution; the amount of the anti diastereomer (retention) follows the order Ph > Me > H, and double substitution is more effective than single. This stereochemical memory is interpreted in terms of the mass effect (inertia) of the substituents and steric interaction (size) between the substituents at the bridgehead and the methylene bridge during the deazetation step of the transient diazenyl diradical conformations (1)DZ (exo-ax) and (1)DZ (exo-eq). These conformers are impulsively generated upon decay of the (1)(n,pi) excited azoalkane, a trajectory assessed through computational work. The new mechanistic feature disclosed by the unprecedented anti stereoselectivity (retention) is the intervention of a puckered 1,3-cyclopentanediyl singlet diradical (1)DR as product bifurcation step, whose conformational relaxation to the planar species (loss of stereochemical memory) is encumbered by bridgehead substitution. PMID- 12371860 TI - Model chemistry calculations of thiophene dimer interactions: origin of pi stacking. AB - The intermolecular interaction energies of thiophene dimers have been calculated by using an aromatic intermolecular interaction (AIMI) model (a model chemistry for the evaluation of intermolecular interactions between aromatic molecules). The CCSD(T) interaction energy at the basis set limit has been estimated from the MP2 interaction energy near the basis set limit and the CCSD(T) correction term obtained by using a medium-size basis set. The calculated interaction energies of the parallel and perpendicular thiophene dimers are -1.71 and -3.12 kcal/mol, respectively. The substantial attractive interaction in the thiophene dimer, even where the molecules are well separated, shows that the major source of attraction is not short-range interactions such as charge transfer but rather long-range interactions such as electrostatic and dispersion. The inclusion of electron correlation increases the attraction significantly. The dispersion interaction is found to be the major source of attraction in the thiophene dimer. The calculated total interaction energy of the thiophene dimer is highly orientation dependent. Although electrostatic interaction is substantially weaker than dispersion interaction, it is highly orientation dependent, and therefore electrostatic interaction play an important role in the orientation dependence of the total interaction energy. The large attractive interaction in the perpendicular dimer is the cause of the preference for the herringbone structure in the crystals of nonsubstituted oligothiophenes (alpha-terthienyls), and the steric repulsion between the beta-substituents is the cause of the pi-stacked structure in the crystals of some beta-substituted oligothiophenes. PMID- 12371861 TI - Mechanisms of tetrazole formation by addition of azide to nitriles. AB - It is well-known that azide salts can engage nitriles at elevated temperatures to yield tetrazoles; however, there is continued debate as to the mechanism of the reaction. Density functional theory calculations with the hybrid functional B3LYP have been performed to study different mechanisms of tetrazole formation, including concerted cycloaddition and stepwise addition of neutral or anionic azide species. The calculations presented here suggest a previously unsuspected nitrile activation step en route to an imidoyl azide, which then cyclizes to give the tetrazole. The activation barriers are found to correlate strongly with the electron-withdrawing potential of the substituent on the nitrile. PMID- 12371862 TI - Mechanistic insight into fragmentation reactions of titanapinacolate complexes. AB - Reactions between terminal alkynes or aromatic ketones and titanapinacolate complexes (DMSC)Ti(OCAr(2)CAr(2)O) (2, Ar = Ph, and 3, Ar = p-MeC(6)H(4); DMSC = 1,2-alternate dimethylsilyl-bridged p-tert-butylcalix[4]arene dianion) occur via rupture of the C-C bond of the titanacycle. Thus, reactions of 2 and 3 with terminal alkynes produce 2-oxatitanacyclopent-4-ene or 2-oxatitanacycloheptadiene complexes along with free Ar(2)CO. These compounds have been characterized spectroscopically and by X-ray crystallography. Because metallapinacolate intermediates have been implicated in important C-C bond-forming reactions, such as pinacol coupling and McMurry chemistry, the mechanism of the fragmentation reactions was studied. Analysis of the kinetics of the reaction of (DMSC)Ti[OC(p MeC(6)H(4))(2)C(p-MeC(6)H(4))(2)O] (3) with Bu(t)Ctbd1;CH revealed that the fragmentation reactions proceed via a preequilibrium mechanism, involving reversible dissociation of titanapinacolate complexes into (DMSC)Ti(eta(2) OCAr(2)) species with release of a ketone molecule, followed by rate-limiting reaction of (DMSC)Ti(eta(2)-OCAr(2)) species with an alkyne or ketone molecule. PMID- 12371863 TI - A general, highly enantioselective method for the synthesis of D and L alpha amino acids and allylic amines. AB - Catalytic and enantioselective synthesis of amino acids is a subject of intense interest in the field of asymmetric catalysis. Traditionally, researchers have concentrated their efforts largely on the design and discovery of enantiopure catalysts for the Strecker reaction, alkylation of tert-butyl gylcinate benzophenone, electrophilic amination of carbonyl compounds, and hydrogenation of N-acyl-aminoacrylic acid; however, the scope of these reactions is limited. In this paper, we report on a different approach to amino acids based on an expeditious route to enantiopure allylic amines. A highly enantioselective and catalytic vinylation of aldehydes leads to allylic alcohols that are then transformed to the allylic amines via Overman's [3,3]-sigmatropic rearrangement of imidates. Oxidative cleavage of the allylic amines furnishes the amino acids in good yields and excellent ee's. The scope and utility of this method are demonstrated by the synthesis of challenging allylic amines and their subsequent transformation to valuable nonproteinogenic amino acids, including both D and L configured (1-adamantyl)glycine. PMID- 12371864 TI - Vapor-liquid interfacial properties of mutually saturated water/1-butanol solutions. AB - Adsorption and ordering at the vapor-liquid interfaces of mutually saturated water/1-butanol solutions at a temperature of 298.15 K were investigated using configurational-bias Monte Carlo simulations in the Gibbs ensemble and compared to the surface properties of neat water and 1-butanol liquids. A dense 1-butanol monolayer is observed at the surface of the water-rich phase, which results in a substantial decrease of its surface tension. In contrast, there is no enrichment of water molecules at the surface of the butanol-rich phase, and its surface tension is not significantly changed. Analysis of the interfacial structures reveals that these systems exhibit orientational ordering and composition heterogeneity. Analysis of the hydrogen-bonding distributions suggests that the formation of the 1-butanol monolayer is driven by an excellent match between water and the primary alcohol; that is, additional hydrogen bonds are formed between the excess free hydrogens of surface water and the excess hydrogen-bond acceptor sites of 1-butanol. PMID- 12371865 TI - A novel layer-by-layer approach to immobilization of polymers and nanoclusters. AB - This paper reports a simple method for the multilayer immobilization of conjugated polymers, gold nanoparticles on solid supports. Poly(phenyenevinylene) functionalized with aldehyde and aminooxy groups was chemoselectively immobilized onto both glass and gold substrates via layer-by-layer deposition. The physical properties of the thin films were characterized by grazing angle IR, TM-AFM, fluorescence, and UV-visible spectroscopy. This methodology was also successfully applied to prepare polymer/gold nanocluster alternating multilayers. The results show that this methodology provides a general route for preparing robust and functionalizable multilayer films on solid substrates with molecular-level thickness control. PMID- 12371866 TI - Perfluoroterephthalate bridged complexes with M-M quadruple bonds: ((t)BuCO(2))(3)M(2)(mu-O(2)CC(6)F(4)CO(2))M(2)(O(2)C(t)Bu)(3), where M = Mo or W. Studies of solid-state, molecular, and electronic structure and correlations with electronic and Raman spectral data. AB - The compounds [((t)BuCO(2))(3)M(2)(mu-O(2)CC(6)F(4)CO(2))M(2)(O(2)C(t)Bu)(3)], M(4)PFT, where M = Mo or W, are shown by model fitting of the powder X-ray diffraction data to have an infinite "twisted" structure involving M.O intermolecular interactions in the solid state. The dihedral angle between the M(2) units of each molecule is 54 degrees. Electronic structure calculations employing density functional theory (Gaussian 98 and ADF2000.01, gradient corrected and time dependent) on the model compounds (HCO(2))(3)M(2)(mu O(2)CC(6)F(4)CO(2))M(2)(O(2)CH)(3), where M = Mo or W, reveal that in the gas phase the model compounds adopt planar D(2)(h) ground-state structures wherein M(2) delta to bridge pi back-bonding is maximized. The calculations predict relatively small HOMO-LUMO gaps of 1.53 eV for M = Mo and 1.22 eV for M = W for this planar structure and that, when the "conjugation" is removed by rotation of the plane of the C(6)F(4) ring to become orthogonal to the M(4) plane, this energy gap is nearly doubled to 2.57 eV for M = Mo and 2.18 eV for M = W. The Raman and resonance Raman spectra of solid M(4)PFT and of Mo(4)PFT in THF solution are dominated by bands assigned to the bridging perfluoroterephthalate (pft) group. The intensities of certain Raman bands of solid W(4)PFT are strongly enhanced on changing the excitation line from 476.5 nm (off resonance) to 676.5 nm, which is on resonance with the W(2) delta --> CO(2) (pft) pi transition at ca. 650 nm. The resonance enhanced bands are delta(s)(CO(2)) (pft) at 518 cm( )(1) and its first overtone at 1035 cm(-)(1), consistent with the structural change to W(4)PFT expected on excitation from the ground to this pi excited state. The electronic transitions for solid Mo(4)PFT (lowest at 410 nm) were not accessible with the available excitation lines (457.9-676.5 nm), and no resonance Raman spectra of this compound could be obtained. For Mo(4)PFT in THF solution, it is the band at 399 cm(-)(1) assigned to nu(MoMo) which is the most enhanced on approach to resonance with the electronic band at 470 nm; combination bands involving the C(6)F(4) ring-stretching mode, 8a, are also enhanced. PMID- 12371867 TI - Single-crystal mesostructured semiconductors with cubic Ia3d symmetry and ion exchange properties. AB - If the full scientific and technological potential of mesostructured materials is to be achieved, systems with continuous domains in the form of single crystals or films must be prepared. Here we report a reliable and facile system for making large single-crystal particles of chalcogenido mesostructured materials with a highly organized cubic structure, accessible pore structure, and semiconducting properties. Building blocks with square planar bonding topology, Pt(2+) and [Sn(2)Se(6)](4)(-), in combination with long-chain pyridinium surfactants (C(n)PyBr, n = 18, 20) favor faceted single-crystal particles with the highest possible space group symmetry Ia3d. This is an important step toward developing large single-domain crystalline mesostructured semiconductors and usable natural self-assembled antidot array systems. The tendency toward cubic symmetry is so strong that the materials assemble readily under experimental conditions that can tolerate considerable variation and form micrometer-sized rhombic dodecahedral cubosome particles. The c-C(n)PyPtSnSe materials are the first to exhibit reversible ion-exchange properties. The surfactant molecules can be ion-exchanged reversibly and without loss of the cubic structure and particle morphology. The cubosomes possess a three-dimensional open Pt-Sn-Se framework with a low-energy band gap of approximately 1.7 eV. PMID- 12371868 TI - Unified synthesis of quinone sesquiterpenes based on a radical decarboxylation and quinone addition reaction. AB - A unified synthesis of several quinone sesquiterpenes is described herein. Essential to this strategy is a novel radical addition reaction that permits the attachment of a fully substituted bicyclic core 16 to a variably substituted quinone 10. The addition product 15 can be further functionalized, giving access to natural products with a high degree of oxygenation at the quinone unit. The quinone addition reaction is characterized by excellent chemoselectivity, taking place only at conjugated and unsubstituted double bonds, and regioselectivity, being strongly influenced by the resonance effect of heteroatoms located on the quinone ring. These features were successfully applied to the synthesis of avarol (1), avarone (2), methoxyavarones (4, 5), ilimaquinone (6), and smenospongidine (7), thereby demonstating the synthetic value of this new method. PMID- 12371869 TI - Participation of the alpha,alpha'-diiminopyridine ligand system in reduction of the metal center during alkylation. AB - The reaction of [[2,6-(i-Pr)(2)PhN=C(Me)](2)(C(5)H(3)N)]MnCl(2) with alkylating agents formed a dinuclear Mn(I) derivative via ligand reductive coupling. In the case of the trivalent Cr analogue, a similar reaction afforded reduction toward Cr(II) but also alkylation at the pyridine ring para position followed by an unprecedented cycloaddition that generated a tricyclic system. PMID- 12371870 TI - Growth of Na-doped Ca(2)CuO(2)Cl(2) single crystals under high pressures of several GPa. AB - Single crystals of Na-doped Ca(2)CuO(2)Cl(2) have been grown for the first time by a flux method under high pressures of up to 5.5 GPa. By changing the Na solubility limit through the applied pressure, the Na content x was successfully controlled without introducing appreciable compositional inhomogeneity within the millimeter-sized crystals. Structural and chemical characterization indicated that the crystals span the phase diagram continuously from the parent antiferromagnetic insulator to an underdoped high-temperature superconductor. Because of the well-defined cleavage plane and resulting high surface quality, these oxychloride single crystals will provide a unique opportunity to explore the electronic evolution of the high-temperature superconductors, using spectroscopic techniques such as scanning tunneling microscopy/spectroscopy and angle-resolved photoemission spectroscopy. PMID- 12371871 TI - The structure of self-assembled multilayers with polyoxometalate nanoclusters. AB - Using electrostatic layer-by-layer self-assembly (ELSA), the formation of multilayers with polyelectrolytes and nanoscopic polyoxometalate (POM) clusters of different sizes and charges is investigated. The multilayers are characterized by UV-vis absorption spectroscopy, optical ellipsometry, cyclic voltammetry, and atomic force microscopy. In all cases, it is possible to find experimental conditions to achieve irreversible adsorption and regular multilayer deposition. Most importantly, the surface coverage is directly related to the total charge of the POM anion and can be controlled from submonolayer to multilayer coverage by adjusting the ionic strength of the dipping solutions. Imaging the interfaces after POM deposition by atomic force microscopy reveals a granular surface texture with nanometer-sized features. The average interfacial roughness amounts to approximately 1 nm. Cyclic voltammetry indicates that the electrochemical properties of the POM clusters are fully maintained in the polyelectrolyte matrix, which opens a route toward practical applications such as sensors or heterogeneous catalysts. Moreover, the permeability toward electrochemically active probe molecules can be tailored through the multilayer architecture and deposition conditions. Finally, we note that despite the low total charge and comparably small size of the discrete POM anions, the multilayers are remarkably stable. This work provides basic guidelines for the assembly of POM-containing ELSA multilayers and provides detailed insight into characteristic surface coverage, permeability, and electrochemical properties. PMID- 12371872 TI - Direct synthesis of gallium oxide tubes, nanowires, and nanopaintbrushes. AB - We demonstrate bulk synthesis of highly crystalline beta-gallium oxide tubes, nanowires, and nanopaintbrushes using molten gallium and microwave plasma containing a mixture of monatomic oxygen and hydrogen. Gallium oxide nanowires were 20-100 nm thick and tens to hundreds of micrometers long. Transmission electron microscopy (TEM) revealed the nanowires to be highly crystalline and devoid of any structural defects. Results showed that multiple nucleation and growth of gallium oxide nanostructures could easily occur directly out of molten gallium exposed to an appropriate composition of hydrogen and oxygen in the gas phase. These gallium oxide nanostructures should be of particular interest for optoelectronic devices and catalytic applications. PMID- 12371873 TI - Mesostructured gamma-Al(2)O(3) with a lathlike framework morphology. AB - A novel three-step assembly pathway is reported for the formation of a mesostructured alumina with framework pore walls made of crystalline, lathlike gamma-Al(2)O(3) nanoparticles. In the initial supramolecular assembly step of the pathway a mesostructured alumina with a wormhole framework morphology and amorphous pore walls is assembled through the hydrolysis of Al(13) oligocations and hydrated aluminum cations in the presence of a nonionic diblock or triblock poly(ethylene oxide) surfactant as the structure-directing porogen. The walls of the initial mesostructure are then transformed in a second hydrolysis step at a higher temperature to a surfactant-boehmite mesophase, denoted MSU-S/B, with a lathlike framework made of boehmite nanoparticles. A final thermal reaction step topochemically converts the intermediate boehmitic mesophase to a mesostructure with crystalline gamma-Al(2)O(3) pore walls, denoted MSU-gamma, with retention of the lathlike framework morphology. The boehmitic MSU-S/B intermediates formed from the chloride salts of aluminum incorporate chloride anions into the mesostructure. Chloride ion incorporation tends to disorder the nanoparticle assembly process, leading to a broadening of the slit-shaped framework pores in the final MSU-gamma phases and to the introduction of intra- and interparticle textural mesopores. However, the well-ordered MSU-gamma phases made from aluminum nitrate as the preferred aluminum reagent exhibit narrow framework pore size distributions and average pore sizes that are independent of the surfactant size and packing parameter, in accord with a lathlike framework assembled from nanoparticles of regular size and connectivity. The high surface areas ( approximately 300-350 m(2)/g) and pore volumes ( approximately 0.45-0.75 cm(3)/g) provided by these mesostructured forms of gamma-Al(2)O(3) should be useful in materials and catalytic applications where the availability of surface Lewis acid sites and the dispersion of supported metal centers govern reactivity. PMID- 12371874 TI - How ions affect the structure of water. AB - We model ion solvation in water. We use the MB model of water, a simple two dimensional statistical mechanical model in which waters are represented as Lennard-Jones disks having Gaussian hydrogen-bonding arms. We introduce a charge dipole into MB waters. We perform (NPT) Monte Carlo simulations to explore how water molecules are organized around ions and around nonpolar solutes in salt solutions. The model gives good qualitative agreement with experiments, including Jones-Dole viscosity B coefficients, Samoilov and Hirata ion hydration activation energies, ion solvation thermodynamics, and Setschenow coefficients for Hofmeister series ions, which describe the salt concentration dependence of the solubilities of hydrophobic solutes. The two main ideas captured here are (1) that charge densities govern the interactions of ions with water, and (2) that a balance of forces determines water structure: electrostatics (water's dipole interacting with ions) and hydrogen bonding (water interacting with neighboring waters). Small ions (kosmotropes) have high charge densities so they cause strong electrostatic ordering of nearby waters, breaking hydrogen bonds. In contrast, large ions (chaotropes) have low charge densities, and surrounding water molecules are largely hydrogen bonded. PMID- 12371875 TI - Orbital symmetry as a tool for understanding the bonding in Krossing's cation. AB - The geometric and electronic structure of Krossing's cation, Ag(eta(2) P(4))(2)(+), which shows an unexpected planar coordination environment at the metal center and D(2)(h) symmetry both in solution and in the solid state, have been investigated using density functional theory and orbital-symmetry-based energy decomposition. The analysis reveals that the contribution from electrostatic interactions to the bond energy is greater than that of orbital interactions. Partitioning of the latter term into the irreducible representations shows that, in addition to the 5s orbital, 5p orbitals of silver act as acceptor orbitals for electron donation from sigma(P-P) orbitals (a(1)(g), b(1)(u)) and n(P) orbitals (b(3)(u)). Back-donation from the 4d(10) closed shell of Ag into sigma orbitals of the pnictogen cages (b(2)(g)) is also important. However, this contribution is shown not to determine the D(2)(h) structure, contradicting conclusions from the pioneering study of the title cation (J. Am. Chem.Soc. 2001, 123, 4603). The contributions from the irreducible representations to the stabilizing orbital interactions in the D(2)(h) structure and in its D(2)(d)-symmetric conformer are analogous, indicating that the planar coordination environment at the metal center in Ag(eta(2)-P(4))(2)(+) is induced by intermolecular rather than by intramolecular interactions. Because ethylene coordination to a metal ion is an elementary reaction step in industrial processes, the bonding in Ag(C(2)H(4))(2)(+) has been analyzed as well and compared to that in Krossing's cation. Surprisingly, similar contributions to the bond energies and an involvement of metal 4d and 5p orbitals have been found, whereas a recent atoms in molecules analysis suggested that the metal-ligand interactions in silver(I) olefin complexes fundamentally differ from those in tetrahedro P(4) complexes. The only qualitative difference between the bonding patterns in Ag(eta(2)-P(4))(2)(+) and Ag(C(2)H(4))(2)(+) is the negligible energy contribution from the b(3)(u) irreducible representation in the ethylene complex because a respective symmetry-adapted linear combination of ligand orbitals is not available. PMID- 12371876 TI - Electronic structure, chemical bond, and optical spectra of metal bis(porphyrin) complexes: a DFT/TDDFT study of the bis(porphyrin)M(IV) (M = Zr, Ce, Th) series. AB - The electronic absorption spectra of the bis(porphyrin) sandwich complexes of the metals Zr, Ce, and Th are studied with time-dependent density functional theory (TDDFT). A ground-state electronic structure analysis reveals that the highest occupied one-electron levels are, as expected, composed of the porphyrin a(1u) and a(2u) highest occupied orbitals (the Gouterman orbitals), but the level pattern is not simply a pair of low-lying nearly degenerate in-phase combinations and a pair of high-lying approximately degenerate antibonding combinations. Instead, the a(1u) split strongly and the a(2u) do not. Since the calculated spectrum agrees very well with experiment, the assignment leaves little doubt that although the experimental spectrum has porphyrin-like features, such as the well-known Q and B bands, the actual composition of the states is rather different from that in porphyrin. In particular the strong mixing of a(1u) --> e(g) and a(2u) --> e(g) is absent, there is mixing with excitations of non Gouterman type, and, in Ce, ring to metal charge-transfer transitions play an important role. The composition of the states as calculated in this work does not lead to a classification of the excitations as purely "excitonic" or "charge resonance". PMID- 12371877 TI - Theoretical study of silylene substituent effects on the abstraction reactions with oxirane and thiirane. AB - The potential energy surfaces for the abstraction reactions of silylenes with oxirane and thiirane have been characterized in detail using density functional theory (B3LYP) as well as the ab initio method (QCISD), including zero-point corrections. Five silylene species including SiH(2), Si(CH(3))(2), Si(NH(2))(2), Si(OH)(2), and SiF(2) have been chosen in this work as model reactants. All the interactions involve the initial formation of a donor-acceptor ylide-like complex followed by a heteroatom shift via a two-center transition state. The complexation energies, activation barriers, and enthalpies of the reactions were used comparatively to determine the relative silylenic reactivity, as well as the influence of substituents on the reaction potential energy surface. As a result, our theoretical investigations suggest that, irrespective of deoxygenation and desulfurization, the alkyl-substituted silylene abstractions are much more favorable than those of the pi donor-substituted silylenes. Moreover, for a given silylene, while both deoxygenation and desulfurization are facile processes, the deoxygenation reaction is more exothermic as well as more kinetically favorable. Furthermore, a configuration mixing model based on the work of Pross and Shaik is used to rationalize the computational results. The results obtained allow a number of predictions to be made. PMID- 12371878 TI - DMSO complexes of trivalent metal ions: first microsolvated trications outside of group 3. AB - The advent of electrospray ionization source opened the door to generation of multiply charged metal ions complexed with organic molecules. A significant amount of work on ligated dications has appeared over the past decade. In contrast, only several microsolvated tripositive ions have been reported, involving solely the few rare earths with the lowest third ionization energies (IEs) of all elements (<23 eV). Here trications of numerous trivalent metals outside of group 3 are shown to coordinate dimethyl sulfoxide (DMSO), an eminent aprotic solvent. These include both main group elements (Al, Ga, In, Bi) and transition metals (V, Fe, Cr) with the third IE up to 31 eV, which is 22 eV above the IE of DMSO. Fragmentation of M(3+)(DMSO)(n) for these metals (plus La, Yb, and Sc) has been characterized in detail using collision-induced dissociation (CID). A rich, highly element specific dissociation chemistry is observed, including the homolytic C-S cleavage in (+3) charge state and various charge reduction processes, such as dissociative electron and proton transfer and heterolytic S=O cleavage with and without a concomitant proton transfer. Characteristic sizes for the charge reduction in M(3+)(DMSO)(n) and M(2+)(DMSO)(n) have been measured as a function of the relevant elemental IE. These reveal no intrinsic gap between the stabilities of dication and trication complexes, once the IE is adjusted for. This, in particular, suggests that even microsolvated tetracations may exist. PMID- 12371879 TI - Reconstructing NMR spectra of "invisible" excited protein states using HSQC and HMQC experiments. AB - Carr-Purcell-Meiboom-Gill (CPMG) relaxation measurements employing trains of 180 degrees pulses with variable pulse spacing provide valuable information about systems undergoing millisecond-time-scale chemical exchange. Fits of the CPMG relaxation dispersion profiles yield rates of interconversion, relative populations, and absolute values of chemical shift differences between the exchanging states, |Deltaomega|. It is shown that the sign of Deltaomega that is lacking from CPMG dispersion experiments can be obtained from a comparison of chemical shifts in the indirect dimensions in either a pair of HSQC (heteronuclear single quantum coherence) spectra recorded at different magnetic fields or HSQC and HMQC (heteronuclear multiple quantum coherence) spectra obtained at a single field. The methodology is illustrated with an application to a cavity mutant of T4 lysozyme in which a leucine at position 99 has been replaced by an alanine, giving rise to exchange between ground state and excited state conformations with a rate on the order of 1450 s(-1) at 25 degrees C. PMID- 12371880 TI - Solvent and guest isotope effects on complexation thermodynamics of alpha-, beta , and 6-amino-6-deoxy-beta-cyclodextrins. AB - The stability constant (K), standard free energy (DeltaG degrees ), enthalpy (DeltaH degrees ), and entropy changes (TDeltaS degrees ) for the complexation of native alpha- and beta-cyclodextrins (CDs) and 6-amino-6-deoxy-beta-CD with more than 30 neutral, positively, and negatively charged guests, including seven fully or partially deuterated guests, have been determined in phosphate buffer solutions (pH/pD 6.9) of hydrogen oxide (H(2)O) or deuterium oxide (D(2)O) at 298.15 K by titration microcalorimetry. Upon complexation with these native and modified CDs, both nondeuterated and deuterated guests examined consistently exhibited higher affinities (by 5-20%) in D(2)O than in H(2)O. The quantitative affinity enhancement in D(2)O versus H(2)O directly correlates with the size and strength of the hydration shell around the charged/hydrophilic group of the guest. For that reason, negatively/positively charged guests, possessing a relatively large and strong hydration shell, afford smaller K(H2O)/K(D2O) ratios than those for neutral guests with a smaller and weaker hydration shell. Deuterated guests showed lower affinities (by 5-15%) than the relevant nondeuterated guests in both H(2)O and D(2)O, which is most likely ascribed to the lower ability of the C-D bond to produce induced dipoles and thus the reduced intracavity van der Waals interactions. The excellent enthalpy-entropy correlation obtained can be taken as evidence for the very limited conformational changes upon transfer of CD complexes from H(2)O to D(2)O. PMID- 12371881 TI - Electrochromic modulation of excited-state intramolecular proton transfer: the new principle in design of fluorescence sensors. AB - Internal Stark effect (or internal electrochromy) consists of the shift of light absorption and emission bands under the influence of electric field produced by proximal charges. In the studies of 3-hydroxyflavone (3HF) derivatives exhibiting the excited-state intramolecular proton transfer (ESIPT), we describe a new phenomenon - a very strong internal electrochromic modulation of this reaction. Fluorescence spectra of 3HF derivatives with charged groups attached to the chromophore from the opposite sides without pi-electronic conjugation, N-[(4' diethylamino)-3-hydroxy-6-flavonyl]methyl-N,N-dimethyloctylammonium bromide and 4 [4-[4'-(3-hydroxyflavonyl)]piperazino]-1-(3-sulfopropyl)pyridinium, were compared with those of their neutral analogues in a series of representative solvents. The introduction of the proximal charge results in shifts of absorption spectrum and of both normal (N) and tautomer (T) emission bands, which correspond to initial and phototautomer states of the ESIPT reaction. The observed shifts are in accordance with the Stark effect theory. The direction of the shift depends on the position of the proximal charge with respect to the chromophore. The magnitude of the shift depends strongly on the solvent dielectric constant and on screening or unscreening produced by addition of the hydrophobic salts. In all of these cases, the spectral shifts are accompanied by extremely strong variations of relative intensities of N and T emission bands. This signifies a strong influence of internal electric field on the ESIPT reaction, which produces a dramatic change of emission color. Thus, the coupling of the initial electrochromic sensory signal with the ESIPT reaction allows for the breaking of the limit in magnitude of response inherent to common electrochromic dyes. This suggests a new principle of designing the ultrasensitive electrochromic two wavelength fluorescence sensors and probes for analytical chemistry, macromolecular science, and cellular biology. PMID- 12371882 TI - Quinonoid oligothiophenes as electron-donor and electron-acceptor materials. A spectroelectrochemical and theoretical study. AB - Two quinonoid bis(dicyanomethylene) oligothiophenes, terthiophene and quaterthiophene analogues of TCNQ, have been investigated by spectroelectrochemical experiments and density functional theory calculations. Electrochemical data show that the molecules can be both reduced and oxidized at relatively low potentials, and that the quaterthiophene derivative forms four stable redox species, the dianion, neutral, cation radical, and dication. The neutral oligomers are characterized by a strong electronic absorption in the red or near-infrared region and can be viewed as structural and electronic analogues of aromatic oligothiophenes in the dication or bipolaron state. Upon reduction, dianions, not anion radicals, are formed which absorb in the visible region. The theoretical calculations show that the dianions have aromatic oligothiophene moieties with two anionic dicyanomethylene groups. The transition from a quinonoid to an aromatic structure is fully supported by UV-vis-near-IR and vibrational spectroscopic data. Oxidation, generating cation radicals and dications, occurs at rather low potentials similar to those reported for oligothiophenes. The electronic spectra of these cations are understood from the calculations, which suggest that the oxidized species are stabilized by the partial aromatization of the oligothiophene backbone. IR spectra of the species, especially the CN stretching frequencies, confirm the structural conclusions and allow comparison with TCNQ and the TCNQ dianion. PMID- 12371883 TI - Introduction: recoverable catalysts and reagents-perspective and prospective. PMID- 12371884 TI - Preparation of polymer-supported ligands and metal complexes for use in catalysis. PMID- 12371885 TI - Recoverable catalysts and reagents using recyclable polystyrene-based supports. PMID- 12371886 TI - ROMPgel reagents in parallel synthesis. PMID- 12371887 TI - Soluble polymers as scaffolds for recoverable catalysts and reagents. PMID- 12371888 TI - Using soluble polymers to recover catalysts and ligands. PMID- 12371889 TI - Recoverable catalysts for asymmetric organic synthesis. PMID- 12371890 TI - Enantioselective catalysis using heterogeneous bis(oxazoline) ligands: which factors influence the enantioselectivity? PMID- 12371891 TI - Supported chiral catalysts on inorganic materials. PMID- 12371892 TI - Incompletely condensed silsesquioxanes: versatile tools in developing silica supported olefin polymerization catalysts. PMID- 12371893 TI - Applications of sol-gel-processed interphase catalysts. PMID- 12371894 TI - Nano-organometallics: heterogenizing homogeneous catalysts via thin film methodology. PMID- 12371895 TI - Design and preparation of organic-inorganic hybrid catalysts. PMID- 12371896 TI - Ordered mesoporous and microporous molecular sieves functionalized with transition metal complexes as catalysts for selective organic transformations. PMID- 12371897 TI - Water-tolerant solid acid catalysts. PMID- 12371898 TI - Ionic liquid (molten salt) phase organometallic catalysis. PMID- 12371899 TI - Tag strategy for separation and recovery. PMID- 12371900 TI - Dendrimers as support for recoverable catalysts and reagents. PMID- 12371901 TI - Reduced transition metal colloids: a novel family of reusable catalysts? PMID- 12371902 TI - Polymeric membranes in catalytic reactors. PMID- 12371903 TI - Photocatalysis with organized systems for the oxofunctionalization of hydrocarbons by O2. PMID- 12371904 TI - Lewis acids as catalysts in oxidation reactions: from homogeneous to heterogeneous systems. PMID- 12371905 TI - Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes. AB - Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase), induced by cell swelling, have been proposed as underlying mechanisms. Because PI 3-kinase controls autophagic proteolysis, we examined the possibility that PI 3-kinase inhibitors interfere with glycogen production due to their anti-proteolytic action. The PI 3-kinase inhibitor wortmannin inhibited endogenous proteolysis, the production of glycogen from glucose and the activity of active (dephosphorylated) GS (GS a ) in the absence of added amino acids. The stimulation by amino acids of glycogen production and of GS a was only slightly affected by wortmannin. These effects of wortmannin could be mimicked by proteinase inhibitors. A combination of leucine, phenylalanine and tyrosine, which we showed previously to stimulate PI 3-kinase-dependent phosphorylation of ribosomal protein S6, did not stimulate glycogen production from glucose. In contrast with wortmannin, LY294002, another PI 3-kinase inhibitor, strongly inhibited both glycogen synthesis and GS a activity, irrespective of the presence of amino acids. Inhibition of glycogen synthesis by LY294002 could be ascribed in part to increased glycogenolysis and glycolysis. It is concluded that, in hepatocytes, activation of PI 3-kinase may not be responsible for the stimulation of glycogen synthesis by amino acids; LY294002 inhibits glycogen synthesis and stimulates glycogen breakdown by a mechanism that is unrelated to its action as an inhibitor of PI 3-kinase. PMID- 12371906 TI - A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers. AB - Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1) binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the 1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1 JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes. PMID- 12371907 TI - p300/cAMP-response-element-binding-protein ('CREB')-binding protein (CBP) modulates co-operation between myocyte enhancer factor 2A (MEF2A) and thyroid hormone receptor-retinoid X receptor. AB - Thyroid hormone receptors (TRs) and members of the myocyte enhancer factor 2 (MEF2) family are involved in the regulation of muscle-specific gene expression during myogenesis. Physical interaction between these two factors is required to synergistically activate gene transcription. p300/cAMP-response-element-binding protein ('CREB')-binding protein (CBP) interacting with transcription factors is able to increase their activity on target gene promoters. We investigated the role of p300 in regulating the TR-MEF2A complex. To this end, we mapped the regions of these proteins involved in physical interactions and we evaluated the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in U2OS cells under control of the alpha-myosin heavy chain promoter containing the thyroid hormone response element (TRE). Our results suggested a role of p300/CBP in mediating the transactivation effects of the TR-retenoid X receptor (RxR) MEF2A complex. Our findings showed that the same C-terminal portion of p300 binds the N-terminal domains of both TR and MEF2A, and our in vivo studies demonstrated that TR, MEF2A and p300 form a ternary complex. Moreover, by the use of CAT assays, we demonstrated that adenovirus E1A inhibits activation of transcription by TR-RxR-MEF2A-p300 but not by TR-RxR-MEF2A. Our data suggested that p300 can bind and modulate the activity of TR-RxR-MEF2A at TRE. In addition, it is speculated that p300 might modulate the activity of the TR-RxR-MEF2A complex by recruiting a hypothetical endogenous inhibitor which may act like adenovirus E1A. PMID- 12371908 TI - The promise of and problems with evidence-based medicine for paediatric asthma management. AB - The concept of 'evidence-based medicine' has now been in widespread use in clinical practice for over a decade. There are different types of clinical study, which may provide evidence on which to base clinical decisions, but some are much less robust than others. In making decisions about treatment, one of the highest levels of evidence in primary research is the randomized controlled trial. This study design has been used in clinical research for over 50 years. Systematic reviews of randomized controlled trial are scientific studies that review, critically appraise and, where appropriate, aggregate results from a number of different randomized controlled trials. They are increasingly being used throughout health care to provide guidance about treatment. Under the auspices of The Cochrane Collaboration, systematic reviews of randomized controlled trials are being conducted across the whole of health care. One of the most active areas for this work is within the field of asthma and clinicians are now able to access a large number of different systematic reviews on The Cochrane Library. If used appropriately, they can aid the clinician in making decisions about individual patients and provide a sound evidence base from which clinical guidelines can be developed. PMID- 12371909 TI - Evidence-based decision making and asthma in the internet age: the tools of the trade. AB - At the dawn of the Information Age, the practice of evidence-based decision making (EBDM) is still hindered by many important barriers related to the decision makers, to the evidence per se or to the health system. Some of these barriers, particularly those related to the distillation, dissemination and packaging of research evidence, could be overcome by recent and ongoing developments in portable/wearable computers, internet appliances, multimedia and wireless broadband internet traffic. This article describes specific EBDM-related tools, with emphasis on internet-enabled "how to" books; and tools to improve the quality of reporting research, to formulate questions; to search for evidence; to access journals, systematic reviews and guidelines; to interact with organizations promoting EBDM; and to tailor evidence to individual cases. However, thinking that all barriers to the practice of EBDM could be solved by fancy information technology is naive. Barriers related to the generation, interpretation, integration and use of the evidence demand more complex and perhaps unfeasible solutions, as overcoming them will require substantial changes in the structure of the health system, in the politics of science and in the way in which humans think and behave. PMID- 12371910 TI - The role of house dust mite elimination in the management of childhood asthma: an unresolved issue. AB - Indoor allergens are likely to be direct environmental causes of asthma and mite exposure, and sensitization is the most important environmental risk factor for childhood asthma in temperate zones. Analagous to occupational asthma, allergen avoidance in asthmatic children sensitized and exposed to mite allergens is associated with a reduction in airway hyperresponsiveness and symptoms associated with improvement in lung function. The long-term effect of this strategy needs to be prospectively evaluated considering both the timing and duration of exposure, as well as the timing and duration of removal. In order to be successful, it is important to achieve and maintain a major reduction on allergen levels, for a long period of time. PMID- 12371911 TI - Efficacy of steroid treatments in the asthmatic preschool child. AB - Asthma represents the most common chronic disease in preschool children. Hospital admission for wheezy disorders is the most common paediatric chronic disease causing hospital admission and more common in young children than later in life. PMID- 12371912 TI - Effectiveness of pharmacotherapy in asthmatic preschool children. AB - The term "effectiveness" relates to the question of whether or not a certain treatment works in practice. Usually, such a treatment was first evaluated under tightly controlled conditions in selected patient populations, and the potential benefits were shown. There is, however, a great difference between the efficacy of a given treatment, indicating its optimal therapeutic action in controlled trials, and its effectiveness when applied to a less well-defined population of patients in daily practice. This is especially relevant for asthma in young children, where many factors are responsible for the difference. Among these are, first of all, the heterogeneity of the wheezing phenotype. Other factors include the compliance with prescribed treatments, as determined by the attitude of doctors and parents towards such treatment, the ease of administration and the perceived effects and side effects. Also, the performance of different inhaler devices may be insufficient for a good, reliable dose deposition in young children in daily life. As a result, the current treatment guidelines for preschool children with recurrent wheeze are probably too optimistic in assuming that inhaled treatment is most effective and feasible at all ages. We propose careful re-evaluation of such recommendations in a first-line setting resembling daily life as closely as possible, and consideration of oral treatments as well. Also, we need methods to separate the different phenotypes within the group of recurrently wheezing preschool children to optimize targeting of asthma treatment to those who have ongoing airway inflammation. PMID- 12371913 TI - Adherence to asthma therapy in the preschool child. AB - Preschool children's adherence to asthma therapy is often sub-optimal and can result in decreased quality of life for children and parents, as well as an increased risk for dangerous asthma exacerbations. Asthma management for the preschool child presents some unique challenges to adherence to therapy, including the child's limited ability to communicate, multiple caregivers responsible for medications, and parental concerns about medications. Parent beliefs, characteristics of the regimen, and family functioning have been associated with adherence levels. Understanding and improving adherence to asthma therapy for the preschool child will necessarily require addressing these age specific concerns. PMID- 12371914 TI - Long-term outcomes in paediatric asthma. AB - Over the years the aims of asthma management have changed markedly from effective prednisolone treatment of symptoms and exacerbations towards more use of continuous prophylactic treatment. With our new understanding of the disease and its management definition of the aims of treatment and assessment of optimal asthma control have become much more complex. Even in times of evidence-based medicine our asthma management is based upon findings of effects on various outcomes in somewhat short-term (<1 year) controlled studies. However, assumptions about long-term effects upon the basis of findings in such studies should be made with great caution. Good examples of this are studies which assess the risk of systemic effects and clinical adverse effects of inhaled corticosteroids. From such studies it has become clear that systemic effects detected in short-term trials may have no predictive value of long-term adverse effects. Thus steroid-induced changes in lower leg growth rates assessed by knemometry do not predict long-term statural growth. Moreover, steroid-induced changes in statural growth over 1 year are not predictive of effects upon attained adult height. In contrast, reduced growth caused by uncontrolled asthma disease also seems to affect attained adult height adversely. These findings suggest that long-term outcomes should play a larger role when future asthma management strategies are decided. Some important long-term outcomes of asthma management in children include cure or remission of the disease, prevention of complications of the disease (airway remodelling, adverse effects upon growth/adult height, peak bone mineral density, physical impairment and psychosocial development) or its pharmacological management (adverse effects upon adult height, peak bone mineral density). More controlled long-term studies (several years) are needed to provide a better understanding of how these outcomes are best achieved. PMID- 12371915 TI - Multiple sclerosis in Stockholm County. A pilot study of utilization of health care resources, patient satisfaction with care and impact on family caregivers. AB - Multiple sclerosis (MS) is a progressive, incurable neurological disease with a large impact on the person/people with MS (PwMS), the family of the PwMS, medical resources and the community. We have explored the feasibility of calculating utilization of health-care resources within Stockholm County and evaluated methods for interviewing PwMS and family caregivers in their homes. Home visits were made to 26 PwMS with different levels of disability, both in ordinary and sheltered living. Questionnaires assessing patient satisfaction, the use of technical aids and home adaptations, help from municipal and family caregivers, and health-related quality of life were administered in the form of structured interviews. Utilization of health-care resources was evaluated with the help of an available computerized register. The study shows that the chosen methods are feasible for PwMS, irrespective of level of disability or form of living. They are well accepted by both PwMS and family caregivers and need only minor modifications in order to be suitable for a population-based study. PMID- 12371916 TI - Sodium valproate in the management of painful neuropathy in type 2 diabetes - a randomized placebo controlled study. AB - OBJECTIVE: To study the effectiveness and safety aspects of sodium valproate in the management of painful neuropathy in patients of type 2 diabetes mellitus. MATERIAL AND METHODS: A randomized double-blind placebo controlled trial of sodium valproate was done in type 2 diabetic patients to assess its efficacy and safety in the management of painful neuropathy. We screened 60 patients but eight patients could not complete the study; hence, the present study was done on 52 patients. Each patient was assessed by clinical examination, pain score by short form of the McGill pain questionnaire (SF-MPQ) and electrophysiological examination, which included motor and sensory nerve conduction velocity, amplitude and H-reflex initially and at the end of 1 month of treatment. RESULTS: Significant improvement was noticed in the pain score of patients receiving sodium valproate in comparison to patients receiving placebo at the end of 1 month (P < 0.05). The changes in electrophysiological data were not significant. The drug was well tolerated by all patients except one who developed a raised aspartate transaminase (AST)/alanine transaminase (ALT) level after 15 days of treatment. CONCLUSION: Sodium valproate is a well-tolerated drug and provides significant subjective improvement in painful diabetic neuropathy. These data provide a basis for future trials of longer duration in a larger group of patients. PMID- 12371917 TI - Peripheral neuropathy in acrodermatitis chronica atrophicans - effect of treatment. AB - Forty-seven patients with the late borrelial manifestation acrodermatitis chronica atrophicans (ACA) and with objective neurological and/or neurophysiological findings were followed up after antibiotic treatment with dermatological, serological, neurological and neurophysiological controls. Despite a good therapeutic effect on ACA lesions, specific antibody values and symptoms of irritative nerve lesions, the objective neurological and neurophysiological findings of nerve deficit remained unchanged. There was no progress of neuropathy findings during the follow-up time. Our interpretation of the results is that the remaining neuropathy signs after treatment of ACA are neurological sequelae and not manifestations of persisting Borrelia infection. PMID- 12371918 TI - Secretory phospholipase A2 and phospholipids in neural membranes in an experimental epilepsy model. AB - OBJECTIVES: Previous studies have revealed an increase in several forms of phospholipase A2 activity associated with cell injury, but the secretory form of phospholipase A2 has not previously been studied in neurological disorders. We investigated the influence of seizures on secretory phospholipase A2 and phospholipid breakdown in synaptosome fractions prepared from rat hippocampus, cortex and cerebellum in pentylenetetrazol-induced epilepsy. MATERIAL AND METHODS: Secretory phospholipase A2 concentration was measured by a photometric enzyme immunoassay. The synaptosomes underwent extraction, and the phospholipids fractions for phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine were recovered from the thin layer chromatography plates. The amount of each phospholipid was quantified using the amount of recovered phosphate in each phospholipid spot. RESULTS: Secretory phospholipase A2 concentration was found to be significantly higher in the epileptic group when compared with the control group. The amounts of phospholipids were found to be highly variable in different brain regions. CONCLUSION: Our results suggest that epileptic seizures enhanced phospholipid breakdown and induced alterations in the distribution of phospholipids in different brain regions. PMID- 12371919 TI - Inter-observer reproducibility and responsiveness of a clinical severity scale in surgically treated carpal tunnel syndrome. AB - OBJECTIVES: To test a recently proposed carpal tunnel syndrome (CTS) clinical severity scale for reproducibility between two observers (neurosurgeon and neurophysiologist) before surgery, for responsiveness to changes in clinical status 6 months after surgery, and for correlations with the electrophysiological findings and 'Boston Carpal Tunnel Syndrome Questionnaire' (BQ). MATERIAL AND METHODS: The tests were applied prospectively to a consecutive series of 254 hands with idiopathic CTS, referred for surgical decompression. The hands belonged to 219 subjects (177 women and 42 men, mean age 55.6). RESULTS: Percentage agreement between the two observers in assigning severity to the same class was 78% and Cohen coefficient kappa was 0.69 (P < 0.001). The scale was found to be responsive to changes in clinical status after surgery. Direct correlations were also found between the scale and patient age, duration of symptoms, BQ scores and the neurophysiological severity scale. The significance of these associations was maintained for 6 months after the operation. CONCLUSION: This clinical severity scale is simple, reproducible and sensitive for evaluating severity of CTS in patients undergoing surgery. PMID- 12371920 TI - Long-term neuropsychological effects and MRI findings in patients with CS2 poisoning. AB - OBJECTIVES: To evaluate the long-term neuropsychological effects and magnetic resonance imaging (MRI) findings among retired patients with a history of exposure to carbon disulfide (CS2). MATERIAL AND METHODS: Seventy-four patients with a history of exposure to CS2 were divided into two equal groups according to their level of exposure, and they completed a questionnaire and were evaluated for neuropsychological symptoms using the Korean version of the revised Wechsler Adult Intelligence Scale. Thirty-one MRI images were reviewed retrospectively. RESULTS: There were no statistically significant differences in total, verbal and performance IQs between high- and low-exposure groups. MRI findings revealed a significantly larger number of cerebral lacunae (five of 12 subjects) in the high exposure group. Periventricular hyperintensities were mostly located in frontal and occipital areas, and white-matter hyperintensities were mostly in frontal and parietal areas. CONCLUSION: The higher prevalence of lacunae in the high-exposure group as revealed by MRI suggests that further MRI studies are needed into long term neuropsychological effects induced by CS2. PMID- 12371921 TI - Prevalence of Parkinson's disease in Estonia. AB - OBJECTIVES: To investigate the prevalence of idiopathic Parkinson's disease (PD) in Tartu district of South Estonia, with a population of 153,240 on prevalence day, 1 January 1996. METHODS: The community-based method of case ascertainment was used, followed by neurologic examination. RESULTS: The age-adjusted prevalence was 152 per 100,000 population, 159 for urban and 139 for the rural group, 154 for men and 153 for women. The age-specific prevalence increased from 22 per 100,000 population in the age group 40-49 years up to 1232 per 100,000 population in the age group 70-79 years. The mean age of PD patients was 71.4 years, the mean age at onset of the symptoms - 66.9 years. CONCLUSIONS: When comparing the prevalence rates with other studies of Caucasian populations in Europe, the results are similar except for slightly but not significantly higher prevalence rates in the urban population in Estonia. PMID- 12371922 TI - Altered calcium in motoneurons by IgG from human motoneuron diseases. AB - OBJECTIVES: The effect of IgG from patients with multifocal motor neuropathy (MMN) on the content and distribution of calcium in spinal motoneurons was compared with the effect of IgG from patients with sporadic amyotrophic lateral sclerosis (SALS) and IgG from normal individuals. MATERIAL AND METHODS: Different purified IgG samples were injected intraperitoneally in mice. Then, the animals were subjected to histochemical techniques to visualize calcium in electron microscopic sections. RESULTS: Quantitative morphometric analysis verified that IgG from MMN decreased the vesicular and axoplasmic calcium content in the axon terminals at the neuromuscular junctions and had no influence on the perikaryon. In contrast to this, IgG from patients with SALS increased the intracellular calcium both in the axon terminal and in the perikaryon. IgG from normal individuals exerted no effect. Elevated intracellular calcium may contribute to motoneuron degeneration. The lack of such effect with MMN immunoglobulins helps to explain the relative sparing of motoneurons in the disease. PMID- 12371923 TI - sPECAM-1 in serum and CSF of acute ischaemic stroke patients. AB - OBJECTIVES: As platelet endothelial cell adhesion molecule-1 (PECAM-1) is one of key mediators of transendothelial migration of leucocytes during inflammation, and inflammatory reaction is observed in cerebral ischaemia, we decided to determine the levels of soluble PECAM-1 (sPECAM-1) in serum and cerebrospinal fluid (CSF) of patients with acute stroke. MATERIAL AND METHODS: Twenty-three patients with first-ever in a lifetime completed ischaemic stroke have been studied. CSF and blood samples were obtained within 24 h of the onset of stroke and the levels of sPECAM-1 in serum and CSF were quantified by ELISA. RESULTS: Stroke patients displayed statistically significant higher levels of sPECAM-1 in sera and CSF in comparison with control group. The levels were significantly higher in serum than in CSF, correlated between each other, and CSF sPECAM-1 fraction was blood-derived. CONCLUSION: Our results indirectly suggest that PECAM 1 may play a role in the pathophysiological events during early phase of ischaemic stroke. PMID- 12371924 TI - Increased plasma TGF-beta1 in patients with amyotrophic lateral sclerosis. AB - OBJECTIVES: To investigate the levels of transforming growth factor-beta1 (TGF beta1) in plasma of patients with amyotrophic lateral sclerosis (ALS). MATERIAL AND METHODS: The TGF-beta1 plasma concentrations were measured with an enzyme linked immunosorbent assay from 11 patients with ALS and 13 age matched healthy controls. RESULTS: The mean TGF-beta1 plasma concentration in the patients with ALS (2.15 +/- 0.80 ng/ml, mean +/- SD) was significantly higher than in the healthy controls (1.59 +/- 0.32 ng/ml) (P=0.031). There was a significant positive correlation between the TGF-beta1 plasma concentration in the patients with ALS and the duration of illness (r=0.66, P=0.028). CONCLUSION: Our findings provide evidence that in ALS the plasma concentration of TGF-beta1 increases significantly with the duration of illness. These results suggest that TGF-beta1 is involved in the disease process of ALS. PMID- 12371925 TI - Lymphocyte subset numbers in cerebrospinal fluid: comparison of tick-borne encephalitis and neuroborreliosis. AB - OBJECTIVE: The aim of this study was to analyze lymphocyte subset numbers in cerebrospinal fluid (CSF) from patients with tick-borne encephalitis (TBE) and acute neuroborreliosis. METHODS: CSF lymphocyte subsets were enumerated in 42 TBE and nine neuroborreliosis patients using flow cytometry. RESULTS: The CSF numbers of CD4+, CD8+, HLA-DR+ and total-T lymphocytes, B lymphocytes, and NK cells were all greater in neuroborreliosis patients than in TBE patients. Neuroborreliosis patients showed positive correlation of CSF protein levels with the numbers of CD4+, HLA-DR+ and total-T lymphocytes. Also, the numbers of CSF B lymphocytes correlated positively with intrathecal Borrelia burgdorferi-specific IgG antibodies. Conversely, TBE patients demonstrated intrathecal protein levels that correlated positively with all investigated CSF lymphocyte subsets. CONCLUSION: These results suggest an intensive recruitment of lymphocyte subsets into the central nervous system (CNS) during acute neuroborreliosis, whereas TBE is characterized by a lower accumulation of lymphocyte subsets in the CSF. PMID- 12371926 TI - MELAS: a neuropsychological and radiological follow-up study. Mitochondrial encephalomyopathy, lactic acidosis and stroke. AB - We report on a patient with long standing, full-blown mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). In contrast to earlier publications, detailed neuropsychological assessment revealed no dementia but a pattern of distinct cognitive deficits with marked impairment of visuo-constructive and executive functions. Focal lesions and progressing atrophy mainly of the basal ganglia and the temporo-parieto-occipital area with preservation of hippocampal and entorhinal structures were present. Furthermore, a 4-year follow-up assessment revealed an increasing deterioration of distinct cognitive functions, including phasic alertness, tactile functions and the discrimination of tone pitch and rhythm. This may be because of chronic regional metabolic disturbances, as there was no further stroke-like episode in that period of time. PMID- 12371927 TI - Cerebral oxygen metabolism monitoring under hypothermia for severe subarachnoid hemorrhage: report of eight cases. AB - OBJECTIVES: The significance of cerebral oxygen metabolism monitoring under hypothermia for severe subarachnoid hemorrhage (SAH) was studied. MATERIAL AND METHODS: Cerebral oxygen metabolism monitoring (jugular venous oxygen saturation: SjO2, arterio-jugular venous difference of oxygen: AJDO2, and oxygen extraction fraction:OEF) during hypothermia (32-34 degrees C) was evaluated in eight patients with SAH (severe vasoapasm group: five patients, and severe brain damage group: three patients). RESULTS: In favorable cases in both groups, each parameter tended to normalize during hypothermia therapy. When changes in SjO2 were normal, however, the value of AJDO2 was low in unfavorable cases in the severe vasospasm group. In unfavorable cases in the severe brain damage group, high level of SjO2 and low level of OEF and AJDO2 were shown even if hypothermia therapy was performed. CONCLUSIONS: The measurement of SjO2 and AJDO2 is useful for estimation of cerebral oxygen metabolism in patients with severe conscious disturbance after SAH under hypothermia therapy. PMID- 12371928 TI - Head tremor in dentatorubral-pallidoluysian atrophy. AB - Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare autosomal-dominant neurodegenerative disorder characterized by variable combination of clinical manifestations including ataxia, myoclonus, seizures, dementia, and choreic movements. Head tremor has been rarely reported. We report a 66-year-old-woman with genetically determined DRPLA who presented with head tremor. A "no-no" type head tremor was the initial and the most prominent symptom, and mild cerebellar signs and choreic movements were also observed later. Neither hand tremor nor dystonia was noted. The patient did not show dementia, myoclonus, or seizures. Surface electromyogram (EMG) revealed 3.5-4 Hz rhythmic EMG bursts in both sternocleidomastoid muscles. DNA analysis disclosed expanded trinucleotide repeats (n = 54) in the DRPLA gene. We suggest that isolated head tremor can be a clinical manifestation of DRPLA. PMID- 12371930 TI - Pancreatic islet xenotransplantation. PMID- 12371931 TI - Regulatory issues in Europe and Canada. PMID- 12371932 TI - Porcine endogenous retrovirus--advances, issues and solutions. PMID- 12371933 TI - Anti-N-glycolylneuraminic acid antibodies identified in healthy human serum. AB - The first and major clinical obstacle in xenotransplantation is antibody-mediated hyperacute rejection. Although human natural antibodies against Galalpha1,3Gal (Gal) antigens, which are common on porcine cells and organs, have been identified to play a major role in hyperacute rejection, other natural antibodies against non-Gal epitopes may be also involved in the process. Here, we present evidence suggesting that the majority of human anti-non-Gal antibodies are specific for carbohydrate structures carrying terminally linked N glycolylneuraminic acid (NeuGc), a xenoantigen existing in almost all animals except humans. Furthermore, this anti-NeuGc activity is detectable in 85% of healthy humans, implicating the involvement of NeuGc in hyperacute rejection and the importance of developing strategies for removing NeuGc for clinical xenotransplantation. PMID- 12371934 TI - High avidity antibodies to fetal pig pancreas endocrine cells transfer rejection but are not normally generated to fetal pig pancreas xenografts. AB - Previous studies on the contribution of T cell-dependent antibody (Ab) to non vascular xenograft rejection have yielded conflicting results, being confounded by the presence of recipient T cells and the use of different tissues and immunizing regimens to generate Ab. In the present study, the effect of adoptive transfer of Ab on fetal pig pancreas (FPP) and pig PK15 cell xenografts was examined in T cell-deficient severe combined immune deficiency (SCID) mice. T cell-dependent Abs raised by hyperimmunization with different cell types and by FPP transplantation were compared. Ab raised by hyperimmunization with pig thymocytes exhibited strong binding to pig thymocytes and PK15 cells but did not transfer FPP rejection. IgG1 and IgM, but not IgG3, Abs bound strongly to FPP exocrine and connective tissue, whereas binding to endocrine cells in vitro and in vivo was weak or absent. This pattern of Ab binding was similar to that observed after transplanting FPP into BALB/c mice. Furthermore, serum recovered from BALB/c mice 20 days after FPP transplantation bound strongly to non endocrine but not endocrine cells and did not transfer FPP rejection. In contrast, serum from mice hyperimmunized with PK15 cells bound strongly to PK15 cells and transferred rejection of intraperitoneal PK15 cells. Furthermore, this serum contained IgG1 and IgM Abs that bound strongly, and IgG3 Abs that bound weakly, to endocrine cells in FPP, and also transferred rejection of FPP in SCID mice. These results indicate that endocrine cells express low concentrations of xenoreactive Ab epitopes and that high Ab concentrations and/or high avidity Abs are required for sufficient endocrine cell binding to cause damage and rejection in the immunodeficient mouse model. Such Abs are not elicited by transplanting FPP into immunocompetent mice. Nevertheless, a contribution of Ab to rejection in immunocompetent mice cannot be excluded. PMID- 12371935 TI - Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect antigen recognition pathway. AB - We have previously demonstrated that human T cells responding to porcine islets are primarily CD4+ and recognized porcine major histocompatibility complex class I molecules through the indirect pathway of antigen presentation. To determine whether this mechanism is responsible for rejection of adult porcine islets xenografts, porcine islets from adult pigs were transplanted under the kidney capsule of streptozotocin-treated CD4-knockout (KO), CD8-KO, Ig-KO and normal C57BL/6 mice. Islet xenografts were acutely rejected with similar kinetics when transplanted into normal C57BL/6 (MST=17.6 +/- 3.5 days) and Ig-KO (MST=19.0 +/- 1.7 days) mice. Interestingly, islet xenografts were rejected significantly earlier when transplanted into CD8-KO mice as compared with normal C57BL/6 (MST=7.0 +/- 0.01 days, P=2 x 10-4). Histopathological analysis revealed classical acute cellular rejection with severe diffuse interstitial cellular infiltrates in all rejected islet xenografts. In contrast, islet xenografts were not rejected when transplanted into CD4-KO mice (MST >/= 100 days, P=1 x 10-9). Histopathological analysis revealed no cellular infiltrates and intact islet xenografts. CD4+ T cells from both normal C57BL/6 and CD8-KO xenograft recipients showed detectable proliferative responses to porcine islets in the presence but not in the absence of syngeneic antigen-presenting cells. In addition, the anti islet proliferative responses observed in normal C57BL/6 mice were significantly lower than those observed in CD8-KO mice. IgG anti-porcine antibodies were readily detected in C57BL/6 and CD8-KO xenograft recipients but not in Ig-KO or CD4-KO recipients. These results indicate that indirectly activated CD4+ T cells mediate acute rejection of adult porcine islet xenografts and that xenoreactive CD8+ T cells and antibodies are not necessary in this process. PMID- 12371936 TI - Promotion of xenogeneic hematopoietic chimerism in rodents by mononuclear phagocyte depletion. AB - The successful establishment of tolerance toward pig tissues in primates through hematopoietic progenitor cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. We developed and tested the hypothesis that phagocytes of the reticuloendothelial system are responsible for the rapid clearance of infused pig hematopoietic cells using a mouse model. Mice received non-myeloablative conditioning and, on various days, were injected with medronate-encapsulated liposomes (M-L) or control blank liposomes, followed by the intravenous infusion of miniature swine hematopoietic cells. M-L were well-tolerated in mice (n=100) at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in normal Balb/c mice as well as in severe combined immune deficient mice increased the accumulation of pig hematopoietic cells in the bone marrow (BM) by 10-fold when measured 24 h after the infusion of the cells. Colony-forming unit analysis showed an increased accumulation of pig hematopoietic progenitors in the BM of mice that were infused with medronate-liposomes. We conclude that depletion of mononuclear phagocytes by M-L has the potential to lower the barrier to the establishment of mixed chimerism and tolerance induction in xenotransplantation. PMID- 12371937 TI - Cloning and potential utility of porcine Fas ligand: overexpression in porcine endothelial cells protects them from attack by human cytolytic cells. AB - Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T-cell line, Jurkat. Anti-human Fas monoclonal antibody (mAb) specifically inhibited this killing in a dose-dependent manner, suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We next cloned porcine FasL, identifying an open reading frame of 849 base pairs predicting a protein of 282 amino acids. The predicted amino acid sequence was 85, 76, and 75% homologous to the predicted amino acid sequences of human, mouse, and rat, respectively, and found that PAEC expressed both FasL mRNA and protein. Transient transfection was used to increase or induce porcine FasL expression in PAEC or COS-7 cells. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human (natural killer) NK cells. Moreover, porcine Fas L-transfected COS-7 cells induced significant apoptosis in Jurkat cells compared with that induced by mock-transfected COS-7 cells. Finally, the overexpression of porcine FasL in PAEC reduced their susceptibility as target cells to lysis by activated human NK or T cells. These findings suggest that porcine FasL overexpression in EC of vascularized xenografts may provide protection from cellular xenograft rejection. PMID- 12371938 TI - Concerns expressed about the virological risks of xenotransplantation. PMID- 12371940 TI - ISPOR (International Society for Pharmacoeconomics and Outcomes Research) 5th Annual European Congress. 3-5 November 2002. Rotterdam, The Netherlands. Abstracts. PMID- 12371944 TI - A trail of research on potassium. AB - A complex pump-leak system involving both active and passive transport mechanisms is responsible for the appropriate distribution of potassium (K) between the intra- and extracellular fluid compartments. In addition, the kidneys, and to a lesser extent the colon, safeguard maintenance of the narrow range of low K concentrations in the extracellular fluid. Early renal clearance studies showed that K is normally both reabsorbed and secreted by renal tubules, and that regulated secretion is the major source of K excretion. Studies at the tubule and cell level have localized secretion and reabsorption of K to principal and intercalated cells in the collecting ducts. Measurements of the electrochemical driving forces across individual cell membranes have permitted the characterization of specific ATPases, K channels and K cotransporters and also provided insights into the molecular structure of individual transporters that regulate K excretion. PMID- 12371953 TI - The elephant in uremia: oxidant stress as a unifying concept of cardiovascular disease in uremia. AB - Cardiovascular disease is the leading cause of mortality in uremic patients. In large cross-sectional studies of dialysis patients, traditional cardiovascular risk factors such as hypertension and hypercholesterolemia have been found to have low predictive power, while markers of inflammation and malnutrition are highly correlated with cardiovascular mortality. However, the pathophysiology of the disease process that links uremia, inflammation, and malnutrition with increased cardiovascular complications is not well understood. We hereby propose the hypothesis that increased oxidative stress and its sequalae is a major contributor to increased atherosclerosis and cardiovascular morbidity and mortality found in uremia. This hypothesis is based on studies that conclusively demonstrate an increased oxidative burden in uremic patients, before and particularly after renal replacement therapies, as evidenced by higher concentrations of multiple biomarkers of oxidative stress. This hypothesis also provides a framework to explain the link that activated phagocytes provide between oxidative stress and inflammation (from infectious and non-infections causes) and the synergistic role that malnutrition (as reflected by low concentrations of albumin and/or antioxidants) contributes to the increased burden of cardiovascular disease in uremia. We further propose that retained uremic solutes such as beta-2 microglobulin, advanced glycosylated end products (AGE), cysteine, and homocysteine, which are substrates for oxidative injury, further contribute to the pro-atherogenic milieu of uremia. Dialytic therapy, which acts to reduce the concentration of oxidized substrates, improves the redox balance. However, processes related to dialytic therapy, such as the prolonged use of catheters for vascular access and the use of bioincompatible dialysis membranes, can contribute to a pro-inflammatory and pro-oxidative state and thus to a pro-atherogenic state. Anti-oxidative therapeutic strategies for patients with uremia are in their very early stages; nonetheless, early studies demonstrate the potential for significant efficacy in reducing cardiovascular complications. PMID- 12371954 TI - Microvascular endothelial injury and dysfunction during ischemic acute renal failure. AB - The pathophysiology of ischemic acute renal failure (ARF) appears to involve a complex interplay between renal hemodynamics, tubular injury, and inflammatory processes. While the current paradigm of the pathophysiology of ischemic ARF invokes both sublethal and lethal tubular injury as being of paramount importance to diminished renal function, a growing body of evidence supports the contribution of altered renal vascular function in potentially initiating and subsequently extending the initial tubular injury. We propose that the "extension phase" of ischemic ARF involves alterations in renal perfusion, continued hypoxia, and inflammatory processes that all contribute to continued tubular cell injury. Vascular endothelial cell injury and dysfunction play a vital part in this extension phase. In the constitutive state the endothelium regulates migration of inflammatory cells into tissue, vascular tone and perfusion, vasopermeability, and prevents coagulation. Upon injury, the endothelial cell loses its ability to regulate these functions. This loss of regulatory function can have a subsequent detrimental impact upon renal function. Vascular congestion, edema formation, diminished blood flow, and infiltration of inflammatory cells have been documented in the corticomedullary junction of the kidney, but linking their genesis to vascular endothelial injury and dysfunction has been difficult. However, new investigative approaches, including multiphoton microscopy and the Tie2-GFP mouse, have been developed that will further our understanding of the roles endothelial injury and dysfunction play in the pathophysiology of ischemic ARF. This knowledge should provide new diagnostic and therapeutic approaches to ischemic ARF. PMID- 12371955 TI - SLC7A9 mutations in all three cystinuria subtypes. AB - BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria (SLC3A1) is associated with the Type I urinary phenotype. A second cystinuria gene (SLC7A9) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well characterized cystinuria probands and their family members. METHODS: Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. RESULTS: Seven mutations were identified. A single bp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G-->A, A354T) were identified on Type III alleles. A nonsense codon (1491G- >T, E436X) and a possible splicing mutation (IVS9-17G-->A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T-->C, I44T and 967C-->T, P261L) linked to Type I alleles. CONCLUSION: Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted. PMID- 12371956 TI - Multiorgan mRNA misexpression in murine autosomal recessive polycystic kidney disease. AB - BACKGROUND: BALB/c mice homozygous for the cpk mutation develop a form of polycystic kidney disease (PKD) with multiorgan pathology similar to human autosomal recessive PKD. Messenger RNA expression in multiple affected organs was analyzed to determine if common gene cascades were misexpressed in the cystic kidney and extrarenal sites of disease. In cystic kidneys, misexpressed mRNAs were found in one of four general groups: proliferation/cell growth, apoptosis, differentiation or extracellular matrix. METHODS: RNA was isolated from kidney, liver and pancreas of cystic and normal BALB/c-cpk mice. Using Northern blot hybridization and ribonuclease protection assays (RPA), the expression of several genes thought to be associated with PKD, namely c-myc, epidermal growth factor receptor (EGF-R) and PKD-1, were evaluated. RPAs were used to assess mRNA expression of cyclins and members of the bax/bcl-2 family. In addition, kidney, liver and pancreas were immunostained for c-Myc and PCNA. RESULTS: Cystic kidney, liver and pancreas all exhibited similar patterns of mRNA misexpression of c-myc, EGF-R and PKD-1. In addition, a number of cell proliferation and apoptosis related mRNAs also were elevated in cystic kidney and pancreas. Renal epithelial cells expressing proliferation-associated proteins [c-Myc and proliferating cell nuclear antigen (PCNA)] were nearly absent in normal kidney; however, cells of cystic and non-cystic renal tubules plus liver and pancreatic cyst exhibited an increased number of nuclei labeled with antibodies to these proteins. CONCLUSIONS: These data suggest that similar pathologic mechanisms (including the expression of c-myc, EGF-R, PKD-1, cyclin, and bax/bcl-2 family mRNAs) may be responsible for the development of cystic changes in kidney, liver and pancreas in murine autosomal recessive PKD. Treatments targeting these similarly misexpressed mRNAs may be efficacious in ameliorating the cystic pathology in the kidney as well as the other affected organs in ARPKD. PMID- 12371957 TI - Gene expression of prostanoid forming enzymes along the rat nephron. AB - BACKGROUND: To obtain information about the general capability of nephron segments to elaborate prostanoids, we determined the gene expression of key enzymes for prostanoid formation. METHODS: For this goal mRNAs were assayed for cyclooxygenases-1 and -2 as well as for the synthases of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostacyclin (PGI2) and thromboxane A2 (TXA2) in microdissected rat nephron segments by RT-PCR. RESULTS: Cyclooxygenase-1 (COX-1) mRNA was strongly expressed in all segments of the collecting ducts and to a lesser extent in glomeruli. COX-2 mRNA was found in the cortical thick ascending limb of Henle, and weaker expression also was detected in glomeruli. The lipocalin-type PGD synthase mRNA displayed a broad expression pattern in the cortex and outer medulla, including proximal convoluted tubule, thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting duct. The hematopoietic PGD synthase mRNA was restricted to the outer medullary collecting duct, and the membrane-associated PGE-synthase mRNA was exclusively expressed in the whole collecting duct system. Prostacylin-synthase mRNA was found in the whole kidney, but not in any microdissected nephron segment analyzed in this study. TXA-synthase mRNA was expressed in glomeruli. CONCLUSION: Given that the existence of cyclooxygenase in combination with the different PG synthases is a prerequisite for the formation of prostanoids, our data suggest that PGD2 is mainly formed in the thick ascending limb and in the collecting duct, while PGE2 appears to be mainly generated by the collecting ducts. Probably no formation of PGI2 occurs within the nephron. Whether TXA2 can be formed by nephron segments remains questionable. PMID- 12371958 TI - Circulating inhibitor of gonadotropin releasing hormone secretion by hypothalamic neurons in uremia. AB - BACKGROUND: Previous studies have suggested a neuroendocrine defect underlying uremic hypogonadism, characterized by a reduced secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). METHODS: We studied the GnRH-producing GT1-7 cell line and the LH-producing LbetaT-2 pituitary cell line under uremic conditions to investigate whether substances circulating in uremic plasma directly affect hypothalamic or pituitary hormone secretion. The cells were incubated with serum from 5/6-nephrectomized or sham-nephrectomized castrated rats, respectively. Furthermore, GT1 cells were incubated with delipidated sera, serum subfractions separated by molecular weight, or several peptide hormones. Cellular viability, apoptosis rate and extracellular hormone degradation were assessed separately. GnRH and LH were measured by RIA in supernatants and cell lysates. GnRH gene expression was assessed by Northern blot. RESULTS: Uremic serum caused a reduction of extracellular GnRH concentration by 31%, whereas intracellular GnRH increased by 12%. This effect was independent of serum lipids and enzymatic GnRH degradation but was abolished by trypsin digestion. Cellular viability, apoptosis rates and GnRH gene expression did not differ between the two groups. The inhibitory activity was recovered from the high-molecular weight fraction, whereas the fraction <5 kD had stimulatory activity. In contrast, uremic serum did not affect LH secretion from LbetaT-2 cells, indicating that the hypoactivity of the hypothalamo-pituitary gonadotrope unit results from an inhibition at the hypothalamic rather than the pituitary level. CONCLUSIONS: Our results suggest that uremic serum contains macromolecular and hydrophilic peptide(s) able to specifically suppress the neurosecretion of GnRH from GT1-7 cells. PMID- 12371959 TI - Differential effects of insulin-like growth factor binding proteins-1, -2, -3, and -6 on cultured growth plate chondrocytes. AB - BACKGROUND: In children with chronic renal failure (CRF), impairment of longitudinal growth is in part due to excess amounts of circulating high-affinity insulin-like growth factor binding proteins (IGFBPs) that might decrease or prevent insulin-like growth factor (IGF) binding to its signaling receptor. However, it appears from the clinical studies that various IGFBPs may have contrasting effects on longitudinal growth. Because of the potential importance of the IGFBPs as modulators of longitudinal growth in pediatric CRF, the aim of the present study was to investigate the biological effects of IGFBP-1, -2, -3, and -6 on cultured growth plate chondrocytes that express the type 1 IGF receptor. METHODS: The effects of exogenous IGFBPs on IGF-independent and IGF dependent proliferation of rat growth plate chondrocytes in primary culture were investigated. Proliferation was assessed by colony formation of agarose stabilized long-term suspension cultures and by the [3H]thymidine assay. The effects of IGFBPs on IGF-I binding and the binding of IGFBPs to chondrocytes were assessed by binding studies with radiolabeled proteins in monolayer culture. RESULTS: Intact IGFBP-1, IGFBP-2 and IGFBP-6 inhibited in equimolar concentration the IGF-I- and IGF-II-stimulated DNA synthesis and cell proliferation, whereas the biological activity of IGFBP-3 was complex. It had an IGF-independent antiproliferative effect and also inhibited IGF-dependent chondrocyte proliferation under coincubation conditions, whereas under preincubation conditions IGFBP-3 enhanced IGF-I-responsiveness. Studies on the mechanism by which IGFBP-3 potentiated IGF activity demonstrated that under preincubation conditions IGFBP-3 is capable to associate with the cell membrane and to facilitate IGF-I cell surface binding. CONCLUSIONS: Intact IGFBP-1, IGFBP-2 and IGFBP-6 act exclusively as growth inhibitors on IGF-dependent proliferation of growth plate chondrocytes. IGFBP-3, however, can either inhibit IGF-independent and IGF-dependent cell proliferation, or enhance IGF responsiveness of chondrocytes dependent on the temporal relationship to the IGF exposure. PMID- 12371960 TI - Early detection of cysteine rich protein 61 (CYR61, CCN1) in urine following renal ischemic reperfusion injury. AB - BACKGROUND: Acute renal failure (ARF) has a high morbidity and mortality. Many therapies have worked in animals but were unsuccessful in clinical trials. The inability to diagnose ARF early may have impaired the success of these trials. METHOD: We screened a subtraction library to search for potential disease markers that would be induced rapidly after renal injury. Mice and rats were subjected to 30 to 40 minutes of bilateral ischemia. RESULTS: mRNA for Cyr61, a secreted growth factor-inducible immediate early gene, was markedly up-regulated at two hours in the kidney but not other organs following renal ischemia. In situ hybridization studies suggested Cyr61 was synthesized in the proximal straight tubule. Cyr61 protein was analyzed by capture with heparin beads followed by Western blotting. Induction of Cyr61 protein could be detected in the kidney within one hour, peaked at four to eight hours, and remained elevated for at least 24 hours following ischemia. Cyr61 protein was detected in urine at three to six hours and peaked at six to nine hours after renal injury. Cyr61 was not detected after volume depletion, which is often difficult to differentiate from ARF. CONCLUSIONS: The secreted, cysteine-rich, heparin binding protein Cyr61 is rapidly induced in proximal straight tubules following renal ischemia, and excreted in the urine where it might serve as an early biomarker of renal injury. PMID- 12371961 TI - Simvastatin increases fibrinolytic activity in human peritoneal mesothelial cells independent of cholesterol lowering. AB - BACKGROUND: The continuous physical and chemical irritation of the peritoneum in peritoneal dialysis patients can result in a nonbacterial serositis with increased fibrin deposition, thus promoting peritoneal fibrosis and adhesion development. By expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), human peritoneal mesothelial cells (HMC) play an important role in regulating peritoneal fibrinolysis. METHODS: Cultured HMC were used to examine the effect of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin, on the expression of t-PA and PAI-1. Antigen concentrations in the cell supernatants were measured by ELISA and Northern blot analysis was conducted for mRNA expression. RESULTS: Simvastatin time- and concentration-dependently increased t-PA and decreased PAI-1 synthesis, reaching maximal effects after 48 hours, when simvastatin (1 micromol/L) increased t-PA levels 5.1 +/- 0.1-fold and suppressed PAI-1 levels 2.6 +/- 0.2-fold. This was accompanied by a twofold increase in mesothelial cell-associated t-PA activity. Qualitatively similar results were obtained in cultured human endothelial cells, but the effects were less pronounced and required higher simvastatin concentrations. Northern blot analysis revealed that the action of simvastatin on t-PA and PAI-1 expression in HMC can be explained by parallel changes in t-PA and PAI-1 mRNA. The effects of simvastatin were prevented in the presence of mevalonate and geranylgeraniol, suggesting that the effect of simvastatin on t-PA and PAI-1 synthesis is mediated through geranylgeranyl-modified intermediates. Experiments using specific inhibitors of geranylgeranylated Rho GTPases excluded a role of members of this family of small GTP-binding proteins in simvastatin action in HMC. The effects of simvastatin on t-PA and PAI-1 expression as well as on cell shape were completely mimicked by cytochalasin D, a disrupter of cellular actin filaments, but not by colchicine, a disrupter of microtubules. CONCLUSIONS: In conclusion, the cholesterol-lowering drug simvastatin is an effective stimulator of local peritoneal fibrinolytic activity, as it increases t-PA and decreases PAI-1 production in mesothelial cells by a mechanism involving geranylgeranyl-modified intermediates and actin skeleton perturbation. These results provide a new rationale to prevent peritoneal fibrin deposition and adhesion development in peritoneal dialysis patients. PMID- 12371962 TI - HSP-25 and HSP-90 stabilize Na,K-ATPase in cytoskeletal fractions of ischemic rat renal cortex. AB - BACKGROUND: We recently designed an in vitro system based on differential Triton extractability of Na,K-ATPase from the cytoskeletal protein fraction isolated from rat renal cortex after renal ischemia. In the present study, we hypothesized that heat shock protein (HSP)-70, HSP-25 and HSP-90 work synergistically to stabilize the cytoskeletal anchorage of Na,K-ATPase. METHODS: Cellular proteins were fractionated by differential centrifugation into cytoskeletal pellets (I PEL) obtained early (exhibiting abnormally high Triton extractability of Na,K ATPase) and non-cytoskeletal supernatants (R-SUP) obtained late (exhibiting high abundance of HSP) after renal ischemia. For assessment of the role of HSP-70, HSP 25 and HSP-90 upon in vitro re-compartmentalization, I-PEL was either incubated in R-SUP with/without HSP antibodies, or in buffer with/without HSPs at different titers and combinations. Effects were evaluated by changes of Triton extractability of Na,K-ATPase after co-incubation. RESULTS: R-SUP was shown to contain significant amounts of HSP-70, HSP-25 and HSP-90. Incubation of I-PEL in R-SUP reduced Triton extractability of Na,K-ATPase. Addition of antibodies against each HSP significantly abolished these effects of R-SUP. Incubation of I PEL with purified HSP-70, HSP-25 or HSP-90 each partly reproduced the effects of R-SUP, whereas the combination of all three HSP demonstrated a strong and more than additive effect on the cytoskeletal stabilization of Na,K-ATPase. CONCLUSIONS: The molecular mechanisms responsible for postischemic re compartmentalization of Na,K-ATPase in rat renal cortex likely involves interactions between HSP-70, HSP-25 and HSP-90, stress proteins known to be induced in the ischemic kidney. PMID- 12371963 TI - Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage. AB - BACKGROUND: Evidence indicates that urinary protein is associated with tubulointerstitial damage and thus it is an aggravating factor for chronic renal disease. As free fatty acids (FFAs) are bound to serum albumin, we hypothesized that FFAs were overloaded to the proximal tubule in massive proteinuria and thus caused tubulointerstitial damage. To test this hypothesis, massive proteinuria was provoked in mice and the renal damage examined. METHODS: Mice were intraperitoneally injected with bovine serum albumin (BSA) replete with FFAs (r BSA group, N = 10), FFA-depleted BSA (d-BSA group, N = 10), or saline (saline group, N = 9) for 14 days. RESULTS: The kidneys of the r-BSA group showed severe tubulointerstitial damage and those of the d-BSA group showed mild tubulointerstitial damage. Urinary excretion of both total protein and mouse albumin were significantly higher in the r-BSA group than in the d-BSA group. To examine the proximal tubular uptake of albumin, the BSA content in the cultured mouse proximal tubules was measured by ELISA after 90 minutes of incubation with each BSA. In terms of the BSA content in the proximal tubules, there was no significant difference between the r-BSA and the d-BSA groups. These results indicate that r-BSA and d-BSA were similarly reabsorbed into the proximal tubule and that r-BSA causes severe tubulointerstitial damage. CONCLUSIONS: It is the FFAs bound to albumin, rather than albumin itself, which cause severe tubulointerstitial damage by being reabsorbed into the proximal tubule. To our knowledge, this is the first in vivo observation in which FFAs have caused severe tubulointerstitial injury. PMID- 12371964 TI - Expression profile of leukocyte genes activated by anti-neutrophil cytoplasmic autoantibodies (ANCA). AB - BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) induce neutrophil activation in vitro with release of injurious products that can mediate necrotizing vasculitis in vivo. The importance of ANCA IgG F(ab')2-antigen binding versus Fcgamma receptor engagement in this process is controversial. We propose that ANCA-antigen binding affects cell signaling pathways that can result in changes of gene expression. METHODS: Microarray GeneChip analysis and real time, quantitative PCR (TaqMan(R)) was used to probe for transcripts in leukocytes from patients (in vivo gene expression study) and in leukocytes treated with ANCA IgG or ANCA-F(ab')2 (in vitro gene expression study). RESULTS: Microarray gene chip analysis showed that ANCA IgG and ANCA-F(ab')2 stimulate transcription of a distinct subset of genes, some unique to whole IgG, some unique to F(ab')2 fragments, and some common to both. DIF-2, COX-2, and IL-8 were identified as genes responsive to ANCA signaling and were selected for in depth evaluation. In vitro DIF-2 and IL-8 were increased by both ANCA IgG and F(ab')2, but COX-2 only by MPO-ANCA F(ab')2. In vivo DIF-2 levels were increased in leukocytes of ANCA patients, which correlated strongly with disease activity and ANCA titer. DIF-2 was not increased in patients in remission or in disease control patients (systemic lupus erythematosus and IgA nephropathy). COX-2 gene expression was significantly increased in patients with active disease, while IL 8 was increased in remission. CONCLUSIONS: The data indicate that leukocyte genes are activated in vitro by both ANCA Fc and ANCA F(ab')2 pathways and that in vitro activation mimics changes in circulating leukocytes of patients with ANCA disease. Increased levels of DIF-2 in patient leukocytes strongly correlate with severity of disease in kidney tissue. The observations indicate a previously unrecognized role for DIF-2 in ANCA-mediated inflammation, which raises the possibility that DIF-2 has an important role in other types of inflammation. PMID- 12371965 TI - Compensatory renal hypertrophy is mediated by a cell cycle-dependent mechanism. AB - BACKGROUND: Two mechanisms exist for inducing renal proximal tubule hypertrophy. One is characterized by regulation of the G1 cell cycle kinase (cell cycle dependent mechanism), while the other mechanism involves an imbalance between rates of protein synthesis and degradation, and occurs independently of cell cycle kinase regulation (cell cycle-independent mechanism). The present studies examined whether the compensatory proximal tubule growth following uninephrectomy is mediated by the cell cycle-dependent or -independent mechanism. METHODS: Studies were done in both rats and C57Bl6 mice on tissue harvested from sham operated or uninephrectomized animals. The magnitude of BrdU incorporation was used as the hyperplasia marker, while the proximal tubule protein: DNA ratio was used as the hypertrophy marker. Cdk4/cyclin D and cdk2/cyclin E kinase activities were assayed on renal cortex (rat studies) or isolated proximal tubules (mouse studies) using an in vitro kinase assay. RESULTS: In both rats and mice, compensatory proximal tubule growth was hypertrophic, not hyperplastic, evidenced by an increase in the protein:DNA ratio without a change in BrdU incorporation. In mice, cdk4/cyclin D kinase activity progressively increased between days 4 and 7, while cdk2/cyclin E kinase activity was decreased at both 4 and 7 days. In rats the development of hypertrophy was associated with an increase in cdk4/cyclin D kinase at days 4, 7, and 10, and an increase in cdk2/cyclin E kinase activity at days 2, 4, and 7. Roscovitine, a cdk2/cyclin E kinase inhibitor, inhibited cdk2/cyclin E kinase activity in both sham and nephrectomized rats; however, it did not prevent the development of proximal tubule hypertrophy. CONCLUSIONS: Uninephrectomy-induced compensatory proximal tubule growth is a hypertrophic form of growth that is mediated by a cell cycle dependent mechanism. PMID- 12371966 TI - P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangial proliferative glomerulonephritis. AB - BACKGROUND: Although extracellular nucleotides have been shown to confer mitogenic effects in cultured rat mesangial cells through activation of purinergic P2 receptors (P2Y receptors), thus far the in vivo relevance of these findings is unclear. Virtually all cells and in particular the dense granules of platelets contain high levels of nucleotides that are released upon cell injury or platelet aggregation. In experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), mesangiolysis and glomerular platelet aggregation are followed by a pronounced mesangial cell (MC) proliferative response leading to glomerular hypercellularity. Therefore, we examined the role of extracellular nucleotides and their corresponding receptors in nucleotide-stimulated cultured mesangial cells and in inflammatory glomerular disease using the P2 receptor antagonist PPADS. METHODS: The effects of PPADS on nucleotide- or fetal calf serum (FCS)-stimulated proliferation of cultured MC were measured by cell counting and [3H]thymidine incorporation assay. After induction of the anti-Thy1 model, rats received injections of the P2-receptor antagonist PPADS at different doses (15, 30, 60 mg/kg BW). Proliferating mesangial and non-mesangial cells, mesangial cell activation, matrix accumulation, influx of inflammatory cells, mesangiolysis, microaneurysm formation, and renal functional parameters were assessed during anti-Thy1 disease. P2Y-mRNA and protein expression was assessed using RT-PCR and real time PCR, Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: In cultured mesangial cells, PPADS inhibited nucleotide, but not FCS stimulated proliferation in a dose-dependent manner. In the anti-Thy1 model, PPADS specifically and dose-dependently reduced early (day 3), but not late (day 8), glomerular mesangial cell proliferation as well as phenotypic activation of the mesangium and slightly matrix expansion. While no consistent effect was obtained in regard to the degree of mesangiolysis, influx of inflammatory cells, proteinuria or blood pressure, PPADS treatment increased serum creatinine and urea in anti-Thy1 rats. P2Y receptor expression (P2Y2 and P2Y6) was detected in cultured MC and isolated glomeruli, and demonstrated a transient marked increase during anti-Thy1 disease. CONCLUSION: These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury. PMID- 12371967 TI - Expression profiling confirms the role of endocytic receptor megalin in renal vitamin D3 metabolism. AB - BACKGROUND: The endocytic receptor megalin constitutes the major pathway for clearance of low-molecular weight plasma proteins from the glomerular filtrate into the renal proximal tubules. Furthermore, the receptor has been implicated in a number of other functions in the kidney including uptake and activation of 25 (OH) vitamin D3, calcium and sodium reabsorption as well as signal transduction. METHODS: We used genome-wide expression profiling by microarray technology to detect changes in the gene expression pattern in megalin knockout mouse kidneys and to uncover some of the renal pathways affected by megalin deficiency. RESULTS: Alterations were identified in several (patho)physiologic processes in megalin-deficient kidneys including the renal vitamin D metabolism, transforming growth factor (TGF)-beta1 signal transduction, lipid transport and heavy metal detoxification. Most importantly, changes were detected in the mRNA levels of 25 (OH) vitamin D-24-hydroxylase and 25-(OH) vitamin D-1alpha-hydroxylase as well as strong up-regulation of TGF-beta1 target genes. Both findings indicate plasma vitamin D deficiency and lack of vitamin D signaling in renal tissues. CONCLUSIONS: Expression profiling confirms a crucial role for megalin in renal vitamin D metabolism. PMID- 12371968 TI - Involvement of reactive oxygen species on gentamicin-induced mesangial cell activation. AB - BACKGROUND: Reactive oxygen species (ROS) have been shown to be involved in the reduction of glomerular filtration rate observed after gentamicin (Genta) treatment in vivo, a phenomenon directly related with mesangial cell (MC) contraction. Our previous study reported that Genta induces concentration dependent MC contraction and proliferation in vitro. METHODS: To study the possible mediation of ROS in the effect of Genta, ROS production was measured in primary cultures of rat MC stimulated with Genta (10-5 mol/L). In addition, the MC response to Genta in the presence of the ROS scavengers superoxide dismutase (SOD) and catalase (CAT) was studied. MC activation and O2- production were studied in the presence of an inhibitor of the NADP(H) oxidase, diphenylene iodinium (DPI), and in the presence of L-NAME, an inhibitor of nitric oxide synthases (NOS). Finally, the effects of Genta on SOD activity and mRNA expression were examined. RESULTS: Genta (10-5 mol/L) induced an increase in O2- production and SOD activity that was neither accompanied by an elevation in cytosolic Cu/Zn-SOD mRNA expression nor by H2O2 accumulation. Genta induced MC contraction and proliferation that were inhibited by SOD plus CAT. Both the extracellular and intracellular ROS donor systems, xantine+xantine oxidase (X+XO) and dimethoxinaphtoquinone (DMNQ), respectively, also stimulated MC contraction and proliferation. Genta-induced MC activation and O2- production were inhibited by DPI. Genta-induced O2- production was inhibited by L-NAME. Furthermore, Genta did not induce detectable changes in membrane fluidity and lipid peroxidation. CONCLUSIONS: These results strongly suggest that an oxidative-mediated pathway exists in Genta-induced MC activation. A portion of the production of O2- may be due to NADP(H) oxidase and NOS activation. The amount of ROS produced, rather than having a toxic effect, might play a role as a mediator of Genta-induced MC activation PMID- 12371969 TI - Atrial natriuretic peptide impairs the stimulatory effect of angiotensin II on H+ ATPase. AB - BACKGROUND: Angiotensin II (Ang II) action on H+-ATPase is not clearly defined, and may vary with renal tubule segment and hormonal doses being studied. Since an increase of cytosolic calcium ([Ca2+]i) can stimulate acid vesicle movement and exocytotic insertion of proton pumps, and it has been shown that Ang II increases [Ca2+]i while atrial natriuretic peptide (ANP) reduces it, there may be some interaction between Ang II and ANP in the regulation of intracellular pH (pHi) mediated by H+-ATPase. METHODS: The effects of Ang II and/or ANP on the regulation of pHi via H+-ATPase and of [Ca2+]i was investigated in Madin-Darby canine kidney cells (MDCK) by the fluorescent probes BCECF-AM and Fluo-4/AM, respectively. The pHi recovery rate was examined following the intracellular acidification after an NH4Cl pulse, in presence of zero Na+ plus Schering 28080, which is a specific inhibitor of H+/K+-ATPase. RESULTS: Ang II (10-12, 10-9 or 10 7 mol/L) increased the rate of pHi recovery and [Ca2+]i in a dose-dependent manner. ANP (10-6 mol/L) or dimethyl-BAPTA/AM (5 x 10-5 mol/L, an intracellular calcium chelator) did not affect the pHi recovery but decreased [Ca2+]i and blocked the stimulatory effect of Ang II on the pHi recovery. CONCLUSIONS: The results suggest that the increase of [Ca2+]i regulates the dose-dependent stimulatory effect of Ang II on H+-ATPase. ANP or dimethyl-BAPTA/AM, by impairing the path causing the increase in [Ca2+]i, blocks this stimulatory effect of Ang II. PMID- 12371970 TI - L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats. AB - BACKGROUND: Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). METHODS: Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. RESULTS: Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. CONCLUSIONS: Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF. PMID- 12371971 TI - Cisplatin-induced inhibition of receptor-mediated endocytosis of protein in the kidney. AB - BACKGROUND: Administration of cisplatin, cis-diamminedichloroplatinum (II) (CDDP), causes a severe impairment of renal function, including increases in urinary excretion of proteins. We recently found that CDDP inhibits vacuolar H+ ATPase, which plays an important role in receptor-mediated endocytosis in the renal proximal tubules. Therefore, CDDP-induced proteinuria may be due to an inhibition of the receptor-mediated endocytosis in the renal proximal tubules following a decrease in vacuolar H+-ATPase activity by the drug. METHODS: Effects of CDDP on receptor-mediated endocytosis of albumin in opossum kidney (OK) epithelial cells, and on urinary excretion of albumin and vitamin D binding protein, which are reabsorbed in the renal proximal tubules by endocytosis, in rats were examined. RESULTS: CDDP inhibited uptake of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, by OK cells in a time- and concentration-dependent fashion. In contrast, CDDP treatment did not affect the uptake of FITC-inulin, a fluid-phase endocytosis marker. CDDP caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. The adenosine 5'-triphosphate (ATP) content in OK cells was not changed by CDDP at concentrations that inhibited FITC-albumin uptake. The endosomal pH in OK cells was increased by CDDP treatment. Administration of CDDP to rats increased the urinary excretion of albumin and vitamin D binding protein. CONCLUSIONS: These results suggest that CDDP decreases the receptor-mediated endocytosis of protein following the inhibition of vacuolar H+-ATPase in the renal proximal tubules, and the inhibition of receptor-mediated endocytosis would be the mechanisms underlying the proteinuria induced by CDDP. PMID- 12371972 TI - Interacting effects of gender and genotype on blood pressure response to hydrochlorothiazide. AB - BACKGROUND: Genetic factors may influence blood pressure (BP) response to diuretic therapy through their effects on activity of the renin-angiotensin aldosterone system (RAAS). The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with variation in serum ACE activity and may influence BP in a gender-specific manner. METHODS: We measured the I/D polymorphism in 206 non-Hispanic women (130 blacks, 76 whites) and 170 non-Hispanic men (62 blacks, 108 whites) with essential hypertension (age 48 +/- 7 years, mean +/- SD) who underwent monotherapy with hydrochlorothiazide (HCTZ) 25 mg daily for four weeks. RESULTS: In both genders, serum ACE activity increased in a co-dominant fashion in association with the D-allele (P < 0.001 for both genders). In regression models that considered the effects of baseline BP, race, gender, age, waist-to-hip ratio, and measures of the RAAS, there was significant interaction between the effects of the ACE genotype and gender on the responses of both systolic and diastolic BP to HCTZ (for systolic BP response, P = 0.03; for diastolic BP response, P = 0.001). Among women, mean declines in systolic and diastolic BP were greater in II than in DD homozygotes; whereas among men, mean declines in systolic and diastolic BP were greater in DD than in II homozygotes. In models that included the effects of race, gender, age, and waist-to-hip ratio, there was also significant interaction between the effects of the ACE genotype and gender on pre-treatment urinary aldosterone excretion (P = 0.01) and change in urinary aldosterone excretion in response to HCTZ (P = 0.007). The genotypes that were associated with the greatest BP responses to HCTZ (II homozygotes in women and DD homozygotes in men) had the lowest pre-treatment urinary aldosterone excretion and the greatest increase in urinary aldosterone excretion in response to HCTZ. CONCLUSION: The relationship between the ACE I/D polymorphism and antihypertensive response to a standard dose of HCTZ differs significantly between women and men. Because the D-allele was associated with significant, co-dominant increases in serum ACE activity in both genders, the gender-specific effects of the I/D polymorphism on BP response to HCTZ may be mediated subsequent to the enzymatic generation of angiotensin II. PMID- 12371973 TI - Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. AB - BACKGROUND: Dialysis patients have accelerated atherosclerosis, with extensive calcification of both the intima and media. Cross-sectional studies have implicated hyperphosphatemia in this process, but the mechanism is unclear. METHODS: To test the hypothesis that hyperphosphatemia and/or uremia induces vascular calcification, bovine vascular smooth muscle cells (BVSMC) were treated with increasing concentrations of beta-glycerophosphate, a phosphate donor, in the presence or absence of inhibitors for sodium/phosphate (Na/Pi) co-transport (foscarnet) or alkaline phosphatase (levamisole) for 48 hours. BVSMC also were incubated for various times with DMEM plus 15% pooled uremic sera from patients with low (LP) or high serum phosphorus (HP), or from pooled healthy control serum. Calcification in BVSMC was examined by quantitation of calcium deposition. Osteopontin expression and alkaline phosphatase activity were assessed by Western blotting and a colorimetric assay. RESULTS: beta-glycerophosphate increased osteopontin expression and alkaline phosphatase activity in BVSMC. Inhibition of either alkaline phosphatase activity or Na/Pi co-transport abolished this effect. Compared to incubation with control human serum, BVSMC cultured with uremic sera had increased mineral deposition. Uremic sera also increased alkaline phosphatase activity and osteopontin expression in BVSMC. The addition of beta glycerophosphate to uremic HP or LP sera did not further augment osteopontin expression. Blocking Na/Pi co-transport or alkaline phosphatase activity only partially inhibited uremic sera-induced osteopontin expression, indicating that other non-Na/Pi co-transport dependent mechanisms also are involved. CONCLUSION: beta-glycerophosphate and uremic sera induce calcification and osteopontin expression in BVSMC. The uremic sera-induced osteopontin expression in BVSMC is partially mediated through alkaline phosphatase activity and a Na/Pi co transporter dependent mechanism. However, other non-Na/Pi dependent mechanisms also contribute to accelerated vascular calcification in patients with ESRD. PMID- 12371974 TI - Determinants of outcome in ANCA-associated glomerulonephritis: a prospective clinico-histopathological analysis of 96 patients. AB - BACKGROUND: The predictive value of clinical and renal histological features for renal outcome in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) associated glomerulonephritis was investigated in a prospective analysis of 96 patients with ANCA-associated vasculitis, and moderate renal involvement (creatinine <500 micromol/L). METHODS: The extent of 39 histological features in 96 biopsies (performed at entry in a clinical trial) was scored by two independent observers, according to a standardized protocol. Age, gender, diagnosis, glomerular filtration rate at entry (GFR0), ANCA-specificity, proteinuria, and treatment of these 96 patients were also taken into account. Treatment was standardized and started after the biopsy was performed. End-points included renal function at 18 months (GFR18), GFR18 corrected for GFR0 (CORGFR18), and the occurrence of relapse or death. RESULTS: Parameters that most strongly correlated with GFR18 were GFR0 (r = 0.67), interstitial fibrosis (r = 0.45), glomerulosclerosis (r = -0.37), and tubular atrophy (r = -0.36). Parameters that most strongly correlated with CORGFR18 were segmental (r = 0.45) and cellular (r = 0.30) crescents, and fibrinoid necrosis (r = 0.46). None of the clinical and histological features predicted the occurrence of relapse or death. By applying a stepwise linear multiple regression analysis, we designed a formula for the estimation of renal function at 18 months: GFR18 (mL/min) = 17 + 0.71 x GFR0 (mL/min) + 0.34 x fibrinoid necrosis (%) + 0.33 x segmental crescents (%), (r2 = 0.60; standard deviation = 19 mL/min). Our results were independent of diagnosis, ANCA-specificity, and treatment limb. CONCLUSIONS: These data suggest that in ANCA-associated glomerulonephritis, GFR0 and predominantly chronic renal lesions are potent predictors of GFR18. Active lesions are associated with renal function recovery and may be reversible. The formula for the estimation of GFR18 shows that a combination of GFR0 and renal histology is a better predictor for GFR18 than GFR0 only. PMID- 12371975 TI - Triglyceride, but not total cholesterol or low-density lipoprotein cholesterol levels, predict development of proteinuria. AB - BACKGROUND: Epidemiological data about the relationship between dyslipidemia and proteinuria are sparse. We conducted a retrospective and longitudinal study in a large screened cohort to evaluate whether triglyceride, high-density lipoprotein (HDL) cholesterol, total cholesterol, and low-density lipoprotein (LDL) cholesterol levels increase the risk of development of proteinuria and loss of renal function. METHODS: Post hoc analysis was performed for 4326 subjects who were free from proteinuria (dipstick 1+ or higher) at baseline (1997) with a follow-up period through 2000. Outcome measures were the development of proteinuria (1+ or higher) and change in glomerular filtration rate (GFR). Multiple logistic analysis and multiple regression analysis were used to analyze baseline characteristics related to the outcome measures. RESULTS: During the observational period, 505 (11.7%) of subjects had one or more episodes of proteinuria (>/=1+). Adjusted relative risk of triglycerides for one or more incidences of proteinuria was 1.007 (95% CI 1.000 to 1.105, P = 0.04) in men and 1.032 (95% CI 1.004 to 1.061, P = 0.02) in women. Total cholesterol, HDL cholesterol, and LDL cholesterol were not significant predictors of proteinuria. The mean change in GFR between 1997 and 2000 was -6.3 (SD = 9.0) mL/min/1.73 m2 in men, and -7.8 (SD = 10.7) mL/min/1.73 m2 in women. HDL cholesterol (beta = 0.04, t = 3.7, P = 0.0002) in men and triglycerides (per 10 mg/dL, beta = -0.09, t = -2.2, P = 0.02) in women were correlated with the change in GFR. CONCLUSIONS: High triglyceride levels predicted a risk of developing proteinuria in both men and women, but not total cholesterol nor LDL cholesterol. High triglyceride in women and low HDL cholesterol in men predicted the decline of renal function. It remains to be determined whether prospective treatment of dyslipidemia will protect against renal injury. PMID- 12371976 TI - The precision of estimating protein intake of patients with chronic renal failure. AB - BACKGROUND: Biochemical methods for estimating protein intake are based on the concept that nitrogen-containing products of protein in diet plus the products arising from endogenous protein are excreted as either urea or non-urea nitrogen (NUN). This formulation is based on the fact that the urea is the principal end product of amino acid degradation and, hence, the urea appearance rate (or net urea production) is parallel to protein intake. The urea nitrogen appearance (UNA) rate is measured as the amount of urea excreted in urine plus the net amount accumulated in body water. A more difficult problem is how to estimate NUN, the sum of fecal nitrogen, and all forms of non-urea urinary nitrogen. Maroni, Steinman, and Mitch (Kidney Int 27:58-65, 1985) proposed estimating nitrogen intake (IN MARONI) from UNA plus NUN excretion rate of 0.031 g nitrogen/kg body weight/day, as they found NUN correlated with body weight but not with dietary nitrogen. Kopple, Gao, and Qing (Kidney Int 27:486-494, 1997) proposed a different equation for estimating nitrogen intake (IN KOPPLE) = 1.20 UNA + 1.74, concluding that dietary nitrogen directly correlates with fecal nitrogen and that NUN is constant for all patients. Their report prompted us to review all nitrogen balance measurements we had conducted in order to address the following questions. Does dietary protein increase fecal nitrogen excretion? Does NUN vary with weight or is it constant? How do the two methods (IN MARONI and IN KOPPLE) compare in estimating dietary protein from UNA? METHODS: We examined nitrogen balance and its components measured in 33 patients with chronic renal failure (CRF) who were eating diets varying from 4.1 to 10.1 g nitrogen/day. We evaluated relationships between dietary nitrogen [intake nitrogen (IN)], NUN, fecal nitrogen, body weight, and the predictability of the two methods. RESULTS: Neither fecal nitrogen nor NUN were significantly correlated with IN (r = 0.04 and r = -0.07, respectively). NUN significantly correlated with body weight (P = 0.008). Measured IN averaged 5.75 +/- 0.41 g nitrogen/day; the estimated IN MARONI value was 5.61 +/- 0.27 g nitrogen/day; the estimated IN KOPPLE was 6.04 +/- 0.44 g nitrogen/day. The prediction errors associated with the IN KOPPLE equation were slightly but not statistically higher than that associated with IN MARONI. CONCLUSION: Fecal nitrogen is not correlated with IN. NUN is not constant but varies with weight, and the traditional method of estimating IN in stable chronic renal insufficiency (CRI) patients from UNA and weight as proposed by Maroni, Steinman, and Mitch is valid. PMID- 12371977 TI - Circulating platelet-derived microparticles with procoagulant activity may be a potential cause of thrombosis in uremic patients. AB - BACKGROUND: Clinical experience indicates that bleeding and thrombotic tendencies co-exist in uremic patients. Numerous studies have shown that platelet functional defects contribute to the bleeding tendency in uremic patients. In contrast, there are no solid studies clarifying the pathogenesis of the prothrombotic state in uremic patients. Platelet-derived microparticles (PMPs), which are small vesicles with procoagulant activity released from activated platelets, are thought to be involved in clinical thrombogenesis. This study addressed the question of why uremic patients are thrombophilic even though they have a bleeding tendency, focusing on the clinical significance of PMPs. METHODS: The subjects were pre-dialyzed patients, patients under hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) therapy, and age-matched healthy controls. Analyses of PMPs were performed using a flow cytometer. Annexin V was used to probe procoagulant activity of PMPs. The impacts of the HD procedure, arteriovenous (AV) fistula, and recombinant human erythropoietin (rHuEPO) treatment on the release of PMPs were additionally assessed. RESULTS: Major results are: (1) PMP counts were significantly greater in each uremic group than in controls. The PMP counts were not different among three types of uremic groups; (2) PMP counts were significantly higher in uremic patients with thrombotic events than in those without thrombotic events; and (3) the HD procedure and existence of AV fistula did not affect PMP counts, but rHuEPO treatment possibly enhanced the PMP release in these patients. CONCLUSIONS: Elevated PMP counts may trigger thrombosis in uremic patients. The primary cause of PMP elevation in uremia was not clarified in this study. PMID- 12371978 TI - Fibrillary glomerulonephritis and immunotactoid (microtubular) glomerulopathy are associated with distinct immunologic features. AB - BACKGROUND: The clinical relevance of distinguishing two types of glomerulonephritis (GN) with non-amyloid organized immunoglobulin (Ig) deposits fibrillary GN (FGN) and immunotactoid (microtubular) GN (IT/MTGN)-on the basis of ultrastructural organization, is debated. METHODS: Twenty-three patients with organized glomerular Ig deposits were classified into two groups based on the fibrillar or microtubular ultrastructural appearance of the deposits. Kidney biopsy samples were studied by immunofluorescence microscopy, using anti-light chain conjugates (all cases) and anti-IgG subclass conjugates (13 patients). In each group, we studied clinicopathological features, associated monoclonal gammapathy (detected by immunoelectrophoresis and/or immunoblot) or B-cell lymphoproliferative disease, effects of chemotherapy and long-term renal outcome. RESULTS: In 14 IT/MTGN and 9 FGN patients, clinical symptoms [hypertension, nephrotic syndrome (NS) and hematuria] and the mean diameters of the substructures were similar. In 13 IT/MTGN patients, glomerular (IgG1, 2 or 3) deposits were monotypic (kappa, 7 cases; lambda, 6 cases). Glomerular deposits were associated with a monoclonal Ig of the same isotype in eight patients, detected in the serum (5 cases), and/or in the cytoplasm of lymphocytes (4 cases), and with lymphoproliferative disease in seven patients. The ultrastructural features of monoclonal Ig inclusions in lymphocytes were similar to those of glomerular microtubular deposits. In contrast, none of the FGN patients presented lymphoplasmocytic proliferation or paraproteinemia. Glomerular Ig deposits were polyclonal in eight cases and contained IgG4 in all three cases studied. Although patient and renal survival did not differ significantly between the two groups, chemotherapy led to remission of NS in ten IT/MTGN patients, with parallel improvement in hematological parameters. CONCLUSIONS: The identification of ultrastructural patterns in these nephropathies is important. GN with organized microtubular monoclonal deposits (GOMMID) probably accounts for a large proportion of immunotactoid (microtubular) GN cases. PMID- 12371979 TI - Impact of renal failure on the risk of myocardial infarction and death. AB - BACKGROUND: It is unclear whether pre-existing cardiovascular disease or predisposition of the uremic state leads to the high cardiovascular morbidity and mortality associated with renal failure. We examined whether renal failure independently increases the risk of myocardial infarction and death. METHODS: A total of 8600 patients with variable glomerular filtration rate (GFR) at the time of coronary angiography participated in the Intermountain Heart Study. Coronary disease was defined as >or=70% stenosis. Modification of Diet in Renal Disease formula was used to calculate glomerular filtration rate (GFR). Cox regression models were used to compare outcomes. RESULTS: The mean GFR was 71 +/- 24 mL/min. There were 1320 (15%) deaths, 657 (9%) myocardial infarctions and 1776 (21%) death or myocardial infarctions over 3.2 +/- 1.9 years. Compared to the highest GFR quartile, the lowest GFR quartile (mean GFR 41 +/- 14 mL/min) was associated with higher risk for myocardial infarction (RR 1.43, 95% CI 1.15 to 1.78), death (RR 2.77, 95% CI 2.32 to 3.30) and death/myocardial infarction (RR 2.13, 95% CI 1.85 to 2.45) in multivariable models adjusted for age, sex, hypertension, hyperlipidemia, smoking, family history of coronary disease and diabetes. Even after further adjustment for coronary angiographic data and the choice of initial therapy, lowest GFR quartile was associated with increased risk of myocardial infarction (RR 1.51, 95% CI 1.21 to 1.88), death (RR 2.60, 95% CI 2.18 to 3.10) and death/myocardial infarction (RR 2.08, 95% CI 1.80 to 2.39). CONCLUSIONS: Even moderate renal failure increases the risk of myocardial infarction and death independent of clinical variables, baseline angiographic evidence of coronary disease and therapy. PMID- 12371980 TI - Blood pressure and long-term mortality in United States hemodialysis patients: USRDS Waves 3 and 4 Study. AB - BACKGROUND: The long-term prognostic associations of pre- and post-dialysis blood pressures, interdialytic weight gain, and antihypertensive use in hemodialysis patients are unclear. METHODS: The United States Renal Data System (USRDS) Dialysis Morbidity and Mortality Waves 3 and 4 Study, a randomly generated sample of 11,142 subjects receiving hemodialysis on December 31, 1993, was examined, with vital status followed until May 2000. RESULTS: Pre- and post-dialysis blood pressure values, interdialytic weight gain and number of antihypertensives averaged 151.8/79.7, 137.0/74, 3.6% and 0.76, respectively. Prognostic discrimination was maximized by considering pre- and post-systolic and diastolic blood pressure values simultaneously, in a pattern suggesting that wide pulse pressures were associated with mortality (P < 0.0001). Comorbidity adjustment markedly affected associations, with low pre-dialysis diastolic (P < 0.05), low post-dialysis dialysis diastolic pressure (P < 0.05), high post-dialysis dialysis systolic pressure (P < 0.05), and high interdialytic weight gains (P = 0.005) associated with mortality. Each class of antihypertensive drug, except angiotensin-converting enzyme (ACE)-inhibitors, was associated with lower mortality in unadjusted models, an effect most pronounced for beta-blockers (hazards ratio 0.72, 95% CI 0.66 to 0.79, P < 0.0001). Comorbidity adjustment eliminated survival associations for each antihypertensive class except beta blockers. CONCLUSIONS: Pre- and post-dialysis blood pressure values have independent associations with mortality, in a way that implicates wide pulse pressures. Much of the adverse prognosis of wide pulse pressures probably reflects older age and cardiovascular comorbidity. Large interdialytic weight gains are associated with shorter survival when comorbidity is taken into account. Beta-blocker use shows a robust association with survival, and may be protective. PMID- 12371981 TI - Inflammation and outcome in end-stage renal failure: does female gender constitute a survival advantage? AB - BACKGROUND: Elevated C-reactive protein (CRP) is a strong predictor of cardiovascular events and all-cause mortality in end-stage renal disease (ESRD) patients. However, although sex hormones may influence serum levels of inflammatory proteins, gender has not been taken into consideration in previous studies of inflammation and outcome in ESRD patients. METHODS: We included 663 (374 males) ESRD patients (59 +/- 1 year) from three European renal centers (Sweden, Germany and Italy) in which CRP levels and outcome data (follow-up 33 +/ 1 months) were available. The relation between outcome and serum levels of the soluble intercellular adhesion molecule (sICAM-1) was evaluated in 312 of the patients. RESULTS: The present study shows that elevated CRP is a strong predictor of outcome, but whereas no difference in all-cause mortality was observed between non-inflamed (CRP C promoter polymorphism of the IL-6 gene. RESULTS: Carriers of the -174C-allele (genotype GC/CC) had an inferior three-year graft survival (71/104 = 68.3%; P = 0.0059) with a 3.7-fold increased relative risk of graft loss compared to carriers of the -174GG-genotype (48/54 = 88.9%). The -174GC/CC genotype retained its negative impact on graft survival when other established prognostic factors and further cytokine polymorphisms (-308TNF-alpha, TGF-beta1 codon 10 & 25, -592/-819/-1082IL-10 and +874IFN-gamma) were considered simultaneously. CONCLUSIONS: Since the clinical impact on transplant outcome seems as important as matching for histocompatibility antigens, genotyping of the IL-6 -174polymorphism may offer a new method for identifying patients at increased risk of allograft loss. PMID- 12371986 TI - Left ventricular mass and hemodynamic overload in normotensive hemodialysis patients. AB - BACKGROUND: It remains uncertain whether the hemodynamic parameters are important determinants of left ventricular mass (LVM) in normotensive chronic hemodialysis (NTHD) patients, as has been found in their hypertensive counterparts. METHODS: Forty NTHD patients (mean age, 53.7 +/- 14.4 years; male/female, 18/22) without the requirement of antihypertensive drugs for at least six months were studied. Controls were 41 hypertensive hemodialysis patients (HTHD) and 46 normotensive subjects with normal renal function (NTNR). The influence of anthropometrics, cardiovascular structure and function, and volume status on LVM (by two dimensional echocardiography) was analyzed by steps of multiple linear regression. RESULTS: As compared with the NTNR and NTHD group, the HTHD group had obvious pressure and volume/flow overload, and greater LV wall thickness, chamber size and mass. In contrast, NTHD subjects had similar blood pressure, large artery function, LV chamber size and stroke volume as the NTNR subjects. However, the NTHD patients still had greater wall thickness and LVM, along with greater cardiac output, lower total peripheral resistance and lower end-systolic meridional stress to volume ratio (ESSV) than the NTNR group. LVM in the NTHD group was significantly positively related to averaged systolic blood pressure (SBPavg), body surface area, extracellular fluid (ECF), carotid intima-media thickness (IMT), aortic pulse wave velocity (PWV), and negatively related to ESSV and Kt/V. The independent significant noncardiac structural determinants of LVM in NTHD subjects were ESSV, SBPavg, PWV and SV (model r2 = 0.617, P < 0.001). CONCLUSIONS: The NTHD patients, without significant pressure and volume overload, still had increased LVM that was partially explained by the persistent flow overload and subclinical LV dysfunction. PMID- 12371987 TI - Effects of prednisone withdrawal on the new metabolic triad in cyclosporine treated kidney transplant patients. AB - BACKGROUND: Cardiovascular disease is a major cause of morbidity and mortality after renal transplantation. Prednisone (Pred) maintenance therapy is associated with risk factors for atherosclerosis. Therefore, we were interested in quantifying the effects of Pred withdrawal on body weight and waist circumference as well as on metabolic markers of coronary heart disease risk. METHODS: Twenty six cyclosporine-treated renal transplant patients (13 men and 13 women) were evaluated before and after at least 11 months (16 +/- 2.9 months) of Pred withdrawal. A complete fasting lipoprotein-lipid profile as well as anthropometric measurements were obtained from each patient. RESULTS: Pred withdrawal was associated with a 6.0% reduction of body weight (-4.34 +/- 5.40 kg; P < 0.05) and with a 7.7% decrease in waist girth (-7.13 +/- 5.75 cm; P < 0.005) in women, whereas no change in these variables were observed in men. In both genders, plasma low-density lipoprotein (LDL) cholesterol and triglyceride concentrations were unaffected by Pred withdrawal, whereas plasma high-density lipoprotein (HDL) cholesterol levels decreased by 14.0% in women (-0.22 +/- 0.22 mmol/L; P < 0.005) and 22.0% in men (-0.36 +/- 0.28 mmol/L; P < 0.005). Pred withdrawal was associated with a significant reduction in plasma apolipoprotein B concentrations in both women (-0.28 +/- 0.15 g/L; -24.6%; P < 0.0001) and men ( 0.22 +/- 0.19 g/L; -20.5%; P < 0.005). A significant reduction in fasting insulin was observed in both women (-27.8 +/- 27.9 pmol/L; -25.3%; P < 0.005) and men ( 25.0 +/- 32.8 pmol/L; -21.4%; P < 0.05), whereas the LDL peak particle size was unaffected by Pred withdrawal. CONCLUSIONS: Pred withdrawal modifies several anthropometric and metabolic cardiovascular risk factors in renal transplant patients. Furthermore, female patients may derive further benefits of Pred withdrawal resulting from the concomitant loss of body weight and abdominal fat. PMID- 12371988 TI - Risk factors for late kidney allograft failure. AB - BACKGROUND: While graft survival rates in the short term have improved dramatically, only a modest improvement has been shown in long-term graft survival rates. We evaluated the causes of late failure in renal allograft recipients treated with cyclosporine A (CsA). METHODS: A total of 864 adults with a functioning graft at one year were evaluated. The end points were dialysis or death with a functioning graft. RESULTS: The 13-year patient and graft survival probabilities were 0.82 and 0.64, respectively. The graft half-life was 20.1 years and the pure graft half-life was 31.1 years. At multivariate analysis, plasma creatinine at one year (P = 0.0006; RR 1.72), low-density lipoproteins (LDL) at one year (P = 0.0014; RR 1.65), older age (P = 0.0128; RR 1.50) and delayed graft function (P = 0.0350; RR 1.45) were associated with the end point. Chronic allograft nephropathy was the cause of failure in 97 patients, death in 70, recurrence of glomerulonephritis in 24, other events in 6. Cardiovascular complications were the most frequent cause of death. Post-transplant cardiovascular events were associated with: pre-transplant cardiovascular events (P = 0.0012; RR 2.65), older age (P = 0.0001; RR 2.46), pre-transplant arterial hypertension (P = 0.0249; RR 1.57), smoking (P = 0.0235; RR 1.29), duration of dialysis (P = 0.0229; RR 1.28). Mean serum cholesterol, LDL and triglycerides were each significantly associated post-transplant cardiovascular events. CONCLUSIONS: The graft half-life was 20 years. Chronic allograft nephropathy was the leading cause of late failure, followed by death. If the data were censored by death, the projected pure graft half-life would be 31.1 years. Pre-transplant selection and preparation of the candidate as well as appropriate life style are recommended to improve life expectancy and extend graft survival. PMID- 12371989 TI - The first international consensus conference on continuous renal replacement therapy. AB - BACKGROUND: Management of acute renal failure (ARF) in the critically ill is extremely variable and there are no published standards for the provision of renal replacement therapy in this population. We sought to review the available evidence, make evidence-based practice recommendations, and delineate key questions for future study. METHODS: We undertook an evidence-based review of the literature on continuous renal replacement therapy (CRRT) using MEDLINE searches. We determined a list of key questions and convened a 2-day consensus conference to develop summary statements via a series of alternating breakout and plenary sessions. In these sessions, we identified supporting evidence and generated practice guidelines and/or directions for future research. RESULTS: Of the 46 questions considered, we found consensus for 20. We found inadequate evidence for 21 questions and for the remaining five we found data but no consensus. Full versions of workgroup findings are available on the Internet at http://www.ADQI.net. CONCLUSIONS: Despite limited data, broad areas of consensus exist for use of CRRT and guideline development appears feasible. Equally broad areas of disagreement also exist and additional basic and applied research in acute renal failure is needed. PMID- 12371990 TI - Permanent hemodialysis vascular access survival in children and adolescents with end-stage renal disease. AB - BACKGROUND: Transplantation is the optimal therapy for pediatric end-stage renal disease (ESRD) patients, but in a subset of patients with peritoneal membrane failure, failed transplants or poor social situations, chronic hemodialysis (HD) remains the only option. Long-term survival of arteriovenous fistulas (AVFs) and arteriovenous grafts (AVGs) in pediatric patients has not been well described. METHODS: We studied the survival of permanent vascular access in 34 pediatric ESRD patients treated with chronic HD at our institution between 1/1/89 and 12/1/95 and followed to 12/31/2000. RESULTS: Twenty-four AVFs and 28 AVGs were created in 19 and 23 patients, respectively. Mean age and weight at insertion were 15.1 years (range 7.1 to 20.9) and 46 kg (18 to 81) for AVFs and 13.3 years (3.8 to 21.1) and 41.5 kg (10.5 to 145) for AVGs. Fifteen patients weighed <35 kg at the time of access creation (7 AVFs in 5 patients, 14 AVGs in 13 patients). Excluding primary failures, one-year, three-year and five-year patency rates for AVFs (74%, 59%, 59%) and AVGs (96%, 69%, 40%) were not significantly different. Patency did not correlate with patient weight or age at access creation. Primary access failure occurred more often (P < 0.01) in AVFs (8/24) compared to AVGs (1/28). Access thrombosis, stenosis and infection occurred more frequently in AVG (P = 0.02). CONCLUSIONS: Both AVF and AVG function well even in small pediatric patients and have survival rates equivalent to adult series and longer than cuffed venous catheters in pediatric patients. Both AVFs and AVGs are preferable for long-term HD access in pediatrics. PMID- 12371991 TI - Total peripheral vascular resistance in pediatric renal transplant patients. AB - BACKGROUND: Abnormal cardiovascular reactivity at rest and during physical exercise may be a risk factor for left ventricular hypertrophy (LVH) in pediatric renal transplanted (Tx) patients. Data on total peripheral vascular resistance (TPR) are not available. METHODS: Eleven renal Tx patients treated with cyclosporine (7 females and 4 males; mean age 14.6 +/- 3.3 years; mean time since transplantation 43 +/- 35 months) were evaluated for 24-hour blood pressure (BP), TPR and echocardiographic left ventricular mass (LVM). TPR values of patients were compared with data of a group of 11 healthy controls matched for sex and age. RESULTS: Twenty-four-hour ambulatory blood pressure monitoring showed that all but one patient had normal daytime BP values and six patients showed a reduced or inverse nocturnal dip. LVH was found in 72% of the patients. In comparison with healthy controls, patients showed significantly elevated TPR at rest and during exercise suggesting an increased vascular tone. The degree of LVH in these patients is severe and appears disproportionate to the BP values. CONCLUSION: The high incidence of LVH can reflect an augmented cardiovascular reactivity associated with a disturbed circadian pattern. The increase in TPR and the reduction of the nocturnal fall of BP also might contribute to the development of LVH in young renal Tx patients. PMID- 12371992 TI - Mortality after kidney transplant failure: the impact of non-immunologic factors. AB - BACKGROUND: One third of cadaveric kidney transplant recipients suffer graft loss within five years of transplantation. Non-immunologic factors that predict mortality among non-transplant patients also may be potentially modifiable risk factors for mortality among patients with transplant failure. METHODS: Applying multivariate survival analysis to data from the United States Renal Data System, we determined the effect of immunologic or transplant related factors and non immunologic factors on mortality in patients who initiated dialysis after kidney transplant failure in the United States between April 1995 and September 1998. RESULTS: A total of 4741 patients were followed for a median +/- standard deviation of 15 +/- 11 months after initiation of dialysis after transplant failure. The majority of the 1016 (21%) deaths were due to cardiac (36%) or infectious (17%) causes. Patients in the following groups had an increased risk for all-cause mortality: older patients [hazard ratio (HR) = 1.04 per year, 95% confidence interval (95% CI) 1.03-1.04], women (HR = 1.31, 95% CI 1.10-1.56), patients of white race (HR = 1.94, 95% CI 1.32-2.84), patients with diabetes (HR = 1.76, 95% CI 1.43-2.16), peripheral vascular disease (HR = 1.94, 95% CI 1.54 2.43), congestive heart failure (HR = 1.26, 95% CI 1.05-1.53), drug use (HR = 2.23; 95% CI 1.08-4.60), smokers (HR = 1.35, 95% CI 1.01-1.81), first transplant recipients (HR = 1.32, 95% CI 1.02-1.69), and patients with a higher glomerular filtration rate (GFR) at dialysis initiation (HR = 1.04 per mL/min higher, 95% CI 1.02-1.06). Those with private insurance (HR = 0.67, 95% CI 0.49-0.93) and higher serum albumin (HR = 0.73 per g/dL higher, 95% CI 0.64-0.83) had a decreased risk for all-cause mortality. Acute rejection, antibody induction, donor source, duration of graft survival and the maximum attained GFR during transplantation did not predict all-cause mortality. CONCLUSIONS: Non-immunologic factors predicted mortality among patients with transplant failure but immunologic and transplant related factors did not. Prevention, early diagnosis and treatment of co-morbid conditions and the complications of chronic kidney disease may improve the survival of patients with transplant failure. PMID- 12371993 TI - Diagnostic value of troponin T for alterations in left ventricular mass and function in dialysis patients. AB - BACKGROUND: Cardiac troponin T (cTnT) is related to left ventricular (LV) mass in patients with end-stage renal disease (ESRD). Furthermore, cTnT reflects the severity of systolic dysfunction in patients with heart diseases. We tested the diagnostic value of cTnT for left ventricular hypertrophy (LVH) and LV systolic dysfunction in a large group of clinically stable hemodialysis patients without heart failure. RESULTS: CTnT was significantly (P < 0.001) higher in patients with LVH than in those with normal LV mass. In a multiple logistic regression model, adjusting for potential confounders (including cardiac ischemia), systolic pressure and cTnT (both P = 0.003) were the strongest correlates of LVH. Similarly, cTnT was significantly higher (P = 0.005) in patients with systolic dysfunction than in those with normal LV function and in a multiple logistic regression model cTnT ranked as the second independent correlate of this alteration after male sex. Serum cTnT had a high positive prediction value for the diagnosis of LVH (87%) but its negative prediction value was relatively low (44%). The positive predictive value of cTnT for LV dysfunction was low (25%) while its negative predictive value was high (93%). A combined analysis including systolic pressure (for the diagnosis of LVH) and sex (for the diagnosis of LV systolic dysfunction) augmented the diagnostic estimates to an important extent (95% positive prediction value for LVH and 98% negative prediction value for LV systolic dysfunction). CONCLUSIONS: CTnT has a fairly good diagnostic potential for the identification of LVH and for the exclusion of LV systolic dysfunction in patients with ESRD without heart failure. This marker may be useful for the screening of alterations in LV mass and function in clinically stable hemodialysis patients. PMID- 12371994 TI - Body size, dialysis dose and death risk relationships among hemodialysis patients. AB - BACKGROUND: The normalized treatment ratio, Kt/V derived from urea kinetic models (UKM), is a commonly used measure of dialysis dose. This measure assumes that smaller patients with low volume of urea distribution (V) require proportionately less total treatment (Kt) than larger patients. The conclusion has been questioned because the UKM use assumptions that could make them invalid for accurately predicting a clinical outcome like survival. It is possible that a relationship exists between Kt and body size whereby a different Kt is required for different sizes. This study therefore explored the relationships among body size, Kt, and death risk focusing on possible interactions between Kt and size. METHODS: The sample included 43,334 patients treated on January 1, 1999. Survival time was modeled using Kt or body size groups to evaluate the shape of the risk profiles. Kt and the size measures were then evaluated together as continuous functions both in main effects (that is, Kt and size) and interaction models to see if the association of Kt with risk might be different for different sizes. The size measures were body weight, weight adjusted statistically for height, body surface area (BSA), weight divided by height (wt/ht) and the body mass index (BMI). RESULTS: The log of risk decreased in rough linear fashion for Kt, weight, weight for height, and BSA. The log-risk relationships were "reverse J-shaped" for wt/ht and BMI. The main effects models suggested improved survival with increasing Kt and all of the size measures. Adding an interaction term increased the benefit associated with increasing Kt and for weight, weight for height and BSA at low values of Kt and size. A significant, positive interaction term mitigated those effects at higher values. Thus, the death risk penalties associated with reducing Kt among small patients were as great as or greater than they were among large patients. A similar pattern was observed for V. Adding the interaction to the BMI model destroyed the main effects, so that there was no significant association between risk and either Kt or BMI. A cross-categorical model of BMI and Kt, however, revealed improving survival with increasing Kt among both low and high BMI patients throughout the range of Kt. CONCLUSIONS: Evidence supporting the intuition that smaller patients require proportionately lower dialysis dose than larger patients was not found. To the contrary, smaller patients suffer as much risk as or more risk than larger patients from reducing Kt. Deciding dialysis treatment using a Kt/V based intuition may lead to avoidable under-dialysis particularly among small patients. PMID- 12371995 TI - Beyond cyclooxygenase. PMID- 12371996 TI - Correlation between hyperhomocysteinemia and interleukin-18 serum levels in maintenance hemodialyzed patients. PMID- 12371997 TI - Uremic myopathy. PMID- 12372000 TI - Regulation of sex differences in progesterone receptor expression in the medial preoptic nucleus of postnatal rats. AB - The medial preoptic nucleus (MPN) of the rat, an excellent model for understanding the mechanisms involved in sexual differentiation, is highly sensitive to gonadal hormones during both pre- and post-natal life. Progesterone receptor (PR) expression is sexually dimorphic in the prenatal MPN. Males have significantly higher levels of PR-immunoreactivity (PRir) than females from approximately embryonic day 19 through at least the day of birth, suggesting that PR may play a role in sexual differentiation. Because the MPN is still sensitive to steroid hormones postnatally, the present study investigated PR expression in the MPN of males and females after birth using immunocytochemistry. Results indicate that a sex difference in PR expression persists until at least postnatal day (P) 28. However, females begin to express PR around P10. Because oestradiol regulates PR expression in the adult brain, this study also examined the influence of gonadal hormones on PR expression in the neonatal male and female MPN. Castration on the day of birth significantly reduced levels of PRir in the MPN by 24 h following surgery. Ovariectomy on P4, before the onset of ovarian steroidogenesis, prevented the induction of PR expression in the female MPN, observed in controls by P13. In both sexes, the presence of PRir in the MPN is dependent on gonadal hormone exposure. These findings suggest that differences in steroid secretion by the neonatal male and female gonads are responsible for producing sex differences in the level of PR expression in the postnatal MPN. PMID- 12372001 TI - Beta-endorphin cells in the arcuate nucleus: projections to the supraoptic nucleus and changes in expression during pregnancy and parturition. AB - Supraoptic nucleus oxytocin neurone activity and secretion are inhibited in late pregnancy and parturition by endogenous opioids. Here, we investigated alterations in the projections and gene expression of beta-endorphin/pro opiomelanocortin neurones in the arcuate nucleus in the pregnant rat. All regions of the arcuate nucleus were found to contain cells immunoreactive for beta endorphin fluorescent microbeads retrogradely transported from the supraoptic nucleus, and double-labelled neurones (beta-endorphin plus microbeads), showing that beta-endorphin neurones throughout the arcuate nucleus project to the supraoptic nucleus. There was an increase in the number of beta-endorphin immunoreactive cells in the arcuate nucleus and an increase in the density of beta-endorphin fibres within the supraoptic nucleus and peri-supraoptic region in late pregnancy and parturition, suggesting enhanced expression of beta-endorphin and increased beta-endorphin innervation of the supraoptic nucleus. Pro opiomelanocortin mRNA expression in the arcuate nucleus increased in late compared to early pregnancy: the number of positive neurones significantly increased in the caudal region. Fos expression (an indicator of neuronal activation) in the arcuate nucleus was colocalized in beta-endorphin neurones in both proestrus and parturient rats, but the number of positive cells did not increase during parturition, suggesting lack of activation of beta-endorphin neurones at birth. Thus, beta-endorphin cells in the arcuate nucleus project to the supraoptic nucleus and increased innervation during pregnancy may explain the enhanced endogenous opioid inhibition of oxytocin neurones. PMID- 12372002 TI - Sauvagine regulates Ca2+ oscillations and electrical membrane activity of melanotrope cells of Xenopus laevis. AB - Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (alpha-MSH) by the neuroendocrine pituitary melanotrope cells of the amphibian Xenopus laevis. These Ca2+ oscillations are built up by discrete increments in the intracellular Ca2+ concentration, the Ca2+ steps, which are generated by electrical membrane bursting firing activity. It has been demonstrated that the patterns of Ca2+ oscillations and kinetics of the Ca2+ steps can be modulated by changing the degree of intracellular Ca2+ buffering. We hypothesized that neurotransmitters known to regulate alpha-MSH secretion also modulate the pattern of Ca2+ oscillations and related electrical membrane activity. In this study, we tested this hypothesis for the secretagogue sauvagine. Using high temporal-resolution Ca2+ imaging, we show that sauvagine modulated the pattern of Ca2+ signalling by increasing the frequency of Ca2+ oscillations and inducing a broadening of the oscillations through its effect on various Ca2+ step parameters. Second, we demonstrate that sauvagine caused a small but significant decrease in K+ currents measured in the whole-cell voltage clamp, whereas Ca2+ currents remained unchanged. Third, in the cell-attached patch-clamp mode, a stimulatory effect of sauvagine on action current firing was observed. Moreover, sauvagine changed the shape of individual action currents. These results support the hypothesis that the secretagogue sauvagine stimulates the frequency of Ca2+ oscillations in Xenopus melanotropes by altering Ca2+ step parameters, an action that likely is evoked by an inhibition of K+ currents. PMID- 12372003 TI - Serotonergic stimulation of corticotropin-releasing hormone and pro opiomelanocortin gene expression. AB - The neurotransmitter serotonin (5-HT) stimulates adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary gland via activation of central 5 HT1 and 5-HT2 receptors. The effect of 5-HT is predominantly indirect and may be mediated via release of hypothalamic corticotropin-releasing hormone (CRH). We therefore investigated the possible involvement of CRH in the serotonergic stimulation of ACTH secretion in male rats. Increased neuronal 5-HT content induced by systemic administration of the precursor 5-hydroxytryptophan (5-HTP) in combination with the 5-HT reuptake inhibitor fluoxetine raised CRH mRNA expression in the paraventricular nucleus (PVN) by 64%, increased pro opiomelanocortin (POMC) mRNA in the anterior pituitary lobe by 17% and stimulated ACTH secretion five-fold. Central administration of 5-HT agonists specific to 5 HT1A, 5-HT1B, 5-HT2A or 5-HT2C receptors increased CRH mRNA in the PVN by 15-50%, POMC mRNA in the anterior pituitary by 15-27% and ACTH secretion three- to five fold, whereas a specific 5-HT3 agonist had no effect. Systemic administration of a specific anti-CRH antiserum inhibited the ACTH response to 5-HTP and fluoxetine and prevented the 5-HTP and fluoxetine-induced POMC mRNA response in the anterior pituitary lobe. Central or systemic infusion of 5-HT increased ACTH secretion seven- and eight-fold, respectively. Systemic pretreatment with the anti-CRH antiserum reduced the ACTH responses to 5-HT by 80% and 64%, respectively. It is concluded that 5-HT via activation of 5-HT1A, 5-HT2A, 5-HT2C and possibly also 5 HT1B receptors increases the synthesis of CRH in the PVN and POMC in the anterior pituitary lobe, which results in increased ACTH secretion. Furthermore, the results indicate that CRH is an important mediator of the ACTH response to 5-HT. PMID- 12372004 TI - Resistance to the satiety action of leptin following chronic central leptin infusion is associated with the development of leptin resistance in neuropeptide Y neurones. AB - Leptin regulates food intake and body weight by acting primarily in the hypothalamus. In humans and rodents, obesity is associated with hyperleptinaemia, suggesting a possible state of leptin resistance. Thus, to begin to examine the mechanisms of leptin resistance, we developed a rat model in which chronic central leptin infusion results in the development of resistance to leptin's satiety action. Adult male rats were infused chronically into the lateral cerebroventricle with leptin (160 ng/h) or phosphate-buffered saline via Alzet pumps for 28 days, followed by artificial cerebrospinal fluid infusion for 3 weeks. After the initial decrease in food intake, rats developed resistance to the satiety action of leptin, and withdrawal of the chronic leptin infusion resulted in hyperphagia. During leptin infusion, body weight was gradually decreased to reach a nadir on day 12, and thereafter, body weight was sustained at a reduced level throughout the entire 28-day infusion, despite normalization in food intake. Body weight was mostly normalized by day 22 postleptin. Since neuropeptide Y (NPY) neurones are one of the targets of leptin signalling in the hypothalamus, we next examined whether the development of resistance to the satiety action of leptin was due to altered NPY gene expression. On day 3-4 of infusion, hypothalamic NPY mRNA levels, as determined by RNAse protection assay (RPA), were significantly decreased in leptin treated rats compared to controls. By contrast, on day 16 of infusion, NPY mRNA levels in the leptin treated group had returned to control levels. In situ hybridization study confirmed the results obtained with RPA and showed further that the effect of chronic leptin infusion on NPY mRNA levels was restricted to the rostral and middle parts of the arcuate nucleus. Overall, the finding that the action of continuous leptin exposure on NPY neurones was not sustained suggests that NPY neurones may be involved in the development of leptin resistance to the satiety action of leptin in the hypothalamus. PMID- 12372005 TI - Expression of cAMP response element binding protein-binding protein in the song control system and hypothalamus of adult European starlings (Sturnus vulgaris). AB - In songbirds, the initiation of song behaviour and the neural substrate of this system are highly influenced by gonadal steroids. Receptors for gonadal steroid hormones, such as androgens and oestrogens, have been localized within select nuclei of the song system. An important step in steroid receptor action is the recruitment of nuclear receptor coactivators. The coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), has been implicated in both androgen and oestrogen receptor transactivation. Although the role of CBP in transcriptional mechanisms has been widely studied, little is known about CBP expression in the brain. The association between the distribution of CBP and oestrogen receptors in the hippocampus has been related to long-term memory. However, the distribution of brain CBP has not been related to the expression of gonadal steroid receptors in a system as relevant to reproductive behaviour as the avian song system. Western immunoblotting of European starling (Sturnus vulgaris) brain tissue reveals a band at 265 kDa. Immunohistochemical localization of CBP in starling brain indicates wide, but heterogeneous expression. CBP-immunoreactive (CBP-ir) cells define the boundaries of song control nuclei. In HVc (sometimes called the High Vocal Center) and the robust nucleus of the archistriatum (RA), there is a higher density of CBP-ir cells within the boundaries of these nuclei than in adjacent neostriatum or archistriatum, for HVc and RA, respectively. We also report that the distribution of CBP-ir cells varies among different nuclei within the song control system. CBP ir cells within area X (also a part of the song system) and HVc are densely packed into clusters, whereas cells can be easily discriminated in RA. CBP is also highly expressed in hypothalamic areas, indicating that areas rich in steroid receptors also contain CBP. These data suggest that CBP is important for modulating transcriptional activities in the song system and other sites in the songbird brain that express gonadal steroid receptors. PMID- 12372006 TI - A novel mechanism for endocrine-disrupting effects of polychlorinated biphenyls: direct effects on gonadotropin-releasing hormone neurones. AB - Polychlorinated biphenyls (PCBs) cause abnormal development and physiology of the reproductive system. We hypothesized that these effects may be mediated, at least in part, by neuroendocrine cells in the hypothalamus that integrate inputs to and outputs from the central nervous system and reproductive systems. The effects of two PCB mixtures, Aroclor 1221 and Aroclor 1254, were tested on the hypothalamic GT1-7 cells, which synthesize and secrete the key hypothalamic hormone, gonadotropin-releasing hormone (GnRH). GT1-7 cells were treated for 24 h in dose response experiments and GnRH gene expression and release were quantified. Aroclor 1221 was stimulatory to GnRH gene expression, particularly at post transcriptional levels (GnRH cytoplasmic mRNA), and increased GnRH peptide levels, suggesting a post-translational regulation of GnRH biosynthesis. It also caused a qualitative increase in GT1-7 neurite outgrowth and cell confluency. Aroclor 1254 had very different effects from Aroclor 1221. It inhibited GnRH nuclear mRNA levels at high dosages, and stimulated GnRH mRNA at low doses, suggesting a post-transcriptional mechanism of regulation. Aroclor 1254 did not alter GnRH peptide levels. Qualitatively, Aroclor 1254 caused a retraction of GT1 7 cell processes and neurotoxicity at high dosages. In order to gauge the involvement of the oestrogen receptor in these responses, the oestrogen receptor antagonist, ICI 182,780 (ICI) was coadministered in other studies with the PCBs. While effects of Aroclor 1221 on GnRH gene expression were not blocked by ICI, its effects on GnRH peptide levels were blocked by ICI, indicating that some but not all of the effects of Aroclor 1221 are mediated by the classical oestrogen receptor alpha and/or beta. The inhibitory effects of Aroclor 1254 on GnRH gene expression were not prevented by ICI, although ICI itself had stimulatory effects on GnRH gene expression that were blocked by cotreatment with Aroclor 1254. These results demonstrate a novel mechanism for effects of the two PCBs directly on GnRH gene expression, and indicate a hypothalamic level for endocrine disruption by these environmental toxicants. PMID- 12372007 TI - In-vitro study of the effect of adrenaline on the functional capacity of human neutrophils: role during exercise. AB - We studied the effect of adrenaline on the capacity of human neutrophils to attach, ingest and destroy Candida albicans. The neutrophils were incubated in vitro in the presence of an adrenaline concentration taken as basal (10-10 m), and another that is referred to as being reached following physical exercise (10 9 m). Two higher concentrations (10-7 m and 10-5 m), which are seldom attained in blood but which can occur at specific locations in the organism, were tested as having a possible pharmacological application. At the two high concentrations (10 7 m and 10-5 m), the capacity to attach, ingest and destroy C. albicans was greater than at the physiological concentrations (10-10 m and 10-9 m). Indeed, the capacity to attach and ingest C. albicans was significantly less after incubation with these physiological concentrations than the control values (incubation in the absence of adrenaline). Hence, high concentrations of adrenaline seem to enhance neutrophils' phagocytic capacity compared to physiological plasma concentrations, whereas small variations such as those caused by physical exercise have no effect on this functional capacity. PMID- 12372008 TI - Oestrogens, via transforming growth factor alpha, modulate basic fibroblast growth factor synthesis in hypothalamic astrocytes: in vitro observations. AB - The data presented here show that, in cultures of type 1 astrocytes obtained from the hypothalamus of neonatal female rat, 17beta-oestradiol is able to increase both the mRNA and the protein levels of basic fibroblast growth factor (bFGF). In particular, after 24 h of exposure to 17beta-oestradiol (10(-9) and 10(-10) m), an increase of messenger levels of bFGF appears in hypothalamic type 1 astrocytes. Similarly, an induction of bFGF protein is also evident at this time of exposure. The effect on the mRNA and protein levels of bFGF is blocked by the presence in the medium of an antibody raised against the transforming growth factor alpha (TGFalpha) receptor. This observation indicates that, TGFalpha, whose synthesis is modulated by oestrogens in hypothalamic astrocytes and which is able to increase, both the mRNA and the protein levels of bFGF in our experimental model, may act as the mediator of the oestrogenic induction of bFGF. Hypothalamic astrocytes, together with hypothalamic neurones synthesizing and secreting luteinizing hormone-releasing hormone (LHRH), form the LHRH network in conjunction with other neuronal systems. Gonadal steroids in general, and oestrogens in particular, play an important role in the control of the activity of this network. In addition, bFGF and TGFalpha, two growth factors released from astrocytes, are able to influence the activity of LHRH neurones. The present observations suggest that oestrogens may also act on LHRH neurones in an indirect fashion (i.e. by modulating the expression of bFGF and TGFalpha in glial cells). PMID- 12372009 TI - Isoform specificity for oestrogen receptor and thyroid hormone receptor genes and their interactions on the NR2D gene promoter. AB - Oestrogens are critical for the display of lordosis behaviour and, in recent years, have also been shown to be involved in synaptic plasticity. In the brain, the regulation of ionotropic glutamate receptors has consequences for excitatory neurotransmission. Oestrogen regulation of the N-methyl-d-aspartate receptor subunit 2D (NR2D) has generated considerable interest as a possible molecular mechanism by which synaptic plasticity can be modulated. Since more than one isoform of the oestrogen receptor (ER) exists in mammals, it is possible that oestrogen regulation via the ERalpha and ERbeta isoforms on the NR2D oestrogen response element (ERE) is not equivalent. In the kidney fibroblast (CV1) cell line, we show that in response to 17beta-oestradiol, only ERalpha, not ERbeta, could upregulate transcription from the ERE which is in the 3' untranslated region of the NR2D gene. When this ERE is in the 5' position, neither ERalpha nor ERbeta showed transactivation capacity. Thyroid hormone receptor (TR) modulation of ER mediated induction has been shown for other ER target genes, such as the preproenkephalin and oxytocin receptor genes. Since the various TR isoforms exhibit distinct roles, we hypothesized that TR modulation of ER induction may also be isoform specific. This is indeed the case. The TRalpha1 isoform stimulated ERalpha mediated induction from the 3'-ERE whereas the TRbeta1 isoform inhibited this induction. This study shows that isoforms of both the ER and TR have different transactivation properties. Such flexible regulation and crosstalk by nuclear receptor isoforms leads to different transcriptional outcomes and the combinatorial logic may aid neuroendocrine integration. PMID- 12372013 TI - Lack of PSD-95 drives hippocampal neuronal cell death through activation of an alpha CaMKII transduction pathway. AB - The PSD-95 protein family organizes the glutamatergic postsynaptic density and it is involved in the regulation of the excitatory signal at central nervous system synapses. We show here that PSD-95 deficiency by means of antisense oligonucleotides induces significant neuronal cell death within 24 h both in primary hippocampal cultures and in organotypic hippocampal slices. On the other hand, cultured cortical neurons are spared by PSD-95 antisense toxicity until they reach a NR2A detectable protein level (24 days in vitro). The neurotoxic event is characterized by increased alpha CaMKII association to NR2 regulatory subunits of NMDA receptor complex. As a direct consequence of alpha CaMKII association, we found increased GluR1 delivery to cell surface in cultured hippocampal neurons paralleled by AMPA-dependent increase in [Na+]I levels. In addition, both CaMKII specific inhibitor KN-93 and AMPA receptor antagonists CNQX and NBQX rescued neuronal survival to control values. On the other hand, both the NMDA channel blocker MK-801 and Dantrolene, an inhibitor of calcium release from ryanodine-sensitive endoplasmic reticulum stores, failed to have any effect on neuronal survival in PSD-95 deficient neurons. Thus, our data provide clues that PSD-95 reduced expression in neurons is responsible for neuronal vulnerability mediated by direct activation of alpha CaMKII transduction pathway in the postsynaptic compartment. PMID- 12372014 TI - Effects of long-term treatment with dopamine receptor agonists and antagonists on terminal arbor size. AB - This study demonstrates that pharmacological manipulation of the dopamine (DA) receptors can modulate the size of the axonal tree of substantia nigra pars compacta (SNpc) neurons in mice. Pharmacological blockade or genetic ablation of the D2 receptor (D2R) resulted in sprouting of DA SNpc neurons whereas treatment with a D2 agonist resulted in pruning of the terminal arbor of these neurons. Agents such as cocaine, that indirectly stimulate D2R, also resulted in reduced terminal arbor. Specific D1 agonists or antagonists had no effect on the density of DA terminals in the striatum. We conclude that the D2 receptor has a central role in regulating the size of the terminal arbor of nigrostriatal neurons. These findings have implications relating to the use of dopaminergic agonists in the management of Parkinson's disease and in controlling plasticity following injury, loss or transplantation of DA neurons. PMID- 12372015 TI - Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve. AB - The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) 'rosettes' and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions. PMID- 12372016 TI - The antidepressant tianeptine persistently modulates glutamate receptor currents of the hippocampal CA3 commissural associational synapse in chronically stressed rats. AB - Recent hypotheses on the action of antidepressants imply a modulation of excitatory amino acid transmission. Here, the effects of long-term antidepressant application in rats with the drug tianeptine were examined at hippocampal CA3 commissural associational (c/a) glutamate receptor ion channels, employing the whole-cell patch-clamp technique. The drug's impact was tested by subjecting rats to daily restraint stress for three weeks in combination with tianeptine treatment (10 mg/kg/day). Whereas stress increased the deactivation time-constant and amplitude of the N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), it did not affect the alpha-amino-3-hydroxy-5 methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated EPSCs. Concomitant pharmacological treatment of stressed animals with tianeptine resulted in a normalized scaling of the amplitude ratio of NMDA receptor to AMPA/kainate receptor-mediated currents and prevented the stress-induced attenuation of NMDA EPSCs deactivation. Both paired-pulse-facilitation and frequency-dependent plasticity remained unchanged. Both in control and stressed animals, however, tianeptine treatment strengthened the slope of the input-output relation of EPSCs. The latter was mimicked by exposing hippocampal slices in vitro with 10 micro m tianeptine, which rapidly increased the amplitudes of NMDA- and AMPA/kainate EPSCs. The enhancement of EPSCs could be blocked by the intracellular presence of the kinase inhibitor staurosporine (1 micro m), suggesting the involvement of a postsynaptic phosphorylation cascade rather then presynaptic release mechanisms at CA3 c/a synapses. These results indicate that tianeptine targets the phosphorylation-state of glutamate receptors at the CA3 c/a synapse. This novel signal transduction mechanism for tianeptine may provide a mechanistic resolution for its neuroprotective properties and, moreover, a pharmacological trajectory for its memory enhancing and/or antidepressant activity. PMID- 12372017 TI - 5-HT4 receptors exert a frequency-related facilitatory control on dorsal raphe nucleus 5-HT neuronal activity. AB - We investigated, using single-unit recordings in chloral hydrate-anaesthetized rats, the role of serotonin 4 (5-HT4) receptors in the control of dorsal raphe nucleus (DRN) 5-HT neuron activity. About one-half (36) of the 76 neurons recorded were affected by either the preferential 5-HT4 agonist cisapride (500 and 1000 micro g/kg, i.v.) or the selective 5-HT4 antagonist, GR 125487 (200- 2000 micro g/kg, i.v.). Responding neurons displayed a significantly higher mean basal firing rate (1.93 +/- 0.1 Hz) than non-responders (1.31 +/- 0.1 Hz). The firing rate of responding 5-HT neurons was enhanced dose-dependently by cisapride (+47 and +94% at 500 and 1000 micro g/kg, respectively), an effect abolished by GR 125487 (500 micro g/kg) and reduced by the 5-HT4 antagonist, SDZ 205557 (500 micro g/kg, i.v). Conversely, GR 125487 induced a dose-dependent inhibition of responders activity, which was almost completely suppressed at the dose of 2000 micro g/kg. In a separate set of experiments, the selective 5-HT4 agonist, prucalopride (500 micro g/kg, i.v), increased the firing activity (+35%) of 5-HT neurons displaying a high basal firing rate; subsequent injection of GR 125487 (500 micro g/kg, i.v.) suppressed this effect. These results indicate that 5-HT4 receptors exert both a tonic and a phasic, positive, frequency-related control on DRN 5-HT neuronal activity. The existence of such a control might open new avenues for therapeutic research in the antidepressant field. PMID- 12372018 TI - Met-enkephalin immunoreactive neurons recruited by acute stress are innervated by axon terminals immunopositive for tyrosine hydroxylase and dopamine-alpha hydroxylase in the anterolateral division of bed nuclei of the stria terminalis in the rat. AB - The bed nuclei of the stria terminalis (BST) are highly heterogeneous forebrain structures, which play a central role in the regulation/modulation of stress responses. Studies using the inducible immediate early gene c-fos as a marker of activated neurons have demonstrated significant stress-induced neuronal activation in this limbic region. The BST also exhibit a dense network of dopamine and noradrenaline immunoreactive (ir) axon terminals. These catecholaminergic projections from various brainstem sources to the BST play an important role in a neurochemically mediated coordination of stress responses. In the anterolateral division of bed nuclei of the stria terminalis, the distribution of several Met-enkephalin immunopositive perikarya overlaps with that of catecholaminergic axon terminals. Both monoaminergic and enkephalinergic structures have been postulated to play a role in the regulation/modulation of the central regulatory pathways of endocrine, behavioural and physiological responses during stress. Therefore the aims of this study were: (i). to study the possible involvement of dopaminergic fibre terminals in stress-induced activation of BST perikarya; (ii). to investigate whether Met-enkephalin-immunoreactive neurons are recruited by acute volumen/osmotic challenge; and (iii). to demonstrate synaptic interactions between Met-enkephalin-ir neurons and fibre terminals immunopositive for dopamine or noradrenaline in the anterolateral division of the BST. From the results of this study we can conclude that depletion of dopamine in fibre terminals completely abolished stress-induced activation of perikarya in the anterolateral division of BST. Furthermore, the innervation of stress-induced Met-enkephalin-ir perikarya by dopaminergic fibre terminals in the oval nucleus of BST was demonstrated, whereas noradrenergic axons contacted enkephalinergic structures in the fusiform and subcomissural nuclei of BST. These interactions can be central in the modulatory control of the major stress regulatory pathway, the limbic hypothalamo-pituitary-adrenal axis. PMID- 12372019 TI - CNS region-specific regulation of glial glutamate transporter expression. AB - The neuronal cell death associated with certain neurodegenerative disorders as well as acute brain injuries is in part due to the reduced expression of glial glutamate transporters and the subsequent accumulation of toxic extracellular glutamate concentrations. Extracellular factors previously found to potently stimulate the expression of the glial glutamate transporters, GLT-1/EAAT2 and GLAST/EAAT1, in astroglial cultures of rat cerebral hemispheres are PACAP, TGF alpha, and EGF. In the present study, we sought to determine whether similar stimulatory influences apply for astroglia from other areas of the central nervous system (CNS). Immunoblot and real-time RT-PCR analysis of striatal astroglial cultures maintained for 72 h with PACAP, TGF alpha, or EGF revealed a prominent increase in GLT-1 and GLAST expression. In apparent contrast, all factors completely failed to affect GLT-1 and GLAST expression in astroglial cultures from the cerebellum, mesencephalon, and spinal cord between 36 h and 7 days. This failure was not due to the absence of functional recognition or transduction machineries for the extracellular factors as suggested by the additional observations that cerebellar, mesencephalic and spinal cord glia were capable of responding to stimulation with PACAP, TGF alpha, or EGF for 10 min with activation of CREB. Moreover, dibutyryl cyclic AMP (dbcAMP) potently promoted GLT-1 and/or GLAST expression in mesencephalic, cerebellar and spinal cord glia, further indicating that extracellular factors regulate glial glutamate transporter expression throughout the CNS. Together these findings identify PACAP, TGF alpha and EGF as potent regulators of glutamate transporter expression in striatal glia. In addition, these findings provide evidence for a CNS region specific regulation of glial glutamate transport. PMID- 12372020 TI - Somatostatin receptor subtypes 2 and 4 affect seizure susceptibility and hippocampal excitatory neurotransmission in mice. AB - We have investigated the role of somatostatin receptor subtypes sst2 and sst4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild type and sst2 knock-out mice. Intrahippocampal injection of three selective sst2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 micro m) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 micro m). Similarly to what was observed in seizures, the effect of L 803087 was reduced by octreotide. In hippocampal slices from sst2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst2 subtypes, suggesting these receptors are functionally coupled. PMID- 12372021 TI - Persistent epileptiform activity induced by low Mg2+ in intact immature brain structures. AB - We have determined the properties of seizures induced in vitro during the first postnatal days using intact rat cortico-hippocampal formations (CHFs) and extracellular recordings. Two main patterns of activity were generated by nominally Mg2+-free ACSF in hippocampal and cortical regions: ictal-like events (ILEs) and late recurrent interictal discharges (LRDs). They were elicited at distinct developmental periods and displayed different pharmacological properties. ILEs were first observed in P1 CHFs 52 +/- 7 min after application of low-Mg2+ ACSF (frequency 1.5 +/- 0.3 h-1, duration 86 +/- 3 s). There is a progressive age-dependent maturation of ILEs characterized by a decrease in their onset and an increase in their frequency and duration. ILEs were abolished by d APV and Mg2+ ions. From P7, ILEs were followed by LRDs that appeared 89 +/- 8 min after application of low-Mg2+ ACSF (frequency approximately 1 Hz, duration 0.66 s, amplitude 0.31 +/- 0.03 mV). LRDs were no longer sensitive to d-APV or Mg2+ ions and persisted for at least 24 h in low-Mg2+ or in normal ACSF. ILEs and LRDs were synchronized in limbic and cortical regions with 10-40 ms latency between the onsets of seizures. Using a double chamber that enables independent superfusion of two interconnected CHFs, we report that ILEs and LRDs generated in one CHF propagated readily to the other one that was being kept in ACSF. Therefore, at a critical period of brain development, recurrent seizures induce a permanent form of hyperactivity in intact brain structures and this preparation provides a unique opportunity to study the consequences of seizures at early developmental stages. PMID- 12372023 TI - Multiple-site optical recording for characterization of functional synaptic organization of the optic tectum of rainbow trout. AB - To map the functional synaptic organization over a wide area in the optic tectum, we directly monitored two-dimensional propagation of postsynaptic depolarization evoked by firing of retinotectal afferents in optic tectum slices prepared from rainbow trout (Oncorhynchus mykiss), using a voltage-sensitive dye and a photodiode array system. The postsynaptic responses to afferent stimulation first propagated in the stratum opticum and stratum fibrosum et griseum superficiale in an anterograde fashion in the afferents and then expanded vertically into the deep layers. This vertical propagation appeared to occur along a bundle-like structure that corresponded well with a cluster of neurons whose somata are located in the stratum periventriculare. Pharmacological studies showed that these postsynaptic responses were mediated by ionotropic glutamate receptors. On the other hand, the optical signals appeared to consist of at least two components (a transient signal and a slow signal). The second transient signal summated with the first slow signal by paired stimulation, suggesting that the transient and slow signals originated from different cell types. Taken together, these results showed that the functional synaptic organization of the teleost optic tectum comprises of two depolarization-signal propagating paths along a horizontal layer structure and a vertical bundle-like structure and that these synaptic responses occur via glutamatergic transmission. PMID- 12372022 TI - Kindling enhances kainate receptor-mediated depression of GABAergic inhibition in rat granule cells. AB - Several lines of evidence indicate a substantial contribution of kainate receptors to temporal lobe seizures. The activation of kainate receptors located on hippocampal inhibitory interneurons was shown to reduce GABA release. A reduced GABA release secondary to kainate receptor activation could contribute to an enhanced seizure susceptibility. As the dentate gyrus serves a pivotal gating function in the spread of limbic seizures, we tested the role of kainate receptors in the regulation of GABA release in the dentate gyrus of control and kindled animals. Application of glutamate (100 micro m) in the presence of the NMDA receptor antagonist d-APV and the AMPA receptor antagonist, SYM 2206 caused a slight depression of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) in control, but a substantial decrease in kindled dentate granule cells. The observation that kainate receptor activation altered paired-pulse depression and reduced the frequency of TTX-insensitive miniature IPSCs without affecting their amplitude is consistent with a presynaptic action on the inhibitory terminal to reduce GABA release. In kindled preparations, neither glutamate (100 micro m) nor kainate (10 micro m) applied in a concentration known to depolarize hippocampal interneurons led to an increase of the TTX-sensitive spontaneous IPSC frequency nor to changes of the postsynaptic membrane properties. Consistently, the inhibitory effect on evoked IPSCs was not affected by the presence of the GABAB receptor antagonist, CGP55845A, thus excluding a depression by an enhanced release of GABA acting on presynaptic GABAB receptors. The enhanced inhibition of GABA release following presynaptic kainate receptor activation favours a use dependent hyperexcitability in the epileptic dentate gyrus. PMID- 12372024 TI - Chronic nicotine treatment changes the axonal distribution of 68 kDa neurofilaments in the rat ventral tegmental area. AB - Region-specific decreases of neurofilament proteins (NF) were described in the ventral tegmental area (VTA) of rats treated chronically with morphine, cocaine or alcohol. In a previous study, we demonstrated that NF levels were also changed in the VTA after chronic treatment with nicotine. The aim of this study was to clarify the submicroscopic basis of decreased immunoreactivity for NF-68, NF-160 and NF-200, as determined by using NR4, BF10 and RT97 antibodies, respectively. Microdensitometric analysis of brain sections showed that immunoreactivity for all NF was reduced in the VTA of animals exposed chronically to nicotine (0.4 mg/kg per day, 6 days of treatment), when compared to rats exposed to saline. Reduction in immunoreactivity was significant for NF-68 (P < 0.05), NF-160 (P < 0.01) and NF-200 (P < 0.05), showing a relative reduction of 34%, 42% and 38%, respectively, when compared to saline-treated rats. No difference was observed for any of the NF under study when immunoreactivity measurements in the substantia nigra were compared. Ultrastructural analysis was applied to evaluate changes in NF-68, NF-160 and NF-200 immunoreactivity in regions of the VTA that contain dopaminergic neurons following chronic nicotine treatment. At the electron microscopic level, no degenerative changes were found in neurons or glial cells of the VTA. With ultrastructural immunohistochemistry, evaluation of the homogeneity parameter of NF distribution showed a loss of homogeneity for NF 68 linked to the nicotine treatment. In areas in which NF organization appeared well preserved, analysis of the numerical density of NF revealed no significant difference for NF-68 (897/ micro m2 vs. 990/ micro m2), NF-160 (970/ micro m2 vs. 820/ micro m2) and NF-200 (1107/ micro m2 vs. 905/ micro m2) in nicotine-treated rats when compared to saline-treated rats. These results confirm that nicotine shares the same properties with cocaine and morphine in reducing NF in the VTA, a key brain structure of the rewards system, and that chronic nicotine treatment changes the axonal distribution of 68 kDa neurofilaments in the rat VTA. PMID- 12372025 TI - Multiple determinants in voltage-dependent P/Q calcium channels control their retention in the endoplasmic reticulum. AB - Surface expression level of voltage-dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage-activated calcium channels requires the association of the pore-forming subunit, Cavalpha, with the auxiliary subunit, Cavbeta. In the absence of this auxiliary subunit, Cavalpha is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8 alpha chimeras, the molecular determinants of Cavalpha2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as 'ER retention'). This study demonstrates that the I-II loop of Cavalpha2.1 contains multiple ER-retention determinants beside the beta subunit association domain. In addition, the I-II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I-II loop, the N- and C-termini of Cavalpha2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I-II loop of Cavalpha1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i). a correct folding of Cavalpha in order to mask several internal ER retention determinants and (ii). the association of other proteins, including the Cavbeta subunit, to suppress the remaining ER-retention determinants. PMID- 12372026 TI - Identification and comparative analysis of differentially expressed proteins in rat striatum following 6-hydroxydopamine lesions of the nigrostriatal pathway: up regulation of amyloid precursor-like protein 2 expression. AB - During neurodegenerative processes, cascades of degeneration and subsequent regeneration are triggered. However, the molecular nature of the factors involved in the neurodegeneration of the CNS remains largely unknown. In this study, the variations of protein expression in the striatum of adult Sprague-Dawley rats following 6-hydroxydopamine lesions were investigated, in order to better understand the molecular events occurring in the denervated target tissue. The rat striatum, ipsilateral to the lesion was analysed by two-dimensional gel electrophoresis followed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Seven proteins were up-regulated (188.1-750% compared to control) in response to the lesion: amyloid precursor-like protein 2 (APLP2), kininogen, glucokinase, tropomyosin alpha chain, type brain-1 and calpactin I light chain; whilst four proteins, neural epidermal growth factor-like 2, minichromosome maintenance 6, and thyroid hormone receptor beta-2, were down regulated (to between 36% and 59% of levels in sham-operated controls). Three proteins that did not match with available data in the SWISS-PROT protein database were also determined. Immunohistochemical analysis demonstrated colocalization of APLP2 and tyrosine hydroxylase in the nigral neurons. Moreover, reduction of APLP2-positive neurons in the substantia nigra pars compacta as well as the increases in the substantia nigra pars reticulata and in the striatum were observed. Furthermore, the conditioned medium of the Chinese hamster ovary cells over-expressing APLP2-751 (chondroitin sulphate-modified), but not APLP2-763 (nonchondroitin sulphate-modified), was able to increase the number of the tyrosine hydroxylase-positive neurons in fetal mesencephalic cultures. These results suggest that the expression of APLP2, a protein that has been thought to be associated with Alzheimer's disease, is up-regulated in the striatum following dopaminergic denervation. They also support the view that chondroitin sulphate modified APLP2 protein may play an important role in the dopaminergic nigrostriatal system. PMID- 12372027 TI - Somatic nociception activates NK1 receptors in the nucleus tractus solitarii to attenuate the baroreceptor cardiac reflex. AB - There is limited information regarding the integration of visceral and somatic afferents within the nucleus of the solitary tract (NTS). We studied the interaction of nociceptive and baroreceptive inputs in this nucleus in an in situ arterially perfused, un-anaesthetized decerebrate preparation of rat. At the systemic level, the gain of the cardiac component of the baroreceptor reflex was attenuated significantly by noxious mechanical stimulation of a forepaw. This baroreceptor reflex depression was mimicked by NTS microinjection of substance P and antagonized by microinjection of either bicuculline (a GABAA receptor antagonist) or a neurokinin type 1 (NK1) receptor antagonist (CP-99994). The substance P effect was also blocked by a bilateral microinjection of bicuculline, at a dose that was without effect on basal baroreceptor reflex gain. Baroreceptive NTS neurons were defined by their excitatory response following increases in pressure within the ipsilateral carotid sinus. In 27 of 34 neurons the number of evoked spikes from baroreceptor stimulation was reduced significantly by concomitant electrical stimulation of the brachial nerve (P < 0.01). Furthermore, the attenuation of baroreceptor inputs to NTS neurons by brachial nerve stimulation was prevented by pressure-ejection of bicuculline from a multi-barrelled microelectrode (n = 8). In a separate population of 17 of 45 cells tested, brachial nerve stimulation evoked an excitatory response that was antagonized by blockade of NK1 receptors. We conclude that nociceptive afferents activate NK1 receptors, which in turn excite GABAergic interneurons impinging on cells mediating the cardiac component of the baroreceptor reflex. PMID- 12372028 TI - Methamphetamine-induced, suprachiasmatic nucleus-independent circadian rhythms of activity and mPer gene expression in the striatum of the mouse. AB - While the suprachiasmatic nucleus (SCN) coordinates the majority of daily rhythms, some circadian patterns of expression are controlled from outside of the SCN. These include responses to daily methamphetamine (MAP) injection, or daily restricted feeding. The mechanisms underlying these SCN-independent circadian rhythms are unknown. A circadian oscillation in the expression of mPer1 and/or mPer2, mouse period, in the SCN is considered necessary to generate an SCN dependent circadian rhythm. Therefore, in this experiment, we examined the association between mPer gene expression and the MAP-induced, SCN-independent circadian rhythm. Acute injection of MAP caused an elevation of mPer1, mBmal1, and mNpas2 gene expression in the striatum and mPer1 in the liver. Daily MAP injection at a fixed time for 6 days shifted the rhythmic mPer1 and mPer2 expression in the striatum from a nocturnal to a diurnal rhythm, but failed to affect that in the SCN. Although lesion of the SCN 'flattened'mPer gene oscillation in the striatum and liver, daily MAP injection caused both behavioural and mPer gene expression rhythms. Daily MAP injection at variable injection intervals (12-36 h) for 6 days, however, failed to produce mPer gene rhythm in the striatum. Daily repeated MAP signals may strengthen the oscillatory force of SCN-independent circadian behavioural and molecular rhythms. The present results suggest that daily oscillation of mPer genes outside the SCN is closely associated with the regulation of SCN-independent rhythms. Thus, the present experiment highlights strongly the important role of clock gene expression, in the brain, that underlies the circadian behavioural rhythm. PMID- 12372029 TI - Speech processing activates visual cortex in congenitally blind humans. AB - Neurophysiological recordings and neuroimaging data in blind and deaf animals and humans suggest that perceptual functions may be organized differently after sensory deprivation. It has been argued that neural plasticity contributes to compensatory performance in blind humans, such as faster speech processing. The present study employed functional magnetic resonance imaging (fMRI) to map language-related brain activity in congenitally blind adults. Participants listened to sentences, with either an easy or a more difficult syntactic structure, which were either semantically meaningful or meaningless. Results show that blind adults not only activate classical left-hemispheric perisylvian language areas during speech comprehension, as did a group of sighted adults, but that they additionally display an activation in the homologueous right hemispheric structures and in extrastriate and striate cortex. Both the perisylvian and occipital activity varied as a function of syntactic difficulty and semantic content. The results demonstrate that the cerebral organization of complex cognitive systems such as the language system is significantly shaped by the input available. PMID- 12372030 TI - The integrative role of the rat medullary subnucleus reticularis dorsalis in nociception. AB - Neurons within the medullary subnucleus reticularis dorsalis (SRD) of the rat convey selectively nociceptive information from all parts of the body. We have sought to define the neuronal networks that convey information from widespread noxious stimuli to the diffuse thalamocortical system and also modulate spinal outflow. The experiments, which were performed in rats, were designed to determine whether efferents from the SRD issue collaterals to the thalamus and spinal cord. Injections of the tracers fluorogold and tetramethylrhodamine labelled dextran were centred stereotaxically in two areas that receive dense projections from the SRD: the cervical spinal cord and the lateral ventromedial thalamus (VMl), respectively. In other experimental series, SRD neurons were characterized electrophysiologically and individually labelled in a Golgi-like manner following juxtacellular iontophoresis of biotin-dextran. More than half reticulothalamic neurons within the SRD provided monosynaptic connections to the spinal cord. SRD neurons that responded to Adelta- or Adelta- and C-fibre activation from any area of the body had axons that gave both ascending and descending collaterals. Because the SRD innervates several areas involved in motor processing and receives strong, direct influences from several cortical regions, it could provide a structural basis for the processing of nociceptive and motor activities. PMID- 12372031 TI - Convergence of parvocellular and magnocellular information channels in the primary visual cortex of the macaque. AB - We investigated whether responses of single cells in the striate cortex of anaesthetized macaque monkeys exhibit signatures of both parvocellular (P) and magnocellular (M) inputs from the dorsal lateral geniculate nucleus (dLGN). We used a palette of 128 isoluminant hues at four different saturation levels to test responses to chromatic stimuli against a white background. Spectral selectivity with these isoluminant stimuli was taken as an indication of P inputs. The presence of magnocellular inputs to a given cortical cell was deduced from its responses to a battery of tests, including assessment of achromatic contrast sensitivity, relative strengths of chromatic and luminance borders in driving the cell at different velocities and conduction velocity of their retino geniculo-cortical afferents. At least a quarter of the cells in our cortical sample appear to receive convergent P and M inputs. We cannot however, exclude the possibility that some of these cells could be receiving a convergent input from the third parallel channel from the dLGN, namely the koniocellular (K) rather than the P channel. The neurons with convergent P and M inputs were recorded not only from supragranular and infragranular layers but also from the principal geniculate input recipient layer 4. Thus, our results challenge classical ideas of strict parallelism between different information streams at the level of the primate striate cortex. PMID- 12372032 TI - Tactile motion activates the human middle temporal/V5 (MT/V5) complex. AB - The human middle temporal/V5 complex (hMT/V5) plays a central role in the perception of visual motion. This region is considered a unimodal visual area with little direct involvement of other sensory modalities. The current study uses H215O PET to test whether tactile motion influences the activity of hMT/V5. Regional cerebral blood flow (rCBF) within hMT/V5 was estimated in eight subjects in separate tactile motion and visual motion conditions, each contrasted with a resting, control. The tactile motion condition involved a brush stroked proximal to-distal along the volar forearm and palm, while the subject attended to the stimulus with closed eyes. The visual motion condition consisted of low contrast, grey-scale rings radiating at 15 degrees /s from a central point, upon which the subject was instructed to fixate. The location of hMT/V5 was defined for each subject separately as the local maximum of rCBF change during the visual motion condition (vs. control). The average change in rCBF within spherical regions of interest at each peak revealed significant bilateral activation of hMT/V5 in the tactile motion condition contrasted with a second, independent set of control scans. Additionally, a single subject received a sufficient number of scans to perform a pixel-wise, within-subject analysis. His functional images were coregistered to his anatomical MRI. In this subject, tactile motion produced a significant increase in rCBF that directly overlapped a region activated by visual motion at the posterior continuance of the inferior temporal sulcus, consistent with the known location of hMT/V5. These results suggest involvement of the hMT/V5 complex in tactile motion processing. PMID- 12372033 TI - Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKC. AB - We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl-sn-glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells. PMID- 12372034 TI - Hippocampal long-term depression as an index of spatial working memory. AB - Long-term potentiation (LTP), a form of synaptic plasticity in the hippocampus, is a cellular model for the neural basis of learning and memory, but few studies have investigated the contribution of long-term depression (LTD), a counterpart of LTP. To address the possible relationship between hippocampal LTD and spatial performance, the spatial cognitive ability of a rat was assessed in a spontaneous alternation test and, thereafter, LTD in response to low-frequency burst stimulation (LFBS) was monitored in the dentate gyrus of the same rat under anaesthesia. To enhance a divergence in the ability for spatial performance, some of the animals received fimbria-fornix (FF) transection 14 days before the experiments. LTD was reliably induced by application of LFBS to the medial perforant path of intact rats, while no apparent LTD was elicited in rats with FF lesions. The behavioural parameters of spatial memory showed a significant correlation with the magnitude of LTD. We found no evidence that the cognitive ability correlated with other electrophysiological parameters, e.g. basal synaptic responses, stimulus intensity to produce half-maximal responses, paired pulse facilitation or paired-pulse depression. These results suggest that the magnitude of LTD in the dentate gyrus serves as a reliable index of spatial cognitive ability, providing insights into the functional significance of hippocampal LTD. PMID- 12372035 TI - Functional anatomy of chemical senses in the alert monkey revealed by positron emission tomography. AB - Functional imaging technique using positron emission tomography (PET) has made it possible to localize functional brain regions in the human brain by detecting changes in regional cerebral blood flow (rCBF). Performing PET studies in the monkey will aid in integrating monkey electrophysiological research with human PET studies. We examined changes in rCBF during olfactory or combined olfactory and gustatory (flavour) stimulation using PET in the alert rhesus monkey. Olfactory or flavour stimulation with acetic acid or apple increased rCBF in the prepyriform area, substantia innominata and amygdala. Besides these areas, flavour stimulation increased rCBF in the anterior insula and frontal operculum, orbitofrontal cortex, inferior frontal gyrus and cerebellum. Apple odour or flavour stimuli increased rCBF in the inferior occipital gyrus in addition to the above areas. These findings suggest that the increases of rCBF in response to neural activities in the primary olfactory and gustatory cortices are detectable by the use of PET. In addition, regions activated by apple stimuli suggest that higher brain function might be detected with PET in the alert monkey. PMID- 12372037 TI - Proceedings of the South-Eastern Organ Procurement Foundation Meeting. 24 January 2002, San Antonio, Texas, USA. PMID- 12372038 TI - Immunobiology and long-term graft function in a transplant heterotopic renal rat model. AB - BACKGROUND: The Th1-Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long-term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre engraftment single dose rapamycin and a 7-d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long-term follow-up (>350 d). METHODS: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 +/- 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI-Lewis) and the isograft group (Lewis Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre-engraftment single dose rapamycin and a 7-d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL-4, IL-10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24-h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukey's test was used; p < 0.05 was considered statistically significant. RESULTS: The mean follow up was 352 +/- 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL-4 and IL-10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI-Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). CONCLUSIONS: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow-up. With the follow-up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance. PMID- 12372039 TI - Flow cytometry beads rather than the antihuman globulin method should be used to detect HLA Class I IgG antibody (PRA) in cadaveric renal regraft candidates. AB - HLA Class I antibody screening can be performed by flow cytometry using a mixture of 30 distinct bead populations each coated with the Class I antigen phenotype derived from different cell lines. In this study we compared the efficacy of Class I antibody screens done by flow cytometry beads with the antihuman globulin (AHG) method for patients awaiting cadaveric renal retransplantation. Class I panel reactive antibody (PRA) screening by flow cytometric beads of 21 regraft serum samples that had all been found to be negative by AHG DTT Class I PRA, revealed that 57.1% (12 of 21) had a flow Class I PRA of > or = 10%. Furthermore, when five regraft sera with an intermediate PRA were screened (mean AHG DTT PRA = 33.2 +/- 13%) the mean flow Class I PRA almost doubled (mean flow PRA = 72.4 +/- 10.2%) (p < 0.01). When active UNOS waiting list regraft candidates, after several months of screening the Class I PRA by flow beads, were divided into the three PRA categories based on their peak flow Class I PRA value (0-20%, 21-79% and > or = 80%), the incidence of a positive flow cross-match was 0%, 72% and 85% and the incidence of retransplantation was 60%, 22% and 10%, in each of these groups, respectively. These data provided our histocompatibility laboratory with the rationale to stop performing the AHG PRA and perform only the flow Class I PRA method for regraft candidates. PMID- 12372040 TI - Conundrums with FlowPRA beads. AB - In 1969, a study by Patel and Terasaki persuaded the renal transplant community that a pre-transplant cross-match should always be performed between donor and recipient to detect HLA antibodies and prevent hyperacute allograft rejection. Although the role of the cross-match among nonsensitized patients is controversial, its importance among sensitized recipients is undeniable. Over the past 30 years, more sensitive techniques, such as the flow cytometric cross-match (FCXM), were developed to identify low levels of antibodies undetectable by other approaches. The clinical relevance of a positive FCXM, however, has been hotly disputed, with some investigators maintaining that the FCXM is 'too sensitive' and rules out acceptable donor-recipient combinations. An alternative explanation is that the FCXM is non-specific, and, at least in certain situations, identifies non-HLA antibodies that are clinically irrelevant. Recently, a solid phase immunoassay utilizing purified HLA Class I or Class II molecules bound to microparticles (FlowPRA) was developed. Ideally, use of the FlowPRA for the identification of HLA antibodies in recipient sera would help ascertain whether a positive FCXM with donor cells was truly the result of an HLA-specific antibody. As shown here, this may not always be true. In this study, two unexpected serum patterns were observed. Pattern 1: FlowPRA beads were positive (with an associated HLA Class I specificity) and the FCXM with cells expressing the HLA antigen(s) to which the antibody was directed, was negative. Sequence analysis of the HLA antigens reactive with this unexpected antibody suggests that the epitope recognized resides on the floor of the groove, a site generally not expected to generate antibody activity. Pattern 2: FlowPRA beads were negative yet the FCXM was T and B cell positive. Further analysis of the FlowPRA negative/FCXM positive sera using a flow cytometric cell-based panel reactive antibody (PRA) approach revealed those sera to have specific anti-HLA Class I activity. We suspect that both types of antibodies described above have clinical relevance. Thus, a negative or positive FCXM (when the FlowPRA against donor antigens is positive or negative, respectively) is not always a straightforward interpretation. PMID- 12372041 TI - Daclizumab induction/tacrolimus sparing: a randomized prospective trial in renal transplantation. AB - Tacrolimus inhibits lymphocyte responses by blocking calcium-dependent signalling pathways important in IL-2 generation. Daclizumab, a humanized monoclonal antibody, binds with high affinity to the Tac subunit of the IL-2 receptor complex. We reasoned therefore that the absence of IL-2R should permit lower doses of tacrolimus and thereby less toxicity. Twenty-eight patients were randomized and followed for 6 months: Group 1, high dose (HD) tacrolimus (trough 12-17 ng/mL; n = 13); Group 2, low dose (LD) tacrolimus (trough 5-10 ng/mL; n = 15). All patients received daclizumab induction (2 mg/kg) on days 0 and 14, mycophenolate mofetil (2 g/d except for one patient who received 1 g) and rapid prednisone taper. Serious infections were minimal in both groups. Hospitalizations, for various reasons, were HD (n = 12) and LD (n = 6). All patients and grafts survived for the 6-month study period. There was one rejection episode in a non-compliant patient at 101 d. LD tacrolimus appears equally effective as HD tacrolimus in preventing rejection episodes and may be associated with fewer adverse events. PMID- 12372042 TI - Thymoglobulin for induction or rejection therapy in pancreas allograft recipients: a single centre experience. AB - PURPOSE: To review the safety and efficacy of thymoglobulin in pancreas transplant patients receiving tacrolimus and mycophenolate mofetil. METHODS: Retrospective, single centre analysis of 45 patients transplanted between 1995 and 2000 who received 54 courses of thymoglobulin, including 36 courses in 29 solitary pancreas transplant recipients (16 pancreas alone, 13 pancreas after kidney transplants) and 18 courses in 16 simultaneous kidney-pancreas transplant patients. Thirty-two patients (71%) were primary pancreas transplants, 10 (22%) were second transplants and three (7%) were third transplants. Of the 54 treatment courses, 19 (35%) were for induction, 27 (50%) were for primary rejection and eight (15%) were rescue therapy for rejection. All rejection episodes were biopsy-proven in at least one organ. RESULTS: The median thymoglobulin dose was 1.5 mg/kg/d with a mean of six doses (range 3-10). Dose reduction or interruption was required in 28 courses (52%), most often due to leukopenia (n = 24), fever (n = 2) and thrombocytopenia (n = 2). Thymoglobulin was resumed in all but three patients, two with persistent fever and one with infection. Infectious complications (n = 25) occurred in 17 patients (38%) within 30 days and included bacterial (n = 16), cytomegalovirus (n = 4), polyoma (n = 1), fungal (Candida albicans, n = 1), toxoplasmosis (n = 1) and ehrlichiosis (n = 2). Post-transplant lymphoproliferative disease occurred in two patients (4%) at a mean of 70 d post-thymoglobulin treatment. In the 19 patients that received thymoglobulin induction, one simultaneous kidney-pancreas transplant, two pancreas alone and four pancreas after kidney transplant recipients developed rejection (37% incidence), while all remaining patients followed by surveillance protocol biopsies were rejection-free. In the 35 patients that received thymoglobulin for rejection, reversal occurred in 26 of the patients (74%). Rejection recurred within 30 d in five patients and post-treatment biopsies revealed persistent rejection in three of 20 pancreas and two of eight renal biopsies. After a mean follow-up of 6 months, the actual patient and pancreas graft survival rates were 93% and 71%, respectively. CONCLUSION: Thymoglobulin was effective as induction therapy in high-risk pancreas transplant recipients, and resulted in initial reversal of rejection in 74% of patients. Dose adjustments were required in over half the cases and were usually due to leukopenia. Infections occurring subsequent to thymoglobulin were not uncommon and reflected the immunosuppressive burden of the patient population. PMID- 12372043 TI - Can a pharmacokinetic approach to immunosuppression eliminate ethnic disparities in renal allograft outcome? AB - Although renal allograft outcome correlates more closely with area under the concentration time curve (AUC) for cyclosporin (CsA) compared with the 12-h trough level (C0), few studies have prospectively evaluated pharmacokinetic monitoring in kidney transplantation. This paper describes a study designed to evaluate the impact of a novel approach to CsA-based immunosuppression on ethnic differences in renal allograft outcome. Sixty (32 African Americans and 28 Caucasians) renal transplant recipients were treated with cyclosporin-based triple therapy. Morning and evening doses were independently adjusted to reach an AUC0-12 of 6600-7200 ng h/mL and a C0 of 250-325 ng/mL, respectively. AUCs were measured within 48 h of starting CsA, and as often as necessary to maintain target levels. Only two patients experienced significant adverse events related to immunosuppression. One (Caucasian) developed haemolytic uremic syndrome and was converted to tacrolimus, while another (African American) developed acute vascular rejection. One graft was lost (Caucasian) due to death with a functioning graft. An average of 8 AUCs (range 5-13) were measured in the first 3 months. AUCs were significantly higher in African Americans compared with Caucasians only in the first and second month. C0 values were similar in both groups throughout the study period. A pharmacokinetic approach to immunosuppression allows individualization of CsA exposure, and appears to reduce ethnic disparities in renal allograft outcome. PMID- 12372044 TI - Calcineurin inhibitor-induced chronic nephrotoxicity in liver transplant patients is reversible using rapamycin as the primary immunosuppressive agent. AB - The purpose of this study was to determine whether calcineurin inhibitor (CNI) induced chronic nephrotoxicity in liver transplant patients is reversible by replacement of the CNI with rapamycin as the primary immunosuppressive agent. CNIs, while providing potent immunosuppression for liver transplant patients, exhibit nephrotoxicity as a major side-effect. Whereas acute CNI-induced nephrotoxicity is reversible by withdrawal of the CNI, chronic nephrotoxicity due to CNIs is a progressive process thought to be irreversible. Eight liver transplant patients with CNI-induced chronic nephrotoxicity were converted to rapamycin as the primary immunosuppressive agent. The CNI was either discontinued (four patients) or the dosage lowered to maintain a subtherapeutic level (four patients). Renal function as assessed by serum creatinine was measured before and after conversion to rapamycin. Two patients progressed to dialysis dependence following conversion to rapamycin. These two patients had been on CNIs for a mean of 112 months (range 93-131 months) prior to conversion to rapamycin. Five patients experienced improvement in renal function. These patients had been on calcineurin inhibitors for a mean of 60 months (range 42-75 months) prior to conversion. One patient with chronic nephrolithiasis as a contributing factor to his renal dysfunction has progressed to dialysis dependence despite conversion to rapamycin following exposure to a CNI for 24 months. In the five patients with improved renal function, serum creatinine levels decreased significantly (2.4 +/- 0.3 mg/dL to 1.5 +/- 0.1 mg/dL, p < 0.05) by a mean of 7.2 months (range 5-10 months) after conversion from CNI to rapamycin-based immunosuppression. Liver function remained stable after conversion to rapamycin. CNI-induced chronic nephrotoxicity can be reversed upon withdrawal of the CNI. Rapamycin is an effective replacement agent as primary immunosuppressive therapy following withdrawal of CNIs in liver transplant patients with CNI-induced chronic nephrotoxicity. PMID- 12372045 TI - Hepatocellular carcinoma: strategy for optimizing surgical resection, transplantation and palliation. AB - In December 1997, a prospective study with informed consent was initiated to test a neoadjuvant treatment of transcatheter hepatic arterial chemo-embolization (TACE) and thermal or chemical ablation followed by transcatheter hepatic arterial chemo-infusion (TACI) in patients with hepatocellular carcinoma (HCC) referred for transplantation (OLT) and for resection. Patients were staged with American Liver Tumor Study Group-modified tumour-node-metastasis (TNM) staging classification using serial 3-6 month physical exam, alphafetoprotein (AFP), abdominal enhanced MRI, chest CT and bone scan. Sixty-five patients with HCC, out of 508 patients referred for OLT, were divided into five clinical groups and an incidental HCC patient group (n = 8), diagnosed on post-transplant explant pathology. The key focus of study was safety, site of HCC recurrence and tumour free survival. One hundred and thirty three ablation, infusion procedures were performed with an overall 24.8% morbidity, including two septic deaths. There were 13 (21.6%) HCC recurrences in 60 patients having one or more ablative treatments with only 23% hepatic HCC recurrences at 43 months of study. Eighteen HCC patients were listed for OLT (Group 3), with 12 patients transplanted after 29-424 d waiting. Two patients were removed from the OLT list due to HCC metastases, waiting a mean of 145 d. Two patients, post-OLT, had their TNM score upgraded from T2, T3 to T4. No Group 3 post-OLT patient has died or had HCC recurrence at mean follow-up of 27 +/- 15 months. No incidental HCC group post OLT patient has died or had HCC recurrence at mean follow-up of 24 +/- 14 months. This neoadjuvant protocol is safe and effective in reducing HCC recurrence prior to and after OLT and resection. PMID- 12372046 TI - Partial splenic embolization for hypersplenism before and after liver transplantation. AB - Partial splenic embolization (PSE) has been demonstrated to be an effective alternative to splenectomy for patients with hypersplenism. Splenectomy in these patients can be associated with an increased risk of perioperative complications, overwhelming post-splenectomy sepsis (OPSS) and mortality. Partial splenic embolization has the advantages of non- operative intervention and resolution of the complications of hypersplenism. We report the use of this technique in patients with portal hypertension and hypersplenism awaiting liver transplant and patients that have undergone othotopic liver transplantation (OLTx) with persistent hypersplenism post-transplant. Six patients--three awaiting liver transplantation and three patients with persistent hypersplenism status post-OLTx -were treated during the period of 1993-99 at the LSUHSC/Willis Knighton Regional Transplant Center in Shreveport, Louisiana. Three patients were male and three female. All six patients had concomitant thrombocytopenia and neutropenia with platelet counts below 50,000. Patients underwent selective arterial catheterization and embolization via a percutaneous approach with Cook microcoils or PVA particles. The lower pole of the spleen was selectively embolized in all patients to achieve a 30-50% reduction in flow as determined by angiography. Patients were followed with routine computed tomography (CT) scans, platelet and WBC counts for a mean of 26 months in the pre-transplant and 37 months in the post-transplant group. In both groups, all patients had persistent resolution of thrombocytopenia and neutropenia after embolization. In the post-transplant group, one patient had persistent splenomegaly and required splenectomy for pain control. No procedure-related complications occurred in any patient. In this limited review, PSE appears to be a safe and effective treatment of persistent hypersplenism in patients with portal hypertension and those who have undergone OLTx. PMID- 12372047 TI - One hundred consecutive living kidney donors: modern issues and outcomes. AB - In order to define current issues and outcomes of living kidney donation, 100 consecutive living donors operated on between July 1996 and March 2001 were evaluated. The 64 women and 36 men ranged in age from 19 to 72 yr (mean 42.5 yr), and 65 were related to the recipient while 35 were unrelated donors. Hospital admission the morning of surgery and use of a minimal open approach to the donor kidney were standard, as were post-operative epidural pain control and plans for short hospital stay. The 100 donors were hospitalized for 2 (25), 3 (48), 4 (18), 5 (8), or 6 (1) days, with an average length of stay of 3.12 d (range 2-6 d). The mean charge for kidney donor hospitalization was 14,470 dollars (range 9671 22,808 dollars). There were no major intra or immediate post-operative complications. Six rehospitalizations occurred for post-donation nausea, vomiting, dehydration (n = 2); spinal headache; pneumonia and wound haematoma; and late wound reexploration (one hernia and one nerve entrapment). All donors returned to pre-operative functional status within 6 d to 6 wk of donation. All kidneys functioned immediately in the 100 recipients (50 women, 50 men) who averaged 46.6 yr of age (range 17-69 yr); recipient length of stay averaged 3.81 d (range 2-15 d). All donors survived in excellent health; recipient graft and patient survival, respectively, are 87 and 90% through the entire 5-yr period. Excellent long-term outcomes for living kidney donors may be accomplished using minimal open surgical technique, post-operative epidural pain control and plans for a brief hospitalization. Expansion of living donor resources in renal transplant programs may grow as unrelated kidney donation and non-directed donation as well as minimally invasive (open and laparoscopic) techniques evolve. PMID- 12372048 TI - Pharmacogenomics: the future of drug therapy. AB - Pharmacogenomics aims to optimize patient management by customizing and synthesizing drugs based on genetic variations in drug response. Polymorphisms affecting metabolism, receptors, and absorption can influence drug sensitivity, toxicity, and dosing. The Human Genome Project, DNA chips, and bioinformatics advance the practice of this field by, respectively, identifying polymorphisms related to drug response, determining an individual's profile of polymorphisms, and integrating data to facilitate clinical decision making. Potential benefits of pharmacogenomics include increasing efficacy and preventing adverse drug reactions, thus improving patient care and decreasing costs. These factors imply that a thorough understanding of the principles and applications of pharmacogenomics will be an indispensable part of the future of drug therapy in clinical medicine. PMID- 12372052 TI - Hair as a diagnostic tool in dysmorphology. AB - Clinical diagnosis in dysmorphology is made by the recognition of a specific pattern of malformations and through an analytic search for discrete features. We present our personal experience regarding the usefulness of hair morphology as a tool for diagnosis in some metabolic and malformation syndromes. These cases represent only a few illustrative examples; an exhaustive review of the topic can be found elsewhere. PMID- 12372053 TI - A study of the influence of different genotypes on the physical and behavioral phenotypes of children and adults ascertained clinically as having PWS. AB - A population-based cohort of people with a clinical diagnosis of Prader-Willi syndrome (PWS) was genetically assessed using molecular diagnostic methods and subsequently divided into the following genetic subtypes involving chromosome 15: 'deletion', 'disomy' and genetically negative (referred to as 'PWS-like'). The physical and behavioral characteristics of the three groups were compared in order to evaluate the unique characteristics of the phenotype resulting from loss of expression of imprinted genes at 15q11q13 (PWS vs. PWS-like cases), the possible effect of either haploid insufficiency of non-imprinted genes (deletion cases), or gain of function of imprinted genes (disomy cases) located within the PWS critical region at 15q11q13. In this study, the main differences between probands with either a deletion or disomy are considered, and the possible involvement of contributing genes discussed. The differences within the PWS group proved difficult to quantify. It would appear that haploid insufficiency or gain of function are more subtle contributors than gender-specific genomic imprinting in the production of the PWS phenotype. PMID- 12372054 TI - Mutation analysis of the STK11/LKB1 gene and clinical characteristics of an Australian series of Peutz-Jeghers syndrome patients. AB - Peutz-Jeghers syndrome (PJS) is a rare cancer predisposition, which is characterized by the presence of hamartomatous polyposis and mucocutaneous pigmentation. A significant proportion of both familial and sporadic forms of this disorder are associated with mutations in the STK11 (serine/threonine kinase 11)/LKB1 gene. In this report we present a series of Australian PJS cases, which suggest that mutations in the STK11 gene do not account for many families or patients without a family history. The most likely explanation is either the presence of another susceptibility gene or genetic mosaicism in the non-familial patients. PMID- 12372055 TI - Acute intermittent porphyria in Sweden. Molecular, functional and clinical consequences of some new mutations found in the porphobilinogen deaminase gene. AB - Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficit of porphobilinogen deaminase (PBGD), the third of eight enzymes in the haem biosynthetic pathway. The overt disease is characterized by neuropsychiatric symptoms that are often triggered by exogenous factors such as certain drugs, stress, and alcohol. The aim of this work has been to identify the underlying genetic defect in each AIP-affected family in order to provide early counselling to assist in the avoidance of precipitating factors. The prevalence of AIP in Sweden is in the order of 1:10 000. The major mutation in Sweden, W198X, is due to a founder effect in the northern part of the country. This mutation, together with a further 11 mutations, have been reported previously. The present communication encompasses the great majority of AIP kindreds in Sweden and includes a further 27 mutations within the PBGD gene. This includes 14 completely new mutations, as well as 11 known mutations detected for the first time in Sweden. The majority of the mutations are located in exons 10 and 12 with fewer in exon 7. The clinical and biochemical outcomes in some patients are described. We also use the three-dimensional structure of the porphobilinogen deaminase enzyme to predict the possible molecular and functional consequences of the new Swedish missense and nonsense mutations. PMID- 12372056 TI - Germline mutations in the PTEN gene in Israeli patients with Bannayan-Riley Ruvalcaba syndrome and women with familial breast cancer. AB - Germline mutations in BRCA1 or BRCA2 account for the majority of inherited breast cancer cases. Yet, in up to 40% of familial breast cancer cases, no mutations can be detected in either gene. Germline mutations in PTEN underlie two inherited syndromes: Cowden disease (CD) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). The known association of CD with breast cancer risk made it plausible that germline mutations within PTEN may play a role in inherited predisposition to breast cancer. The nine coding exons of the PTEN gene were screened for harboring germline mutations using denaturing gradient gel electrophoresis (DGGE) complemented by sequencing, in two subsets of Israeli patients: 12 patients clinically diagnosed with BRRS, and 89 women with an apparent inherited predisposition to breast cancer, some with salient features of CD. Two of three familial BRRS patients exhibited novel germline mutations in PTEN: a missense mutation changing methionine to arginine at codon 134, and insertion of two nucleotides (CA) at cDNA position 1215 resulting in a frameshift at codon 61 and a premature stop at codon 99. Among 89 high-risk women, two missense mutations were detected in exon 4: A to C change at cDNA position 1279 resulting in a change of aspargine to threonine at codon 82 (N82T), and a G to an A alteration in 1269 which alters threonine to alanine at codon 78 (T78A), a non-conservative missense mutation. This study suggests that PTEN does not play a major role in predisposing to hereditary breast cancer in Israeli women, and that detection of PTEN mutations in BRRS patients is more likely in familial cases. PMID- 12372057 TI - The A1555G mtDNA mutation in Danish hearing-impaired patients: frequency and clinical signs. AB - The A1555G mutation of the mtDNA is associated with both aminoglycoside-induced and non-syndromic hearing loss. The A1555G is relatively frequent in the Spanish and some Asian populations, but has only been reported rarely in other populations, possibly because of ascertainment bias. We studied 85 Danish patients with varying degrees of hearing impairment and found two patients with the A1555G mutation (2.4%). Neither had received aminoglycosides. Our study indicates that the mutation might not be uncommon in Danish patients with hearing impairment. PMID- 12372059 TI - Homologous telomere association of 19q in a female with premature ovarian failure. AB - Premature ovarian failure (POF) may be due to a variety of genetic mechanisms. We report here, for the first time, telomere association of the long arms of chromosome 19, identified at low frequency (1%) in the peripheral blood cultures of a 30-year-old female with POF. Repeat cultures identified, in addition, the presence of 16q and 22q associations at a lower frequency (0.5%). These consistent observations are suggestive of a non-random event. Their association with POF may just be coincidental or may hypothetically explain it by an abnormal mechanism of chromosome separation, a constitutional telomere anomaly or an unidentified chromosome instability disorder. PMID- 12372058 TI - The novel R75Q mutation in the GJB2 gene causes autosomal dominant hearing loss and palmoplantar keratoderma in a Turkish family. AB - Dominant mutations in the GJB2 gene encoding connexin 26 (Cx26) can cause non syndromic hearing impairment alone or in association with palmoplantar keratoderma (PPK). We have identified the novel G224A (R75Q) mutation in the GJB2 gene in a four-generation family from Turkey with autosomal dominant inherited hearing impairment and PPK. The age of onset and progression of hearing loss were found to be variable among affected family members, but all of them had more severe impairment at higher hearing frequencies. Interestingly, the novel R75Q mutation affects the same amino acid residue as described recently in a small family (R75W) with profound prelingual hearing loss and PPK. However, the R75W mutation was also observed in a control individual without PPK and unknown hearing status. Therefore, the nature of the R75W mutation remains ambiguous. Our molecular findings provide further evidence for the importance of the conserved R75 in Cx26 for the physiological function of the inner ear and the epidermal cells of the skin. PMID- 12372060 TI - A new case of dup(3q) syndrome due to a pure duplication of 3qter. AB - The characteristic clinical features of the dup(3q) syndrome include typical facial features, mental and growth retardation, and (often) congenital heart anomalies. However, pure duplication of 3qter is rare because most of the reported cases are patients who carry an unbalanced translocation and, in addition to the duplication for 3qter, have a deletion for another chromosomal segment. A new case with a pure duplication of 3q detected in a 2-month-old boy is presented here. Extensive cytogenetic analysis revealed an inverted duplication of the distal part of 3q (chromosomal band 3q26.3 up to the telomere), with no (detectable) loss of the original telomeric sequences. Clinical evaluation revealed several phenotypic hallmarks characteristic for the dup(3q) syndrome. By comparing the duplicated region of this patient with the duplicated regions of the other patients with a pure duplication of 3q, we were able to localize the critical region for the dup(3q) phenotype to band 3q26.3. Alongside this new case with a pure duplication of 3q, an overview of six previous cases is given. PMID- 12372061 TI - Genetic and clinical analysis of spinocerebellar ataxia type 8 repeat expansion in Yugoslavia. AB - Spinocerebellar ataxia type 8 (SCA8) is a slowly progressive ataxia causally associated with untranslated CTG repeat expansion on chromosome 13q21. However, the role of the CTG repeat in SCA8 pathology is not yet well understood. Therefore, we studied the length of the SCA8 CTA/CTG expansions (combined repeats, CRs) in 115 patients with ataxia, 64 unrelated individuals with non triplet neuromuscular diseases, 70 unrelated patients with schizophrenia, and 125 healthy controls. Only one patient with apparently sporadic ataxia was identified with an expansion of 100 CRs. He had inherited the expansion from his asymptomatic father (140 CRs) and transmitted the mutation to his son (92 CRs). Paternal transmission in this family produced contractions of 40 and 8 CRs, respectively. None of the subjects from other studied groups had an expansion at the SCA8 locus. In the control group the number of CRs at the SCA8 locus ranged from 14 to 34. Our findings support the notion that allelic variants of the expansion mutation at the SCA8 locus can predispose to ataxia. PMID- 12372062 TI - Analysis of the two common alpha-1-antitrypsin deficiency alleles PiMS and PiMZ as modifiers of Pseudomonas aeruginosa susceptibility in cystic fibrosis. AB - Lung disease is the direct cause of death in more than 90% of cystic fibrosis (CF) patients. Proteinase-antiproteinase imbalances are common in CF and alpha-1 antitrypsin (AAT) deficiency. We investigated the hypothesis that the AAT deficiency alleles PiS and PiZ contribute to pulmonary prognosis in CF. Two hundred and sixty-nine CF patients from Southern Germany were included in this study. The serum concentrations of AAT and C-reactive protein (CRP) were determined by nephelometry, and patients were screened by polymerase chain reaction (PCR) and restriction enzyme digest for the common AAT deficiency alleles PiS and PiZ. The onset of chronic bacterial colonization by Pseudomonas aeruginosa (Pae) was correlated with the AAT phenotypes PiMM, PiMS and PiMZ. Only three out of nine CF patients (33%) diagnosed with either PiMS or PiMZ had developed chronic Pae lung infection earlier in their lives. The remaining six patients showing a PiMS or PiMZ phenotype showed a later onset of chronic Pae lung infection. Our results indicate that PiMS and PiMZ are not associated with worse pulmonary prognosis in CF. These data need to be confirmed in studies with a much larger number of cases. PMID- 12372063 TI - Association of angiotensin-converting-enzyme gene polymorphism with the depressor response to mild exercise therapy in patients with mild to moderate essential hypertension. AB - We studied the association of angiotensin I-converting enzyme (ACE) gene polymorphism with the depressor response to exercise therapy in 64 Japanese subjects with mild to moderate essential hypertension. Each subject performed 10 weeks of mild (lactate threshold intensity: approximately 50% maximum oxygen consumption) exercise therapy on a bicycle ergometer. Systolic blood pressure (SPB), diastolic blood pressure (DPB), and mean arterial pressure (MAP) were significantly decreased by exercise therapy in subjects with the ACE-II and ID genotypes but not in DD subjects. The time-by-genotype interaction effects were significant for DBP and MAP. According to a multiple logistic regression analysis, the age- and baseline plasma renin activity-adjusted relative risk (odds ratio) for the lack of a depressor response conferred by the D allele (assuming an additive effect) was 2.72 [95% confidence interval (CI), 1.07-6.91; p = 0.034]; for DD genotypes, as compared with the DI and II genotypes (assuming that the D allele is recessive), it was 11.7 (95% CI, 2.25-60.6; p = 0.003). ACE gene I/D polymorphism is associated with the depressor response of essential hypertensives to mild exercise therapy, which suggests that genetic features may underlie, at least in part, the heterogeneity of the depressor response in essential hypertensives to mild exercise therapy. PMID- 12372064 TI - A novel frameshift founder mutation in the cytochrome P450 1B1 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco. AB - Primary congenital glaucoma (PCG) is a heterogeneous autosomal recessive disorder caused by unknown developmental defect(s) of the anterior chamber of the eye. A member of the cytochrome P450 gene family, CYP1B1, was found to be mutated in PCG patients in different populations, albeit to a variable extent. In this study, CYP1B1 mutations were searched for in 32 unrelated PCG patients from Morocco. Two mutations were detected in 11 (34%) patients. One, 4339delG, is novel and causes a frameshift at residue 179. The other, G61E, was previously found in patients from Turkey and Saudi Arabia. Seven patients were homozygous for 4339delG and two other patients for G61E, whereas the two remaining patients were compound heterozygotes. The close association of 4339delG with a rare allele of D2S177, a microsatellite marker located 270 kb upstream of CYP1B1, strongly suggested a founder effect for 4339delG. The occurrence of this mutation was tentatively dated at between 900 and 1700 years ago. Typing 4339delG and G61E mutations should help to prevent blindness resulting from a delayed diagnosis of PCG in Morocco. PMID- 12372065 TI - A 4-Mb critical region for intrauterine growth retardation at 15q26. PMID- 12372075 TI - Sporotrichosis. AB - Sporotrichosis is a cutaneous fungal infection with a global distribution. The disease has several clinical forms, primarily cutaneous with associated lymphadenopathy. However, dissemination to osteoarticular structures and viscera may occur, both in healthy and immunosuppressed individuals; such disseminated forms usually follow spread after inhalation of fungal spores. Cutaneous infection is usually associated with trauma during the course of outdoor work, and treatment is required for the majority of patients. Potassium iodide (the treatment of choice in endemic areas) is an effective and inexpensive therapy; however, its adverse effects and complicated dosage regimen often weigh against its use in developed countries, where itraconazole is the antimycotic of choice. PMID- 12372076 TI - Management of patients taking anticoagulant, aspirin, non-steroidal anti inflammatory and other anti-platelet drugs undergoing dermatological surgery. AB - An increasing number of patients who require surgical treatment for skin tumours also take anticoagulants or anti-platelet drugs to prevent thromboembolic events. Stopping therapy places the patient at risk from a thromboembolic event, whilst continuing treatment places them at risk from bleeding complications during surgery. We have reviewed all of the published studies of the effects of warfarin, aspirin, non-steroidal anti-inflammatory and other anti-platelet drugs on surgical outcome in patients having skin surgical procedures. There is little evidence that continuing with treatment is harmful in any of these groups with the exception of warfarin therapy, where the risk of haemorrhagic complications is presumably dependent on the degree of anticoagulation. PMID- 12372077 TI - The accuracy of clinical diagnosis of suspected premalignant and malignant skin lesions in renal transplant recipients. AB - Renal transplant recipients have a greatly increased risk of nonmelanoma skin cancer. We investigated the accuracy of diagnosis of suspected skin malignancies in this population by prospectively recording all lesions detected in a specialist clinic over 5 months and comparing the provisional diagnosis with the histological diagnosis. The diagnostic accuracy was 54% in the 102 lesions that were biopsied, with the highest accuracy for the head and neck (67%). The diagnostic accuracy for squamous cell carcinoma was 48.7% (sensitivity 90.5%, specificity 75.3%) and for basal cell carcinoma 40.0% (sensitivity 66.6%, specificity 85.6%). The overall low accuracy rate implies the need for biopsy of any suspicious lesions in the renal transplant population. PMID- 12372078 TI - Laser treatment improves quality of life of hirsute females. AB - Hirsutism has a significant impact on the quality of life of affected patients. We report a prospective study of 45 hirsute females attending a laser clinic. Of these, 15 patients completed a pair of modified dermatology life quality index (DLQI) questionnaires, immediately before and at varying intervals (up to 6 months) after laser treatment. The mean DLQI score before treatment was 12.8 (median = 9.0, SD = 8.5). The mean DLQI score at 1-2 months was 7.0 (median = 2.5, SD = 10.0, P = 0.06), at 2-4 months it was 9.2 (median = 10.0, SD = 10.0, P = 0.48) and at 4-6 months it was 11.5 (median = 10.5, SD = 8.0, P = 0.88). There was a major improvement in DLQI score at 1-2 months but longer-term benefit was not observed. In a separate questionnaire, hirsute females (n = 45) reported a high level of patient satisfaction (71.1%) and willingness to have further treatment (77.8%) despite the fact that 97.1% had unwanted hair back at pretreatment levels at 6 months. PMID- 12372079 TI - Sun awareness and behaviour in healthcare professionals and the general public. AB - The knowledge, attitudes and behaviour of healthcare professionals and the general public towards issues of sun awareness were assessed in this questionnaire-based study. The findings indicated that junior doctors and nurses were no different from the general public when it came to seeking a tan or experiencing sunburn. The increased level of knowledge in doctors was not sufficient to have changed their behaviour and the medical profession should be targeted in education campaigns at an early stage in career development. PMID- 12372080 TI - Late onset vulvar steatocystoma multiplex. AB - Steatocystoma multiplex is a rare, autosomal dominant condition characterized by multiple cysts, usually arising on the trunk and proximal extremities at puberty. We present two unrelated women with sporadic steatocystoma multiplex strictly confined to the vulva and arising at an older age. These cases represent an unusual localized form of the disorder. PMID- 12372081 TI - A case of childhood sarcoidosis. AB - Cutaneous sarcoidosis is rare in children. We report a case of a 5-year-old Bangladeshi girl who presented with fever, a papular eruption on the lower limbs and trunk, malaise, anorexia and weight loss. There was multisystem involvement with marked hepatosplenomegaly, generalized lymphadenopathy, parotid fullness and chronic uveitis. Pulmonary infiltrates were seen on the chest X-ray. Histology of a skin biopsy showed naked noncaseating granulomata and PCR for Mycobacterium tuberculosis was negative. A clinical diagnosis of sarcoidosis was made. The patient was treated with oral prednisolone (2 mg/kg per day). An excellent clinical response with resolution of the rash and improvement of extracutaneous signs was noted within 3 months and she remains well on low-dose prednisolone on alternate days. We discuss the presentation and management of sarcoidosis in children, and highlight the potential difficulty in differentiating this from disseminated tuberculosis. PMID- 12372082 TI - Leg ulcers in active lepromatous leprosy associated with cryoglobulinaemia. AB - A 40-year-old male agricultural labourer presented with active lepromatous leprosy and painful leg ulcers of 2 months' duration. Biopsy from the ulcer showed nonspecific changes. Raised erythrocyte sedimentation rate and positive rheumatoid factor made us suspect underlying cryoglobulinaemia. Presence of cryoprecipitate in the serum, demonstration of cryoglobulins by serum electrophoresis and raised cryocrit were compatible with cryoglobulinaemia as the cause of atypical leg ulcers in this case. The ulcers healed with bed rest, aspirin and specific anti-leprosy treatment. Though 95% of lepromatous leprosy patients can have cryoglobulinaemia, the presence of atypical ulcers as seen in our patient has not previously been related to the presence of cryoglobulinaemia. PMID- 12372083 TI - Papular elastorrhexis: a distinct variant of connective tissue nevi or an incomplete form of Buschke-Ollendorff syndrome? AB - Papular elastorrhexis is a rare entity, possibly a form of multiple elastic tissue naevi. The cutaneous lesions in this disorder are characterized by multiple white papules usually occurring on the trunk. These tend to be nonfollicular and scattered evenly over the affected area. Histopathologically, there is a decrease of elastic fibres, that may also appear thin and fragmented. Most reported cases are sporadic but familial occurrence has been described and some authors believe that papular elastorrhexis may represent a mild form of Buschke-Ollendorff syndrome. We report an 18-year-old woman whose clinical and histopathological features were compatible with papular elastorrhexis. There was no evidence of skeletal changes or relevant family history. PMID- 12372084 TI - Disappearance of pili annulati following an episode of alopecia areata. AB - Pili annulati is a distinctive autosomal dominant hair shaft disorder that produces alternating light and dark bands that can give a spangled appearance to the hair. The literature contains three case reports of patients in whom the condition has disappeared following recovery from alopecia totalis. None of these reports contain a direct microscopic comparison of pre- and post-regrowth hairs. We report a 6-year-old girl who was first noted to have pili annulati at the age of 2 years and who developed alopecia totalis at the age of 3 years. When the hair regrew spontaneously, 18 months later, the pili annulati was no longer visible. Hair samples obtained before and after the episode of alopecia areata were compared by normal and cross-polarized light microscopy. While not apparent on careful clinical examination, banding was present on light microscopy in 20% of the hairs. Eighty per cent of the affected hairs displayed banding throughout their entire length. In contrast, prior to the episode of alopecia totalis, when the pili annulati was clearly visible, 50% of the hair obtained was banded on microscopy and 90% of the affected hairs showed banding throughout their microscopic length. PMID- 12372085 TI - Tattoo ink darkening of a yellow tattoo after Q-switched laser treatment. AB - The popularity of tattoos is burgeoning with 20-30 million tattooed individuals in the Western World. Requests for removal can be expected to rise concurrently with increased applications. Laser removal of tattoos is potentially a more cosmetically acceptable method of removing tattoos than surgical excision or dermabrasion. Nevertheless, complications and side-effects can result from laser treatment and include scarring, hypopigmentation, hyperpigmentation, partial removal, infection, bleeding and tattoo ink darkening. The latter has been reported for flesh-toned and red tattoos. Such a complication has never been reported for the laser treatment of a yellow tattoo in the dermatological literature. We describe a case of tattoo ink darkening of a yellow tattoo after treatment with the 532 nm quality-switched Neodymium : Ytrrium-Aluminium Garnet laser to highlight clinicopathological features. The mechanism by which some tattoos darken after laser treatment is not clearly understood. We review darkening of tattoos after laser treatment to raise awareness of this important complication. This paper will help to facilitate discussions with the patient and in obtaining informed consent prior to commencing treatment. Tattoo ink darkening of a yellow tattoo adds to the growing list of complications resulting from attempts at tattoo removal. PMID- 12372086 TI - Fixed drug eruption following metronidazole therapy and the use of topical provocation testing in diagnosis. AB - Fixed drug eruption is characterized by recurrent well-defined lesions appearing in the same location each time the drug responsible is taken. A number of agents have been implicated. Metronidazole, a nitroimidazole agent widely used for its antibacterial and antiprotozoal activity, has been reported only rarely as the causative agent. We describe a patient with FDE due to metronidazole in whom we were able to induce the clinical and histological features of FDE by topical provocation testing. In agreement with the published literature we commend the use of topical provocation testing as a possible first-line investigation in the diagnosis of FDE. This may avoid the need for subsequent oral provocation testing and therefore the prevention of possible adverse sequelae. PMID- 12372087 TI - Linear oro-facial lichen sclerosus. AB - Lichen sclerosus is a depigmenting mucocutaneous disorder that most frequently affects the female genitalia. Lichen sclerosus affecting the oral mucosa is extremely rare. Oral lesions are asymptomatic but cosmetically unacceptable. We report here a case of lichen sclerosus presenting with a linear lesion over the nose that extended to involve the philtrum and the upper lip with intraoral extension up to the gingiva. Treatment with a short course of oral and intralesional corticosteroids resulted in partial resolution of the lesions. PMID- 12372088 TI - Haemorrhage into chronic plaque psoriasis as a consequence of disseminated intravascular coagulation. AB - We describe a patient with chronic plaque psoriasis who developed haemorrhage into pre-existing lesions during an episode of disseminated intravascular coagulation secondary to sepsis. Disseminated intravascular coagulation is a complex disorder characterized by widespread intravascular deposition of fibrin with consumption of coagulation factors and platelets and occurs as a consequence of many disorders that release procoagulant material into the circulation or cause widespread endothelial damage or platelet aggregation. As both disseminated intravascular coagulation and psoriasis occur relatively frequently in the general population we were surprised to find no previous reports of this phenomenon in the literature. PMID- 12372089 TI - Subacute cutaneous lupus erythematosus associated with phenytoin. AB - Subacute cutaneous lupus erythematosus is a well recognized subset of systemic lupus erythematosus. It is characterized by a nonscarring, papulosquamous or annular eruption in a photosensitive distribution. Several cases, thought to be caused by drugs, have been reported. We report a case of subacute cutaneous lupus erythematosus caused by phenytoin, which has not previously been associated with this condition. PMID- 12372090 TI - Dyschromatosis universalis hereditaria. AB - Dyschromatosis universalis hereditaria is a clinically heterogenous disorder. We report two unrelated Indian patients with dyschromatosis universalis hereditaria, who had generalized and progressive reticulate hyper- and hypo-pigmentation of the skin. The oral mucosa and tongue also showed mottled pigmentation. Intriguingly, the palms and soles were also affected with a diffuse hyper pigmentation interspersed with spotty de-pigmented macules. Dystrophic nail changes with pterygium formation were seen in one case. Histopathology revealed a variable degree of pigmentary incontinence. Although the precise aetiology of this disorder is not yet known, the clinicopathological findings implicate an inherent abnormality of melanosomes or melanin processing. PMID- 12372091 TI - Direct injection of naked DNA and cytokine transgene expression: implications for keratinocyte gene therapy. AB - Intradermally injected DNA diffuses into the epidermis and can then enter keratinocytes and become expressed by these cells. Using this method, plasmids containing cytokine genes that have been introduced into keratinocytes can induce a level of cytokine expression sufficient to provide biological effects in the treated skin. Furthermore, transgenic cytokines released from the transduced keratinocytes can also enter the circulation and have downstream effects on other target organs. Thus far, naked DNA injection appears to be a safe, simple, and relatively efficient method that enables genes to be expressed in transplanted human skin on immunosuppressed animals. In humans, keratinocyte gene therapy using the cytokine gene DNA injection method has the potential to become a powerful therapeutic tool for dermatologists in the management of certain inflammatory and other dermatoses. PMID- 12372092 TI - Dermatological applications of DNA array technology. AB - The Human Genome Project and other large scale sequencing consortia continue to generate huge amounts of DNA sequence data. Despite the identification of specific disease-related genes and genetic markers, we still appear to know little about how diverse gene products actually interact with each other or respond to other chemical or biological stimuli. Such information is of course fundamental to understanding complex disease pathways and biochemical processes and, as such, has spawned new fields of investigative genetics, that of functional genomics and proteomics. DNA array technology is emerging as a powerful, high-throughput and versatile tool that can be applied to the study of functional genomics. This article reviews the methodology involved in array analysis and provides insight into how, as an investigative tool, DNA arrays are becoming increasingly useful in understanding fundamental abnormalities in dermatological disease and also in refining the management of patients with certain skin disorders. PMID- 12372093 TI - Pain caused by photodynamic therapy of skin cancer. AB - Pain resulting from photodynamic therapy (PDT) of skin cancer was investigated. The study included 69 lesions (60 patients) with different types of skin tumours or precursors. Protoporphyrin IX, which is produced by the topical application of delta-aminolevulinic acid, was used as a photosensitizing agent. Twenty-three of the lesions (19 patients) were examined with a fluorescence imaging system which demarcates the tumour area from the healthy skin and visualizes the contrast between the fluorescence from healthy skin and that from the tumour. EMLA is used on all patients as part of our routine PDT protocol but despite this the major side-effect of PDT is pain during treatment. There is a large variation in pain intensity experienced by the patients, as measured by a visual analogue scale (VAS). Patients with actinic keratoses experienced more pain than those with Bowen's disease or basal cell carcinoma. The mean VAS score was higher when treating lesions located on the head than when treating lesions on the torso or the extremities. Also, treatment of large skin areas resulted in more pain than treatment of small areas, and men experienced more pain than women. The pain experienced by the patients did not correlate with treatment dose, Fitzpatrick skin type, age or fluorescence intensity. PMID- 12372094 TI - Association of class I HLA antigens with the clinical manifestations of Turkish patients with Behcet's disease. AB - Genetic factors appear to be important in the pathogenesis of Behcet's disease. Although it is known to be strongly associated with HLA-B 51, the association of HLA class I antigens with specific clinical findings of the disease has not been studied extensively and the few studies are conflicting. The aim of this study was to investigate the association of HLA class I alleles with the manifestations of Behcet's disease in Turkish patients. Eighty-five patients with Behcet's disease were typed for HLA-A, B, and C antigens with the serologic, standard microlymphocytotoxicity technique. Possible associations of the HLA complex with clinical findings of Behcet's disease were examined. Statistically significant findings are as follows (P < 0.05): increased HLA-B 51 and decreased HLA-B35 frequency in patients with thrombophlebitis, increased HLA-A29 and decreased HLA Bw6 frequency in patients with ocular involvement, decreased HLA-Cw2 frequency in patients with erythema nodosum, and decreased HLA-Cw 7 frequency in patients with genital ulceration. Of particular note, the results of this study suggest that the presence of HLA-B 51 and the absence of HLA-B35 can be regarded as laboratory risk factors of venous thrombosis in patients with Behcet's disease. PMID- 12372095 TI - Urinary N-methylhistamine as an indicator of bone marrow involvement in mastocytosis. AB - Thirty-seven patients with mastocytosis and unexplained elevated levels of urinary N-methylhistamine who were undergoing bone marrow biopsy were studied with respect to the diagnosis of mastocytosis and the manifestations of the disease. These patients were from a group of 66 patients from whom a bone marrow biopsy was obtained and urinary N-methylhistamine levels were measured in the period 1990-1998. In seven (19%) of the 37 patients, mastocytosis was limited to the skin. Five (14%) of the 37 patients showed accumulation of mast cells in the bone marrow without characteristic skin lesions, whereas seven (19%) of the 37 patients showed increased numbers of mast cells both in the skin and the bone marrow. Eighteen (49%) of the 37 patients with elevated N-methylhistamine did not have mast cell accumulation in either the skin or the bone marrow biopsy. The median level of N-methylhistamine in the urine of patients with mastocytosis limited to the skin was 245 micro mol/mol creatinine. The average level of N methylhistamine was 509 micro mol/mol creatinine in patients with mast cell accumulation in the bone marrow and cutaneous mastocytosis. There was a significant difference in the levels of N-methylhistamine in patients with mast cell accumulation in the bone marrow biopsy compared with those without. The likelihood of mastocytosis with mast cell accumulation in the bone marrow biopsy at a given level of N-methylhistamine was calculated. It was established that an N-methylhistamine level of 297 micro mol/mol creatinine or higher may be considered as a threshold indicator for obtaining a bone marrow biopsy in patients suspected of mastocytosis with mast cell accumulation in the bone marrow. For practical purposes, we propose to consider the cut-off level of approximately 300 micro mol/mol N-methylhistamine creatinine for this assay. PMID- 12372096 TI - Effects of antioxidants on pyridinoline cross-link formation in culture supernatants of fibroblasts from normal skin and hypertrophic scars. AB - Free radicals are normally generated in many metabolic pathways. They are closely associated with inflammatory diseases. The aim of this study was to investigate the effects of free radicals and their antioxidants on the formation of pyridinoline using human fibroblasts from normal skin and hypertrophic scars. The significance of the increase in pyridinoline cross-links is that large quantities have been found in hypertrophic scars formed post-burn than in normal skin, and that catalase was effective in reducing the pyridinoline cross-link formation in hypertrophic scars. The pyridinoline cross-link concentration expressed in nM/ micro g hydroxyl proline was found to be higher in the culture supernatants of the fibroblasts from hypertrophic scars (9.04 +/- 2.74) than that of normal skin (7.55 +/- 2.1). When the human fibroblasts from normal skin and hypertrophic scar were subject to hydroxyl radicals generated by the Fenton reaction, there was no significant increase in pyridinoline cross-link concentration (nm/ micro g hydroxyl proline) in the supernatants compared with the control. When the controls plus various treatments with free radicals were subject to different antioxidants, including superoxide dismutase, catalase, glutathione peroxidase, and desferrioxamine, it was found that catalase alone was most effective in scavenging hydroxyl radicals as determined by the decrease in pyridinoline cross links. PMID- 12372097 TI - Aloesin inhibits hyperpigmentation induced by UV radiation. AB - Skin hyperpigmentation is caused by the overproduction of melanin pigment, which is synthesized by the action of tyrosinase. We recently reported that aloesin inhibits tyrosinase activity. The present study was undertaken to test the inhibitory effect of aloesin on pigmentation in human skin after UV radiation. Experimental subjects were UV-irradiated (210 mJ) on the inner forearm. UV irradiated regions were assigned to four groups: vehicle control, aloesin treated, arbutin treated, and aloesin and arbutin treated. Aloesin and/or arbutin were administered four times a day for 15 days. Aloesin treatment suppressed pigmentation by 34%, arbutin by 43.5%, and the cotreatment by 63.3% compared with the control (n = 15; P < 0.05). Moreover, aloesin treatment showed pigmentation suppression in a dose-dependent manner (n = 7; P < 0.05). These results raise the possibility that aloesin may be used as an agent that inhibits melanin formation induced by UV radiation. PMID- 12372098 TI - Topical 5-ALA photodynamic therapy for the treatment of cutaneous T-cell lymphoma. AB - Therapeutic options for cutaneous T cell lymphoma (CTCL) include topical steroids, topical chemotherapy and phototherapy. Patients with limited disease that is unresponsive to these therapies present a particular challenge. We report successful treatment of a patient with two plaques of CTCL using topical photodynamic therapy (PDT). 5-aminolaevulinic acid (5-ALA) was applied 6-24 h preillumination with 100 J/cm2 red light. Treatment was repeated on four occasions with clinical and histological clearance. ALA-PDT may be a useful addition to the therapeutic options for CTCL. Further studies are required to define optimal treatment protocols. PMID- 12372099 TI - Audit of admissions to dermatology beds in Greater Manchester. AB - We present the results of a prospective questionnaire-based audit of admissions to dermatology beds. We examined the admission practices of clinicians and the outcome in terms of benefit to patients. The majority of patients (90%) were admitted because of the severity of their skin disease but other contributing factors included: inability to cope (40%); need for further investigation or observation (33%); coexisting medical factors (17%); poor social support; transport and psychological factors. Most (87%) patients benefited from admission and the dermatology life quality index improved by 42%. We demonstrate that inpatient treatment is effective and improves patients' quality of life. PMID- 12372100 TI - E. coli cellulitis. PMID- 12372101 TI - Baboon syndrome following oral roxithromycin. PMID- 12372102 TI - Gabapentin -- a promising treatment in glossodynia. PMID- 12372103 TI - Allotropic cutaneous amyloidosis: coincidence or progression? PMID- 12372104 TI - Methotrexate is of limited value in the treatment of hidradenitis suppurativa. PMID- 12372107 TI - Case 1: lipoid proteinosis (hyalinosis cutis et mucosae; Urbach-Wiethe disease). PMID- 12372108 TI - Case 2: fibrous hamartoma of infancy. PMID- 12372109 TI - Case 3: vulval crohn's disease (VCD). PMID- 12372110 TI - A memorable patient - A nasty surprise. PMID- 12372111 TI - Prolonged remissions of cystic acne and conglobate acne with 13-cis-retinoic acid Peck GL, Olsen TG, Yoder FW et al. PMID- 12372114 TI - Outdoor air pollution, climate and allergic respiratory diseases: evidence of a link. PMID- 12372115 TI - ST2: marker, activator and regulator of Th2 immunity? PMID- 12372116 TI - MAPPIT: a cytokine receptor-based two-hybrid method in mammalian cells. AB - Identifying novel targets for therapy in allergic disease: protein interactions inside the cell Therapy of allergic disease currently relies on pharmacological manipulation of mediators or immunotherapy. Drugs have been developed to target specific mediators and their receptors: for example antihistamines blocking the H1 receptor have been refined to maximize antagonism and reduce central side effects or adverse effects of activity on other receptors such as muscarinic cholinergic receptors. Traditional pharmacological approaches identify new surface receptors against which chemists will then design or screen compounds for activity: examples are H3 or H4 histamine receptors. With the advent of the sequenced human genome we are faced with a vast array of genes and proteins that interact to define normal physiology or indeed pathology. A major challenge to biotechnology is to evolve novel techniques to understand the function and interaction of these myriad proteins. One particular area of current interest is the signalling cascades downstream of surface receptors. For many years pathways have appeared overlapping and to offer little chance of specific intervention. However, greater understanding of the complexity and integration of signalling, together with the possibility of directing drugs to specific cells has aroused considerable interest in this area for novel therapeutics. Indeed, targeting events within the cell has been done for many years with steroids. Here, Jan Tavernier and colleagues describe some signalling pathways relevant to allergic disease and potential methods for understanding protein interactions that allow mapping of the cascades. In particular they describe an elegant new system of analysis of protein-protein interactions in a mammalian system, which they have developed, termed MAPPIT. The basis of the system is an engineered receptor with JAK kinase but which lacks STAT activation sites. To the cytoplasmic end of the receptor is added a bait protein of interest, and the cell line can then be transduced with plasmid containing 'prey' cDNA from a library of interest linked to an active STAT binding site. If this cDNA encodes a protein which, upon expression, is activated and recruited to the membrane complex, it will bind to the receptor via the bait, then STAT activation will occur and activate a reporter gene system such as luciferase or puromycin resistance. This novel system allows study of known protein-protein interactions by targeted mutagenesis, or screening for novel interactions. It has the advantage over existing systems such as yeast 2 hybrid that it uses mammalian cells and thus can reproduce the physiological conditions for protein processing or activation. As new genes and proteins are linked to the atopic phenotypes, systems such as this hold promise of rapidly defining their function and interacting proteins and may be important in linking genomics and proteomics with function and pharmacology in the future. PMID- 12372117 TI - The impact of climate and traffic-related NO2 on the prevalence of asthma and allergic rhinitis in Italy. AB - BACKGROUND: Environmental factors are likely to be involved in explaining the wide geographical variation in asthma and atopic diseases that has been documented in many recent epidemiological studies. AIM: To evaluate to what extent climate and outdoor NO2 pollution can explain the geographical variation in the prevalence of asthma and allergic rhinitis, and to estimate the relative risk for exposure to different levels of these two factors. METHODS: The impact of climate and long-term exposure to nitrogen dioxide (NO2) pollution on asthma and allergic rhinitis was assessed in a cross-sectional study, carried out during 1998 to 2000 on young adults aged 20 to 44 years (n = 18 873), living in 13 areas from two different Italian climatic regions (subcontinental and Mediterranean). RESULTS: Mediterranean areas had a significantly higher prevalence of asthma-like symptoms (P < 0.001), higher annual mean temperature (16.2 degrees C vs. 12.9 degrees C), lower temperature range (16.0 C degrees vs. 22.1 degrees C) and lower NO2 levels (31.46 microg/m3 vs. 57.99 microg/m3) than subcontinental ones. Mediterranean climate was associated with an increased risk of wheeze (OR = 1.23; 95% CI 1.13 to 1.35), tightness in the chest (OR = 1.21; 95% CI 1.11 to 1.33), shortness of breath (OR = 1.21; 95% CI 1.08 to 1.36) and asthma attacks (OR = 1.19; 95% CI 1.07 to 1.31). After adjusting for climate, an increase of 18.3 microg/m3 in NO2 levels moderately increased the risk of asthma attacks (OR = 1.13; 95% CI 0.98 to 1.32), tightness in the chest (OR = 1.11; 95% CI 0.98 to 1.26) and wheeze (OR = 1.11; 95% CI 0.96 to 1.28). When the levels of outdoor NO2 exposure rose, the prevalence of allergic rhinitis increased significantly in the Mediterranean region (OR = 1.38; 95% CI 1.12 to 1.69), but not in the subcontinental one (OR = 1.03; 95% CI 0.83 to 1.28). CONCLUSION: Our results show that the prevalence of asthma increases when annual mean temperature increases and temperature range decreases. Furthermore, climate interacts with NO2 outdoor exposure, increasing the risk for allergic rhinitis in people exposed to high stable temperatures. A long-term role for the effect of traffic pollution on asthma is also suggested. PMID- 12372118 TI - Household characteristics and mite allergen levels in Manchester,UK. AB - BACKGROUND: Mite allergen levels vary enormously between different homes in the same geographical area. No large scale studies of mite levels in Manchester homes has been conducted to identify factors associated with higher levels. OBJECTIVES: To quantify exposure to mite allergens and to identify characteristics associated with higher Der p 1 levels in a large sample of homes in Manchester, UK. METHODS: Der p 1 was measured in dust from the living room floor, sofa, bedroom floor and mattress in 564 homes. Data on household characteristics were collected by administering a questionnaire. Univariate and multivariate analyses were performed to identify household characteristics associated with higher mite allergen levels. RESULTS: Der p 1 levels were highest in the mattress (GM 1.19 microg/g, 95% CI 0.98-1.45, P < 0.001). Two-thirds of homes contained Der p 1 levels > 2 microg/g in at least one dust reservoir, and 40.3% contained Der p 1 greater than 10 microg/g. There was a large range in Der p 1 levels between homes (> 10(3)-fold). Damp and condensation were common findings in homes. In the multivariate analyses, factors associated with higher Der p 1 levels in more than one dust reservoir were older homes, older living room carpets, damp, condensation and mixed glazing. Older mattresses were associated with higher mite allergen levels in the mattress where the age of the mattress was recorded. Twenty-four homes contained no detectable mite allergen, six of which reported damp. CONCLUSIONS: Mite allergen levels are high enough in two of every three homes to be associated with an increase in the risk of sensitization to mite. Factors such as older homes, carpets and mattresses, damp and condensation are associated with higher mite allergen. However, mite allergen levels are occasionally unpredictably very low in homes with risk factors for high levels. PMID- 12372119 TI - Atopic allergy and delayed type hypersensitivity in Estonian children. AB - BACKGROUND: A shift in the balance ofT helper (Th) cell subsets towards a polarized Th2 population is generally accepted to occur in atopic disease, however, both Th1 and Th2 disorders have increased over the past decades in Western communities. OBJECTIVE: The aim of our study was to investigate delayed type hypersensitivity (DTH) response in atopic and non-atopic children in a population with a low prevalence of allergic disorders. METHODS: Skin prick tests (SPT) were performed with fresh egg white and extracts of five inhalant allergens, i.e. cat, dog, house dust mite (Dermatophagoides pteronyssinus), birch and timothy, and DTH response was evaluated by Multitest CMI in 72 Estonian 4- to 6-year-old children. RESULTS: The frequency of response to diphtheria was significantly increased in SPT-positive children (55% vs. 26%, chi2 = 5.5; P = 0.038). The induration to diphtheria (2.4 +/- 0.5 vs. 0.9 +/- 0.2 mm; P = 0.004), and tetanus (3.5 +/- 0.6 vs. 2.1 +/- 0.3 mm; P = 0.025) was significantly greater in the SPT-positive children. The cumulative size of induration in the positive DTH tests was significantly greater in the SPT-positive children (9.0 +/- 1.2 vs. 5.2 +/- 0.6 mm, P = 0.01). CONCLUSION: In this group of children our findings do not support the hypothesis of an immune deviation with decreased Th1 and increased Th2 responses leading to atopic disease, but rather a process of immune modulation whereby both Th1 and Th2 responses are increased in atopic subjects. PMID- 12372120 TI - Prevalence of symptoms, sensitization to rats, and airborne exposure to major rat allergen (Rat n 1) and to endotoxin in rat-exposed workers: a cross-sectional study. AB - OBJECTIVE: To analyse the relation between airborne exposure to major rat allergen and to endotoxins in exclusively rat-exposed workers and the prevalence of rat-related symptoms and sensitization. METHODS: A total of 113 workers answered a standardized questionnaire on their atopy status, occupational exposure to rats, and possible work-related symptoms. Specific IgE against rat urinary proteins (RUP) was measured for 73 subjects. Individual airborne exposure to Rat n 1 and endotoxin were determined with static (n = 256) samplings. Rat n 1 was measured with enzyme-linked immunosorbent assay (ELISA) and endotoxin by the Limulus method. RESULTS: Forty-four of 113 subjects (38.9%) reported at least one rat-related symptom: asthma (4.4%), rhinitis (34%) and conjunctivitis (16%). Twelve per cent were sensitized to RUP (specific IgE > 0.35 KU/L). But only 30.8% of all symptomatic subjects were sensitized to rat allergens. Airborne Rat n 1 levels were not related to symptoms in workers. Symptomatic patients not sensitized to rats were exposed to higher endotoxin levels, but airborne exposure to endotoxins did not significantly protect against or increase sensitization to RUP or rat-related symptoms. CONCLUSION: Most symptomatic workers were not sensitized to rat allergen; but no significant relation between rat-related symptoms and endotoxin levels was found. This suggests that more studies are needed to determine causes other than rat allergens or endotoxins that may be responsible for symptoms in rat-exposed workers. PMID- 12372121 TI - Severe respiratory syncytial virus infection in early life is associated with increased type 2 cytokine production in Gambian children. AB - BACKGROUND: Severe respiratory syncytial virus (RSV) infection in early childhood has been associated with subsequent wheezing and atopy. The aim of this study was to test if severe RSV infection in early life was associated with an increase in type 2 cytokine production and atopy in Gambian children 5 years later. METHODS: A cohort of children with severe RSV infection during the first year of life ('cases', n = 66) and without ('controls', n = 122) was followed-up at 5 years of age. Immediate hypersensitivity to common allergens, airway reactivity, serum IgE concentration and the production of IFN-gamma, IL-5 and IL-13 by lymphocytes activated in vitro with RSV F-G or control antigens was determined. RESULTS: After adjustment for confounders, cases produced significantly higher concentrations of IL-13 in response to RSV F-G and of IL-5 and IL-13 in response to tuberculin. Cases were more likely to have presented with a wheezy lower respiratory tract infection in the first 3 years of life (adjusted odds ratio = 9.9; 95% CI 1.6-61.0), but not thereafter. Cases and controls had similar skin response to allergens, airway reactivity and serum IgE concentrations. CONCLUSION: Severe RSV infection in early life is associated with a higher production of type 2 cytokines in Gambian children at 5 years of age. However this does not appear to result in increased risk of atopy or clinical allergy at that age. PMID- 12372122 TI - Atypical nasal challenges in patients with idiopathic rhinitis: more evidence for the existence of allergy in the absence of atopy? AB - BACKGROUND: The pathophysiology of idiopathic rhinitis is unknown although evidence is accumulating to suggest that many patients may have a localized form of allergic rhinitis in the absence of other atopic symptoms and markers. This study compares detailed nasal challenge results obtained from patients with idiopathic rhinitis to those of atopic and normal controls. METHODS: Patients with idiopathic rhinitis (n = 23), perennial allergic rhinitis (n = 8) and normal controls (n = 8) underwent a normal saline challenge to exclude hyper-reactivity and then bilateral nasal allergen challenges. Nasal patency was assessed by anterior active rhinomanometry. RESULTS: All of the patients with atopic rhinitis demonstrated positive bilateral allergen challenges. All normal control subjects had bilateral negative challenges. Two patients in the idiopathic group tested positively to saline and were excluded from further study with 62% of the remainder testing positive to allergens. Of the idiopathic patients testing positive, 85% were sensitive to house dust mite. CONCLUSION: A significant proportion of patients with idiopathic rhinitis have positive nasal challenges, the vast majority to house dust mite allergen. These findings add to the weight of evidence that suggests 'localized allergy' may exist in the absence of systemic atopic markers. PMID- 12372123 TI - Lower airway inflammatory responses to repeated very-low-dose allergen challenge in allergic rhinitis and asthma. AB - BACKGROUND: Low-dose allergen challenge (LDAC) may be a useful tool for studying the capacity of allergens to induce airway inflammation in atopic subjects. OBJECTIVE: To evaluate lower airway inflammatory changes following repeated inhalation of very low doses of allergen (VLDAC) in non-asthmatic subjects with allergic rhinitis (NAAR) compared with mild allergic asthmatic subjects (AA). METHODS: Fourteen NAAR and 11 AA were seen out of the pollen season and had skin prick tests with common aeroallergens. Baseline spirometry (S) and methacholine challenge (MC) were done and blood and induced sputum (IS) differential cell counts were obtained. Each subject underwent VLDAC on four consecutive mornings with a relevant allergen. S, MC, and blood and IS samplings were repeated 6 h after the second and fourth VLDAC and one week later. RESULTS: Although there were, as expected, no changes in FEV1 or PC20 in either group, mean percentage eosinophils on IS were significantly increased in NAAR on day 2 of VLDAC and decreased in all but one subject on day 4, with a tendency to return to baseline levels one week later. In AA, there was a non-significant trend for sputum eosinophils to increase on day 2; four subjects showed a decrease of eosinophils on day 4 of VLDAC. There was a correlation between eosinophil cationic protein (ECP) levels and eosinophil counts in NAAR throughout the study. There were no variations in other sputum cells or blood inflammatory cells. CONCLUSION: VLDAC can increase the percentage of eosinophils in IS of NAAR subjects without associated respiratory symptoms nor physiological modifications. A reduction in eosinophilic response despite repeated exposure, more common in NAAR subjects, suggests an adaptation process that needs to be further evaluated. PMID- 12372124 TI - Nasal challenges with recombinant derivatives of the major birch pollen allergen Bet v 1 induce fewer symptoms and lower mediator release than rBet v 1 wild-type in patients with allergic rhinitis. AB - BACKGROUND: Genetic engineering of the major birch pollen allergen (Bet v 1) has led to the generation of recombinant Bet v 1 derivatives with markedly reduced IgE-binding capacity, but with retained T cell activating ability. OBJECTIVE: To compare the mucosal reactivity to rBet v 1 derivatives with rBet v 1 wild-type as basis for new therapeutic strategies for birch pollen allergy based on mucosal tolerance induction. METHODS: Outside the pollen season, 10 patients with birch pollen allergic rhinitis and mild asthma underwent four nasal challenge-sessions in a randomized, double-blind, and cross-over design, employing increasing doses of rBet v 1 fragment mix, rBet v 1 trimer, rBet v 1 wild-type and diluent (albumin). Nasal lavage fluids (NAL) were collected before the challenge-series as well as 10 min, 4 and 24 h thereafter. Nasal lavage fluid levels of tryptase as well as EPO and ECP were measured as indices of mast cell and eosinophil activity, respectively. RESULTS: All 10 patients tolerated the highest accumulated dose, 8.124 microg, when challenged with rBet v 1 trimer, eight with rBet v 1 fragments compared to one when challenged with rBet v 1 wild-type. No late phase reactions were observed. The change in tryptase levels (pre-challenge vs. 10 min) was significantly lower after challenges with rBet v 1 trimer and rBet v 1 fragments than with rBet v 1 wild-type. The change in EPO/ECP concentration pre-challenge versus 4 h post-challenge was lower for rBet v 1 trimer and the change was significantly lower when pre-challenge versus 24 h post challenge to rBet v 1 fragments and rBet v 1 wild-type was examined. CONCLUSION: The derivatives induced significantly fewer symptoms and lower mast cell and eosinophil activation than rBet v 1 wild-type upon application to the nasal mucosa. They could in the future be candidates for immunotherapy based on mucosal tolerance induction. PMID- 12372125 TI - Threshold levels of purified natural Bos d 2 for inducing bronchial airway response in asthmatic patients. AB - BACKGROUND: Provocation tests are invaluable in establishing threshold levels and a causal relationship between atopic asthma and a certain allergen source, especially in relation to work-associated exposure. Purified major allergens open possibilities for a more accurate assessment of sensitization. OBJECTIVE: To determine the threshold dose of purified major bovine dander allergen Bos d 2 in bronchial provocation in comparison with the standard allergen and a set of other parameters of allergy. METHOD: Nine consecutive patients referred to hospital for confirming the bovine origin of their occupational asthma were subjected to bronchial provocation tests with purified natural Bos d 2 and a standard bovine dander allergen. Additional tests included bronchial histamine challenge, measurements of total IgE, specific IgE antibody determinations and skin prick tests (SPT) with both allergens. RESULTS: In the provocation tests with Bos d 2, a 15% decrease in the forced expiratory volume in 1 s (FEV1) and/or peak expiratory flow (PEF) values in eight out of nine patients confirmed the predominant role of Bos d 2 in the sensitization. The threshold dose of Bos d 2 varied from 0.1 microg to > 100 microg (median +/- median absolute deviation = 4.5 +/- 3.9 microg). A positive SPT was induced by a median dose of 13.9 +/- 9.8 microg of Bos d 2. Bronchial response to histamine and IgE antibodies against Bos d 2 showed the highest correlations to the provocations results. CONCLUSIONS: The efficacy of Bos d 2 in bronchial provocation in patients with occupational cattle associated asthma was confirmed and the range of the threshold level was determined. There were individual variations, but the response in provocation remains the reference method for identification of the cause of occupational atopic asthma. SPT and the measurement of specific IgE antibodies, preferably with purified or recombinant major allergens, increase the accuracy of the diagnosis. PMID- 12372126 TI - Genetic variation of Der p 2 allergens: effects on T cell responses and immunoglobulin E binding. AB - BACKGROUND: Der p 2 is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variations on T cell and antibody responses has not been compared. OBJECTIVES: To compare IgE antibody binding and T cell proliferation and cytokine release induced by variants of Der p 2. METHODS: Peripheral blood mononuclear cells (PBMC) from 19 allergic and 15 non-allergic people were stimulated with recombinant variants of Der p 2. IL-5, IL-10, IL-13 and IFN-gamma were measured by a time resolved fluorescence (TRF) assay. Serum IgE antibody was measured using a solid-phase TRF assay. RESULTS: Overall the most prevalent variant of Der p 2 (Der p 2. 0101) was the highest or approximately equal highest inducer of T cell proliferation and IL-5, IL-10, IL 13 and IFN-gamma release. The most divergent variant 0104 induced the next highest responses. The variants 0107 and 0108 showed interesting changes especially when the allergic status was considered. Responses to 0107 showed poor Th1/Th2 polarization and, except for IL-10 release, cytokine responses to 0108 were low for non-allergic subjects. The variant 0101 showed similar monoclonal antibody binding but moderately less IgE binding than the other variants. CONCLUSIONS: The most prevalent variant, Der p 2. 0101, was the most active for T cell stimulation and although its IgE binding was slightly less than other variants that was highly correlated. The variant Der p 2. 0104 which contains the known common polymorphic changes had a response which was similar to Der p 2. 0101 and thus these two variants were the most stimulatory representations of Der p 2. The T cell responses to the less common variants 0107 and 0108 however, showed consistent differences demonstrating that changes in the sequence could change the cytokine response. PMID- 12372127 TI - The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level. AB - BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development. PMID- 12372128 TI - Are Ca2+-binding motifs involved in the immunoglobin E-binding of allergens? Olive pollen allergens as model of study. AB - BACKGROUND: Several Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. OBJECTIVE: To study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. METHODS: Two olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunoblotting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. RESULTS: Thirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. CONCLUSION: EF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross reactivity among members of the same family. PMID- 12372129 TI - A comparative study of eicosanoid concentrations in sputum and urine in patients with aspirin-intolerant asthma. AB - BACKGROUND: Although many studies have assumed that the overproduction of cysteinyl- leukotrienes (cys-LTs) and an imbalance of arachidonic acid metabolism may be plausible causes for the pathogenesis of aspirin-intolerant asthma (AIA), there has been little experimental evidence to substantiate this notion in lower airways of patients with AIA. OBJECTIVES: The purpose of this study was to compare the eicosanoid concentrations in sputum and urine from patients with AIA. METHODS: The concentrations of sputum cys-LTs, prostaglandin E2 (PGE2), PGF2alpha, PGD2 and thromboxane B2 were measured to assess local concentrations of eicosanoids in patients with AIA and in those with aspirin-tolerant asthma (ATA). The concentrations of two urinary metabolites, leukotriene E4 (LTE4) and 9alpha11betaPGF2, were also measured to corroborate the relationship between the eicosanoid biosynthesis in the whole body and that in lower airways. RESULTS: The concentration of PGD2 in sputum was significantly higher in patients with AIA than in those with ATA (median, 5.3 pg/mL vs. 3.1 pg/mL, P < 0.05), but there was no significant difference in the concentration of the corresponding metabolite, 9alpha11betaPGF2, between the two groups. No differences were noted in the concentrations of other prostanoids in sputum between the two groups. The sputum cys-LT concentrations showed no differences between the two groups, in spite of the observation that the concentration of urinary LTE4 was significantly higher in patients with AIA than in those with ATA (median, 195.2 pg/mg-cre vs. 122.1 pg/mg-cre, P < 0.05). There was a significant correlation among the concentration of cys-LTs, the number of eosinophils and the concentration of eosinophil-derived neurotoxin (EDN) in sputum. CONCLUSION: The urinary concentration of LTE4 does not necessary reflect cys-LT biosynthesis in lower airways. A significantly higher concentration of PGD2 in sputum from patients with AIA suggests the possible ongoing mast cell activation in lower airways. PMID- 12372130 TI - The effect of leukotriene-modifier drugs on aspirin-induced asthma and rhinitis reactions. AB - BACKGROUND: Leukotrienes (LTs) appear to be crucial mediators of aspirin (ASA) induced lower respiratory tract reactions. Therefore, it is logical to assume that leukotriene-modifier drugs (LTMDs) might block these reactions. OBJECTIVE: The aim of this study was to determine whether concomitant treatment with LTMDs was associated with a reduction of ASA-provoked lower respiratory tract reactions in patients with aspirin-exacerbated respiratory disease (AERD), when compared to AERD patients who were not treated with LTMDs. Secondly, if ASA-induced lower respiratory tract reactions were prevented in LTMD-treated patients, was there then a higher prevalence of upper respiratory reactors or, alternatively, a higher prevalence of blocked reactions ('non-reactors') in this group. METHODS: Of 271 patients suspected by history of having AERD, 96 were taking cys-LT receptor antagonists (cys-LTRAs) and 12 were taking zileuton at the time of oral ASA challenges. A matched control group of 163 patients was not receiving LTMDs. All subjects underwent standard oral ASA challenges. Reactions were classified as follows: classic [naso-ocular combined with a 20% or > decline in forced expiratory volume of 1 s (FEV1)]; pure lower (20% or > decline in FEV1 without naso-ocular); partial asthma (naso-ocular + 15-20% decline in FEV1); upper only (naso-ocular with < 15% decline in FEV1); negative (no reactions). RESULTS: In patients treated with cys-LTRAs, there were significant reductions in numbers of patients with ASA-induced bronchospastic reactions and a concomitant increase in upper respiratory reactors. There were no significant differences in mean provoking doses of ASA or the percent changes in FEV1 values in both groups. In the 12 patients receiving zileuton, no reactions to ASA (16%) were similar to the cys-LTRA-treated group (11%) and the control group (15%). CONCLUSION: During oral ASA challenges, LTMD treatment appeared to shift target organ responses from both upper and lower respiratory tracts to upper tract alone. LTMD blocking of the entire respiratory tract did not appear to occur. PMID- 12372131 TI - Influence of different dosage schedules of inhaled fluticasone propionate on peripheral blood cytokine concentrations in childhood asthma. AB - BACKGROUND: Asthma is characterized by eosinophilic airways inflammation with elevated levels of IL-4, IL-5 and sICAM-1, and reduced levels of IL-10 and IFN gamma. Inhaled corticosteroids powerfully reduce airways inflammation. OBJECTIVE: To investigate if eosinophil counts, serum eosinophilic cationic protein (ECP) and sICAM-1 levels, as well as serum and production of cytokines (IL-4, IL-5, IL 10, IFN-gamma) by peripheral blood monocytes (PBMCs) are useful markers to monitor therapy with inhaled fluticasone propionate (FP) in asthmatic children. METHODS: In a double-blind, 1-year study, 55 asthmatic children (aged 6-10 years) stopped inhaled corticosteroids for a mean period of 24 days and were randomized to receive either FP 200 microg/day (constant dose group), or a starting dose of FP 1000 microg/day with two monthly reductions to 500, 200 and 100 microg/day (stepdown group). Hyper-responsiveness, symptom scores and blood sampling were performed at 2-month intervals. RESULTS: Symptoms and hyper-responsiveness improved significantly in both treatment groups after reintroduction of FP. Eosinophil counts decreased significantly more during the first 2 months of FP in the stepdown group than in the constant dose group (P = 0.03). We found a trend towards a dose-dependent response in changes of eosinophil counts and serum ECP levels during treatment. Serum IL-4 and IL-5 levels were undetectable in the majority of children. No significant effect of the dose of FP on the release of IL-4, IL-5, IL-10 or IFN-gamma by Con A stimulated PBMCs was found. sICAM-1 levels did not significantly differ at any time point between the two groups. CONCLUSION: Serum ECP as well as peripheral blood eosinophils, cytokine production by PBMCs and sICAM-1 levels are insensitive markers in titrating and monitoring therapy with inhaled corticosteroids over a wide dose range in childhood asthma. PMID- 12372132 TI - Effects of fexofenadine and desloratadine on subjective and objective measures of nasal congestion in seasonal allergic rhinitis. AB - BACKGROUND: In vitro studies have shown much higher H1-receptor antagonist potency with desloratadine (DL) compared to fexofenadine (FEX), although it is unclear whether this has any clinical relevance on disease control parameters in seasonal allergic rhinitis (SAR), especially for nasal congestion. OBJECTIVE: To compare the relative efficacy between presently recommended doses of DL and FEX on daily measurements of peak nasal inspiratory flow (PNIF) and nasal symptoms in SAR. METHODS: Forty-nine patients with SAR were randomized into a double-blind, placebo-controlled cross-over study during the grass pollen season, comparing 2 weeks of once daily treatment with (a) 180 mg FEX or (b) 5 mg DL, taken in the morning. There was a 7-10 day placebo run-in and washout prior to each randomized treatment. Measurements were made in the morning (AM) and in the evening (PM) for PNIF (the primary outcome variable), nasal and eye symptoms. The average of AM/PM values were used for analysis. RESULTS: There were significant (P < 0.05) improvements, compared to placebo, with FEX and DL, for PNIF, nasal blockage, nasal irritation, and total nasal symptoms, but not nasal discharge or eye symptoms. There were no significant differences between active treatments. Values for PNIF (L/min) for mean placebo baseline, mean difference from baseline (95% CI for difference) were 126, 10 (4-16) for FEX; and 122, 11 (4-17) for DL. The mean difference (95% CI) between FEX vs. DL was 1 L/min (-7-8). Values for total nasal symptoms (out of 12) were: 3.2, 0.7 (0.2-1.2) for FEX; and 3.4, 0.9 (0.3-1.5) for DL, and for nasal blockage (out of 3) were: 1.1, 0.2 (0.1-0.4) for FEX; and 1.2, 0.3 (0.1-0.5) for DL. The mean difference (95% CI) in total nasal symptoms and nasal blockage between FEX vs. DL was 0.1 (-0.6-0.8) and 0.1 (-0.2-0.3), respectively. CONCLUSIONS: Recommended once daily doses of fexofenadine and desloratadine were equally effective in improving nasal peak flow and nasal symptoms in SAR. PMID- 12372133 TI - Gelatin-specific cellular immune responses persist for more than 3 years after priming with gelatin containing DTaP vaccine. AB - BACKGROUND: Gelatin-specific cell-mediated immunity develops in subjects inoculated with gelatin containing DTaP vaccine. However, it is not yet known whether such established sensitization to gelatin disappears or persists with time. OBJECTIVE: The aim of this study was to follow the patients with gelatin sensitization elicited by DTaP vaccination for their lymphocyte responsiveness and IgE, IgG antibody specific to gelatin over several years and to compare the activities with those at the time of enrollment into the study. METHODS: We studied 28 subjects who developed positive lymphocyte proliferation test (LPT) after receiving gelatin containing DTaP vaccine and eight subjects who had a negative LPT after inoculation of non-gelatin DTaP. Determination of IgE, IgG antibodies and specific lymphoproliferative response directed against gelatin were performed at enrollment and on follow up. RESULTS: None of the subjects had antibody to gelatin at enrollment and none developed gelatin IgE or IgG during follow-up. There was no significant difference in the SIs of the subjects receiving gelatin DTaP (P = 0.150, 95% CI, -0.198-0.032), whereas lymphocyte activity to gelatin increased between enrollment and follow-up in the subjects with non-gelatin DTaP (P = 0.011, 95% CI, 0.063-0.338). CONCLUSION: Gelatin specific lymphocyte activity persists at comparable levels for more than 3 years in subjects who acquire a positive LPT response to gelatin after receiving primary DTaP vaccine containing gelatin. Furthermore, five out of eight subjects initially with negative LPT to gelatin have been shown to acquire specific LPT with time. PMID- 12372134 TI - High incidence of adverse reactions to egg challenge on first known exposure in young atopic dermatitis children: predictive value of skin prick test and radioallergosorbent test to egg proteins. AB - BACKGROUND: Egg skin prick test (SPT) and/or radioallergosorbent test (RAST) positivity has been described in infants and children with a food allergy, or in infants at high risk of atopy who have never eaten eggs. Clinical reactions are also observed when some of these children or infants eat eggs for the first time. OBJECTIVE AND METHOD: A prospective study was made of 107 atopic dermatitis (AD) children (66 boys, 41 girls) aged 1-19 months (median 5 months) who had never ingested egg, to compare the outcome of a first oral egg challenge and the results of albumen and yolk SPTs and RASTs. RESULTS: The egg challenge (conducted at age 12-24 months: mean 16 months, median 15 months) was positive in 72/107 children (67.3%). The reactions were immediate or early (first 6 h) in 56/72 (77.8%). The most severe (all within the first 6 h) were one case of anaphylactic shock (1.4%), three cases of laryngeal oedema (4.1%) and one serious attack of asthma (1.4%). The skin weal diameter at and above which reactions always occurred was 5 mm for both albumen and yolk. They were, however, also observed in the complete absence of a weal. The outcome of the challenge was always positive when the specific IgEs (sIgE) for albumen and yolk were > 99 KU/L and > or = 17.5 KU/L, respectively. Here, too, reactions were noted even when sIgE levels were < 0.35 KU/L. CONCLUSION: AD children who have never eaten eggs may be sensitized and display reactions at the first ingestion. The percentage of reactions in this series was by no means negligible. These findings were observed in children with mild as well as moderate-severe AD when first examined. SPT for albumen and yolk diameter > or = 5 mm, and sIgE for albumen > 99 KU/L and for yolk > or = 17.5 KU/L were 100% specific in predicting the outcome of the challenge. It may thus be concluded that children with AD whose SPT and/or RAST for albumen and/or yolk are equal to or higher than these cut-off values should not be subjected to the oral challenge when consideration is given to the introduction of egg in their diet. Even when these cut-offs are not reached, however, clinical reactions to the challenge cannot be ruled out a priori, and it should be preferably performed in a protected environment, such as a hospital. PMID- 12372135 TI - Expression and function of the ST2 gene in a murine model of allergic airway inflammation. AB - BACKGROUND: We have recently reported that soluble ST2 protein levels are elevated in the sera of patients with asthma, and correlate well with the severity of asthma exacerbation. However, the role, function, and kinetics of soluble ST2 expression in asthma remain unclear. OBJECTIVE: The objective of the present study was to clarify the function and kinetics of soluble murine (m) ST2 expression in a murine asthma model. METHODS: We analyzed the kinetics of gene and protein expression of mST2 in sera or lung tissue after allergen (ovalbumin; OVA) challenge in a murine model of allergic airway inflammation, the effects of mST2 protein on OVA-induced Th2 cytokine production in vitro from splenocytes of sensitized mice, and the effects of soluble mST2 on Th2-dependent allergic airway inflammation by in vivo gene transfer of mST2. RESULTS: Serum mST2 protein levels increased to the maximal level 3 h after the allergen challenge, before serum IL 5 levels peaked. The mRNA expression of mST2 in lung tissue was induced after the allergen challenge, while that in the spleen was constitutively detected. Furthermore, pre-treatment with mST2 protein significantly inhibited the production of IL-4 and IL-5, but not IFN-gamma, from OVA-stimulated splenocytes in vitro, and intravenous mST2 gene transfer resulted in a drastic reduction in the number of eosinophils and in the levels of IL-4 and IL-5 in bronchoalveolar lavage fluid, compared with those in response to transfer of non-coding plasmid vector or of lipid alone. CONCLUSION: These results suggest that increases in endogenous mST2 protein after allergen exposure may modulate Th2-mediated airway inflammation, and that in vivo gene transfer of mST2 can be applicable to use in a novel immunotherapy for allergic diseases. PMID- 12372136 TI - The involvement of matrix metalloproteinases in basement membrane injury in a murine model of acute allergic airway inflammation. AB - BACKGROUND: Airway remodelling in asthma such as subepithelial fibrosis is thought to be the repair process that follows the continuing injury as of chronic airway inflammation. However, how acute allergic inflammation causes tissue injury in the epithelial basement membrane in asthmatic airways remains unclear. Matrix metalloproteinases (MMPs) capable of degrading almost all of the extracellular matrix components have been demonstrated to be involved in cell migration through the basement membrane in vivo and in vitro. OBJECTIVE: We investigated the alterations of matrix construction and the role of MMPs in matrix degradation in the subepithelium during acute allergic airway inflammation. METHODS: Airway inflammation, the ultrastructure of the subepithelium and injury of types III and IV collagen in tracheal tissues from ovalbumin (OVA)-sensitized mice after OVA inhalation with or without the administration of tissue inhibitor of metalloproteinase-2 (TIMP-2) and dexamethasone were evaluated by cell counting in bronchoalveolar lavage (BAL) fluids, electron microscopy and immunohistochemistry, respectively. RESULTS: The disruption of the lamina densa and matrix construction and the decrease of the immunoreactivity for type IV collagen in subepithelium were observed in association with the accumulation of inflammatory cells in airways 3 days after OVA inhalation. This disorganization of the matrix components in the subepithelium, as well the cellular accumulation, was abolished by the administration of TIMP-2 and dexamethasone. The immunoreactivity for type IV collagen in the subepithelium in OVA-inhaled mice returned to the level of that in saline-inhaled mice 10 days after inhalation in association with a decrease of the cell numbers in the BAL fluid. The immunoreactivity for type III collagen was changed neither 3 nor 10 days after OVA inhalation. CONCLUSION: These results suggest that epithelial basement membrane gets injured by, at least in part, MMPs as a consequence of cell transmigration through the membrane during acute allergic airway inflammation. PMID- 12372138 TI - An Asilomar moment. AB - As governments in the US and Europe contemplate legislation that would divert funding of some genomics-driven research to Offices of Homeland Security and the like, and that would restrict the freedom of biologists to publish and share some of their data, we of the scientific community are facing a crisis, brought on by fears of bioterrorism, that eerily mirrors the early days of recombinant DNA research. PMID- 12372139 TI - On the importance of being finished. AB - The publication of an increasing number of draft genome sequences presents problems that will only be resolved by improved search tools and by complete finishing of the sequences - and their deposition in publicly accessible databases. PMID- 12372140 TI - Molecular archeology of L1 insertions in the human genome. AB - BACKGROUND: As the rough draft of the human genome sequence nears a finished product and other genome-sequencing projects accumulate sequence data exponentially, bioinformatics is emerging as an important tool for studies of transposon biology. In particular, L1 elements exhibit a variety of sequence structures after insertion into the human genome that are amenable to computational analysis. We carried out a detailed analysis of the anatomy and distribution of L1 elements in the human genome using a new computer program, TSDfinder, designed to identify transposon boundaries precisely. RESULTS: Structural variants of L1 elements shared similar trends in the length and quality of their target site duplications (TSDs) and poly(A) tails. Furthermore, we found no correlation between the composition and genomic location of the pre insertion locus and the resulting anatomy of the L1 insertion. We verified that L1 insertions with TSDs have the 5'-TTAAAA-3' cleavage site associated with L1 endonuclease activity. In addition, the second target DNA cut required for L1 insertion weakly matches the consensus pattern TTAAAA. On the other hand, the L1 internal breakpoints of deleted and inverted L1 elements do not resemble L1 endonuclease cleavage sites. Finally, the genome sequence data indicate that whereas singly inverted elements are common, doubly inverted elements are almost never found. CONCLUSIONS: The sequence data give no indication that the creation of L1 structural variants depends on characteristics of the insertion locus. In addition, the formation of 5' truncated and 5' inverted L1s are probably not due to the action of the L1 endonuclease. PMID- 12372141 TI - Long terminal repeat retrotransposons of Oryza sativa. AB - BACKGROUND: Long terminal repeat (LTR) retrotransposons constitute a major fraction of the genomes of higher plants. For example, retrotransposons comprise more than 50% of the maize genome and more than 90% of the wheat genome. LTR retrotransposons are believed to have contributed significantly to the evolution of genome structure and function. The genome sequencing of selected experimental and agriculturally important species is providing an unprecedented opportunity to view the patterns of variation existing among the entire complement of retrotransposons in complete genomes. RESULTS: Using a new data-mining program, LTR_STRUC, (LTR retrotransposon structure program), we have mined the GenBank rice (Oryza sativa) database as well as the more extensive (259 Mb) Monsanto rice dataset for LTR retrotransposons. Almost two-thirds (37) of the 59 families identified consist of copia-like elements, but gypsy-like elements outnumber copia-like elements by a ratio of approximately 2:1. At least 17% of the rice genome consists of LTR retrotransposons. In addition to the ubiquitous gypsy- and copia-like classes of LTR retrotransposons, the rice genome contains at least two novel families of unusually small, non-coding (non-autonomous) LTR retrotransposons. CONCLUSIONS: Each of the major clades of rice LTR retrotransposons is more closely related to elements present in other species than to the other clades of rice elements, suggesting that horizontal transfer may have occurred over the evolutionary history of rice LTR retrotransposons. Like LTR retrotransposons in other species with relatively small genomes, many rice LTR retrotransposons are relatively young, indicating a high rate of turnover. PMID- 12372142 TI - Genomic analysis of membrane protein families: abundance and conserved motifs. AB - BACKGROUND: Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families. RESULTS: Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels. CONCLUSIONS: We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families. PMID- 12372143 TI - Mining microarray expression data by literature profiling. AB - BACKGROUND: The rapidly expanding fields of genomics and proteomics have prompted the development of computational methods for managing, analyzing and visualizing expression data derived from microarray screening. Nevertheless, the lack of efficient techniques for assessing the biological implications of gene-expression data remains an important obstacle in exploiting this information. RESULTS: To address this need, we have developed a mining technique based on the analysis of literature profiles generated by extracting the frequencies of certain terms from thousands of abstracts stored in the Medline literature database. Terms are then filtered on the basis of both repetitive occurrence and co-occurrence among multiple gene entries. Finally, clustering analysis is performed on the retained frequency values, shaping a coherent picture of the functional relationship among large and heterogeneous lists of genes. Such data treatment also provides information on the nature and pertinence of the associations that were formed. CONCLUSIONS: The analysis of patterns of term occurrence in abstracts constitutes a means of exploring the biological significance of large and heterogeneous lists of genes. This approach should contribute to optimizing the exploitation of microarray technologies by providing investigators with an interface between complex expression data and large literature resources. PMID- 12372144 TI - Analysis of EF-hand-containing proteins in Arabidopsis. AB - BACKGROUND: In plants, calcium (Ca2+) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca2+]cyt), which in turn is thought to regulate cellular and developmental processes via Ca2+-binding proteins. Three out of the four classes of Ca2+-binding proteins in plants contain Ca2+-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca2+ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins. RESULTS: A maximum of 250 proteins possibly having EF-hands were identified. Diverse proteins, including enzymes, proteins involved in transcription and translation, protein- and nucleic-acid-binding proteins and a large number of unknown proteins, have one or more putative EF-hands. Phylogenetic analysis identified six major groups that contain some families of proteins. CONCLUSIONS: The presence of EF-hand motif(s) in a diversity of proteins is consistent with the involvement of Ca2+ in regulating many cellular and developmental processes. Thus far, only 47 of the possible 250 EF-hand proteins have been reported in the literature. Various domains that we identified in many of the uncharacterized EF hand-containing proteins should help in elucidating their cellular role(s). Our analyses suggest that the Ca2+ messenger system is widely used in plants and that EF-hand-containing proteins are likely to be the key transducers mediating Ca2+ action. PMID- 12372145 TI - Conservation of long-range synteny and microsynteny between the genomes of two distantly related nematodes. AB - BACKGROUND: Comparisons between the genomes of the closely related nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal high rates of rearrangement, with a bias towards within-chromosome events. To assess whether this pattern is true of nematodes in general, we have used genome sequence to compare two nematode species that last shared a common ancestor approximately 300 million years ago: the model C. elegans and the filarial parasite Brugia malayi. RESULTS: An 83 kb region flanking the gene for Bm-mif-1 (macrophage migration inhibitory factor, a B. malayi homolog of a human cytokine) was sequenced. When compared to the complete genome of C. elegans, evidence for conservation of long range synteny and microsynteny was found. Potential C. elegans orthologs for II of the 12 protein-coding genes predicted in the B. malayi sequence were identified. Ten of these orthologs were located on chromosome I, with eight clustered in a 2.3 Mb region. While several, relatively local, intrachromosomal rearrangements have occurred, the order, composition, and configuration of two gene clusters, each containing three genes, was conserved. Comparison of B. malayi BAC-end genome survey sequence to C. elegans also revealed a bias towards intrachromosome rearrangements. CONCLUSIONS: We suggest that intrachromosomal rearrangement is a major force driving chromosomal organization in nematodes, but is constrained by the interdigitation of functional elements of neighboring genes. PMID- 12372146 TI - Asymmetric directional mutation pressures in bacteria. AB - BACKGROUND: When there are no strand-specific biases in mutation and selection rates (that is, in the substitution rates) between the two strands of DNA, the average nucleotide composition is theoretically expected to be A = T and G = C within each strand. Deviations from these equalities are therefore evidence for an asymmetry in selection and/or mutation between the two strands. By focusing on weakly selected regions that could be oriented with respect to replication in 43 out of 51 completely sequenced bacterial chromosomes, we have been able to detect asymmetric directional mutation pressures. RESULTS: Most of the 43 chromosomes were found to be relatively enriched in G over C and T over A, and slightly depleted in G+C, in their weakly selected positions (intergenic regions and third codon positions) in the leading strand compared with the lagging strand. Deviations from A = T and G = C were highly correlated between third codon positions and intergenic regions, with a lower degree of deviation in intergenic regions, and were not correlated with overall genomic G+C content. CONCLUSIONS: During the course of bacterial chromosome evolution, the effects of asymmetric directional mutation pressures are commonly observed in weakly selected positions. The degree of deviation from equality is highly variable among species, and within species is higher in third codon positions than in intergenic regions. The orientation of these effects is almost universal and is compatible in most cases with the hypothesis of an excess of cytosine deamination in the single-stranded state during DNA replication. However, the variation in G+C content between species is influenced by factors other than asymmetric mutation pressure. PMID- 12372147 TI - Are Drosophila telomeres an exception or the rule? AB - At the ends of eukaryotic chromosomes are telomeres, specialized structures with unusual properties. Specific efforts to compare sequences and properties of telomeres across species can reveal the generalities of telomere properties. PMID- 12372148 TI - Large-scale gene-expression studies and the challenge of multiple sclerosis. AB - In multiple sclerosis, a complex neurodegenerative disorder, a combination of genetic and environmental factors results in inflammation and myelin damage. Recent transcription-profiling studies have found distinct gene-expression patterns in diseased tissue; such large-scale studies at different stages of the disease are contributing to understanding multiple sclerosis and developing effective therapy. PMID- 12372149 TI - A genomic approach to studying cell-size homeostasis in yeast. AB - Using a complete set of budding-yeast mutants bearing deletions of all known open reading frames, a recent study has revealed multiple overlapping pathways that coordinately regulate cell-cycle progression with ribosome biogenesis and translation efficiency, providing new insights into the mechanisms governing cell size homeostasis in eukaryotes.. PMID- 12372150 TI - Variations in abundance: genome-wide responses to genetic variation and vice versa. AB - How do naturally occurring polymorphisms in DNA sequence relate to variation in gene expression? Recent work to map genetic sources of expression variation has shown a surprising balance between cis and trans effects. Other work suggests some chromosomal clustering of genes by expression pattern. A synthesis of approaches may provide new insight in to adaptive mechanisms in evolution and the population basis of complex traits. PMID- 12372151 TI - Ascidian gene-expression profiles. AB - With the advent of gene-expression profiling, a large number of genes can now be investigated simultaneously during critical stages of development. This approach will be particularly informative in studies of ascidians, basal chordates whose genomes and embryology are uniquely suited for mapping developmental gene networks. PMID- 12372152 TI - Histidine protein kinases: key signal transducers outside the animal kingdom. AB - Histidine protein kinases (HPKs) are a large family of signal-transduction enzymes that autophosphorylate on a conserved histidine residue. HPKs form two component signaling systems together with their downstream target proteins, the response regulators, which have a conserved aspartate in a so-called 'receiver domain' that is phosphorylated by the HPK. Two-component signal transduction is prevalent in bacteria and is also widely used by eukaryotes outside the animal kingdom. The typical HPK is a transmembrane receptor with an amino-terminal extracellular sensing domain and a carboxy-terminal cytosolic signaling domain; most, if not all, HPKs function as dimers. They show little similarity to protein kinases that phosphorylate serine, threonine or tyrosine residues, but may share a distant evolutionary relationship with these enzymes. In excess of a thousand known genes encode HPKs, which are important for multiple functions in bacteria, including chemotaxis and quorum sensing, and in eukaryotes, including hormone dependent developmental processes. The proteins divide into at least 11 subfamilies, only one of which is present in eukaryotes, suggesting that lateral gene transfer gave rise to two-component signaling in these organisms. PMID- 12372153 TI - Livelihoods, nutrition and health in Dhaka slums. AB - OBJECTIVES: To identify groups within Dhaka slums that report similar patterns of livelihood, and to explore nutritional and health status. DESIGN: A random sample of households participated in a longitudinal study in 1995-1997. Socio-economic and morbidity data were collected monthly by questionnaire and nutritional status was assessed. Cluster analysis was used to aggregate households into livelihood groups. SETTING: Dhaka slums, Bangladesh. SUBJECTS: Five-hundred and fifty-nine households. MAIN OUTCOME MEASURES: Socio-economic and demographic variables, nutritional status, morbidity. RESULTS: Four livelihood groups were identified. Cluster 1 was the richest cluster with land, animals, business assets and savings. Loans as well as income were higher, which shows that this group was credit-worthy. The group was mainly self-employed and worked more days per month than the other clusters. The cluster had the second highest body mass index (BMI) score, and the highest children's nutrition status. Cluster 2 was a poor cluster and was mainly dependent self-employed. Savings and loans were lower. Cluster 3 was the most vulnerable cluster. Members of this group were mainly casual unskilled, and 40% were female-headed households. Total income and expenditure were lowest amongst the clusters. BMI and children's nutritional status were lowest in the slum. Cluster 4 was the second richest cluster. This group comprised skilled workers. BMI was the highest in this cluster and children's nutritional status was second highest. CONCLUSIONS: Cluster analysis has identified four groups that differed in terms of socio-economic, demographic and nutritional status and morbidity. The technique could be a practically useful tool of relevance to the development, monitoring and targeting of vulnerable households by public policy in Bangladesh. PMID- 12372154 TI - Acceptability of the use of iron cooking pots to reduce anaemia in developing countries. AB - OBJECTIVE: To evaluate acceptability, compliance and attitude towards the use of iron pots compared with aluminium pots, for cooking in a community that traditionally did not use iron pots. DESIGN: Randomised trial. SETTING: Two rural Malawian villages. SUBJECTS: Fifty-two households received iron pots and 61 aluminium pots. RESULTS: Pot characteristics were assessed by a questionnaire after 3, 6, 11 and 20 weeks of use. Within households using iron pots there was a significant decrease in acceptability score with usage, from an initial value of 13.7 to 11.4 (range 1-20) Answers to questions concerning cooking characteristics showed that after 3 weeks' use the aluminium pot scored better, whereas after 20 weeks fewer answers differed between the iron and aluminium pot groups. Almost a third of the households planned to continue using iron pots daily after 20 weeks, although they had ready access to their former aluminium pot. The presence of a group of consistent pot users suggests that if households were convinced about daily use, then they were likely to maintain consistent use. Some householders considered that iron pots required less firewood for cooking than aluminium pots. The main problems related to lower acceptability were rusting and pot weight. About 25% of problems with iron pots were unrelated to their cast iron characteristics. Overall 23.4% of the households indicated they would buy an iron pot. CONCLUSIONS: The low acceptability of iron pots for cooking could limit their value as an intervention to control iron-deficiency anaemia. Design modifications and better instructions on pot use should improve acceptability. The study highlights the need to assess the acceptability of interventions in order to facilitate their adoption in traditional communities. PMID- 12372155 TI - Free school meals and children's social and nutritional status in Trinidad and Tobago. AB - OBJECTIVE: To evaluate the provision of free school meals in Trinidad and Tobago in relation to children's social and nutritional status. DESIGN AND METHODS: Cross-sectional survey of a nationally representative sample of 66 government schools, including children in the admissions classes (aged 4 to 7 years) and classes for 'rising nines' (aged 7-10 years). Data included questionnaire details of free school meals and children's social background, and measurements of children's heights, weights and skinfold thicknesses. RESULTS: Of 6731 eligible children, data were analysed for 5688 (85%). There were 2386 (42%) children receiving free meals provided at school. At different schools the proportion of all children receiving free meals ranged from 20% to 100%, Receipt of free meals was associated with larger family size (one child, 32% received free meals; > or =6 children, 63%), lower paternal educational attainment (primary, 52% free; university, 30%), father's employment (employed, 39% free meals; unemployed >12 months, 59%) as well as maternal education and employment and household amenities. After adjusting for age, sex and ethnic group, children who received free meals were shorter (mean difference in height standard deviation score (SDS) -0.12, 95% confidence interval (CI) -0.17 to -0.06), lighter (body mass index SDS -0.21, -0.28 to -0.14) and thinner (subscapular skinfold SDS -0.13, -0.18 to 0.09). CONCLUSIONS: Free school meals were widely available, with some targeting of provision to children with less favourable social and nutritional status. Greater universality would reduce inequity, but more stringent targeting and reduction of school-level variation would increase efficiency. PMID- 12372156 TI - Long-term effects of breast-feeding in a national birth cohort: educational attainment and midlife cognitive function. AB - OBJECTIVE: A recent meta-analysis showed that breast-feeding confers a 3.2 point increment in cognitive function through adolescence. Little is known, however, about possible longer-term effects of breast-feeding. We investigated the effect of breast-feeding on educational attainment, and on a range of cognitive skills in midlife, in the British 1946 birth cohort. DESIGN: Regression analyses were used to test the association between breast-feeding, likelihood of obtaining advanced educational qualifications by age 26 years, and three cognitive test scores at age 53 years: i.e. reading ability (NART), timed visual search and verbal memory. These associations were then adjusted for social confounding variables and for cognitive ability at age 15 years. SETTING AND SUBJECTS: One thousand seven hundred and thirty-nine male and female participants in the MRC National Survey of Health and Development, also known as the British 1946 birth cohort, distributed throughout England, Wales and Scotland. RESULTS: Breast feeding was significantly and positively associated with educational attainment, an effect that was independent of early social background, but largely accounted for by cognitive ability at age 15 years. Breast-feeding was significantly and positively associated with the NART at 53 years, an effect that was independent of early social background, educational attainment and adult social class, but, again, largely accounted for by cognitive ability at 15 years. There was no independent effect of breast-feeding on timed visual search or verbal memory at 53 years. CONCLUSION: The benefit of breast-feeding has long-term potential impact across the life course through its influence on childhood cognition and educational attainment. PMID- 12372157 TI - Food insecurity among refugee families in East London: results of a pilot assessment. AB - OBJECTIVE: To identify child hunger and examine its association with family factors, receipt of benefits, housing conditions and social support among recently arrived refugee families with young children. DESIGN: Structured and semi-structured questionnaire administered to a service-based, purposive sample of caregivers. SETTING: East London, United Kingdom. SUBJECTS: Thirty households with children <5 years old, resident in the UK for <2 years. RESULTS: All households sampled were food-insecure, and 60% of index children were experiencing hunger as defined on the Radimer/Cornell scale. Child hunger was significantly associated with recent arrival, marginally significantly associated with receipt of fewer benefits and younger parenthood, and not associated with maternal education or self-efficacy score, household size or composition, or measures of social support. CONCLUSIONS: A community-based, participatory approach for rapid assessment of the prevalence, extent and causes of child hunger among newly arrived asylum seekers recently arrived in Britain is feasible, and preliminary results suggest a programmatic need for a broader, population-based assessment of food insecurity in this rapidly growing population group. PMID- 12372158 TI - Hypertension and blood pressure among meat eaters, fish eaters, vegetarians and vegans in EPIC-Oxford. AB - OBJECTIVE: To compare the prevalence of self-reported hypertension and mean systolic and diastolic blood pressures in four diet groups (meat eaters, fish eaters, vegetarians and vegans) and to investigate dietary and other lifestyle factors that might account for any differences observed between the groups. DESIGN: Analysis of cross-sectional data from participants in the Oxford cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC Oxford). SETTING: United Kingdom. SUBJECTS: Eleven thousand and four British men and women aged 20-78 years at blood pressure measurement. RESULTS: The age adjusted prevalence of self-reported hypertension was significantly different between the four diet groups, ranging from 15.0% in male meat eaters to 5.8% in male vegans, and from 12.1% in female meat eaters to 7.7% in female vegans, with fish eaters and vegetarians having similar and intermediate prevalences. Mean systolic and diastolic blood pressures were significantly different between the four diet groups, with meat eaters having the highest values and vegans the lowest values. The differences in age-adjusted mean blood pressure between meat eaters and vegans among participants with no self-reported hypertension were 4.2 and 2.6 mmHg systolic and 2.8 and 1.7 mmHg diastolic for men and women, respectively. Much of the variation was attributable to differences in body mass index between the diet groups. CONCLUSIONS: Non-meat eaters, especially vegans, have a lower prevalence of hypertension and lower systolic and diastolic blood pressures than meat eaters, largely because of differences in body mass index. PMID- 12372159 TI - Anthropometric indices predict physical function and mobility in older Australians: the Australian Longitudinal Study of Ageing. AB - OBJECTIVE: To evaluate, in terms of function and mobility, the predictive value of commonly adopted anthropometric 'definitions' used in the nutritional assessment of older adults, in a cohort of older Australians. DESIGN: Prospective cohort study - Australian Longitudinal Study of Ageing (ALSA). SETTING: Adelaide, South Australia (1992-1994). SUBJECTS: Data were analysed from 1272 non institutionalised (685 males, 587 females) older adults > or =70 years old in South Australia. Seven 'definitions' commonly used in the anthropometric assessment of both under- and overnutrition (including four using body mass index (BMI), waist-to-hip ratio, waist circumference and percentage weight change) were evaluated at baseline, for their ability to predict functional and mobility limitation assessed (by self-report questionnaire) at two years follow-up. All questionnaires were administered and anthropometry performed by trained investigators. The associations between the definitions and decline in mobility and physical function were evaluated over two years using multiple logistic regression. RESULTS: A BMI >85th percentile or >30 kg m-2 or a waist circumference of >102 cm in males and >88 cm in females increased risk of functional and mobility limitations. Over two years, a loss of 10% body weight significantly increased the risk of functional and mobility limitations. CONCLUSION: Maintaining weight within older adults, irrespective of initial body weight, may be important in preventing functional and mobility limitations. Excessive weight is associated with an increased risk of limitation in function and mobility, both key components of health-related quality of life. PMID- 12372160 TI - Socio-economic differences in fruit and vegetable consumption among Australian adolescents and adults. AB - OBJECTIVES: To determine whether socio-economic groups differ in their fruit and vegetable consumption, and the variety eaten, and whether socio-economic differences are similar for adolescents and adults. The study also examined whether socio-economic groups vary in their reported desire to increase the amount of fruit and vegetables consumed, and the perceived barriers to achieving this. DESIGN, SETTING AND SUBJECTS: The 1995 Australian National Nutrition Survey collected fruit and vegetable intake data from adolescents aged 13-17 years and adults 18-64 years using a 24-hour dietary recall. Gross annual household income was used to measure socio-economic position. RESULTS: Approximately 44% of males and 34% of females did not consume fruit in the 24 hours preceding the survey, and 20% of males and 17% of females did not consume vegetables. Among adolescents and adults, fruit and vegetable consumption was positively related to income. The only exception was vegetable consumption among adolescent males, which did not vary by income. Lower-income adults consumed a smaller variety of fruits and vegetables than their higher-income counterparts. Fruit and vegetable variety did not vary by income among adolescents. Lower-income adults expressed less desire to increase their fruit and vegetable consumption, and were more likely to report that price and storage were barriers to doing so. Socio-economic differences in consumption and variety were more apparent for adults than for adolescents. CONCLUSIONS: In addition to increasing the consumption of fruits and vegetables among the general population, nutrition interventions, programmes and policy aiming to improve diet should target adolescents and adults from low socio economic groups. Strategies should address price and storage barriers. PMID- 12372162 TI - Exploring predictors of eating behaviour among adolescents by gender and socio economic status. AB - OBJECTIVE: Guided by theory, this study explored cross-sectional differences in factors influencing adolescent eating behaviour including gender and socio economic status (SES), and subsequently tested the longitudinal predictive power of the models. DESIGN/SETTING/SUBJECTS: Data were collected by questionnaires in a longitudinal study of adolescents (age 13 years at baseline) and their parents from Hordaland County, Norway. Association of personal and environmental variables (family, friends, school/society) with the consumption of fruit and vegetables (FV) and selected sources of fat and of sugar were assessed at age 15 The final cross-sectional models were subsequently employed in groups stratified by gender/SES and to predict consumption at age 21 RESULTS: The model explained more of the variation in the sugar score (21%) and the FV score (13.5%) than in the fat score (5%). SES was associated with both the sugar and FV scores. The strongest associations with the sugar score and FV were for antisocial behaviour and evaluation of own diet, respectively. The former association was significant in all gender/SES groups, whereas the latter association was only significant in the low SES groups. For all three types of food, the strongest significant predictors in the longitudinal models were frequency of consumption at age 15. CONCLUSION: The model's ability to explain variation in eating behaviours differed by food type, and possibly by gender/SES, but previous eating behaviour was an important predictor for all three foods. Prospective studies should carefully operationalize theoretical constructs when further investigating the influences of and interrelationships between these factors and gender/SES on the development of eating behaviours. PMID- 12372163 TI - Severe underreporting of energy intake in normal weight subjects: use of an appropriate standard and relation to restrained eating. AB - OBJECTIVE: To assess the influence of different standards and restrained eating on underreporting in healthy, non-obese, weight-stable young subjects. DESIGN AND SUBJECTS: Eighty-three young adults (20-38 years, 55 women, 28 men) were assessed under weight-stable conditions with a 7-day dietary record and the three-factor eating questionnaire by Stunkard and Messick. Resting energy expenditure (REE; indirect calorimetry) plus data derived from physical activity records (PA) (Standard 1) or REE times an activity factor (AF) (Standard 2) was used as standard for total energy expenditure (TEE). For comparison, doubly labelled water (DLW) was used to measure TEE in a subgroup of subjects. RESULTS: There was an association between self-reported energy intake and Standard 2 but not with Standard 1. When compared with DLW both calculated standards were inaccurate, but Standard 2 avoided high levels of overreporting. Using Standard 2 to identify 'severe' underreporting (SU; as defined by a deviation of energy intake (EI) and TEE of >20%), SU was seen in 37% of all subjects. It was more frequently found in women than in men (49% of women, 14.3% of men, ). Underreporting subjects had a reduced EI but there were no significant differences in nutritional status (body weight and height, body mass index, fat mass and fat-free mass), energy expenditure and the proportion of energy from macronutrients between normal and underreporting subjects. However, high restraint was associated with a higher degree of underreporting in the total group, whereas disinhibition had an influence only in men. CONCLUSIONS: A high prevalence of SU is seen in non-obese subjects. Characteristics of eating behaviour (restraint and disinhibition) were associated with underreporting but seemed to have a different influence in men and women. PMID- 12372164 TI - Validation of a semi-quantitative food-frequency questionnaire for use among adults in Guatemala. AB - OBJECTIVE: The purpose of the study was to assess the validity of a 52-item semi quantitative food-frequency questionnaire (FFQ) by comparing it with multiple 24 hour dietary recalls. DESIGN: Three non-consecutive 24-hour dietary recalls and one FFQ were administered over a one-month period. SETTING: Four communities of El Progreso, Guatemala. SUBJECTS: Seventy-three individuals aged 22-55 years. RESULTS: : Intakes of energy and other nutrients as measured by the FFQ were higher than intakes measured by 24-hour recalls. Energy was overestimated by 361 kcal, and nutrient overestimates were particularly great for vitamin C and iron. Pearson correlation coefficients for crude energy and nutrients intakes ranged from 0.64 for energy to 0.12 for vitamin C. Exact agreement for both methods (measured by the concordance correlation coefficient) ranged from 0.59 (fat) to 0.06 (vitamin C). Pearson correlation coefficients for energy-adjusted nutrients ranged from 0.59 (carbohydrates) to 0.11 (thiamin). Pearson correlation coefficients for the proportion of total energy derived from specific foods ranged from 0.59 (tortillas) to 0.01 (sugared beverages). Cross-classification of quartiles of crude nutrient intakes for both methods indicated that <11% were grossly misclassified; after adjusting for energy intake, <13% were grossly misclassified. CONCLUSIONS: This FFQ provides good measures of energy and macronutrient intakes and a reasonably reliable measure of micronutrient intake, indicating its suitability for comparing exposures within a study population in reference to heath-related endpoints. Our results highlight the need to adapt any FFQ to specific cultural needs - in this case, the Guatemalan 'core foods' (tortilla, bread and beans), for which inter-individual variability in intake is high. PMID- 12372165 TI - Development of a short questionnaire to assess the dietary intake of heterocyclic aromatic amines. AB - OBJECTIVE: Development and validation of a short instrument to assess the dietary intake of heterocyclic aromatic amines (HCA). DESIGN: At first, a longer instrument asking for the consumption of 11 meat and fish items and different preparation methods was developed. The degree of browning of these foods was assessed by means of photos. This questionnaire was sent to 500 participants of the European Prospective Investigation into Cancer and Nutrition (EPIC) in Heidelberg, Germany, in June 1999. Using 385 completed questionnaires, a short questionnaire was developed covering just seven food items, which was sent to the participants again. Of these, 344 were returned within four months. Total dietary intake of HCA as well as the intake of different HCA were calculated and compared between both versions. RESULTS: Median dietary intake of total HCA was 103 ng day 1 as assessed with the short version; the intakes of 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5 f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) were 63, 34 and 2 ng day-1, respectively. These results did not differ significantly from those obtained with the longer version. Spearman rank correlation coefficients between the long and the short version ranged from 0.46 to 0.6. In quartile cross-classification, 70-78% of the participants were assigned into the same or an adjacent quartile while categorisation into opposite quartiles was < or =3.5%. CONCLUSION: The short version of the HCA questionnaire demonstrates good validity compared with the longer version. The intake of HCA as assessed with the short questionnaire is comparable to that found in other studies using a short questionnaire. PMID- 12372166 TI - [Antibiotic resistance in blood isolates of Staphylococcus aureus in 31 Spanish hospitals participating in the European Antimicrobial Resistance Surveillance System (2000)]. AB - BACKGROUND: In 1998, the European Union funded the European Antimicrobial Resistance Surveillance System. We present here Staphylococcus aureus data in 2000 in Spain. MATERIAL AND METHOD: 31 hospitals were involved, covering almost 25% Spanish total population. All nosocomial blood isolates of S. aureus were included. Each laboratory carried out microbiological studies with its own usual methods. Quality control was carried out by UK National External Quality (NEQAS). A questionnaire including hospital, patient and specimen data was filled out for each isolate. Results were registered in a single database and analyzed and validated with Whonet. 5 software. RESULTS: Invasive S. aureus was isolated in 903 patients. Overall incidence was 1.45/1000 admitted patients. Resistance was 28.1% (95% CI, 25.2-31.1) to oxacillin (O), 26.6% to ciprofloxacin (C), 23.8% to erythromycin (E) and 16.6% to gentamicin (G). Multiresistance was noticed in 80% oxacillin-resistant strains. More frequent multiresistance profiles were OECG (11.3% of all isolates) and OEC (6.3%). Oxacillin resistance was higher in ICU units (44.5%) than in other medical departments (27.4%) (p < 0.001). Hospitals with 500 beds or more showed 36.4% prevalence of O resistance, while in hospitals having less than 500 beds it was 18.8% (p < 0.001). A decreased susceptibility to vancomycin was not detected. CONCLUSIONS: In Spain, invasive S. aureus shows a high prevalence of resistance to oxacillin, ciprofloxacin, erythromycin and gentamicin. Most oxacillin-resistant strains were resistant to three or more antibiotics. Nearly 50% oxacillin-resistant isolates were susceptible to gentamicin. Oxacillin resistance and resistance to multiple antibiotics was more frequent in ICU units and in hospitals with 500 beds or more. PMID- 12372167 TI - [Spanish version of a scale for the assessment of mania: validity and reliability of the Young Mania Rating Scale]. AB - BACKGROUND: The Young Mania Rating Scale is the most widely used tool for the assessment of the intensity of manic symptoms. Unfortunately, to date, there was no Spanish validated version available. This study validated the Spanish version of the YMRS. PATIENTS AND METHOD: A sample of 541 DSM-IV manic or hypomanic bipolar patients were recruited in 56 different psychiatric settings in Spain and assessed with the YMRS by 112 clinicians specifically trained in its use on days 1, 7, 14, 30, 45 and at 3 and 6 months. The mania subscale of the Clinical Global Impression for Bipolar Disorders was also performed in order to have a standard measure to compare our results. Feasibility, reliability, validity and sensitivity of the YMRS were analysed. RESULTS: The YMRS Spanish version showed reliability index of 0.88 (internal consistency) and 0.76 (test-retest reliability), and good internal validity and external (p < 0.001) when compared to the mania subscale of the Modified Clinical Global Impression. The results also showed good sensitivity and feasibility. CONCLUSIONS: The YMRS Spanish Version is a useful, valid and reliable tool for the assessment of manic symptoms. PMID- 12372168 TI - [Imported paludism: an emerging illness]. AB - BACKGROUND: We aimed at knowing the epidemiological and clinical characteristics of imported malaria in Maresme county (Barcelona), Spain. PATIENTS AND METHOD: A descriptive and retrospective study of patients diagnosed with imported malaria at the Hospital de Mataro (HM) (1982-2000). RESULTS: 64 cases of malaria were diagnosed, which supposed a significant increase in its incidence over the last decade. The disease mostly affected men (83%) as well as young and sub-Saharan immigrants (72%). 17% of affected people were immigrants' children. 50% cases were diagnosed in August-September. 80% of species corresponded to Plasmodium falciparum, either alone or in association with other species. Only 10% of patients underwent a correct chemoprophylaxis. CONCLUSIONS: Imported malaria is an emergent, potentially mortal, illness, which has to be taken into account in high-rate immigration areas. Prophylaxis compliance and knowledge is low. PMID- 12372169 TI - [Designer drugs]. PMID- 12372170 TI - [Forecasting the impact of hepatitis C in Catalonia (Spain)]. PMID- 12372171 TI - [How lymphangioleiomyomatosis can be recognized and treated?]. PMID- 12372172 TI - [Confusional syndrome in the elderly]. PMID- 12372173 TI - [Lipodystrophies]. PMID- 12372174 TI - [Neuro-ophthamologic manifestations as unusual presentation of Lyme disease]. PMID- 12372175 TI - [Effect of STI-571, a c-kit tyrosine kinase inhibitor, in mestastasic gastrointestinal tumor]. PMID- 12372176 TI - [Confounding factors and automatic selection of variables]. PMID- 12372177 TI - [Dalteparin induced thrombocytopenia. Study of a case]. PMID- 12372178 TI - [Antiretroviral therapy with nelfinavir for patient infected, by HIV and secondary diabetes mellitus due to indinavir]. PMID- 12372179 TI - [Useful foreign drugs for the critically ill patient]. PMID- 12372180 TI - [The principle of precaution: implications for public health]. PMID- 12372181 TI - [The need to look in depth into the debate and analysis of waiting lists]. PMID- 12372182 TI - [Profile of the hospital case mix of the immigrant population in Barcelona, Spain]. AB - OBJECTIVE: Although the immigrant population in cities such as Barcelona has tripled in the last five years, until now the impact of this group on the health system has not been rigorously evaluated. The aim of this study was to compare hospital resource utilization among the immigrant population with that among the native population through case mix, demographic characteristics and hospital day use. MATERIAL AMD METHODS: We analyzed 15,057 discharges from Hospital del Mar in Barcelona in 2000. This hospital attends 60% of admissions from the Ciutat Vella district. In 2000, 21% of the population of this district were immigrants. Socio demographic patient characteristics and case mix were compared between the immigrant and the native population. Hospital resource use was compared according to age, case mix (diagnosis related groups) and seriousness (severity, complications and comorbidities) of the events requiring medical care. RESULTS: The case mix of the immigrant population differed from that of the autochthonous population due to pronounced ge differences and a higher fertility rate. Thirty three percent of immigrant admissions were for deliveries. The mean cost of discharge of immigrants from low-income countries was 30% lower than that for the remaining discharges. After adjusting for age, case mix and severity, length of stay among the immigrant population was significantly shorter. A 5% reduction was found after adjusting for case mix and a 10% reduction was found when all the factors were considered. CONCLUSIONS: Case mix differences are due to age and socio-cultural factors. Immigrants are rejuvenating the ageing native population and the role of gynecology-obstetrics and pediatrics needs to be increased. The finding that resource use per discharge is lower among immigrants from low-income countries contradicts the expectation that lower socioeconomic status leads to higher hospital resource use intensity. Therefore, new hypotheses and analyses that explain this situation should be put forward. PMID- 12372183 TI - [Invasive pneumococcal disease in children in the region of Murcia (Spain)]. AB - OBJECTIVE: Because of the availability of a conjugate pneumococcal vaccine, the incidence and characteristics of invasive pneumococcal disease in children in the region of Murcia should be determined. This would provide information that could be useful for properly establishing the indications for vaccination. METHODS: A retrospective search was conducted for cases of invasive Streptococcus pneumoniae in children aged less 15 years old treated in hospitals in Murcia from 1991-2000. The data sources were the databases of the microbiology services, the Minimum Data Set, the Pediatric Admissions Register and the EDO Register. RESULTS: The incidence rate for the period 1996-2000 was 18.25 per 105 children per year for children aged under 1 year in the case of invasive disease (10.6 for meningitis), 13.6 for those under 2 years for invasive disease (6 for meningitis), 8.9 for those under 5 years (1.35 for meningitis) and 3.7 for those under 15 years (1.3 for meningitis). Twenty-eight percent of the patients presented risk factors. Complications occurred in 35.2% and sequelae occurred in 5%. The mortality rate was 11.8%. The prevalent serogroups were 19, 6, 18, 5, 14 and 23. CONCLUSIONS: The high percentage of patients with risk factors for invasive pneumococcal disease suggests the need to implement vaccination programs aimed at risk groups. Although the incidence of invasive pneumococcal disease in the region of Murcia differs from that in other areas, the incidence of meningitis is similar to that reported by other studies. Because of the severity of the disease, cost effectiveness studies to evaluate the possible incorporation of the vaccine in the vaccination calendar are justified. PMID- 12372184 TI - [Cost-effectiveness of 23-valent antipneumococcical vaccination in Catalonia (Spain)]. AB - OBJECTIVES: Pneumococcal vaccination is an effective procedure for preventing pneumococcal pneumonia. In this study we evaluate the cost-effectiveness of pneumococcal vaccination strategies (23 serotypes) in the population aged 5 years and older in Catalonia. METHODS: Cost-effectiveness was evaluated in terms of cost per year of life gained (YLG) by comparing the net cost of the vaccination program with its effectiveness. The net cost of the vaccination program was calculated by subtracting 70% of the population from the vaccination costs, representing the reduction in health costs due to pneumococcal pneumonia that can ve achieved with vaccination. Vaccination costs were estimated based on a price of 12.41 euros (1,915 ptas.) for pneumococcal vaccine. The costs and benefits of the vaccination program were updated for 1996 by using a discount rate of 5%. RESULTS: A cost-effectiveness ratio of 9,023.27 euros per YLG was achieved for universal vaccination of the population. Cost-effectiveness was 11,3177.12 euros per YLG in individuals aged 5-24 years, 19,482.51 euros per TLG in those aged 25 44 years, 7,122.80 euros per YLG in those aged 45-64 years and less than 0 in those aged 65 years and older. In this group the reduction in cost of the disease was greater than the vaccination costs with a cost-benefit ratio of 1.58. The results of the cost-efecctiveness analysis were sensitive to vaccine costs and efficacy and the percentage of pneumonias caused by pneumococcus but were less sensitive to the costs of pneumococcal pneumonia, the rate of hospital admission among patients with community-acquired pneumonia and vaccine coverage. CONCLUSION: The results of this study show that pneumococcal vaccination should be a priority in individuals aged 65 years and older and in those aged 45-64 years. PMID- 12372185 TI - [Primary care: decentralization and efficiency]. AB - OBJECTIVE: The purpose of this study was to evaluate whether the productive behavior of health centers in autonomous communities with competence in health is more efficient than that among centers belonging to Spanish public health system (INSALUD). METHODS: The technical efficiency of 66 health centers in Alava, Navarre and La Rioja was analyzed. Centers in autonomous communities that in 1997 had been granted complete authority from the central government to manage their healthcare services were compared with centers whose administration, in the same year, was still in the hands of INSALUD. The method used to measure and quantify the efficiency of these centers was data envelopment analysis. RESULTS: Nonparametric contrast of the health centers' mean efficiency rates revealed no significant differences in the (in)efficiency of centers from La Rioja, Navarre and Alava. CONCLUSIONS: The results obtained from the model of efficiency measurement used did not indicate that decentralization improves the productive efficiency of primary care centers. PMID- 12372186 TI - [Qualitative analysis of organizational innovations in Spanish public hospitals]. AB - OBJECTIVE: To determine the opinion of chief executive officers (CEOs) and physicians in public hospitals concerning new managerial trends. METHODS: We performed a qualitative study designed to determine the opinion of CEOs and physicians on the organizational innovations that affect more than one level of health management intervention. In-depth semi-structured interviews were conducted to identify behavior, experiences, opinions, knowledge and other personal and institutional aspects related to the study's aim. Focus groups (two study groups and one control group) were also used. Interaction between groups was used to obtain different types of information on the development of ideas, operational capacity, and the degree of consensus and disagreement on the subjects discussed. RESULTS: Comparison between the control and the study groups revealed that the new management trends added value in the following areas: economy of contracts, delegation, administrative decentralization, incentives, risk avoidance, process re-engineering, heath care continuity, competitiveness, leadership, information systems and client centeredness. CONCLUSIONS: Physicians are showing increased interest in organizational innovations while CEOs are ambivalent about their changing role and respective responsibilities. There is evidence of resistance to change. There is no single institutional model; institutional design depends on internal factors (cohesion and leadership) and external factors (environment, size and technology). The incipient development of innovations reveals the need for changes in the style and characteristics of management structure (composition, functions, responsibilities). PMID- 12372187 TI - [Opinions of primary care managers on sources of influence on medical practice. Differences with physicians' opinions]. AB - OBJECTIVES: To determine the opinion of primary healthcare managers on the importance and legitimacy of different sources of influence in medical practice, and to compare the results with the opinions of physicians in healthcare teams. MATERIAL AND METHODS: DESIGN: cross-sectional study. POPULATION: primary healthcare managers in the Spanish public health system (area managers, medical and nursing directors) and in the Andalusian health service (district director, nursing coordinators and epidemiology and program coordinators). The sample comprised the total population of 302. As dependent variables, a series of questions was designed to gather the interviewees' opinions on different strategies, institutions and/or collectives that exert some kind of influence on medical practice. The degree of importance of each factor was summarized into a set of 9 items. The subjects were asked to score each item from 1 (most important) to 9 (least important). To assess the legitimacy of these scores, 16 items were presented measured using a Likert-type 7-point scale (1: not at all legitimate; 7: very legitimate). A self-administered questionnaire was used, sent by mail. Non-parametric tests (Friedman and Kruskall-Wallis) were used for statistical analysis of the data. RESULTS: The response rate was 79.8%. Using the Friedman test for an ordinal 9-point scale, analysis of the mean ranges for each item revealed that the most important sources of influence for the primary healthcare managers interviewed were: the devising of management protocols by the doctors themselves; discussion with colleagues; feedback from patients, and attending training courses, and reading articles and scientific journals. The institutions or groups with the greatest legitimacy to influence medical practice were: users or citizens; internal audits; peers; scientific associations, and the managers themselves. CONCLUSIONS: The sources of influence considered to have the greatest importance and legitimacy in influencing medical practice concern the professional medical system (self-defined protocols, discussion with colleagues, etc.). Managers accept the use of business managerial tools as well as the influence of social actors to a greater extent than do physicians. This finding could indicate differences in the value systems between primary healthcare physicians and managers. PMID- 12372188 TI - [Tobacco taxes, prices and demand for tobacco products: a comparative analysis]. AB - This paper analyzes the extent to which an increase in tobacco taxes affects the demand for tobacco products, especially for cigarettes. Comparison of the studies reviewed revealed that higher tobacco taxes result in higher tobacco prices. The price-elasticity of cigarette demand in low- and middle-income countries is about double that in high-income countries, about 0.4. Furthermore, because of the addictive nature of tobacco use, demand for tobacco products is more elastic in the long run than in the short run. The effect of higher tobacco taxes is greater on the young, among whom demand is more sensitive to price than among adults. The empirical evidence for Spain estimates the price elasticity of cigarette demand in the short run to be in the range of 0.5 to 0.3, a result which is similar to other studies. These results do not suggest that tax policy is an effective tool for tobacco control, although taxes are useful for their revenue generating potential and for compensating the external costs generated by tobacco consumption. Furthermore, when the possibilities of substitutions among brands and the strategies of the tobacco industry to compensate for the effects of taxes (lowering prices and encouraging cigarette smuggling) are considered, the panorama is even more pessimistic PMID- 12372189 TI - [Has the time arrived for the management of waiting lists?]. AB - Individuals on the waiting list frequently suffer an additional risk caused by the mean time until they receive treatment; however, other individuals do not need the treatment for which they are waiting.Both arguments, which can be contrasted with empirical evidence, would be sufficient to affirm that waiting list management should be implemented, leaving aside policies that are more of less opportunistic. Opportunistic policies are understood as those providing misinformation on waiting lists or their "manipulation", and using programs of auto-coordination with the sole aim of reaching the end of the year without a waiting list of not more than six months, etc. The panorama is not completely bleak. Some management initiatives (and even Politics with a capital P) are opening the way forward and may enter the Agenda in the next few years. In this context, the application of guaranteed times of medical care or the prioritization of waiting lists according to explicit criteria should be highlighted. It is worth remembering that, except for the queues in the waiting rooms of health centers and emergency departments, waiting lists are mediated by the decision of the physician. Therefore, an essential strategy for managing waiting lists consists of attenuating the problems caused by uncertainty (or ignorance) of the patient's diagnosis or prognosis. PMID- 12372190 TI - [Management of surgical waiting lists by health centers and health professionals]. AB - Waiting lists for non-urgent medical care, in diagnostic or therapeutic procedures, occur mainly in public health systems such as those found in Spain and many other European countries. If waiting lists are moderate they can be useful in the process of managing these patients and are accepted by health professionals and health services users. Waiting lists for surgical procedures can be interpreted, erroneously, as a simple imbalance between the supply and demand for a particular procedure. If that were the case, we would only have to progressively increase resources until eliminating the lists.However, considerable evidence suggests that the isolated increase of resources does not solve the problem since the mean waiting time is reduced but the waiting list becomes longer. Therefore, other management measures are required. The management of waiting lists is necessary at the levels of society, health administration and especially health centers. Clinical management by departments and individual health professionals is essential, using the criteria of inclusion of scientific evidence in the indication for treatment and in the results expected for each patient (effectiveness of the procedure) as well as ethical criteria and considerations of resource use efficiency. Prioritizing patients according to severity, probability of improvement and social criteria is an unavoidable obligation in improving the problem of waiting lists. In this process of prioritization, society should also be able to voice an opinion since non-medical factors may influence the distribution and prioritization of resources and in this context the experience of other countries should be analyzed. Finally, as Archie Cochrane said "all effective treatment should be free" which, put another way would be: in a public system, the financing of procedures that do not provide significant benefits to patients is not justified. PMID- 12372192 TI - [Point processes as a tool for analyzing possible sources of contamination]. AB - Point pattern analysis pattern comprises a series of techniques that enables the distribution of a series of events occurring in the vicinity of a particular region of a map to be studied. In epidemiology, this problem arises when a potential source of environmental contamination, possibly leading to cases of a specific disease, is investigated.The present study provides a brief description of point pattern analysis. The approach is illustrated through determination of the environmental source and study of the areas of greatest risk of incidence of an outbreak of legionella pneumonia that occurred between the middle of September and beginning of October in the city of Alcoi in Alicante (Spain).Point pattern analysis was able to confirm the environmental source of the outbreak and identify the areas of the city at greatest risk. This provided the justification for an exhaustive inspection of the installations generating aerosols after which, to date, the epidemics ceased. PMID- 12372193 TI - [Diagnosis of HIV infection in primary care]. PMID- 12372194 TI - [New applications of magnetic resonance imaging for the thorax]. PMID- 12372195 TI - [Non-invasive continuous positive airways pressure for post-extubation laryngitis in pediatric patients]. AB - Post-extubation laryngeal edema (PLE) is a common complication (10-15%) in pediatric intensive care units, and some authors have reported high failure rates for conventional treatment. HYPOTHESIS: Non-invasive continuous positive airways pressure (CPAP) in children with PLE may have a lower failure rate than conventional management. PATIENTS AND METHOD: Twenty-five patients were needed to detect a difference between the two treatment groups. The patients were assigned to receive the conventional treatment (nebulized epinephrin and humidified oxygen) or the experimental treatment (non-invasive CPAP treatment for 18 hours), using a randomized block design (10-patient blocks). After 9 months, the study was halted when a significant difference emerged between the two groups. RESULTS: Of 270 children extubated during the study, 28 (10.3%) developed PLE and 25 met the enrollment requirements. Thirteen were assigned to conventional therapy and 12 to CPAP. General characteristics, time of intubation, FiO2 upon admission, use of corticoids before extubation and scores for respiratory difficulty upon admission were similar in the two groups (p > 0.05). With conventional therapy, 5 (38.5%) children improved and 8 patients worsened or remained the same. Eleven (91.7%) of the patients receiving CPAP improved (p = 0.01). Those who failed on conventional treatment were prescribed CPAP and all then improved, although one had to be re-intubated. CONCLUSION: The hypothesis was confirmed. CPAP treatment reduced the failure rate by 53.2% in comparison with conventional therapy. PMID- 12372196 TI - [Perception of improvement in asthma patients]. AB - The objective of this study was to investigate the ability of patients with stable asthma to recognize improvement in bronchial obstruction with treatment. We enrolled 75 stable asthmatics (44 women and 31 men, mean age 43 17 years) who reported baseline dyspnea on a modified Borg scale. Acute bronchodilation of 15% was provoked in the laboratory, after which the patients were asked if there was a change in dyspnea. Our results were as follows. 1) Overall, 19 asthmatics (25%) failed to perceive improvement in dyspnea with bronchodilation. 2) The mean change in dyspnea was 1.17 1.11, although the change was greater in patients with more severe asthma (0.60 0.5 for mild asthmatics, 1.05 1.07 for moderate asthmatics and 1.93 1.4 for severe asthmatics; p < 0.0001). 3) Perception of improvement was significantly related to level of the patient's emotional balance (anxiety-depression), quality of life, education, socioeconomic level, age, age of onset, severity, baseline dyspnea and obstruction, thoracic pressure and number of visits to the doctor in the preceding year. 4) The variables entered into the stepwise regression model were baseline dyspnea, depression, thoracic pressure and age. 5) Generally, young asthmatics whose disease appeared at a younger age and who also had less ventilatory obstruction and greater quality of life, showed a tendency to underestimate the beneficial effect of bronchodilator treatment. Moreover, when asthma was severe, non-perceptive individuals had significantly more admissions to intensive care units due to asthma exacerbation.In conclusion, 25% of our asthmatics are unable to recognize whether their bronchia dilate as a result of treatment, meaning that they would delay the start of rescue medication during an exacerbation. Such patients should be identified in order to establish therapeutic guidelines in function of objective home criteria (peak-flow monitoring). PMID- 12372197 TI - [Analysis of admissions for chronic obstructive pulmonary disease in Andalusia in 2000]. AB - OBJECTIVE: To analyze the impact of admissions for chronic obstructive pulmonary disease (COPD) in Andalusia during 2000. METHODS: All patients with DRG codes 088 and 541, which would receive ICD-9 codes 491, 492, 493.2, 494 and 496 in the cause of admission field, were extracted from the Minimum Basic Data Set for Andalusia. We compiled descriptive statistics from these data, calculated the cost per day of hospitalization for our own hospital, and then extrapolated to estimate the cost for Andalusia. RESULTS: COPD exacerbations generated 10,386 admissions in 2000, leading to 117,011 days of hospitalization. Eighty-three percent of the patients were men and the mean age was 70 12 years. The average hospital stay was 11 10 days. Huelva was the province with the shortest hospital stay (9 days). Mortality was 6.7%. The minimum expenditure generated was E 27 million, not counting the cost of intensive care unit admissions. CONCLUSIONS: Admissions due to COPD have great impact on the Andalusian health care system. Further studies are needed to evaluate alternatives to hospitalization. PMID- 12372198 TI - [Thoracocentesis for the assessment of lung cancer with pleural effusion]. AB - OBJECTIVE: To analyze the pleural and mediastinal effect of thoracentesis tumor positive cytology in pleural effusions (PE) detected by chest X ray of lung cancer patients. PATIENTS AND METHODS: The study was performed in patients with lung cancer for whom PE was evident in chest X ray films, who then underwent thoracentesis followed by video-assisted thoracoscopy (VAT) to evaluate direct pleural tumor infiltration, mediastinal node involvement and the existence of pleural metastasis. Patients without contraindication underwent the procedure, even if tumor positive cytology was present. When pleural metastasis was found the treatment employed was talc pleurodesis and chemotherapy. Descriptive statistics were compiled and the validity of VAT for pleural metastasis diagnosis, of thoracentesis pleural cytology to detect infiltration of the tumor adyacent pleura, N2 disease and pleural metastasis were calculated. Survival was also analyzed. RESULTS: PE was present in 188 of 971 consecutive lung cancer patients. Seventy two PEs were visible in the chest X ray films. Volume exceeded 425 mL. Tumor positive pleural cytology was detected in 29 cases (40%). Pleural metastasis were found in 54 patients, 23 of whom had tumor positive pleural cytology. In the other 6 patients with positive cytology the primary neoplasm infiltrated the visceral pleura, completely in 5. In 4 of those 5, the mediastinal pleura was also involved. The primary tumor and diseased lymph nodes were removed from 11 patients, 3 of them with tumoral pleural cytology. Visual pleural inspection by VAT had a sensitivity of 93%, specificity of 82%, positive predicted value (PPV) of 94% and negative predicted value (NPV) of 78% for the diagnosis of pleural metastasis. Thoracentesis cytology showed a sensitivity of 43%, specificity of 67%, PPV of 79% and NPV of 28% for pleural metastasis. For the evaluation of adjacent pleura infiltration, without pleural metastasis, the sensitivity of cytology was 40%, specificity 100%, PPV 100% and NPV 25%. For mediastinal node invasion clinically evaluated, the sensitivity of cytology was 55%, specificity of 62%, PPV 18% and NPV 90%. Survival after thoracotomy was 39% after 2 years, and the median survival time was 14.5 months. In the 11 resected patients, survival was 53% at two years. The difference in survival between patients treated by thoracotomy and those treated by talc pleurodesis after VAT was significant (p < 0.01). The 3 resected patients with pleural tumor-positive cytology survived 84, 39 and 25 months. CONCLUSIONS: Nineteen percent of patients with lung cancer have PE, of which 7% can be seen in chest X ray films. In such patients the likelihood of pleural metastasis is 75%. Pleural metastasis is not necessarily present when PE cytology indicates that tumor is present. VAT can be considered the ideal technique for the assessment of direct pleural invasion by the tumor or of pleural metastasis. PMID- 12372199 TI - [Dyspnea and quality of life in chronic obstructive pulmonary disease]. PMID- 12372200 TI - [The history of chest drainage]. PMID- 12372201 TI - [Lung toxicity due to thalidomide]. AB - Although the side effects of thalidomide are well known, lung toxicity has not been reported. We describe the case of a 65-year-old man with multiple myeloma (IgG kappa) in stage IA who, on the thirty-seventh day of treatment with thalidomide, developed acute coughing, general malaise, dyspnea at rest and sudoresis. Blood pressure was 90/60 mm Hg and temperature was normal. An interstitial and alveolar pattern was visible on the right side of a chest film and arterial blood gases indicated partial respiratory insufficiency (pH 7.40, PaCO2 40 mmHg, PaO2 47 mmHg). Blood analysis showed alterations expected for multiple myeloma and microbiology was negative (sputum and blood cultures and urinary antigen detection for Streptococcus pneumoniae and Legionella pneumophila). After thalidomide was withdrawn and oxygen and intravenous corticoids were administered, outcome was good. A chest film 4 days later was normal and arterial blood gases showed that respiratory insufficiency had disappeared. We conclude that severe lung toxicity should be included among the potential adverse effects of thalidomide. PMID- 12372202 TI - [Antisynthetase syndrome and interstitial lung involvement. Report of 6 cases]. AB - The cases of 6 patients (4 men, 2 women) with antisynthetase syndrome are reported. The mean age was 60 years and the most frequent symptom was increasing dyspnea (4 patients). One of the remaining 2 patients had hemoptysis and the last was asymptomatic. Systemic symptoms included Raynaud's phenomenon (2 patients), arthritis in hands (3) and muscle impairment (4). Chest films showed linear interstitial infiltrates of varying severity in 5 patients; the patient without such infiltrates also suffered silicosis. Functional assessment showed restrictive impairment in 4 patients; of the remaining 2 patients, 1 had chronic obstructive pulmonary disease and 1 had normal function. The antisynthetase antibody (ASAB) detected was anti-Jo-1 in 4 cases, anti-PL-12 in 1 case, and unidentified in 1 case. The course of disease was satisfactory for 5 patients. ASAB analysis is useful for studying idiopathic interstitial lung disease. PMID- 12372203 TI - [Spontaneous pneumomediastinum: diagnostic difficulties]. PMID- 12372204 TI - [Surgical treatment of the fibrosarcomatous variant of giant dermatofibrosarcoma protuberans]. PMID- 12372205 TI - [Bilateral Pancoast syndrome]. PMID- 12372206 TI - [Doctor, I want to give up smoking]. PMID- 12372207 TI - [Pneumococcal vaccine effectiveness in the elderly. Systematic review and meta analysis]. AB - AIM: Estimate pneumococcal vaccine effectiveness in preventing Streptococcus pneumoniae illness in the elderly. DESIGN: Systematic review and meta-analysis. DATA SOURCE. MEDLINE, years 1964 to the 2000; EMBASE, from 1988 to the 2000; Cochrane Library, identified studies and previously published systematic reviews citations peruse, and contacts with field experts. STUDY SELECTION: Clinical trials, cohort and case-control studies, published in Spanish, English or French, that estimated pneumococcal disease rates in vaccinated or not vaccinated elderly. DATA EXTRACTION: The studies were valued independently by four investigators with predefined criteria of validity, such as results comparing rates of disease caused by serotypes included in the vaccine, random allocation, double blind design, included subjects pertaining to the same study base, and losses of less than 10% in clinical trials and 20% in observational studies. RESULTS: Eight clinical trials considered the relative risk (RR) of pneumococcal pneumonia, three did not make estimations on pneumonia originated by serotypes included in the vaccine and only one study fulfilled all the inclusion criteria. Vaccinated versus not vaccinated pneumococcal pneumonia RR was 0.86 (95%CI, 0.24 to 2.99). Vaccine effectiveness was 14% (95%CI, -199 to 76%). Ten studies performed estimations on the effectiveness of the vaccine on invasive disease by vaccine serotypes. Of these, two clinical trials and two observational studies fulfilled the required quality criteria. RR of invasive disease was of 0.68 (95%CI, 0.39-1.18); vaccine effectiveness was 32% (95%CI, 18-61%). CONCLUSIONS: No evidence was found supporting pneumococcal vaccine effectiveness to reduce or avoid S. pneumoniae disease in the elderly. PMID- 12372209 TI - [Oral anticoagulation treatment in patients with non-valvular auricular fibrillation]. AB - OBJECTIVES: To evaluate the knowledge, attitudes and difficulties of family doctors in the indication of oral anti-coagulation treatment (OCT) in patients with non-valvular auricular fibrillation (NVAF). DESIGN: Transversal descriptive study.Setting. Area 11 of Madrid primary care.Participants. 250 doctors by simple randomised sampling. MAIN MEASUREMENTS: After a pilot study at a health centre, mailing of a questionnaire with a subsequent re-mailing. This collected social and personal details, knowledge of the question, attitudes and difficulties. RESULTS: 157 (62.8%) replied; 91 were women (58.0%); mean age was 39 (SD, 6.0). 97 had reviewed the question recently (61.8%). 110 thought that the anti aggregation criteria were clear (70.1%; CI, 62.2-77.0%), 107 that the oral anti coagulation criteria were (68.2%; CI, 60.2-75.2%), 132 that the OCT risks were (84.1%; CI, 77.2-89.2%), and 74 that risk factors of cerebrovascular accident were clear (47.1%; CI, 39.2-55.2%). Initially 96 doctors gave anti-aggregants and referred to cardiology (61.1%; CI, 53.0-68.7%), and 29 began OCT (18.5%; CI, 12.9 25.6%). 134 thought that we avoided initiating OCT (85.3%; CI, 78.6-90.3%), giving as the main reasons the difficulty of monitoring and of requesting further tests, the risks involved and OCT not being up-to-date. CONCLUSIONS: Most professionals have the criteria for OCT in NVAF clear, although they continue to avoid the initiation of OCT. The majority approach is to give anti-aggregants and refer to Cardiology, given the risk of the therapy and the difficulties involved in monitoring and requesting further tests. PMID- 12372210 TI - [Erectile dysfunction in primary care as possible marker of health status: associated factors and response to sildenafil]. AB - OBJECTIVES: To find the factors linked to erectile dysfunction, to evaluate this as a possible marker of health status, to analyse the evolution of clinical parameters of associated illnesses and the response to Viagra.Design. Intervention study without a control. SETTING: Alguazas Health Centre (Murcia). PARTICIPANTS: All the patients in the programme (125), with a figure on the sexual health in men index (SHIM) below 21.Interventions. Health education and administration of Viagra. MAIN MEASUREMENTS: Concomitant illnesses, pathologies previously unknown to the patient, changes in erectile function valued on the international index of erectile function (IIEF), and changes in blood pressure, glucaemia and lipids. RESULTS: Factors linked to erectile dysfunction were diabetes (50.4%), hypertension (33.6%), hypercholesterolaemia (22.4%), urological pathology (12.8%) and mental health disorders (33.6%). The hidden pathology detected was 15 cases of hypertension, 3 diabetes, 2 cardiopathies, 20 dyslipaemias, 3 depressions, 13 anxiety and 5 urological problems. The variations in clinical parameters at 3 months were: glucaemia, -38.3 mg (P<.001, Student s t=-5.186); HbA1c, -0.9 (P<.05, Student s t=-2.16); systolic blood pressure, -16 mm Hg (P<.01, Student s t=-3.486) and diastolic pressure -13 mm Hg (P<.001, Student s t=-4.594); and total cholesterol, -14.2% (P<.001, Student s t=7.01). Erectile function improved by 74% with Viagra. CONCLUSIONS: 2 out of every 3 patients with erectile dysfunction presented associated diseases; one in every 3 were ignorant of their health problem. Monitoring of chronic illnesses improved significantly. Finally, 3 in every 4 responded to Viagra. PMID- 12372211 TI - [Factors that affect the prescription of benzodiazepines and actions to improve their use: a Delphi study of primary care doctors]. AB - OBJECTIVES: To identify the factors that affect the prescription of benzodiazepines and similar drugs and the actions that can be taken to reduce their prescription.Design. Consensus method. Delphi technique. SETTING: Four primary care areas in Asturias. Participants. 39 doctors from primary care teams agreed voluntarily to take part in the study, and 32 completed the study. They belonged to 20 health centres. METHOD: They were sent by mail three questionnaires one after the other. The second and third questionnaires were worked out on the basis of the analysis of the information from the replies to the preceding questionnaire. Those who did not send in a reply were reminded by phone. RESULTS: The 5 most influential factors in benzodiazepine prescription were agreed: reduction in the threshold of tolerance of emotional discomfort; increase of the prevalence of pathologies; lack of time in the consulting-room; social and economic conditioning factors; properties of the benzodiazepine family. The 5 most important actions that could reduce prescription of these drugs were agreed: general health education; reduction in case loads; making doctors more conscious of prescribing correctly; strengthening the social support network; doctors fomenting use of effective alternative treatments. CONCLUSIONS: The prescription of benzodiazepines and their analogues is a multi-factorial action with social and psychological roots. The action most voted on to reduce their prescription was general health education. PMID- 12372212 TI - [Home care as an alternative to conventional hospital admission]. PMID- 12372213 TI - [Recommendations on the diagnostic and therapeutic approach to smokers. Consensus document]. PMID- 12372214 TI - [Palliative care: care in the final days]. PMID- 12372215 TI - [Treatment of urinary incontinence]. PMID- 12372216 TI - [Bronchial asthma crises and failures of oral contraceptives]. PMID- 12372217 TI - [Study of urinary infection in a district of Terrassa]. PMID- 12372218 TI - [Adolescents or under-age?]. PMID- 12372219 TI - [What is diabetics' real cardiovascular risk?]. PMID- 12372221 TI - [Influence of serotonergic transmission on response to olanzapine]. AB - INTRODUCTION: This study aimed to investigate associations between the response to olanzapine and genetic variations (polymorphisms) in serotonergic transmission related genes in a sample of prospectively studied schizophrenic patients treated with this drug. METHODOLOGY: A total of 51 non-related patients with a DSM-IV diagnosis of schizophrenia were treated with olanzapine (mean dose: 12 mg/day; range: 5-25 mg) and followed-up for at least three months. Response to olanzapine was measured by the difference between baseline and post-treatment scores on the PANSS and GAS scales. The following polymorphisms were studied: serotonin receptor 5-HT2A (102-T/C, His452Tyr), serotonin receptor 5-HT2C (Cys23Ser, -330 GGT/-244-CT), and serotonin transporter (VNTR, 5-HTTLPR). RESULTS: Global clinical improvement, measured with both the GAS and PANSS total scores, was observed. When patients were divided into responders and non-responders, the distribution of genotypic and allelic frequencies was similar to the one observed in previous studies with clozapine. When regression analyses were undertaken, polymorphism 330-GT/-244-CT of the 5-HT2C serotonin receptor and 5-HTTLPR of the serotonin transporter showed a tendency towards the association to olanzapine response. CONCLUSIONS: The present study provides preliminary evidence of the important role of variations in serotonin transmission related genes in determining clinical response to olanzapine. Considering previous studies, it can also be concluded that olanzapine and clozapine may have similar affinities to serotonin receptors. PMID- 12372222 TI - [Drop-out from out-patient treatment for alcohol dependence: a two-year prospective study]. AB - INTRODUCTION: Treatment compliance in alcohol-dependent patients seems closely related to abstinence rates, so it could be an outcome measure. The aim of our study was to identify which sociodemographic and clinical characteristics of alcohol-dependent patients are associated to high drop-out rates. METHOD: 165 alcohol-dependent out-patients were assessed by means of a structured questionnaire and followed for two years. RESULTS: It was shown a predictive value for personal history of suicidal intents or affective and anxiety disorders, comorbidity with affective and personality disorders, family history of affective disorders and psychiatric and somatic complications of dependence, and past or present abstinence. DISCUSSION: Implications of these findings and inconsistencies between previous studies and these results are discussed. PMID- 12372223 TI - [Predictors for hospitalization rates of patients with schizophrenia]. AB - INTRODUCTION: For clinicians and care providers, the use of hospital facilities by patients with schizophrenia is one of the areas of most concern in the field of mental health studies. The objective of this study is to determine those characteristics which relate to hospital admissions for this group of patients. METHOD: A one year follow up study for a group of 83 patients with schizophrenia in contact with mental health services was carried out to identify the socio demographic, clinical and care needs' variables related to hospital admissions. RESULTS: As a result of the multivariate models applied, male patients presenting hyperactivity, thought disturbance, deliria and exaggerated interpretation, socially embarrassing behaviour and use of drugs were shown to be those with a greater probability of hospital admission. PMID- 12372224 TI - [Paroxetine treatment for children and adolescents with anxiety disorders]. AB - BACKGROUND: Paroxetine has become an effectiveness treatment in anxiety disorders in adults. Despite the fact that this is an especially prevalent psychiatrist disorder in children and adolescents, there are very few studies in this population. This study examines the effectiveness of paroxetine in children and adolescents with anxiety disorders. METHODOLOGY: Fifteen children and adolescents with ICD-criteria for anxiety disorder were selected. Anxiety measurement was taken with STAI scale and was filled out before treatment and 6 months later (mean). We have used descriptive parameters and t Student test for the analysis of dependent samples. Statistic work was done with SPSS 8.0. RESULTS: On first testing, the mean score for State Factor was 41.8 (ds: 5.9) and on second after treatment- it was 24.66 (ds: 9.8). Trait Factor was 43.53 (ds: 8.27) on first testing and 25 (ds: 8.91) on second. These differences in mean scores for both State and Trait factors were significant (alpha=0.05, p= 0.000). CONCLUSIONS: Our results support the hypothesis of clinical improvement at Anxiety Disorders in children and adolescent using Paroxetine. It seems logical to continue the study increasing sample size and evaluation time. PMID- 12372225 TI - [Influence of behaviour, attitudes and childrearing on the development of the child in mothers with postpartum depression]. AB - INTRODUCTION: Various studies find an association between the postpartum depression and child development; nevertheless, it exists few uniformity in the method in which are accomplished these. The objective of this study is to analyze the influence of the postpartum depression on the development of the children during the first 28 months. Also, it is studied the childrearing of the mother, as possible mechanism for which exercises this influence. METHOD: Longitudinal and prospective study of 205 primiparous women and their children. The definitive sample understands 23 depressed women and 37 belonging to the group control that they are followed during the first 28 months postpartum. RESULTS: It is obtained a 13.5% from women with postpartum depression. The duration of the depression is related to a childrearing characterized by a under affection and care. The childrearing low in affection is associated negatively with the cognitive and social development of the son during the first year of life. The association of a greater duration of the depression with a childrearing low in affected during the first year has a negative affection on the cognitive development of the son to the 28 months. CONCLUSIONS: The mothers with postpartum depression accomplish a childrearing on their children characterized by a lower affection and care. The variable that more weigh develops on the development of the child during the period of study is the childrearing low in affection. PMID- 12372226 TI - [A user-friendly scale for the short and long term outcome of bipolar disorder: the CGI-BP-M]. AB - Bipolar disorder is a cyclic and polymorphic disease. Patients may show manic, hipomanic, depressive or mixed symptoms, and they may be in partial or complete remission. For this reason, the assessment of the course, severity and outcome of the disorder is very complex. Most of the available psychometric instruments have been designed for the assessment of acute episodes of specific polarity. METHODS: We present the modified version of the Clinical Global Impression for Bipolar Disorder (CGI-BP-M) a condensed version of the CGI-BP, which is also an adaptation of the CGI for bipolar patients. RESULTS: The CGI-BP-M takes only a few minutes to complete, and it has proved to be useful in the assessment of the short and long-term efficacy of several treatments such as olanzapine and quetiapine. Significant improvements in several subescales of the CGI-BP-M after treatment with these compounds are shown. CONCLUSIONS: The CGI-BP-M, a user friendly scale for the assessment of manic, hypomanic, depressive or mixed symptoms, and long-term outcome of bipolar disorder, is a useful tool for the assessment of the efficacy of several treatments. PMID- 12372227 TI - [Bipolar disorder: helping the treatment]. AB - INTRODUCTION: [corrected] Psychoeducation programs for bipolar patients try to give them a theoretical and practical system to understand and cope with the consequences of this disorder. The published studies reviewed support the idea that patients included in this therapeutic approach present higher treatment compliance. METHODOLOGY: The purpose of our study is to specify the benefits of the psychoeducation programs, comparing the degree of knowledge of the illness (Measured by means of the Understanding Mood Disorder Questionnaire Gavazzi et al, 1997, Spanish version of Livianos and Rojo, 1998) of 10 patients included in our program with that of another 10 patients who attended the same lithium clinic, but were not included in the psychoeducation program. RESULTS: There are statistically significant differences in the total of the scale as well as on the mania scale and known facts, but no differences on the depression scale. CONCLUSION: The results allow us to stress the impact of the psychoeducation programs on the knowledge of illness. PMID- 12372228 TI - [Functionality as a goal in the treatment of schizophrenia]. AB - Rational use of new atypical antipsychotics have allowed clinicians to have a more optimistic view, on functional outcome in Schizophrenia. Functional outcome is revised here as an essential concept to be kept in mind in the treatment and control of schizophrenic symptoms. This view allows for a better evaluation of the clinical meaning of symptoms and signs, and the impact on daily functioning of unwelcome side effects of some antipsychotics; and finally the impact of all these upon social functioning and may allow the clinician to implement some interventions in the clinical setting taking into account the ultimate and realistic goal in the treatment of schizophrenic patients: their functional outcome. PMID- 12372229 TI - [Cognitive impairment in a 35 years-old male]. AB - Cognitive impairment is a syndrome with multiple causes, presenting frequently neuropsychiatric symptoms. In these cases, the psychiatrist role is essential, including both diagnosis and therapy.HIV-associated dementia usually includes psychiatric symptoms, even in the absence of neurological symptoms in initial stages. Moreover, the prolonged life span of patients with HIV leaves the possibility that prevalence or HIV-associated neurologic disease increases in coming years. We report a case of a HIV male with insidious cognitive impairment, making diagnosis difficult due to ambiguous symptoms. The patient begins with depressive symptoms and slightly develops cognitive impairment. The presence of cognitive impairment in a young person must alert us think about HIV pathology, because it is one of the leading causes of dementia in the young. PMID- 12372230 TI - [Clozapine-induced obsessive-compulsive disorder: a case report]. AB - Since 1992 it has been reported that obsessive-compulsive behavior may emerge in patients treated with clozapine. We would like to report a case of obsessive compulsive behavior emerging after initiation of treatment with clozapine in a 22 year-old man with a diagnosis of schizoaffective disorder that was successfully treated with fluoxetine. PMID- 12372231 TI - [Structured interruptions in antiretroviral treatment: a new therapeutic strategy?]. PMID- 12372232 TI - [Parvovirus B19 outbreak in a rural community in Alicante]. AB - BACKGROUND: Parvovirus B19 (PVB19) has been identified as the cause of erythema infectiosum. The epidemiology of PVB19 has not been extensively studied in Spain or in the autonomic community of Valencia. The aim of this work is to describe an outbreak of PVB19 infection occurring in the area of Monforte del Cid, Alicante. METHODS: A probable case was defined as: all subjects living in Monforte who presented a rash (mainly facial) and/or arthralgia starting from November 1999. A confirmed case was defined as: a probable case confirmed by laboratory analysis or a case having an epidemiological link. Laboratory confirmation included specific IgG or IgM antibodies to PVB19. Cases were mainly detected through the Monforte del Cid Primary Health Care Center. RESULTS: The outbreak occurred from November 1999 to August 2000. A total of 118 cases were detected, giving an overall attack rate (AR) of 23.2 cases per 1,000 inhabitants. The highest rates were in the age groups of 0-4 years old (AR 5 114.5 per 1,000) and 5-9 years old (AR 5 180.3 per 1,000). By gender, the AR per 1,000 inhabitants was 26.9 in men and 16.7 in women. Two of the cases were pregnant women and one of them had a miscarriage. CONCLUSIONS: The outbreak of erythema infectiosum lasted 10 months and mainly affected children under 14 years old. Active surveillance was focussed on women in the first three months of pregnancy. PMID- 12372233 TI - [Detection of Enterococcus with high-level aminoglycoside and glycopeptide resistance in Lactuca sativa (lettuce)]. AB - BACKGROUND: The aim of this study was to assess the presence of enterococci highly resistant to aminoglycosides and glycopeptides in foods eaten uncooked, in order to evaluate their role as reservoirs of antimicrobial resistance. METHODS: We isolated 92 strains of enterococci from 79 samples of lettuce grown on farms near the city of Corrientes (Argentina). Enterococci were identified by standard methods. Antimicrobial susceptibility was determined by growth in azide dextrose broth supplemented with 500 microg/ml of gentamicin and 2,000 microg/ml of streptomycin, and by disk diffusion technique using disks with high levels of aminoglycosides, vancomycin and teicoplanin, in Muller Hinton agar. RESULTS: The most frequently detected species was E. faecium (32.61%), followed by E. faecalis (21.74%), E. gallinarum (13.04%), E. casseliflavus and E. mundtii (7.60%), E. hirae, (6.52%), E. durans (4.35%), E. raffinossus and E. saccharolyticus (2.17%), E. avium and E. malodoratus (1.10%). High-level resistance to streptomycin and gentamicin was found in 2 strains of E. Faecium. Resistance to streptomycin alone was observed in 2 strains of E. faecium, in 3 of E. hirae and in 1 of E. mundtii. Resistance only to gentamicin was not observed in any strain. None of the isolates showed glycopeptide resistance, except the intrinsically resistant species (E. gallinarum and E. casseliflavus). CONCLUSION: These results confirm the presence of enterococci within the community having susceptibility profiles similar to those of strains found in hospitals. PMID- 12372234 TI - [Evolution of the use of antibiotics in a hospital long-term care center in Catalonia]. AB - INTRODUCTION: Advanced age, together with immune system changes, malnutrition, chronic disease, and the institutional environment, all contribute to a higher risk of acquiring infection in the elderly. Antibiotics are widely used in geriatric centers, but often their use is not optimal. MATERIAL AND METHODS: Study carried out during the period 1992-1999 in Centro Sociosanitario Albada (Sabadell, Spain). Data were taken from the Pharmacy Department's unidose registry. We determined the most frequently used antibiotics, the hospital units with highest consumption, the variation in these factors over time, and related costs. RESULTS: A progressive increase in overall antibiotic consumption was observed during the first 5 years of the study with subsequent stabilization. The units showing highest consumption were the Moderate and Highly-Dependent Chronic Unit, the Palliative Care Unit and the Convalescence and Rehabilitation Unit, with significant increases in the Palliative Care Unit in the last two years of the study. Amoxicillin-clavulanate, ciprofloxacin and norfloxacin were the most extensively used antibiotics. Cost increases were seen in the last three years despite the stabilization of antibiotic use. CONCLUSION: We observed a change in the consumption and profile of the antimicrobial agents used in our setting, probably related to changes in the population, increases in parenteral treatment and changes in the criteria for treatment of terminal patients. The establishment of controls for antibiotic use in long-term care centers would lead to improvements in the quality of the care provided. PMID- 12372235 TI - [Use of MPO chromogenic culture medium for routine processing of urine cultures]. AB - BACKGROUND: The chromogenic culture medium, MPO, was compared to culture on CLED (cystein, lactose, electrolyte-deficient) agar for the detection, enumeration and identification of urinary tract pathogens. METHODS: A total of 1,080 clinical urine specimens were assessed. All samples were inoculated in MPO and CLED using the calibrated loop method. RESULTS: Among 145 positive urine samples, 171 strains of bacteria were isolated (111 Escherichia coli, 26 Enterococcus spp., 12 Proteus spp., 10 Enterobacteriaceae from the Klebsiella-Enterobacter-Serratia group, 5 Pseudomonas aeruginosa, 4 Streptococcus agalactiae, 3 Staphylococcus spp. and 4 Candida albicans. For all samples, enumeration of microorganisms was comparable with the two media studied. Identification was also similar, except for 6 cases in which Enterococcus spp. were only detected with the chromogenic medium. CONCLUSIONS: Overall urine culture results with MPO chromogenic medium were similar to those obtained with CLED, making it a feasible alternative to the standard medium. Moreover, use of a chromogenic technique implies a significant reduction in workload, since additional tests to identify the microorganisms isolated are not needed in most cases. PMID- 12372236 TI - [Recommendations for non-occupational postexposure HIV prophylaxis. Spanish Working Group on Non-Occupational Postexposure HIV Prophylaxis of the Catalonian Center for Epidemiological Studies on AIDS and the AIDS Study Group]. AB - Evidence is lacking on the possible efficacy and effectiveness of non occupational postexposure prophylaxis (PEP). However, because of its biological plausibility, the use of antiretroviral (ARV) drugs to prevent the development of infection in certain cases of accidental or sporadic exposure has begun to be considered as common clinical practice. Previous studies performed in Spain have demonstrated both the demand and the prescription of ARV as PEP and especially the diversity and inconsistency in the criteria used. In this context, in April of 2000 the Centre for Epidemiological Studies on AIDS of Catalonia (CEESCAT) (Department of Health and Social Security of the Autonomous Government of Catalonia), in collaboration with the National AIDS Plan and the AIDS Study Group (GESIDA), promoted the creation of a working group for the drafting of recommendations for PEP against HIV outside the occupational health context. The recommendations have been made bearing in mind the exceptional character of the exposure, the time elapsed since exposure, as well as evaluation of the risk of infection according to the type of exposure and the information available on the source of infection. In addition, the recommendations include the immediate measures necessary, as well as the preventive measures and clinical follow-up required both for HIV and for other infectious agents. All PEP regimens should be started within 72 hours of exposure and appropriate daily doses of two nucleoside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor (PI), or two NRTIs and a non-nucleoside reverse transcriptase inhibitor (NNRTIs), should be administered for four weeks, bearing in mind the pharmacological and clinical situation of the source person. These recommendations should be updated periodically. PMID- 12372237 TI - [Respiratory and renal insufficiency in a COPD patient receiving corticoid treatment]. PMID- 12372238 TI - [Movies as a teaching resource for infectious diseases and clinical microbiology]. AB - Since its inception, the cinema has constantly provided a reflection of infectious diseases because of their omnipresence in life and their importance to individuals and society. Few infectious diseases escape its eye, to the extent that the cinema constitutes an authentic treatise on these phenomena. The cinema is a very valuable educational resource, able to supplement classical teaching methods and to encourage critical thinking among students. The enormous flow of information, images, sounds, consequences, situations, and points of view that it provides should not be wasted and can be of great use, both in the spread of ideas and in training in infectious diseases and clinical microbiology. PMID- 12372239 TI - [Pharmacokinetic and pharmacodynamic concepts for an interpretative reading of the antibiogram]. AB - Both microbiological and epidemiological reading of the antibiogram can be performed without additional pharmacokinetic or pharmacodynamic concepts. However, when the aim is a clinical reading of the antibiogram, knowledge of the pharmacokinetics of antimicrobial agents and of all phenomena occurring between antimicrobial agents and microorganisms is imperative. Pharmacokinetics includes the study of absorption, distribution, metabolism and elimination of drugs. These data provide information on the active concentrations of antimicrobial agents in blood and other fluids as well as in the tissues where infection may develop. Knowledge of metabolism and of elimination pathways complete the main pharmacokinetic parameters of antimicrobial agents. The bactericidal effect of antimicrobial agents can be either concentration- or time-dependent. In the former, high concentrations of antimicrobial agents, much higher than their corresponding MICs, are needed, while in the latter, maintaining the concentration of antimicrobial agents at levels slightly higher than their MICs over time is more important. With these concepts, a microbiological pharmacological reading of the antibiogram, taking into account dosage, administration pathway and location of the infectious process among other factors, can be achieved. Most working groups, either national or from abroad, have considered both pharmacokinetics and pharmacodynamics in interpretative reading of the antibiogram. In all cases, breakpoints for susceptibility and resistance should be corrected on the basis of data from well designed and performed clinical trials. Clinically oriented breakpoints do not necessarily have to be the same as those based on microbiological or epidemiological concepts, but clinical microbiologists should be able to give an appropriate response to the question and to the application based on that response. PMID- 12372240 TI - [Seroprevalence of hepatitis A]. PMID- 12372241 TI - [Discrepancies between the Coombs anti-Brucella test and the Brucallacapt test]. PMID- 12372242 TI - [Encephalitis as the first manifestation of herpes zoster]. PMID- 12372243 TI - [Endocarditis due to Bartonella henselae on a native valve. A new case with some notable aspects]. PMID- 12372244 TI - [Babesia microti: a new form of human babesiosis in Europe?]. PMID- 12372245 TI - [Complete auriculoventricular block in a patient treatment with Lopinavir/Ritonavir]. PMID- 12372246 TI - Evolution of yellow gene regulation and pigmentation in Drosophila. AB - BACKGROUND: Changes in developmental gene expression are central to phenotypic evolution, but the genetic mechanisms underlying these changes are not well understood. Interspecific differences in gene expression can arise from evolutionary changes in cis-regulatory DNA and/or in the expression of trans acting regulatory proteins, but few case studies have distinguished between these mechanisms. Here, we compare the regulation of the yellow gene, which is required for melanization, among distantly related Drosophila species with different pigment patterns and determine the phenotypic effects of divergent Yellow expression. RESULTS: Yellow expression has diverged among D. melanogaster, D. subobscura, and D. virilis and, in all cases, correlates with the distribution of black melanin. Species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression. Evolutionary changes in the activity of orthologous cis-regulatory elements are responsible for differences in abdominal Yellow expression; however, cis-regulatory element evolution is not the sole cause of divergent Yellow expression patterns. Transformation of the D. melanogaster yellow gene into D. virilis altered its expression pattern, indicating that trans-acting factors that regulate the D. melanogaster yellow gene have also diverged between these two species. Finally, we found that the phenotypic effects of evolutionary changes in Yellow expression depend on epistatic interactions with other genes. CONCLUSIONS: Evolutionary changes in Yellow expression correlate with divergent melanin patterns and are a result of evolution in both cis- and trans-regulation. These changes were likely necessary for the divergence of pigmentation, but evolutionary changes in other genes were also required. PMID- 12372247 TI - The gibberellin pathway mediates KNOTTED1-type homeobox function in plants with different body plans. AB - BACKGROUND: The shoot apical meristem (SAM) is an indeterminate structure that gives rise to the aerial parts of higher plants. Leaves arise from the differentiation of cells at the flanks of the SAM. Current evidence suggests that the precise regulation of KNOTTED1-like homeobox (KNOX) transcription factors is central to the acquisition of leaf versus meristem identity in a wide spectrum of plant species. Factors required to repress KNOX gene expression in leaves have recently been identified. Additional factors such as the CHD3 chromatin remodeling factor PICKLE (PKL) act to restrict meristematic activity in Arabidopsis leaves without repressing KNOX gene expression. Less is known regarding downstream targets of KNOX function. Recent evidence, however, has suggested that growth regulators may mediate KNOX activity in a variety of plant species. RESULTS: Here we show that reduced activity of the gibberellin (GA) growth regulator pathway promotes meristematic activity, both in the natural context of KNOX function in the SAM and upon ectopic KNOX expression in Arabidopsis leaves. We show that constitutive signaling through the GA pathway is detrimental to meristem maintenance. Furthermore, we provide evidence that one of the functions of the KNOX protein SHOOTMERISTEMLESS (STM) is to exclude transcription of the GA-biosynthesis gene AtGA20ox1 from the SAM. We also demonstrate that AtGA20ox1 transcript is reduced in the pkl mutant in a KNOX independent manner. Moreover, we show a similar interaction between KNOX proteins and GA-biosynthesis gene expression in the tomato leaf and implicate this interaction in regulation of the dissected leaf form. CONCLUSIONS: We suggest that repression of GA activity by KNOX transcription factors is a key component of meristem function. Transfer of the KNOX/GA regulatory module from the meristem to the leaf may have contributed to the generation of the diverse leaf morphologies observed in higher plants. PMID- 12372248 TI - Transcriptional profile of aging in C. elegans. AB - BACKGROUND: Numerous gerontogene mutants leading to dramatic life extensions have been identified in the nematode Caenorhabditis elegans over the last 20 years. Analysis of these mutants has provided a basis for understanding the mechanisms driving the aging process(es). Several distinct mechanisms including an altered rate of aging, increased resistance to stress, decreased metabolic rate, or alterations in a program causing organismic aging and death have been proposed to underlie these mutants. RESULTS: Whole-genome analysis of gene expression during chronological aging of the worm provides a rich database of age-specific changes in gene expression and represents one way to distinguish among these models. Using a rigorous statistical model with multiple replicates, we find that a relatively small number of genes (only 164) show statistically significant changes in transcript levels as aging occurs (<1% of the genome). Expression of heat shock proteins decreases, while expression of certain transposases increases in older worms, and these findings are consistent with a higher mortality risk due to a failure in homeostenosis and destabilization of the genome in older animals. Finally, a specific subset of genes is coordinately altered both during chronological aging and in the transition from the reproductive form to the dauer, demonstrating a mechanistic overlap in aging between these two processes. CONCLUSIONS: We have performed a whole-genome analysis of changes in gene expression during aging in C. elegans that provides a molecular description of C. elegans senescence. PMID- 12372249 TI - Rhythms of mammalian body temperature can sustain peripheral circadian clocks. AB - BACKGROUND: Low-amplitude temperature oscillations can entrain the phase of circadian rhythms in several unicellular and multicellular organisms, including Neurospora and Drosophila. Because mammalian body temperature is subject to circadian variations of 1 degrees C-4 degrees C, we wished to determine whether these temperature cycles could serve as a Zeitgeber for circadian gene expression in peripheral cell types. RESULTS: In RAT1 fibroblasts cultured in vitro, circadian gene expression could be established by a square wave temperature rhythm with a (Delta)T of 4 degrees C (12 hr 37 degrees C/12 hr 33 degrees C). To examine whether natural body temperature rhythms can also affect circadian gene expression, we first measured core body temperature cycles in the peritoneal cavities of mice by radiotelemetry. We then reproduced these rhythms with high precision in the liquid medium of cultured fibroblasts for several days by means of a homemade computer-driven incubator. While these "in vivo" temperature rhythms were incapable of establishing circadian gene expression de novo, they could maintain previously induced rhythms for multiple days; by contrast, the rhythms of control cells kept at constant temperature rapidly dampened. Moreover, circadian oscillations of environmental temperature could reentrain circadian clocks in the livers of mice, probably via the changes they imposed upon both body temperature and feeding behavior. Interestingly, these changes in ambient temperature did not affect the phase of the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. CONCLUSIONS: We postulate that both endogenous and environmental temperature cycles can participate in the synchronization of peripheral clocks in mammals. PMID- 12372250 TI - A common cortical substrate activated by horizontal and vertical sound movement in the human brain. AB - Perception of movement in acoustic space depends on comparison of the sound waveforms reaching the two ears (binaural cues) as well as spectrotemporal analysis of the waveform at each ear (monaural cues). The relative importance of these two cues is different for perception of vertical or horizontal motion, with spectrotemporal analysis likely to be more important for perceiving vertical shifts. In humans, functional imaging studies have shown that sound movement in the horizontal plane activates brain areas distinct from the primary auditory cortex, in parietal and frontal lobes and in the planum temporale. However, no previous work has examined activations for vertical sound movement. It is therefore difficult to generalize previous imaging studies, based on horizontal movement only, to multidimensional auditory space perception. Using externalized virtual-space sounds in a functional magnetic resonance imaging (fMRI) paradigm to investigate this, we compared vertical and horizontal shifts in sound location. A common bilateral network of brain areas was activated in response to both horizontal and vertical sound movement. This included the planum temporale, superior parietal cortex, and premotor cortex. Sounds perceived laterally in virtual space were associated with contralateral activation of the auditory cortex. These results demonstrate that sound movement in vertical and horizontal dimensions engages a common processing network in the human cerebral cortex and show that multidimensional spatial properties of sounds are processed at this level. PMID- 12372251 TI - Virtual-reality techniques resolve the visual cues used by fruit flies to evaluate object distances. AB - Insects can estimate distance or time-to-contact of surrounding objects from locomotion-induced changes in their retinal position and/or size. Freely walking fruit flies (Drosophila melanogaster) use the received mixture of different distance cues to select the nearest objects for subsequent visits. Conventional methods of behavioral analysis fail to elucidate the underlying data extraction. Here we demonstrate first comprehensive solutions of this problem by substituting virtual for real objects; a tracker-controlled 360 degrees panorama converts a fruit fly's changing coordinates into object illusions that require the perception of specific cues to appear at preselected distances up to infinity. An application reveals the following: (1) en-route sampling of retinal-image changes accounts for distance discrimination within a surprising range of at least 8-80 body lengths (20-200 mm). Stereopsis and peering are not involved. (2) Distance from image translation in the expected direction (motion parallax) outweighs distance from image expansion, which accounts for impact-avoiding flight reactions to looming objects. (3) The ability to discriminate distances is robust to artificially delayed updating of image translation. Fruit flies appear to interrelate self-motion and its visual feedback within a surprisingly long time window of about 2 s. The comparative distance inspection practiced in the small fruit fly deserves utilization in self-moving robots. PMID- 12372252 TI - Metalloproteinase shedding of Fas ligand regulates beta-amyloid neurotoxicity. AB - Extracellular deposits of beta-amyloid (Abeta) peptide closely match areas of neuronal loss in, and are a postmortem diagnostic indicator of, Alzheimer's disease. Neuronal cultures treated with fibrillar Abeta can be protected from neurotoxicity by caspase-8 inhibition or the expression of dominant-negative FADD, both of which are components of the Fas death receptor pathway, and neurons with defective Fas and FasL are resistant to Abeta neurotoxicity. The receptor binding region of FasL can be shed from cells by metalloproteinases, and this process greatly reduces its proapoptotic activity. Here, we show that factors affecting the shedding of membrane-bound FasL significantly impact Abeta neurotoxicity. A broad-spectrum metalloproteinase inhibitor, GM6001/Ilomastat, acted synergistically with Abeta to enhance neurotoxicity through a FasL dependent mechanism. The disruption of ADAM-based metalloproteinase activity was likely responsible, as MMP-inhibiting TIMPs had no such effect. In contrast, enhanced FasL shedding, by recombinant MMP-7, completely protected neurons from Abeta neurotoxicity. These findings suggest that factors that affect metalloproteinase-mediated shedding of FasL may play a role in the etiology of Alzheimer's disease and may provide an avenue for therapeutic intervention. PMID- 12372253 TI - Mouse gridlock: no aortic coarctation or deficiency, but fatal cardiac defects in Hey2 -/- mice. AB - Gridlock (grl) is one of the first mutations characterized from the large zebrafish mutagenesis screens, and it results in an arterial (aortic) maturation defect, which was proposed to resemble aortic coarctation, a clinically important human malformation. While the grl mutation appears to be a hypomorph, grl knockdown experiments have shown even stronger effects on arterial development. We have generated a knockout of the murine Hey2 (gridlock) gene to analyze the mammalian phenotype. Surprisingly, Hey2 loss does not affect aortic development, but it instead leads to a massive postnatal cardiac hypertrophy with high lethality during the first 10 days of life. This cardiomyopathy is ameliorated with time in surviving animals that do not appear to be manifestly impaired during adult life. These differences in phenotypes suggest that changes in expression or function of genes during evolution may lead to quite different pathological phenotypes, if impaired. PMID- 12372254 TI - Tetralogy of fallot and other congenital heart defects in Hey2 mutant mice. AB - Congenital malformations of the heart and circulatory system are the most common type of human birth defect. Recent studies have implicated the Notch signaling pathway in human cardiac development by demonstrating abnormalities of the JAG1 gene as the basis for Alagille syndrome and some cases of isolated tetralogy of Fallot or pulmonic stenosis. How the Notch pathway acts in cardiac development remains unknown, but the Hey family of basic helix-loop-helix (bHLH) transcription factors are candidates for mediating Notch signaling in the developing cardiovascular system. Here, we use gene targeting to determine the developmental functions of mouse Hey2, a Hey family member that is expressed during the embryonic development of the heart, arteries, and other organs. Homozygotes for the Hey2 mutant allele display a spectrum of cardiac malformations including ventricular septal defects, tetralogy of Fallot, and tricuspid atresia, defects that resemble those associated with mutations of human JAG1. These results establish Hey2 as an important regulator of cardiac morphogenesis and suggest a role for Hey2 in mediating or modulating Notch signaling in the developing heart. PMID- 12372255 TI - Drosophila atonal fully rescues the phenotype of Math1 null mice: new functions evolve in new cellular contexts. AB - Many genes share sequence similarity between species, but their properties often change significantly during evolution. For example, the Drosophila genes engrailed and orthodenticle and the onychophoran gene Ultrabithorax only partially substitute for their mouse or Drosophila homologs. We have been analyzing the relationship between atonal (ato) in the fruit fly and its mouse homolog, Math1. In flies, ato acts as a proneural gene that governs the development of chordotonal organs (CHOs), which serve as stretch receptors in the body wall and joints and as auditory organs in the antennae. In the fly CNS, ato is important not for specification but for axonal arborization. Math1, in contrast, is required for the specification of cells in both the CNS and the PNS. Furthermore, Math1 serves a role in the development of secretory lineage cells in the gut, a function that does not parallel any known to be served by ato. We wondered whether ato and Math1 might be more functionally homologous than they appear, so we expressed Math1 in ato mutant flies and ato in Math1 null mice. To our surprise, the two proteins are functionally interchangeable. PMID- 12372256 TI - The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs. AB - A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, we still lack the exact nature of its binding site. We have now identified and characterized the N-WASP WH1 binding motif in WIP in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif we have identified is conserved in WIP, CR16, WICH, and yeast verprolin. PMID- 12372257 TI - Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo. AB - The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP 2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs. PMID- 12372260 TI - Back to the public over the GM crop battle. PMID- 12372258 TI - Mouse Dispatched homolog1 is required for long-range, but not juxtacrine, Hh signaling. AB - Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh. PMID- 12372261 TI - Hidden success of German university reform. PMID- 12372262 TI - Ciona. PMID- 12372263 TI - The Drosophila clock protein Timeless is a member of the Arm/HEAT family. PMID- 12372264 TI - Germ cell migration: as slow as molasses. AB - In Drosophila embryos, germ cells and somatic cells are formed separately. A recent analysis of the slow as molasses (slam) gene provides a potential link between somatic cell formation and germ cell migration. PMID- 12372265 TI - Insect vision: controlling actions through optic flow. AB - Insects depend upon optic flow to supply much of their information about the three-dimensional structure of the world. Many insects use translational flow to measure the distance of objects from themselves. A recent study has provided new insights into the way Drosophila use optic flow to pick out a close target to approach. PMID- 12372266 TI - Centriole duplication: centrin in on answers? AB - Many aspects of centriole biology remain mysterious. A new study has shed light on the role of the centriolar protein centrin-2: reducing levels of centrin-2 in HeLa cells has been found to block centriole duplication, eventually leading to cell death. PMID- 12372267 TI - Transcription activation: unveiling the essential nature of TFIID. AB - The TAF subunits of TFIID mediate activation of subsets of the eukaryotic genome. Recent results demonstrate that TFIID is recruited to promoters in an activator specific manner involving functional interaction between upstream regulatory elements and the core promoter, thereby coordinating the expression of distinct sets of genes. PMID- 12372268 TI - Ribosome biogenesis: ribosomal RNA synthesis as a package deal. AB - Ribosome biogenesis encompasses a complicated series of events involving hundreds of transiently interacting components. Insight into a mechanism for coordinating some of these events may come from characterization of a functional processing complex. PMID- 12372269 TI - Retinal processing: smaller babies thrown out with bathwater. AB - Rod bipolar cells in the mammalian retina receive synaptic input from many noisy rod photoreceptors. When photons are scarce, linear addition of inputs would swamp signals with noise. A nonlinearity at the synapse optimizes the signal to noise ratio. PMID- 12372270 TI - Limb patterning: reports of model's death exaggerated. AB - The progress zone model for the specification of positional values for patterning the proximodistal axis of the vertebrate limb has been questioned, but the results can be largely reconciled with the old model. PMID- 12372271 TI - Glutamate receptors: desensitizing dimers. AB - Recent structural studies show, not only how the desensitization of a ligand gated ion channel with bound agonist can be rationalized in terms of subunit subunit instability, but also how a previously unknown mode of interaction may provide clues into how the receptor is tetramerically assembled in vivo. PMID- 12372272 TI - New directions for fluorescent speckle microscopy. AB - Fluorescent Speckle Microscopy (FSM) is a technology for analyzing cytoskeleton dynamics, giving novel insight into their roles in living cells. New applications of FSM, together with the development of computer-based FSM image analysis, will make FSM the first microscopy-based method to deliver quantitative kinetic readouts at high spatial and temporal resolution for a wide variety of macromolecular systems. Here, we review the most recent applications and developments and give a glimpse of future directions and potentials of FSM. PMID- 12372273 TI - Lurcher, nPIST, and autophagy. AB - Previous work has shown that neurodegeneration in the lurcher mouse is due to a mutation in the GluRdelta2 gene that results in a constitutively active glutamate receptor ion channel. Characterization of the cell death pathway in these animals reported by Yue et al. in this issue of Neuron provides important insight into the toxicity induced by the abundant transmitter glutamate. Through protein protein interactions, the GluRdelta2(Lc) mutant channel activates autophagy. PMID- 12372274 TI - Trapping the sensor. AB - Voltage-gated ion channels open in response to a change in membrane potential. The "sensor," or the channel's molecular entity responsible for the detection of voltage change, is formed by a transmembrane element, rich with basic residues, called the "voltage sensor" or the "S4 domain." The movement of the S4 drives a global conformational change leading to the opening of the permeation pathway and ion conduction. In this issue of Neuron, Schonherr and colleagues show that physical constrains of the "gating canal," or the crevice through which the S4 moves, determines whether voltage-gated potassium channels open quickly or slowly. PMID- 12372275 TI - Forgetting those painful moments. AB - We all know that memories fade-although not always as quickly as we would like. What molecular and cellular processes underlie forgetting? In this issue of Neuron, Schwaerzel et al. indicate that extinction of an odor memory in Drosophila may involve the same neurons as those involved in forming the memory. PMID- 12372276 TI - Frontally mediated control processes contribute to source memory retrieval. AB - Remembering is a cognitively demanding task that requires the strategic selection of information from memory. In this issue of Neuron, Dobbins et al. present functional MRI (fMRI) data that shed insight into the specific, dissociated contributions of frontal regions to remembering. PMID- 12372277 TI - Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington's disease and related disorders. AB - The mechanisms of neurodegeneration in the CAG repeat polyglutamine diseases, including Spinal and Bulbar Muscular Atrophy (SBMA), Huntington's disease (HD), DentatoRubral and PallidoLuysian Atrophy (DRPLA), and Spino-Cerebellar Ataxia (SCA), have been controversial. Issues have included the role of polyglutamine aggregation and possible amyloid formation, localization in the cell nucleus, and possible proteolytic processing. Proposed mechanisms have included activation of caspases or other triggers of apoptosis, mitochondrial or metabolic toxicity, and interference with gene transcription. Recent studies using transgenic mouse and Drosophila models have helped resolve some of these issues and raise hopes for development of therapeutic targets. PMID- 12372278 TI - Fine-tuning motor neuron properties: signaling from the periphery. AB - Our understanding of motor neuron differentiation is rapidly evolving. New studies demonstrate that cells in the periphery of the embryo provide feedback signals for spinal cord motor neurons that are instrumental in the timing and regulation of their development. Two papers in this issue of Neuron identify a motor neuron survival factor, GDNF, and the ETS transcription factor, PEA3, as key components of a signal transduction pathway whose goals are 2-fold: to cluster motor pool-specific cell bodies and to promote axon arborization. PMID- 12372279 TI - Making connections in the fly visual system. AB - Understanding the molecular mechanisms that regulate formation of precise patterns of neuronal connections within the central nervous system remains a challenging problem in neurobiology. Genetic studies in worms and flies and molecular studies in vertebrate systems have led to an increasingly sophisticated understanding of how growth cones navigate toward their targets and form topographic maps. Considerably less is known about how growth cones recognize their cellular targets and form synapses with them. Here, we review connection formation in the fly visual system, the methodological approaches used to study it, and recent progress in uncovering the molecular basis of connection specificity. PMID- 12372280 TI - Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy. AB - Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. We generated a transgenic mouse model carrying a full-length AR containing 97 CAGs. Three of the five lines showed progressive muscular atrophy and weakness as well as diffuse nuclear staining and nuclear inclusions consisting of the mutant AR. These phenotypes were markedly pronounced in male transgenic mice, and dramatically rescued by castration. Female transgenic mice showed only a few manifestations that markedly deteriorated with testosterone administration. Nuclear translocation of the mutant AR by testosterone contributed to the phenotypic difference with gender and the effects of hormonal interventions. These results suggest the therapeutic potential of hormonal intervention for SBMA. PMID- 12372281 TI - Androgen-dependent neurodegeneration by polyglutamine-expanded human androgen receptor in Drosophila. AB - Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset, neurodegenerative disorder affecting only males and is caused by expanded polyglutamine (polyQ) stretches in the N-terminal A/B domain of human androgen receptor (hAR). Although no overt phenotype was detected in adult fly eye photoreceptor neurons expressing mutant hAR (polyQ 52), ingestion of androgen or its known antagonists caused marked neurodegeneration with nuclear localization and structural alteration of the hAR mutant. Ligand-independent toxicity was detected with a truncated polyQ-expanded A/B domain alone, which was attenuated with cytosolic trapping by coexpression of the unliganded hAR E/F ligand binding domain. Thus, our findings suggest that the full binding of androgen to the polyQ expanded hAR mutants leads to structural alteration with nuclear translocation that eventually results in the onset of SBMA in male patients. PMID- 12372282 TI - LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal. AB - Adult neural stem cells are rare, and little is known about their unique characteristics, leaving their in vivo identity enigmatic. We show that Lewis X (LeX), a carbohydrate expressed by embryonic pluripotent stem cells, is made by adult mouse subventricular zone (SVZ) stem cells and shed into their environment. Only 4% of acutely isolated SVZ cells are LeX(+); this subpopulation, purified by FACS, contains the SVZ stem cells. Ependymal cells are LeX(-), and purified ependymal cells do not make neurospheres, resolving the controversial claim that these are stem cells. Thus, LeX expression by adult CNS stem cells aids their in vivo identification, allows their enrichment, and raises new questions about the role of this unusual carbohydrate in stem cell biology. PMID- 12372283 TI - ETS gene Pea3 controls the central position and terminal arborization of specific motor neuron pools. AB - The projection of developing axons to their targets is a crucial step in the assembly of neuronal circuits. In the spinal cord, the differentiation of specific motor neuron pools is associated with the expression of ETS class transcription factors, notably PEA3 and ER81. Their initial expression coincides with the arrival of motor axons in the vicinity of muscle targets and depends on limb-derived signals. We show that in Pea3 mutant mice, the axons of specific motor neuron pools fail to branch normally within their target muscles, and the cell bodies of these motor neurons are mispositioned within the spinal cord. Thus, the induction of an intrinsic program of ETS gene expression by peripheral signals is required to coordinate the central position and terminal arborization of specific sets of spinal motor neurons. PMID- 12372284 TI - GDNF acts through PEA3 to regulate cell body positioning and muscle innervation of specific motor neuron pools. AB - Target innervation by specific neuronal populations involves still incompletely understood interactions between central and peripheral factors. We show that glial cell line-derived neurotrophic factor (GDNF), initially characterized for its role as a survival factor, is present early in the plexus of the developing forelimb and later in two muscles: the cutaneus maximus and latissimus dorsi. In the absence of GDNF signaling, motor neurons that normally innervate these muscles are mispositioned within the spinal cord and muscle invasion by their axons is dramatically reduced. The ETS transcription factor PEA3 is normally expressed by these motor neurons and fails to be induced in most of them in GDNF signaling mutants. Thus, GDNF acts as a peripheral signal to induce PEA3 expression in specific motor neuron pools thereby regulating both cell body position and muscle innervation. PMID- 12372285 TI - Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex. AB - Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A. PMID- 12372286 TI - A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice. AB - Autophagy is a pathway for bulk degradation of subcellular constituents that is hyperactivated in many neurodegenerative conditions. It has been considered a second form of programmed cell death. Death of cerebellar Purkinje cells in lurcher animals is due to a mutation in GluRdelta2 that results in its constitutive activation. Here we have identified protein interactions between GluRdelta2, a novel isoform of a PDZ domain-containing protein (nPIST) that binds to this receptor, and Beclin1. nPIST and Beclin1 can synergize to induce autophagy. GluRdelta2(Lc), but not GluRdelta2(wt), can also induce autophagy. Furthermore, dying lurcher Purkinje cells contain morphological hallmarks of autophagic death in vivo. These results provide strong evidence that a direct link exists between GluRdelta2(Lc) receptor and stimulation of the autophagic pathway in dying lurcher Purkinje cells. PMID- 12372287 TI - Conformational switch between slow and fast gating modes: allosteric regulation of voltage sensor mobility in the EAG K+ channel. AB - Voltage-gated EAG K+ channels switch between fast and slow gating modes in a Mg2+ dependent manner by an unknown mechanism. We analyzed molecular motions in and around the voltage-sensing S4 in bEAG1. Using accessibility and perturbation analyses, we found that activation increases both the charge occupancy and volume of S4 side chains in the gating canal. Fluorescence measurements suggest that mode switching is due to a motion of the S2/S3 side of the gating canal. We propose that when S4 is in the resting state and its thin end is in the gating canal, a conformational rearrangement of S2/S3 narrows the canal around S4, forming the Mg2+ binding site. Binding of Mg2+ is proposed to stabilize this conformation and to slow opening of the gate by impeding S4's voltage-sensing outward motion. PMID- 12372288 TI - Extinction antagonizes olfactory memory at the subcellular level. AB - Memory loss occurs by diverse mechanisms, as different time constants of performance decrement and sensitivities to experimental manipulations suggest. While the phenomena of memory decay, interference, and extinction are well established behaviorally, little is known about them at the circuit or molecular level. In Drosophila, odorant memories lasting up to 3 hr can be localized to mushroom body Kenyon cells, a single neuronal level in the olfactory pathway. The plasticity underlying this memory trace can be induced without Kenyon cell synaptic output. Experimental extinction, i.e., presentation of the conditioned stimulus without the reinforcer, reduces memory performance and does so at the same circuit level as memory formation. Thus, unreinforced presentation of learned odorants antagonizes intracellularly the signaling cascade underlying memory formation. PMID- 12372289 TI - Saccadic eye movements modulate visual responses in the lateral geniculate nucleus. AB - We studied the effects of saccadic eye movements on visual signaling in the primate lateral geniculate nucleus (LGN), the earliest stage of central visual processing. Visual responses were probed with spatially uniform flickering stimuli, so that retinal processing was uninfluenced by eye movements. Nonetheless, saccades had diverse effects, altering not only response strength but also the temporal and chromatic properties of the receptive field. Of these changes, the most prominent was a biphasic modulation of response strength, weak suppression followed by strong enhancement. Saccadic modulation was widespread, and affected both of the major processing streams in the LGN. Our results demonstrate that during natural viewing, thalamic response properties can vary dramatically, even over the course of a single fixation. PMID- 12372290 TI - Neural correlates of visual working memory: fMRI amplitude predicts task performance. AB - We used fMRI to investigate how moment-to-moment neural activity contributes to success or failure on individual trials of a visual working memory (WM) task. We found that different nodes of a distributed cortical network were activated to a greater extent for correct compared to incorrect trials during stimulus encoding, memory maintenance during delays, and at test. A logistic regression analysis revealed that the fMRI signal amplitude during the delay interval in a network of frontoparietal regions predicted successful performance on a trial-by-trial basis. Differential delay activity occurred even for only those trials in which BOLD activity during encoding was strong, demonstrating that it was not a simple consequence of effective versus ineffective encoding. Our results indicate that accurate memory depends on strong sustained signals that span the delay interval of WM tasks. PMID- 12372291 TI - Executive control during episodic retrieval: multiple prefrontal processes subserve source memory. AB - During recognition, one may sense items as familiar (item memory) and additionally recollect specific contextual details of the earlier encounters (source memory). Cognitive theory suggests that, unlike item memory, source memory requires controlled cue specification and monitoring processes. Functional imaging suggests that such processes may depend on left prefrontal cortex (PFC). However, the nature and possible anatomical segregation of these processes remains unknown. Using functional magnetic resonance imaging, we isolated distinct response patterns in left PFC during source memory consistent with semantic analysis/cue specification (anterior ventrolateral), recollective monitoring (posterior dorsolateral and frontopolar), and phonological maintenance/rehearsal (posterior ventrolateral). Importantly, cue specification and recollective monitoring responses were not seen during item memory and were unaffected by retrieval success, demonstrating that the mere attempt to recollect episodic detail engages multiple control processes with different left PFC substrates. PMID- 12372292 TI - Anatomical correlates of learning novel speech sounds. AB - We examined the relationship between brain anatomy and the ability to learn nonnative speech sounds, as well as rapidly changing and steady-state nonlinguistic sounds, using voxel-based morphometry in 59 healthy adults. Faster phonetic learners appeared to have more white matter in parietal regions, especially in the left hemisphere. The pattern of results was similar for the rapidly changing but not for the steady-state nonlinguistic stimuli, suggesting that morphological correlates of phonetic learning are related to the ability to process rapid temporal variation. Greater asymmetry in the amount of white matter in faster learners may be related to greater myelination allowing more efficient neural processing, which is critical for the ability to process certain speech sounds. PMID- 12372293 TI - Rothman and Schekman SNAREd by Lasker for trafficking. AB - This year, the recipients of the Lasker Award for Basic Medical Research are James Rothman and Randy Schekman. This highly anticipated honor highlights their unique contributions to our understanding of the mechanisms of membrane traffic. PMID- 12372294 TI - Structures of SET domain proteins: protein lysine methyltransferases make their mark. AB - Proteins bearing the widely distributed SET domain have been shown to methylate lysine residues in histones and other proteins. In this issue, three-dimensional structures are reported for three very different SET domain-containing proteins. The structures reveal novel folds for several new domains, including SET, and provide early insights into mechanisms of catalysis and molecular recognition in this family of enzymes. PMID- 12372295 TI - Identification of TOR signaling complexes: more TORC for the cell growth engine. AB - The Target of Rapamycin (TOR) proteins function in signaling pathways that promote protein synthesis and cell growth. In yeast, TOR signaling is regulated by nutrient availability, whereas in metazoan cells TOR activities may be controlled by both nutrients and growth factors. The recent identification of novel TOR-interacting proteins has provided crucial insights into TOR regulation and function. PMID- 12372296 TI - Sequence first. Ask questions later. AB - Comparative sequence analyses of eukaryotic genes and genomic regions are beginning to provide a wealth of information that is directly relevant to human biology. Functional changes that set us apart from apes are identifiable, as are functional constraints in proteins and genomic elements that arose in our relatively distant phylogenetic past. PMID- 12372297 TI - Coordinate regulation of sugar flux and translation by PAS kinase. AB - PAS kinase is a serine/threonine kinase regulated in cis by a PAS domain. A genetic study of the two PAS kinase genes in budding yeast gave evidence of the involvement of these enzymes in the control of sugar metabolism and translation. Using a biochemical screen for PAS kinase substrates, three translation factors were identified as direct phosphorylation targets. PAS kinase was also found to phosphorylate UDP-glucose pyrophosphorylase and glycogen synthase, the enzymes catalyzing the two final steps in the glycogen biosynthetic pathway. Genetic, biochemical, and physiological data provide evidence that both of these enzymes are inhibited by PAS kinase-dependent phosphorylation, thereby downregulating carbohydrate storage. These studies provide evidence of a cell-autonomous signaling system that both controls and connects the balance of fuel consumption/storage to protein synthesis. PMID- 12372298 TI - The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. AB - We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen. PMID- 12372299 TI - The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. AB - The Period2 gene plays a key role in controlling circadian rhythm in mice. We report here that mice deficient in the mPer2 gene are cancer prone. After gamma radiation, these mice show a marked increase in tumor development and reduced apoptosis in thymocytes. The core circadian genes are induced by gamma radiation in wild-type mice but not in mPer2 mutant mice. Temporal expression of genes involved in cell cycle regulation and tumor suppression, such as Cyclin D1, Cyclin A, Mdm-2, and Gadd45alpha, is deregulated in mPer2 mutant mice. In particular, the transcription of c-myc is controlled directly by circadian regulators and is deregulated in the mPer2 mutant. Our studies suggest that the mPer2 gene functions in tumor suppression by regulating DNA damage-responsive pathways. PMID- 12372300 TI - Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage. AB - The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. Here, we show that a component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL, and we demonstrate that deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. Our results suggest that the regulation of Bcl-xL deamidation has a critical role in the tumor-specific activity of DNA-damaging antineoplastic agents. PMID- 12372301 TI - Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched. AB - The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins. PMID- 12372302 TI - Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning. AB - A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+ ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity. PMID- 12372303 TI - Structure and catalytic mechanism of a SET domain protein methyltransferase. AB - Protein lysine methylation by SET domain enzymes regulates chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. The 2.6 A resolution structure of Rubisco large subunit methyltransferase in a pseudo-bisubstrate complex with S-adenosylhomocysteine and a HEPES ion reveals an all-beta architecture for the SET domain embedded within a larger alpha-helical enzyme fold. Conserved regions of the SET domain bind S adenosylmethionine and substrate lysine at two sites connected by a pore. We propose that methyl transfer is catalyzed by a conserved Tyr at a narrow pore connecting the sites. The cofactor enters by a "back door" on the opposite side of the enzyme from substrate, promoting highly specific protein recognition and allowing addition of multiple methyl groups. PMID- 12372304 TI - Crystal structure and functional analysis of the histone methyltransferase SET7/9. AB - Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate. PMID- 12372306 TI - Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing. AB - Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation. The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA. Directed hydroxyl radical probing of 16S and 23S rRNA from Fe(II) tethered to ten positions on the surface of E. coli RRF constrains it to a well-defined location in the subunit interface cavity. Surprisingly, the orientation of RRF in the ribosome differs markedly from any of those previously observed for tRNA, suggesting that structural mimicry does not necessarily reflect functional mimicry. PMID- 12372305 TI - Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. AB - AdoMet-dependent methylation of histones is part of the "histone code" that can profoundly influence gene expression. We describe the crystal structure of Neurospora DIM-5, a histone H3 lysine 9 methyltranferase (HKMT), determined at 1.98 A resolution, as well as results of biochemical characterization and site directed mutagenesis of key residues. This SET domain protein bears no structural similarity to previously characterized AdoMet-dependent methyltransferases but includes notable features such as a triangular Zn3Cys9 zinc cluster in the pre SET domain and a AdoMet binding site in the SET domain essential for methyl transfer. The structure suggests a mechanism for the methylation reaction and provides the structural basis for functional characterization of the HKMT family and the SET domain. PMID- 12372308 TI - Reflections on the musculoskeletal therapists' multifaceted role and influences on treatment outcomes. PMID- 12372309 TI - Achilles tendinopathy. AB - Achilles tendon injury (tendinopathy) and pain occur in active individuals, when the tendon is subject to high or unusual load. Achilles tendinopathy can be resistant to treatment, and symptoms may persist despite both conservative and surgical intervention. The pathology of overuse tendinopathy is non-inflammatory, with a degenerative or failed healing tendon response. The diagnosis of Achilles tendinopathy requires excellent differential diagnosis and an understanding of the role of tendon imaging. Conservative treatment must include exercise, with a bias to eccentric contractions. Surgical treatment is effective after complete tendon rupture, but may not assist recovery from overuse tendinopathy. Further research into the clinical aspects of Achilles tendinopathy is required. PMID- 12372310 TI - Manual therapy for mechanical neck disorders: a systematic review. AB - Neck disorders are common, disabling and costly. Randomized trials were reviewed using a Cochrane format, to determine if manual therapy improves pain, function and patient satisfaction in adults suffering from neck disorders with and without radicular findings or headache. Sequenced computerized searches ended in December 1997. Two independent reviewers extracted data while three assessed trial quality. Standard mean difference and relative risks were translated to number needed to treat (NNT) and the percent treatment advantage. The 20 selected trials' quality was 2.4 (SD: 1.04) on the 5-point scale described by Jadad. Trials were clinically heterogenous. Manipulation alone, mobilizations alone, manipulation/mobilization and treatments including massage consistently showed similar effects to placebo, wait period or control. Multimodal manual therapy care including exercise were superior to a control, to certain physical medicine methods and to rest for pain and patient satisfaction. The NNT for a clinically important reduction in pain varied from 2 to 11 and treatment advantage from 6% to 41% at the cost of benign transient side-effects. While results remain inconclusive, some clinical themes have emerged. For mechanical neck disorder with or without headache, it appears that to be most beneficial, manual therapies should be done with exercise for improving pain and patient satisfaction. Manipulation and mobilization alone appear to be less effective. Factorial design would help delineate the magnitude of effect for each component of care. PMID- 12372311 TI - The effect of soft tissue deloading tape on thoracic spine pressure pain thresholds in asymptomatic subjects. AB - The application of tape to deload soft tissue is used in the management of thoracic spine pain. A reported clinical feature of this treatment is reduced tenderness of the spine during postero-anterior mobilizations. A randomized, single blind, placebo controlled, repeated measures design study was employed to investigate the effects of deloading tape on pressure pain threshold measurements at the level of the T7 spinous process in an asymptomatic group of 24 subjects. Pressure pain thresholds were assessed prior to and following the application of deloading tape, placebo sham tape and no-tape control conditions. All subjects received all three conditions in a randomized order on three separate days. Differences between the pre- and post-measurements were used as indicators of change in a subject's pressure pain threshold. No significant change in pressure pain threshold measurements was found between conditions. In summary, this study demonstrated that deloading tape applied to the level of the T7 spinous process did not significantly change pressure pain threshold measurements in asymptomatic subjects, raising the possibility that any pain relieving effect may well be conditional upon pain being present. PMID- 12372312 TI - Does taping influence electromyographic muscle activity in the scapular rotators in healthy shoulders? AB - Although taping techniques are commonly used in addition to exercise programmes in the rehabilitation of shoulder instability and secondary subacromial or internal impingement, few studies exist on the effect of taping on the muscle activity of the scapular rotators. The purpose of our study was to examine the influence of one particular tape on muscular activity in scapular muscles. Twenty healthy shoulders were examined with surface EMG recordings on the three parts of trapezius and serratus anterior muscle during dynamic full range of motion abduction and forward flexion. The movement direction, and tape and no-tape conditions were randomized. The statistical analyses with ANOVA repeated Measures (GLM model) showed significant differences among the means between the four muscles (P<0.05), two movement directions (P<0.05), applied resistance (P<0.01), and movement period (P<0.01). However, no significant difference was observed based on the application of tape. The results of our investigation revealed no significant influence of tape application on EMG activity in the scapular muscles in healthy subjects. Future research will be necessary to examine other parameters of neuromuscular control in order to determine possible proprioceptive changes in muscle recruitment with tape application. PMID- 12372313 TI - Thoracic pain in a collegiate runner. AB - This case study describes the process of examination, re-examination, and intervention for a collegiate runner with mechanical thoracic pain preventing athletic participation and limiting daily function. Unimpaired function fully returned in less than 3 weeks with biweekly sessions to re-establish normal and painfree thoracic mechanics via postural hygiene, exercise, mobilization, and manipulation. The outcome of this case study supports the original hypothesis that the pattern of impairments was in fact responsible for the functional limitations and disability in this athlete. At the time of publication the athlete was without functional limitations and had fully returned to competitive sprinting for the university track team. PMID- 12372315 TI - X-ray absorption microtomography (microCT) and small beam diffraction mapping of sea urchin teeth. AB - Two noninvasive X-ray techniques, laboratory X-ray absorption microtomography (microCT) and X-ray diffraction mapping, were used to study teeth of the sea urchin Lytechinus variegatus. MicroCT revealed low attenuation regions at near the tooth's stone part and along the carinar process-central prism boundary; this latter observation appears to be novel. The expected variation of Mg fraction x in the mineral phase (calcite, Ca(1-x)Mg(x)CO(3)) cannot account for all of the linear attenuation coefficient decrease in the two zones: this suggested that soft tissue is localized there. Transmission diffraction mapping (synchrotron X radiation, 80.8 keV, 0.1 x 0.1mm(2) beam area, 0.1mm translation grid, image plate area detector) simultaneously probed variations in 3-D and showed that the crystal elements of the "T"-shaped tooth were very highly aligned. Diffraction patterns from the keel (adaxial web) and from the abaxial flange (containing primary plates and the stone part) differed markedly. The flange contained two populations of identically oriented crystal elements with lattice parameters corresponding to x=0.13 and x=0.32. The keel produced one set of diffraction spots corresponding to the lower x. The compositions were more or less equivalent to those determined by others for camarodont teeth, and the high Mg phase is expected to be disks of secondary mineral epitaxially related to the underlying primary mineral element. Lattice parameter gradients were not noted in the keel or flange. Taken together, the microCT and diffraction results indicated that there was a band of relatively high protein content, of up to approximately 0.25 volume fraction, in the central part of the flange and paralleling its adaxial and abaxial faces. X-ray microCT and microdiffraction data used in conjunction with protein distribution data will be crucial for understanding the properties of various biocomposites and their mechanical functions. PMID- 12372316 TI - Effects of the osmolyte trimethylamine-N-oxide on conformation, self-association, and two-dimensional crystallization of myelin basic protein. AB - The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination. PMID- 12372317 TI - Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms. AB - Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma. PMID- 12372318 TI - Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite. AB - The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase. PMID- 12372319 TI - Preliminary crystallographic analysis of the bacteriophage P22 portal protein. AB - Portal proteins are components of large oligomeric dsDNA pumps connecting the icosahedral capsid of tailed bacteriophages to the tail. Prior to the tail attachment, dsDNA is actively pumped through a central cavity formed by the subunits. We have studied the portal protein of bacteriophage P22, which is the largest connector characterized among the tailed bacteriophages. The molecular weight of the monomer is 82.7 kDa, and it spontaneously assembles into an oligomeric structure of approximately 1.0 MDa. Here we present a preliminary biochemical and crystallographic characterization of this large macromolecular complex. The main difficulties related to the crystallization of P22 portal protein lay in the intrinsic dynamic nature of the portal oligomer. Recombinant connectors assembled from portal monomers expressed in Escherichia coli form rings of different stoichiometry in solution, which cannot be separated on the basis of their size. To overcome this intrinsic heterogeneity we devised a biochemical purification that separates different ring populations on the basis of their charge. Small ordered crystals were grown from drops containing a high concentration of the kosmotropic agent tert-butanol and used for data collection. A preliminary crystallographic analysis to 7.0-A resolution revealed that the P22 portal protein crystallized in space group I4 with unit cell dimensions a=b=409.4A, c=260.4A. This unit cell contains a total of eight connectors. Analysis of the noncrystallographic symmetry by the self-rotation function unambiguously confirmed that bacteriophage P22 portal protein is a dodecamer with a periodicity of 30 degrees. The cryo-EM reconstruction of the dodecahedral bacteriophage T3 portal protein will be used as a model to initiate phase extension and structure determination. PMID- 12372320 TI - Crystallization and characterization of PU.1/IRF-4/DNA ternary complex. AB - PU.1 and IRF-4, members of the Ets and interferon regulatory families of transcription factors, respectively, form a cooperative ternary complex that is implicated in the regulation of B-cell-specific gene expression. A portion of the cooperativity involves interaction between the PU.1 and IRF-4 DNA-binding domains. We report here the crystallization and preliminary characterization of PU.1 and IRF-4 DNA-binding domains bound to a 21-mer DNA site from the lambdaB element of immunoglobulin light chain lambda(2-4) enhancer. The crystals belong to space group P2(1) with unit cell dimensions of a=40.7A, b=62.3A, c=67.9A, beta=95.6 degrees with one complex molecule per asymmetric unit. Crystals diffract to a resolution of 3A using X-rays from a rotating anode generator but improve to 2.3A with synchrotron radiation. The crystals are highly mosaic, but the mosaicity can be improved following a series of steps involving the addition of additives, use of peritone oil as a cryoprotectant, slow flash-cooling in the cryostream, and several cycles of crystal annealing. PMID- 12372321 TI - Characterization of the performance of a 200-kV field emission gun for cryo electron microscopy of biological molecules. AB - The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10A barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high resolution performance tests for electron microscopes are made close to focus, at several hundreds of A underfocus at a magnification of 500,000x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30,000 to 60,000x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300A in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode. PMID- 12372322 TI - Microbiological commissioning and monitoring of operating theatre suites. PMID- 12372323 TI - Importance of focus identification in the treatment of Staphylococcus aureus bacteraemia. AB - Staphylococcus aureus bacteraemia increases in frequency, and it is still a life threatening disease. In recent years, some interesting studies such as the need for focus identification and the focus eradication have been performed. The aim of this review is to present an up-to-date assessment of the current challenges in the management of S. aureus bacteraemia in order to improve the outcome. PMID- 12372324 TI - A clone of coagulase-negative staphylococci among patients with post-cardiac surgery infections. AB - Coagulase-negative staphylococci (CoNS) are important causes of hospital-acquired infections such as infections after cardiac surgery. Efforts to reduce these infections are hampered by the lack of knowledge concerning the epidemiology of CoNS in this setting. Forty strains of CoNS collected during the surgical revision of 27 patients operated on between 1997 and 2000 were analysed. Strains were also collected from the ambient air in the operating suite. Their pulsed field gel electrophoresis (PFGE) characteristics and antibiotic resistance were analysed. Using PFGE 19 of 40 strains from 15 of 27 patients were shown to belong to one clone, and strains from this clone were also isolated from the ambient air. This clone had caused infections throughout the period. Antibiotic resistance did not correlate with PFGE patterns. Using PFGE one clone could be identified that caused 56% of the CoNS infections during this period. A strain from this clone was also found in the air of the operating suite suggesting the origin of the CoNS causing infections was the hospital environment. PMID- 12372325 TI - Genetic relationship between Escherichia coli strains isolated from the intestinal flora and those responsible for infectious diseases among patients hospitalized in intensive care units. AB - The exact origin of strains of Escherichia coli responsible for infectious diseases in intensive care units (ICUs) remains partly unknown. Our aim was to determine the nature of the link between strains from the intestinal flora of hospital staff, strains from the intestinal flora of patients hospitalized in ICUs and strains isolated from ICU patients with invasive diseases. For this purpose, 77 strains of E. coli were genetically characterized by exploring their entire genomes by random amplified polymorphism of DNA (RAPD), and by determining their phylogenetic position in ECOR (E. coli reference) groups, the virulence factors harboured (pap, sfa, afa, hly, aer and cnf) and their ability to mutate. The strains isolated from the intestinal flora of hospital staff were found to constitute a genetically heterogeneous population compared with the strains isolated from ICU carriers, which were highly clustered. The latter strains harboured numerous virulence factors, and 80% belonged to the group ECOR B2. The strains isolated from infected patients harboured fewer virulence factors than those from the ICU carriers, and only half belonged to ECOR B2. Moreover, these strains were more genetically related to strains from hospital staff than to strains from ICU carriers. Thus, the exogenous origin of the E. coli strains is probably almost as important as translocation from intestinal flora in ICUs. Moreover, a strong mutator phenotype had a minor, or no, role in the rapid adaptation to modifications in the ecological environment. PMID- 12372326 TI - Bacterial strike-through of re-usable surgical drapes: the effect of different wetting agents. AB - Wound contamination and the resultant postoperative infection is a major problem in all forms of surgery. Air contamination, gloves, surgical instruments and drapes have all been investigated as sources of wound contamination. We investigated the effect of different wetting agents on strike-through rate of bacteria through re-usable polyester/cotton surgical drapes using a newly described method. Within 30 min bacterial strike-through of dry surgical drapes occurs. Wetting drapes with blood or normal saline enhances the strike-through rate of bacteria. Wetting drapes with iodine or chlorhexidine diminishes, but does not stop, bacterial strike-through. The use of re-usable polyester/cotton drapes is a potential source of wound contamination especially when wetted with blood or normal saline. PMID- 12372327 TI - Surveillance of hospital-acquired infections in an intensive care department-the benefit of the full-time presence of an infection control nurse. AB - In the 42-bed intensive care department of a teaching hospital, the creation of a full-time infection control nurse post was followed by a 42% reduction in device related hospital-acquired infection rates over a period of three years, and 33% reduction over a period of five years. Permanent surveillance accompanied by revision of procedures and bedside teaching were key factors in the improvement of quality of care. In the specific setting of an intensive care department, this study validates the previous conclusions reached in the SENIC study and emphasizes the essential role played by the infection control nurse in the care of critically ill patients. PMID- 12372328 TI - Hospital-acquired Aspergillus fumigatus infection: can molecular typing methods identify an environmental source? AB - Aspergillus fumigatus infection in hospitalized immunocompromised patients often raises suspicion regarding the potential for hospital acquisition. Hospital staff have an important responsibility in implementing preventive measures, especially since the advent of current legislation concerning hospital-acquired infections. There have been high expectations that molecular typing methods might determine the source of Aspergillus fumigatus, a ubiquitous mould. The aim of the present epidemiological study, was therefore, to identify the origin(s) of Aspergillus infection in six well-documented patients. All the clinical strains (N=33), and those from hospital (N=14) and home environments (N=34) were isolated according to a standardized protocol and typed by sequence-specific DNA primer analysis. The results confirmed the huge biodiversity of the A. fumigatus population, and consequently the difficulty in ascertaining a hospital source of the infection, as opposed to infections due to other Aspergillus species less frequently encountered. PMID- 12372329 TI - Effect of jewellery on surface bacterial counts of operating theatres. AB - Twenty items of three jewellery types were studied. Finger rings, nose and ear piercings increased local surface bacterial counts when in situ, and especially after removal (P<0.0001). Although in the UK the National Association of Theatre Nurses' guidelines suggest that all jewellery should be removed before scrubbing, we suggest that jewellery worn on noses and ears should be left in situ and covered by masks and hats, respectively. The effect of jewellery on skin disinfection needs further study before guidelines can be made concerning finger rings. PMID- 12372330 TI - Risk to immune suppressed patients from Aspergillus spp. growing in fridge condensate trays. PMID- 12372331 TI - Enterobacter cloacae infection and colonization in a neonatal intensive care unit. PMID- 12372332 TI - Alcaligenes xylosoxidans subspecies denitrificans pseudobacteraemia in a university children's hospital. PMID- 12372333 TI - Use of adult isolation facilities in a UK infectious diseases unit. PMID- 12372334 TI - Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement. AB - Bombesin-like peptides, including the mammalian homologue gastrin-releasing peptide, are highly expressed and secreted by neuroendocrine cells in prostate carcinoma tissues and are likely to be related to the progression of this neoplastic disease. Previously, we demonstrated that bombesin increased migration and protease expression in androgen-independent cells. In this work we show that bombesin is able to activate pro-MMP-9 through a mechanism involving the beta1 integrin subunit. In fact, MMP-9 processing was evident only when beta1 integrin was engaged with specific adhesive substrates, such as type I collagen, or when cells were seeded on dishes coated with antibodies against beta1 integrin, resulting in activation of the surface ligand. When exogenous pro-MMP-9 was added to PC3 cells, MMP-9 active forms were produced within 30 min by bombesin-treated cultures while control cultures expressed activated forms only after a longer time and at lower levels. MMP-9 activation required cytoskeleton integrity since this effect was abolished by cytochalasin D. Engagement of beta1 integrin caused an increased membrane-linked uPA activity which was required for MMP-9 activation. The cross talk between bombesin- and beta1-integrin-engaged signals seems to be crucial for the modulation of both membrane-linked uPA activity and MMP-9 activation and triggers complex intracellular signaling pathways requiring activation of tyrosine kinase activity, including that of src and PI3K. The beta1 integrin may be considered an important mechanism by which bombesin induces MMP-9 activation. This finding supports the idea that cellular responses to growth factors may be driven by cell-matrix interactions and stresses the role of neuroendocrine factors in prostate carcinoma progression. PMID- 12372335 TI - Suppression of kinesin expression disrupts adenomatous polyposis coli (APC) localization and affects beta-catenin turnover in young adult mouse colon (YAMC) epithelial cells. AB - Mutational inactivation of the adenomatous polyposis coli (APC) protein initiates most hereditary and sporadic colon cancers. The tumor-suppressive effect of APC is mediated by promoting degradation of the oncogenic transcriptional activator beta-catenin, and loss of APC function often results in nuclear accumulation of beta-catenin in cancer cells. APC is a nuclear-cytoplasmic shuttling protein and moves along microtubules in the cytoplasm. However, the molecular motor proteins responsible for APC translocation and the implications of APC trafficking on beta catenin turnover are unknown. Here we show that APC protein is associated with microtubules and is colocalized with kinesin heavy chain (KHC) and beta-catenin to clusters of puncta at the tip regions of cellular extensions in a conditionally immortalized mouse colon epithelial cell line, young adult mouse colon (YAMC, APC+/+). Inhibition of KHC expression using an antisense oligonucleotide disrupts peripheral translocation of APC and induces nucleocytoplasmic accumulation of beta-catenin. These data indicate that KHC mediated APC translocation is tightly coordinated with beta-catenin turnover in the cell. PMID- 12372336 TI - Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells. AB - Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL 6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells. PMID- 12372337 TI - Induction of the IFN-gamma gene and protein is linked to the establishment of cell polarity in a porcine epithelial cell line. AB - We report here original properties of a porcine trophectoderm cell line, TBA B4 3, that developed a polarized phenotype with high transepithelial electrical resistance (TER) values and functional tight junctions (TJs) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with a strong protein kinase C (PKC) agonist, phorbol 12-myristate-13-acetate (PMA), induced 3-4 days later a transient interferon-gamma (IFN-gamma) mRNA expression and vectorial IFN-gamma protein secretion toward the apical side of the monolayer. Exposure of TBA B4-3 cells to PMA first resulted in a rapid and profound disorganization of the monolayer structure mainly characterized by the appearance of multilayered polyp-like foci structures, a strong decrease of the TER, and a increase of permeability correlated with changes in the organization and localization of the TJ-associated proteins (ZO-1 and occludin) and filamentous actin (f-actin). After PMA removal, spontaneous return to the initial polarized monolayer state occurred, characterized by TER rising to prestimulation values, TJ protein relocalization, and multilayered cell structures fading. This return was strictly correlated with transient IFN-gamma gene induction. Our report represents the first example of an inducible expression of IFN-gamma by a polarized epithelial cell. After PMA treatment, the close correlation between establishment of cell polarity and IFN-gamma gene expression suggests a link between these phenomena. This also suggests a novel biological mechanism by which transient and reversible disorganization of a polarized monolayer of epithelial cells could trigger regulated expression of a cytokine gene by these cells. PMID- 12372338 TI - Unique behavior of a dictyostelium homologue of TRAP-1, coupling with differentiation of D. discoideum cells. AB - Dd-TRAP1 is a Dictyostelium homologue of TRAP-1, a human protein that binds to the type 1 tumor necrosis factor (TNF) receptor. TRAP-1 has a putative mitochondrial localization sequence and shows significant homology to members of the HSP90 family. Although TRAP-1 is mainly localized to mitochondria in several mammalian cells, in certain tissues it is also localized at specific extramitochondrial sites. In Dictyostelium cells, Dd-TRAP1 is predominantly located in the cell membrane/cortex during growth and just after starvation. Double staining of vegetatively growing cells with the anti-Dd-TRAP1 antibody and TRITC-phalloidin has demonstrated colocalization of Dd-TRAP1 and F-actin at the leading edge of cortical protrusions such as pseudopodes. Coupled with differentiation, however, Dd-TRAP1 located at the cortical region is translocated to mitochondria in spite of the absence of the mitochondrial localization sequence at its N-terminus. The translocation of this protein raises interesting and fundamental questions regarding possible mechanisms by which Dd-TRAP1 is involved in cellular differentiation. PMID- 12372339 TI - Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders. AB - Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL 15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and alpha-actin accumulation in differentiated myotubes. Secreted factors from IL-15 overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions. PMID- 12372340 TI - Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells. AB - Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells. PMID- 12372341 TI - Novel activities of pro-IGF-I E peptides: regulation of morphological differentiation and anchorage-independent growth in human neuroblastoma cells. AB - Insulin-like growth factor-I (IGF-I) is translated as a pre-pro-peptide that is posttranslationally processed to its mature form by proteolytic removal of the signal peptide and the E-domain peptide. Contrary to the mature human (h) IGF-I, the recombinant rtEa4 -peptide significantly reduced the anchorage-independent cell growth in human neuroblastoma cells (SK-N-F1), shown by colony formation assay in vitro. Significant inhibition of colony formation is also observed in SK N-F1 cells stably transfected with a bicistronic expression construct encoding a secretory form of the rtEa4 peptide. Furthermore, treatment with the recombinant rtEa4 peptide, but not the mature hIGF-I, resulted in morphological differentiation of SK-N-F1 cells characterized by long neurite outgrowth. Similar morphological differentiation is also observed in SK-N-F1 cell clones stably transfected with the rtEa4 peptide expression construct. A spectrum of biological activities similar to those of rtEa4 peptide is also observed in the synthetic hEb peptide, but not-the hEa peptide. Results of further characterization reveal that neurites induced by rtEa4 or hEb peptide contain neuronal-specific MAP-2, Tau, and neurofilament (NF-160), accompanied by an increased expression of the neuronal marker gene neuropeptide tyrosine (NPY). Furthermore, effects of signal transduction inhibitors are indicative of the involvement of MAP-kinase PI-3 kinase cascades. The activation of ERK-1/-2 is markedly increased in response to rtEa-4 or hEb peptide stimulation, further indicating the involvement of MAPK signaling cascade. These unique biological activities exhibited by the rtEa4 or hEb peptide suggest that E peptide of the pro-IGF-I may play distinct roles in regulating cell growth and differentiation in neuroblastoma cells. PMID- 12372342 TI - Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells. AB - Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage. PMID- 12372343 TI - Epidermal growth factor up-regulates the transcription of mouse lon homology ATP dependent protease through extracellular signal-regulated protein kinase- and phosphatidylinositol-3-kinase-dependent pathways. AB - Epidermal growth factor (EGF) induces tumorigenic transformation of mouse epidermal cells (JB6 P(+)). We cloned a full-length EGF-responsive cDNA in JB6P(+) cells; EGF up-regulated mRNA expression of this gene 5- to 6-fold. The deduced amino acid sequence of this cDNA exhibited 84 and 96% homology with human and rat Lon homology ATP-dependent protease, respectively, and all conserved domains of Lon, such as ATPase/protease domains, are present in the mouse gene, indicating that this gene is mouse Lon. EGF increased the transcriptional rate without affecting the mRNA stability of m-Lon. The level of m-Lon in irreversibly transformed mouse epidermal cells (JB7) was 3.4-fold higher than that in parental JB6 P(+) cells. Similarly, human mammary epithelial cells overexpressing the proto-oncogene ErbB2 expressed significantly higher levels of Lon than normal mammary epithelial cells. EGF failed to regulate Lon expression in ERK-deficient JB6 P(-) cells or cells that expressed the dominant-negative p85 P13-K regulatory unit. Furthermore, selective chemical blockers for MEK1 and P13-K (PD98059 and LY294002) inhibited EGF-mediated induction. Mitochondria-localized Lon protease plays a critical role in the regulation of mitochondrial gene expression and genome integrity. Disruption of mitochondrial homeostasis is a general characteristic of tumorigenic transformation. Thus, the role of Lon in tumor promotion warrants further study. PMID- 12372344 TI - Constitutive and inducible expression of the epithelial antigen MUC1 (CD227) in human T cells. AB - MUC1 (CD227) is a large glycoprotein normally produced by epithelial tissue and expressed aberrantly in carcinomas. Here we show that resting human T cells express basal levels of MUC1 mRNA and protein forms with molecular masses of approximately 150 and approximately 250 intracellularly, but lack surface expression. Mitogenic stimulation induces the appearance of new MUC1 mRNA and >300-kDa MUC1 forms. Concomitantly, MUC1 is translocated to the outer cell membrane and its density is continuously modulated according to the cycling status. Inhibitors of mRNA and protein synthesis and of Golgi-dependent protein transport prevent MUC1 induction. Ligation of surface MUC1 has no effect on T cell proliferation. Also, altering the overall protein structure by preventing glycosylation has no effect. Sizable amounts of >300-kDa glycosylated MUC1 forms are shed by proliferating T cells. This soluble MUC1 does not appear to influence T-cell response, and we found no evidence for MUC1 binding sites on T cells or for transfer of the protein on cell-cell contact. We therefore suggest that MUC1 fulfills the criteria for an early T-cell activation marker but its function remains to be determined. Finally, although we found that cancer- and T cell associated MUC1 expose common protein core and sialylated epitopes, there is a peptide region, accessible in carcinomas due to an aberrant glycosylation, that is stably not accessible in T cells with potential implications for cancer immunotherapy. PMID- 12372345 TI - Regulation of epithelial cell migration and tumor formation by beta-catenin signaling. AB - Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. beta-catenin plays a dual role in this process: as a member of adherens junctions it is essential to link cadherins to the cytoskeleton thereby allowing tight intercellular adhesion, and as a member of the Wnt-signaling pathway, beta-catenin is translocated into the nucleus and serves together with the LEF1/TCF-transcription factors to drive gene expression necessary for the epithelial-to-mesenchymal transition (EMT). Activated beta catenin signaling has been implicated in the genesis of a variety of tumors. Here we demonstrate a pivotal function for beta-catenin signaling in epithelial cell migration and tumorigenesis. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) induce beta-catenin signaling under conditions where they stimulate cell motility. Ectopic expression of either stabilized beta-catenin or a regulatable form of activated beta-catenin induces cell migration in different cell types and cooperates with EGF and HGF in this process. Activation of beta catenin signaling induces expression of the new target gene osteopontin during migration. Cells expressing stabilized beta-catenin also exhibit significantly increased capability to form tumors in a nude mouse xenograft model. The data suggest that a critical threshold of beta-catenin signaling, activated by cooperative mechanisms, may be important during the EMT and tumorigenesis. PMID- 12372346 TI - Caveolin-1 phosphorylation in human squamous and epidermoid carcinoma cells: dependence on ErbB1 expression and Src activation. AB - Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression. PMID- 12372347 TI - The man who executed "imagined" movements: evidence for dissociable components of the body schema. AB - We examined the nature of representations underlying motor imagery and execution in a patient (CW) with bilateral parietal lesions. When imagining hand movements, CW executed the imagined motor act but was unaware of the movements. These movements were significantly more accurate than volitional movements for the left but not right hand. CW also exhibited preserved motor imagery for the left but not right hand. Consistent with previous accounts, these findings suggest that motor imagery may normally involve the inhibition of movements. CW's unawareness of movements during motor imagery may reflect inattention or misattribution of the unexpected sensory feedback. Furthermore, in line with current models of motor control, motor imagery may depend on the integrity of a "forward model" derived from motor outflow information to generate a prediction of the consequences of a motor command. Such predictions appear to be preserved for imagery of left but not right hand movements in CW. Action may additionally depend on precise updating of effector position derived from the comparison of predicted and actual sensory information. We propose that CW's impaired volitional movements may be attributable to the degradation of such an updating mechanism. PMID- 12372348 TI - Bisecting and behavior: lateral inattention predicts 8-week academic performance. AB - Converging evidence supports a left hemisphere role in defensive repression and sensation seeking. This led to the hypothesis that students with a relatively active left hemisphere would perform poorly during 8 weeks of a college class. The measure of relative hemispheric activation was the visual line-bisecting task given early in the course. The hypothesis was supported. Previous evidence that activation asymmetry is stable over time was supported because the single measurement of line bisecting was a longitudinal predictor of multiple behaviors. A temporal pattern of increasing correlation between the bisecting and performance measures favors a feedback repression model. Alternative explanations based on sensation seeking, subject-matter repression, and cooperation were considered but not eliminated. PMID- 12372349 TI - Reaction time slowing in adults with HIV: results of a meta-analysis using brinley plots. AB - A meta-analysis in the form of Brinley plots was conducted on the mean reaction times of HIV+ and HIV- groups. HIV+ reaction times were regressed on HIV- reaction times across 122 task conditions from 29 studies, producing a highly linear function (r(2) = .94), with a slope b = 1.039, which was not significantly different than b = 1.00. Asymptomatic and symptomatic HIV+ reaction times were also separately regressed on HIV- reaction times across 67 task conditions from 13 studies. Both functions were highly linear (r(2) = .97 and.90 respectively), with slopes of 1.034 and 1.117 respectively. Only the slope for symptomatic HIV+ groups was significantly larger than b = 1.00, suggesting a modest generalized slowing of cognition in these adults. PMID- 12372350 TI - A serial test of the laterality of familiar face recognition. AB - The purpose of the present study was to address the issue of laterality of familiar face recognition. Seventy-two participants judged familiar faces presented laterally or centrally for their "faceness," familiarity, occupation, and name (which represent four stages of familiar face processing) using one of three response modes-verbal, manual, or combined. The pattern of reaction times (RTs) implied a serial process of familiar face recognition. Centrally presented stimuli were recognized faster than laterally presented stimuli. No RT differences were found between the left and right visual fields (VFs) across all judgments and response modes. The findings were interpreted as supporting the notion that there are no significant hemispheric differences in familiar face recognition. PMID- 12372351 TI - Dissociating perceptual and conceptual implicit memory in multiple sclerosis patients. AB - Previous studies indicate that Multiple Sclerosis (MS) patients exhibit deficits in tests of explicit memory such as free recall, but show normal priming on implicit tests of memory such as word stem completion. However, the memory performance of patients with different MS disease subtypes has not been fully examined. In the current study, memory was assessed in Primary Progressive (PPMS), Relapsing Remitting (RRMS), and Secondary Progressive (SPMS) MS subgroups. Explicit memory as well as perceptual and conceptual implicit memory were examined using free recall, word fragment completion, and exemplar generation tests, respectively. All three groups of MS patients exhibited free recall deficits and normal priming on the exemplar generation test. However, the PPMS group exhibited a deficit in word fragment completion priming, whereas the RRMS and SPMS groups exhibited normal levels of priming on this task. Lesion load was assessed using magnetic resonance imaging and was negatively correlated with explicit memory performance, but it did not account for the observed deficits in perceptual implicit memory. The results indicate that PPMS patients exhibit a pattern of memory impairment that is distinct from that of the RRMS and SPMS groups. Moreover, the results indicate that perceptual implicit memory can be neurologically dissociated from conceptual implicit memory. PMID- 12372352 TI - Division of the corpus callosum into subregions. AB - Various attempts have been made to subdivide the corpus callosum (CC) into anatomically and functionally distinct subareas. A promising current approach is the use of factor analytic techniques in conjunction with traced MRI images. The traced images are divided into 99 percentile slices, where the widths of the percentile slices are used as variables that are entered into the analysis (Denenberg, Kertesz & Cowell, 1991). Studies that use this technique agree broadly between 6 and 7 factors, but available factor solutions contain inconsistencies and large gaps, which arise when many of the percentile slices do not load appreciably on any of the factors. The present study uses a larger number of brains (N = 184), all normalized, and some methodological refinements in the analysis of the traced MRI images of the CC. A stable 7 factor solution was found, and the factor structure for males and females was very similar. PMID- 12372353 TI - Cognitive efficiency on a match to sample task decreases at the onset of puberty in children. AB - Electrocortical evidence indicates that a wave of synaptic proliferation occurs in the frontal lobes around the age of puberty onset. To study its potential influence on cognition, we examined 246 children (10-17 years) and 49 young adults (18-22 years) using a match-to-sample type of task to measure reaction times to assess emotionally related information. Based upon the instruction set, subjects made a yes/no decision about the emotion expressed in a face, a word, or a face/word combination presented tachistoscopically for 100 ms. The faces were images of a single individual with a happy, angry, sad or neutral expression. The words were 'happy,' 'angry,' 'sad,' or 'neutral,' In the combined stimulus condition, subjects were asked to decide if the face and word matched for the same emotion. Results showed that compared to the previous year, reaction times were significantly slower for making a correct decision at 11 and 12 years of age in girls and boys, the approximate ages of puberty onset. The peripubertal rise in reaction time declined slowly over the following 2-3 years and stabilized by 15 years of age. Analyses of the performance of 15-17 year olds revealed significantly longer reaction times in females to process both faces and words compared to males. However, this sex difference in late puberty appeared to be transient since it was not present in 18-22 year olds. Given the match-to-sample nature of the task employed, the puberty related increases in reaction time may reflect a relative inefficiency in frontal circuitry prior to the pruning of excess synaptic contacts. PMID- 12372354 TI - Frontal and striatal brain lesions increase susceptibility to masking in perceptual decisions. AB - Recent data suggest that perceptual decisions on masked stimuli involve neural activity in frontal cortex. We examined the effect of damage to frontal and striatal brain regions in man on the susceptibility to backward masking in rapid stimulus streams. Patients with unilateral frontal excisions and patients with early Huntington's disease were compared to controls in the identification of a brief white letter embedded in short streams of black letters at two presentation rates: (a) 9 letters/s; (b) 12.5 letters/s and also in a control condition in which the first post-target masking letter was absent. Patients could identify the target when the post-target mask was absent, but reducing the delay between stimuli significantly increased the error rates in patients. Intrusion errors often involved reporting post-target or pre-target distractors instead of the target. These results suggest that fronto-striatal lesions increase the period during which perceptual decisions are susceptible to perturbation. This deficit is compatible with a functional role of frontal systems in the cognitive control of brief perceptual decisions. PMID- 12372355 TI - Visual field asymmetries and allocation of attention in visual scenes. AB - Single items such as objects, letters or words are often presented in the right or left visual field to examine hemispheric differences in cognitive processing. However, in everyday life, such items appear within a visual context or scene that affects how they are represented and selected for attention. Here we examine processing asymmetries for a visual target within a frame of other elements (scene). We are especially interested in whether the allocation of visual attention affects the asymmetries, and in whether attention-related asymmetries occur in scenes oriented out of alignment with the viewer. In Experiment 1, visual field asymmetries were affected by the validity of a spatial precue in an upright frame. In Experiment 2, the same pattern of asymmetries occurred within frames rotated 90 degrees on the screen. In Experiment 3, additional sources of the spatial asymmetries were explored. We conclude that several left/right processing asymmetries, including some associated with the deployment of spatial attention, can be organized within scenes, in the absence of differential direct access to the two hemispheres. PMID- 12372356 TI - The relationship between testosterone and route-learning strategies in humans. AB - The relationships between route-learning strategies and circulating testosterone and estradiol levels were investigated in men and women. Testosterone and estradiol concentrations were measured by salivary assays and route-learning strategies were assessed using a direction-giving paradigm based on a novel map. Testosterone was positively correlated with the use of male-biased route-learning strategies in men, but not in women. These findings suggest sex-specific patterns of relationships between circulating testosterone and spatial processing, which apply to everyday spatial behavior. PMID- 12372357 TI - Sex differences in face recognition--women's faces make the difference. AB - Sex differences favoring women have been found in face recognition tasks as well as in verbal episodic memory tasks. Women's higher face recognition performance was hypothesized to be related to either their higher verbal ability or to their superiority in recognizing female faces, rather than faces in general. Results showed that whereas there were no differences between men and women in the recognition of male faces, or in verbal ability, women performed at a higher level than men in the recognition of female faces. Verbal ability did not influence women's face recognition performance. Potential explanations for this pattern of data, such as sex differences in interest and prior knowledge, are discussed. PMID- 12372359 TI - Effects of orientation on Rey complex figure performance. AB - An experiment was performed that examined the impact of stimulus orientation on performance on the Rey complex figure. A total of 48 undergraduates (24 men, 24 women) were randomly assigned to one of four Rey figure orientation groups (0 degrees, 90 degrees, 180 degrees, and 270 degrees ). Participants followed standard procedures for the Rey figure, initially copying it in whatever orientation group they were assigned to. Next, all participants performed a 15-20 min lexical decision experiment, used as a filler task. Finally, and unbeknownest to them, participants were asked to recall as much of the figure as they could. As expected, results revealed a main effect of Task (F = 83.92, p < .01), in which copy performance was superior to recall performance. However, the main effect for orientation was not significant, nor did orientation interact with task (Fs < .68, ps > .57). The results are important from an applied setting, especially if testing conditions are less than optimal and a fixed stimulus position is not possible (e.g., testing at the bedside). PMID- 12372358 TI - Neuropsychological consequences of right thalamic haemorrhage: case study and review. AB - The neuropsychological performance of a right-handed man is examined following haemorrhage from the anterior sections of the right thalamus. A pattern of temporally graded retrograde amnesia, global anterograde amnesia, impaired short term memory, behavioural changes, and severe executive deficits were identified. The deficits evident in this case are discussed in reference to existing neuropsychological literature regarding the consequences of thalamic infarction. It is proposed that damage to the anterior thalamic nuclei results in a frontal dysexecutive syndrome and that such a dysexecutive syndrome can explain the neuropsychological deficits observed in this case. PMID- 12372360 TI - Cognitive status in Down syndrome individuals with sleep disordered breathing deficits (SDB). AB - Twelve subjects with Down syndrome underwent polysomnographic studies during night sleep and performed the Mini-Mental state test and the Raven Progressive Matrices (RPM), sets A, B, and B(1). Sleep-disordered breathing (SDB) deficits were observed in Down syndrome individuals and their Mini-Mental and RPM scores were extremely low. Regression analysis of the results revealed that the number of apneas per hour was related with the results of the RPM, set A, which were also related with the orientation of Mini-Mental test, indicating that the more apneas an individual has the more difficulties he has in the kind of visuoperceptual skills, including orientation, associated with normal right hemisphere functioning, which are tested by set A of the RPM. PMID- 12372361 TI - On the ability of children with developmental coordination disorder (DCD) to inhibit response initiation: the simon effect. AB - The ability of children with developmental coordination disorder (DCD) to inhibit the initiation of an incorrect manual response urge was examined using a typical Simon task. While the size of the Simon effect was the same for both DCD (N = 20) and Control (N = 20) groups of children, showing no difference with respect to the time needed to complete the inhibition an unwanted response, children with DCD produced significantly more errors of the type which reflected a reduced ability to successfully effect the inhibition operation when it was required. This result is consistent with some earlier findings pointing to an inhibitory deficit for children with DCD (Wilson & Maruff, 1999). PMID- 12372363 TI - Management of regional lymph nodes in patients with malignant melanoma: questionnaire survey of UK current practice. AB - In 2001, a short postal questionnaire regarding the management of regional lymph nodes in patients with cutaneous malignant melanoma was sent to 69 NHS departments of plastic and reconstructive surgery in the UK. Questionnaires were returned by 53 units, giving a response rate of 76.8%. Of these 53 units, 49 reported that they treat patients with primary malignant melanoma. There was considerable variation in the number of melanoma patients managed by each unit. This survey confirmed that elective lymph-node dissection is not routinely practiced in the UK; observation and therapeutic lymph-node dissection for patients who develop regional metastasis is the preferred pattern of care. The majority of centres in the UK do not use sentinel lymph node mapping: only 15 of the 49 units do so (30.6%). The number of sentinel lymph node biopsies performed in each unit varied significantly. There was considerable variation in the materials used and the process of care for sentinel lymph node biopsy. On the basis of this current practice, we recommend the setting up of a prospective clinical melanoma register to record the surgical treatment of melanoma patients. PMID- 12372364 TI - Prediction of the position of the intraparotid portion of the facial nerve on MRI and CT. AB - Despite developments in imaging technology, visualisation of the intraparotid portion of the facial nerve is not possible. Three separate radiological techniques have been described to predict the position of the facial nerve: Conn's arc; a plane extending posteriorly from the outer surface of the mandibular ramus; and soft-tissue structures, including the posterior belly of the digastric muscle, the retromandibular vein and the lateral border of the masseter muscle. We investigated the reliability of these techniques in predicting the relationship of tumours to the facial nerve. Cross-sectional imaging of the parotid glands was performed prior to the removal of a parotid mass in 26 patients. Twenty patients underwent MRI, and six had CT scans. We removed 14 malignant neoplasms, nine benign lesions and three non-neoplastic lesions. The relationship of the tumour to the facial nerve was assessed radiologically by each of the three techniques, and compared with the findings at surgery. In 18 patients the tumour involved the parotid gland deep to the facial nerve. The above techniques predicted the position of the facial nerve in 69%, 58% and 46% of cases, respectively. When planning parotid surgery, it is important that the surgeon understands the advantages and limitations of the radiological assessment of the position of parotid tumours in relation to the facial nerve. PMID- 12372365 TI - Surgical correction of cauliflower ear. AB - We have classified the cauliflower ear into different types according to the zone and the degree of deformity. One major group is deformity without change in the outline of the ear, and this is divided into four subgroups according to the zone. All of these subgroups can be treated by shaving the deformed cartilage through suitable incision lines. For deformities accompanied by a skin deficit, a postauricular skin flap should be used. The other major group is deformity accompanied by a change in the outline of the ear, which is divided into two subgroups. If the ear is rigid, a conchal cartilage graft is used. If the structural integrity of the ear is poor, costal cartilage is used to provide rigidity. PMID- 12372366 TI - Management of chondrodermatitis helicis by protective padding: a series of 12 cases and a review of the literature. AB - There are numerous theories concerning the aetiology of chondrodermatitis, and many authors have suggested that pressure is a significant factor. We prospectively gathered information from 14 patients with a clinical diagnosis of this condition. Many of the patients had a physical condition that forced them to lie on the side of the affected ear. Patients were advised to use protective padding of the ear at night. Most patients were rapidly relieved of their symptoms, although healing was frequently prolonged. This positive response rate and the high recurrence rate after surgery suggest that this condition should be primarily treated conservatively; they also support the theory that pressure on the ear is the main aetiological factor. Biopsies in two patients who did not respond to conservative treatment led to an altered diagnosis. PMID- 12372367 TI - Thoracodorsal artery perforator (TAP) flap: report of our experience and review of the literature. AB - We report our experience with the thoracodorsal artery perforator flap. Its use and pitfalls are critically highlighted, especially in terms of its application as an island flap. Publications on this recently described flap are scarce, possibly because it is rarely used as a result of the high level of skill needed in its preparation. In our hospital, we have used this flap twice as an island flap, both of which failed, and ten times as a free flap, one of which failed. Transfer as a free flap is a delicate procedure involving transmuscular vessel preparation; transposition as an island flap is limited by range. Based on our limited experience, we recommend this flap only in very selected cases. This procedure has to be performed by experienced microsurgeons, after training on cadavers, and after preoperative colour Doppler imaging to determine the precise location of the main perforators. Use of this perforator flap is indicated in cases where a long vascular pedicle for an appropriate free tissue transfer is necessary, and where aesthetic appearance and minimising donor-site morbidity are more important than a potentially high risk of failure. PMID- 12372368 TI - An anatomical and clinical study of the dorsal intercostal cutaneous perforators, and application to free microvascular augmented subdermal vascular network (ma SVN) flaps. AB - We report a two-part anatomical and clinical study whose aim was to map the dominant dorsal intercostal cutaneous perforators (DICPs), which are useful for microvascular augmentation of flaps raised from the skin of the back called subdermal vascular network (SVN) flaps, and to test their reliability in the clinical setting. In the anatomical arm of the study, using preserved cadavers, we macroscopically confirmed the location of DICPs, and performed micro angiography of the dorsal skin to find each dominant DICP. In the clinical arm of the study, we confirmed the location of the dominant DICP during microvascular augmented SVN flap transfer. Postoperatively, posteroanterior radiographs of the chest were taken to locate vessel clips used to ligate the DICPs. The combined study results showed that the dominant DICP is the sixth or seventh in most instances, but there are some anatomical variations. If no dominant DICP is found in the sixth or seventh spaces, at least one DICP that is of sufficient calibre for microvascular augmentation can usually be found in the general vicinity, such as the fifth, eighth or ninth spaces. The clinical application of microvascular augmented SVN flaps, both pedicled and free, is presented. PMID- 12372369 TI - The microvascular augmented subdermal vascular network (ma-SVN) flap: its variations and recent development in using intercostal perforators. AB - In 1994 we reported the use of the microvascular augmented occipito-cervico dorsal 'super-thin' flap for reconstruction of the cervical region in three cases. Since this preliminary report, we have performed a further 17 flaps, and the usefulness of the flap in the treatment of anterior cervical scar contractures in extensively burned patients has become apparent. Moreover, we have devised flaps with not only a narrow skin pedicle but also myocutaneous or island vascular pedicles. Various augmentation vessels, including myocutaneous perforators of the intercostal spaces in the back and chest, have also been used successfully. Here, we describe the microvascular augmented subdermal vascular network flaps that we have devised. PMID- 12372370 TI - Free radial forearm adipofascial flaps raised through limited incisions. AB - Free radial forearm adipofascial flaps were used for limb reconstruction in four cases. The flaps were harvested through limited skin incisions with the aid of lighted retractors. The mean surface area was 96 cm(2). The donor sites and the transferred tissue healed satisfactorily in all cases. Although the dissection through limited incisions is slightly awkward and prolongs the operating time, the technique offers significant benefits in terms of donor-site morbidity and aesthetics. PMID- 12372371 TI - Patient outcome following a thoracodorsal to musculocutaneous nerve transfer for reconstruction of elbow flexion. AB - This study reports patient outcome following a thoracodorsal to musculocutaneous nerve transfer. We retrospectively reviewed the charts of six patients who had undergone transfer of the thoracodorsal nerve to the musculocutaneous nerve for reconstruction of elbow flexion. The mean age was 47 years (standard deviation: 24 years; range: 17-72 years). The mean time from injury to surgery was 3 months (standard deviation: 2 months; range: 1-5 months). In all cases, the biceps muscle was successfully reinnervated; in one case the Medical Research Council (MRC) muscle grade was grade 5, in four cases it was grade 4, and in one case it was grade 2. No patients complained of functional weakness with shoulder adduction and/or internal rotation. In the majority of cases, transfer of the thoracodorsal nerve to the musculocutaneous nerve provides excellent recovery of elbow flexion. PMID- 12372372 TI - A conservative approach for deep dermal burn wounds using polarised-light therapy. AB - This article reports a clinical study investigating the role of polarised-light therapy in the conservative treatment of deep dermal burn wounds. In 22 out of 67 patients with deep dermal burn wounds, clinical evaluation revealed only a very limited potential for spontaneous healing, and, despite the fact that the majority of the surgeons (four out of six) would have recommended surgery, these patients were treated conservatively with polarised-light therapy (400-2000 nm, 40 m W cm(-2), 2.4 J cm(-2)) until complete closure. Evaluation by a panel of four surgeons, all experts in burn surgery, revealed that conservative treatment of these deep dermal wounds with polarised-light irradiation resulted in a significantly shorter healing time, with almost no hypertrophic scarring, and optimal aesthetic and functional results at long-term follow-up. No extension of the hospital stay was required. Polarised-light therapy may be a valuable way of avoiding surgery in patients with deep dermal burns. PMID- 12372373 TI - Research options for plastic surgical trainees. AB - A period of formal research training has become a popular option for many plastic surgical trainees. In this article, we discuss the range of postgraduate research options available and provide information that will allow plastic surgical trainees to make an informed choice about their relative suitability. PMID- 12372374 TI - The role of pre-ischaemic application of the nitric oxide donor spermine/nitric oxide complex in enhancing flap survival in a rat model. AB - Spermine/nitric oxide complex (Sper/NO) is a new nitric oxide (NO) donor with a long half-life providing controlled biological release of NO in vivo. The purpose of this study was to determine whether flap survival could be improved by pre ischaemic or post-ischaemic intravenous administration of Sper/NO. We divided 37 male Wistar rats into four experimental groups. An extended epigastric adipocutaneous flap was raised in each animal. The mean area of flap necrosis was assessed for all groups on the fifth postoperative day, using planimetry software. The average area of flap necrosis was mean +/- s.d. = 68.2%+/-18.1% in the control group, and 29.7% +/- 13.3% in the non-ischaemic controls. The group with pre-ischaemic application of Sper/NO demonstrated an average flap necrosis of mean+/-s.d. = 11.2%+/-5.9%, whereas this increased to 59.2%+/-14.4% in the group receiving Sper/NO 5 min prior to reperfusion. The group with pre-ischaemic application of Sper/NO showed a significantly lower area of flap necrosis than either of the control groups or the group receiving Sper/NO just prior to reperfusion (P < 0.05). The group receiving Sper/NO just prior to reperfusion demonstrated a significantly higher mean area of flap necrosis than the non ischaemic controls (P < 0.05), but did not differ significantly from the control group. Our data show that pharmacological preconditioning and enhancement of flap survival can be achieved by intravenous administration of Sper/NO. The application of Sper/NO at the end of the ischaemia period or in the early reperfusion period provides no protection against ischaemia-reperfusion injury. PMID- 12372375 TI - A snake in the clinical grass: late compartment syndrome in a child bitten by an adder. AB - Snakebite envenomation is an uncommon condition in the UK, but requires vigilance with regard to both the systemic effects of the venom and the locoregional impact on the soft tissues. We describe a case requiring delayed fasciotomies for closed compartment syndrome of the leg and thigh, and discuss in detail the controversies surrounding decompression in such a case. Adder bites are uncommon in the UK, but can result in envenomation of varying severity. Apart from the numerous possible systemic effects that require attention, there are local effects that, very rarely, can be limb threatening. Of these, elevated limb compartment pressures are of paramount importance, and recognition of closed compartment ischaemia is vital if the limb is to be saved by surgical decompression. Guidelines on threshold compartment pressures and fasciotomies are indistinct regarding snakebite, with diagnostic emphasis still placed on clinical signs and symptoms. In the paediatric setting, measurement of compartment pressures is a valuable adjunct to clinical suspicion in the diagnosis of acute compartment syndrome secondary to snakebite. PMID- 12372376 TI - Free allogeneic muscle transfer for cranial reconstruction. AB - An allogeneic muscle transfer was used to cover a large cranial defect in one of a pair of craniopagus twin separated a decade ago. Both separated twins died, 7 months apart, with the twin that had received the transfer dying first. The cause of death was generalised cytomegalovirus infection. An autopsy showed extensive brain necrosis caused by vascular insufficiency, a result of the abnormal vascular anatomy at birth. The muscle allograft showed no signs of rejection. Progress in immunosuppressive treatment over the last decade, which has enabled successful allogeneic nerve grafts and composite-tissue transplantations, might make muscle transplantation for the coverage of large defects, with and without functional demands, feasible in the future. PMID- 12372377 TI - Cross-limb vascular shunting as an auxiliary to major limb revascularisation. AB - A 40-year-old male motorcyclist suffered a near-total amputation of his right foot. His limb was successfully salvaged with the aid of a cross-leg vascular shunt. Temporary arterial flow from the contralateral limb was transmitted via a pressure monitor tube to perfuse the avulsed part. This allowed the surgeon to carry out unhurried wound debridement, dissection of vital structures and skeletal fixation. The cannulation port was placed well distal to the proposed definitive anas<$>tomosis, to reduce damage to the endothelium. This procedure could be a valuable adjunct in major limb replantation, particularly in cases of prolonged ischaemia. PMID- 12372378 TI - A neonate with a giant congenital naevus: new treatment option with the erbium:YAG laser. AB - We report a neonate with a giant congenital naevus on the scalp, who was treated with the erbium:YAG laser when she was 9 days old. This treatment option proved to be a valuable alternative approach to this problem. PMID- 12372379 TI - Malignant melanoma metastasis to the sentinel node in the popliteal fossa. AB - Malignant melanoma metastasis to regional nodes is a well-recognised clinical event. Increasingly, sentinel lymph-node biopsy is being advocated for diagnostic and prognostic purposes. The lymphatic spread of tumour from the lateral aspect of the lower leg and foot is classically described as draining directly to the groin. We discuss the role of lymphoscintigraphy and popliteal dissection with reference to a recent case of a patient with a malignant melanoma at the level of the lateral malleolus that was shown to drain directly to a sentinel node in the popliteal fossa. PMID- 12372380 TI - Giant basal cell carcinoma affecting the lower abdominal, genital and bilateral inguinal regions. AB - We describe a giant basal cell carcinoma, measuring 40 cm x 20 cm, of the lower abdominal, genital and bilateral inguinal regions. The rectus abdominis muscle and the adductor magnus muscle were exposed centrally, and the penis and scrotum were completely destroyed. Reconstruction was performed with a fillet thigh flap, and an excellent result was obtained 1 year after surgery. PMID- 12372381 TI - Squamous cell carcinoma of the penile skin in a neovagina 20 years after male-to female reassignment. AB - We present a patient who underwent male-to-female reassignment, and then developed squamous cell carcinoma during a complicated long-term follow-up. In very rare cases, squamous cell carcinoma may be considered in the differential diagnosis of sustained ulceration in neovaginas constructed by inverting the penile skin in male-to-female reassignments, in particular because clinical examination may be hampered by contractile scar formation of the neovaginal canal. Despite the lack of statistical evidence, it may be assumed that the heterotopic penile skin is at an increased risk of developing HPV-induced squamous cell carcinoma, especially if, over the years, there is a personal history of venereal warts. PMID- 12372382 TI - Staging of melanoma, and the UK guidelines. PMID- 12372383 TI - Ambiguity in the UK guidelines for the management of cutaneous melanoma. PMID- 12372384 TI - The bra-strap injury. PMID- 12372385 TI - Incidental improvement of breast capsular contracture following treatment of arthritis with glucosamine and chondroitin. PMID- 12372386 TI - Capsule within a capsule: an unusual entity. PMID- 12372387 TI - Successful treatment of an allergic reaction in a red tattoo with the Nd-YAG laser. PMID- 12372388 TI - An improved technique for the application of topical anaesthetic cream to skin graft donor sites. PMID- 12372389 TI - Plastic surgery waiting lists--is anyone listening? The static work activity profile (SWAP). PMID- 12372390 TI - Use of a scar simulator to aid patients' understanding of postoperative scar outcome. PMID- 12372391 TI - The turnaround suture to maintain direction in continuous areolar sutures. PMID- 12372393 TI - The pillowcase sling after hand surgery. PMID- 12372392 TI - Innovative use of the breast balloon dissector as a sizer. PMID- 12372394 TI - A conversation with Wan Kyoo Cho. Interview by K. Noelle Gracy. PMID- 12372395 TI - Novel human prostate-specific cDNA: molecular cloning, expression, and immunobiology of the recombinant protein. AB - The differential display-polymerase chain reaction technique was employed to obtain a prostate-specific approximately 300-bp cDNA fragment. On screening the human prostate-lambdagt10 library with this fragment, a full-length approximately 1.5-kb cDNA encoding for a prostate antigen, designated as human novel prostate specific antigen (hNPSA), was found. Extensive database searches revealed that the hNPSA cDNA is a novel sequence. It has an open reading frame (ORF) of 735-bp encoding for 245 amino acids (aa), with a calculated molecular mass of approximately 27kDa. Hydrophilicity analysis of the deduced aa sequence indicated that hNPSA is a membrane-anchored peptide. Analysis for tissue-specificity by Northern blot and RT-PCR-Southern blot procedures indicated that hNPSA is specifically expressed only in human prostate. The hNPSA (ORF) was subcloned into pET22b(+) vector and expressed using the histidine-tagged gene fusion system. The recombinant (r) protein of approximately 27kDa was purified and antibodies (Ab) were raised in rabbits. The rhNPSA Ab recognized a specific protein band of approximately 35kDa in solubilized human prostate tissue and not in any of the other 10 human tissues tested in the Western blot procedure. The hNPSA expression is upregulated 2.5- to 3-fold, both at the mRNA and protein levels in androgen dependent LNCaP cells, as compared to normal whole prostate tissue. Antisense, but not the sense, phosphothiorate-conjugated oligonucleotides based on the hNPSA cDNA sequence significantly (p<0.001) inhibited proliferation of LNCaP cells in a concentration-dependent manner. Thus, the novel hNPSA, which has prostate specific expression and seems to be involved in carcinogenesis, may have applications in the specific diagnosis and treatment of prostate cancer. PMID- 12372396 TI - Crystallization and characterization of Smaug: a novel RNA-binding motif. AB - During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3(')UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of a=b=129.3A, c=33.1A, alpha=beta=90 degrees, gamma=120 degrees and diffract to 1.80A with synchrotron radiation. Initial characterization of this domain suggests that it encodes a novel RNA-binding motif. PMID- 12372397 TI - Identification and characterization of a third gastrin response element (GAS-RE3) in the human histidine decarboxylase gene promoter. AB - In human gastric cancer cells the human histidine decarboxylase gene is regulated by gastrin through two overlapping cis-acting elements known as gastrin response elements 1&2 (GAS-RE1, GAS-RE2) [J. Biol. Chem. 274 (1999) 20961]. Here, we report the identification and characterization of a third element GAS-RE3 that was localized to a region +28 to +48 downstream of the transcriptional start site (+1). Gastrin stimulation induced a rapid increase in binding to the element of a novel nuclear factor named gastrin response element-binding protein 3 (GAS REBP3). Block mutations in the GAS-RE3 sequence (+38GTGCG(+42) to +38TAAGT(+42)) led to reduced promoter activity and decreased binding in EMSA. UV cross-linking studies and Southwestern blot analysis with wildtype and mutant GAS-RE3 showed that GAS-REBP3 was a approximately 110kDa protein. Thus, gastrin-mediated regulation of HDC gene expression appears to be mediated by a complex cis-acting element, which binds at least three distinct nuclear factors. PMID- 12372398 TI - Change of product specificity of hexaprenyl diphosphate synthase from Sulfolobus solfataricus by introducing mimetic mutations. AB - The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) prenyl diphosphate synthase, hexaprenyl diphosphate synthase from a thermoacidophilic archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes. PMID- 12372399 TI - Prostaglandin F(2alpha) stimulates tyrosine phosphorylation of phospholipase C gamma1. AB - In this study, we investigated the ability of prostaglandin F(2alpha) (PGF(2alpha)) to induce tyrosine phosphorylation of phospholipase C-gamma1 (PLC gamma1) in cat iris sphincter smooth muscle (CISM) cells. PGF(2alpha)(1 microM) stimulated PLC-gamma1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF(2alpha), suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF(2alpha). Furthermore, PGF(2alpha)-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF(2alpha) stimulates tyrosine phosphorylation of PLC-gamma1 in CISM cells and indicate that PGF(2alpha)-stimulated tyrosine phosphorylation is responsible for an early signal transduction event. PMID- 12372400 TI - Leucine stimulates the secretion of hepatocyte growth factor by hepatic stellate cells. AB - Branched-chain amino acids (BCAAs) modulate various cellular functions, in addition to providing substrates for the production of proteins. In this study, we examined the effect of BCAAs on the secretion of hepatocyte growth factor (HGF) by hepatic stellate cells. A hepatic stellate cell clone was cultured in medium supplemented with various concentrations of valine, leucine, or isoleucine. Of these BCAAs, leucine markedly induced an increase in the levels of HGF in the medium in a dose-dependent manner. The addition of valine or isoleucine had no significant effect on HGF levels in the medium. The difference in levels of HGF in the medium between leucine-treated and non-treated cells was enhanced by the incubation period. These results demonstrate that, among BCAAs, leucine stimulates the secretion of HGF by cultured hepatic stellate cells. PMID- 12372401 TI - Ligand-specific control of src-suppressed C kinase substrate gene expression. AB - The src-suppressed C-kinase substrate, SSeCKS, is now recognized as a key regulator of cell signaling and cytoskeletal dynamics. However, few ligands that control SSeCKS expression have been identified. We report that platelet-derived growth factor-BB (PDGF-BB), lysophosphatidic acid (LPA), and eicosapentaenoic acid (EPA) potently modulate SSeCKS gene expression in cultured smooth muscle (RASM) cells relative to other bioactive ligands tested. In addition, EPA dependent regulation of SSeCKS expression correlates with distinct changes in cell morphology and adhesion in RASM cells. Independent evidence that ligand specific control of SSeCKS expression links to the regulation of cell adhesion and morphology was obtained using ras-transformed fibroblasts, KNRK. Sodium butyrate (NaB) upregulates SSeCKS mRNA and protein expression corresponding to increased cell-spreading and adhesion. In addition, ectopic expression of recombinant SSeCKS recapitulates attributes of NaB-induced morphogenesis in KNRK cells. The data provide novel evidence that SSeCKS functions in PDGF-BB-, LPA-, EPA-, and NaB-mediated cell signaling. PMID- 12372402 TI - Delivery of nucleic acid into mammalian cells by anthrax toxin. AB - Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells. PMID- 12372403 TI - Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398. AB - Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS 398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC. PMID- 12372404 TI - Splicing mutation of the prostacyclin synthase gene in a family associated with hypertension. AB - Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patient's family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension. PMID- 12372405 TI - Characterization of the short neuropeptide F receptor from Drosophila melanogaster. AB - A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs. PMID- 12372406 TI - Porphyromonas gingivalis lipopolysaccharide interferes with salivary mucin synthesis through inducible nitric oxide synthase activation by ERK and p38 kinase. AB - Porphyromonas gingivalis is a Gram-negative periodontopathic bacterium colonizing the oral cavity and its lipopolysaccharide (LPS) is a key factor in the development of periodontitis. We investigated the effect of P. gingivalis LPS on the cellular responses associated with mucin synthesis in sublingual salivary gland acinar cells. Exposure of the acinar cells to the LPS led to a dose dependent decrease in mucin synthesis and was accompanied by a massive induction in inducible nitric oxide synthase (NOS-2) activity and the increase in NO production, caspase-3 activity and apoptosis. Inhibition of extracellular signal regulated kinase (ERK) with PD98059 accelerated the LPS-induced decrease in the glycoprotein synthesis and caused further increase in apoptosis and NOS-2 activity, while the blockade of p38 mitogen-activated kinase (MAPK) with SB203580 countered the LPS-induced reduction in the glycoprotein synthesis and obviated the induced increases in NOS-2 and apoptosis. Introduction of NOS-2 inhibitor, L NAME, not only countered the LPS-induced increase in NO generation, caspase-3 activity and apoptosis, but caused the impedance of the LPS inhibition on mucin synthesis. The findings point to the upregulation in NOS-2 expression by P. gingivalis LPS as a key detrimental culprit affecting salivary mucin synthesis. PMID- 12372407 TI - Molecular model of cyclin-dependent kinase 5 complexed with roscovitine. AB - Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives. PMID- 12372408 TI - Novel human cDNAs homologous to Drosophila Orct and mammalian carnitine transporters. AB - The molecular basis of the transport of organic ions (which include such medically important compounds as drugs, toxins, and metabolites) has been intensively studied ever since the identification of the prototypical anion and cation transporters, OAT1 (originally cloned by us as NKT) and OCT1. Here we report the cloning of two novel putative organic ion transporters with 12 predicted membrane spanning segments that are most homologous to mammalian OCTNs (carnitine transporters) and to the Drosophila putative transporter, Orct, an intriguing correspondence that led us to name our sequences Fly-like putative transporters (Flipts). Another transporter we cloned has recently been identified as OAT5. Inclusion of Flipts reveals that the organic ion transporter family tree has trifurcated into three branches, one bearing Flipts, OCTNs, and fly transporters, and the other two bearing OATs and OCTs. Flipts are widely expressed in adult kidney, brain, muscle, and other tissues; in contrast, OAT1 is largely in kidney, and OAT5, in liver. In the embryo as well, Flipts are broadly distributed, whereas OAT5 was found only in fetal liver. Flipt expression patterns resemble those of the phylogenetically related OCTNs, suggesting that Flipts might also participate in carnitine transport, particularly in brain, which has relatively high Flipt expression, including EST matches from amygdala, hippocampus, and hypothalamus. PMID- 12372409 TI - Reconstruction of glycosaminoglycan chains in decorin. AB - The glycosaminoglycan chain of decorin from human spinal ligaments was digested using the hydrolysis of bovine testicular hyaluronidase. As a result, decorin with hexasaccharide, octasaccharide, and decasaccharide including the linkage region, GlcA-Gal-Gal-Xyl, was obtained. The obtained decorin as an acceptor and hyaluronic acid as a donor were incubated with bovine testicular hyaluronidase under the condition of transglycosylation reaction. The transglycosylation reaction product had hexasaccharide to triacontasaccharide. Judging from the analysis of glycosaminoglycan chain in the transglycosylation reaction product, it was confirmed that hyaluronic acid chain as a donor was transferred to the retained glycosaminoglycan chain of decorin as an acceptor. Similarly, it was possible to reconstruct the glycosaminoglycan chain in decorin to chondroitin, chondroitin 4-sulfate or chondroitin 6-sulfate. Therefore, we succeeded in synthesizing an artificial family of decorins. PMID- 12372410 TI - Identification of the hRDH-E2 gene, a novel member of the SDR family, and its increased expression in psoriatic lesion. AB - To identify novel psoriasis-associated genes, we focused on several ESTs (expressed sequence tags) whose expression was predominantly increased in the affected skin in patients with psoriasis vulgaris, as assessed by microarray assay. In this paper, a full-length cDNA corresponding to one of those ESTs (AI440266) was isolated by screening of cultured human keratinocyte cDNA libraries. This cDNA has an open reading frame of a 309-amino-acid protein, sharing significant homology to one of the short-chain alcohol dehydrogenase/reductase (SDR) families that can catalyze the first and rate limiting step that generates retinaldehyde from retinol. So, this gene was designated as hRDH-E2 (human epidermal retinal dehydrogenase 2). The hRDH-E2 gene has a single functional copy on chromosome 8q12.1, spanning approximately 20kb with seven exons. The deduced amino acid sequence contains three motifs that are conserved in the SDR family. Qualitative RT-PCR demonstrated that the mRNA levels of hRDH-E2 were significantly elevated in the affected skin in psoriasis patients as compared to the unaffected skin in patients and the normal skin in healthy individual. These results suggest that hRDH-E2 may be involved in the pathogenesis of psoriasis through its critical role in retinol metabolism in keratinocyte proliferation. PMID- 12372411 TI - Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit CBP-NF-kappaB interaction in activated microglia. AB - The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP), two immunomodulatory neuropeptides, act as anti inflammatory factors for activated microglia, by inhibiting the production of pro inflammatory factors, mainly mediated through the inhibition of NF-kappaB nuclear translocation and DNA binding. An additional regulatory element in the NF-kappaB transcriptional activity is the coactivator CBP, which links p65 with components of the basal transcriptional machinery. The present report demonstrates that VIP and PACAP inhibit the formation of p65/CBP complexes and that this event is directly related to the neuropeptide inhibition of NF-kappaB transcriptional activity. Since CBP is in limiting amounts in the nucleus and is capable of interacting with several transcriptional factors, competition for CBP provides another mechanism for transcriptional regulation. VIP and PACAP increase CBP binding to CREB, replacing p65/CBP with CREB/CBP complexes in activated microglia. This is due to VIP/PACAP-induced increases in CREB phosphorylation/activation and is mediated through the specific VPAC1 receptor and the cAMP/PKA pathway. The VIP/PACAP interference with the p65/CBP interaction in activated microglia may represent a significant element in the regulation of the inflammatory response in the CNS by the endogenous neuropeptides. PMID- 12372412 TI - Species-specific differences in the usage of several caspase substrates. AB - The activation of caspases cleaving a plethora of specific substrates is pivotal for initiation as well as execution of apoptosis. The recognition motif for caspases is a tetrapeptide sequence containing an essential aspartic acid residue at the fourth position (often DXXD). Here, we report that the caspase cleavage sites of most identified substrates show a high degree of conservation between different species. However, we have identified differences in the cleavage sites of five substrates between murine and human proteins leading to either select processing in only one species or to different cleavage patterns. Finally, we provide evidence that murine c-Abl but not its human homolog serves as efficient substrate during apoptosis. PMID- 12372413 TI - Association of human RAD52 protein with transcription factors. AB - The human RAD52 protein has been implicated in DNA homologous recombination. Four major functional domains have been identified: a DNA binding domain (amino acids 1-85), a self-association and UBC9-interacting domain (amino acids 85-159), an RPA-interacting domain (amino acids 221-280), and a RAD51-interacting domain (amino acids 287-330). However, it is uncertain about the functional roles of the C-terminal region of RAD52 protein. In this report, we demonstrate an association of a C-terminal domain of human RAD52 (amino acids 302-418) with the XPB and XPD subunits of transcription factor TFIIH and RNA polymerase II (RNAPII). Using a Gal-4 binding based transcription assay, we further show that this C-terminal domain activates transcription. However, the RAD52 self-association domain suppresses transcription, resulting in an overall activity of transcriptional suppression by the full-length RAD52 protein. These results suggest a novel activity of RAD52 in transcription regulation and may further imply a functional role of RAD52 in targeting DNA damage on transcription active loci to recombinational repair. PMID- 12372414 TI - A new class of tetraspanins in fungi. AB - Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins. PMID- 12372415 TI - Histamine H(2) receptor-mediated modulation of local cytokine expression in a mouse experimental tumor model. AB - Accumulating evidence indicates that histamine is involved in the modulation of cytokine expression patterns. We previously reported that daily treatment with the H(2) receptor antagonist, cimetidine, suppressed tumor growth through alteration of the local cytokine expression pattern. In this study, we used a mouse strain genetically lacking histidine decarboxylase (HDC), to evaluate the role of endogenous histamine synthesis on cytokine expression and tumor development. In the mutant mice, cimetidine had no effect on tumor growth, whereas an H(2) agonist, dimaprit, significantly enhanced tumor growth. When the HDC-deficient mice were implanted with mutant CT-26 cells stably expressing HDC, drastic suppression of tumor growth by cimetidine was observed, which was accompanied by augmentation of mRNA expression of LT-beta, TNF-alpha, and IFN gamma in the tumor tissues. These results suggest that endogenous histamine synthesis in tumor tissues suppresses local tumor immunity via the H(2) receptors, resulting in tumor growth promotion. PMID- 12372416 TI - Vitamin D(3) and analogues modulate the expression of CSF-1 and its receptor in human dendritic cells. AB - The active vitamin D(3)-metabolite 1,25(OH)(2)D(3) inhibits the interleukin 4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF)-induced differentiation of human monocytes into dendritic cells without altering survival. Colony-stimulating factor 1 (CSF-1) is an important survival factor for cells of the monocytic lineage. We therefore investigated whether the inhibitory activity of 1,25(OH)(2)D(3) is paralleled by a regulation of CSF-1 and its receptor. Purified human monocytes were cultured together with IL-4/GM-CSF in the presence of 1,25(OH)(2)D(3), its analogue tacalcitol, the low-affinity vitamin D receptor ligand 24,25(OH)(2)D(3), or the solvent ethanol for up to 5 days. Expression of CSF-1, CSF-1R, and GM-CSF mRNA was measured by RT-PCR. Protein secretion for CSF-1 was measured by ELISA, expression of CSF-1R by flow cytometry. The results showed that 1,25(OH)(2)D(3) and tacalcitol significantly up-regulated CSF-1 mRNA-expression and protein secretion in a dose-dependent manner. The effect of 1,25(OH)(2)D(3) occurred already after 1h of pre-treatment. In contrast, CSF-1R mRNA- and cell surface-expression was down-regulated simultaneously. The solvent ethanol and 24,25(OH)(2)D(3) were without effect. GM CSF mRNA expression was not modulated in 1,25(OH)(2)D(3)-treated cells. These data point towards a distinct and specific regulation of CSF-1 and its receptor by 1,25(OH)(2)D(3) and its analogue tacalcitol in human monocytes which parallels the inhibition of differentiation into dendritic cells without altering survival. PMID- 12372417 TI - NF-kappaB activates fibronectin gene expression in rat hepatocytes. AB - Fibronectin (FN) plays a role in various biological processes such as fibrosis and tumor metastasis. In this study, we investigated the regulation of FN gene expression by NF-kappaB transcription factor. Transient expression of NF-kappaB p65 increased FN promoter activity in rat hepatocytes. Deletion analysis of FN promoter localized the NF-kappaB-responsive region at the position between -1214 and -1126. Mutations in a putative NF-kappaB element (5(')-GAGAATTTCC-3(')) at 1180 blocked most of the p65-induced promoter activity. Electromobility shift assays showed that the expression of p65 induced the binding of the p65/p65 homodimer to the NF-kappaB site at -1180. Stably p65-expressing cells showed increase of promoter activity, FN protein, and its mRNA levels over control cells. Furthermore, treatment of cells with interleukin-1beta, a NF-kappaB stimulating cytokine, also increased promoter activity, FN production, and mRNA levels. These results show that NF-kappaB activates FN gene expression by binding to the responsive element at -1180 as the p65/p65 homodimer in rat hepatocytes. PMID- 12372419 TI - Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells. AB - The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14). PMID- 12372418 TI - Defining the boundaries of the testis angiotensin I-converting enzyme ectodomain. AB - Numerous cytokines, receptors, and ectoenzymes, including angiotensin I converting enzyme (ACE), are shed from the cell surface by limited proteolysis at the juxtamembrane stalk region. The membrane-proximal C domain of ACE has been implicated in sheddase-substrate recognition. We mapped the functional boundaries of the testis ACE ectodomain (identical to the C domain of somatic ACE) by progressive deletions from the N- and C-termini and analysing the effects on catalytic activity, stability, and shedding in transfected cells. We found that deletions extending beyond Leu37 at the N-terminus and Trp616 at the C-terminus abolished catalytic activity and shedding, either by disturbing the ectodomain conformation or by inhibiting maturation and surface expression. Based on these data and on sequence alignments, we propose that the boundaries of the ACE ectodomain are Asp40 at the N-terminus and Gly615 at the C-terminus. PMID- 12372420 TI - Nitration and chlorination of folic acid by peroxynitrite and hypochlorous acid, and the selective binding of 10-nitro-folate to folate receptor beta. AB - The aim of this work was to characterize folates modified by ONOO(-) and HOCl and to evaluate the binding capacity of folates modified by ONOO(-) to folate receptor alpha and beta. For the modification of folate by ONOO(-), folic acid was reacted with the combination of PMA activated PMN and PAPA NONOate or chemically synthesized ONOO(-). For the modification of folate by HOCl, folic acid was reacted with the combination of MPO and H(2)O(2) or NaOCl. The structures of products were determined by 1H-NMR and MALDI-TOF mass. Nitrated folate species were identified as 10-nitro-folate and 12-nitro-folate, and chlorinated folate was identified as 12-chloro-folate. The 10-nitro-folate showed the selective binding to FR-beta, compared to folic acid. PMID- 12372421 TI - The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells. AB - IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter. PMID- 12372422 TI - Genomic organization, chromosomal localization, and alternative splicing of the human phosphodiesterase 8B gene. AB - We have characterized the gene for human phosphodiesterase 8B, PDE8B, and cloned the full-length cDNA for human PDE8B (PDE8B1) and two splice variants (PDE8B2 and PDE8B3). The PDE8B gene is mapped to the long arm of chromosome 5 (5q13) and is composed of 22 exons spanning over approximately 200kb. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. PDE8B1 encodes an 885 amino acid enzyme, containing an N-terminal REC domain, a PAS domain, and a C-terminal catalytic domain. PDE8B2 and PDE8B3 both have deletion in the PAS domain and encode 838 and 788 amino acid proteins, respectively. RT-PCR analysis revealed that while PDE8B1 is the most abundant variant in thyroid gland, PDE8B3, but not PDE8B1, is the most abundant form in brain. These findings suggest that selective usage of exons produces three different PDE8B variants that exhibit a tissue-specific expression pattern. PMID- 12372423 TI - Effects of leptin, troglitazone, and dietary fat on stearoyl CoA desaturase. AB - Leptin, troglitazone, and high fat feeding profoundly influence the lipid content of various tissues. To determine if they affect the expression of stearoyl CoA desaturase (SCD)-1 and -2, their mRNA was measured in livers of normal, hyperleptinemic, troglitazone-treated, and fat-fed rats. Hyperleptinemia, which reduces tissue TG by downregulating lipogenic enzymes and upregulating fatty acid oxidation, lowered SCD-1 96% below controls and reduced SCD-2 slightly. By contrast, hepatic SCD-1 mRNA of leptin-resistant fa/fa rats was five times wild type controls, but SCD-2 mRNA was 66% lower. High fat feeding lowered SCD-1 by 80%, possibly by inducing hyperleptinemia. Troglitazone treatment, which reduces nonadipose tissue TG of fa/fa rats without downregulating lipogenic enzymes, raised SCD-2 13-fold but lowered SCD-1 by 25%. The findings suggest that leptin controls SCD-1 expression and that troglitazone's antilipotoxic action may involve SCD-2 upregulation. PMID- 12372424 TI - Phosphatase inhibition leads to activation of IkappaB kinase in murine macrophages. AB - We have been interested in elucidating the role of intracellular phosphatase activity in the regulation of immune cell activation. To this end, we treated RAW 264.7 murine macrophages with the phosphatase inhibitor, calyculin-A. Treatment with calyculin-A led to activation of IkappaB kinase, degradation of IkappaBalpha, and induced nuclear translocation and DNA binding of NF-kappaB. Each of these effects occurred in both a time- and dose-dependent manner. In addition, each of these effects was negatively modulated by prior induction of the heat-shock response. Despite clear activation of the IkappaB kinase/IkappaBalpha/NF-kappaB pathway, however, phosphatase inhibition did not lead to increased expression of NF-kappaB-dependent genes. Thus, intracellular phosphatase activity is a central regulator of the NF-kappaB signal transduction pathway and is negatively modulated by heat shock. Inhibition of intracellular phosphatase activity with calyculin-A is not sufficient to induce NF-kappaB dependent gene expression, demonstrating the complexity of NF-kappaB regulation in immune cells. PMID- 12372425 TI - Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin induced angiogenesis. AB - In skeletal muscles, angiogenesis can be induced by increases in wall shear stress. To identify molecules involved in the angiogenic process, a method based on the use of BS-1 lectin-coated magnetic beads was developed to isolate a cellular fraction enriched in microvascular endothelial cells which are directly exposed to wall shear stress. Using such cellular fractions from skeletal muscles of C57 mice in which angiogenesis was induced by administration with the alpha(1) adrenergic antagonist prazosin, we found the concentration of vascular endothelial growth factor (VEGF) increased in correlation to the duration of the prazosin stimulus. In contrast, the angiopoietin-2/tie-2 system was not changed even after 4days of prazosin treatment. In neuronal nitric oxide synthase (nNOS) knockout mice, the VEGF concentration was also elevated after prazosin treatment but remained almost unchanged in endothelial nitric oxide synthase (eNOS) knockout mice. However, eNOS (and not nNOS) knockout mice expressed higher levels of VEGF under non-stimulated conditions as compared to C57 mice. These results suggest that VEGF produced in endothelial cells is involved in angiogenesis in skeletal muscles of mice responding to the administration of systemic vasodilators. NO derived from eNOS and nNOS may be an important regulator of the angiogenic response in skeletal muscles in vivo. PMID- 12372426 TI - TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways. AB - TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex. PMID- 12372427 TI - Cloning and characterization of the highly expressed ETEA gene from blood cells of atopic dermatitis patients. AB - Analysis of patients with atopic dermatitis (AD) for differential expression of genes, as compared to normal individuals, will be useful for understanding the molecular pathogenesis of AD. We found that the expression of the gene ETEA in human peripheral blood CD3-positive cells from patients with atopic dermatitis was significantly higher than in normal individuals. Eosinophils from AD patients expressed ETEA at a significantly higher level than the healthy controls. The overall sequence of the 445 aa deduced polypeptide from the cloned ETEA cDNA showed homology to human Fas-associated factor 1 (FAF1), which is involved in Fas mediated apoptosis. However, the interaction of ETEA with the Fas death domain was weaker than that of FAF1, as studied in yeast two-hybrid experiments. The ETEA-EGFP fusion protein was expressed in cytoplasm. During the course of activation-induced cell death of primary T cells, transcription levels of ETEA and FAF1 were upregulated with similar kinetics. The enhanced expression of ETEA may play a role in the regulating the resistance to apoptosis that is observed in T cells and eosinophils of AD patients. PMID- 12372428 TI - Intracellular fatty acid downregulates ob gene expression in 3T3-L1 adipocytes. AB - The effect of intracellular free fatty acid (FFA) accumulation on ob gene expression in adipocytes was examined. In fully differentiated 3T3-L1 adipocytes, triacsin C, a specific acyl CoA synthetase inhibitor with a K(i) of 8.97 microM, inhibited ob gene expression by 20% at 5 x 10(-5)M. At this concentration, triacsin C induced accumulation of intracellular FFA. Treatment with both chylomicron and triacsin C reduced ob gene expression more than treatment with triacsin C alone. Treatment with 2-bromopalmitate, a poorly metabolizable palmitate analog, reduced ob gene expression by 50% at 10(-4)M, but palmitate at the same concentration had no effect. This is the first demonstration that the ob gene is downregulated by intracellular FFA accumulation, thereby raising the possibility that ob product is regulated in response to lipolysis. PMID- 12372429 TI - Enhancer elements in the mouse Cyp1a2 gene for constitutive expression. AB - CYP1A2 is one of the major hepatic cytochrome P450s that is involved in the metabolism of many drugs, as well as in the activation of chemical carcinogens. To elucidate the transcriptional regulation of the constitutive expression of the mouse Cypla2 gene, the 4.8-kbp 5(')-flanking region of the gene was analyzed for transcriptional activity using a primary cultured mouse hepatocyte system. With 5(')- and 3(')-deletion analysis, two enhancer elements, i.e., a 20-bp DNA fragment (E1) from -4401 to -4382 and a 9-bp (E2) from -4300 to -4292, were identified. E1 and E2 contain a phorbol 12-O-tetradecanoate-13-acetate (TPA) responsive element (TRE) and TRE-like element, respectively. Electrophoretic mobility shift assay confirmed specific binding between these two enhancer elements and nuclear proteins. Site-directed mutagenesis assay suggested that the TRE element in E1 is essential for constitutive expression of the mouse Cypla2 gene. PMID- 12372430 TI - Transcriptional activation of the human stress-inducible transcriptional repressor ATF3 gene promoter by p53. AB - Activating transcription factor 3 (ATF3) is an immediate early response gene that is induced in cells exposed to a variety of stress stimuli. In this report, upon exposure of cells to ultraviolet (UV) or proteasome inhibitor MG132, ATF3 protein was induced more efficiently in cells with intact p53 allele than in those with null mutant p53 allele. In Saos-2 cells harboring the temperature-sensitive mutant p53(Val-138), the expression of ATF3 gene was more significant at permissive temperature of 32.5 degrees C than at non-permissive 37.5 degrees C. Reporter assay of the human ATF3 gene promoter identified two p53-responsive elements at -379 to -370 and -351 to -342 from the transcriptional start site. These elements were capable of conferring p53 responsiveness to a heterologous promoter and specifically bound p53 protein in electrophoretic mobility shift assay. Furthermore, ATF3 gene promoter was more significantly activated by UV in cells with wild p53 allele. These results clearly show that the human ATF3 gene is one of the target genes directly activated by p53 and may suggest a functional link between stress-inducible transcriptional repressor ATF3 and p53. PMID- 12372432 TI - Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus. AB - The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha. PMID- 12372431 TI - Effects of resveratrol on the G(0)-G(1) transition and cell cycle progression of mitogenically stimulated human lymphocytes. AB - Resveratrol (RSV) has been suggested to have cancer preventive properties, on the basis that it suppresses proliferation and induces apoptosis in various tumor cells. Here we test its cytostatic effects on peripheral blood human lymphocytes. RSV (up to 50 microM) had no detectable effects on resting lymphocytes. With the mitogen phytohemagglutin (PHA), however, RSV elicited concentration- and time dependent responses in lymphocytes. RSV (>/=50 microM) prevented cell entry into the cell cycle, resulting in 99% suppression at 100 microM. The arrested lymphocytes following 24h treatment with 50 microM RSV had minimal RNA content, the feature characteristic of G(0) cells, and were blocked at the stage past the induction of cyclins D2 and D3 and prior to induction of cyclin E. Prolonged treatment (72h) of PHA-stimulated lymphocytes with 100 microM RSV showed a pronounced decrease in the expression of pRb, cyclins E and B, and reduction in p34cdc2 and PCNA. The activation-induced apoptosis was also reduced in the presence of >/=50 microM RSV. These data suggest that studies designed to test RSV efficacy as a chemopreventive agent should include evaluation of its immunomodulatory effect revealed by suppression of lymphocyte stimulation as well as its effect on apoptosis of stimulated lymphocytes. PMID- 12372433 TI - Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation. AB - Integrin-mediated adhesion induces the formation of focal adhesions that link the extracellular matrix and intracellular actin cytoskeletal networks. We previously showed that integrin-linked kinase (ILK), which can interact with beta1 and beta3 integrins, and its interacting protein, affixin, play an essential role in the initial assembly of focal adhesion structures and actin stress fibers. Although the relevant structures are also observed in integrin alphaIIbbeta3 in platelets, the precise underlying molecular mechanism remains unclarified. Here, we found that ILK stably forms a complex with ss-affixin in platelets. Thrombin stimulation induces their association with integrin beta3, which is followed by their incorporation into the Triton-insoluble membrane-cytoskeletal fraction. During the course of thrombin-induced platelet aggregation, ILK activity was enhanced within 90s to 2.1-fold of the basal level, independent of phosphatidylinositol 3-kinase. Taken together with the observation that the treatment with an anti-integrin beta3 antibody stimulates ILK activity without inducing platelet aggregation, these results suggest that the outside-in signaling induced by fibrinogen binding to integrin enhances ILK activity and results in the initial phase to reorganize the actin cytoskeleton. PMID- 12372434 TI - Characterization of transgenic rats constitutively expressing vitamin D-24 hydroxylase gene. AB - Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D(3) [25(OH)D(3)] to 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in the kidney. CYP24 is also involved in the breakdown of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the active form of vitamin D(3). In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH)(2)D(3). Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism. PMID- 12372435 TI - Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice. AB - In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET 1 gene expression increases significantly, parallel to the increase in beta casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice. PMID- 12372436 TI - Location of 2(')-O-methyl nucleotides in 26S rRNA and methylation guide snoRNAs in Caenorhabditis elegans. AB - Many nucleotides in rRNAs are modified. We devised a method to locate 2(')-O methyl nucleotide residues using a conventional DNA sequencer. We found 38 2(')-O methyl nucleotides in the 26S rRNA of Caenorhabditis elegans using this method. Fourteen of the 38 residues are conserved in both human and yeast rRNAs and 14 residues are conserved in either human or yeast rRNA. The remaining 10 nucleotides are uniquely methylated in C. elegans 26S rRNA. We searched the C. elegans genomic sequence for small nucleolar RNAs (snoRNAs), which guide the methylation of ribose residues, and predicted 18 snoRNA sequences that are expected to guide the methylation of some of these nucleotide residues. PMID- 12372437 TI - Inhibition of Candida albicans secreted aspartic protease by a novel series of peptidomimetics, also active on the HIV-1 protease. AB - Nineteen reduced amide, monohydroxy- or dihydroxyethylene-based transition-state peptidomimetics, known to be good inhibitors of the aspartic protease of HIV-1, were tested against a secreted aspartic protease (Sap2), purified from the culture medium of a virulent strain of Candida albicans. Ten of these compounds exhibited IC(50)s against Sap2 lower than 15 microM; the best inhibitor, Kyn-Val Phe-Psi[OH-OH]-Phe-Val-Kyn, when added to the C. albicans culture, repressed the hydrolysis of bovine serum albumin (BSA), contained in the culture medium, and inhibited the growth of the fungus. PMID- 12372438 TI - Proceedings from the VIII World Congress on Endometriosis. February 2002. PMID- 12372439 TI - Endometriosis: novel etiopathogenetic concepts and clinical perspectives. AB - OBJECTIVE: To discuss current ideas about therapy for endometriosis derived from new observations generated by using molecular biology techniques and in vivo animal models of disease. METHOD(S): The MEDLINE database was reviewed for English-language articles on new drugs that affect the endocrine or immunologic system, the possibility that endometriosis has multiple forms, and the association of endometriosis with cancer. Specific attention was given to in vivo studies in animals or humans. CONCLUSION(S): Among the novel potential candidate drugs, aromatase inhibitors and raloxifene should be considered for treatment of postmenopausal women with endometriosis. Notable observations have emerged from studies of immunomodulators and antiinflammatory agents in animal models of disease. These findings must be confirmed in women. The histogenesis of ovarian endometriomas is still unclear, thus limiting new experimental approaches to this form of disease. Given the low but established risk for malignant transformation of endometriosis, efforts should be directed toward identification of susceptibility loci for the disease and its potential transformation into cancer. PMID- 12372440 TI - The International Endogene Study: a collection of families for genetic research in endometriosis. AB - OBJECTIVE: The aim of the International Endogene Study is to discover genes that influence susceptibility to endometriosis. DESIGN: The study brings together two research groups based in Australia and the United Kingdom that independently have been collecting families for linkage analysis and candidate gene studies. Both groups used similar methods to recruit families, obtain clinical notes, assign disease status based on the operative records and available histology, and collect common clinical data including age at onset of symptoms, age at diagnosis, and symptoms experienced. SETTING: Recruitment has been mainly from Australia, the United Kingdom, and the United States. PATIENT(S): All affected participants have surgically confirmed disease. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical and epidemiological data. RESULT(S): To date, >1,100 families with affected sisters have been recruited, and >1,200 triads (affected women and both parents), for case-control studies. CONCLUSION(S): We have created the largest resource yet assembled of clinical data and DNA for linkage and association studies in endometriosis. The increase in power to detect susceptibility genes vindicates the decision to merge the two studies and demonstrates the value of large-scale international collaboration. PMID- 12372441 TI - Was Sampson wrong? AB - OBJECTIVE: Sampson's theory of reflux menstruation suggests that endometriosis is one form of a condition known as an autotransplant. This study seeks to characterize autotransplants as they are described in the literature and to determine whether endometriosis resembles an autotransplant. DESIGN: Literature review of published studies containing the following types of information: [1] characterization of the histologic features, immunohistochemistry, or structural function of autotransplants; and [2] comparisons of endometriosis with endometrium. MAIN OUTCOME MEASURE(S): Characteristics of multiple types of autotransplants were noted. Similarity or dissimilarity of endometriosis and endometrium was tabulated to judge qualitatively whether the bulk of the evidence supports the notion that endometriosis is an autotransplant. RESULT(S): Autotransplants remain very similar or identical to eutopic tissues of origin, regardless of the length of time following autotransplantation. Endometriosis differs in many profound and fundamental ways from eutopic endometrium, including clonality of origin, enzymatic activity, protein expression, and histologic and morphologic characteristics. A minority of studies has found similarities between endometriosis and eutopic endometrium. CONCLUSION(S): Endometriosis is dissimilar to eutopic endometrium and therefore lacks characteristics of an autotransplant. Sampson's theory of origin of endometriosis is not supported by the results of this study. Studies of experimental endometriosis that have not used menstrual endometrium may be misleading. PMID- 12372442 TI - Emerging role of genomics in endometriosis research. AB - OBJECTIVE: Rapidly evolving methods in genomics and proteomics research already are changing the nature of biomedical investigation. In this report we briefly review some of the seminal technological advances that inspired the genomics revolution and describe their application in the field of endometriosis research. DESIGN: Review of different techniques successfully applied to endometriosis research. SETTING: Collaborative investigation in academic and pharmaceutical industry laboratories. PATIENT(S): Biopsies were obtained from consenting ovulatory women on no medications, with or without laparoscopically proven endometriosis. RESULT(S): Genomics techniques have confirmed gene products shown to be abnormally expressed in endometriotic tissues by prior studies. In addition, identification of novel transcripts not previously appreciated in this condition were discovered. Examples of dysregulated genes in endometriosis include glycodelin, complement, early growth response-1 and transducer of erbB-1. CONCLUSION(S): Global gene profiling studies are poised to revolutionize the diagnosis and treatment of endometriosis and other human diseases. PMID- 12372443 TI - Effect of prolonged gonadotropin-releasing hormone agonist therapy on the outcome of in vitro fertilization-embryo transfer in patients with endometriosis. AB - OBJECTIVE: To evaluate the effect of a 3-month course of GnRH agonist administered immediately before IVF-ET in infertile patients with endometriosis. DESIGN: Prospective, randomized trial. SETTING: Three tertiary care assisted reproductive technology programs. PATIENT(S): IVF-ET candidates with surgically confirmed endometriosis. INTERVENTION(S): Twenty-five patients received three courses of a long-acting GnRH agonist, 3.75 mg i.m. every 28 days, followed by standard controlled ovarian hyperstimulation. Twenty-six patients received standard controlled ovarian hyperstimulation with mid-luteal phase GnRH agonist down-regulation or microdose flare regimens. MAIN OUTCOME MEASURE(S): Response to controlled ovarian hyperstimulation, ongoing pregnancy rates per cycle, group implantation rates, and implantation rate per embryo transfer procedure. RESULT(S): The extent of surgically confirmed endometriosis was greater in patients who received the long-acting GnRH regimen for 3 months before IVF-ET. The groups did not differ significantly in terms of dose or duration of gonadotropin stimulation, number of oocytes retrieved, fertilization rate, or number of embryos transferred. Patients who received the long-acting GnRH regimen had significantly higher ongoing pregnancy rates (80% vs. 53.85%) and a trend toward higher implantation rates (42.68% vs. 30.38%). CONCLUSION(S): Prolonged use of GnRH agonist before IVF-ET in patients with endometriosis resulted in significantly higher ongoing pregnancy rates than did standard controlled ovarian hyperstimulation regimens. No deleterious effect on ovarian response was observed. PMID- 12372444 TI - Use of intraperitoneal interferon alpha-2b therapy after conservative surgery for endometriosis and postoperative medical treatment with depot gonadotropin releasing hormone analog: a randomized clinical trial. AB - OBJECTIVE: To evaluate the possible therapeutic effects of interferon alpha-2b left in the peritoneum after surgery, followed by or not followed by treatment with GnRH analogs. DESIGN: A prospective, randomized clinical trial. SETTING: University hospital. PATIENT(S): Fifty-two infertile patients with moderate or severe endometriosis. INTERVENTION(S): Laparotomic conservative surgery and either interferon alpha-2b or saline alone left in the pouch of Douglas followed by administration of either GnRH analogs depot or oral indomethacin with transvaginal echography and analysis of CA-125, immunoglobulins, and lymphocyte populations. MAIN OUTCOME MEASURE(S): Recurrence of endometriosis was considered clinically, echographically, and laparoscopically. RESULT(S): Recurrence of endometriosis in four cases without interferon (15.4%) versus 11 patients (42.3%) with interferon alpha-2b. Life table analysis showed significant differences between the groups with and without interferon 21 months after conservative surgery. There were no differences in the recurrence between the groups with or without GnRH analogs. Likewise, there were no significant changes in immunoglobulins and lymphocyte populations among patients with and without recurrence of endometriosis. The patients that received GnRH analogs depot showed a decrease in the number of CD16 and an increase of CD11b cells after treatment. CONCLUSION(S): The use of interferon alpha-2b within the peritoneal cavity after conservative surgery may be inappropriate because it increased later recurrence of endometriosis. The postoperative treatment with GnRH analogs did not significantly reduce the recurrence rate. Immunoglobulins and lymphocyte populations did not change in relation to the recurrence of endometriosis. PMID- 12372445 TI - Iron overload in the peritoneal cavity of women with pelvic endometriosis. AB - OBJECTIVE: To examine the possible involvement of iron in the physiopathology of endometriosis. DESIGN: Prospective study. SETTING: Department of gynecology in a university hospital. PATIENT(S): Seventy patients undergoing laparoscopy. INTERVENTION(S): Collection of peritoneal fluid (n = 57), blood samples, and biopsy samples from endometrium (n = 62) and from endometriotic (n = 33) and normal-appearing peritoneum (n = 53). MAIN OUTCOME MEASURE(S): Measurement of iron and ferritin in serum and peritoneal fluid and staining of iron deposits with Prussian blue in tissues. RESULT(S): Iron and ferritin concentrations were significantly higher in the peritoneal fluid of patients with endometriosis compared with controls during the secretory phase. Higher rates of ferritin and hemosiderin deposits were observed in the peritoneum adjacent to red (100%), black (57%), and white (62%) lesions compared with normal-appearing peritoneum (25%). Deposits were more frequent during the secretory phase than the proliferative phase in healthy peritoneum from controls, whereas they were found throughout the cycle in the vicinity of lesions in patients with endometriosis. Similar rates of iron deposition were observed in the stroma of black and white lesions and in eutopic endometrium from patients with endometriosis. CONCLUSION(S): Iron overload was observed in the cellular and peritoneal fluid compartments of the peritoneal cavity of women with endometriosis. Iron deposits seem to be related to the presence of lesions, suggesting that iron may be involved in the pathogenesis of endometriosis. PMID- 12372446 TI - Relation between pain symptoms and the anatomic location of deep infiltrating endometriosis. AB - OBJECTIVE: To investigate whether specific types of pelvic pain are correlated with the anatomic locations of deeply infiltrating endometriosis (DIE). DESIGN: Retrospective data analysis. SETTING: University tertiary referral center. PATIENT(S): Two hundred and twenty-five women with pelvic pain symptoms and DIE. INTERVENTION(S): During surgery, we recorded the anatomic locations of DIE implants and associated endometriosis. MAIN OUTCOME MEASURE(S): We studied the incidence of pelvic pain symptoms including severe dysmenorrhea, deep dyspareunia, noncyclic chronic pelvic pain, painful defecation during menstruation, urinary tract symptoms, and gastrointestinal symptoms as related to the location of DIE. RESULT(S): The frequency of severe dysmenorrhea increased with Douglas pouch adhesions and decreased with parity. The frequency of dyspareunia increased with a uterosacral ligament DIE location and decreased when it involved the bladder. The frequency of noncyclic chronic pelvic pain was higher when it involved the bowel and was lower for women who were treated for infertility. The frequency of painful defecation during menstruation was higher when DIE involved the vagina; lower urinary tract symptoms were more frequent when DIE involved the bladder and less frequent in women with a lower body mass index. Gastrointestinal symptoms were associated with bowel or vaginal DIE locations. CONCLUSION(S): The types of pelvic pain are related to the anatomic location of DIE. Knowledge of the characteristics of pelvic pain symptoms is important in the preoperative assessment of patients with suspected DIE. PMID- 12372447 TI - Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-alpha in women with endometriosis. AB - OBJECTIVE: To determine the effect of autologous peritoneal fluid and tumor necrosis factor-alpha (TNF-alpha) on proliferation of endometrial cells from women with endometriosis. DESIGN: Endometrial cells from eutopic and ectopic endometrium were cultured in vitro with peritoneal fluids or recombinant TNF alpha for 72 hours before DNa synthesis determination by 3H-thymidine labeling and liquid scintillation counting. SETTING: An institute for the study and treatment of endometriosis and university-based research laboratories. PATIENT(S): Thirty-five women with endometriosis and 17 controls without endometriosis. MAIN OUTCOME MEASURE(S): In vitro incorporation of 3H-thymidine in endometrial cells was examined. RESULT(S): Peritoneal fluid from women with endometriosis enhanced proliferation of autologous and heterologous endometrial cell cultures from women with endometriosis. The soluble TNF-receptor etanercept blocked the ability of peritoneal fluid from women with endometriosis to enhance proliferation of eutopic or ectopic endometrial cells. Recombinant TNF-alpha also enhanced proliferation of eutopic and ectopic endometrial cells from women with endometriosis. In contrast, autologous peritoneal fluid, heterologous peritoneal fluid from women with endometriosis, and recombinant TNF-alpha failed to enhance, and often inhibited, the proliferation of eutopic endometrial cells from controls without endometriosis. CONCLUSION(S): Endometrial cells from women with endometriosis can utilize factors in peritoneal fluids, such as TNF-alpha, to facilitate proliferation in ectopic environments. Endometrial cells from women without endometriosis do not share this ability, suggesting that this abnormality is etiologically related to development of the disease. Therapy with agents that block the effects of TNF-alpha may be warranted. PMID- 12372448 TI - Usefulness of CA19-9 versus CA125 for the diagnosis of endometriosis. AB - OBJECTIVE: To investigate the clinical value of the serum CA19-9 level in comparison with the serum CA125 level for diagnosing and determining the severity of endometriosis. DESIGN: Retrospective study. SETTING: Department of Comprehensive Reproductive Medicine in a university hospital. PATIENT(S): One hundred one women with endometriosis and 22 without endometriosis participated in this study. INTERVENTION(S): Blood samples were collected before the operation (laparoscopy, oophrectomy, cystectomy, and/or hysterectomy), and tissue samples of ovarian chocolate cysts were collected during the operation. MAIN OUTCOME MEASURE(S): The serum CA19-9 and CA125 levels and the localization of these antigens in ovarian chocolate cysts. RESULT(S): The mean serum CA19-9 levels in patients at all stages of endometriosis were significantly higher than those in patients without endometriosis, and serum CA19-9 levels significantly correlated with the Revised American Fertility Society classification scores. Intense staining of CA19-9 was observed in 15 of the 20 samples of ovarian chocolate cysts. CONCLUSION(S): CA19-9 is a useful marker for determining the severity of endometriosis. PMID- 12372449 TI - Anatomopathological lesions of bladder endometriosis are heterogeneous. AB - OBJECTIVE: To present the anatomopathological characteristics of deep bladder endometriosis. DESIGN: Descriptive anatomapathological study. SETTING: A university hospital department of gynecological surgery. PATIENT(S): Eleven consecutive patients complaining of pelvic pain and painful urinary functional symptoms. INTERVENTION(S): Laparoscopic partial cystectomy. MAIN OUTCOME MEASURE(S): Macroscopic and microscopic characteristics of deep bladder endometriosis lesions. RESULT(S): Deep bladder endometriosis lesions were extremely heterogeneous, not only in any one patient but also from one patient to another. Bladder muscularis propria presented three aspects: [1] hyperplasia of the fibromuscular tissue (4/11); [2] simple dissociation of the smooth muscle fiber bundles with no veritable "disorganization" (4/11); [3] simple thickening of the interstitial collagen network, or sclerosis (3/11). A histological adenomyotic nodule aspect was only observed in one patient (9%). CONCLUSION(S): Bladder endometriosis is an enigmatic disease. No hypothesis can be proposed as a single explanation for its pathogenesis. PMID- 12372450 TI - Location, color, size, depth, and volume may predict endometriosis in lesions resected at surgery. AB - OBJECTIVE: To correlate the diagnosis of endometriosis in lesions excised at laparoscopy with pathologic diagnosis. DESIGN: Prospective study. SETTING: U.S. government research hospital. PATIENT(S): Women with chronic pelvic pain thought to be due to endometriosis. INTERVENTION(S): Excision of lesions suspicious for endometriosis. MAIN OUTCOME MEASURE(S): Histologic examination of lesions for color, width, depth, and location of endometriosis. Lesion colors were grouped as black, red, white, mixed color, or endometriomas. RESULT(S): Sixty-five women with a surgical diagnosis of endometriosis had minimal (n = 22), mild (n = 25), moderate (n = 9), or severe disease (n = 9) according to the revised American Fertility Society classification. Endometriosis was confirmed in all but seven patients with minimal and one with severe disease. Twelve other patients did not have endometriosis. Of 314 lesions excised, 189 (61%) were endometriotic. Black or red lesions were less often histologically confirmed to be endometriosis than were white lesions, mixed-color lesions or endometriomas. Lesions > 5 mm wide or deep were more likely to be endometriosis than were narrower or shallower implants. Endometriomas deeper than 1 cm were histologically confirmed to be endometriosis, and 50% of peritoneal windows contained endometriosis. CONCLUSION(S): White lesions, mixed-color lesions, endometriomas, and larger lesions by depth or width were more likely to be histologically confirmed endometriosis than were smaller, black, or red lesions. PMID- 12372451 TI - Cycle-specific and cumulative fecundity in patients with endometriosis who are undergoing controlled ovarian hyperstimulation-intrauterine insemination or in vitro fertilization-embryo transfer. AB - OBJECTIVE: To compare controlled ovarian hyperstimulation-intrauterine insemination (COH-IUI) or IVF-ET pregnancy rates per cycle (PR) and cycle and cumulative fecundity (f and cf) with COH-IUI or IVF-ET in endometriosis. DESIGN: Retrospective analysis. SETTING: Endometriosis research institute. PATIENT(S): Women with endometriosis and infertility (n = 313) who underwent consecutive COH IUI (202 patients, 648 cycles), IVF-ET (111 patients, 139 cycles), or IVF-ET after failed COH-IUI (56 patients, 68 cycles). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Crude PR and life table-estimated f and cf. RESULT(S): With COH-IUI, 69 patients conceived; 65 conceived with IVF-ET; and 30 conceived with IVF-ET after COH-IUI (PR 11%, 47%, and 44%). With COH-IUI, six-cycle cf was 41%, and f for cycles 1-6 was 15%, 12%, 8%, 7%, 7%, and 0. With IVF-ET, three-cycle cf was 73%, whereas f for cycles 1-3 was 47%, 27%, and 33%. First-cycle f with IVF ET was significantly higher than cf of six COH-IUI cycles. When the data were stratified according to the stage of endometriosis and women's age, the benefit of IVF over COH was even more pronounced. Prior COH-IUI failure did not adversely affect IVF-ET outcome. CONCLUSION(S): In endometriosis, PR, f, and cf are significantly higher with IVF-ET than COH-IUI, especially in stage IV and in women >38 years of age. Considering adverse effects of prolonged ovarian stimulation on endometriosis, IVF-ET should be the first-line approach in the management of infertility in this disease. If COH-IUI is attempted, it should not exceed three to four cycles. PMID- 12372452 TI - Increased pregnancy rates after ultralong postoperative therapy with gonadotropin releasing hormone analogs in patients with endometriosis. AB - OBJECTIVE: To examine whether ultralong GnRH analog (GnRH-a) therapy after surgical treatment of endometriosis and before ART influences the pregnancy rate. DESIGN: Prospective, randomized, controlled study. SETTING: University clinic for reproductive medicine and gynecologic endocrinology. PATIENT(S): One hundred ten patients with stage II to IV endometriosis according to ASRM criteria. INTERVENTION(S): Fifty-five patients received GnRH-a for 6 months after surgery and subsequently underwent up to 3 cycles of ART, and 55 patients received 3 cycles of ART alone immediately after surgery. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates. RESULTS: The pregnancy rate per patient was higher among patients who received follow-up treatment with GnRH-a. The same results were found in patients with stage III or IV endometriosis who were undergoing IUI or IVF/ICSI. CONCLUSION(S): Ultralong GnRH-a therapy increases the pregnancy rate of ART in patients with severe endometriosis. PMID- 12372453 TI - Peritoneal defects and the development of endometriosis in relation to the timing of endoscopic surgery during the menstrual cycle. AB - OBJECTIVE: In vitro studies demonstrated that implantation on membranes (peritoneum, amniotic membranes) can take place if there are defects on the surface of the membranes. If these mechanisms play a role for the development of endometriosis in vivo, then patients with surgical treatment of peritoneal endometriosis in the luteal phase must have a high recurrence rate. DESIGN: Retrospective analysis of operation charts and follow-up data. SETTING: Department of gynecology, in a hospital-based endometriosis treatment center. PATIENT(S): Two hundred twenty premenopausal women. INTERVENTION(S): Laparoscopic treatment for peritoneal endometriosis, stage I and II by revised American Society for Reproductive Medicine guidelines. MAIN OUTCOME MEASURE(S): During the follow-up period of 2 years, symptoms and gynecological and sonographic findings were documented. In case of suspected recurrence a repeat laparoscopy with biopsy was performed to prove the recurrent endometriosis macroscopically and histologically. RESULT(S): The total recurrence rate after 2 years was 9.6%. The recurrence rate of group III (15%) was twice as high as those of group I (7%) and group II (8%), as indicated by subjective complaints, clinical findings, macroscopy, and histology; no differences were found between groups I and II. CONCLUSION(S): Endoscopic surgery for the treatment of peritoneal endometriosis should not be performed in the luteal phase. PMID- 12372454 TI - Characteristics of patients with endometriosis in the United States and the United Kingdom. AB - OBJECTIVE: To investigate differences in characteristics of patients with endometriosis in the United States and the United Kingdom. DESIGN: Patient questionnaire. SETTING: Two university-based endometriosis referral centers. PATIENT(S): Women with surgically diagnosed endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Patient demographics, menstrual and obstetric history, contraceptive use, medical history, risk factors, family history, endometriosis diagnosis, and current pain status and treatment. RESULT(S): Most demographic characteristics were similar between groups. However, patients in the United States were diagnosed at a younger age than were patients in the United Kingdom (25.6 +/- 6.7 years vs. 28.0 +/- 7.1 years) and more commonly presented with an ovarian mass. More U.K. women used oral contraceptives before diagnosis and were younger at first use. U.K. patients underwent fewer additional surgeries than U.S. patients but reported that surgery alone provided the best relief of symptoms, whereas most U.S. patients reported that surgical and medical therapy together provided the best relief of symptoms. CONCLUSION(S): The many similarities in demographics and symptoms among women with endometriosis in the U.S. and the U.K. support the universality of the disease process. Despite a variety of treatments, most patients from both groups still experienced pain from their endometriosis at the time of the survey. PMID- 12372455 TI - A long-term follow-up study of women with asymptomatic endometriosis diagnosed incidentally at sterilization. AB - OBJECTIVE: To evaluate whether asymptomatic endometriosis diagnosed in connection with tubal sterilization is likely to cause symptoms later in the woman's life. DESIGN: Controlled, clinical follow-up study of women who were examined for endometriosis in connection with tubal sterilization performed between 1986 and 1989. SETTING: University hospital. PATIENT(S): Thirty-nine women with mostly minimal endometriosis discovered at sterilization and 157 control women with no endometriosis discovered at sterilization. INTERVENTION(S): Interview in 2001 by a posted questionnaire. MAIN OUTCOME MEASURE(S): Report on pain, pelvic operations, menopausal status, and use of hormone replacement therapy. RESULT(S): Pelvic pain was more frequently reported by controls than by women with endometriosis (28% vs. 6%). There was no significant difference between the groups concerning dysmenorrhea, premenstrual pain, or dyspareunia, nor was there any significant difference in the hysterectomy rate. CONCLUSION(S): There is little risk that asymptomatic, minimal endometriosis found incidentally will become symptomatic. PMID- 12372456 TI - Development of a Web site for the genetic epidemiology of endometriosis. AB - OBJECTIVE: Endometriosis is a complex trait, in which genetic and environmental factors act together to produce the phenotype. So far, research into candidate genes has largely been based on biological and clinical hypotheses. Results of these studies and the wealth of gene and marker sequence information from the Human Genome Project could--when brought together--provide the researcher with new etiological avenues to explore. DESIGN: Online review. SETTING: The Web site being developed draws together evidence of genetic variants associated with endometriosis and new etiological hypotheses. It incorporates links to up-to-date genomic information relevant to the candidates from a range of bioinformatics databases. PATIENT(S): Endometriosis cases and controls in association studies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Allele and genotype frequencies. RESULT(S): The Web site summarizes the main hypotheses for endometriosis etiology that provide the basis for the search for genes involved, together with [1] the existing evidence of associations with candidate genes, with links to the relevant publications; [2] details of these candidate genes and the surrounding chromosomal area (location, function, polymorphisms, marker maps); [3] molecular biological findings, from studies of aberrant gene and protein expression in relevant tissues; and [4] chromosomal regions that have been implicated. CONCLUSION(S): This Web site should provide a useful information tool for the endometriosis researcher. We encourage researchers worldwide to use it, contribute to it, and share their knowledge about the condition. PMID- 12372457 TI - Pathological evaluation of the rat endometriosis model. AB - OBJECTIVE: To observe in detail the morphology of experimental rat endometriosis, specifically in peritoneum adjacent to uterine transplants attached via autotransplantation. DESIGN: Light and electron microscopic study. SETTING: Tochigi Institute of Clinical Pathology, Japan. ANIMAL(S): Female-SD rats maintained on a schedule of 12 hours of light and 12 hours of dark for 2 weeks. INTERVENTION(S): Uterine transplants were attached to rat peritoneum via the surgical autotransplantation technique. The implanted area of peritoneum, including abdominal muscle, were excised from anesthetized rats at four (n = 10), seven (n = 10), and 14 (n = 10) days after uterine autotransplantation. The mesenteries were autotransplanted as a comparative control. MAIN OUTCOME MEASURE(S): We examined the morphologic alterations of uterus-attached peritoneum following the time interval after the implantation. RESULT(S): In rat endometriosis models, the stromal tissue of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophils, plasma cells, lymphocytes, and macrophages. These lesions increased with time after implantation; however, ultimately these infiltrating cells disappeared and proliferation declined. CONCLUSION(S): Our findings suggest that uterine autotransplantation induces the infiltration of allergic inflammatory-related cells and proliferative lesions in peritoneal stroma attached endometrium. These data should prove useful for investigations of human endometriosis. PMID- 12372458 TI - Matrix metalloproteinase-2, membranous type 1 matrix metalloproteinase, and tissue inhibitor of metalloproteinase-2 expression in ectopic and eutopic endometrium. AB - OBJECTIVE: To investigate expression of matrix metalloproteinase-2 (MMP-2), membranous type 1 matrix metalloproteinase (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2) in ectopic and eutopic endometrium from women with and without endometriosis throughout the menstrual cycle. DESIGN: Molecular studies in human tissue. SETTING: Reproductive immunology laboratory of a university medical center. PATIENT(S): Fifty-three premenopausal woman (23 with endometriosis and 30 without endometriosis) undergoing laparoscopic surgery. Endometrium and ectopic endometriosis tissue were obtained at the time of surgery. MAIN OUTCOME MEASURE(S): Messenger RNA and protein expression from eutopic and ectopic endometrium was analyzed by using quantitative competitive polymerase chain reaction, zymography, and Western blot assay. RESULT(S): Uterine endometrium from women with endometriosis expressed higher levels of MMP-2 and MT1-MMP and lower levels of TIMP-2 than did endometrium from normal women. CONCLUSION(S): Eutopic endometrium from patients with endometriosis may be more invasive and prone to peritoneal implantation because of greater expression of MMP-2 and MT1-MMP and lower expression of TIMP-2 messenger RNA, compared with endometrium from women without endometriosis. Thus, increased proteolytic activity may help to explain the invasive factors that result in endometriosis. PMID- 12372459 TI - The alpha(2)beta(1) and alpha(3)beta(1) integrins do not mediate attachment of endometrial cells to peritoneal mesothelium. AB - OBJECTIVE: To evaluate the possible role of mesothelial alpha(2)beta(1) and alpha(3)beta(1) integrins in the attachment of endometrial stromal cells (ESCs) and endometrial epithelial cells (EECs). DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Women of reproductive age (n = 26). MAIN OUTCOME MEASURE(S): Mesothelial cells were grown on collagen IV. Endometrial stromal cells and EECs were plated on mesothelial cells for 1 hour. Before plating, mesothelial cells or endometrial cells were incubated with antibodies to alpha2, alpha3, and beta1 integrin subunits. The effect of these antibodies on ESC and EEC binding to collagen IV and collagen I was also examined. The expression of collagen I, collagen IV, fibronectin, and laminin by cultured ESCs and EECs was examined. RESULT(S): The anti-integrin antibodies had no effect on endometrial binding to mesothelium. The beta1 integrin antibody decreased binding of ESCs and EECs to the collagen matrices. In culture, ESCs and EECs expressed collagen I, collagen IV, fibronectin, and laminin to varying degrees. CONCLUSION(S): The initial adhesion of ESCs and EECs to mesothelium is not mediated by beta1 integrins. In contrast, ESC and EEC attachment to collagen IV and collagen I, which are present in the submesothelial extracellular matrix, is mediated by beta1 integrins. PMID- 12372460 TI - Involvement of catalase in the endometrium of patients with endometriosis and adenomyosis. AB - OBJECTIVE: To determine the distribution of catalase in eutopic and ectopic endometria in patients with endometriosis or adenomyosis. DESIGN: Retrospective randomized study. SETTING: Department of obstetrics and gynecology in a university hospital. PATIENT(S): Thirty-three patients with endometriosis, 36 with adenomyosis, and 47 fertile controls (total, 116 women). MAIN OUTCOME MEASURE(S): Semiquantitative immunostaining of endometrial cells obtained by biopsy sampling, followed by calculation of an evaluation nomogram score. RESULT(S): The score of catalase in the glandular epithelium of controls group fluctuated during the menstrual cycle; it was lowest in the early proliferative phase and peaked in the late secretory phase. In patients with endometriosis, catalase scores did not fluctuate during the cycle, and scores were high compared with controls throughout the menstrual cycle. Catalase scores did not vary in patients with adenomyosis, and scores in this group were consistently higher than those in patients with endometriosis throughout the cycle. CONCLUSION(S): Abnormal expression of catalase in the eutopic and ectopic endometrium strongly suggests pathologic involvement of free radicals in endometriosis and adenomyosis. PMID- 12372461 TI - Endometriotic haptoglobin binds to peritoneal macrophages and alters their function in women with endometriosis. AB - OBJECTIVE: To evaluate the effects of endometriotic haptoglobin on peritoneal macrophage function. DESIGN: Prospective laboratory study. SETTING: School of medicine. PATIENT(S): Twenty-three women with and without endometriosis. INTERVENTION(S): Peritoneal macrophages cultured without or with haptoglobin. MAIN OUTCOME MEASURE(S): Peritoneal macrophage haptoglobin immunoreactivity, adhesion, and interleukin-6 (IL-6) production. RESULT(S): In vivo, significantly more peritoneal macrophages from women with endometriosis bound haptoglobin and exhibited reduced adhesion compared to women without endometriosis. In vitro, haptoglobin treatment significantly decreased peritoneal macrophage adherence only in women without endometriosis; this effect was not seen in women with endometriosis, probably owing to in vivo haptoglobin saturation. Conversely, haptoglobin treatment robustly increased IL-6 production only by macrophages from women with endometriosis, suggesting differential immune response in these women. CONCLUSION(S): Endometriotic lesions synthesize and secrete a unique form of haptoglobin (endometriosis protein-I) that is up-regulated by IL-6. This study shows that haptoglobin adheres to peritoneal macrophages; decreases adhesion, which may influence phagocytic function; and up-regulates IL-6 production. Hence, a feed-forward loop is proposed whereby endometriotic lesion haptoglobin decreases macrophage phagocytic function while increasing IL-6 production, which in turn increases endometriotic haptoglobin and promotes establishment of endometriosis. PMID- 12372462 TI - Comparative immunohistochemical studies of endometriosis lesions and endometriotic cysts. AB - OBJECTIVE: To compare immunohistochemical staining patterns in noncystic and cystic endometriosis lesions. DESIGN: Experimental. SETTING: Archived pathology material in an academic research environment. PATIENT(S): Endometriosis tissues from the pathology archives including slide tissue sections and blocks. INTERVENTION(S): None; this was a retrospective study. MAIN OUTCOME MEASURE(S): Immunohistochemical staining of the tissues was performed using anti-bcl-2, anti p53, anti-matrix metalloproteinase IX, and anti-collagen VI antibodies. Staining was qualitatively assessed in terms of extent and intensity. RESULT(S): p53 showed no staining in both groups. Anti-bcl-2 stained 100% (30/30) of endometriosis lesions compared with only 23% (7/30) of endometriotic cysts (P<.0001), and anti-matrix metalloproteinase IX stained 85% (23/27) of endometriosis lesions and only 39% (14/36) of endometriotic cysts (P=.0003). Anti collagen VI, however, stained only 6% (2/35) of endometriosis lesions and 75% (21/28) of endometriotic cysts (P<.0001). CONCLUSION(S): Compared with endometriosis lesions, endometriotic cysts display different expression of proteins with relative overexpression of collagen VI and underexpression of bcl-2 and metalloproteinase IX. This report is the first comparative immunohistochemical study showing these differences. PMID- 12372463 TI - Aromatase P450 messenger RNA expression in eutopic endometrium is not a specific marker for pelvic endometriosis. AB - OBJECTIVE: To determine whether expression of aromatase P450 mRNA in eutopic endometrium is predictive of the presence of pelvic endometriosis. DESIGN: A prospective, multicenter, observational study. SETTING: Four tertiary centers for reproductive medicine. PATIENT(S): Sixty subjects of reproductive age undergoing laparoscopy for subfertility exploration, pain assessment, or sterilization. INTERVENTION(S): Endometrial biopsy at time of laparoscopy. MAIN OUTCOME MEASURE(S): The expression of aromatase P450 mRNA in endometrial specimens was determined by single-tube reverse transcription-polymerase chain reaction (RT PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified in parallel to exclude amplification failure. RESULT(S): The RT-PCR amplification was successful in 56 of the 60 biopsies (93%). Pelvic endometriosis was diagnosed in 34 patients (61%) and was strongly associated with aromatase P450 mRNA expression in eutopic endometrium. As a diagnostic marker for endometriosis, aromatase P450 mRNA expression yielded a sensitivity of 82%, a specificity of 59%, a positive predictive value of 76%, and a negative predictive value of 67%. If additional uterine pathology was taken in account, the sensitivity increased to 84%, the specificity to 72%, the positive predictive value to 87%, but the negative predictive value remained unchanged (67%). CONCLUSION(S): Although endometrial aromatase P450 gene expression is highly predictive of the presence of pelvic disease, the relative high incidence of false-negative results and lack of specificity is likely to impair clinical application. PMID- 12372464 TI - Relationship between apoptosis and the number of macrophages in eutopic endometrium from women with and without endometriosis. AB - OBJECTIVE: To investigate the relationship between apoptotic cells and macrophages in the eutopic endometrium of women with and without endometriosis. DESIGN: Retrospective analysis of archival uterine endometrial biopsy specimens. SETTING: Institute for the Study and Treatment of Endometriosis, and university based pathology and research laboratories. PATIENT(S): Fifty-one women with endometriosis and 24 healthy control subjects without endometriosis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The number of TUNEL+ (terminal deoxynucleotide transferase [TdT]-mediated deoxyuridine triphospate [dUTP] nick end-labeling-positive) (apoptotic) cells and CD68+ (CD68 positive) (macrophages). RESULT(S): Apoptotic cells and macrophage numbers were positively correlated in the eutopic endometrium of women with and without endometriosis. However, the number of apoptotic cells and the macrophage content in the endometrium of women with endometriosis was significantly reduced compared with that of healthy control subjects without endometriosis. Differences between apoptosis and macrophage numbers between the two populations were observed predominantly during the early proliferative phase of the menstrual cycle. CONCLUSION(S): The reduction in apoptosis described for endometrial cells in women with endometriosis may be related to reduced macrophage trafficking into the eutopic endomtrium during the early-proliferative phase of the menstrual cycle. PMID- 12372465 TI - Soluble interleukin-1 receptor type II blocks monocyte chemotactic protein-1 secretion by U937 cells in response to peripheral blood serum of women with endometriosis. AB - OBJECTIVE: To assess the ability of peripheral blood serum from women with endometriosis to induce monocyte chemotactic protein-1 (MCP-1) secretion by monocytes and the putative role of the interleukin-1 (IL-1) system in endometriosis-associated monocyte activation. DESIGN: Cultures of U937 monocytic cells exposed to serum from normal women (control group) or women with endometriosis. SETTING: Gynecology clinic and human reproduction research laboratory. PATIENT(S): Seventy-nine women with endometriosis and 38 control women with no evidence of endometriosis at laparoscopy. INTERVENTION(S): Peripheral blood obtained a few days before laparoscopy. MAIN OUTCOME MEASURE(S): MCP-1 secretion in the culture medium and serum concentrations of soluble IL-1 receptor type II (sIL-1RII), IL-1beta, and IL-1alpha by ELISA or by enzyme immunometric assay. RESULT(S): Serum concentrations of sIL-1RII were significantly lower in women with stage I-II endometriosis than in control women, whereas serum concentrations of IL-1beta and IL-1alpha were comparable between the two groups. The serum of women with endometriosis induced higher secretion of MCP-1 by U937 cells than that of control women, particularly in the initial stages of endometriosis (stages I-II), and recombinant IL-1RII (rIL-1RII) significantly blocked that secretion. CONCLUSION(S): These findings point toward a deficiency in the mechanisms involved in the down-regulation of IL-1 actions at the systemic level and reveal sIL-1RII as a key factor involved in that process. PMID- 12372466 TI - Induction of monocyte chemotactic protein-1 in peritoneal mesothelial and endometrial cells by oxidized low-density lipoprotein and peritoneal fluid from women with endometriosis. AB - OBJECTIVE: To elucidate the effect of oxidized low-density lipoprotein (LDL) and peritoneal fluid of women with endometriosis on monocyte chemotactic protein-1 (MCP-1) production by peritoneal mesothelial cells and endometrial cells. DESIGN: In vitro study. SETTING: University medical center. PATIENT(S): Five women undergoing surgery for pelvic pain, infertility, or endometriosis; five women without endometriosis who were undergoing tubal ligation were the controls. INTERVENTION(S): Mesothelial cells and endometrial cells in culture were treated with oxidized LDL and peritoneal fluid from control and endometriosis patients, then MCP-1 levels were measured. MAIN OUTCOME MEASURE(S): ELISA was used to measure MCP-1 in the culture supernatants exposed to oxidized LDL and peritoneal fluid from control and endometriosis patients. Cellular MCP-1 messenger RNA expression was evaluated by reverse transcription-polymerase chain reaction (RT PCR) assay. RESULT(S): Treatment with oxidized LDL caused an increase in accumulation of immunoreactive MCP-1 in the medium of cultured mesothelial and endometrial cells (primary endometrial stromal cells and endometrial cell line EM42). The mesothelial cells secreted more MCP-1 than did endometrial cells under the culture condition. The EM42 cells cultured in the presence of peritoneal fluid from endometriosis patients secreted more MCP-1 than those cultured with peritoneal fluid from normal women. However, no differences were found in MCP-1 levels in the supernatant of endometrial stromal cells cultured with peritoneal fluid. CONCLUSION(S): This is the first report of MCP-1 expression in mesothelial cells induced by oxidized LDL, and provides direct evidence of inflammatory action of peritoneal fluid of women with endometriosis. PMID- 12372468 TI - Elevated soluble Fas ligand levels may suggest a role for apoptosis in women with endometriosis. AB - OBJECTIVE: To evaluate soluble Fas ligand concentrations in serum and peritoneal fluid from women with endometriosis and from fertile controls without endometriosis, and to study levels of soluble Fas ligand in conditioned media of cultured endometrial stromal cells. DESIGN: Prospective, experimental trial. SETTING: Two academic IVF centers. PATIENT(S): Twenty-nine fertile women without endometriosis and 57 infertile women with endometriosis (32 with stage I or II disease and 25 with stage III or IV disease). MAIN OUTCOME MEASURE(S): Enzyme linked immunosorbent assay was used to measure soluble Fas ligand concentrations in paired samples of serum and peritoneal fluid from women with and without endometriosis. Concentrations were also measured in conditioned media of cultured endometrial stromal cells at basal conditions and after stimulation with interleukin-8 (0.001-10 ng/mL) and tumor necrosis factor-alpha (1-10 ng/mL). RESULT(S): Compared with fertile controls and women with early-stage of endometriosis, women with moderate to severe endometriosis had elevated serum (87.2 +/- 6.4, 88.2 +/- 6.9, and 162.3 +/- 7.8 pg/mL, respectively) and peritoneal fluid (81.0 +/- 6.0, 80.5 +/- 6.8, and 166.2 +/- 10.3 pg/mL, respectively) concentrations of soluble Fas ligand. Serum levels of soluble Fas ligand positively correlated with levels in peritoneal fluid. Comparison of patients in the same menstrual cycle in each group revealed that increased levels of soluble Fas ligand in patients with advanced endometriosis were not attributable to the difference in cycle phases. Soluble Fas ligand was not detected in conditioned media of endometrial stromal cells under baseline conditions or after stimulation. CONCLUSION(S): Serum and peritoneal fluid of women with moderate to severe endometriosis contain elevated concentrations of soluble Fas ligand compared to women with minimal or mild endometriosis and women without endometriosis. These findings suggest a role for apoptotic dysregulation in the pathophysiology of endometriosis. PMID- 12372467 TI - Altered expression of a cell-cycle suppressor gene, Tob-1, in endometriotic cells by cDNA array analyses. AB - OBJECTIVE: Interleukin (IL)-1beta, a product of activated peritoneal macrophages, is a central cytokine coordinating neovascularization and monocyte chemotaxis in endometriotic implants. To evaluate the effects of this cytokine on normal endometrial stromal cells and endometriotic stromal cells we performed cDNA expression array analyses before and after exposure to IL-1beta. DESIGN: Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING: Reproductive endocrinology clinic at a university hospital. PATIENT(S): Endometriosis and normal endometrial biopsies from eight patients were used to prepare stromal cell cultures from which mRNA was extracted. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Commercially available expression arrays (Atlas Human cDNA Expression Array, Clontech, representing 597 individual genes) were used to screen for mRNAs whose expression was affected by 12 hours of exposure to IL-1beta (10 ng/mL). Northern blotting and subsequent quantitative densitometric evaluation was done to confirm steady-state levels of Tob-1 mRNA transcripts. RESULT(S): Array analyses revealed a cell-cycle regulatory gene, Tob-1, which was differentially expressed by the two cell types after incubation with IL-1beta. Tob-1 was reduced 48% in endometriotic stromal cells exposed to IL-1beta, but there was only a 16% reduction in normal endometrial stromal cells. Replicate Northern analyses (n = 4) showed that exposure to IL-1beta for 12 hours resulted in a 25% +/- 5% diminution of Tob-1 mRNA in endometriotic stromal cells. In contrast, no significant decrease (<3%) was observed in IL-1beta exposed normal endometrial stromal cells. CONCLUSION(S): Tob-1, a cell-cycle inhibitor gene is differentially responsive to IL-1beta in endometriotic stromal cells compared to normal endometrial stromal cells. IL 1beta down-regulated Tob-1 in endometriotic stromal cells, but had no significant effect on normal endometrial stromal cells. Our results suggest that IL-1beta promotes growth of endometriotic lesions through inhibition of Tob-1. These findings are the first to associate IL-1beta with an alteration of cell-cycle gene expression in cells derived from endometriotic implants. PMID- 12372469 TI - Alterations in expression of endometrial endothelial nitric oxide synthase and alpha(v)beta(3) integrin in women with endometriosis. AB - OBJECTIVE: To determine the expression of endometrial endothelial nitric oxide synthase (eNOS) protein and alpha(v)beta(3) integrin in patients with and without endometriosis. DESIGN: Case-control cohort study. SETTING: University-based tertiary care center. PATIENT(S): Endometrial biopsy samples were obtained from 9 fertile women with regular cycles and 30 infertile women with varying severity of endometriosis. Peritoneal fluid levels of nitric oxide were determined in 13 infertile women with a normal pelvis and 12 infertile women with endometriosis. MAIN OUTCOME MEASURE(S): Expression of eNOS and alpha(v)beta(3) integrin protein in the endometrium and peritoneal fluid levels of nitric oxide. RESULTS: In patients with endometriosis, expression of eNOS was significantly increased in the glandular and luminal epithelium, with no significant changes in the stroma. Peritoneal fluid levels of nitric oxide were unchanged, and expression of alpha(v)beta(3) integrin expression in glandular and luminal epithelium was significantly decreased compared with controls. A significant negative correlation was observed between luminal expression of eNOS and alpha(v)beta(3) integrin and between glandular expression of eNOS and luminal expression of alpha(v)beta(3) integrin. CONCLUSION(S): The nitric oxide pathway may play a role in the pathogenesis of endometriosis. PMID- 12372470 TI - Influence of severe endometriosis on gene expression of vascular endothelial growth factor and interleukin-6 in granulosa cells from patients undergoing controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer. AB - OBJECTIVE: To evaluate how endometriosis affects expression of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in granulosa cells. DESIGN: Prospective study. SETTING: IVF-ET program at Osaka Medical College. PATIENT(S): Seventeen patients with revised American Fertility Society stage IV endometriosis and 17 patients with tubal infertility and no endometriosis. INTERVENTION(S): Granulosa cells obtained at oocyte retrieval were examined for VEGF and IL-6 gene expression. MAIN OUTCOME MEASURE(S): Serum E(2) and P levels at hCG administration, number of oocytes, fertilization rate, high-quality embryo rate, and pregnancy rate, and expression of VEGF and IL-6 genes. RESULT(S): Total hMG and FSH levels were statistically significantly higher in patients with endometriosis; however, the number of retrieved oocytes and the fertilization rate were lower compared with patients with tubal infertility. Serum E(2) levels and expression of VEGF in patients with tubal infertility were statistically significantly higher than those in patients with endometriosis. Interleukin-6 gene expression did not differ between the groups. CONCLUSION(S): In severe endometriosis, lower VEGF gene expression in granulosa cells may adversely affect oocyte development and maturation. PMID- 12372471 TI - Laparoscopic management of 15 patients with infiltrating endometriosis of the bladder and a case of primary intravesical endometrioid adenosarcoma. AB - OBJECTIVE: To report laparoscopic management of 15 patients with infiltrative bladder wall endometriosis and to report a case of endometrioid adenosarcoma. DESIGN: Prospective chart review. SETTING: Referral center for endometriosis. PATIENT(S): Fifteen women with infiltrating endometriosis of the bladder. INTERVENTION(S): Laparoscopic segmental cystectomy and pathologic review of endometriotic bladder nodules in 15 patients. MAIN OUTCOME MEASURE(S): Location and characteristics of endometriotic bladder nodules. RESULT(S): Laparoscopic and cystoscopic evaluation confirmed that the endometriotic lesions were penetrating through the bladder wall. In 8 patients, the lesions were located in the dome of the bladder. In the remaining 7, the lesions were in the posterior wall, above the trigone. It was possible to treat all the lesions by performing a laparoscopic partial cystectomy. No intraoperative complications occurred. Deeply infiltrating endometriosis was confirmed on histologic evaluation in 14 cases. One patient was diagnosed with endometriosis on frozen section, but the final pathology revealed an adenosarcoma of the bladder. CONCLUSION(S): Surgical excision of deeply infiltrating endometriosis of the bladder wall can be performed laparoscopically and offers the benefit of a definitive pathologic diagnosis to rule out an occult malignancy. PMID- 12372472 TI - Laparoscopic ovarian cystectomy of endometriomas does not affect the ovarian response to gonadotropin stimulation. AB - OBJECTIVE: To evaluate the ovarian response cycles of IVF-ET in patients who previously underwent laparoscopic cystectomy for endometriomas. DESIGN: Retrospective study with prospective selection of participants and controls. SETTING: Instituto de Ginecologia y Fertilidad Buenos Aires, Argentina. PATIENT(S): Thirty-nine patients underwent an operation for ovarian endometriomas by atraumatic removal of the pseudocapsule with minimal bipolar cauterization of small bleeders and an IVF-ET cycle (group A) and 39 control patients of similar age underwent an IVF-ET cycle for tubal factor infertility (group B). INTERVENTION(S): Laparoscopic endometrioma cystectomy, IVF-ET cycle. MAIN OUTCOME MEASURE(S): E(2) levels, number of gonadotropin ampoules, follicles, oocytes retrieved, number and quality of embryos transferred, and clinical pregnancy rate. RESULT(S): There were no differences in all the parameters studied (E(2) levels, number of follicles, oocytes retrieved, number and quality of embryos transferred, and clinical pregnancy rate) except for the number of gonadotropin ampoules needed for ovarian hyperstimulation, which was significantly higher in group A than in group B. CONCLUSION(S): Our results indicate that laparoscopic cystectomy for endometriomas is an appropriate treatment since it did not negatively affect the ovarian response for IVF-ET. PMID- 12372473 TI - Massive and acute hemoperitoneum due to rupture of the uterine artery by erosion from an endometriotic lesion. AB - OBJECTIVE: To report a case of acute hemoperitoneum due to erosion of the uterine artery by an endometriotic lesion of the left ovary. DESIGN: Case report and review of literature. SETTING: University medical center. PATIENT(S): A 39-year old nulliparous woman with stage 3 endometriosis. INTERVENTION(S): Operative laparoscopy followed by laparotomy, oophorectomy, and ligation of the bleeding uterine artery. RESULT(S): Patient is fully recovered and is attempting to conceive. CONCLUSION(S): An endometriotic lesion eroded the wall of the uterine artery, causing massive, acute hemoperitoneum. Such an event may be overlooked during laparotomy and attributed to the trauma of surgery. PMID- 12372474 TI - The difference that makes a difference? PMID- 12372476 TI - The difference that makes a difference? PMID- 12372477 TI - Clarification of efficacy! PMID- 12372480 TI - Important effects of cyproterone acetate on endometriosis? PMID- 12372481 TI - The spectrum of embryo transfer days. PMID- 12372483 TI - Estrogen levels and thrombophilia--an intervening variable or a confounder? PMID- 12372485 TI - Estrogen levels and thrombophilia-an intervening variable or a confounder? PMID- 12372486 TI - Metformin and BMI? PMID- 12372488 TI - Clinical decision-making--when science is uncertain. PMID- 12372490 TI - Clinical decision-making--when science is uncertain. PMID- 12372494 TI - Novel semi-synthetic glycopeptide antibiotics active against methicillin resistant staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE): doubly-modified water-soluble derivatives of chloroorienticin B. AB - A series of N-alkylated and aminomethylated derivatives of chloroorienticin B, a vancomycin-related glycopeptide antibiotic, were synthesized. Doubly-modified derivatives having both hydrophobic and hydrophilic substituents exhibited potent antibacterial activity against MRSA and VRE along with considerable water solubility. PMID- 12372495 TI - An efficient and practical method for solid-phase synthesis of tripeptide-bearing glycopeptide antibiotics: combinatorial parallel synthesis of carboxamide derivatives of chloroorienticin B. AB - An efficient and practical method was established for solid-phase parallel synthesis of the peptide-bearing carboxamide derivatives of chloroorienticin B, and over 80 compounds were synthesized simultaneously. Among the derivatives prepared, compounds having both tryptophan and tyrosine residues (1-3) were found to possess potent antibacterial activity against VRE. PMID- 12372496 TI - Exiguamide, a new spirocyclic sesquiterpene from the marine sponge Geodia exigua that inhibits cell fate specification during sea urchin embryogenesis. AB - A new nitrogen-containing bicyclic spirosesquiterpene designated exiguamide which inhibited cell fate specification during sea urchin embryogenesis has been isolated from the marine sponge Geodia exigua. Its structure was determined by interpretation of spectral data and X-ray crystallographic analysis. PMID- 12372497 TI - Structure-Activity relationships of 2-substituted 5,7-Diarylcyclopenteno[1,2 b]pyridine-6-carboxylic acids as a novel class of endothelin receptor antagonists. AB - Synthesis and structure-activity relationships of 2-substituted-5,7 diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids, a novel class of endothelin receptor antagonists, were described. Derivatization of a lead structure 1 (IC(50)=2.4nM, 170-fold selectivity) by incorporating a substituent such as an alkyl, alkoxy, alkylthio, or alkylamino group into the 2-position of the cyclopenteno[1,2-b]pyridine skeleton was achieved via the key intermediate 8. Introduction of an alkyl group led to the identification of potent ET(A)/ET(B) mixed receptor antagonists, a butyl (2d: IC(50)=0.21nM, 52-fold selectivity) and an isobutyl (2f: IC(50)=0.32nM, 26-fold selectivity) analogue. In contrast, installment of a primary amino group resulted in ET(A) selective antagonists, a propylamino 2p (IC(50)=0.12nM, 520-fold selectivity) and an isopropylamino 2q (IC(50)=0.10nM, 420-fold selectivity) analogue. These results suggested that a substituent at the 2-position of the 5,7-diarylcyclopenteno[1,2-b]pyridine-6 carboxylic acids played a key role in the binding affinity for both ET(A) and ET(B) receptors. PMID- 12372498 TI - alpha-bromoacetophenone derivatives as neutral protein tyrosine phosphatase inhibitors: structure-Activity relationship. AB - A series of alpha-haloacetophenone derivatives was tested for inhibition of protein tyrosine phosphatases SHP-1 and PTP1B. The results show that the bromides are much more potent than the corresponding chlorides, whereas the phenyl ring is remarkably tolerant to modifications. Derivatization of the phenyl ring with a tripeptide Gly-Glu-Glu resulted in a potent, selective inhibitor against PTP1B. PMID- 12372499 TI - A series of C-terminal amino alcohol dipeptide A beta inhibitors. AB - Potent, small molecule A beta inhibitors have been prepared that incorporate an alanine core bracketed by an N-terminal arylacetyl group and various C-terminal amino alcohols. The compounds exhibit stereospecific inhibition as demonstrated in an in vitro assay. PMID- 12372500 TI - Synthesis and evaluation of potent pyrrolidine H(3) antagonists. AB - The synthesis and biological evaluation of novel antagonists of the rat H(3) receptor are described. These compounds differ from prototypical H(3) antagonists in that they do not contain an imidazole moiety, but rather a substituted aminopyrrolidine moiety. A systematic modification of the substituents on the aminopyrrolidine ring was performed using pre-formatted precursor sets, where applicable, to afford several compounds with high affinity and selectivity for the H(3) receptor. PMID- 12372502 TI - Novel nucleotide phosphonate analogues with potent antitumor activity. AB - We have identified several nucleotide phosphonates demonstrating in vitro antiproliferative activity in several human cancer cell lines with IC(50) values in the microM range. The synthesis as well as structure-activity relationship are described. PMID- 12372501 TI - Synthesis and biological activity of potent heterocyclic thiol-based inhibitors of endothelin-converting enzyme-1. AB - Directed screening of metalloprotease inhibitors identified CGS 30084 (1) as a potent inhibitor of endothelin-converting enzyme-1 (ECE-1) in vitro (IC(50)=77 nM). Herein we report the syntheses and biological activities of analogues containing modified biphenyl moieties, bearing heterocyclic proximal rings. Compound 20, the thioacetate ethyl ester prodrug derivative of compound 19a, was found to be an orally active and potent inhibitor of ECE-1 activity in rats. PMID- 12372503 TI - bis-Azaaromatic quaternary ammonium analogues: ligands for alpha4beta2* and alpha7* subtypes of neuronal nicotinic receptors. AB - A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N,N'-Decane 1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [(3)H]nicotine binding sites (K(i)=330 nM), but did not inhibit [(3)H]methyllycaconitine binding (K(i) >100 microM), indicative of an interaction with alpha4beta2*, but not alpha7* receptor subtypes, respectively. Also, bNDI inhibited (IC(50)=3.76 microM) nicotine-evoked (86)Rb(+) efflux from rat thalamic synaptosomes, indicating antagonist activity at alpha4beta2* nAChRs. N,N' Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [(3)H]methyllycaconitine binding sites (K(i)=1.61 microM), but did not inhibit [(3)H]nicotine binding (K(i)>100 microM), demonstrating an interaction with alpha7*, but not alpha4beta2* nAChRs. Thus, variation of N-n-alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the alpha4beta2* nAChR subtype, as well as ligands with selectivity at alpha7* nAChRs. PMID- 12372504 TI - Parallel solution- and solid-phase synthesis of spiropyrrolo-pyrroles as novel neurokinin receptor ligands. AB - The combination of a 3,5-bis(trifluoromethyl)phenyl needle with the spiropyrrolo pyrrole motive as a privileged GPCR scaffold was the basis for designing a focused combinatorial library targeted towards the neurokinin-1 receptor. A solution- and solid-phase method is described and binding affinities of representative compounds are presented. PMID- 12372505 TI - Aminoalkoxybiphenylnitriles as histamine-3 receptor ligands. AB - Biaryl nitrile amines were prepared and found to have high affinity and selectivity for human and rat histamine H(3) receptors. PMID- 12372506 TI - Bridged bicyclic vasopressin receptor antagonists with V(2)-selective or dual V(1a)/V(2) activity. AB - The synthesis and biological testing of a novel series of nonpeptide vasopressin receptor antagonists, containing a bridged bicyclic nucleus, are reported. Variation of substituents (R(1)-R(3)) in general formula 3, and the configuration of the stereocenter, resulted in potent V(2)-selective (e.g., 5) and balanced dual V(1a)/V(2) (e.g., 10) compounds. Data from receptor binding, cell-based functional, and in vivo assays are presented [corrected] PMID- 12372507 TI - Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding. AB - The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme. PMID- 12372508 TI - Hydroxamate based inhibitors of adenylyl cyclase. Part 2: the effect of cyclic linkers on P-site binding. AB - The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Previous work in our group identified novel inhibitors which possess an adenine ring joined to a metal-coordinating hydroxamic acid through flexible linkers. Considering the spatial positioning of the metals with respect to the adenine binding site coupled with potentially favorable entropic factors, conformational restriction of the tether through a stereochemistry based SAR employing a rigid cyclic scaffold was explored. PMID- 12372509 TI - A general synthesis of specifically deuterated nucleotides for studies of DNA and RNA. AB - An efficient procedure is described for the preparation of ribonucleotides and deoxyribonucleotides with deuterium incorporated at the 1', 4', or 5' position. Three intermediates-[1-2H]-D-ribose, [4-2H]-D-ribose, and [5-2H(2)]-D-ribose-were prepared by chemical synthesis and subsequently converted to ribonucleotides and deoxyribonucleotides via enzymatic reactions. Milligram quantities of the desired products were obtained with an average deuterium content of 96+/-1%. PMID- 12372510 TI - Synthesis and evaluation of 5-HT(2A) and 5-HT(2C) receptor binding affinities of novel pyrimidine derivatives. AB - In an effort to find potential anxiolytic and/or antipsychotic agents with selective 5-HT(2C) affinity a series of new pyrimidine derivatives was prepared and the binding affinities for 5-HT(2A) and 5-HT(2C) receptors were determined. PMID- 12372511 TI - Novel lopinavir analogues incorporating non-Aromatic P-1 side chains--synthesis and structure--activity relationships. AB - The HIV protease inhibitor Lopinavir has a pseudosymmetric core unit incorporating benzyl groups at both P-1, P-1' positions. A series of analogues incorporating non-aromatic side chains at the P-1 position were synthesized and the structure-activity relationships explored. PMID- 12372512 TI - Indoline and piperazine containing derivatives as a novel class of mixed D(2)/D(4) receptor antagonists. Part 1: identification and structure-activity relationships. AB - Optimization of the lead compound 2-[-4-(4-chloro-benzyl)-piperazin-1-yl]-1-(2,3 dihydro-indol-1-yl)-ethanone 1 by systematic structure-activity relation (SAR) studies lead to two potent compounds 2-[-4-(4-chloro-benzyl)-piperazin-1-yl]-1-(2 methy-2,3-dihydro-indol-1-yl)-ethanone 2n and 2-[-4-(4-chloro-benzyl)-piperazin-1 yl]-1-(2-methy-2,3-dihydro-indol-1-yl)-ethanone 7b. Their related synthesis was also reported. PMID- 12372514 TI - Inhibition of HIV-1 nuclear import via schiff base formation with arylene bis(methylketone) compounds. AB - Arylene bis(methylketone) compounds specifically block nuclear translocation of the HIV-1 pre-integration complex by forming Schiff-base adducts with contiguous lysines within nuclear localization signal. PMID- 12372513 TI - Indoline and piperazine containing derivatives as a novel class of mixed D(2)/D(4) receptor antagonists. Part 2: asymmetric synthesis and biological evaluation. AB - A series of chiral benzylpiperazinyl-1-(2,3-dihydro-indol-1-yl)ethanone derivatives were prepared and examined for their affinity at dopamine D(2) and D(4) receptors. Three compounds having D(2)/D(4) affinity ratios approximating that found for the atypical neuroleptic clozapine were further evaluated in behavioral tests of antipsychotic efficacy and motor side effects. PMID- 12372515 TI - 1,8-Naphthyridin-2,7-(1,8H)-dione is an effective mimic of protonated cytosine in peptide nucleic acid triplex recognition systems. AB - A novel bicyclic mimic of protonated cytosine [1,8-naphthyridin-2,7-(1,8H)-dione, (K)] for Hoogsteen type triplex recognition of guanine has been designed for incorporation into peptide nucleic acids. Bis-PNA clamps with the K base incorporated in the Hoogsteen strand showed a significant stabilization of the triplexes at pH 7 as compared to similar triplexes with PNA oligomers containing either cytosine (6.7 degrees C per unit) or pseudoisocytosine (1.5 degrees C per unit). Cooperative stabilization was observed when the K units were placed in adjacent positions ( approximately 3 degrees C per unit). PMID- 12372516 TI - A survey of cyclic replacements for the central diamide moiety of inhibitors of inosine monophosphate dehydrogenase. AB - A series of heterocyclic replacements for the central diamide moiety of 1, a potent small molecule inhibitor of inosine monophosphate dehydrogenase (IMPDH) were explored The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for these new series of inhibitors is given. PMID- 12372517 TI - Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease. AB - Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+) dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes. PMID- 12372518 TI - DNA Interaction and photonicking properties of DNA-targeted acridine (2,2' Bipyridine)platinum(II) complexes. AB - Synthesis of two (2,2'-bipyridine)platinum(II) complexes tethered to one or two acridine chromophores is reported. These acridine complexes efficiently unwind and photocleave supercoiled plasmid DNA under physiological conditions of temperature and pH. PMID- 12372519 TI - Synthesis and structure--activity relationship of novel aminotetralin derivatives with high micro selective opioid affinity. AB - Several novel racemic aminotetralin derivatives have been prepared using a stereoselective aziridine ring opening reactions and were evaluated for their micro-opioid receptor binding affinity. Selectivity index towards other opioid receptors and antinociceptive activity in mice have been evaluated for the most potent derivatives. PMID- 12372520 TI - Synthesis and biological activity of retinoic acid receptor-alpha specific amides. AB - Retinoids are analogues of all-trans-retinoic acid, a powerful hormone that mediates many fundamental biological processes. Cancer and other serious hyperproliferative diseases are attractive therapeutic targets for retinoids, but the therapeutic use of retinoids is limited due to severe toxicity. We report here the design of retinoid receptor-alpha specific ligands with growth inhibitory activity in breast cancer cell lines, and which do not cause the cutaneous toxicity associated with the currently available nonselective retinoid agonists. PMID- 12372521 TI - Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors. AB - We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7). PMID- 12372522 TI - Synthesis and SAR of novel imidazoquinoxaline-based Lck inhibitors: improvement of cell potency. AB - A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement of the cellular activity was achieved over the initial lead, compound 2. PMID- 12372523 TI - Synthesis and structure-activity relationships of aminoalkylazetidines as ORL1 receptor ligands. AB - A series of aminoalkylazetidines has been discovered as novel ORL1 receptor ligands. Structure-activity relationships have been investigated at the azetidine N and the alkyl side chain sites. Several potent and selective analogues have been identified. PMID- 12372524 TI - Synthesis and NK(1)/NK(2) binding activities of a series of diacyl-substituted 2 arylpiperazines. AB - The synthesis and binding affinity for hNK(1) and hNK(2) receptors of a series of diacyl substituted 2-aryl piperazines are described. SAR evaluation led to the racemic derivative 11g as an apparent dual inhibitor. Chiral chromatographic separation of 11g led to the observation that NK(1) activity was shown by one enantiomer (13a) and NK(2) activity was shown by the other enantiomer (13b). X ray crystallographic analysis of the crystalline di-BOC derivative of the NK(2) active piperazine (15) showed that the 2R configuration was associated with NK(2) activity. Further derivatization indicated that dual NK(1)/NK(2) activity could be built into the 2R series. PMID- 12372526 TI - The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase. AB - The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin. PMID- 12372525 TI - A mechanism-based probe for gp120-Hydrolyzing antibodies. AB - An antigenic peptide analogue consisting of HIV gp120 residues 421-431 (an antigen recognition site probe) with diphenyl amino(4 amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies. PMID- 12372527 TI - Opioid activity of 4-imidazolidinone positional analogues of Leu-Enkephalin. AB - Modulation of opioid activity was accomplished for analogues of Leu-enkephalin through incorporation of a 4-imidazolidinone moiety. The peptide backbone was constrained via a methylene bridge between two neighboring amides within its regular peptide sequence, which was expected to disrupt the secondary structure of the original molecule. Five positional analogues of Leu-enkephalin based on the same sequence and different location of the imidazolidinone-constrict were designed, synthesized, and examined for their affinity to micro-, delta- and kappa-opioid receptors. PMID- 12372528 TI - New irreversible adenosine A(1) antagonists based on FSCPX. AB - FSCPX (1) and its amide analogue (2) have been reported to exhibit potent and selective irreversible antagonism of the A(1) adenosine receptor (A(1)AR) when used in in vitro biological preparations. In order to obtain an irreversible A(1)AR antagonist with improved stability, analogues of FSCPX incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine pharmacophore by a ketone linkage were explored. Compounds 4a-c exhibited improved affinity for the A(1)AR and concentration-dependent irreversible binding to the A(1)AR. PMID- 12372529 TI - Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity. AB - A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice. PMID- 12372530 TI - Substituted uracil derivatives as potent inhibitors of poly(ADP-ribose)polymerase 1 (PARP-1). AB - A new class of PARP-1 inhibitors, namely substituted fused uracil derivatives were synthesised. Starting from a derivative with an IC(50)=2microM the chemical optimisation program led to compounds with more than a 100-fold increase in potency (IC(50)<20nM). Additionally, physicochemical and pharmacokinetic properties were evaluated. It could be shown that compounds bearing a piperazine or phenyl substituted betaAla-Gly side chain exhibited the best overall profile. PMID- 12372531 TI - Synthesis and mechanism of action of novel pyrimidinyl pyrazole derivatives possessing antiproliferative activity. AB - Pyrimidinyl pyrazole derivatives 1-4, prepared as a new scaffold of an anti-tumor agent, showed antiproliferative activity against human lung cancer cell lines and inhibited tubulin polymerization. Furthermore, it was found that compound 2 bound at the colchicine site on tubulin, but the tubulin binding pattern was different from that of colchicine. Here, we describe the synthesis of the derivatives and the differences of the action mechanism on tubulin polymerization inhibition between compound 2 and colchicine. PMID- 12372532 TI - Dual NK(1) antagonists--serotonin reuptake inhibitors as potential antidepressants. Part 2: SAR and activity of benzyloxyphenethyl piperazine derivatives. AB - The synthesis, structure-affinity relationship and activity of benzyloxyphenethyl piperazine derivatives combining NK(1) antagonism and serotonin reuptake inhibition is described. Compound 7u was shown to be active in animal models of 5 HT reuptake inhibition and central NK(1) receptor blockade, and was demonstrated to be orally active in an integrated model sensitive to both mechanisms. This class of compounds potentially represents a new generation of antidepressants. PMID- 12372533 TI - P1 Phenethyl peptide boronic acid inhibitors of HCV NS3 protease. AB - A series of peptide boronic acids containing extended, hydrophobic P1 residues was prepared to probe the shallow, hydrophobic S1 region of HCV NS3 protease. The p-trifluoromethylphenethyl P1 substituent was identified as optimal with respect to inhibitor potency for NS3 and selectivity against elastase and chymotrypsin. PMID- 12372534 TI - Two selective novel triterpene glycosides from sea cucumber, Telenata Ananas: inhibitors of chemokine receptor-5. AB - The aqueous methanolic extract of a sea cucumber was found to contain two triterpene glycosides 1 and 2. The structures of 1 and 2 were established based on high-resolution NMR studies. Compounds 1 and 2 exhibited inhibitory activity (K(i)) of 30 and 5microM, respectively, in a chemokine receptor subtype 5 (CCR5) assay. Both compounds did not show any significant inhibition in a CXCR2 assay at 50microM, suggesting their selectivity for the CCR5 receptor. PMID- 12372535 TI - An efficient protocol for solution- and solid-phase end-group differentiation of spermidine. AB - The end-group differentiation of a selectively protected spermidine was achieved through a short sequence of steps. The functionalization of spermidine in solid phase was monitored by FT-IR. PMID- 12372537 TI - Identification and structure-activity studies of novel ultrashort-acting benzodiazepine receptor agonists. AB - The synthesis and evaluation of novel ultrashort-acting benzodiazepine (USA BZD) agonists is described. A BZD scaffold was modified by incorporation of amino acids and derivatives. The propionate side chain of glutamic acid tethers an enzymatically labile functionality where the metabolite carboxylic acid displays markedly reduced BZD receptor affinity. The USA BZDs were characterized by full agonism profiles. Copyright2000 Elsevier Science Ltd. PMID- 12372536 TI - Synthesis and preliminary evaluation of trans-3,4-conformationally-restricted glutamate and pyroglutamate analogues as novel EAAT2 inhibitors. AB - Select trans-4,5-[bi]cyclohexenylglutamic and pyroglutamic acids (3,4-substituted glutamates) were synthesized in three steps and were screened as potential inhibitors of the sodium dependent excitatory amino acid transporters 2 (EAAT2) and 3 (EAAT3), the chloride dependent glial cystine/glutamate exchanger system x(c)(-), and the glutamate vesicular transport system (VGLUT). Two glutamate analogues and one pyroglutamate analogue were found to inhibit EAAT2 with activity comparable to dihydrokainate. PMID- 12372538 TI - Relating the structure, activity, and physical properties of ultrashort-acting benzodiazepine receptor agonists. AB - The ultrashort-acting benzodiazepine (USA BZD) agonists reported previously have been structurally modified to improve aqueous solubility. Lactam-to-amidine modifications, replacement of the C5-haloaryl ring, and annulation of heterocycles are presented. These analogues retain BZD receptor potency and full agonism profiles. PMID- 12372539 TI - Conformational restraint is a critical determinant of unnatural nucleotide recognition by protein kinases. AB - This report describes the synthesis of N(4)-(benzyl) AICAR triphosphate, a conformationally restrained analogue of N(4)-(benzyl) ribavirin triphosphate. Both of these nucleotides were evaluated as phosphodonors for wild-type p38MAP kinase and T106G p38MAP kinase, a designed mutant with expanded nucleotide specificity. The conformationally restrained nucleotide, N(4)-(benzyl) AICAR triphosphate, is orthogonal to (not accepted as a substrate by) wild-type p38MAP kinase, in contrast to N(4)-(benzyl) ribavirin triphosphate. Furthermore, N(4) (benzyl) AICAR triphosphate, is accepted as a substrate by T106G p38MAP kinase, in contrast to N(4)-(benzyl) ribavirin triphosphate. We hypothesize that the presence of an internal hydrogen bond in N(4)-(benzyl) AICAR and its absence in N(4)-(benzyl) ribavirin triphosphate is the main determinant for their differing structure-activity relationships. PMID- 12372540 TI - Synthesis and SAR of 4-carboxy-2-azetidinone mechanism-based tryptase inhibitors. AB - A series of N1-activated C4-carboxy azetidinones was prepared and tested as inhibitors of human tryptase. The key stereochemical and functional features required for potency, serine protease specificity and aqueous stability were determined. From these studies compound 2, BMS-262084, was identified as a potent and selective tryptase inhibitor which, when dosed intratracheally in ovalbumin sensitized guinea pigs, reduced allergen-induced bronchoconstriction and inflammatory cell infiltration into the lung. PMID- 12372541 TI - Synthesis of potent and highly selective inhibitors of human tryptase. AB - The serine protease tryptase has been implicated in allergic and inflammatory diseases and associated with asthma. The synthesis and SAR of a series of N1 activated-4-carboxy azetidinones are described, resulting in identification of BMS-363131 (2) as a potent inhibitor of human tryptase (IC(50)<1.7 nM) with high selectivity (>3000-fold) for tryptase versus related serine proteases including trypsin. PMID- 12372542 TI - Some contributions of Rafael Lorente de No to neuroscience: a reminiscence. AB - Rafael Lorente de No is one of the towering neuroscientists of the 20th century. He was born in Zaragoza, Spain, in 1902. In 1920, he moved to Madrid where he became the youngest, and eventually the best known, of Ramon y Cajal's disciples. In his youth, Lorente de No worked with Oskar and Cecile Vogt in Germany and with Robert Barany in Sweden. In 1934, he moved to the United States, where he first worked at the Central Institute for the Deaf (CID); in 1936, he was invited by Herbert S. Gasser to work at the Rockefeller Institute. After his formal retirement from this institute in 1972, Lorente de No spent 5 years at the Head and Neck Surgery Department and Brain Research Institute of the University of California at Los Angeles. He died in Tucson, Arizona, in 1990. Lorente de No was a gifted person: a polyglot with a remarkable memory and a versatile intellect, which together with his strong, almost belligerent character, made him a rather controversial human. The strength of his scientific contributions is evident by their current impact. Among these are: the modular (i.e., columnar) organization of the cerebral cortex, the synaptic delay, nerve volume conduction, synaptic summation, and the cybernetic (feed-back) neuron circuit. The present article provides a comment upon some of his neurohistological studies (including the cerebral isocortex, Ammon's horn, brainstem, and spinal cord), highlighted by transcripts from taped conversations with him, and illustrated by reproductions of some of his original figures. PMID- 12372543 TI - Alphaviral gene transfer in neurobiology. AB - Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus are simple, enveloped plus-strand RNA viruses belonging to the Alphavirus genus of the Togaviridae family. They have been developed into expression vectors that infect a wide host cell range and cause rapid and high level transgene expression. Their easy and fast generation, classification into biosafety levels 1 and 2, and preferential transduction of neurons in cell and tissue cultures makes them an increasingly used gene transfer system. This review summarizes the alphaviral replication and expression, the replicon system, and its application in neurobiology. Alphaviral vectors can introduce multiple transgenes into host cells, and mutants with low or absent cytotoxicity and increased or decreased transgene expression levels are available. Temperature dependent mutants permit to control the host cell specificity as well as the on- and offset of gene expression. These features, together with the transduction characteristics revealed in a direct comparison of alphaviral and other viral vectors in hippocampal slice cultures, make SFV and SIN vectors a powerful tool for neurobiological studies. PMID- 12372544 TI - Effect of cryoinjury on the contractile parameters of bladder strips: a model of impaired detrusor contractility. AB - In anesthetized Sprague-Dawley rats, the bladder was exposed and cryoinjury was induced by abruptly freezing the serosal side of the bladder wall with a chilled aluminum rod previously placed on dry ice (-40 degrees C). Five days later, the rats were euthanized, and strips were prepared from the area adjacent to the injury. Neurally and alpha,beta methylene-ATP (alpha,beta m-ATP; 50 microM) evoked contractions were measured in bladder strips from cryoinjured or intact bladders prepared from sham-operated rats. Cryoinjured bladder strips produced significantly lower contractile forces than intact strips to electrical stimulation at higher (10-40 Hz) frequencies. The maximal rate of the neurally evoked contractions was slower in the cryoinjured bladders. The contractile response to alpha,beta m-ATP was smaller in the cryoinjured preparations indicating that the changes may have also occurred at the postjunctional site. In addition, atropine was more effective at inhibiting the neurally evoked contractions in the cryoinjured bladder strips suggesting that a cholinergic dominance occurs after cryoinjury. It is concluded that cryoinjury is a viable method of causing a defined, reproducible injury to the urinary bladder resulting in impaired function of both the cholinergic transmission and the smooth muscle. The bladder cryoinjury can be used as a model for studying impaired bladder compliance and detrusor contractility as well as treatments that may improve bladder function such as tissue engineering. PMID- 12372545 TI - The effects of beta-endorphin (beta-END) on cardiovascular and behavioral dynamics in conscious rats. AB - Beta-endorphin (beta-END) a product of the proopiomelanocortin (POMC) has been demonstrated to play a role in the regulation of metabolic and autonomic responses. Recent studies have suggested the involvement of the endogenous opioid system in cardiovascular control. Previous studies conducted in our laboratory using anesthetized animals investigated the actions of beta-END and other POMC derived peptides on sympathetic and cardiovascular dynamics. In this study, we determined both the acute and chronic effects of beta-END on cardiovascular and behavioral dynamics in conscious unrestrained rats using radio-telemetry. Animals were instrumented with a radio-telemetry transmitter in the abdominal cavity and the attached catheter inserted into the femoral artery for recording of cardiovascular dynamics and activity. They were subsequently implanted with intracerebroventricular (ICV) cannulas. The acute ICV administration of beta-END significantly increased the mean arterial pressure (MAP) and heart rate (HR) compared to controls. The cardiovascular responses returned toward control levels after 2 h. In contrast, the chronic infusion of beta-END significantly decreased the MAP and HR during both the active and inactive phase. Chronic beta-END administration also decreased physical activity. Food intake was increased initially and later declined and water consumption followed a similar pattern. We conclude that in the conscious unrestrained animal the acute administration of beta-END increases MAP and HR while the chronic infusion of beta-END decreases MAP, HR, physical activity, and stimulate a short-term increase in food and water intake. PMID- 12372546 TI - Protective effect of the antiepileptic drug candidate talampanel against AMPA induced striatal neurotoxicity in neonatal rats. AB - 2,3-Benzodiazepines represent a family of specific, noncompetitive AMPA receptor antagonists with anticonvulsant and neuroprotective properties. In this study, the antiexcitotoxic potency of the clinical antiepileptic drug candidate, talampanel (4 x 2 mg/kg), and that of two related 2,3-benzodiazepines, 5-(4 aminophenyl)-8-methyl-9H-1,3-dioxolo[4,5-h][2,3]-benzodiazepine (GYKI 52466) (4 x 10 mg/kg) and GYKI 53784 (4 x 2 mg/kg), was investigated in 7-day-old rats. The AMPA antagonists were applied in four consecutive i.p. injections at 1-h intervals, the first dosage was given shortly after the intrastriatal injection of (S)-alpha-amino-3-hydroxy-5,7-methylisoxazole-4-propionic acid (AMPA) (2.5 nmol). All tested compounds protected animals from brain damage induced by AMPA as assessed 5 days later by using a tissue volume determination method based on computer-aided serial section reconstruction. GYKI 53784 (56.1 +/- 5.0% protection) and talampanel (42.5 +/- 5.3% protection) were more potent neuroprotective agents than GYKI 52466 (21.8 +/- 2.8% protection). Furthermore, the three compounds attenuated the unilateral AMPA injection-induced turning behavior and seizure-like events.Our present findings are in agreement with those of other investigators who found talampanel neuroprotective in various in vivo experimental models. These data indicate that besides being a promising antiepileptic drug candidate talampanel may have a value in the pharmacotherapy of acute and chronic neurodegenerative diseases, including perinatal ischemia/hypoxia-induced brain injuries, as well. PMID- 12372547 TI - In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase. AB - Cholinesterases are a large family of enzymatic proteins widely distributed throughout both neuronal and non-neuronal tissues. In Alzheimer's disease (AD), analytical as well as epidemiological studies suggest an implication of an abnormal focal accumulation of aluminum in the brain. In this devastating disease, aluminum may interfere with various biochemical processes including acetylcholine metabolism, and can thus act as a possible etiopathogenic cofactor. Acetylcholinesterase (AChE) exists in several molecular forms that differ in solubility and mode of membrane attachment rather than in catalytic activity. Mice were treated orally with aluminum chloride or aluminum lactate (Al(lac)(3)), and AChE activity in their brain homogenates was then assayed. Results showed that this in vivo treatment augmented the activity of the enzyme. An activating effect was also observed in vitro, when the aluminum compounds were added directly to mouse brain homogenates. However, the activating effect observed in vivo was much more marked than that observed in vitro. In addition, the activation produced by Al(lac)(3) was higher than that obtained after aluminum chloride treatment. Kinetics measurements of AChE activity in the absence and presence of treatment with aluminum both in vivo and in vitro are reported. The influence of the metal speciation on enzymatic activity is discussed in relation to a possible implication of aluminum in some neurodegenerative diseases. PMID- 12372548 TI - Deficit in hippocampal long-term potentiation in monosodium glutamate-treated rats. AB - Rats subjected to monosodium glutamate (MSG) administration during the neonatal period present chronic neuroendocrine dysfunction associated with marked cognitive deficits. Long-term potentiation (LTP) in the hippocampus provides a model suited for the study of mammalian brain plasticity and memory formation. In the present work, we used the LTP protocol to investigate the synaptic plasticity in the hippocampal CA1 area of adult rats subjected to MSG treatment during the first 10 days of life. Synaptic transmission in CA1 area was analyzed using extracellular field recordings in response to Schaffer's collateral fiber stimulation in hippocampal slices. Animals injected with MSG exhibited a dramatic decrement of LTP field excitatory postsynaptic potentials (fEPSPs) compared to control group. Analysis of percent enhancement of fEPSP slope at 2 min after high frequency stimulation (HFS) increased by 189.3 +/- 33.2% in slices from control rats and 129.45 +/- 18.5% (p < 0.01) in slices from MSG-treated rats. Additionally, MSG-treated animals failed to maintain or consolidate LTP as revealed by a significant reduction in fEPSP slope enhancement over time after HFS. The mean fEPSP slope, 60 min after HFS, was 154.28 +/- 21% of the average baseline slope in control slices versus only 124.4 +/- 15% in MSG-treated rats (p < 0.01). At 90 min after HFS, slices from controls reached a potentiation of 44.5 +/- 2.9%, whereas the MSG group displayed an overall response enhancement of 17.65 +/- 2.7% of basal levels (p < 0.01). These findings indicate that MSG treated rats display a chronic impairment of CA1 synaptic plasticity. PMID- 12372549 TI - Decreased gene expression of calretinin and ryanodine receptor type 1 in tottering mice. AB - Tottering mice are a spontaneously occurring animal model of human absence epilepsy. They carry a mutation in the P/Q-type calcium channel alpha1A subunit gene which is highly expressed by cerebellar Purkinje cells. In this study, we investigated the role of calretinin and ryanodine receptor type 1 (RyR1) gene expression in the cerebellum of tottering mice. Cerebellar tissue specimens from four experimental groups were processed for in situ hybridization histochemistry (ISHH): (1) wild-type (+/+); (2) heterozygous (tg/+) and two homozygous groups; either (3) without occurrence of an episode of paroxysmal dyskinesia (tg/tg-N); or (4) after an episode of paroxysmal dyskinesia (tg/tg-P) that lasted about 45 min on average. Quantitative analysis showed a statistically significant decrease (p = 0.0001, ANOVA) of calretinin gene expression at the level of the simple lobule of the cerebellum in both homozygous groups compared to the wild-type and heterozygous groups. RyR1 was decreased in the flocculus of the cerebellum in both the tg/tg-N and tg/tg-P groups compared to wild type (p = 0.0174, ANOVA). These results suggest that calretinin gene expression, as well as other genes involved in regulation of calcium homeostasis, such as RyR1, may play a role in the biochemical functional alterations present in tottering mice. PMID- 12372550 TI - Vestibular-evoked myogenic potentials: a method to assess vestibulo-spinal conduction in multiple sclerosis patients. AB - Vestibular-evoked myogenic potentials (VEMPs), elicited by acoustic stimulation, have been proposed in the assessment of the vestibulo-cervical reflex pathways. The procedure has been previously validated in several otovestibular disorders. The aim of this study was to investigate patients affected by multiple sclerosis (MS) in the attempt to clarify the underlying physiopathogenetic mechanisms and the clinical utility of VEMPs in detecting vestibulospinal involvement in this disease. VEMPs were obtained according to the technique described by Colebatch and Halmagyi [Neurology 42 (1992) 1635]. We averaged the surface tonic electromyogram from right and left sternocleidomastoid muscle, after bilateral click stimulation (click duration 0.1 ms, repetition rate 3 Hz, intensity 140 dBSPL, 256 stimuli, repeated at least twice). In all cases, we obtained the biphasic, initially positive, p13-n23 wave pattern. P13 peak latency was bilaterally or unilaterally delayed in 8 out of 15 patients (mean delay: 2.2 ms; p < 0.01 on right and <0.05 on left side) and peak-to-peak amplitude significantly reduced (mean amplitude loss: 130 microV; p < 0.01 on right and <0.05 on left side). Their overall diagnostic yield resulted in 60%. In conclusion, the present findings prove that VEMPs are delayed in p13 component and altered in amplitude in MS patients. We hypothesise that these changes might be the result of a conduction impairment in vestibulo-spinal fibres, producing a morphologic alteration of the myogenic responses. PMID- 12372551 TI - Fos and FRA protein expression in rat nucleus paragigantocellularis lateralis during different space flight conditions. AB - The nucleus paragigantocellularis lateralis (LPGi) exerts a prominent excitatory influence over locus coeruleus (LC) neurons, which respond to gravity signals. We investigated whether adult albino rats exposed to different gravitational fields during the NASA Neurolab Mission (STS-90) showed changes in Fos and Fos-related antigen (FRA) protein expression in the LPGi and related cardiovascular, vasomotor, and respiratory areas. Fos and FRA proteins are induced rapidly by external stimuli and return to basal levels within hours (Fos) or days (FRA) after stimulation. Exposure to a light pulse (LP) 1 h prior to sacrifice led to increased Fos expression in subjects maintained for 2 weeks in constant gravity (either at approximately 0 or 1 G). Within 24 h of a gravitational change (launch or landing), the Fos response to LP was abolished. A significant Fos response was also induced by gravitational stimuli during landing, but not during launch. FRA responses to LP showed a mirror image pattern, with significant responses 24 h after launch and landing, but no responses after 2 weeks at approximately 0 or 1 G. There were no direct FRA responses to gravity changes. The juxtafacial and retrofacial parts of the LPGi, which integrate somatosensory/acoustic and autonomic signals, respectively, also showed gravity-related increases in LP induced FRA expression 24 h after launch and landing. The neighboring nucleus ambiguus (Amb) showed completely different patterns of Fos and FRA expression, demonstrating the anatomical specificity of these results. Immediate early gene expression in the LPGi and related cardiovascular vasomotor and ventral respiratory areas may be directly regulated by excitatory afferents from vestibular gravity receptors. These structures could play an important role in shaping cardiovascular and respiratory function during adaptation to altered gravitational environments encountered during space flight and after return to earth. PMID- 12372552 TI - Abnormal brain energy metabolism shown by in vivo phosphorus magnetic resonance spectroscopy in patients with chronic liver disease. AB - We used phosphorus magnetic resonance spectroscopy (31P-MRS) to assess in vivo the brain bioenergetics of 28 patients with liver cirrhosis. Seven had clinical hepatic encephalopathy (HE), nine hepatocellular carcinoma. 31P-MRS was performed by the DRESS localisation technique on occipital lobes. Brain phosphocreatine was significantly reduced in patients with or without overt HE, and inorganic phosphate was increased in both groups of patients. The cytosolic phosphorylation potential (PP), the relative rate of oxidative metabolism and the regulatory [ADP] were all abnormal. Brain PP was inversely correlated with serum ammonia concentration only in patients without liver cancer. The degree of bioenergetic failure was significantly higher in the presence of overt encephalopathy. We conclude that patients with liver cirrhosis had a derangement of brain energy metabolism, and that 31P-MRS offers a non-invasive method for investigating the underlying mechanisms of HE, with relevant implications in the identification and management of this condition. PMID- 12372553 TI - Bartolomeo Panizza's "Observations on the optic nerve" (1855). AB - Bartolomeo Panizza was the first person to produce experimental and clinical evidence for a visual area in the posterior cerebral cortex. We here provide the first translation of this work entitled "Observations on the optic nerve" originally published in Italian in 1855. We also review the state of knowledge of the brain around Panizza's time, summarize his career, and consider why his work was ignored. PMID- 12372554 TI - Pyridoxine sometimes fails to be activated to pyridoxal phosphate. PMID- 12372556 TI - Controversies in neuroscience. PMID- 12372557 TI - Clinical neurotransplantation: core assessment protocol rather than sham surgery as control. AB - Basic neurotransplantation research evoked clinical trials of restorative brain surgery. Parkinson's disease was the first and primary test bed for this putative new therapeutic method. Various centers performed the grafting surgery and the behavioral evaluations in different ways, and observed a varying degree of symptomatic relief. This led to a plea for double blind placebo-controlled clinical trials, which have since been performed and of which the first outcomes were recently published. In the present paper this approach of experimental neurotransplantation in brain diseases is discussed and rejected. Neural grafting in the central nervous system is irreversible and is therefore not suitable for experimental approaches originally designed for and best suited to drug studies. For Parkinson's disease in particular, the technique is far from optimized to perform large-scale studies at this stage. Moreover, previous negative results of adrenal medulla tissue implantation in the brain of patients make placebo effects rather unlikely. Moral arguments concerning the validity of the informed consent, therapeutic misconception, and the risk/benefit ratio can be added in the plea against this control surgery. Finally, a recommendation is made for study designs that apply a disease-dedicated core assessment protocol (CAP) that can evaluate the period from pre-operative to post-convalescent stages quantitatively, and therefore, unbiased. The strength of these CAPs is that they allow comparisons of different grafting techniques, of results between centers and of other types of interventions and invasive treatments such as deep brain stimulation. On ethical grounds, it is unacceptable not to use a study design that circumvents sham or imitation surgery. It is a challenge for the neuroscience community to develop CAPs for brain diseases that are eligible for neurotransplantation in the future. PMID- 12372558 TI - tGLP-1 action on the isolated hypothalamo-neurohypophysial system under glutamate receptor blockade. AB - The isolated rat hypothalamo-neurohypophysial system was used to investigate possible mechanisms of glucagon-like peptide-1 (7-36) amide (tGLP-1) effects on the vasopressin/oxytocin (AVP/OXY) release. The non-selective inhibition of synaptic transmission as brought about by excess of MgSO(4) in the incubation medium completely abolished the tGLP-1-induced AVP release and attenuated OXY secretion. The non-specific blockade of excitatory amino acid receptors with kynurenic acid (KA) completely suppressed the tGLP-1-induced AVP but not OXY release. Specific inhibition of NMDA receptors suppressed the tGLP-1-evoked AVP release without affecting tGLP-1-induced OXY secretion. Selective blockade of non NMDA receptors did not affect either tGLP-1-induced AVP or OXY release. It is concluded that tGLP-1 can influence the function of AVP neurons indirectly, most probably via the glutamatergic system through NMDA receptors. On the other hand, tGLP-1-evoked activation of OXY neurons, at least in part, seems to be a result of direct tGLP-1 activation of these neurons. PMID- 12372559 TI - Differential effect of peripheral glutamate (NMDA, non-NMDA) receptor antagonists on bee venom-induced spontaneous nociception and sensitization. AB - This study aimed to investigate the role of peripheral N-methyl-d-aspartate (NMDA) and non-NMDA receptor on (1). spontaneous nociception and (2). on sensitization induced by subcutaneous (s.c.) injection of bee venom (0.2mg/50 micro l) in rats. Peripheral s.c. administration of the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5), the non-competitive NMDA receptor channel blocker MK-801, and the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were performed before (pre-treatment) and after (post-treatment) bee venom-induced inflammation. Pre-treatment with AP5 (10mM, 50 micro l) and both pre-treatment and post-treatment with MK-801 (2mM, 50 micro l) into the same area of the bee venom injection site markedly reduced the bee venom-increased spontaneous responses of wide-dynamic range (WDR) neuron of the spinal cord. Post-treatment with the same dose of AP5 as well as pre treatment and post-treatment with CNQX (5mM, 50 micro l) did not produce any inhibitory effects. Additionally, the role of peripheral NMDA and non-NMDA receptors on bee venom-induced mechanical allodynia and hyperalgesia were investigated and assessed by the paw withdrawal reflex to the innocuous and noxious mechanical stimulation. Peripheral administration of AP5, but not CNQX, reduced mechanical allodynia and hyperalgesia. The data suggest that the peripheral NMDA receptor, but not non-NMDA receptor, plays a pivotal role in the bee venom-induced persistent nociception and hyperexcitability. PMID- 12372560 TI - Depth amplitude and phase profiles of carbachol-induced theta in hippocampal formation slices. AB - Phase and amplitude depth profiles of EEG theta-like activity (TLA) induced by bath perfusion of 50 micro M of the cholinergic agonist carbachol were investigated using rat hippocampal formation slices. The data showed that the amplitude and phase of laminar profiles of TLA recorded in vitro were the same as the type III theta profiles recorded in in vivo rat preparations. These results supported the conclusion that the neural mechanisms involved in the generation of theta field activity in in vivo preparations and the generation of TLA in in vitro preparations were similar. The study provided further validation for the use of carbachol in vitro slice preparation as a model for studying the neural circuitry underlying theta generation in the hippocampal formation. PMID- 12372561 TI - Effects of hypergravity on the morphological properties of the vestibular sensory epithelium. II. Life-long exposure of rats including embryogenesis. AB - Rats were exposed to a hypergravity (HG) level of 2.5 x g from conception until the age of 14 weeks. The vestibular epithelia of four of these animals and four control animals were immunohistochemically labeled for actin and tubulin. The apical cross-sectional area of epithelial cells of HG exposed rats appeared to be larger in all end organs. Area increase was 7.0% in the utricle (p<0.005) and 8.2% in the crista (p<<0.001). Hair cells and supporting cells appeared to be intact. The cellular arrangement and the proportion of different cell types within the epithelia was normal. PMID- 12372562 TI - Effects of hippocampal lesions on patterned motor learning in the rat. AB - Motor skill learning in rats has been linked to cerebellar function as well as to cortical and striatal influences. The present study evaluated the contribution of the hippocampus to motor learning. Adult male rats received electrolytic lesions designed to selectively destroy the hippocampus; a sham-lesioned group of animals served as a control. The animals with hippocampal lesions acquired a patterned motor learning task as well as sham controls. In contrast, rats with hippocampal lesions were impaired in spatial, but not cued, learning in the Morris water maze. In addition, lesioned rats showed profound impairment in the novel object recognition memory task, when a 1-h delay was used between training and testing. Taken together, these results suggest that the hippocampus is not necessary during acquisition of the motor learning task. PMID- 12372563 TI - Protection of cultured oligodendrocytes against tumor necrosis factor-alpha by the antioxidants coenzyme Q(10) and N-acetyl cysteine. AB - Tumor necrosis factor-alpha (TNF-alpha) retards the rate of terminal maturation of oligodendrocytes in vitro. The following respective compounds were used along with TNF-alpha in order to try and restore the normal rate of maturation: (1). the antioxidant, coenzyme Q(10) (CoQ(10)); (2). the antioxidant, N-acetyl cysteine (NAC); (3). creatine, which helps to preserve cellular energy; and (4) S adenosyl methionine (SAM), which contributes to the biosynthesis of lipids and proteins. Of these compounds, only CoQ(10) or NAC was able to restore the numbers of mature myelin basic protein-positive cells and the ability of the oligodendrocytes to form membrane sheets. If TNF-alpha treatment causes oxidative damage by compromising oxidative metabolism in oligodendrocytes, increasing products of lipid peroxidation and/or generating radical oxygen species that can interfere with maturation signals, CoQ(10) and NAC may protect oligodendrocytes by reversing one or more of those destructive processes during terminal maturation. PMID- 12372564 TI - Serotonin potentiation of glycine-activated whole-cell currents in the superficial laminae neurons of the rat spinal dorsal horn is mediated by protein kinase C. AB - The modulatory effects of serotonin (5-HT) on glycine (Gly)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (I and II) of the rat spinal dorsal horn using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Our results demonstrate that (1). Gly acted on strychnine (STR)-sensitive Gly receptors and elicited inward Cl(-) currents (I(Gly)) at a holding potential of -40 mV; (2). 5 HT potentiated I(Gly) without affecting the reversal potential of I(Gly); (3). the agonist (alpha-methyl-5-HT) and antagonist (ketanserine) of 5-HT(2) receptor mimicked and blocked the potentiating effect of 5-HT on I(Gly), respectively; (4). bisindolylmaleimide I (BIM), a selective inhibitor of protein kinase C (PKC), reduced the potentiating effect of 5-HT on I(Gly); and (5). 5-HT-induced enhancement of I(Gly) was not affected by pretreatment with 1,2-bis-(2 aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA AM), a Ca(2+) chelator. These results indicate that (1). the potentiation of 5-HT on I(Gly) is mediated by 5-HT(2) receptor and through Ca(2+)-independent PKC intracellular signal transduction pathway; and (2). the interactions between 5-HT and Gly might modulate the transmission of nociceptive information through the spinal cord. PMID- 12372565 TI - The antinociceptive effect of mirtazapine in mice is mediated through serotonergic, noradrenergic and opioid mechanisms. AB - The antinociceptive effects of the noradrenergic and specific serotonergic antidepressant (NaSSA) drug mirtazapine and its interaction with various opioid receptor subtypes were evaluated in mice with a hotplate analgesicmeter. Mirtazapine elicited an antinociceptive effect in a dose-dependent manner following doses from 1 to 7.5mg/kg. As the mirtazapine dose increased beyond 10mg/kg latencies returned to baseline, yielding a biphasic dose-response curve. The effect of opioid, adrenergic, and serotonergic receptor antagonists was examined as to their ability to block mirtazapine antinociception. Mirtazapine (at 10mg/kg)-induced antinociception was significantly inhibited by naloxone, nor BNI, and naltrindole, but neither by beta-FNA nor by naloxonazine, implying the involvement of kappa(1)- and delta-opioid mechanisms. When adrenergic and serotonergic antagonists were used, both metergoline and yohimbine, decreased antinociception elicited by mirtazapine, implying a combined serotonergic and noradrenergic mechanism of antinociception. When mirtazapine was administered together with various agonists of the opioid receptor subtypes, it significantly potentiated antinociception mediated only by kappa(3)-opioid receptor subtypes. Summing up these results we conclude that the antinociceptive effect of mirtazapine is mainly influenced by the kappa(3)-opioid receptor subtype combined with both serotonergic and noradrenergic receptors. These results suggest a potential use of mirtazapine in the management of some pain syndromes, and raise questions regarding a possible indirect opioid-dependence induced by mirtazapine. However, further research is needed in order to establish both the exact clinical indications and the effective doses of mirtazapine when prescribed for pain. PMID- 12372566 TI - Estradiol and progesterone modify microtubule associated protein 2 content in the rat hippocampus. AB - The molecular mechanisms involved in the regulation of synaptic plasticity and neuroprotection by estradiol (E(2)) and progesterone (P(4)) are unknown. Because these processes involve changes in cytoskeleton organization, we studied the effects of E(2) and P(4) in the expression of two cytoskeletal proteins: microtubule associated protein 2 (MAP2) and tau in the hippocampus and the frontal cortex of ovariectomized adult rats. While tau expression was unaffected by E(2) and P(4), an increase in MAP2 protein content in the hippocampus but not in the cortex was observed after E(2) and P(4) treatments. Interestingly, these steroids did not modify MAP2 mRNA content in the hippocampus. These data suggest that MAP2 is involved in the structural changes induced by E(2) and P(4) in the rat hippocampus, and that MAP2 expression is regulated by these steroid hormones at a postranscriptional level. PMID- 12372567 TI - Prognosis in the thrombolysis in myocardial ischemia III registry according to the Braunwald unstable angina pectoris classification. AB - The unstable angina pectoris (UAP) classification proposed by Braunwald in 1989, although often used, has never been validated in a large, prospective multicenter study in which all subgroups of patients were included. Patients with UAP or non ST-elevation myocardial infarction (NSTEMI) were enrolled in the Thrombolysis In Myocardial Ischemia III Registry and classified according to the Braunwald classification for UAP. Clinical end points were compared at 6 weeks and 1 year. Of 3,318 patients, those with primary UAP had lower rates of recurrent myocardial infarction (MI) or death when compared with patients with secondary UAP and post MI UAP at 6 weeks (4.1% vs 6.4% vs 13.4%, respectively; p <0.001) and 1 year (9.7% vs 16.7% vs 19.7%; p <0.001). Recurrent ischemia at 6 weeks followed the same gradient (13.2% vs 18.5% vs 20.8%; p <0.001). Patients with secondary UAP had similar extent of disease at angiography as primary UAP. Patients with nonresting UAP had lower rates of death or MI than patients with UAP at rest (3.0% vs 5.6%, p = 0.011 at 6 weeks, and 8.2% vs 12.5%, p = 0.004 at 1 year). Patients with ST-segment deviation and those who had received prior antianginal medical treatment also had worse outcomes. Thus, the Braunwald classification of UAP predicts prognosis with secondary UAP, post-MI UAP, and patients with pain at rest who have a higher risk for death or recurrent cardiac events. Given their high risk for adverse events, patients with secondary UAP should be treated aggressively. PMID- 12372568 TI - Prevalence and prognostic value of perfusion defects detected by stress technetium-99m sestamibi myocardial perfusion single-photon emission computed tomography in asymptomatic patients with diabetes mellitus and no known coronary artery disease. AB - Coronary artery disease (CAD) is the leading cause of morbidity and mortality in diabetics. Early diagnosis of CAD and identification of high-risk subgroups, followed by appropriate therapy, may therefore enhance survival. This study sought to determine the value of stress myocardial perfusion single-photon emission computed tomography (SPECT) with technetium-99m sestamibi to detect perfusion defects and predict cardiac events in asymptomatic diabetics. One hundred eighty asymptomatic diabetics without known CAD who underwent 2-day stress technetium-99m sestamibi SPECT were followed up for 36 +/- 18 months. End points were defined as hard (myocardial infarction or cardiac death) or total events (myocardial infarction, cardiac death, or late revascularization). Logistic regression analysis evaluated clinical variables, type of stress, exercise treadmill test (ETT), and SPECT as predictors of end points. Perfusion defects were found in 26% of patients (15% reversible, 6% mixed, and 5% fixed). Clinical or ETT variables were not associated with perfusion defect type or with hard events. However, male gender predicted total events (chi-square 3.3; p = 0.01). An abnormal SPECT significantly increased the risk of hard events (chi square 5.4; p = 0.001) and total events (chi-square 7.4; p = 0.0001). Extensive defects determined the highest risk of total events (chi-square 18.8; p = 0.0001). Event rates increased according to SPECT: 2% of hard events per year and 5% of total events per year in patients with normal SPECT versus 9% per year and 38% per year, respectively, in those with abnormal SPECT. Importantly, a normal SPECT identified a relatively low-risk subgroup of patients. Thus, stress technetium-99m sestamibi SPECT was useful in evaluating asymptomatic diabetics for the presence of CAD, and effectively risk-stratified this population. PMID- 12372569 TI - Persistent negative T waves in the infarct-related leads as an independent predictor of poor long-term prognosis after acute myocardial infarction. AB - This study sought to determine the long-term prognostic significance of persistent or transient negative T waves in infarct-related leads. After acute myocardial infarction (AMI), QRS and T wave alterations may resolve. No clinical study has investigated the prognostic importance of persistent versus transient negative T waves. We studied 147 consecutive patients with first AMI and >/=2 negative T waves in the infarct-related leads on the electrocardiogram. One hundred twenty patients developed Q waves. Patients were followed clinically for 60 +/- 21 months. T-wave normalization was observed early (before hospital discharge) in 34 patients and late (at 4 +/- 1 months) in 65. Thirty patients had Q-wave regression. Adverse outcome occurred in 57 patients. There were 23 hard events (cardiac death in 12 patients and nonfatal AMI in 11). Patients with early or late T-wave normalization had similar event-free survival curves that diverged rapidly from that of patients with persistent negative T waves, who had a worse outcome (p <0.0001). Patients with or without Q-wave regression had similar survival curves. Using multivariate Cox regression analysis, higher end-systolic volume (hazard ratio [HR] 1.01, p = 0.007), the presence of multivessel disease (HR 3.33, p = 0.009), and persistent negative T waves (HR 2.92, p = 0.024) predicted hard events. Persistent negative T waves 4 months after first AMI were independently associated with a worse outcome, whereas Q-wave regression has no long-term prognostic importance. PMID- 12372570 TI - Revascularization, stenting, and outcomes of patients with acute myocardial infarction complicated by cardiogenic shock. AB - Randomized clinical trials have demonstrated a reduction in mortality with early revascularization of patients with acute myocardial infarction (AMI) complicated by cardiogenic shock, and recent single-center studies have particularly suggested further benefit for coronary stenting. The purpose of this study was to examine the use of revascularization and coronary stenting for patients with shock from a multicenter, international perspective. Patients with AMI complicated by cardiogenic shock (n = 583) who enrolled between April 1999 and June 2001 were prospectively identified from the large, multinational, observational Global Registry of Acute Coronary Events. We examined the use of coronary reperfusion strategies, adjunctive therapy, and hospital mortality in this group of patients. Cardiac catheterization (52%) and revascularization (43%) were performed in approximately half of the cardiogenic shock patients. Elderly patients (age >/=75 years) comprised 40% of the shock cohort. Regional differences were seen in the use of revascularization, adjunctive medical therapy, and type of revascularization used (coronary stenting). Total hospital mortality was 59%, but case fatality rates ranged from 35% for patients who underwent coronary stenting to 74% for patients who did not undergo any cardiac catheterization. Percutaneous coronary intervention with coronary stenting was the most powerful predictor of hospital survival (odds ratio 3.99, 95% confidence interval 2.41 to 6.62). Thus, cardiogenic shock continues to be a devastating complication of AMI, and relative underuse of a revascularization strategy may be related to the large proportion of elderly patients in this population. In this multinational registry study, coronary stenting was the most powerful independent predictor of hospital survival. PMID- 12372571 TI - Angiographic analysis of the angioplasty versus rotational atherectomy for the treatment of diffuse in-stent restenosis trial (ARTIST). AB - Patients with diffuse in-stent restenoses (ISRs) are at high risk for recurrent restenosis after percutaneous transluminal balloon angioplasty (PTCA). Percutaneous transluminal rotational ablation (PTCR) has proved effective in removing neointimal burden in ISRs. This study compares the acute and long-term results of PTCA and PTCR for the treatment of diffuse ISR in a randomized, multicenter investigation. The primary end point was the comparison of the minimum luminal diameter (MLD) between both groups at 6-month follow-up. Patients with symptomatic, diffuse, or high-grade ISRs were included; 146 patients were randomized to PTCA and 152 patients to PTCR. Diameter stenosis was reduced from 80 +/- 12% to 29 +/- 10% and from 80 +/- 11% to 28 +/- 12%, respectively, and MLD increased from 0.55 +/- 0.3 to 1.9 +/- 0.3 mm in the PTCA group and from 0.54 +/- 0.3 mm to 1.9 +/- 0.4 mm in the PTCR group. Spasm in the treated vessel and an intermittent slow flow phenomenon occurred more often after rotational ablation (17.7% vs 8.6%, p = 0.001; 5.3% vs 0%, p = 0.007). Minimum stenosis diameter at 6 month follow-up was smaller in the PTCR group than in the PTCA group (1.0 +/- 0.6 vs 1.2 +/- 0.6 mm, p = 0.008) and the restenosis rate was higher (64.9% vs 51.2%, p = 0.027). Procedural factors did not influence long-term outcome. In the PTCR group, the restenosis rate increased with decreasing vessel size, whereas this was not seen in the PTCA group. The lesion length and the baseline diameter stenosis were found to be predictive of restenosis with both treatment strategies; however, a residual diameter stenosis of <30% predicted absence of a restenosis only in the PTCR group. Thus, PTCA and PTCR of diffuse ISRs yield comparable acute angiographic results. The recurrence of a restenosis is higher after PTCR than after PTCA. PMID- 12372572 TI - Heart rate recovery after treadmill exercise testing and risk of cardiovascular disease events (The Framingham Heart Study). AB - A delayed heart rate (HR) recovery after graded exercise testing has been associated with increased all-cause mortality in clinic-based samples. No prior study has examined the association of HR recovery after exercise with the incidence of coronary heart disease (CHD) and cardiovascular disease (CVD) events. We evaluated 2,967 Framingham study subjects (1,400 men, mean age 43 years) who were free of CVD and underwent a treadmill exercise test (Bruce protocol) at a routine examination. We examined the relations of HR recovery indexes (decrease in HR from peak exercise) to the incidence of a first CHD or CVD event and all-cause mortality, adjusting for established CVD risk factors. During follow-up (mean 15 years), 214 subjects experienced a CHD event (156 men), 312 developed a CVD event (207 men), and 167 died (105 men). In multivariable models, continuous HR recovery indexes were not associated with the incidence of CHD or CVD events, or with all-cause mortality. However, in models evaluating quintile-based cut points, the top quintile of HR recovery (greatest decline in HR) at 1-minute after exercise was associated with a lower risk of CHD (hazards ratio vs bottom 4 quintiles 0.54, 95% confidence interval [CI], 0.32 to 0.93) and CVD (hazards ratio 0.61, 95% CI 0.41 to 0.93), but not all-cause mortality (hazards ratio 0.99, 95% CI 0.60 to 1.62). In our community-based sample, HR recovery indexes were not associated with all-cause mortality. A very rapid HR recovery immediately after exercise was associated with lower risk of CHD and CVD events. These findings should be confirmed in other settings. PMID- 12372573 TI - Intravenous lidocaine versus intravenous amiodarone (in a new aqueous formulation) for incessant ventricular tachycardia. AB - The effectiveness of intravenous amiodarone for the treatment of incessant (shock resistant) ventricular tachycardia (VT) has not been established. This study evaluated the efficacy of a water-soluble amiodarone preparation or lidocaine for the treatment of shock-resistant VT. The trial was a double-blinded parallel design. Patients were randomized to receive up to 2 boluses of either 150 mg intravenous amiodarone or 2 boluses of 100 mg lidocaine followed by a 24-hour infusion. If the first assigned medication failed to terminate VT, the patient was crossed over to the alternative therapy. Twenty-nine patients were randomized to the study (18 received amiodarone and 11 received lidocaine). There were no significant differences between groups with regard to baseline characteristics. Immediate VT termination was achieved in 14 patients (78%) with amiodarone versus 3 patients (27%) on lidocaine (p <0.05). After 1 hour, 12 patients (67%) on amiodarone and 1 patient (9%) on lidocaine were alive and free of VT (p <0.01). Amiodarone had a 33% drug failure rate, whereas there was a 91% drug failure rate for lidocaine. The 24-hour survival was 39% on amiodarone and 9% on lidocaine (p <0.01). Drug-related hypotension with aqueous amiodarone was less frequent than with lidocaine. This study found that amiodarone is more effective than lidocaine in the treatment of shock-resistant VT. PMID- 12372574 TI - Coronary flow reserve and myocardial diastolic dysfunction in arterial hypertension. AB - The aim of this study was to assess the relation between coronary blood flow and left ventricular (LV) myocardial diastolic dysfunction in arterial hypertension. The study population included 30 hypertensive patients who were free of coronary artery disease and pharmacologic therapies. They underwent standard Doppler echocardiography and color tissue Doppler of the middle posterior septum at baseline and with high-dose dobutamine, and second-harmonic Doppler flow analysis of the distal left anterior descending coronary artery at baseline and after vasodilation by dipyridamole (0.56 mg/kg IV in 4'). Coronary flow reserve (CFR) was estimated as the ratio of hyperemic and baseline diastolic flow velocities. According to CFR, hypertensives were divided into 2 groups: 15 patients with normal CFR (>/=2) and 15 patients with reduced CFR (<2). The 2 groups were comparable for sex, age, body mass index, baseline heart rate, and blood pressure. LV mass index was greater in hypertensives with reduced CFR (p <0.01). By color tissue Doppler, baseline and high-dose dobutamine septal systolic velocities did not differ between the 2 groups. The ratio between myocardial velocities in early diastole and at atrial contraction (E(m)/A(m) ratio) was lower in patients with reduced CFR, both at baseline (p <0.05) and with high-dose dobutamine (p <0.00001). After adjusting for age, body mass index, LV mass index, and both high-dose dobutamine diastolic blood rate and heart rate by a multiple linear regression analysis, E(m)/A(m) ratio at high-dose dobutamine was independently associated with CFR in the overall population (beta 0.62, p <0.0005) (cumulative R(2) 0.38, p <0.0005). In conclusion, this study provides evidence of an independent association between CFR and myocardial diastolic function. In hypertensive patients without coronary artery stenosis, CFR alteration may be a determinant of myocardial diastolic dysfunction or diastolic impairment that should be taken into account as possibly contributing to coronary flow reduction. PMID- 12372575 TI - Comparison of transcatheter closure of secundum atrial septal defect using the Amplatzer septal occluder associated with deficient versus sufficient rims. AB - To evaluate the feasibility of transcatheter closure of secundum atrial septal defects (ASDs) associated with deficient rims (<5 mm) using the Amplatzer septal occluder (ASO), 23 patients (median age 10.7 years) underwent an attempted transcatheter closure. The patients had a deficient anterior rim of 0 to 4 mm (n = 20), an inferior rim of 2 mm (n = 2), or a posterior rim of 4 mm (n = 1) as assessed by transesophageal echocardiography (TEE) or intracardiac echocardiography (ICE). Forty-eight patients with sufficient rims (>5 mm) who underwent closure served as controls. There were no differences between the 2 groups in ASD stretched diameter and device size (p >0.05). Of 23 patients with deficient rims, 17 (74%) had immediate complete closure compared with 44 of 48 patients (92%) with sufficient rims (p <0.05). At 24-hour and 6-month follow-up, the complete closure rates were not significantly different between the 2 groups (91% for patients with deficient rims vs 94% for patients with sufficient rims at 24 hours and 100% vs 93% at 6 months, respectively). The fluoroscopic time and procedure time were longer in patients with deficient rims (13 +/- 7 and 72 +/- 26 minutes, respectively) compared with those with sufficient rims (10 +/- 4 and 61 +/- 22 minutes, respectively). No major complications were encountered either during or after the closure procedure in both groups. Thus, transcatheter closure of ASDs associated with small anterior, inferior, or posterior rims is feasible using an ASO. Long-term follow-up data are still needed to assess long-term safety and efficacy. PMID- 12372576 TI - Usefulness of lipid-lowering therapy in elderly patients. PMID- 12372577 TI - Effects of atorvastatin 80 mg daily early after onset of unstable angina pectoris or non-Q-wave myocardial infarction. PMID- 12372578 TI - Long-term prognosis of patients with clinical unstable angina pectoris without elevation of creatine kinase but with elevation of cardiac troponin i levels. PMID- 12372579 TI - Comparison of positron emission tomography with the resting electrocardiogram for assessing viable myocardium in chronic ischemic cardiomyopathy involving the anterior left ventricular wall. PMID- 12372580 TI - Utility of immediate exercise treadmill testing in patients taking beta blockers or calcium channel blockers. PMID- 12372581 TI - Minimally invasive direct coronary artery bypass (MIDCAB) versus coronary artery stenting for elective revascularization of the left anterior descending artery. PMID- 12372582 TI - Effectiveness of multiple antilipidemic agents on Vertical Auto Profile II guided treatment of dyslipoproteinemia. PMID- 12372583 TI - Efficacy of biphasic waveform cardioversion for atrial fibrillation and atrial flutter compared with conventional monophasic waveforms. PMID- 12372584 TI - Frequency of aspirin resistance in patients with congestive heart failure treated with antecedent aspirin. PMID- 12372585 TI - Prognostic value of treatment-induced changes in twenty-four-hour mean and pulse pressures in adult hypertensive patients. PMID- 12372586 TI - Left ventricular hypertrabeculation/noncompaction and association with additional cardiac abnormalities and neuromuscular disorders. PMID- 12372587 TI - Reducing radiation dose in the cardiac catheterization laboratory by design alterations and staff education. PMID- 12372588 TI - Acute effects of caffeine on heart rate variability. PMID- 12372589 TI - An unanticipated effect of meditation on cardiovascular pharmacology and physiology. PMID- 12372590 TI - Residual plaque burden after stenting: does it matter? PMID- 12372591 TI - HU: promoting or counteracting DNA compaction? AB - The role of HU in Escherichia coli as both a protein involved in DNA compaction and as a protein with regulatory function seems to be firmly established. However, a critical look at the available data reveals that this is not true for each of the proposed roles of this protein. The role of HU as a regulatory or accessory protein in a number of systems has been thoroughly investigated and in many cases has been largely elucidated. However, almost 30 years after its discovery, convincing evidence for the proposed role of HU in DNA compaction is still lacking. Here we present an extensive literature survey of the available data which, in combination with novel microscopic insights, suggests that the role of HU could be the opposite as well. The protein is likely to play an architectural role, but instead of being responsible for DNA compaction it could be involved in antagonising compaction by other proteins such as H-NS. PMID- 12372592 TI - How the lipid-free structure of the N-terminal truncated human apoA-I converts to the lipid-bound form: new insights from NMR and X-ray structural comparison. AB - The X-ray structure of the N-terminal truncated human apoA-I [Borhani et al., Proc. Natl. Acad. Sci. USA 94 (1997) 12291] and the NMR structure of intact human apoA-I [Okon et al., FEBS Lett. 517 (2002) 139] found similar repeating helices. The crystal structure is a twisted circular four-helix bundle, consisting of four molecules of apoA-I(44-243), where four copies of the lecithin:cholesterol acyltransferase (LCAT)-activating domains are located outside the ring structure, while the aromatic-rich strong lipid-binding domains are inside. This architecture suggests a lipid-binding mechanism that lipids directly enter the hole of the crystal structure. Indeed, four copies of Trp50 and Trp72 are exposed and oriented toward the center of the ring, initiating lipid binding. This is followed by the inside-out rotations of the terminal helices to make a belt with all the hydrophobic faces of the helices facing inward. Such lipid-binding induced rotations have an impact on the conformation of the lipid-free form. Indeed, the structure of residues 78-81 changes from helical (free) to disordered (bound) while the structure of residues 221-227 changes from extended to helical. PMID- 12372593 TI - p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families. AB - We identified families of proteins characterized by the presence of a domain similar to human p23 protein, which include proteins such as Sgt1, involved in the yeast kinetochore assembly; melusin, involved in specific interactions with the cytoplasmic integrin beta1 domain; Rar1, related to pathogenic resistance in plants, and to development in animals; B5+B5R flavo-hemo cytochrome NAD(P)H oxidoreductase type B in humans and mice; and NudC, involved in nucleus migration during mitosis. We also found that p23 and the HSP20/alpha-crystallin family of heat shock proteins, which share the same three-dimensional folding, show a pattern of conserved residues that points to a common origin in the evolution of both protein domains. The p23 and HSP20/alpha-crystallin phylogenetic relationship and their similar role in chaperone activity suggest a common function, probably involving protein-protein interaction, for those proteins containing p23-like domains. PMID- 12372594 TI - B plexins activate Rho through PDZ-RhoGEF. AB - Plexins are receptors for the repulsive axon guidance molecules semaphorins. Previously, we have shown that plexin-B1 binds activated Rac, but that clustering of plexin-B1 causes Rho activation, resulting in stress fiber formation. Using the yeast two-hybrid system, we found that the C-terminus of B plexins interacted directly with Rho-specific exchange factors, via their PDZ domain. Mutation of the carboxy-terminal amino acids of plexin-B1 or coexpression of a dominant negative PDZ-RhoGEF abrogated the ability of plexin-B1 to cause stress fiber formation. Our results demonstrate a role for PDZ-RhoGEF in B plexin-mediated activation of Rho/Rho kinase signaling, implicated in the regulation of axon guidance and cell migration. PMID- 12372595 TI - Quantitative proteomics: the copy number of pyruvate dehydrogenase is more than 10(2)-fold lower than that of complex III in human mitochondria. AB - Pyruvate dehydrogenase (PDH) and complex III are two key protein complexes in mitochondrial metabolic activity. Using a novel quantitative Western blotting method, we find that PDH and complex III exist at a steady-state ratio of 1:100, 1:128 and 1:202 in HeLa cell extracts, fibroblast mitochondria and heart tissue mitochondria, respectively. This difference in stoichiometry is reflected in the immunogold labeling intensities of the two complexes and by the much more sparse distribution of PDH in fluorescence microscopy. In Rho0 fibroblasts there is a 64% reduction of complex III but the concentration of PDH remains the same as wild-type. PMID- 12372597 TI - Interaction of ALG-2 with ASK1 influences ASK1 localization and subsequent JNK activation. AB - ALG-2 (apoptosis linked gene-2) is an essential protein for the execution of apoptosis whose function is largely unknown. Here, we demonstrate that ALG-2 could interact with the C-terminus (amino acids 941-1375) of the apoptosis signal regulating kinase 1 (ASK1) in BOSC23 cells as well as in vitro. ASK1 failed to bind to an isotype of ALG-2 found in the liver, ALG-2,1, in which two amino acids (Gly-121 and Phe-122) are deleted. This implies that the interaction is very specific. Cotransfection with ALG-2 resulted in the nuclear presence of ASK1 and inhibited the activation of c-Jun N-terminal kinase (JNK) by ASK1 in BOSC23 cells. This study reports that ALG-2 could regulate the subcellular localization and the JNK activity modulation of ASK1 by direct interaction. PMID- 12372598 TI - Heterogeneous exchange behavior of Samia cynthia ricini silk fibroin during helix coil transition studied with (13)C NMR. AB - The structure and structural transition of the glycine residue adjacent to the N terminal alanine residue of the poly(L-alanine), (Ala)(12-13), region in Samia cynthia ricini silk fibroin was studied using (13)C nuclear magnetic resonance (NMR). Most of the glycine carbonyl peaks in the (13)C solution NMR spectrum of [1-(13)C]glycine-silk fibroin could be assigned to the primary structure from the comparison of the (13)C chemical shifts of seven glycine-containing tripeptides. The slow exchange between helix and coil forms in the NMR time scale was observed with increasing temperature exclusively for the underlined glycine residue in the Gly-Gly-(Ala)(12-13) sequence during fast helix-coil transition of the (Ala)(12 13) region. PMID- 12372596 TI - Prediction of structure and functional residues for O-GlcNAcase, a divergent homologue of acetyltransferases. AB - N-Acetyl-beta-D-glucosaminidase (O-GlcNAcase) is a key enzyme in the posttranslational modification of intracellular proteins by O-linked N acetylglucosamine (O-GlcNAc). Here, we show that this protein contains two catalytic domains, one homologous to bacterial hyaluronidases and one belonging to the GCN5-related family of acetyltransferases (GNATs). Using sequence and structural information, we predict that the GNAT homologous region contains the O GlcNAcase activity. Thus, O-GlcNAcase is the first member of the GNAT family not involved in transfer of acetyl groups, adding a new mode of evolution to this large protein family. Comparison with solved structures of different GNATs led to a reliable structure prediction and mapping of residues involved in binding of the GlcNAc-modified proteins and catalysis. PMID- 12372599 TI - Stimulation and inhibition of fibril formation by a peptide in the presence of different concentrations of SDS. AB - Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially alpha-helical but converts rapidly to a beta-sheet structure and large quantities of fibrils form. In SDS above the critical micellar concentration the peptide adopts a stable alpha-helical structure and no fibrils are observed. These findings demonstrate the sensitivity of fibril formation to solution conditions and suggest a possible role for membrane components in amyloid fibril formation in living systems. PMID- 12372600 TI - H(2)O(2) generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis. AB - Characterization of lignified Zinnia elegans hypocotyls by both alkaline nitrobenzene oxidation and thioacidolysis reveals that coniferyl alcohol units are mainly found as part of 4-O-linked end groups and aryl-glycerol-beta-aryl ether (beta-O-4) structures. Z. elegans hypocotyls also contain a basic peroxidase (EC 1.11.1.7) capable of oxidizing coniferyl alcohol in the absence of H(2)O(2). Results showed that the oxidase activity of the Z. elegans basic peroxidase is stimulated by superoxide dismutase, and inhibited by catalase and anaerobic conditions. Results also showed that the oxidase activity of this peroxidase is due to an evolutionarily gained optimal adaptation of the enzyme to the microM H(2)O(2) concentrations generated during the auto-oxidation of coniferyl alcohol, the stoichiometry of the chemical reaction (mol coniferyl alcohol auto-oxidized/mol H(2)O(2) formed) being 0.496. These results therefore suggest that the H(2)O(2) generated during the auto-oxidation of coniferyl alcohol is the main factor that drives the unusual oxidase activity of this highly conserved lignin-synthesizing class III peroxidase. PMID- 12372601 TI - Coexpression of a Ca(v)1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity. AB - L-type Ca(2) channels play a critical role in many types of cells, including nerve, muscle and endocrine cells. The most popular and effective tools for analyzing the roles of L-type calcium channels (L-channels) are specific antagonists such as dihydropyrigines. With these drugs however, it is difficult to target specific cells. One solution is to develop a genetically targetable inhibitor coded by DNA. As a candidate for such an inhibitor, a dominant negative mutant of Ca(v)1.2 was designed by mimicking an ascidian 3-domain-type alpha1 subunit (that inhibits the full-length subunit's current). The 3-domain-type Ca(v)1.2 subunit significantly inhibited wild-type Ca(v)1.2 current, but not other ionic currents such as Ca(v)2.1 and Na(v) channels in Xenopus oocyte expression systems. Western blot analysis showed that the expression of the wild type protein into the plasma membrane was significantly suppressed on coexpression with the truncated protein. These findings support that an N terminus-truncated mutant could serve as a specific genetically encoded inhibitor for L-channels. PMID- 12372602 TI - A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: characterisation of recombinant sulphite:cytochrome c oxidoreductase from Starkeya novella. AB - The phototrophic purple non-sulfur bacterium Rhodobacter capsulatus expresses a wide variety of complex redox proteins in response to changing environmental conditions. Here we report the construction and evaluation of an expression system for recombinant proteins in that organism which makes use of the dor promoter from the same organism. A generic expression vector, pDorEX, was constructed and used to express sulphite:cytochrome c oxidoreductase from Starkeya novella, a heterodimeric protein containing both molybdenum and haem c. The recombinant protein was secreted to the periplasm and its biochemical properties were very similar to those of the native enzyme. The pDorEX system therefore seems to be potentially useful for heterologous expression of multi subunit proteins containing complex redox cofactors. PMID- 12372603 TI - A simple method for isolating import-competent Arabidopsis chloroplasts. AB - We present a simple, rapid and low-cost method for isolating a high yield of Arabidopsis chloroplasts that can be used to study chloroplast protein import. Efficiency of chloroplast isolation was dependent upon the ratio between amount of plant tissue and the buffer volume, the size and speed of the homogenisation equipment, and the size of the homogenisation beaker. The import method proved useful when characterising different precursor proteins, developmental stages and import-defective mutants. Time-course experiments enabled the measurement of import rates in the linear range. Compared to protoplastation, this isolation method has significant time and cost savings (approximately 80% and approximately 95%, respectively), and yields chloroplasts with a higher capacity to import proteins. PMID- 12372604 TI - Nitric oxide synthesis is involved in arterial haptoglobin expression after sustained flow changes. AB - The acute phase protein haptoglobin is highly expressed in arteries after sustained flow changes and involved in cell migration and arterial restructuring. In the liver, haptoglobin expression is mainly regulated by interleukin-6 (IL-6). In the artery, shear stress and NO influence IL-6 expression. In the present study, we demonstrate that NO synthesis is involved in the regulation of arterial haptoglobin expression after sustained flow changes. Decreased haptoglobin expression after NO inhibition coincided with decreased IL-6 levels. However, IL 6 knockout mice had normal arterial haptoglobin expression levels after sustained flow changes suggesting that other mediators may provide compensatory mechanisms for the regulation of arterial haptoglobin expression. PMID- 12372605 TI - 3'-end processing of precursor M1 RNA by the N-terminal half of RNase E. AB - M1 RNA, the catalytic component of Escherichia coli RNase P, is derived from the 3'-end processing of precursor M1 RNA, a major transcript of the rnpB gene. In this study, we investigated the mechanism of 3'-end processing of M1 RNA using the recombinant N-terminal half RNase E. The cleavage site preference of RNase E differed from that of the 40% ammonium sulfate precipitate (ASP-40), a partially purified cell extract containing processing activity. However, the addition of a trace amount of ASP-40 changed the cleavage site preference of RNase E to that of ASP-40 suggesting the involvement of a soluble factor in cleavage site preference. PMID- 12372606 TI - Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma. AB - To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin-induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial-mediated apoptosis could play a role in oxaliplatin resistance. PMID- 12372607 TI - Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli. AB - Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications. PMID- 12372608 TI - Dap-SL: a new site-directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins. AB - A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more flexible than the modified Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3(10) helix and alpha-helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe. PMID- 12372609 TI - Caspase 9 activation by the dsRNA-dependent protein kinase, PKR: molecular mechanism and relevance. AB - The double-stranded RNA-dependent protein kinase (PKR) induces apoptosis by activation of the FADD/caspase 8 pathway. Here we show that upon PKR expression, caspase 9 is processed and activated, correlating with the translocation of cytochrome c to the cytoplasm and breakdown of mitochondrial potential upon Bax insertion. However, treatment of cells with an inhibitor of caspase 9 could not prevent PKR-induced apoptosis. During PKR-induced apoptosis, caspase 9 is activated downstream of caspase 8. Our findings revealed that caspase 9, although dispensable, is a mediator of PKR-induced cell death. PMID- 12372610 TI - Most of the structural elements of the globular domain of murine prion protein form fibrils with predominant beta-sheet structure. AB - The conversion of the cellular prion protein into the beta-sheet-rich scrapie prion protein is thought to be the key step in the pathogenesis of prion diseases. To gain insight into this structural conversion, we analyzed the intrinsic structural propensity of the amino acid sequence of the murine prion C terminal domain. For that purpose, this globular domain was dissected into its secondary structural elements and the structural propensity of the protein fragments was determined. Our results show that all these fragments, excepted that strictly encompassing helix 1, have a very high propensity to form structured aggregates with a dominant content of beta-sheet structures. PMID- 12372611 TI - Identification of the ice-binding face of antifreeze protein from Tenebrio molitor. AB - The beetle Tenebrio molitor produces several isoforms of a highly disulfide bonded beta-helical antifreeze protein with one surface comprised of an array of Thr residues that putatively interacts with ice. In order to use mutagenesis to identify the ice-binding face, we have selected an isoform that folds well and is tolerant of amino acid substitution, and have developed a heating test to monitor refolding. Three different types of steric mutations made to the putative ice binding face reduced thermal hysteresis activity substantially while a steric mutation on an orthogonal surface had little effect. NMR spectra indicated that all mutations affected protein folding to a similar degree and demonstrated that most of the protein folded well. The large reductions in activity associated with steric mutations in the Thr array strongly suggest that this face of the protein is responsible for ice binding. PMID- 12372612 TI - The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells. AB - The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells. PMID- 12372613 TI - The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin. AB - The vasodilator-stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain. Point mutations within this motif drastically weaken VASP/G-actin interactions, thereby abolishing any actin nucleating activity of VASP. Additionally, actin nucleation was found to depend on VASP oligomerisation since VASP monomers fail to induce the formation of actin filaments. Phosphorylation negatively affects VASP/G-actin interactions preventing VASP-induced actin filament formation. PMID- 12372614 TI - Sorsby's fundus dystrophy mutant tissue inhibitors of metalloproteinase-3 induce apoptosis of retinal pigment epithelial and MCF-7 cells. AB - C-terminal domain tissue inhibitor of metalloproteinases-3 (TIMP-3) mutations cause the rare hereditary blindness Sorsby's fundus dystrophy (SFD), which involves loss of retinal pigment epithelial (RPE) cells. Since wild-type TIMP-3 causes apoptosis, we investigated whether SFD TIMP-3 might kill RPE and other cells. Plasmid-mediated overexpression of Ser-156, Gly-167, Tyr-168 and Ser-181 SFD mutant TIMP-3 decreased RPE viability to 22+/-8, 20+/-6, 32+/-5, 30+/-12% (SFD mutants all P<0.01 versus wild-type 50+/-8%) and similarly increased propidium iodide staining and in situ end labelling. Adenovirus-mediated overexpression of the Gly-167 mutant also caused RPE apoptosis dose-dependently. Apoptosis of RPE cells might therefore contribute to the pathology of SFD. PMID- 12372615 TI - Induction of human inhibitor of apoptosis protein-2 by shear stress in endothelial cells. AB - To disclose the anti-atherosclerotic mechanisms of steady laminar shear stress, we analyzed the expression of human inhibitor of apoptosis protein-2 (HIAP-2), whose gene was selected from a cDNA library of sheared endothelial cells (ECs), on ECs. HIAP-2 was dose-independently and time-dependently induced in ECs by shear stress, regulated at the transcriptional level. HIAP-2 expression was also identified in vivo. Shear stress-mediated inhibition of EC apoptosis was associated with the inhibition of caspase-3 activity, suggesting that the shear stress prevents EC apoptosis via negative regulation of caspase-3 by the increment of HIAP-2. PMID- 12372616 TI - Study of the cytotoxic effect of a peptidic inhibitor of the p53-hdm2 interaction in tumor cells. AB - Inhibitors of the p53-hdm2 interaction are attractive molecules for stimulating the p53 pathway in tumors. In this report, an inhibitor of the p53-hdm2 interaction, the AP peptide, is used to activate p53 in tumor cells expressing various levels of hdm2 protein. It induces apoptosis only in cells expressing high endogenous levels of hdm2 protein. The absence of apoptosis in tumor cells with low hdm2 levels is due not to alterations in the p53-dependent apoptotic pathway but to a different regulation of this pathway. The peptide is also less toxic for non-tumor cells than for tumor cells overexpressing the hdm2 protein. PMID- 12372617 TI - Solvent environment conducive to protein aggregation. AB - The effect of solvent structuring induced by molecular crowding is elucidated within a competitive situation involving protein folding and aggregation. Two patterned fragments of amyloidogenic proteins are chosen as study cases and analyzed by molecular dynamics with an implicit treatment of the solvent. The extent of crowding needed to induce aggregation is determined. The results constitute a first step to assess the relevance of in vivo environments in understanding fibrillogenesis. The approach is independently validated by satisfactorily reproducing the results of an all-atom explicit solvent trajectory. PMID- 12372618 TI - Regulatory expression of MDP77 protein in the skeletal and cardiac muscles. AB - The mdp77 gene was first cloned from the cDNA library of denervated chick muscles, while its role(s) in vivo was unknown. In the present study, using specific polyclonal antibodies against MDP77, we show that MDP77 was expressed specifically in the skeletal and cardiac muscle, and confirm its presence in the cytoplasm of the extrafusal muscle fibers. In mature muscles, MDP77 immunoreactivity was observed in a repetitive manner along the sarcomere. The onset of MDP77 expression occurred just after myotube formation both in vivo and in vitro. Furthermore, MDP77 was enriched in the intrafusal muscle fibers. Our findings suggest that MDP77 plays an important role(s) in the differentiation, maturation and function of both the skeletal and cardiac muscles. PMID- 12372619 TI - The prolyl 4-hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate generates effective iron deficiency in cultured cells. AB - Ethyl-3,4-dihydroxybenzoate (EDHB) is commonly utilized as a substrate analog and competitive inhibitor of prolyl 4-hydroxylases. These iron-dependent enzymes have received a lot of attention for their involvement in crucial biochemical pathways such as collagen maturation and oxygen sensing. Since EDHB is also capable of chelating the enzyme-bound iron, we study here its function as a chelator. We show that the affinity of EDHB for ferric iron is significantly lower than that of desferrioxamine. Nevertheless, EDHB is sufficient to promote effective iron deficiency in cells, reflected in the activation of the iron-responsive element/iron regulatory protein regulatory network. Thus, treatment of B6 fibroblasts with EDHB results in slow activation of iron regulatory protein 1 accompanied by an increase in transferrin receptor levels and reduction of the ferritin pool. PMID- 12372620 TI - Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle. AB - Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441-444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold acclimated chickens. The results obtained here show that suppression of palmitate induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle. PMID- 12372621 TI - WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis. AB - Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus. PMID- 12372622 TI - The novel endoplasmic reticulum (ER)-targeted protein HAP induces cell apoptosis by the depletion of the ER Ca(2+) stores. AB - HAP, a novel human apoptosis-inducing protein, was identified to localize exclusively to the endoplasmic reticulum (ER) in our previous work. In the present work, we reported that ectopic overexpression of HAP proteins caused the rapid and sustained elevation of the intracellular cytosolic Ca(2+), which originated from the reversible ER Ca(2+) stores release and the extracellular Ca(2+) influx. The HeLa cells apoptosis induced by HAP proteins was not prevented by establishing the clamped cytosolic Ca(2+) condition, or by buffering of the extracellular Ca(2+) with EGTA, suggesting that the depletion of ER Ca(2+) stores rather than the elevation of cytosolic Ca(2+) or the extracellular Ca(2+) entry contributed to HAP-induced HeLa cells apoptosis. Caspase-3 was also activated in the process of HAP-triggered apoptotic cell death. PMID- 12372623 TI - Recombinant Escherichia coli biotin synthase is a [2Fe-2S](2+) protein in whole cells. AB - EPR and Mossbauer spectroscopies have been used to determine the type and properties of the iron-sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification. Difference EPR spectra of samples of whole cells from a strain over-expressing E. coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the [2Fe-2S](+) cluster of the E. coli iron-sulfur cluster assembly 2Fe ferredoxin, based on principal g values, linewidths and relaxation behavior. Comparison of the Mossbauer spectra of whole cells with and without the bioB insertion revealed that the E. coli cells with over-expressed BioB contain an additional species that exhibits a spectrum identical to that of the [2Fe-2S](2+) cluster in purified recombinant BioB. The concentration of this [2Fe-2S](2+) species in the whole cell sample was quantified using a Mossbauer standard and found to be approximately 260 microM, which was comparable to the BioB protein concentration estimated for the cell paste. The results demonstrate that the [2Fe-2S](2+) cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over-expressed in an inactive form during aerobic growth. PMID- 12372624 TI - Eukaryotic initiation factor 4GI is a poor substrate for HIV-1 proteinase. AB - Eukaryotic initiation factor (eIF) 4GI is efficiently cleaved during picornaviral replication. eIF4GI processing has also recently been observed during HIV-1 replication. We have compared the efficiency of eIF4GI proteolysis in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L(pro)) or the HIV-1 proteinase (HIV-1(pro)). L(pro) cleaved 50% eIF4GI within 12 min whereas HIV-1(pro) required 4 h; the concentrations were 2 pg/microl (0.1 nM) for L(pro) and 60 pg/microl (2.66 nM) for HIV-1(pro). HIV-1(pro) processing of eIF4GI is therefore not quantitatively analogous to that of L(pro), suggesting that the primary function of eIF4GI cleavage in HIV-1 replication may not be protein synthesis inhibition. PMID- 12372625 TI - N-terminal acetylation of ectopic recombinant proteins in Escherichia coli. AB - N-terminal acetylation is a protein modification common in eukaryotes, but rare in prokaryotes. Here, we characterized five mammalian stathmin-like subdomains expressed in Escherichia coli by matrix-assisted laser desorption/ionization time of-flight mass spectrometry and nanoESI Q-TOF tandem mass spectrometry. We revealed that RB3(SLD) and RB3'(SLD) are N(alpha)-acetylated, whereas SCG10(SLD) and SCLIP(SLD), although identical up to residue 6, are not, as well as stathmin. To assess the influence of the N-terminal sequences on N(alpha)-acetylation, we exchanged residues 7 and 8 between acetylated RB3(SLD) and unacetylated SCG10(SLD), and showed that it reversed the acetylation pattern. Our results demonstrate that ectopic recombinant proteins can be extensively N(alpha) acetylated in E. coli, and that the rules governing N(alpha)-acetylation are complex and involve the N-terminal region, as in eukaryotes. PMID- 12372626 TI - Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes. AB - The mitochondrial membrane bound dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme was unaccessible to date for in vitro characterization. Structure activity relationship studies explained that potent inhibitors of animal DHODH do not significantly affect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools. PMID- 12372627 TI - Swelling of mitochondria in cultured rat hippocampal astrocytes is induced by high cytosolic Ca(2+) load, but not by mitochondrial depolarization. AB - The influence of cytosolic Ca(2+) load and of mitochondrial membrane potential change on mitochondrial morphology was investigated in cultured rat hippocampal astrocytes. The uncoupler FCCP, applied together with oligomycin, depolarized mitochondria rapidly but did not change their morphology. Depolarization was associated with a moderate cytosolic [Ca(2+)](i) rise of up to 0.3 microM. Only high cytosolic Ca(2+) load (above a threshold of 50 microM), which was evoked by application of the ionophore 4-Br-A23187 in Ca(2+)-containing medium, caused drastic change of mitochondrial morphology. The shape change from the typical rod like to a spherical shape, indicating mitochondrial swelling, was associated with depolarization. Cyclosporin A sensitivity suggests involvement of permeability transition. Thus, a dramatic cytosolic [Ca(2+)](i) rise is required to induce mitochondrial swelling and depolarization. A large but still moderate [Ca(2+)](i) rise evoked by physiological stimulation, however, has no comparable effect. PMID- 12372628 TI - Transfection of a phosphatidyl-4-phosphate 5-kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and alpha-adrenergic agonists. AB - GIRK (G protein-activated inward-rectifying K(+) channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with betagamma subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P(2) levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl-4-phosphate 5-kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M(2) receptors but completely abolished inhibition of guanosine triphosphate-gamma-S activated current by endothelin-1 or alpha-adrenergic agonists. We conclude that though PtIns(4,5)P(2) is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by alpha(1A) receptors and endothelin-A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P(2). PMID- 12372629 TI - Thrombopoietin acts synergistically with LIF to maintain an undifferentiated state of embryonic stem cells homozygous for a Shp-2 deletion mutation. AB - Thrombopoietin (Tpo) and its receptor, c-mpl, are expressed in murine embryonic stem (ES) cells. ES cells are maintained in a pluripotent state by leukemia inhibitory factor (LIF) via activation of the Janus kinase (Jak)-STAT3 signaling pathway. Tpo, like LIF, activates STAT3. We report that Tpo increases the number of undifferentiated colonies derived from wild type or Shp-2 mutant (Shp 2(Delta46-110)) ES cells. Tpo plus LIF acted synergistically on the Shp-2(Delta46 110) ES cells to maintain undifferentiated colonies but no evidence of synergism via Jak-STAT3 activation was detected. Collectively, these data suggest that Tpo can play a role in preventing ES cell differentiation via Jak-STAT3 activation and perhaps via novel pathways that are enhanced in the absence of functional Shp 2. PMID- 12372630 TI - Ethacrynic acid rapidly and selectively abolishes blood flow in vessels supplying the lateral wall of the cochlea. AB - The mechanisms underlying the ototoxicity of ethacrynic acid (EA) are not fully understood. Previous studies have focused on morphologic and enzymatic changes in the stria vascularis. The current experiment shows that one of the earliest effects of EA is ischemia, resulting from impaired blood flow in vessels supplying the lateral wall of the cochlea. Inner ear microcirculation, endocochlear potentials, compound action potentials (CAP), cochlear microphonics (CM) and summating potentials (SP) were monitored over time in chinchillas following a single injection of EA (40 mg/kg i.v.). At all times after EA injection, blood vessels supplying the spiral lamina, modiolus, and vestibular end organs appeared normal. In contrast, lateral wall (spiral ligament and stria vascularis) vessels were poorly stained with eosin 2 min after EA injection, and devoid of red blood cells at 30 min post EA. Decline, but not recovery, of CAP, CM and SP followed the microcirculation changes in the lateral wall. Reperfusion was delayed in stria vascularis arterioles relative to other lateral wall vessels. The ischemia-reperfusion caused by EA would be expected to generate large quantities of free radicals, which may trigger or contribute to the cellular, enzymatic, and functional pathologies that have been described in detail previously. PMID- 12372631 TI - An immunogold investigation of the distribution of calmodulin in the apex of cochlear hair cells. AB - Calmodulin is found in the mechanosensitive stereociliary bundle of hair cells where it plays a role in various calcium-sensitive events associated with mechanoelectrical transduction. In this study, we have investigated the ultrastructural distribution of calmodulin in the apex of guinea-pig cochlear hair cells, using post-embedding immunogold labelling, in order to determine in more detail where calmodulin-dependent processes may be occurring. Labelling was found in the cuticular plate as well as the hair bundle, the rootlets of the stereocilia being more densely labelled than the surrounding filamentous matrix. In the bundle, labelling was found almost exclusively at the periphery rather than over the centre of the actin core of the stereocilia, and was clearly associated with the attachments of the lateral links that connect them to their nearest neighbours. It was also found to be denser towards the tips of stereocilia compared to other stereociliary regions and occurred consistently at either end of the tip link that connects stereocilia of adjacent rows. The contact region between stereocilia that is found just below the tip link was also clearly labelled. These concentrations of labelling in the bundle are likely to indicate sites where calmodulin is associated with calcium/calmodulin-sensitive proteins such as the various myosin isoforms and the plasma membrane ATPase (PMCA2a) that are known to occur there, and possibly with the transduction channels themselves. At least one of the myosin isoforms, myosin 1c, is thought to be associated with slow adaptation, and PMCA2a with control of calcium levels in the bundle. The concentration of calmodulin in the contact region further supports the suggestion that this is a functionally distinct region rather than a simple geometrical association between adjacent stereocilia. PMID- 12372632 TI - Addition of noise enhances neural synchrony to amplitude-modulated sounds in the frog's midbrain. AB - The ability of 109 single units in the midbrain acoustic centre of frogs (Rana ridibunda, Rana temporaria) to reproduce 10%, 20 Hz sinusoidal amplitude modulation of a long-duration characteristic frequency tone was studied. The sinusoidal modulation was presented either in isolation or summed with a low frequency (0-50 Hz) noise. Recordings were obtained in the adapted state. The magnitude of the 20 Hz periodic response component was estimated by means of the synchronisation coefficient and the amplitude of the sine modulation of the instantaneous spike rate. In many units, addition of noise modulation produced considerable enhancement of both the mean discharge rate and the discharge rate synchronised to the 20 Hz amplitude modulation. This enhancement phenomenon is interpreted in the context of stochastic resonance theory. PMID- 12372633 TI - Modeling the relation between head orientations and otolith responses in humans. AB - We have performed a finite element simulation of realistic displacements of otolith membranes by static linear accelerations. The simulations were based on accurate measurements of the surfaces of human utricular and saccular maculae, which indicate a clear curvature of these surfaces. The results show that this curvature, a feature probably found in all mammals, has no effect on the mechanics of the structure as a whole since the elastic coupling in the otolith membrane is insufficient. Hair cell excitations on any place of the macula are only affected by the local orientation of the macula with respect to acceleration. Based on the displacements of the otolith membrane, we also calculated the induced activation patterns on the otolith epithelia. These patterns provide for the first time a complete image of peripheral otolith activity. The individual activation patterns at selected locations on the macula correspond well with single cell recordings of actual peripheral otolith neurons. PMID- 12372634 TI - Ultrastructural transynaptic effects of unilateral cochlear ablation in the gerbil medial superior olive. AB - This study investigated the long-term effects of unilateral hearing loss on the structure of synapses within the gerbil medial superior olivary (MSO) nuclei. Five animals had complete (surgical) left cochlear ablation at postnatal day 18. Previous studies have shown this to produce, within 3 days, significant transneuronal atrophy in the left dendritic field of both MSOs. Electron micrographs from sagittal ultrathin sections through the MSOs of the cochlear ablated animals were compared to those from unoperated normals. Qualitatively, the ultrastructural features were similar. Most of the axodendritic terminals were R-type (round-type vesicles, putative excitatory) whereas, in the central part of the nucleus, predominated by neuron soma profiles, terminals of P- and F type (pleomorphic- and flattened-type vesicles, putative inhibitory) were present in equal numbers with R-type terminals. F-type terminals were infrequent and occurred most around lateral parts of the MSO somata. These three types of terminals seen around the somata and proximal dendrites all had extended profiles with multiple, discontinuous appositions. Quantitative analysis revealed that R type axodendritic terminals became smaller and less densely populated with vesicles where they synapsed onto the remaining dendrites arrayed towards the ablated side of both MSOs, and axosomatic P-type afferent terminals were smaller in the contralateral nuclei. A significant reduction in the number of terminals and synapses occurred in the central, somatic, region of the ipsilateral MSO. However, the terminal vesicle concentration in the remaining terminals increased. The results indicate that cochlear ablation can induce transynaptic reduction in the size of afferent axon terminals within the MSO, and alter their vesicle concentration. These changes are likely to affect the probability of transmitter release and thus influence their signaling power within the nucleus. PMID- 12372635 TI - Localization of biotinidase in the brain: implications for its role in hearing loss in biotinidase deficiency. AB - Biotinidase deficiency is an autosomal recessively inherited disorder characterized by neurological and cutaneous features, including sensorineural hearing loss. Although many of the features of the disorder are reversible following treatment with biotin, the hearing loss appears to be irreversible. To better characterize the nature of the hearing loss in this disorder, location of the expression and presence of biotinidase within the brain was examined using Northern blot analysis, in vitro hybridization of a cDNA panel, and immunohistochemical staining. Results indicate low, but detectable expression of biotinidase throughout the brain, but increased concentrations of biotinidase within the dorsal cochlear nucleus, ventral cochlear nucleus, and superior olivary complex of the brainstem, as well as, in the hair cells and spiral ganglion of the cochlea. These findings suggest that biotinidase and possibly biotin plays an important role in hearing. PMID- 12372636 TI - Perception of the periodicity strength of complex sounds by the chinchilla. AB - The perception of periodicity strength was studied in chinchillas using a stimulus generalization paradigm in an operant-conditioning, positive reinforcement behavioral task. Stimuli consisted of cosine-phase and random-phase harmonic complex tones, infinitely iterated rippled noises, and wideband noise. These stimuli vary in periodicity strength as measured by autocorrelation functions and are known to generate a continuum in the perception of pitch strength in human listeners. Chinchillas were trained to discriminate a cosine phase harmonic tone complex from wideband noise and tested in the generalization paradigm using random-phase tone complexes and iterated rippled noises as probe stimuli. Chinchillas were tested in three different conditions in which the periods of the fundamental frequencies of the tone complexes were fixed at 2 ms, 4 ms, or 8 ms. Behavioral responses obtained from chinchillas were related to stimulus periodicity strength. For most animals, the behavioral responses to random-phase tone complexes were smaller than those to cosine-phase tone complexes. The behavioral responses were analyzed in terms of the Auditory Image Model of Patterson et al. [Patterson, R.D., Allerhand, M.H., Giguere, C., J. Acoust. Soc. Am. 98 (1995) 1890-1894], and the results suggest that the periodicity information in the stimulus envelope has a large influence in controlling the behavioral response of the chinchilla. Comparison of the generalization data obtained in the present study to magnitude estimation data obtained previously in human subjects suggests a greater influence of stimulus envelope for the perception of periodicity strength in chinchillas than for the perception of pitch strength in human listeners. PMID- 12372638 TI - Axonal injury in auditory nerve observed in reversible latency changes of brainstem auditory evoked potentials (BAEP) during cerebellopontine angle manipulations in rats. AB - Intraoperative monitoring of brainstem auditory evoked potentials (BAEP) has been widely utilized to reduce the incidence of postoperative hearing disturbance due to cerebellopontine angle manipulations. The prolongation of wave V of BAEP is usually used as a criterion to warn the surgeons to modify their surgical maneuvers. However, it is not known whether all neuropathological changes are avoided if BAEP latency intraoperatively returns to the baseline level or some neuropathological changes 'silently' occur even if BAEP normalizes. The aim of this study was to experimentally clarify this point that would be important for the long-term prognosis of patients' hearing. The cerebellopontine angle portion of the auditory nerve was quantitatively compressed in the rats and reversible prolongation of BAEP latency was reproduced just as it occurs during surgery in humans. Twenty-four hours after the compression, the auditory nerve was removed for beta-APP immunostaining to investigate the degree of axonal injury. The results of the present study disclosed that axonal injury occurred even in the cases where the intraoperative normalization of prolonged wave IV (equivalent to wave V in humans) latency had been obtained. Therefore, the interpretation of BAEP changes based only on the prolongation of the latency of BAEP was not enough to prevent the auditory nerve from developing morphological changes. Changes in the amplitude of wave V of BAEP appears to be more sensitive than its latency change as an intraoperative indicator for axonal injury in the auditory nerve. PMID- 12372637 TI - Immunohistochemical localization of calcium-binding proteins in the brainstem vestibular nuclei of the jaundiced Gunn rat. AB - Vestibular gaze and postural abnormalities are major sequelae of neonatal hyperbilirubinemia. The sites and cellular effects of bilirubin toxicity in the brainstem vestibular pathway are not easily detected. Since altered intracellular calcium homeostasis may play a role in neuronal cell death, we hypothesized that altered expression of calcium-binding proteins may occur in brainstem vestibular nuclei of the classic animal model of bilirubin neurotoxicity. The expression of the calcium-binding proteins calbindin-D28k and parvalbumin in the brainstem vestibular pathways and cerebellum of homozygous recessive jaundiced (jj) Gunn rats was examined by light microscopy and immunohistochemistry at 18 days postnatally and compared to the findings obtained from age-matched non-jaundiced heterozygous (Nj) littermate controls. Jaundiced animals exhibited decreased parvalbumin immunoreactivity specifically in synaptic inputs to superior, medial, and inferior vestibular nuclei, and to oculomotor and trochlear nuclei, whereas the neurons retained their normal immunoreactivity. Jaundiced animals also demonstrated a decrease in calbindin expression in the lateral vestibular nuclei and a paucity of calbindin-immunoreactive synaptic endings on the somata of Deiters' neurons. The involved regions are related to the control of the vestibulo-ocular and vestibulospinal reflexes. Decreased expression of calcium binding proteins in brainstem vestibular neurons may relate to the vestibulo ocular and vestibulospinal dysfunction seen with clinical kernicterus, and may provide a sensitive new way to assess bilirubin toxicity in the vestibular system. PMID- 12372639 TI - Distortion product otoacoustic emissions in frogs: correlation with middle and inner ear properties. AB - Four frog species, Rana pipiens, Scaphiopus couchii, Xenopus laevis and Bombina orientalis, were examined for distortion product otoacoustic emissions (DPOAE). These species were chosen for their diverse otic morphologies. Rana has a well developed caudal extension of the amphibian papilla within the inner ear; Scaphiopus, Xenopus and Bombina do not. Rana and Scaphiopus have a tympanic middle ear, Xenopus has a subcutaneous tympanic disk and Bombina has only an operculum. DPOAEs were present in Rana and Xenopus, with amplitudes up to 55 and 20 dB SPL, respectively. DPOAEs could be detected in neither Scaphiopus nor Bombina. These results show that (1) a well-developed caudal extension is not necessary for generation of DPOAEs, and (2) a tympanic middle ear is neither required nor sufficient to have DPOAEs. PMID- 12372640 TI - Expression and localization of heat shock factor (Hsf) 1 in the rodent cochlea. AB - Activation of heat shock factors (Hsfs) is one of the potential mechanisms for regulating the transcription of the heat shock proteins (Hsps) and certain other stress-responsive genes. Reverse transcription polymerase chain reaction (RT PCR), Western blot and immunocytochemistry were used to examine the expression and localization of Hsf1, the stress-responsive member of the Hsf family, in the rat and mouse cochlea. Cerebellum was used as a positive control. Semi quantitative RT-PCR of cochlear RNA revealed that Hsf1 was more highly expressed in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus, with the alpha splice form predominant over the beta in both subfractions. Immunocytochemistry showed selective staining in the rodent cochlea. Hsf1 immunostaining was found in the nuclei of inner and outer hair cells in the organ of Corti, spiral ganglion cells in the modiolus, and cells in the marginal and intermediate layers of the stria vascularis. This is largely consistent with where Hsp70 induction is reported. Hsf1 activation following heat shock was examined by Western blot. Hyperthermia resulted in stress-induced Hsf1 hyperphosphorylation in cochlea as well as cerebellum. This hyperphosphorylation as well as the correlation of its localization with Hsp70 induction supports a role for Hsf1 in the cochlear stress response. PMID- 12372641 TI - Representation of whispered word-final stop consonants in the auditory nerve. AB - Recent physiological results from the auditory nerve suggest that specific response patterns for word-initial /d/ and /t/ are present across acoustic variations. In this study, single cell recordings from the auditory nerve of anesthetized chinchillas in response to the stop consonants /d/ and /t/ presented in a variety of acoustic contexts were analyzed. Consonants had variable word positions, vowel contexts, types of phonation, and speakers. The response patterns from individual auditory nerve fibers did not reliably differentiate the consonants /d/ and /t/. Global average peristimulus time histograms (GAPSTs) contained invariant patterns for all tokens of each word-final consonant, regardless of context. Ensemble responses to word-final consonants had similarities in their temporal patterns to those in GAPSTs for word-initial consonants. The similar representations in the ensemble auditory nerve response for consonants with different acoustic content suggest a possible substrate for perceptual normalization. Both invariant and variable elements of speech can be computed from the ensemble response of the auditory nerve. PMID- 12372642 TI - The origin of the 900 Hz spectral peak in spontaneous and sound-evoked round window electrical activity. AB - We have monitored the spectrum of the (spontaneous) neural noise at the round window (RW) and on the surface of the antero-ventral cochlear nucleus (CN) and the dorsal CN (DCN) of anaesthetised guinea pigs. We have also obtained the average gross extracellular waveform evoked by 20 kHz tone-bursts (0.25 ms and 25 ms) at each of these recording sites, and calculated the spectrum of the average waveforms (SAW). With these tone-bursts, only a small population of neurones in the extreme basal turn of the cochlea near the RW electrode responds, presumably with only a single action potential for each 0.25 ms tone-burst. The RW waveforms recorded between 20 dB and 60 dB SPL were very similar, and are therefore presumably a simple estimate of the shape of the contribution of the firing of a single neurone to the gross RW signal (the unitary potential or UP). In normal animals, the SNN and the SAW were remarkably similar, with peaks at 900 Hz and at 2400 Hz, suggesting that they are not due to neural synchronisation (as suggested previously by others), but are due to an oscillatory waveform produced by each single fibre action potential. Abolition of all spike activity by RW tetrodotoxin left a waveform with only a summating potential and a dendritic potential, and no 900 Hz peak in the SAW or SNN, indicating that the spectral peak is due to neural spiking only. Abolition of the CN contribution to the RW waveforms by CN application of lignocaine or sectioning of the cochlear nerve at the internal meatus (by focal aspiration of the DCN and underlying cochlear nerve) showed that the 900 Hz peak was not simply due to the addition of a delayed and inverted CN contribution: mathematical modelling shows that this would produce a broad spectral peak at about 1200 Hz. Moreover, the 900 Hz spectral peak remains after complete abolition of the CN contribution, although reduced in amplitude. This residual 900 Hz peak can be traced to an oscillation in the gross waveform due to the presence of two peaks (P(1)* and N(2)*) which follow the intact N(1) peak. The P(1)* and N(2)* peaks were present at the RW, but not at the cochlear nerve as it exits the internal meatus, suggesting that they were not due to double spiking of some of the neurones, but were probably due to a sub-threshold electrical resonance in the peripheral dendrites. We have successfully modelled the production of the SNN and the compound action potential and SAW in response to 0.25 ms and 25 ms tone-bursts at 20 kHz by including only a damped 900 Hz resonance in the UP, without refractory effects, preferred intervals or synchronisation in the timing of neural spike generation. Such resonances in other neurones are known to be due to the activation kinetics of the voltage controlled sodium (Na(+)) channels of these neurones. The presence of such sub threshold oscillations probably indicates that the peripheral dendrites are devoid of stabilising potassium (K(+)) channels. We also discuss the role of this membrane resonance in generating burst-firing of the cochlear nerve (as with salicylate) and the role of such burst-firing in generating tinnitus. PMID- 12372643 TI - Outer hair cells functionally and structurally deteriorate during reperfusion. AB - Transient ischemia of the cochlea was induced in 65 albino guinea pigs by pressing the labyrinthine artery, and the effects of cochlear reperfusion on cochlear potentials (endocochlear potential, compound action potential and cochlear microphonics (CM)) and structural changes in hair cells were examined. Although 15 min ischemia did not elevate the post-ischemic CM pseudo-threshold as compared with the pre-ischemic value, ischemia of 30 min or longer significantly elevated the CM pseudo-threshold. CM amplitude tended to progressively decrease during the reperfusion period in the animals subjected to 45 or 60 min ischemia. After transient ischemia, outer hair cells (OHCs) were swollen and exhibited alterations of the nucleus. Severer structural deterioration of OHCs was induced by 4 h reperfusion than ischemia itself when the ischemic period was 45 or 60 min. Perilymphatic perfusion of dimethylthiourea, a hydroxyl radical scavenger, partially ameliorated the elevation of the CM pseudo-thresholds and the structural changes of OHCs. These results indicate that cochlear reperfusion induces functional and structural deterioration of OHC probably by hydroxyl radical generation. PMID- 12372644 TI - Antioxidant status and hearing function in noise-exposed workers. AB - The cellular antioxidant system appears to protect cochlear hair cells from oxidative stress due to noise and aging. The role of individual metabolic variables remains poorly understood, however. We examined the role of a number of metabolic factors on human cochlear function in noise-exposed individuals. In 58 factory workers we measured audiometry and distortion product otoacoustic emissions prior to a workshift. Simultaneously we measured levels of vitamin E, vitamin C, and polymorphism status for two metabolic genes related to glutathione S-transferase function (GSTM1 and GSTT1). Age and total noise exposure were predictive of hearing status. Vitamin E levels were negatively correlated with hearing function, and this effect was partly explained by an increase in vitamin E levels with age. No effect was found for vitamin C. Individuals possessing the GSTM1 gene had significantly better high frequency otoacoustic emissions compared to GSTM1 null individuals. The protective effect of GSTM1 was present even after adjusting for age, race, sex, and years of noise exposure. GSTT1 did not exhibit a similarly protective effect. While the cross-sectional nature of the study precludes drawing conclusions about causation, these data suggest that GSTM1, an antioxidant enzyme which is found in the mammalian cochlea, may play a protective role in humans against hair cell damage due to noise or aging. PMID- 12372645 TI - Changes in cat primary auditory cortex after minor-to-moderate pure-tone induced hearing loss. AB - In this paper we present findings in the primary auditory cortex of cats exposed for 2 h to a 115 dB SPL, 6 kHz tone at 36 days, 56 days or 118 days after birth. We evaluate the effects of age at exposure, amount of hearing loss, and time after induction of trauma on the functional reorganization of the cortical tonotopic map. We found a fairly sharp demarcation in the amount of hearing loss (20-25 dB) that caused cortical reorganization. For localized hearing losses, unmasking of excitatory contributions of neighboring frequency regions was found. For cats showing reorganization of the tonotopic map, the frequency-tuning curve bandwidth at 20 dB above threshold at CF (BW(20dB)) increased with increasing threshold at CF. Threshold at CF, and BW(20dB) increased with time after exposure. Minimum spike latency was initially increased, but subsequently decreased with time after exposure at a rate that was two times faster in cats with reorganized cortex than in cats with normal tonotopic maps, to reach the same asymptotic value. Thresholds at CF were correlated with the peripheral hearing loss at near CF frequencies as estimated from ABR measurements. The correlation between BW(20dB) and CF threshold suggests that part of the reorganization could be due to 'residual' sensitivity of the high frequency neurons to not-affected lower or higher frequencies. However, for CFs above 6 kHz, the BW(20dB) for cats with reorganization of the tonotopic map was significantly lower (on average 0.3 octave, P<0.05) than for cats with normal tonotopic maps. This is not what one would expect in cases of pseudo-plasticity characterized by concurrent shifts in BW(20dB) and CF as a result of residual sensitivity to lower frequencies. PMID- 12372646 TI - Gene transfer into supporting cells of the organ of Corti. AB - To utilize the rapidly accumulating genetic information for developing new therapeutic technologies for inner ear disease, it is necessary to design technologies for expressing transgenes in the inner ear, especially in the organ of Corti. We examined the outcome of an adenovirus gene transfer into the organ of Corti via the scala media in guinea pigs. The transgene insert is the bacterial lacZ gene driven by a cytomegalovirus promoter. We demonstrate that the inoculation is detrimental to the hair cells that surround the site of inoculation, but the supporting cells in the organ of Corti survive and retain the ability to express the reporter transgene beta-gal. The ability to deliver transgenes that are expressed in the supporting cells is an important step in the development of clinically applicable treatments that involve hair cell regeneration. PMID- 12372647 TI - Local administration of antioxidants to the inner ear. Kinetics and distribution(1). AB - Round window (r.w.) administration of drugs involves the delivery of medication directly into the inner ear via the r.w. membrane, avoiding a systemic effect of the therapy. Earlier experimental studies suggest that a number of antioxidants and scavengers hold promise for ameliorating the tissue damaging capacity of reactive oxygen species in some acquired cochlear disorders. D-Methionine and thiourea are two small sulfur-containing molecules with an antioxidative and scavenging effect. The passage through the r.w. of radioactive D-methionine and thiourea administered by 1 h infusion to the r.w. was studied in a rat model. Levels of the tracers were measured in scala tympani perilymph (PLT) 17-254 min after r.w. administration. Both tracers pass the r.w. membrane readily. Peak levels were found in the earliest taken samples after the administration. The radioactivity in PLT of the basal turn reached a peak to about 1.5-1.9% of the irrigating medium radioactivity. Following the r.w. administration, the concentration of radioactive D-methionine and thiourea declined with a terminal half-life of 0.57 and 0.77 h, respectively. The distribution of the tracers at the cellular level was analyzed by autoradiography. The most intense expression was found in the lateral wall of the cochlea. It can be postulated that local delivery to the cochlea of D-methionine and thiourea via the r.w. gives high local concentrations of the substances in PLT in the basal turn. PMID- 12372648 TI - Children, stress, and sensitization: an integration of basic and clinical research on emotion? PMID- 12372649 TI - Maternal stress beginning in infancy may sensitize children to later stress exposure: effects on cortisol and behavior. AB - BACKGROUND: Preclinical studies demonstrate that the neonatal environment can permanently alter an individual's responses to stress. To demonstrate a similar phenomenon in humans, we prospectively examined the relationships of maternal stress beginning in infancy and concurrent stress on preschoolers' hypothalamic pituitary-adrenal activity and later mental health symptoms. METHODS: Salivary cortisol levels were assessed in 282 4.5-year-old children and 154 of their siblings. Maternal reports of stress were obtained when the children were ages 1, 4, and 12 months, and again at 4.5 years. Children's mental health symptoms were assessed in first grade. RESULTS: A cross-sectional analysis revealed that preschoolers exposed to high levels of concurrent maternal stress had elevated cortisol levels; however, a longitudinal analysis revealed that concurrently stressed children with elevated cortisol also had a history of high maternal stress exposure in infancy. Importantly, children exposed only to high levels of concurrent or early stress had cortisol levels that did not significantly differ from those never exposed to stress. Further analysis of the components of stress indicated that maternal depression beginning in infancy was the most potent predictor of children's cortisol. We also found that preschoolers with high cortisol levels exhibited greater mental health symptoms in first grade. CONCLUSIONS: These results link the findings of preclinical studies to humans by showing that exposure to early maternal stress may sensitize children's pituitary adrenal responses to subsequent stress exposure. PMID- 12372650 TI - Smaller prefrontal and premotor volumes in boys with attention deficit/hyperactivity disorder. AB - BACKGROUND: Anatomic magnetic resonance imaging (MRI) studies of attention deficit/hyperactivity disorder (ADHD) have been limited by use of callosal rather than sulcal/gyral landmarks in defining cerebral lobes and functionally relevant sublobar regions (e.g., prefrontal cortex). We present an investigation of cerebral volumes in ADHD using a Talairach-based approach that uses cortical landmarks to define functionally relevant regions. METHODS: Volumes were compared between groups of 12 boys with ADHD and 12 age- and gender-matched control subjects, using a series of multiple analyses of variance. RESULTS: Boys with ADHD had (on average) 8.3% smaller total cerebral volumes. Significant reductions in lobar volumes were seen only for the frontal lobes. Within the frontal lobes, a reduction was seen in both gray and white matter volumes, with some evidence suggesting lateralization of these findings: reduction in frontal white matter volume was specific to the left hemisphere; there was a bilateral reduction in frontal gray matter volume but more so in the right hemisphere. Subparcellation of the frontal lobe revealed smaller prefrontal, premotor, and deep white matter volumes. CONCLUSIONS: Findings suggest that ADHD is associated with decreased frontal lobe gray and white matter volumes. More than one subdivision of the frontal lobes appears to be reduced in volume, suggesting that the clinical picture of ADHD encompasses dysfunctions attributable to anomalous development of both premotor and prefrontal cortices. PMID- 12372651 TI - Effects of event probability and sequence on children with attention deficit/hyperactivity, reading, and math disorder. AB - BACKGROUND: We investigated the impact of stimulus probability and sequence on performance and event-related potentials of 310 children classified into 12 combinations of attention-deficit/hyperactivity disorder (Not-attention deficit/hyperactivity disorder, Inattentive and Combined subtypes) with presence/absence of reading disorder and math disorder. METHODS: Subjects pressed buttons to displays of the letters O and X, which were presented with probabilities of either .17/.83 or .50/.50. Greater response selection was required in the .17/.83 condition. RESULTS: Stimulus probability had comparable effects on all diagnostic groups. The extent of mismatch between a stimulus and preceding events elicited less systematic increases in errors, P3b latency, and P3b amplitude among both attention-deficit/hyperactivity disorder subtypes than controls. Mismatch with preceding trials more greatly reduced math disorder and reading disorder + math disorder children's speed in the Rare task and accuracy in both conditions. Math disorder and reading disorder + math disorder subjects also registered less the effects of alternations of the infrequent O on N2 amplitude and on P3b latency. CONCLUSIONS: Math disorder and reading disorder + math disorder youngsters' lower sensitivity to sequence irregularity in their event-related potentials along with greater disruption of performance suggest working memory deficits that adversely affected response selection. Comorbidity of attention-deficit/hyperactivity disorder and reading disorder did not affect the results. PMID- 12372652 TI - Glutamic acid decarboxylase 65 and 67 kDa proteins are reduced in autistic parietal and cerebellar cortices. AB - BACKGROUND: A limited number of reports have demonstrated abnormalities involving the glutamate and gamma amino butyric acid systems in blood and platelets of subjects with autism. To further investigate these studies, brain levels of rate limiting enzyme, glutamic acid decarboxylase, which is responsible for normal conversion of glutamate to gamma amino butyric acid in the brain, were investigated. METHODS: Postmortem cerebellar and parietal cortices of age (mean +/- SD for controls 23 +/- 4.2, autistic 25.2 +/- 5.2 cerebellum; controls 23.5 +/- 4.8, autistic 21.6 +/- 3.8 parietal cortex), gender and postmortem interval matched autistic and control subjects (n = 8 control, n = 5 autism, cerebellum; n = 4 control, n = 5 autism, parietal cortex) were subjected to SDS-PAGE and western blotting. Brain levels of glutamic acid decarboxylase proteins of 65 and 67 kDa and beta-actin were determined. RESULTS: Glutamic acid decarboxylase protein of 65 kDa was reduced by 48% and 50% in parietal and cerebellar (p <.02) areas of autistic brains versus controls respectively. By the same token, glutamic acid decarboxylase protein of 67 kDa was reduced by 61% and 51% in parietal (p <.03) and cerebellar areas of autistic brains versus controls respectively. Brain levels of beta-actin were essentially similar in both groups. CONCLUSIONS: The observed reductions in glutamic acid decarboxylase 65 and 67 kDa levels may account for reported increases of glutamate in blood and platelets of autistic subjects. Glutamic acid decarboxylase deficiency may be due to or associated with abnormalities in levels of glutamate/gamma amino butyric acid, or transporter/receptor density in autistic brain. PMID- 12372653 TI - Monoamine oxidase inhibition during brain development induces pathological aggressive behavior in mice. AB - BACKGROUND: Monoamine oxidase (MAO) is historically a focus of concern in research on impulsive and aggressive behavior. Recent studies in a single kindred with a point mutation in the MAO-A gene, together with phenotypic evaluations of MAO-A knockout mice, have sharpened this interest. The goal of this study was to investigate the behavioral consequences of MAO inhibition during brain development and to determine the extent to which specific effects could be attributed to MAO- A versus MAO-B. METHODS: MAO-A and B inhibitors were administered, separately or in combination, during gestation and lactation. Behavioral evaluations included neurologic testing, delay of rewarded response, and the resident-intruder aggression paradigm, conducted before and after an acute pharmacologic challenge. RESULTS: Total prenatal MAO inhibition produced a pervasive increase in aggressive behavior, whereas MAO-B inhibited mice demonstrated a similar pattern of lower intensity. Aggression was elevated in MAO A inhibited mice only after acute pharmacologic challenge, suggesting prenatal sensitization. CONCLUSIONS: Developmental inhibition of MAO activity engenders behavioral effects that parallel those observed in animals with genetic ablation of MAO function. These data underscore the importance of neurochemical changes during development and provide a possible model for disinhibited aggression, common in clinical populations. PMID- 12372654 TI - Correlations between peripheral polyunsaturated fatty acid content and in vivo membrane phospholipid metabolites. AB - BACKGROUND: There is evidence for membrane abnormalities in schizophrenia. It is unclear whether the observed membrane deficits in peripheral cells parallel central membrane phospholipid metabolism. To address this question we examined the relations between red blood cell polyunsaturated fatty acids and brain phospholipid metabolites from different regions of interest in schizophrenia and healthy subjects. METHODS: Red blood cell membrane fatty acids were measured by capillary gas chromatography and in vivo brain phospholipid metabolite levels were measured using a multi-voxel (31)P Magnetic Resonance Spectroscopy technique on 11 first-episode, neuroleptic-naive schizophrenic subjects and 11 normal control subjects. RESULTS: Both the total polyunsaturated fatty acids and the individual 20:4(n-6) contents were significantly correlated with the freely mobile phosphomonoester [PME(s-tau(c))] levels (r =.5643, p =.0062 and r =.6729, p =.0006, respectively). The 18:2(n-6) polyunsaturated fatty acids content correlated positively with freely-mobile phosphodiester [PDE(s-tau(c))] levels (r =.5573, p =.0071). The above correlations were present in the combined right and left prefrontal region of the brain, while other regions including the basal ganglia, occipital, inferior parietal, superior temporal and centrum semiovale yielded no significant correlations. CONCLUSIONS: Our preliminary data support the association between the decreased red blood cell membrane phospholipid polyunsaturated fatty acids content and the decreased building blocks [PME(s tau(c))] and breakdown products [PDE(s-tau(c))] of membrane phospholipids in the prefrontal region of first-episode, neuroleptic-naive schizophrenic subjects. PMID- 12372655 TI - Beta power in the EEG of alcoholics. AB - BACKGROUND: In this study, the magnitude and spatial distribution of beta power in the resting electroencephalogram (EEG) were examined to address the possibility of an excitation-inhibition imbalance in the central nervous system of alcoholics. METHODS: Log transformed absolute power in the Beta 1 (12.5-16 Hz), Beta 2 (16.5-20 Hz), and Beta 3 (20.5-28 Hz) bands in the eyes-closed EEG of 307 alcohol-dependent subjects and 307 unaffected age- and gender-matched control subjects were compared using a multivariate repeated measures design. Effect of gender, age, and drinking variables was examined separately. RESULTS: Increased Beta 1 (12.5-16 Hz) and Beta 2 (16.5-20 Hz) absolute power was observed in alcohol-dependent subjects at all loci over the scalp. The increase was most prominent in the central region. Increased Beta 3 (20.5-28 Hz) power was frontal in the alcoholics. Age and clinical variables did not influence the increase. Male alcoholics had significantly higher beta power in all three bands. In female alcoholics the increase did not reach statistical significance. CONCLUSIONS: Beta power in all three bands of resting EEG is elevated in alcoholics. This feature is more prominent in male alcoholics. The increased beta power in the resting EEG may be an electrophysiological index of the imbalance in the excitation inhibition homeostasis in the cortex. PMID- 12372656 TI - Early physical and sexual abuse associated with an adverse course of bipolar illness. PMID- 12372658 TI - Non-invasive neuroimaging using near-infrared light. AB - This article reviews diffuse optical brain imaging, a technique that employs near infrared light to non-invasively probe the brain for changes in parameters relating to brain function. We describe the general methodology, including types of measurements and instrumentation (including the tradeoffs inherent in the various instrument components), and the basic theory required to interpret the recorded data. A brief review of diffuse optical applications is included, with an emphasis on research that has been done with psychiatric populations. Finally, we discuss some practical issues and limitations that are relevant when conducting diffuse optical experiments. We find that, while diffuse optics can provide substantial advantages to the psychiatric researcher relative to the alternative brain imaging methods, the method remains substantially underutilized in this field. PMID- 12372659 TI - Magnetic resonance spectroscopic approaches to studying neuronal: glial interactions. AB - In vivo magnetic resonance spectroscopy (MRS) is a noninvasive technique for the measurement of the concentration and synthesis of metabolites in the brain. Application of the state-of-the-art in vivo (13)C and (15)N MRS techniques to studying the synthesis of glutamate and glutamine has revealed that the glutamate glutamine cycle between neurons and glia is a major metabolic flux, with a flux rate of 60%-80% relative to neuronal oxidative glucose metabolism in the resting human cerebral cortex. The MRS studies leading to the quantification of the glutamate-glutamine cycling flux are reviewed here. The advantages and limitations of different strategies are also discussed. PMID- 12372660 TI - Neurocognitive correlates of the COMT Val(158)Met polymorphism in chronic schizophrenia. AB - BACKGROUND: Neurocognitive deficits are recognized as a cardinal feature of schizophrenia, but the determinants of these deficits remain unknown. Recent reports have suggested that a functional polymorphism, Val(158)Met in exon III of the catechol-O-methyltransferase gene, shares approximately 4% variance with performance on the Wisconsin Card Sorting Test. These findings led to suggestions that the catechol-O-methyltransferase polymorphism may exert its effects by modulating prefrontal dopamine function, but few other neurocognitive measures have been examined, leaving open questions about phenotypic specificity. METHODS: We examined the effects of the catechol-O-methyltransferase Val(158)Met polymorphism in 58 individuals with chronic schizophrenia who completed a battery of 15 neurocognitive tests, which were reduced to four reliable neurocognitive domain scores. We examined the effects of genotype on these four domains and on global neurocognitive ability. RESULTS: The Met allele was associated with better performance in the Processing Speed and Attention domain, but not with other domain scores measuring executive and visuoperceptual functions, declarative verbal learning and memory, simple motor ability, or global neurocognitive function. Genotype shared approximately 11% of variance with Processing Speed and Attention scores, and approximately 2% of variance with Wisconsin Card Sorting Test scores. CONCLUSIONS: The findings provide independent support for the hypothesis that the catechol-O-methyltransferase Val(158)Met polymorphism influences neurocognitive function in schizophrenia, and suggest that the functional effects may be expressed on measures of Processing Speed and Attention. This information may prompt reconsideration of the "prefrontal dopamine" hypothesis and invites examination of a broader range of effects in efforts to refine the neurocognitive phenotype that is most relevant to variation in catechol-O-methyltransferase expression. PMID- 12372661 TI - Selective deficits in prefrontal cortical GABAergic neurons in schizophrenia defined by the presence of calcium-binding proteins. AB - BACKGROUND: Postmortem studies have provided evidence for abnormalities of the gamma-aminobutyric acid (GABA)-ergic system in schizophrenia, including deficits of GABA-containing interneurons. The calcium-binding proteins parvalbumin, calbindin, and calretinin can be used as markers for specific subpopulations of cortical GABAergic interneurons. METHODS: Following our previous observation of a reduction in the density of parvalbumin- but not calretinin-immunoreactive cells in the prefrontal cortex (Brodmann area 10) in schizophrenia, we have quantified the laminar density of neurons immunoreactive for the calcium-binding proteins parvalbumin, calbindin, and calretinin in a further prefrontal cortical region (Brodmann area 9) in patients with schizophrenia, bipolar disorder, major depression, and in matched control subjects (each group n = 15). RESULTS: Initial statistical analysis revealed reductions in the total cortical density of parvalbumin- and calbindin- but not calretinin-immunoreactive neurons in schizophrenia relative to control subjects. Further analysis comparing individual laminar densities between groups indicated that, following correction for multiple comparisons, only a reduction in calbindin-immunoreactive neurons in cortical layer II in the schizophrenic group attained statistical significance. CONCLUSIONS: These findings suggest that deficits of specific GABAergic neurons, defined by the presence of calcium-binding proteins, are present in schizophrenia. Trends toward similar reductions are observed in bipolar disorder. PMID- 12372662 TI - Craniofacial dysmorphogenesis in fetally irradiated nonhuman primates: implications for the neurodevelopmental hypothesis of schizophrenia. AB - BACKGROUND: Craniofacial abnormalities arising from gestational disturbances have been documented in some schizophrenic patients. Reduction of thalamic neurons, a key feature of the neuropathology of schizophrenia, could also have a prenatal origin via disruption of thalamic neurogenesis. This study investigates whether craniofacial dysmorphology and thalamic neuron loss might be associated manifestations of a disruption in embryonic development. METHODS: Thalamic neurons were deleted by exposing fetal macaques to x-rays during thalamic genesis (E33-42). Another group of macaques was irradiated after thalamic genesis (E70 81). Body, head, and facial measurements were obtained from the early irradiated (EX), late irradiated (LX), and control animals at adulthood. RESULTS: Head width, distance between outer eye edges, and ear width were smaller in EX macaques compared with control animals. The LX macaques exhibited only reduced ear width compared with control animals. CONCLUSIONS: These findings indicate that certain features of thalamic neuropathology and craniofacial dysmorphogenesis observed in schizophrenic patients may have a common etiology. PMID- 12372663 TI - The effects of nicotine on specific eye tracking measures in schizophrenia. AB - BACKGROUND: The role of neuronal nicotinic receptors in the etiology and pathophysiology of schizophrenia has been suggested by postmortem findings as well as by linkage analysis implicating chromosome 15q14, the region where the alpha-7 nicotinic receptor gene is located. In addition, drug probe studies show that acute nicotine administration reverses sensory gating and eye-tracking deficits associated with the genetic liability for schizophrenia. The purpose of the current study was to examine the effects of acute administration of nicotine on specific measures of smooth pursuit eye movements and visual attention. METHODS: Twenty nine subjects with schizophrenia (15 smokers and 14 nonsmokers), and 26 healthy comparison subjects (15 smokers and 11 nonsmokers) completed testing. The effects of 1 mg of nicotine, administered by nasal spray, on smooth pursuit initiation, pursuit maintenance, and predictive pursuit were examined. RESULTS: Nicotine significantly improved eye acceleration during smooth pursuit initiation in both smoker and nonsmoker patients but had no effects in healthy subjects. The fact that patient initiation eye acceleration in response to nicotine was significantly higher than in healthy subjects suggests that the lack of effect in healthy subjects was not due to ceiling effects. Nicotine significantly improved pursuit gain during maintenance at a target velocity of 18.7 deg/sec. There were no effects of nicotine on visually guided and memory saccades, or visual attention (d' from a continuous performance task). CONCLUSIONS: Nicotine showed differential effects in schizophrenic patients compared to healthy subjects. These effects of nicotine were unlikely the result of differences in vigilance or sustained attention, because saccadic peak velocity, a sensitive measure of vigilance, and continuous performance task measures were not affected by nicotine. These findings are not thought to be an artifact of nicotine withdrawal effects at baseline, because the abstinence period was very short, and there were similar effects of nicotine on initiation in nonsmoker patients. These findings suggest an abnormality in neuronal nicotinic system functioning in schizophrenic patients. PMID- 12372664 TI - Effects of typical and atypical antipsychotics on prepulse inhibition and latent inhibition in chronic schizophrenia. AB - BACKGROUND: Prepulse inhibition and latent inhibition are the two animal paradigms currently dominating neuropharmacological research on attentional deficits in schizophrenia. Both paradigms have been shown to have a reasonable amount of face, predictive, and construct validity, but responsiveness to typical and atypical antipsychotics differs between the two, as indicated by animal and human studies. The relationship between the paradigms in schizophrenic patients is still unclear. METHODS: We tested prepulse inhibition and auditory latent inhibition in a sample of 33 chronic schizophrenic patients medicated either with atypical (n = 17) or typical (n = 16) antipsychotics. RESULTS: Latent inhibition was found to be intact in both patient groups. Prepulse inhibition was intact in the group receiving atypicals, but deficient in the group receiving typicals (at 60 msec lead interval condition). CONCLUSIONS: The direct comparison supports the hypothesis that atypical and typical antipsychotics have different effects on prepulse inhibition than on latent inhibition in schizophrenic patients; however, the results may also be explained by a greater sensitivity of the prepulse inhibition method. Because it is crucial to understand why there are considerable differences between the two paradigms and between human and animal studies, research should focus more strongly on comparative approaches. PMID- 12372665 TI - Dopaminergic abnormalities in amygdaloid nuclei in major depression: a postmortem study. AB - BACKGROUND: A deficiency of mesolimbic dopamine (DA) is a leading candidate for the etiology of certain symptoms of depression (e.g., anhedonia and loss of motivation). Here we show amounts of dopaminergic proteins in the amygdala, a key brain structure involved in the integration of emotions and stress, in subjects with major depression and in psychiatrically normal control subjects. METHODS: The specific binding of [(125)I]RTI 55 to the DA transporter, [(3)H]SCH 23390 to the D1 receptor and [(125)I]epidepride to D2/D3 receptors were measured in the right amygdaloid complex in postmortem brains from 11 subjects with major depression and 11 matched control subjects. RESULTS: The binding of [(125)I]RTI 55 to DA transporter was significantly lower in the basal and central amygdaloid nuclei, whereas the binding of [(125)I]epidepride to D2/D3 receptors was significantly higher in the basal, central, and lateral amygdaloid nuclei in major depression compared with control subjects. No difference in the binding of [(3)H]SCH 23390 to D1 receptors was observed. CONCLUSIONS: Given that DA depletion in rats can induce a reduction in the DA transporter and an upregulation of D2/D3 receptors, our data are consistent with the hypothesis that major depression is associated with a deficiency of mesolimbic DA. PMID- 12372666 TI - Duration mismatch negativity in biological relatives of patients with schizophrenia spectrum disorders. AB - BACKGROUND: One of the most consistent findings in schizophrenia research over the past decade is a reduction in the amplitude of an auditory event-related brain potential known as mismatch negativity (MMN), which is generated whenever a deviant sound occurs in a background of repetitive auditory stimulation. The reduced amplitude of MMN in schizophrenia was first observed for deviant sounds that differ in duration relative to background standard sounds, and similar findings have been observed for sounds that are deviant in frequency. The aim of this study was to determine whether first-degree relatives of schizophrenia patients show a similar reduction in MMN amplitude to duration deviants. METHODS: We measured MMN to duration increments (deviants 100 msec vs. standards 50 msec) in 22 medicated patients with a diagnosis in the schizophrenia spectrum, 17 individuals who were first-degree unaffected relatives of patients, and 21 healthy control subjects. RESULTS: Mismatch negativity amplitude was reduced in patients and relatives compared with control subjects. There were no significant differences between patients and relatives. In contrast, the subsequent positive component, P3a, was larger in relatives compared with patients. CONCLUSIONS: These findings suggest that a reduced MMN amplitude may be an endophenotype marker of the predisposition to schizophrenia. PMID- 12372667 TI - Sex differences in striatal presynaptic dopamine synthesis capacity in healthy subjects. AB - BACKGROUND: There are sex differences in the clinical features of several neuropsychiatric illnesses associated with dopamine dysfunction. The effects of sex on brain dopaminergic function have been sparsely studied in human subjects using modern imaging techniques. We have previously reported that the apparent affinity of [(11)C]raclopride for striatal D(2) dopamine receptors in vivo is lower in women than in men, whereas D(2) receptor density is not different. This finding indirectly suggests that women have a higher synaptic concentration of dopamine in the striatum. We explored further the basis of this phenomenon in an independent study and hypothesized that striatal presynaptic dopamine synthesis capacity would also be elevated in women. METHODS: A total of 23 healthy men and 12 healthy women (age range 20-60 years) were studied using positron emission tomography and [(18)F]fluorodopa. RESULTS: Women had significantly higher striatal [(18)F]fluorodopa uptake (Ki values) than men. The difference was more marked in the caudate (+26%) than in the putamen (+12%). In addition, there was a negative correlation between striatal [(18)F]fluorodopa Ki values and age in men but not in women. CONCLUSIONS: The results further substantiate sex differences in striatal dopaminergic function in humans. This finding may be associated with sex differences in vulnerability and clinical course of neuropsychiatric disorders with dopaminergic dysregulation, e.g., schizophrenia, alcohol dependence, and Parkinson's disease. PMID- 12372668 TI - Focal temporoparietal slow activity in Alzheimer's disease revealed by magnetoencephalography. AB - BACKGROUND: Patients suffering from Alzheimer's disease exhibit more activity in the conventional electroencephalographic delta and theta bands. This activity concurs with atrophy and reduced metabolic and perfusion rates, particularly in temporoparietal structures. METHODS: Whole-head magnetoencephalographic recordings were obtained from 15 patients diagnosed with Alzheimer's disease and 19 healthy control subjects during a resting condition. The generators of focal magnetic slow waves were located employing a single moving dipole model. RESULTS: Dipole density in the delta and theta bands was enhanced in the Alzheimer's disease group compared with healthy control subjects. Slow-wave activity differed significantly between groups in temporoparietal regions of both hemispheres. Right temporoparietal slow-wave activity covaried with cognitive performance, whereas left temporal delta activity varied with a functional status scale. CONCLUSIONS: Our results support the predominant role of the temporoparietal areas in the diagnosis of Alzheimer's disease. Magnetoencephalography and the source analysis of focal slow activity in particular provide interesting and potentially clinically useful tools to assess functional modifications of patients' brain and to evaluate its relationship with the cognitive status. PMID- 12372669 TI - Ambulatory pulse pressure as predictor of outcome in older patients with systolic hypertension. AB - We enrolled 808 older patients with isolated systolic hypertension (160 to 219/71 <95 mm Hg) to investigate whether ambulatory measurement of pulse pressure and mean pressure can refine risk stratification. The patients (> or =60 years) were randomized to nitrendipine (10 to 40 mg/day) with the possible addition of enalapril (5 to 20 mg/day) or hydrochlorothiazide (12.5 to 25 mg/day) or to matching placebos. At baseline, pulse pressure and mean pressure were determined from six conventional blood pressure (BP) readings and from 24-h ambulatory recordings. With adjustment for significant covariables, we computed mutually adjusted relative hazard rates associated with 10 mm Hg increases in pulse pressure or mean pressure. In the placebo group, the 24-h and nighttime pulse pressures consistently predicted total and cardiovascular mortality, all cardiovascular events, stroke, and cardiac events. Daytime pulse pressure predicted cardiovascular mortality, all cardiovascular end points, and stroke. The hazard rates for 10 mm Hg increases in pulse pressure ranged from 1.25 to 1.68. Conventionally measured pulse pressure predicted only cardiovascular mortality with a hazard rate of 1.35. In the active treatment group compared with the placebo patients, the relation between outcome and ambulatory pulse pressure was attenuated to a nonsignificant level. Mean pressure determined from ambulatory or conventional BP measurements was not associated with poorer prognosis. In conclusion, in older patients with isolated systolic hypertension higher pulse pressure estimated by 24-h ambulatory monitoring was a better predictor of adverse outcomes than conventional pulse pressure, whereas conventional and ambulatory mean pressures were not correlated with a worse outcome. PMID- 12372670 TI - Incomplete benefit of antihypertensive therapy on stroke reduction in older hypertensives with abnormal nocturnal blood pressure dipping (extreme-dippers and reverse-dippers). AB - To determine whether the benefits of antihypertensive treatment vary according to dipper status, 811 asymptomatic elderly Japanese hypertensives underwent 24-h ambulatory blood pressure monitoring. During a mean follow-up period of 41 months, 32 stroke events were observed in patients who remained nonmedicated (n = 385), and in 27 patients in the medicated group (n = 426), indicating a 24% lower rate of stroke as a result of antihypertensive therapy. Patients were divided into a white-coat hypertensive (WCHT) group (ambulatory blood pressure <130/80 mm Hg; n = 236) and a sustained hypertensive (SHT) group (n = 575). Sixty-one percent of SHT and 32% of WCHT patients were being medicated. In the SHT group, the stroke rates were 12.4% in nonmedicated and 7.4% in medicated group (P =.04), whereas in the WCHT group the stroke rates were 2.5% in nonmedicated and 1.3% in medicated group (P = not significant). The SHT were further classified according to their nocturnal systolic blood pressure (BP) decrease, as follows: 97 extreme dippers with >20% nocturnal systolic BP decrease; 230 dippers with >10% but <20% decrease; 185 nondippers with >0% but <10% decrease; 63 reverse-dippers with <0% decrease. In the dipping groups of SHT, the stroke rates were similar according to medication versus no-medication in extreme-dippers (12% v 13%), and reverse dippers (23% v 22%), but in nondippers there was a significantly lower rate (by 65%, P =.038) in the medicated (4.4%) than the nonmedicated (13%) groups. In dippers, the stroke rate was also lower in the medicated than the nonmedicated patients (4.7% v 8.8%), a decrease of 47% (P =.217), although the difference was not significant. In conclusion, in older SHT subjects, antihypertensive therapy using clinic BP may be less effective for the groups with extremely abnormal diurnal BP patterns (extreme-dippers and reverse-dippers) than those with relatively normal patterns (dippers and nondippers). Patients with WCHT also showed no benefit. PMID- 12372671 TI - Carotid hemodynamic alterations in hypertensive patients with insulin resistance. AB - BACKGROUND: Insulin resistance (IR) is implicated in the pathogenesis of atherosclerosis. One mechanism is thought to be the impaired vasodilation, which can lead to a reduction in peripheral blood flow. Hypertensive patients with IR have greater intima-media thickness (IMT) in the common carotid artery (CCA) than those without IR. However, the relationship between IR and hemodynamic alterations of the CCA has not been clarified. METHODS: Seventy patients with essential hypertension (EHT) and 11 normotensive controls (NT) participated in this study. The IMT, number of plaques, and internal dimensions of the CCA were evaluated by ultrasound imaging. Mean diastolic (Vd) and systolic (Vs) velocity were determined by the Doppler flow method, and other parameters such as Vd/Vs and the cross-sectional distensibility coefficient (CSDC) were further calculated. When the homeostasis model assessment (HOMA) index exceeded 2.0, the subject was considered to have IR. RESULTS: The IMT was positively correlated with the HOMA index in all subjects. The Vd/Vs and CSDC were significantly decreased in EHT patients with IR compared to NT and EHT patients without IR. The Vd/Vs and CSDC were negatively correlated with the HOMA index. A stepwise regression analysis revealed that age, HDL-cholesterol, and the HOMA index were independently associated with IMT in patients with EHT. Age, the HOMA index, and mean blood pressure (MBP) were independently associated with CSDC, and the first two factors were independently associated with Vd/Vs. CONCLUSIONS: Decreased distensibility of the arterial wall and ensuing low diastolic perfusion are possible mechanisms of atherosclerotic changes in the CCA in EHT patients with IR. PMID- 12372672 TI - Essential hypertension in adolescents: association with insulin resistance and with metabolism of homocysteine and vitamins. AB - BACKGROUND: Although insulin resistance and elevated plasma homocysteine are associated with hypertension in adults, the role of these conditions in the initial phase of hypertension is largely unknown. We examined whether insulin resistance and disturbed homocysteine metabolism are present in young adults at the early stages of essential hypertension. METHODS: We measured physical characteristics, plasma levels of insulin, lipids, total homocysteine, and vitamins in 164 patients with essential juvenile hypertension (median age, 19 years; 92% males) and in 173 controls (median age, 18 years; 66% males). Furthermore, we analyzed the prevalence of six polymorphisms in four genes of the methionine cycle. RESULTS: Patients with hypertension and controls differed significantly (P <.05) in body mass index, levels of insulin, high-density lipoprotein-cholesterol, fasting and post-load plasma homocysteine, and folates. Systolic blood pressure was correlated with homocysteine levels and inversely correlated with plasma folates. Logistic regression showed that fasting homocysteine, vitamin B(12), and low-density lipoprotein-cholesterol were associated with a significantly increased risk of juvenile hypertension. In contrast, the birth length, polymorphism c.2756 A-->G in the MTR gene and plasma folate were associated with a significantly decreased risk of juvenile hypertension. CONCLUSIONS: Our study showed that essential hypertension in adolescents is associated with lower folate and higher homocysteine levels, and with signs of insulin resistance. These data suggest that hypertension in young individuals may be a part of early manifestation of insulin resistance syndrome, and that disturbed folate and homocysteine metabolism may play a role in the early stages of hypertension. PMID- 12372673 TI - Stress-induced hemodynamic responses are associated with insulin resistance in mild hypertensives. AB - BACKGROUND: High blood pressure (BP) and pulse pressure (PP) are recognized as independent risk factors for cardiovascular diseases, whereas insulin resistance (IR) is often associated with hypertension. The purpose of the study was to verify whether PP, taken at the doctor's office and during laboratory stimuli, might be predictive of IR. METHODS: Homeostasis model assessment insulin resistance index (HOMA) was calculated in 75 grade 1 hypertensives (148 +/- 2/92 +/- 1 mm Hg). Then, patients underwent hemodynamic reactivity study, induced by color word stroop, cold pressor, and handgrip tests. Stress response was calculated as total area (value x time) - baseline area (baseline value x time). RESULTS: Patients with similar age, history of hypertension, blood lipids and office blood pressure, but different HOMA (IR-low: 36.3 +/- 1.7 v IR-medium: 62.6 +/- 1.6, P <.001; IR-high: 123.1 +/- 12.8, P <.001 v IR-low and IR-medium), were divided in tertiles. They demonstrated a significant reactivity of systolic BP (IR-low: 225 +/- 58 v IR-medium: 448 +/- 43, P <.01; IR-high: 625 +/- 55, P <.001 v IR-low and P <.01 v IR-medium), PP (IR-low: -8 +/- 19 v IR-medium: 83 +/- 13, P <.001; IR-high: 201 +/- 19, P <.001 v IR-low and IR-medium), and stroke volume (SV) (IR-low: -138 +/- 43 v IR-medium: 1 +/- 27, P <.01; IR-high: 28 +/- 24, P <.001 v IR-low), but similar arterial stiffness (PP/SV) response. Partial correlation between IR and hemodynamic measurements showed a significant association only for systolic BP (0.54, P <.001), PP (0.686, P <.001), and SV (0.384, P <.001) reactivity, but not for office and baseline values. Stepwise multiple regression showed that only PP (beta: 0.634, P <.001) and, among hemodynamic determinants, SV (beta: 0.401, P <.001) response entered into the equation. CONCLUSIONS: The findings demonstrate that increased stress-induced PP, maintained by SV response, is the most predictive hemodynamic variable of reduced insulin sensitivity in mild hypertensives. PMID- 12372674 TI - Effects of enalapril on the vascular wall in an experimental model of syndrome X. AB - Evidence links the insulin resistance syndrome with endothelial dysfunction. Previously, we have described a decreased endothelial nitric oxide synthase (eNOS) activity in both aortic endothelium and cardiac tissue, and an increased proliferation of aortic primary cultured vascular smooth muscle cells (pC-VSMCs), obtained from fructose-fed rats (FFR), an experimental model of syndrome X. Because the participation of the renin-angiotensin system (RAS) in this model is still unclear, the present study examined the effect of chronic administration of an angiotensin converting enzyme (ACE) inhibitor enalapril (E) on pC-VSMCs proliferation and eNOS activity in a conduit artery (aorta) and in resistance vessels (mesenteric vascular bed) from fructose-fed rats. Male Wistar rats were used: Control, FFR, Control + E, and FFR + E (n = 8 in each group). After 8 weeks, tissue samples were obtained and 10% fetal calf serum (FCS) proliferative effect was examined in pC-SMCs of aortic and mesenteric arteries by [(3)H]thymidine incorporation. The eNOS activity was estimated in endothelial lining from both origins by conversion of [(3)H]arginine into [(3)H]citrulline. The FFR aortic and mesenteric pC-VSMCs showed a significantly increased 10% FCS induced [(3)H]thymidine incorporation compared to controls. The FFR aortic and mesenteric endothelium eNOS activity was significantly decreased. Chronic treatment with E abolished the increased proliferation and restored eNOS activity. These data confirm that changes in VSMCs proliferation and endothelial dysfunction at different levels of the vascular system are involved in syndrome X, and that the inhibition of angiotensin II production can revert those changes, suggesting an important role for RAS and possibly kinins, in the physiopathologic mechanism of this model of syndrome X. PMID- 12372675 TI - Ramipril treatment alters Ca(2+) and K(+) channels in small mesenteric arteries from Wistar-Kyoto and spontaneously hypertensive rats. AB - Numerous studies have emphasized the important role of altered Ca(2+) channel function in hypertension. We previously showed that Ca(2+) currents measured in myocytes isolated from both Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) small mesenteric arteries closely correlated with systolic blood pressure (BP) during normal development. The purpose of the present experiments was to determine whether antihypertensive therapy with an angiotensin converting enzyme inhibitor normalizes Ca(2+) channel function in SHR myocytes along with BP. Ramipril (3.5 mg/kg/day) was added to the drinking water of 12-week-old male WKY and SHR for 8 weeks. Segments of small mesenteric arteries were used for isometric contraction studies, and for isolation of myocytes for measurement of Ca(2+) and K(+) currents (I(Ca) and I(K)) by patch clamp methods. Ramipril treatment decreased systolic pressure in WKY and SHR, decreased heart weight and heart weight-to-body weight ratio in SHR, and decreased body weight in WKY. Maximum contractile responses to Bay k 8644 in SMA from ramipril-treated SHR were smaller compared to untreated SHR (10% +/- 2% v 55% +/- 7% of the response to 120 mmol/L KCl). The smaller responses in WKY were not affected by ramipril treatment (11% +/- 4% v 8% +/- 3%). Contractile responses to 10 mmol/L tetraethylammonium (TEA) were not different in untreated versus ramipril-treated SHR (65% +/- 6% v 82% +/- 8%) but were increased in treated WKY (4% +/- 1% v 35% +/- 9%). Ramipril treatment decreased peak I(Ca) and equalized the voltage-dependence of I(Ca) activation between SHR and WKY. The I(K) measured from holding potentials of -60 and -20 mV were significantly smaller in treated SHR and WKY compared to their untreated counterparts, as was the component of I(K) measured in the presence of 100 nmol/L iberiotoxin. These results show that ramipril treatment decreases arterial pressure and Ca(2+) channel function in SHR as expected but unexpectedly also decreases I(K) in both WKY and SHR. These results suggest that angiotensin may have a BP independent effect on ion channel function in arterial smooth muscle. PMID- 12372676 TI - Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species. AB - Hypertension and coronary artery disease are intimately connected. The migration of circulating monocytes into the subendothelial occurs through the expression of some adhesion molecules on endothelial cells. The nuclear factor (NF)-kappaB, a redox-sensitive element, plays a key role in adhesion molecule gene induction. In this study we have compared the effects of two different angiotensin converting enzyme (ACE) inhibitors, one possessing an active sulfhydryl group (zofenopril) and one lacking this group (enalapril) on the cellular redox state (monitored by measuring intracellular reactive oxygen species and thiol status), expression of adhesion molecules, and activation of NF-kappaB in human umbilical vein endothelial cells (HUVECs). Zofenoprilat, the active form of zofenopril, significantly and dose dependently reduced the intracellular reactive oxygen species (ROS) and superoxide formation induced by oxidized low-density lipoprotein (ox-LDL) (P <.001) and tumor necrosis factor-alpha (TNF-alpha) (P <.001). Enalaprilat, the active form of enalapril, was ineffective. Zofenoprilat but not enalaprilat also decreased the consumption of the intracellular GSH induced by ox-LDL (P <.01) and TNF-alpha (P <.01). Although zofenoprilat significantly and dose dependently reduced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), and E-selectin induced by ox-LDL (P <.01) and TNF-alpha (P <.01) on HUVECs, enalaprilat did not. Ox-LDL and TNF-alpha increased the activation of NF-kappaB and the preincubation of HUVECs with zofenoprilat, but not with enalaprilat, dose dependently reduced its activation (P <.001). The conclusion is that the sulfhydryl (SH)-containing ACE inhibitors may be useful in inhibiting foam cell formation and thus slow the development of atherosclerosis. PMID- 12372677 TI - High prevalence of primary aldosteronism using postcaptopril plasma aldosterone to renin ratio as a screening test among Italian hypertensives. AB - The prevalence of primary aldosteronism (PA) was assessed in a specialized hypertension center. Baseline and postcaptopril (50 mg orally) aldosterone to plasma renin activity ratio (A/R) as a screening tool were preliminarily tested in a sample including 22 patients with histories of PA and 53 patients with low renin essential hypertension (EH). Sensitivity and specificity of A/R > or =35 were 95.4% and 28.3% at baseline, compared with 100% and 67.9% after captopril. Using postcaptopril A/R > or =35 and confirmation by acute saline loading, a PA prevalence of 6.3% was found among 1046 consecutive hypertensive patients with normal renal function. Of those 66 PA patients, 16 (24.2%) had a unilateral adenoma, whereas 50 (75.8%) had idiopathic hyperaldosteronism. At presentation, 45.4% of the PA and 16.3% of EH patients were treated with two or more antihypertensive drugs (chi(2) = 33.117, P <.0001). However, among untreated patients (n = 553), the prevalence of mild-to-moderate hypertension (ie, <180/110 mm Hg) was not different between patients with PA and those with EH (70.6% v 76.7%, chi(2) = 0.086, P =.770). Serum potassium > or =3.6 mEq/L was found in 60.6% of PA patients. In conclusion, we observed the following: 1) postcaptopril compared with baseline A/R is a better screening tool for PA; 2) PA is relatively frequent among hypertensive individuals; 3) PA is not necessarily associated with severe hypertension; and 4) hypokalemia is an insensitive screening criterion for PA. PMID- 12372678 TI - Impaired stress-induced pressure natriuresis increases cardiovascularload in African American youths. AB - BACKGROUND: We hypothesized that impaired stress-induced pressure natriuresis increases blood pressure (BP) load. METHODS: The 118 African American youths were brought into similar levels of sodium balance. The protocol consisted of a 2-h baseline period, a 1-h stress period (competitive video games), and a 2-h recovery period. RESULTS: Normal pressure natriuresis (n = 80) resulted from a resistance-mediated (r = 0.23; P <.03) increase in BP (P <.001). In contrast, impaired pressure natriuresis (n = 38), leading to an extended period of elevated BP (P <.05), resulted from a volume-mediated (r = 0.55; P <.002) increase in BP (P <.001). CONCLUSIONS: Impaired stress-induced pressure natriuresis may contribute to the development of essential hypertension, particularly in African Americans. PMID- 12372679 TI - Positron emission tomography, echo-doppler, and exercise studies of functional capacity in hypertensive heart disease. AB - BACKGROUND: This study examines the relationship between functional capacity, left ventricular diastolic function, and myocardial perfusion reserve (MPR) in patients with left ventricular hypertrophy (LVH). METHODS: We studied 16 patients with LVH and 10 controls. Functional capacity was assessed by cardiopulmonary exercise, MPR by positron emission tomography, and left ventricular diastolic function by echo-Doppler. RESULTS: Functional capacity and MPR were significantly lower in the patients. Functional capacity correlated positively with MPR and left ventricular diastolic function. CONCLUSIONS: Diminished functional capacity in patients with hypertension-induced LVH is related to the impairment in MPR and left ventricular diastolic function. PMID- 12372680 TI - Antihypertensive effects of angiotensin converting enzyme inhibition by lisinopril in post-transplant patients. AB - BACKGROUND: It is not known whether strict control of blood pressure (BP) in mild post-transplant hypertension gives any benefit. Our primary objective was to test the antihypertensive effects of lisinopril added to standard therapy on ambulatory BP (ABP) of post-transplant patients. The secondary objective was to monitor echocardiographic and hemodynamic end points. METHODS: Post-transplant patients with an abnormality of the 24-h ABP recording were recruited to this double-blind randomized prospective study that started 2 to 3 months after transplantation. Patients were then evaluated at 6, 12, 18, and 24 months after transplantation. RESULTS: Lisinopril decreased the clinic BP and ABP, the latter from 134/85 to 126/82 mm Hg at 6 months (P =.01 v placebo) and 121/79 mm Hg after 2 years (P =.03 v placebo). Fewer patients in the lisinopril group required added amlodipine to control the BP (P =.01). Data on left ventricular (LV) mass are difficult to interpret because by coincidence in this small study, the lisinopril group had lower initial values than placebo. However, in the lisinopril group mean LV mass decreased by 10% (P =.02) and mass index by 13% (P =.01), whereas placebo LV mass and index did not change. The LV end-diastolic diameter increased only in the placebo group (P =.008). There were no significant changes in any of the other secondary outcomes, including the cardiac index and systemic vascular resistance. CONCLUSIONS: Thus, in these post-transplant patients, stricter BP control to normal levels by the addition of lisinopril to existing therapy, reduced BP and modestly decreased LV mass without altering cardiac hemodynamic function. PMID- 12372681 TI - Hypertension guidelines: criteria that might make them more clinically useful. AB - Cardiovascular disease prevention depends on reduction of risk factors, including hypertension. Guidelines designed to improve management of hypertension are widely available. Their purpose is to assemble the available data from basic biomedical science, epidemiology, and clinical science in an accessible form with which physicians and patients can make reasoned decisions for individual cases. However, guidelines have been neither widely accepted, nor effectively implemented. We recommend a strategy for guideline preparation designed to yield a product more user friendly, accessible, and effective. Guideline recommendations and the evidence used to make them should be based on an explicit grading system. Relevant clinical as well as nonclinical factors must be considered. Moreover, because the goal of antihypertensive therapy is to prevent cardiovascular events, and the likelihood of such events is determined by multifactor or absolute risk assessment, risk, rather than level of blood pressure (BP), should determine the need for therapy. Similarly, the benefit of therapy must be assessed by reduction in cardiovascular disease morbidity and mortality. PMID- 12372682 TI - Deficient oxygen transport: an alternative mechanism for the development of hypertension? PMID- 12372683 TI - Essential hypertension is associated with Chlamydia pneumoniae but not Epstein Barr antibodies. PMID- 12372684 TI - International recommendations for paternity testing standards. PMID- 12372685 TI - Paternity Testing Commission of the International Society of Forensic Genetics: recommendations on genetic investigations in paternity cases. AB - The International Society for Forensic Genetics (ISFG) has established a Paternity Testing Commission (PTC) with the purpose of formulating international recommendations concerning genetic investigations in paternity testing. The PTC recommends that paternity testing be performed in accordance with the ISO 17025 standards. The ISO 17025 standards are general standards for testing laboratories and the PTC offers explanations and recommendations concerning selected areas of special importance to paternity testing. PMID- 12372686 TI - Allelic alterations at the STR markers in the buccal tissue cells of oral cancer patients and the oral epithelial cells of healthy betel quid-chewers: an evaluation of forensic applicability. AB - Although cancerous specimens are usually not used in forensic DNA typing, they might be forcibly employed under certain instances. On the other hand, though the oral epithelial samples have been applied to forensic identification, the great popularity of betel quid (BQ)-chewing in Taiwan, which is known to be a risk factor leading to an oral cancer, makes this application questionable. The DNA stability of nine short tandem repeat (STR) markers (the AmpFlSTR kit) was first investigated and then used to evaluate the forensic appropriateness of the oral samples of both healthy BQ-chewers and the archived clinical specimens from oral cancer patients. The analyses were performed on buccal samples from 100 BQ chewers and 100 oral cancer patients, as well as their paired peripheral blood samples, and a group of 100 non-BQ-chewers were used for the control. In the group of 100 oral cancer patients, two types of DNA instability were found. They were major allelic imbalance, and allelic alterations including the expansion, the contraction and the un-classified type (i.e. can not be confirmed as the expansion or the contraction). The overall percentage of the cancerous subjects demonstrating DNA instability was 33% (five patients possessing both types of DNA instability). Both types of DNA instability showed a tendency of increasing with the severity of the pathological stage of oral cancer. Forty-four occurrences of major allelic imbalance were found from 21 cancer patients. The statistical result revealed that there was no significant difference in the allelic imbalanced occurrence among the nine STR loci. Allelic alterations were found in 17 patients, within which 12 individuals had the expansion, five had the contraction, and three were the un-classified type. Further, among these 17 patients, three were found to acquire multiple allelic alterations at multiple loci. In the group of 100 unrelated healthy BQ-chewers, two loci with major allelic imbalance were detected. However, the two imbalanced alleles were virtually half lost, and could still be recognized as heterozygous alleles. The statistical results of ANOVA, chi(2), and Scheffe tests indicated that the means of allelic imbalance at the nine STR loci of the oral cancerous group revealed a significant difference from those in the control group. Our results suggest that oral cancer tissues cannot be used as references for forensic purposes using the PCR-based STR systems, whereas the oral swabs from healthy BQ-chewers can be employed, but should be done with caution. PMID- 12372687 TI - Hair-MAP: a prototype automated system for forensic hair comparison and analysis. AB - This paper demonstrates the feasibility of the automation of forensic hair analysis and comparison task using neural network explanation systems (NNESs). Our system takes as input microscopic images of two hairs and produces a classification decision as to whether or not the hairs came from the same person. Hair images were captured using a NEXTDimension video board in a NEXTDimension color turbo computer, connected to a video camera. Image processing was done on an SGI indigo workstation. Each image is segmented into a number of pieces appropriate for classification of different features. A variety of image processing techniques are used to enhance this information. Use of wavelet analysis and the Haralick texture algorithm to pre-process data has allowed us to compress large amounts of data into smaller, yet representative data. Neural networks are then used for feature classification. Finally, statistical tests determine the degree of match between the resulting collection of hair feature vectors. An important issue in automation of any task in criminal investigations is the reliability and understandability of the resulting system. To address this concern, we have developed methods to facilitate explanation of neural network's behavior using a decision tree. The system was able to achieve a performance of 83% hair match accuracy, using 5 of the 21 morphological characteristics used by experts. This shows promise for the usefulness of a fuller scale system. While an automated system would not replace the expert, it would make the task easier by providing a means for pre-processing the large amount of data with which the expert must contend. PMID- 12372688 TI - Fatal malignant hyperthermia--delayed onset and atypical course. AB - A case of malignant hyperthermia (mh) in a 27-year-old man is described. In a first anaesthesia using isoflurane and succinylcholine, the end-tidal CO(2) rose from 39 to 49 mmHg 2.75 h post-intubation and the body temperature rose to 39.8 degrees C 14 h post-intubation but was normal again the next day. In a second anaesthesia using the same medication, the maximal end-tidal CO(2) was 44 mmHg and the body temperature rose to 39 degrees C after 9 h. After 4 days, the fever rose to 40 degrees C, and to 42 degrees C when death occurred 10 days after the second anaesthesia. Masseter spasms or muscle rigidity were never present. According to the death certificate, death was due to multi-organ failure from sepsis. At autopsy, the skeletal muscles were pale and oedematous. Histology demonstrated focal necroses in the skeletal muscles, shock kidneys with myoglobin excretion and myoglobin clots in small blood vessels of the lungs. Hence, the postmortem diagnosis "malignant hyperthermia" was established but accusations of medical maltreatment were rejected because of the atypical and protracted clinical course and because uncharacteristic signs of malignant hyperthermia were attributable to the clinically suspected sepsis. PMID- 12372689 TI - Suicide by a power drill. AB - This is the report of a 62-year-old man who committed suicide by drilling through his anterior chest wall with an electric power drill. Death was caused by pericardial tamponade combined with bleeding into the pleural cavity. The skin lesion at the left hemithorax was similar to a bullet entrance wound. PMID- 12372690 TI - 4-Dimethylaminopyridine as a catalyst in heroin synthesis. AB - In this paper, we describe an acetylating method for fast synthesis of heroin from morphine in the presence of 4-dimethylaminopyridine (4-DMAP) as a catalyst. In the reaction which led to heroin formation, the morphine base was subjected to a solution made up of 4-DMAP (catalyst), methylene chloride (solvent) and acetic anhydride (acetylating agent). We showed that in comparison with classic acetylating procedures, reaction time can be reduced from at least several hours at elevated temperatures to <10 min at room temperature. In general, reaction time is dependent on the molar concentration ratio between morphine and 4-DMAP. PMID- 12372691 TI - Estimation of length of calcaneum and talus from their bony markers. AB - Since hardly a report is available on estimation of length of calcaneum and talus from a fragment of them, a fresh study was made on a present day south Indian population. A total of 110 calcanei (55 right and 55 left), and 70 tali (35 right and 35 left), all unpaired, dry, and devoid of gross pathology, were used. Maximum anteroposterior length of the bone was measured in millimeter using an anthropometric board, and linear measurements of the other bony markers were measured in millimeter using a sliding caliper. Bony markers of calcaneum were maximum anteroposterior length, maximum transverse width, length, width and depth of groove on the sustentaculum tali, and length, width, and depth of the sulcus calcanei. Bony markers of talus were maximum anteroposterior length, maximum transverse width, length and width of articular surface for the lateral malleolus, length and width of articular surface for the medial malleolus, vertical width and transverse width of articular surface of the head, width and depth of groove for tendon of the flexor hallucis longus, and length, width, and depth of the sulcus tali. Simple regression suggested that maximum length of the calcaneum regressed significantly with maximum transverse width, length, width and depth of groove on the sustentaculum tali, and length, width, and depth of the sulcus calcanei and that maximum length of the talus regressed significantly with maximum transverse width, length and width of the lateral articular surface, length of the medial articular surface, vertical and transverse diameters of the head, and depth of the sulcus tali. Maximum length of calcaneum and talus is derived from the regression values, to predict the stature of the person from available stature equations in the literature. PMID- 12372692 TI - The calcaneus: sex assessment of prehistoric New Zealand Polynesian skeletal remains. AB - The increasing pace of urbanisation has meant that prehistoric Polynesian skeletal remains are frequently being recovered in New Zealand. Since such material must often be reinterred quickly, it is important that the sex of individuals be determined from the remains in a relatively short time. For this purpose, discriminant function analysis was utilised for sex determination of prehistoric adult New Zealand Polynesian calcanei (26 male and 22 female). Five measurements were taken and subjected to Statistical Package for the Social Sciences (SPSS) discriminant function analysis. For the discriminant functions derived, accuracy of sex determination ranged from 88.4 to 93.5%. Reduction in error over random assignment by sex ranged from 77 to 87%. PMID- 12372693 TI - Sequencing strategy of mitochondrial HV1 and HV2 DNA with length heteroplasmy. AB - We describe a method to obtain reliable mitochondrial DNA (mtDNA) sequences downstream of the homopolymeric stretches with length heteroplasmy in the sequencing direction. The method is based on the use of junction primers that bind to a part of the homopolymeric stretch and the first 2-4 bases downstream of the homopolymeric region. This junction primer method gave clear and unambiguous results using samples from 21 individuals with length heteroplasmy in the hypervariable regions HV1, HV2 or both. The method is of special value for forensic casework, because sequencing of both strands of an mtDNA region is preferable in order to reduce ambiguities in sequence determination. PMID- 12372694 TI - STR data for the AmpFLSTR Profiler loci from Sinkiang (NW China). AB - Allele frequencies for nine STRs included in the APMF1STR kit were obtained from blood samples of 100 unrelated individuals born in Sinkiang Uygur Autonomy Region of China (NW China). PMID- 12372695 TI - Genetic data on eight STRs (D5S818, D7S820, F13B, LPL, TH01, TPOX, VWA31, CSF1PO) from a Colombian population. AB - Allele frequencies for eight short tandem repeats (STRs) (D5S818, D7S820, F13B, LPL, TH01, TPOX, VWA31 and CSF1PO) were estimated from a sample of 155 unrelated individuals living in different departments of the southwest of Colombia, Caqueta, Cauca, Huila, Narino, Putumayo and Cauca Valley. PMID- 12372696 TI - Galanin and galanin receptors in embryonic stem cells: accidental or essential? PMID- 12372697 TI - Langerhans cell expression of neuropeptide Y and peptide YY. AB - Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides with a variety of known functions. The role of these peptides in the skin is largely unknown, although NPY-like immunoreactivity has been reported in the epidermis. The recent report that these peptides have antimicrobial properties suggests that NPY and PYY may contribute to the skin's defense mechanisms against invading microorganisms. We have demonstrated that Langerhans cells (LC) and a certain BALB/c epidermis-derived dendritic cell line contain mRNA for NPY and PYY using RT-PCR. Furthermore, this dendritic cell line as well as an epidermis-derived dendritic cell line from A/J mice were found to produce NPY and PYY and LC produced PYY, as assessed by radioimmunoassay. These data suggest that the protective function of LC include not only antigen presentation, but also production of antimicrobial peptides. PMID- 12372698 TI - Substance P release in the spinal dorsal horn following peripheral nerve injury. AB - Spinal microdialysis was used to study the potassium induced in vivo release of substance P (SP) in the rat dorsal horn at different time points (3, 14, and 60 days) following partial sciatic nerve ligation (PNL) or sciatic nerve axotomy. The withdrawal threshold to innocuous mechanical stimuli was investigated with von Frey filaments in the PNL rats prior to microdialysis. The release of SP was significantly elevated at 60 days following PNL but not following complete nerve injury. However, the PNL rats in all time groups displayed mechanical hypersensitivity, which implies that this late change in SP release seems to be unrelated to the development of neuropathy. The present results indicate that there is an increase of the releasable pool of SP in the dorsal horn at late post operative times after PNL. This change in SP release may reflect an altered sensory processing or may instead relate to adaptive responses to promote recovery. PMID- 12372699 TI - Bradykinin-induced neuropeptide Y release by human pheochromocytoma tissue. AB - Neuropeptide Y (NPY) and noradrenaline (NA) are frequently co-localized and co released in the sympathetic nervous system. Since bradykinin (BK) is known to stimulate neurotransmitter release as NA in adrenal glands, we therefore hypothesized that BK might also be involved in the release of NPY. The effect of BK(1-9) on immunoreactive NPY (Ir-NPY) release was investigated in superfused human pheochromocytoma tissue. BK(1-9) (10(-7)-10(-5) M) was shown to induce a rapid Ir-NPY release in a concentration-dependent manner. This effect of BK(1-9) (10(-6) M) was mimicked by the B2 agonist [Phe(8)(CH(2)NH)Arg(9)]-bradykinin (10( 5) M) and blocked by the selective B2-receptor antagonist HOE140 (10(-5) M). Increasing Ir-NPY release was probably not mediated by nitric oxide (NO) since the outflow of Ir-NPY was not influenced by the NO synthase inhibitor N-omega nitro-L-arginine methyl ester (L-NAME) (10(-4) M). In presence of bapta-AM (10( 5) M), a chelator of cytosolic calcium, W7 (10(-5) M), a calmodulin inhibitor, TMB-8 (10(-5) M), a blocker of intracellular calcium mobilization and ryanodine (10(-5) M), a selective inhibitor of the Ca(2+)-induced release mechanism, the NPY release by BK(1-9) was significantly inhibited by 126%, 98%, 91%, and 94%, respectively. These results indicate that BK increased the release of NPY by the tumor acting through the interaction with the BK-B2 receptor and request intracellular calcium mobilization independently of a NO mechanism. PMID- 12372700 TI - The ability of hippocampal CA1 area for induction of long-term potentiation is persistently reduced by prior treatment with cysteamine: an in vitro study. AB - Using field potential recording in the CA1 region of hippocampal slices from rats injected with cysteamine (200 mg/kg, s.c.), changes in activity and plasticity of Schaffer collateral-CA1 pyramidal cell synapses were examined. Extracellular field potential recording prior to and following either theta-pattern primed bursts (PBs), perfusion with low Mg(2+) or with high Ca(2+), indicated long-term potentiation (LTP) of population spikes amplitude (PSA). The extent of LTP of PSA was significantly lower in cysteamine-treated rats. It is concluded that cysteamine can entail lasting modifications in susceptibility of hippocampal CA1 for synaptic plasticity induced by tetanus. Similarly, disability in function of CA1 synapses can be traced by other protocols of LTP induction. The relevancy of the results to the facilitatory role of endogenous somatostatin in the function of Schaffer collateral-CA1 pyramidal cell synapses is also discussed. PMID- 12372701 TI - Chemokine release is associated with the protective action of PACAP-38 against HIV envelope protein neurotoxicity. AB - The envelope protein (gp120) of the human immunodeficiency virus produces neuronal cell death in cultures that can be prevented by co-treatment with pituitary adenylate activating peptide-38 (PACAP-38) or chemokines. To investigate the hypothesis that a functional relationship exists between these two protectants, the release of chemokines was measured in rat astrocyte cultures after PACAP-38 treatment. Chemokine analyses were performed by immunoaffinity capillary electrophoresis. Bell-shaped dose-responses for PACAP-mediated release of chemokines into the culture medium were observed with EC(50)'s of 3 x 10(15) M (RANTES: regulated upon activation normal T cell expressed and secreted), 3 x 10( 11) M (MIP-1 beta) and 10(-7)M (MIP-1 alpha). In addition, PACAP-mediated depletion of chemokines from cultured astrocytes exhibited inverted bell-shaped curves, with similar EC(50)'s to those observed for chemokine measurements of the medium. Comparative studies with structurally related peptides (vasoactive intestinal peptide [VIP] and secretin) revealed that PACAP was the most potent secretagogue for RANTES on astrocyte cultures. Gp120-mediated neuronal cell death was prevented by co-treatment with PACAP-38, although the efficacy of protection varied significantly among the gp120 isolates. A bi-model dose-response was observed with EC(50)'s of 3 x 10(-15) and 3 x 10(-11) M. Co-treatment with neutralizing antiserum to RANTES attenuated PACAP-mediated protection from toxicity associated with gp120. In contrast to previous studies with VIP and gp120 toxicity, co-treatment with anti-MIP-1 alpha did not affect PACAP-induced protection. These studies support the hypothesis that PACAP produces neuroprotection from gp120 toxicity, in part, through the release of RANTES and this mechanism is distinct from that observed with VIP. PMID- 12372702 TI - Distribution of leucine-enkephalin in bone and joint tissues. AB - The distribution and concentration of leu-enkephalin in periosteum, cortical bone, bone marrow and synovial membrane of normal rats were analysed. Periosteum, cortical bone and bone marrow of the rat femurs were collected as well as the ankles. The distribution of leu-enkephalin was analysed by immunoelectron microscopy and the concentration of leu-enkephalin was measured with radioimmunoassay. Immunoelectron microscopy showed that leu-enkephalin is abundant in monocytes of bone marrow, nerve fibers and endothelial cells in the periosteum and also in macrophage-like-cells of the synovial membrane. The concentration of leu-enkephalin measured by RIA showed highest concentration in bone marrow followed by periosteum and cortical bone. The study supports that leu enkephalin is present and can be quantified in different compartments of bone and joint tissues suggesting that leu-enkephalin may be involved in the physiological regulation of nociception and immunoregulation. PMID- 12372703 TI - Effects of vasoconstriction on the acute anterior pituitary hormonal response to head injury. AB - Since cerebral vasoconstriction alone may impair the hypothalamic and pituitary circulation, we planned to investigate whether the hormonal response to the vasoconstriction that may be induced by the head injury is a significant component of the general acute hormonal response to head injury. Although diffuse adrenocorticotropic hormone immunohistochemical staining of the adenohypophysis of rabbits was observed in the head trauma administered group, only mild positive staining was present in the Endothelin-1 administered group. However, decreased prolactin staining was found in both of the groups. It is postulated that trauma induced vasoconstriction may not be an important manipulating factor in the corticotrophic hormone response to injury, while it may be responsible for the decreased prolactin response. PMID- 12372704 TI - Chronic dexamethasone-treatment alters mineralocorticoid receptor, truncated trkB and selected glutamate receptor subunit mRNA expression in the porcine hippocampus. AB - Prepubertal boars (n = 4/treatment) were killed 24 h after a 5 day course of intravenous injections of dexamethasone (Dex, 1 and 5 mg kg(-1)), or saline vehicle. Gene expression was quantified in brain sections following in situ hybridisation histochemistry. The objective was to determine whether chronic glucocorticoid treatment would alter the expression of mRNAs for gluco- and mineralocorticoid receptors (GR and MR), brain-derived neurotrophic factor (BDNF), its receptor, trkB, and selected ionotropic glutamate receptor (iGluR) subunits in the hippocampus. Although Dex did not alter GR message, the higher dose reduced MR mRNA in all hippocampal subfields studied. There was no effect of Dex on the expression of BDNF, or the full-length form of its receptor but there was evidence to suggest that mRNA for the truncated form of trkB was increased. Expression of mRNA for glutamate receptor subunits was either unaffected (NR1) or decreased (GluR2 and GluR3). These findings indicate that acute and chronic glucocorticoid treatment has differential effects on hippocampal gene expression in the porcine brain. PMID- 12372705 TI - Clear skies not so clean. PMID- 12372706 TI - 10 years and counting: the hype and hope of cancer biotechnology. PMID- 12372707 TI - "Inject-a-gel" chemotherapy for advanced head and neck cancer. PMID- 12372708 TI - Surprise phase III failure for ZD1839. PMID- 12372711 TI - Cervical cancer research focuses on the HPV E7 gene. PMID- 12372718 TI - Postoperative radiotherapy in completely resected, early-stage, non-small-cell lung cancer: reflections and future directions. PMID- 12372720 TI - Anticoagulants as anticancer agents? PMID- 12372721 TI - Neoadjuvant and adjuvant therapy for resectable hepatocellular carcinoma: review of the randomised clinical trials. AB - Hepatocellular carcinoma (HCC) is common worldwide, and its incidence is increasing. Liver resection or transplantation is potentially curative, although subsequent recurrence and death are common. We reviewed randomised trials on the role of adjuvant therapy in resectable HCC. We identified 13 randomised trials with recurrence or survival endpoints reported at 3 years or longer. Three studies involved predominantly systemic adjuvant chemotherapy; four involved predominantly hepatic-artery-based chemotherapy or embolisation; and six used other therapeutic modalities including immunological, radiation, and differentiation agents. A therapeutic benefit in terms of disease-free or overall survival was noted in six trials, five of which involved modalities other than systemic or hepatic-artery chemotherapy or embolisation. We conclude that systemic and hepatic-artery chemotherapy or chemoembolisation have not been shown to improve overall or disease-free survival after resection of HCC, although there has been no definitive trial comparing adjuvant systemic chemotherapy with no treatment. Other adjuvant modalities (mostly tested in small, preliminary settings) may confer benefit after potentially curative resection of HCC. PMID- 12372722 TI - Dynamism of tumour vasculature in the early phase of cancer progression: outcomes from oesophageal cancer research. AB - The vascular structure of cancer changes during tumour progression. A particularly dramatic change occurs during the early phase of progression when in situ tumour is transformed to invasive cancer. Recent advances in morphological investigations have made it possible to visualise and characterise the microvascular-network alterations. In addition, laboratory studies have also revealed the molecular profile--the changing levels of expression of different proteins--of cancer progression, which has helped to advance understanding of the mechanism of carcinogenesis. In this review, we discuss recent outcomes of research of oesophageal cancer and consider the responses of the vascular network and the development of new blood vessels during the early phase of cancer progression. Such considerations will be useful not only for understanding vascular biology but also for exploring novel diagnostic, therapeutic, and preventive approaches targeting early stage or latent phase human cancer. PMID- 12372723 TI - Hormonal risk factors for breast cancer: identification, chemoprevention, and other intervention strategies. AB - Breast cancer remains a leading cause of female morbidity and mortality worldwide. Many hormonal and genetic risk factors have been identified and have led to the development of mathematical models that can be used in the clinic to give a woman an estimate of her individual risk of developing breast cancer. These models can also be used to identify women who might benefit from breast cancer chemoprevention with tamoxifen or be suitable for entry into trials with new agents. In this review, we discuss the relative merits of the Gail and Claus risk models. The Claus model is based mainly on family history, whereas the Gail model also includes simple markers of oestrogen exposure. We explore more sophisticated measures of lifetime oestrogen exposure that can be used to improve the discriminatory ability of these models. We also appraise the four trials of breast-cancer chemoprevention, including the trial that has led to licensing of tamoxifen for this indication in the USA. Finally, we discuss other agents and interventions that could be used in the future to improve the efficacy and tolerability of breast-cancer risk reduction. PMID- 12372724 TI - Quality of life in patients undergoing systemic therapy for advanced breast cancer. AB - To date no published reviews have examined the effects of systemic therapy on health-related quality of life (HRQOL) in patients with advanced breast cancer. We did a systematic review identifying 19 randomised controlled trials, with 5732 participants. Most of the trials (12) involved chemotherapy, but six involved hormonal therapies, and one a biological therapy. 15 studies assessed HRQOL as a secondary endpoint; only seven reported any significant differences in HRQOL between treatment groups. We identified several limitations with methods. Most studies reported problems with withdrawal of patients, which reduces statistical power and can lead to bias. Baseline characteristics of patients were not reported in many cases, and only three studies examined clinical significance. We conclude that HRQOL data provide some invaluable insights into the treatment and care of patients, but future studies should address several common problems with methods. We propose some approaches to overcome these limitations and improve future study designs. PMID- 12372725 TI - Informing and involving cancer patients in their own care. AB - Doctors have long feared that disclosure of a cancer diagnosis may harm the patient. However, the vast majority of cancer patients in more developed countries prefer to have as much information as possible, regardless of whether it is good or bad. Moreover, these patients are often dissatisfied with the amount and quality of information they receive. Additionally, many patients are unable to participate to the extent that they wish in decisions about their own care, and doctors frequently fail to recognise or appreciate the role that their patients prefer in decision-making. Various information resources have been developed to meet the needs of patients and their families. This paper discusses the information needs and participation preferences of patients with cancer and the consequences of not meeting these expectations. The paper then reviews the types of information resources that have been developed focusing on their reported effectiveness. The review concludes with suggestions for future research. PMID- 12372726 TI - Beyond futility: to what extent is the concept of futility useful in clinical decision-making about CPR? AB - The concept of futility has often been invoked to justify abstention from treatment and decisions such as 'do not attempt resuscitation' (DNAR). In this capacity, futility has played an important part in the development of several sets of official clinical guidelines. In this paper, we examine the nature of futility and question whether it is a sufficiently robust concept to meet the ethical and clinical demands placed upon it. Although the concept of futility promises simplicity, it cannot stand alone as a satisfactory framework for clinical decision-making. Practitioners and policy makers should be cautious about their use of the concept. PMID- 12372729 TI - The clinical relevance and scientific potential of ultra high-field-strength MR imaging. PMID- 12372730 TI - Toward an evidence-based approach in the management of concussion: the role of neuroimaging. PMID- 12372731 TI - Diffusion-tensor MR imaging of gray and white matter development during normal human brain maturation. AB - BACKGROUND AND PURPOSE: Conventional MR imaging findings of human brain development are thought to result from decreasing water content, increasing macromolecular concentration, and myelination. We use diffusion-tensor MR imaging to test theoretical models that incorporate hypotheses regarding how these maturational processes influence water diffusion in developing gray and white matter. METHODS: Experimental data were derived from diffusion-tensor imaging of 167 participants, ages 31 gestational weeks to 11 postnatal years. An isotropic diffusion model was applied to the gray matter of the basal ganglia and thalamus. A model that assumes changes in the magnitude of diffusion while maintaining cylindrically symmetric anisotropy was applied to the white matter of the corpus callosum and internal capsule. Deviations of the diffusion tensor from the ideal model predictions, due to measurement noise, were estimated by using Monte Carlo simulations. RESULTS: Developing gray matter of the basal ganglia and developing white matter of the internal capsule and corpus callosum largely conformed to theory, with only small departures from model predictions in older children. However, data from the thalamus substantially diverged from predicted values, with progressively larger deviations from the model with increasing participant age. CONCLUSION: Changes in water diffusion during maturation of central gray and white matter structures can largely be explained by theoretical models incorporating simple assumptions regarding the influence of brain water content and myelination, although deviations from theory increase as the brain matures. Diffusion-tensor MR imaging is a powerful method for studying the process of brain development, with both scientific and clinical applications. PMID- 12372732 TI - Neonatal alloimmune thrombocytopenia: antenatal and postnatal imaging findings in the pediatric brain. AB - BACKGROUND AND PURPOSE: Neonatal alloimmune thrombocytopenia (NAIT) is a maternal fetal platelet antigen incompatibility disorder estimated to occur in one of 2000 to 5000 neonates. The diagnosis is made serologically by showing parental platelet antigen incompatibility and the presence of maternal platelet antibodies. In the absence of formalized antenatal screening, the radiologist has an important role to play in the recognition of this disorder, which has significant implications for the index case and any subsequent offspring. Our aim was to characterize the neuroradiologic findings and identify, if possible, a consistent pattern of neurologic injury typical of NAIT. METHODS: We retrospectively reviewed the ultrasonograms, CT scans, MR images, and medical histories of six patients (21 weeks gestation to 9 years old) with intracranial injury secondary to serologically proved NAIT. RESULTS: Hemispheric porencephalic cysts (n = 6), primarily located within a temporal lobe with extension into other lobes, were seen on the ultrasonograms, CT scans, and MR images of all six children. In five cases, this was thought to represent encephaloclastic porencephaly and, in one case, schizencephaly (agenetic porencephaly). Six children had ventriculomegaly of varying degrees and severity and asymmetry. Extra-axial hemorrhages (n = 2), intraventricular hemorrhage (n = 1), acute parenchymal hemorrhage (n = 2), and neuronal migrational disorder (n = 1) occurred with varying frequency. CONCLUSION: Antenatal or early postnatal neuroradiologic imaging showing hemispheric porencephaly and lateral ventriculomegaly is a recognizable pattern of cerebral injury suggestive of the diagnosis of NAIT. In the absence of a cost-effective screening program of primiparous women and neonates for this disease, the radiologist has an important role to play in the recognition of this disease entity. It is crucial for the reporting radiologist to consider the possibility of NAIT in any child with antenatal hemorrhage and, more importantly, with the pattern of cerebral injury described above. Because a high percentage of subsequent pregnancies might be equally or more severely affected, antenatal management directed at preventing intracranial hemorrhages in utero has become of significant clinical importance. PMID- 12372733 TI - New syndrome characterized by hypomyelination with atrophy of the basal ganglia and cerebellum. AB - BACKGROUND AND PURPOSE: Leukoencephalopathies of unknown origin constitute a considerable problem in child neurology. The purpose of our ongoing study of the subject was to define new disease entities among them by using primarily MR imaging pattern recognition. METHODS: We identified seven unrelated patients with a distinct MR imaging pattern consisting of hypomyelination and atrophy of the basal ganglia (neostriatum) and cerebellum (H-ABC). We reviewed the clinical, MR imaging, MR spectroscopic, and laboratory data. RESULTS: Clinically, the patients' diseases were characterized by variably disturbed early development followed by increasing extrapyramidal movement abnormalities, ataxia, and spasticity. Mental capacity was variably affected, but it appeared to be relatively preserved. Parents were not related, and none of their siblings were affected. No metabolic defect was found. Follow-up MR imaging demonstrated atrophy of the cerebral white matter, neostriatum, and cerebellum, which was most pronounced in the most clinically severe cases. Single-voxel proton MR spectroscopic results were normal in the parietal cortex. In the cerebral white matter, myo-inositol and creatine levels were elevated; this finding was compatible with gliosis. N-acetylaspartate and choline levels were normal, suggesting that neither axonal loss nor active demyelination occurred. Proton MR spectroscopic imaging revealed relatively decreased N-acetylaspartate levels in the frontal region. CONCLUSION: The uniform and highly characteristic MR imaging findings, in combination with the similarities in the clinical findings, provide evidence of a distinct nosologic entity. The acronym H-ABC is offered to indicate patients sharing these clinical and MR imaging features. PMID- 12372734 TI - Ectopic posterior pituitary lobe and periventricular heterotopia: cerebral malformations with the same underlying mechanism? AB - BACKGROUND AND PURPOSE: Ectopic posterior pituitary lobe often occurs in children with growth hormone deficiency and is part of the spectrum associated with septo optic dysplasia. Some cases of septo-optic dysplasia are caused by homozygous mutations in the homeobox gene HESX1, whereas heterozygous mutations are associated with milder phenotypes. To date, HESX1 is the only gene associated with ectopic posterior pituitary lobe. We describe an association between ectopic posterior pituitary lobe and periventricular heterotopia in four children without classic features of septo-optic dysplasia and suggest possible mechanisms on the basis of a review of pituitary embryology and recent molecular genetic advances. METHODS: Among 20 children with ectopic posterior pituitary lobe, four had associated periventricular heterotopia. We herein review the clinical and MR imaging findings of these four children. Mutation screening of HESX1 was performed in two. RESULTS: All four children had growth hormone deficiency. None had visual or neurologic disturbances. MR images showed a range of pituitary appearances, with scattered discrete periventricular heterotopia in each case. Other abnormalities were limited to small suprasellar lipomas and callosal dysgenesis. A heterozygous HESX1 mutation was present in one case. CONCLUSION: The coexistence of ectopic posterior pituitary lobe and periventricular heterotopia suggests they have a common underlying genetic basis that is due to gene expression at different locations and stages of development. The presence of a heterozygous HESX1 mutation in one case suggests this gene is important in the development of both ectopic posterior pituitary lobe and periventricular heterotopia and supports their place in the spectrum of septo-optic dysplasia. Further analysis of HESX1 and other genes in related developmental pathways will elucidate their roles in the development of both malformations. PMID- 12372735 TI - Spontaneous regression of an infantile dural sinus fistula with pial recruitment. AB - Infantile dural sinus fistulas are rare and generally have a poor prognosis unless treatment can be undertaken. We report a unique case of an infantile dural sinus fistula with secondary pial recruitment that was managed conservatively. Subsequent spontaneous regression of the lesion occurred over 11 months. The clinical and angiographic features that indicated a probable favorable prognosis are discussed. PMID- 12372736 TI - Comparative study of MR sialography and digital subtraction sialography for benign salivary gland disorders. AB - BACKGROUND AND PURPOSE: MR sialography has become an alternative imaging technique for ductal salivary gland diseases. We compared the diagnostic accuracies of MR sialography and digital subtraction sialography in patients with successful completion of both examinations and benign salivary gland disorders. METHODS: In a prospective study, we attempted to examine salivary glands in 80 patients with clinically suspected diagnoses of sialadenitis and/or sialolithiasis. Each patient underwent digital subtraction sialography and MR sialography. MR sialography was obtained with a T2-weighted single-shot turbo spin-echo sequence (TR/TE 2800/1100 msec, acquisition time 7 seconds), with use of a quadrature head coil. Final diagnoses were confirmed by clinical follow-up and results of biopsy (n = 9) or surgery (n = 19). RESULTS: Failure rate was 5% (four of 80) for MR sialography and 14% (11 of 80) for digital subtraction sialography. Eighty-one salivary glands (48 parotid, 33 submandibular) in 65 patients were successfully visualized with both modalities. MR sialography depicted the main ductal system and first- and second-order branches, whereas digital subtraction sialography was able to depict third-order branches. Sensitivity and specificity to diagnose chronic sialadenitis were 70% and 98% with MR and 96% and 100% with digital subtraction sialography. MR sialography enabled diagnosis of sialolithiasis with a sensitivity of 80% and a specificity of 98% versus 90% and 98% for each with digital subtraction sialography. CONCLUSION: MR sialography with a heavily T2-weighted sequence is highly successful in the noninvasive visualization of the ductal system of major salivary glands. It is useful for diagnosing sialolithiasis and sialadenitis. Digital subtraction sialography, an invasive technique, had a substantial procedural failure rate, particularly for the submandibular duct. However, because of its higher spatial resolution, successfully completed digital subtraction sialography achieved superior diagnostic information compared with that of MR sialography. PMID- 12372737 TI - Intracanalicular meningioma mimicking vestibular schwannoma. AB - SUMMARY: Three cases of intracanalicular meningioma mimicking vestibular schwannoma are presented. In each case, a contrast-enhancing mass filling the internal auditory canal was identified on MR images and was originally diagnosed as a vestibular schwannoma. Although it is difficult to differentiate definitively between these lesions preoperatively, imaging findings inconsistent with a diagnosis of vestibular schwannoma can be identified. Preoperative identification of intracanalicular meningiomas permits alterations in surgical planning that allow for the more complete resection of these rare tumors. PMID- 12372738 TI - Granulocytic sarcoma of the temporal bone. AB - We report an unusual case of granulocytic sarcoma involving the temporal bone. The occurrence of this tumor usually heralds acute myelogenous leukemia or the onset of the blastic phase of chronic myelogenous leukemia. Recognition of this rare entity is important, because early aggressive chemotherapy can cause regression of the tumor, as in our case, and thus improve patient longevity. PMID- 12372739 TI - The craniocervical venous system in relation to cerebral venous drainage. AB - BACKGROUND AND PURPOSE: Passing from the supine to the upright position favors cerebral venous outflow into vertebral venous systems rather than into the internal jugular veins. We sought to determine venous connections between dural venous sinuses of the posterior cranial fossa and craniocervical vertebral venous systems. METHODS: Corrosion casts of the cranial and cervical venous system were obtained from 12 fresh human cadavers, and anatomic confirmation was made by dissection of three previously injected fresh human specimens. MR venography was performed to provide radiologic correlation. RESULTS: The lateral, posterior, and anterior condylar veins and the mastoid and occipital emissary veins were found to represent the venous connections between the dural venous sinuses of the posterior cranial fossa and the vertebral venous systems. This study revealed the nearly constant presence of the anterior condylar confluent (ACC) located on the external orifice of the canal of the hypoglossal nerve. The ACC offered multiple connections with the dural venous sinuses of the posterior cranial fossa, the internal jugular vein, and the vertebral venous system. All these structures were shown by MR venography. CONCLUSION: The lateral, posterior, and anterior condylar veins and the mastoid and occipital emissary veins connect the dural venous sinuses of the posterior cranial fossa with the vertebral venous systems. These connections are clinically relevant, because encephalic drainage occurs preferentially through the vertebral venous system in the upright position. The ACC is a constant anatomic structure that may play an important role in the redirection of cerebral blood in the craniocervical region. PMID- 12372740 TI - Brain atrophy in mild or moderate traumatic brain injury: a longitudinal quantitative analysis. AB - BACKGROUND AND PURPOSE: Although mild or moderate traumatic brain injury (TBI) is known to cause persistent neurologic sequelae, the underlying structural changes remain elusive. Our purpose was to assess decreases in the volume of brain parenchyma (VBP) in patients with TBI and to determine if clinical parameters are predictors of the extent of atrophy. METHODS: We retrospectively assessed the total VBP in 14 patients with mild or moderate TBI at more than 3 months after injury and in seven patients at two time points more than 3 months apart. VBP was calculated from whole-brain MR images and then normalized by calculating the percent VBP (%VBP) to correct for intraindividual variations in cranial size. Clinical parameters at the time of trauma were evaluated for potential predictors of atrophy. Findings were compared with those of control subjects of similar ages. RESULTS: In the single time-point analysis, brain volumes, CSF volumes, and %VBP were not significantly different between patients and control subjects. In the longitudinal analysis, the rate of decline in %VBP (0.02 versus 0.0064 U/day, P =.05) and the change in %VBP between the first and second time points (-4.16 +/ 1.68 versus -1.49 +/- 1.7, P =.022 [mean +/-SD]) were significantly greater in patients. Change in %VBP was significantly greater in patients with loss of consciousness (LOC) than in those without LOC (P =.023). CONCLUSION: Whole-brain atrophy occurs after mild or moderate TBI and is evident at an average of 11 months after trauma. Injury that produces LOC leads to more atrophy. These findings may help elucidate an etiology for the persistent or new neurologic deficits that occur months after injury. PMID- 12372741 TI - Experimental cerebral fat embolism: embolic effects of triolein and oleic acid depicted by MR imaging and electron microscopy. AB - BACKGROUND AND PURPOSE: In fat embolism, free fatty acid is more toxic than neutral fat in terms of tissue damage. We evaluated the hyperacute embolic effects of triolein and oleic acid in cat brains by using MR imaging and electron microscopy. METHODS: T2-weighted imaging, diffusion-weighted imaging, and contrast-enhanced T1-weighted imaging were performed in cat brains after the injection of triolein (group 1, n = 8) or oleic acid (group 2, n = 10) into the internal carotid artery. MR images were quantitatively assessed by comparing the signal intensity ratios of the lesions with their counterparts on T2-weighted images, apparent diffusion coefficient (ADC) maps, and contrast-enhanced T1 weighted images. Electron microscopic findings in group 1 were compared with those in group 2. RESULTS: Qualitatively, MR images revealed two types of lesions. Type 1 lesions were hyperintense on diffusion-weighted images and hypointense on ADC maps. Type 2 lesions were isointense or mildly hyperintense on diffusion-weighted images and isointense on ADC maps. Quantitatively, the signal intensity ratios of type 1 lesions in group 2 specimens were significantly higher on T2-weighted images (P =.013)/(P =.027) and lower on ADC maps compared with those of group 1. Electron microscopy of type 1 lesions in both groups revealed more prominent widening of the perivascular space and swelling of the neural cells in group 2, in contrast to notable endothelial defects in group 1. CONCLUSION: MR and electron microscopic data on cerebral fat embolism induced by either triolein or oleic acid revealed characteristics suggestive of both vasogenic and cytotoxic edema in the hyperacute stage. Tissue damage appeared more severe in the oleic acid group than in the triolein group. PMID- 12372742 TI - Reproducibility of primary motor cortex somatotopy under controlled conditions. AB - BACKGROUND AND PURPOSE: The somatotopic organization of the contralateral primary motor cortex (M1) and its intra- and intersubject reproducibility has been the subject of many investigations and controversies. A potential explanation for a least some of the conflicting results could be the lack of movement control in the studies performed. The purpose of this study was to investigate these issues under controlled experimental conditions. METHODS: Functional MR imaging was performed in 12 healthy volunteers performing hand, finger, wrist, elbow, foot, and tongue movements. Two experimental sessions were separated by 2 weeks. Controlled conditions were achieved by means of a custom-designed arm and hand manipulandum providing standardization of the movements within and across subjects. RESULTS: The experiments revealed a clear large-scale somatotopy of the contralateral M1 with distinct subregions controlling the foot, arm, and tongue. Despite considerable overlap of the volumes, geometric centers of gravity (COGs) showed statistically significant differences in coordinates between the elbow, wrist, fingers, and hand. COGs showed a high degree of intra- and interindividual reproducibility, particularly for the upper limb movements, in contrast to the activation volumes that proved to be unreliable parameters, despite the controlled conditions. CONCLUSION: These findings support the existence of a gross-scale somatotopic organization yet also demonstrate a clear, fine-scale somatotopy of the within-arm representations. Furthermore, they reveal high reproducibility of the COGs when standardized conditions are applied. This observation highlights the need for movement control to allow for intra- and intersubject comparison. PMID- 12372743 TI - Quantitative diffusion-weighted MR imaging in transient ischemic attacks. AB - BACKGROUND AND PURPOSE: The risk of stroke after a transient ischemic attack (TIA) is high. Appropriately directed therapies may reduce this risk. However, sensitive means of detecting the presence of subtle neuronal ischemia are lacking. We investigated the potential use of quantitative diffusion-weighted (DW) MR imaging in the detection of deficits produced by transient cerebral ischemia. METHODS: Twenty-eight patients who came to the stroke service from the emergency room of a tertiary teaching hospital with the final diagnosis of transient cerebral ischemia underwent conventional MR imaging, MR angiography, and DW MR imaging within 24 hours of presentation. Fifteen patients had normal conventional DW images confirmed by a staff neuroradiologist and neurologist. For these patients, absolute quantitative diffusion values were subsequently calculated for the clinically relevant brain region and were compared with the values calculated for the corresponding contralateral unaffected brain region. Thirteen patients had conventional DW images positive for lesions and were not studied. RESULTS: Quantitative DW imaging enabled detection of abnormal decreases (9-26%, P <.05) in the diffusion constant in brain regions suspected to be clinically involved by ischemia, when compared with the contralateral clinically unaffected brain tissue as well as with two other internal controls. CONCLUSION: Quantitative DW imaging depicts diffusion deficit in patients with TIA. Quantitative DW imaging may have better sensitivity compared with conventional DW imaging in detecting transient cerebral ischemia. PMID- 12372744 TI - Changes in brain size with treatment in patients with hyper- or hypothyroidism. AB - BACKGROUND AND PURPOSE: Although neuropsychological symptoms and signs are common in thyroid disease, their organic substrate is unknown. We performed brain MR imaging in patients with hyperthyroidism or hypothyroidism before and after treatment and correlated the results with hormonal markers. METHODS: Eight patients with hyperthyroid disease and three with hypothyroid disease underwent imaging within 1-2 days of a thyroid hormone testing. Images were registered, and brain and ventricular sizes were measured by using a semiautomated contour and thresholding technique. Changes in brain and ventricular volume were correlated with serum levels of total thyroxine (T(4)), unbound triiodothyronine (free T(3)), and thyroid-stimulating hormone (TSH) before and after treatment. RESULTS: With treatment, brain size decreased by 6,329-31,183 mm(3) in the hyperthyroid group and increased by 2,599-48,825 mm(3) in the hypothyroid group. Conversely, with treatment, ventricular size increased by 325-6,279 mm(3) in the hyperthyroid group and decreased by 760-2,376 mm(3) in the hypothyroid group. There was a highly significant correlation between reduction in brain size and reduction in T(4), as well as between the increase in ventricular size and reduction in T(4). There was a significant correlation between reduction in ventricular size and reduction in free T(3). There were highly significant correlations between reduced levels of TSH and increase in brain size, as well as between increased levels of TSH and increase in ventricular size. CONCLUSION: In thyroid disease, the size of the brain and ventricles significantly change after treatment, and these changes are correlated with T(4), free T(3), and TSH levels. The mechanism of these changes is uncertain, but it may involve osmolyte regulation, the sodium and water balance, and alterations in cerebral hemodynamics. PMID- 12372745 TI - Comparison of 2D and 3D digital subtraction angiography in evaluation of intracranial aneurysms. AB - BACKGROUND AND PURPOSE: Although digital subtraction angiography (DSA) is considered the criterion standard for depiction of intracranial aneurysms, it is often difficult to determine the relationship of overlapping vessels to aneurysms when using 2D DSA. We compared 2D and 3D DSA in evaluation of intracranial aneurysms. METHODS: Thirty-six consecutive patients with cerebral aneurysms underwent 2D and 3D DSA. After standard 2D DSA, rotational DSA was performed. Maximum intensity projection (MIP) and shaded surface display (SSD) images were created from the rotational DSA data sets. All images were assessed randomly for overall image quality, presence of aneurysm, presence of aneurysmal lobulation, visualization of aneurysmal neck, and relationship to adjacent vessels. Data analysis was conducted for 40 aneurysms treated by clip placement. RESULTS: One aneurysm that was not detected at 2D DSA was classified as uncertain on the basis of rotational DSA. All aneurysms were classified as probably or definitively present on the basis of MIP and SSD findings. Overall image quality of rotational DSA, MIP, and SSD was statistically inferior to that of the standard 2D DSA for visualization of distal arteries. However, MIP and SSD images were significantly superior to those of standard 2D DSA for all other evaluations. For detection of lobulation, SSD images were significantly superior to other images, and for visualization of aneurysmal neck and relationship to neighboring arteries, SSD images were significantly superior to those of rotational DSA. For evaluation of the relationship to neighboring arteries, MIP images were significantly superior to those of rotational DSA. CONCLUSION: Three-dimensional DSA, especially SSD, provided more detailed information for evaluating cerebral aneurysms than did standard 2D and rotational DSA. PMID- 12372746 TI - Visualization of microvascularity in glioblastoma multiforme with 8-T high spatial-resolution MR imaging. AB - We used 8-T high-spatial-resolution gradient-echo MR imaging to directly visualize microvascularity in pathologically proved glioblastoma multiforme. Images were compared with 1.5-T high-spatial-resolution fast spin-echo T2 weighted images and digital subtraction angiograms. Preliminary data indicate that 8-T high-spatial-resolution MR imaging may enable the identification of areas of abnormal microvascularity in glioblastoma multiforme that are not visible with other routine clinical techniques. PMID- 12372747 TI - (31)phosphorous and single voxel proton MR spectroscopy and diffusion-weighted imaging in a case of star fruit poisoning. AB - We herein describe the case of a patient with chronic renal failure complicated by star fruit poisoning. T2-weighted and diffusion-weighted MR imaging showed hyperintense lesions at the thalami and right temporo-occipital cortex. Single voxel proton MR spectroscopy revealed elevation of lactate and (31)phosphorous MR spectroscopy revealed elevation of inorganic phosphate and decrease of phosphocreatine and nucleoside triphosphates. The imaging and metabolic changes indicated energy deprivation, with subsequent cortical necrosis proved at autopsy. PMID- 12372748 TI - Primary angiitis of the central nervous system and silent cortical hemorrhages. AB - Primary angiitis of the CNS is histopathologically characterized by ischemic lesions and small petechial hemorrhages. Unlike CT or conventional MR imaging, gradient-echo MR imaging depicts these chronic petechial hemorrhages. We herein report the case of biopsy-proved primary angiitis of the CNS in a 42-year-old man; whom gradient-echo MR imaging revealed multiple petechial hemorrhages in the cortical-subcortical brain regions. The identification of petechial hemorrhages by gradient-echo MR imaging promises to be a valuable surrogate marker supporting the diagnosis of primary angiitis of the CNS. PMID- 12372749 TI - Medulloblastoma with extensive nodularity: single photon emission CT study with iodine-123 metaiodobenzylguanidine. AB - We present the case of an infant with medulloblastoma with extensive nodularity, which had been called cerebellar neuroblastoma. MR imaging clearly revealed a nodular enhancement, which appeared as a grape-like lesion. Single photon emission CT revealed markedly high iodine-123 metaiodobenzylguanidine uptake in the enhancing tumor. Iodine-123 metaiodobenzylguanidine single photon emission CT may be useful in the diagnosis of medulloblastoma with extensive nodularity, which has been considered to be a subgroup of medulloblastoma with extensive neuronal differentiation. PMID- 12372750 TI - Thrombus formation at the neck of cerebral aneurysms during treatment with Guglielmi detachable coils. AB - BACKGROUND AND PURPOSE: Thromboembolic events are a common source of complications during Guglielmi detachable coil (GDC) treatment of intracranial aneurysms. Thrombus formation at the coil-parent artery interface is not commonly reported but is an important potential source of emboli. We describe nine cases in which thrombus propagated from GDCs into the parent artery during coil therapy of cerebral aneurysms and subsequent treatment of the thrombus. METHODS: A retrospective review of a procedural database was performed to identify cases in which thrombus occurred during GDC treatment of cerebral aneurysms during a 30 month period. All images were reviewed at the time of the procedure. Nine cases of thrombus forming at the coil-parent artery interface and five cases of distal emboli were identified among 210 cases. All patients underwent anticoagulation with heparin during GDC treatment procedures. RESULTS: Thrombus was identified at the coil-parent artery interface during GDC treatment in nine (4.3%) of 210 cases. In each case, the thrombus was recognized before distal embolic complication occurred and was successfully treated with heparin alone (five patients) or with heparin plus a glycoprotein IIb-IIIa inhibitor (four patients). CONCLUSION: Potential clinical complications can be avoided by early recognition of thrombus at the coil-parent artery interface and by administering appropriate medical therapy. PMID- 12372751 TI - Rapid enlargement of a posterior communicating artery aneurysm after Guglielmi detachable coil treatment of ipsilateral carotid artery aneurysms. AB - This case illustrates rapid aneurysm enlargement, presumably due to altered hemodynamics resulting from endovascular treatment of aneurysms on the same artery. We postulate that increased hemodynamic force directed to the inflow zone of the posterior communicating artery aneurysm was caused by the treatment of the two ophthalmic artery aneurysms. Originally, many of the flow vectors may have been directed into the larger ophthalmic segment aneurysm, located on the outside of the curve of the internal carotid artery. After treatment, flow may have been directed more smoothly around the carotid siphon and into the posterior communicating artery aneurysm. PMID- 12372752 TI - New expandable hydrogel-platinum coil hybrid device for aneurysm embolization. AB - This study introduces a new, hybrid embolic device that in addition to offering all the important attributes of existing detachable platinum coils also shows an enhanced ability to fill aneurysm cavities. The device consists of a carrier platinum coil coupled to an expandable hydrogel material, which undergoes a ninefold increase in volume when placed into a physiological environment. Distinct from previous devices aimed at speeding the organization of thrombus, the new device has been designed to entirely fill the aneurysm cavity, with complete or near-complete exclusion of thrombus. Unlike thrombus, the hydrogel material is stable and unaffected by natural thrombolytic processes and thus may diminish observed rates of aneurysm recanalization. We report the angiographic and histologic findings of the new, hybrid device used to treat experimental aneurysms in rabbits. PMID- 12372753 TI - Bare stent-graft technique: a new method of endoluminal vascular reconstruction for the treatment of giant and fusiform aneurysms. AB - We present our initial experience with a newly developed endovascular stent graft technique in the treatment of two patients with giant aneurysms. In both of these patients, surgery and conventional endovascular techniques were likely to fail. The technique resulted in the successful management of the aneurysms in both cases. Our technique is described, and related experiences in the literature are discussed. PMID- 12372754 TI - Complete recovery after early intraarterial recombinant tissue plasminogen activator thrombolysis of carotid T occlusion. AB - Carotid T occlusion (intracranial carotid bifurcation occlusion with involvement of A1 and M1 segments) is associated with poor outcome. In most cases, treatment with intraarterial thrombolysis within a 6-hour window has been unsuccessful. We describe the case of a 26-year-old woman who presented with severe neurologic deficits (National Institutes of Health Stroke Scale score of 23) secondary to angiographically proved right carotid T occlusion. She was treated with intraarterial infusion of recombinant tissue plasminogen activator that was started less than 3 hours after symptom onset (26 mg administered during 2 hours 15 minutes). Thrombolysis resulted in recanalization of all major intracranial vessels and complete neurologic recovery. Early intraarterial thrombolysis may be effective in the treatment of patients with carotid T occlusion and should be considered for appropriate candidates. PMID- 12372755 TI - Separate origins of the left internal and external carotid arteries from the aortic arch: MR angiographic findings. AB - Agenesis of the left common carotid artery with independent origins of the internal and external carotid arteries arising directly from the aortic arch is an extremely rare congenital anomaly. We present MR angiographic findings in a case with agenesis of left common carotid artery that, to our knowledge, is unique by virtue of its detection and description with MR angiography. PMID- 12372756 TI - MR imaging findings of spinal dural involvement with Wegener granulomatosis. AB - Involvement of the brain and meninges is rare in cases of Wegener granulomatosis, occurring in 2% to 8% of cases. Meningeal involvement in association with Wegener granulomatosis has scarcely been reported as being confined to the dura mater of brain on images and is thought to represent granulomatous infiltration. There are a few reported cases of Wegener granulomatosis that document involvement of dura at the level of the spinal cord. We present the case of a 52-year-old man with Wegener granulomatosis involving the cervical spinal dura and include detailed MR imaging findings. PMID- 12372757 TI - MR imaging of the spine in epidermal nevus syndrome. AB - We report two cases of epidermal nevus syndrome (ENS) involving the spine. MR imaging of the spine demonstrated intraspinal lipomas in both cases. Abnormal, enhancing, enlarged cervical and lumbosacral nerve roots were present in one patient. Spinal imaging for patients with ENS may help in the diagnosis of subtle intracranial manifestations, as it did in both of our cases. ENS has features similar to those of other neurocutaneous syndromes, such as neurofibromatosis type 1 and encephalocraniocutaneous lipomatosis. PMID- 12372760 TI - Dural arteriovenous fistulae: noninvasive diagnosis with dynamic MR digital subtraction angiography. PMID- 12372761 TI - Obstructive nephropathy and renal fibrosis. AB - Interstitial fibrosis has a major role in the progression of renal diseases. Several animal models are available for the study of renal fibrosis. The models of aminonucleoside-induced nephrotic syndrome, cyclosporin nephrotoxicity, and passive Heyman nephritis are characterized by molecular and cellular events similar to those that occur in obstructive nephropathy. Additionally, inhibition of angiotensin-converting enzyme exerts salutary effects on the progression of renal fibrosis in obstructive nephropathy. Unilateral ureteral obstruction (UUO) has emerged as an important model for the study of the mechanisms of renal fibrosis and also for the evaluation of the impact of potential therapeutic approaches to ameliorate renal disease. Many quantifiable pathophysiological events occur over the span of 1 wk of UUO, making this an attractive model for study. This paper reviews some of the ongoing studies that utilized a rodent model of UUO. Some of the findings of the animal model have been compared with observations made in patients with obstructive nephropathy. Most of the evidence suggests that the rodent model of UUO is reflective of human renal disease processes. PMID- 12372762 TI - Structural determinants and significance of regulation of electrogenic Na(+) HCO(3)(-) cotransporter stoichiometry. AB - Na(+)-HCO(3)(-) cotransporters play an important role in intracellular pH regulation and transepithelial HCO(3)(-) transport in various tissues. Of the characterized members of the HCO(3)(-) transporter superfamily, NBC1 and NBC4 proteins are known to be electrogenic. An important functional property of electrogenic Na(+)-HCO(3)(-) cotransporters is their HCO(3)(-):Na(+) coupling ratio, which sets the transporter reversal potential and determines the direction of Na(+)-HCO(3)(-) flux. Recent studies have shown that the HCO(3)(-):Na(+) transport stoichiometry of NBC1 proteins is either 2:1 or 3:1 depending on the cell type in which the transporters are expressed, indicating that the HCO(3)( ):Na(+) coupling ratio can be regulated. Mutational analysis has been very helpful in revealing the molecular mechanisms and signaling pathways that modulate the coupling ratio. These studies have demonstrated that PKA-dependent phosphorylation of the COOH terminus of NBC1 proteins alters the transport stoichiometry. This cAMP-dependent signaling pathway provides HCO(3)(-) transporting epithelia with an efficient mechanism for modulating the direction of Na(+)-HCO(3)(-) flux through the cotransporter. PMID- 12372763 TI - Ca(2+)/calmodulin-dependent and cAMP-dependent kinases in induction of c-fos in human mesangial cells. AB - Mesangial cell proliferation is an early event in several progressive renal diseases. When mesangial cells in culture are rendered quiescent by serum starvation and subsequently stimulated to proliferate, induction of c-fos is an early indicator of entry into the cell cycle. Several heparin-sensitive signals transduce these events. We have examined the potential roles of CaMK and PKA. Selective stimulation of CaMK with Ca(2+) ionophores and of PKA with forskolin or dibutyryl cAMP both result in induction of c-fos mRNA. CaMK but not PKA signaling is suppressed by low concentrations of heparin. Cross talk between the pathways has been demonstrated in some cells, with evidence of CaMK phosphorylating cAMP response element binding protein (CREB) at an inhibitory site and PKA suppressing CaMK-dependent signaling. However, in the present study, both pathways phosphorylated CREB on Ser(133) and induced c-fos in an additive manner. Serum, ionomycin, and forskolin all caused a rapid decline in cyclin D1 levels, but only serum effected a subsequent increase, indicative of cell cycle progression. We conclude that, in human mesangial cells, CaMK and PKA can both contribute to cell cycle entry, and, although induction of c-fos by CaMK requires active PKA, neither pathway antagonizes or synergizes c-fos induction by the other. PMID- 12372764 TI - Low-calcium diet in hypercalciuric enuretic children restores AQP2 excretion and improves clinical symptoms. AB - In this study, we analyzed the effect of a therapeutic intervention in 46 enuretic children, 26 (57%) of whom were hypercalciuric. All the patients (n = 46) were treated with DDAVP for 3-6 mo. The hypercalciuric patients (n = 26) received a low-calcium diet (approximately 500 mg/day) for the same period. After the therapy, the bed-wetting episodes stopped in 80% of the 46 patients tested. In those patients having low-AVP levels before the therapy, circulating AVP concentration returned to normal (>4 pg/ml), and the hypercalciuria was resolved in the hypercalciuric patients (calcium/creatinine ratio <0.2). Urinary aquaporin 2 (AQP2) levels were semiquantified by densitometric scanning and reported as a ratio between the intensity of the signal in the day vs. the night urine samples (day/night AQP2 ratio). In the hypercalciuric patients, the day/night AQP2 ratio returned to values close to those found in the healthy children (from 1.19 +/- 0.20 before to 0.69 +/- 0.10 after the treatment, n = 26, P = 0.03). In contrast, in the normocalciuric children we saw no significant modulation of AQP2 excretion (from 1.07 +/- 0.14 before to 0.99 +/- 0.14 after the treatment, n = 20). This study clearly demonstrates that urinary calcium levels modulate AQP2 excretion and is likely to be useful for treatment of children with enuresis. PMID- 12372765 TI - NF-kappaB inhibits transcription of the H(+)-K(+)-ATPase alpha(2)-subunit gene: role of histone deacetylases. AB - The H(+)-K(+)-ATPase alpha(2) (HKalpha(2)) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the kappaB DNA binding element at -104 to -94 influences basal HKalpha(2) gene transcription in these cells. Recombinant NF-kappaB p50 footprinted the region 116/-94 in vitro. Gel shift and supershift analysis revealed NF-kappaB p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated -104/-94 NF-kappaB element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-kappaB p50, p65, or their combination trans-repressed the HKalpha(2) promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-kappaB-mediated trans-repression of the HKalpha(2) promoter. HDAC6 overexpression inhibited HKalpha(2) promoter activity, and HDAC6 coimmunoprecipitated with NF-kappaB p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-kappaB to suppress HKalpha(2) transcription and identify NF-kappaB p50 and p65 as novel binding partners for HDAC6. PMID- 12372766 TI - UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney. AB - UT-B1 is the facilitated urea transporter of red blood cells (RBCs) and endothelial cells of descending vasa recta in the kidney. Immunoblotting with a polyclonal antibody against the C-ter sequence of rat UT-B1 revealed UT-B1 as both nonglycosylated (29 kDa) and N-glycosylated (47.5 and 33 kDa) proteins in RBC membranes, kidney medulla, brain, and bladder in rat. In testis, UT-B1 was expressed only as a nonglycosylated protein of 47.5 kDa. Immunocytochemistry confirmed that the location of UT-B1 is restricted to descending vasa recta. In brain, UT-B1 protein was found in astrocytes and ependymal cells. Cell bodies and perivascular end feet of astrocytes were labeled in brain cortex, whereas astrocyte cell processes were labeled in corpus callosum. Flow cytometry analysis of RBCs revealed a good cross-reactivity of the antibody with mouse and human UT B1. UT-B1 protein expression in rat kidney medulla was downregulated greatly by long-term [deamino-Cys(1),D-Arg(8)]vasopressin infusion and moderately by furosemide treatment. This study discloses an uneven distribution of UT-B1 protein within astrocytes and the regulation of renal UT-B1 protein by antidiuretic hormone. PMID- 12372767 TI - Sodium transporter abundance profiling in kidney: effect of spironolactone. AB - Renal tubule profiling studies were carried out to investigate the long-term effects of administration of spironolactone, a mineralocorticoid receptor antagonist, on abundances of the major Na transporter and Na channel proteins along the rat renal tubule. Oral administration of spironolactone for 7 days to NaCl-restricted rats did not significantly alter abundances of Na transporters expressed proximal to the macula densa, while substantially decreasing the abundances of the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha-subunit of the amiloride-sensitive epithelial Na channel (ENaC), and the 70-kDa form of the gamma-subunit of ENaC. A dependency of NCC expression on aldosterone was confirmed by showing increased NCC expression in response to aldosterone infusion in adrenalectomized rats. Immunoperoxidase labeling of ENaC in renal cortex confirmed that dietary NaCl restriction causes a redistribution of ENaC to the apical domain of connecting tubule cells and showed that high-dose spironolactone administration does not block this apical redistribution. In contrast, spironolactone completely blocked the increase in alpha-ENaC abundance in response to dietary NaCl restriction. We conclude that the protein abundances of NCC, alpha-ENaC, and the 70-kDa form of gamma-ENaC are regulated via the classical mineralocorticoid receptor, but the subcellular redistribution of ENaC in response to dietary NaCl restriction is not prevented by blockade of the mineralocorticoid receptor. PMID- 12372769 TI - Phospholipid metabolite production in human urothelial cells after protease activated receptor cleavage. AB - Our laboratory demonstrated previously that stimulation of protease-activated receptors (PARs) on the human urothelial carcinoma cell line RT4 results in activation of a calcium-independent phospholipase A(2) (iPLA(2)), leading to arachidonic acid and PGE(2) release. In this study, we have examined PAR activation in normal human urothelial cells (HUR) leading to the production of inflammatory or cytoprotective phospholipid metabolites. The presence of both PAR 1 and PAR-2 on HUR was confirmed by immunoblotting. Stimulation of PAR-1 with thrombin or PAR-2 by tryptase leads to activation of a membrane-associated iPLA(2) and the production of platelet-activating factor, arachidonic acid, and PGE(2). These responses were all blocked by pretreatment with the iPLA(2) selective inhibitor bromoenol lactone. Thus stimulation of PAR-1 or PAR-2 on HUR leads to iPLA(2)-catalyzed phospholipid hydrolysis, resulting in the production of metabolites that may mediate inflammation or provide cytoprotection to the bladder. PMID- 12372768 TI - Estrogen upregulates renal angiotensin II AT(2) receptors. AB - AT(2) receptors may act in opposition to and in balance with AT(1) receptors, their stimulation having beneficial effects. We found renal AT(2) receptor expression in female mice higher than in male mice. We asked the question of whether such expression might be estrogen dependent. In male, female, ovariectomized, and estrogen-treated ovariectomized mice, we studied renal AT(1) and AT(2) receptors by immunocytochemistry and autoradiography, AT(2) receptor mRNA by RT-PCR, and cAMP, cGMP, and PGE(2) by RIA. AT(1) receptors predominated. AT(2) receptors were present in glomeruli, medullary rays, and inner medulla, and in female kidney capsule. AT(1) and AT(2) receptors colocalized in glomeruli. Female mice expressed fewer glomerular AT(1) receptors. Ovariectomy decreased AT(1) receptors in medullary rays and capsular AT(2) receptors. Estrogen administration normalized AT(1) receptors in medullary rays and increased AT(2) receptors predominantly in capsule and inner medulla, and also in glomeruli, medullary rays, and inner stripe of outer medulla. In medullas of estrogen treated ovariectomized mice there was higher AT(2) receptor mRNA, decreased cGMP, and increased PGE(2) content. We propose that the protective effects of estrogen may be partially mediated through enhancement of AT(2) receptor stimulation. PMID- 12372770 TI - Formate-stimulated NaCl absorption in the proximal tubule is independent of the pendrin protein. AB - A significant fraction of active chloride reabsorption across the apical membrane of the proximal tubule is mediated by a chloride/formate exchange process, whereby intracellular formate drives the transport of chloride into the cell. When chloride/formate exchange operates in parallel with Na(+)/H(+) exchange and H(+)-coupled recycling of formate, the net result is electroneutral NaCl reabsorption. Pendrin is the protein product of the PDS gene (SLC26A4) and functions in several different anion exchange modes, including chloride/formate exchange. Pendrin is expressed in the kidney and may serve as the transporter responsible for formate-dependent NaCl reabsorption. In the present study, Pds knockout mice were used to determine the role of pendrin in proximal tubule chloride reabsorption. We show that formate-dependent NaCl absorption in microperfused proximal tubules is similar between wild-type and pendrin-deficient mice. In addition, there is no difference in the rate of formate-mediated chloride transport in brush-border membrane vesicles isolated from wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not responsible for formate-dependent NaCl reabsorption in the proximal tubule. PMID- 12372771 TI - Superoxide stimulates NaCl absorption by the thick ascending limb. AB - The thick ascending limb of the loop of Henle (THAL) plays an important role in the regulation of NaCl and water reabsorption. In vivo studies have shown that the free radical superoxide (O) stimulates Na and water reabsorption by the kidney. However, it is not known whether O regulates transport along the nephron in general or in the THAL specifically. We hypothesized that O stimulates THAL NaCl reabsorption. Cl absorption was measured in isolated, perfused THALs from Sprague-Dawley rats. First, we tested whether extracellular O stimulates Cl absorption. Addition of the O-generating system xanthine oxidase/hypoxanthine increased Cl absorption from 112.7 +/- 12.0 to 146.2 +/- 13.9 pmol. mm(-1). min( 1), a 33% increase (P < 0.03). When superoxide dismutase (300 U/ml) was present in the bath, addition of xanthine oxidase/hypoxanthine did not significantly increase Cl absorption (116.9 +/- 13.8 vs. 102.5 +/- 8.5 pmol. mm(-1). min(-1)). Furthermore, adding 200 nM H(2)O(2) to the bath did not significantly affect Cl absorption (from 130.3 +/- 13.7 to 125.3 +/- 19.6 pmol. mm(-1). min(-1)). Because extracellular O stimulated Cl absorption, we next tested whether endogenously produced O could stimulate transport. Under basal conditions, THALs produced detectable amounts of O, as measured by lucigenin-enhanced chemiluminescence. Adding the O scavenger tempol to the bath decreased Cl absorption from 198.1 +/- 35.4 to 132.4 +/- 23.5 pmol. mm(-1). min(-1), a 31% decrease (P < 0.02). To make sure tempol was not exerting cytotoxic effects, we tested whether its effect was reversible. With tempol in the bath, Cl absorption was 117.2 +/- 9.3 pmol. mm( 1). min(-1). Sixty minutes after tempol was removed from the bath, Cl absorption had increased to 149.2 +/- 9.1 pmol. mm(-1). min(-1) (P < 0.05). We concluded that both exogenous and endogenous O stimulate THAL NaCl absorption. To our knowledge, these are the first data showing a direct effect of O on nephron transport. PMID- 12372772 TI - Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells. AB - Medullary thick ascending limb (mTAL) cells in primary culture express the Ca(2+) sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca(2+) (Ca(o)(2+)) concentration, resulting in increases of intracellular Ca(2+) concentration and PKC activity. Exposure of mTAL cells to either Ca(o)(2+) or the CaR-selective agonist poly-L-arginine increased TNF-alpha synthesis. Moreover, the response to Ca(o)(2+) was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca(o)(2+), suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca(2+)-mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE(2) synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE(2) production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca(2+)-mediated TNF production. Significant inhibition of COX-2 protein expression and PGE(2) synthesis also was observed when cells were challenged with Ca(o)(2+) in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion. PMID- 12372773 TI - Induction of a laminin isoform and alpha(3)beta(1)-integrin in renal ischemic injury and repair in vivo. AB - Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of approximately 220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the alpha(3)-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and alpha(3)beta(1) integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium. PMID- 12372774 TI - Interaction of MAPK and 12-lipoxygenase pathways in growth and matrix protein expression in mesangial cells. AB - The lipoxygenase (LO) pathway of arachidonate metabolism and mitogen-activated protein kinases (MAPKs) can mediate cellular growth and ANG II effects in vascular smooth muscle cells. However, their role in renal mesangial cells (MC) is not very clear. ANG II treatment of rat MC significantly increased 12-LO mRNA expression and formation of the 12-LO product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE; P < 0.03]. ANG II-induced [(3)H]leucine incorporation was blocked by an LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (P < 0.02). 12(S) HETE and ANG II directly induced cellular hypertrophy and fibronectin (FN) expression (P < 0.01) to a similar extent. ANG II and 12(S)-HETE led to activation of p38(MAPK) and its target transcription factor cAMP-responsive element-binding protein (CREB). ANG II- and 12(S)-HETE-induced CREB activation and [(3)H]leucine incorporation were blocked by the p38(MAPK) inhibitor SB 202190. A specific molecular inhibitor of rat 12-LO mRNA, namely, a novel ribozyme, could attenuate ANG II-induced FN mRNA. Thus p38(MAPK)-dependent CREB activation may mediate ANG II- and LO product-induced FN expression and cellular growth in rat MC. ANG II effects may be mediated by the LO pathway. These results suggest a novel interaction between LO and p38(MAPK) activation in MC matrix synthesis associated with renal complications. PMID- 12372775 TI - Effects of dietary salt changes on renal renin-angiotensin system in rats. AB - Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in the kidney (cortex and medulla) and plasma of Wistar-Kyoto rats on a low-sodium (LS; 0.025% NaCl; n = 8), normal-sodium (NS; 1% NaCl; n = 7), or high-sodium (HS; 8% NaCl; n = 7) diet for 21 days. RA, ANG I, and ANG II levels increased in a manner inversely related to sodium content of the diet in both plasma and renal tissues. The LS diet resulted in a 16-, 2.8-, and 1.8-fold increase in plasma RA, ANG I, and ANG II levels, respectively, compared with those in HS rats. In the renal cortex and medulla, RA, ANG I, and ANG II levels were also increased by diminution of dietary salt content but, in contrast to plasma, ANG II levels increased much more than RA or ANG I levels [5.4 (cortex)- and 4.7 (medulla)-fold compared with HS rats]. In summary, we demonstrated variations of ANG II levels in the kidney during dietary salt modifications. Our results confirm that RA and ACE activity are not the steps limiting intrarenal ANG II levels. Nevertheless, despite RA and ACE activity differences between renal cortex and medulla, ANG I and ANG II levels are equivalent in these two tissues; these results argue against a compartmentalization of RAS in these two intrarenal areas. PMID- 12372776 TI - Contribution of angiotensin II internalization to intrarenal angiotensin II levels in rats. AB - This study was designed to determine the involvement of AT(1) receptors in the uptake of ANG II in the kidney of rats exposed to differing salt intake. Male Wistar-Kyoto rats were treated with a normal-salt (NS; 1% NaCl, n = 7) or a low salt (LS; 0.025% NaCl, n = 7) diet combined with (LS+Los, n = 7; NS+Los, n = 7) or without losartan (30 mg. kg(-1). day(-1)), an AT(1) receptor antagonist. Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in plasma, renal cortex, and medulla. In LS rats, in both plasma and renal cortex, the increase in RA was associated with an increase in ANG I and ANG II levels compared with NS rats, but intrarenal ANG II levels increased more than ANG I levels. In NS+Los rats, the increase in RA in plasma was followed by a marked increase in plasma ANG I and ANG II levels compared with NS rats whereas in the kidney the increase of renal RA was followed by a decrease of the levels of these peptides. The same pattern was observed in LS+Los rats, but the decrease in renal ANG II levels was much more pronounced in LS+Los rats than in NS+Los rats. Our results suggest that the increase in renal ANG II levels after salt restriction results mainly from an uptake of ANG II, via AT(1) receptors. Such elevated intrarenal ANG II levels could contribute to maintain sodium and fluid balance and arterial blood pressure during salt deficiency states. PMID- 12372777 TI - Regulation of expression of the SN1 transporter during renal adaptation to chronic metabolic acidosis in rats. AB - During chronic metabolic acidosis, renal glutamine utilization increases markedly. We studied the expression of the system N1 (SN1) amino acid transporter in the kidney during chronic ammonium chloride acidosis in rats. Acidosis caused a 10-fold increase in whole kidney SN1 mRNA level and a 100-fold increase in the cortex. Acidosis increased Na(+)-dependent glutamine uptake into basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated from rat cortex (BLMV, 219 +/- 66 control vs. 651 +/- 180 pmol. mg(-1). min(-1) acidosis; BBMV, 1,112 +/- 189 control vs. 1,652 +/- 148 pmol. mg(-1). min(-1) acidosis, both P < 0.05). Na(+)-independent uptake was unchanged by acidosis in BLMV and BBMV. The acidosis-induced increase in Na(+)-dependent glutamine uptake was eliminated by histidine, confirming transport by system N. SN1 protein was detected only in BLMV and BBMV from acidotic rats. After recovery from acidosis, SN1 mRNA and protein and Na(+)-dependent glutamine uptake activity rapidly returned to control levels. These data provide evidence that regulation of expression of the SN1 amino acid transporter is part of the renal homeostatic response to acid-base imbalance. PMID- 12372778 TI - Three GADD45 isoforms contribute to hypertonic stress phenotype of murine renal inner medullary cells. AB - Mammalian renal inner medullary (IM) cells routinely face and resist hypertonic stress. Such stress causes DNA damage to which IM cells respond with cell cycle arrest. We report that three growth arrest and DNA damage-inducible 45 (GADD45) isoforms (GADD45alpha, GADDD45beta, and GADD45gamma) are induced by acute hypertonicity in murine IM cells. Maximum induction occurs 16-18 h after the onset of hypertonicity. GADD45gamma is induced more strongly (7-fold) than GADD45beta (3-fold) and GADD45alpha (2-fold). GADD45alpha and GADD45beta protein induction is more pronounced and stable compared with the corresponding transcripts. Hypertonicity of various forms (NaCl, KCl, sorbitol, or mannitol) always induces GADD45 transcripts, whereas nonhypertonic hyperosmolality (urea) has no effect. Actinomycin D does not prevent hypertonic GADD45 induction, indicating that mRNA stabilization is the mechanism that mediates this induction. GADD45 induction patterns in IM cells exposed to 10 different stresses suggest isoform specificity, but similar functions, of individual isoforms during hypertonicity, heat shock, and heavy metal stress, when GADD45gamma induction is strongest (17-fold). These data associate all known GADD45 isoforms with the hypertonicity phenotype of renal IM cells. PMID- 12372779 TI - Phosphatase inhibitors increase the open probability of ENaC in A6 cells. AB - We studied the cellular phosphatase inhibitors okadaic acid (OKA), calyculin A, and microcystin on the epithelial sodium channel (ENaC) in A6 renal cells. OKA increased the amiloride-sensitive current after approximately 30 min with maximal stimulation at 1-2 h. Fluctuation analysis of cell-attached patches containing a large number of ENaC yielded power spectra with corner frequencies in untreated cells almost two times as large as in cells pretreated for 30 min with OKA, implying an increase in single channel open probability (P(o)) that doubled after OKA. Single channel analysis showed that, in cells pretreated with OKA, P(o) and mean open time approximately doubled. Two other phosphatase inhibitors, calyculin A and microcystin, had similar effects on P(o) and mean open time. An analog of OKA, okadaone, that does not inhibit phosphatases had no effect. Pretreatment with 10 nM OKA, which blocks protein phosphatase 2A (PP2A) but not PP1 in mammalian cells, had no effect even though both phosphatases are present in A6 cells. Several proteins were differentially phosphorylated after OKA, but ENaC subunit phosphorylation did not increase. We conclude that, in A6 cells, there is an OKA-sensitive phosphatase that suppresses ENaC activity by altering the phosphorylation of a regulatory molecule associated with the channel. PMID- 12372780 TI - Functional and molecular characterization of the shark renal Na-K-Cl cotransporter: novel aspects. AB - The Na-K-Cl cotransporter isoform 1 (NKCC1) has been isolated from several species, including Squalus acanthias. A second kidney-specific isoform (NKCC2) has been cloned mainly from higher vertebrates. Here, we have isolated the S. acanthias NKCC2 and found that it is produced in at least four spliced variants (saNKCC2A, saNKCC2F, saNKCC2AF, and saNKCC2AFno8) of approximately 1,090 residues. Expression of these transcripts in Xenopus laevis oocytes revealed that only the A and F variants are functional and that they are more active after incubation in low-Cl or hyperosmolar media. Rates of activation after exposure to these media were exceptionally rapid, demonstrating for the first time that the NKCC2 itself represents an important site of regulation by Cl and that extracellular domains are involved. Another remarkable finding in this study was the failure to identify NKCC2B, a variant found in the kidney of higher vertebrates and expressed specifically in macula densa cells. This result, in conjunction with the fact that the shark kidney lacks a well-developed juxtaglomerular apparatus, suggests that the B exon evolved as a result of selective pressure (presumably by exon duplication) and that a restricted relationship exists between NKCC2B and macula densa. PMID- 12372781 TI - Effect of renal nerve stimulation on responsiveness of the rat renal vasculature. AB - When the renal nerves are stimulated with sinusoidal stimuli over the frequency range 0.04-0.8 Hz, low (< or =0.4 Hz)- but not high (> or =0.4 Hz)-frequency oscillations appear in renal blood flow (RBF) and are proposed to increase responsiveness of the renal vasculature to stimuli. This hypothesis was tested in anesthetized rats in which RBF responses to intrarenal injection of norepinephrine and angiotensin and to reductions in renal arterial pressure (RAP) were determined during conventional rectangular pulse and sinusoidal renal nerve stimulation. Conventional rectangular pulse renal nerve stimulation decreased RBF at 2 Hz but not at 0.2 or 1.0 Hz. Sinusoidal renal nerve stimulation elicited low frequency oscillations (< or =0.4 Hz) in RBF only when the basal carrier signal frequency produced renal vasoconstriction, i.e., at 5 Hz but not at 1 Hz. Regardless of whether renal vasoconstriction occurred, neither conventional rectangular pulse nor sinusoidal renal nerve stimulation altered renal vasoconstrictor responses to norepinephrine and angiotensin. The RBF response to reduction in RAP was altered by both conventional rectangular pulse and sinusoidal renal nerve stimulation only when renal vasoconstriction occurred: the decrease in RBF during reduced RAP was greater. Sinusoidal renal nerve stimulation with a renal vasoconstrictor carrier frequency results in a decrease in RBF with superimposed low-frequency oscillations. However, these low-frequency RBF oscillations do not alter renal vascular responsiveness to vasoconstrictor stimuli. PMID- 12372782 TI - Structure/function analysis of Na(+)-K(+)-ATPase central isoform-specific region: involvement in PKC regulation. AB - Specific functions served by the various Na(+)-K(+)-ATPase alpha-isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH(2) terminus and a 10-residue region near the center of each molecule (isoform specific region; ISR). Although the NH(2) terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat alpha(1)- and alpha(2)-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K(+) deocclusion pattern and their response to PKC activation. Comparisons with rat full-length alpha(1)- and alpha(2)-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K(+) deocclusion. PMID- 12372783 TI - Existence of a regulatory loop between MCP-1 and TGF-beta in glomerular immune injury. AB - Glomerular upregulation of monocyte chemotactic protein-1 (MCP-1), followed by an influx of monocytes resulting eventually in extracellular matrix deposition is a common sequel of many types of glomerulonephritis. However, it is not entirely clear how early expression of MCP-1 is linked to the later development of glomerulosclerosis. Because transforming growth factor-beta (TGF-beta) is a key regulator of extracellular matrix proteins, we hypothesized that there might be a regulatory loop between early glomerular MCP-1 induction and subsequent TGF-beta expression. To avoid interference with other cytokines that may be released from infiltrating monocytes, isolated rat kidneys were perfused with a polyclonal anti thymocyte-1 antiserum (ATS) and rat serum (RS) as a complement source to induce glomerular injury. Renal TGF-beta protein and mRNA expressions were strongly stimulated after perfusion with ATS-RS. This effect was attenuated by coperfusion with a neutralizing anti-MCP-1 but was partly mimicked by perfusion with recombinant MCP-1 protein. On the other hand, renal MCP-1 expression and production were stimulated by administration of ATS-RS. Additional perfusion with an anti-TGF-beta antibody further aggravated this increase, whereas application of recombinant TGF-beta protein reduced MCP-1 formation. Our data demonstrate an intrinsic regulatory loop in which increased MCP-1 levels stimulate TGF-beta formation in resident glomerular cells in the absence of infiltrating immune competent cells. PMID- 12372784 TI - Sustained activation of MAPK/ERKs signaling pathway in cystic kidneys from bcl-2 /- mice. AB - Cell proliferation, survival, and differentiation are carefully orchestrated processes during nephrogenesis that become aberrant during renal cyst formation. Signaling through focal adhesion kinase (FAK) impacts these processes, although its role during nephrogenesis requires further delineation. We previously demonstrated that phosphorylation of FAK and paxillin is not downregulated in cystic kidneys from B cell lymphoma/leukemia-2 (bcl-2) -/- mice. Here we examine whether FAK downstream signaling pathways are affected in these cystic kidneys. Cystic kidneys from bcl-2 -/- mice exhibited sustained phosphorylation of Src and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK, ERK1). However, similar levels of expression were noted for phosphorylated c-Jun NH(2)-terminal kinase, phosphatidylinositol-3-kinase, and its target protein kinase B/ATP-dependent tyrosine kinase in kidneys from postnatal day 20 bcl-2 +/+ and bcl-2 -/- mice. We also examined expression of the adapter protein Shc, implicated in growth and apoptosis. Expression of p66(Shc) decreases to low levels in postnatal kidneys, whereas p52/p46(Shc) was constitutively expressed during nephrogenesis. Shc expression was similar in normal and cystic kidneys. Therefore, sustained activation of MAPK/ERKs through the Src/FAK pathway may contribute to the hyperproliferation observed in cystic kidneys from bcl-2 -/- mice. PMID- 12372785 TI - Acute acidosis-induced alteration in bone bicarbonate and phosphate. AB - During an acute fall in systemic pH due to a decrease in the concentration of serum bicarbonate ([HCO(3)(-)]), metabolic acidosis, there is an influx of hydrogen ions into the mineral phase of bone, buffering the decrement in pH. When bone is cultured in medium modeling acute metabolic acidosis, the influx of hydrogen ions is coupled to an efflux of sodium and potassium and a depletion of mineral carbonate. These ionic fluxes would be expected to neutralize some of the excess hydrogen ions and restore the pH toward normal. Approximately one-third of bone carbonate is located on the hydration shell of apatite, where it is readily accessible to the systemic circulation, whereas the remainder is located in less accessible areas. We hypothesize that the surface of bone would respond to acidosis in a different manner than the interior of bone, with depletion of carbonate preferentially occurring on the bone surface. We utilized a high resolution scanning ion microprobe with secondary ion mass spectroscopy to localize the changes in bone carbonate, as measured by HCO(3)(-), and phosphate and determine their relative contribution to the buffering of hydrogen ions during acute metabolic acidosis. Neonatal mouse calvariae were incubated in control medium (pH approximately 7.44, [HCO(3)(-)] approximately 27 mM) or in medium acidified by a reduction in [HCO(3)(-)] (pH approximately 7.14, [HCO(3)( )] approximately 13). Compared with control, after a 3-h incubation in acidic medium there is a fivefold decrease in surface HCO(3)(-) with respect to the carbon-carbon bond (C(2)) and a threefold decrease in surface HCO(3)(-) with respect to the carbon-nitrogen bond (CN) with no change in cross-sectional HCO(3)(-). Compared with control, after a 3-h incubation in acidic medium there is a 10-fold decrease in cross-sectional phosphate with respect to C(2) and a 10 fold decrease in cross-sectional phosphate with respect to CN, with no change in surface phosphate. On the bone surface, there is a fourfold depletion of HCO(3)( ) in relation to phosphate, and, in cross section, a sevenfold depletion of phosphate in relation to HCO(3)(-). Thus acute hydrogen ion buffering by bone involves preferential dissolution of surface HCO(3)(-) and of cross-sectional phosphate. PMID- 12372786 TI - Apical H(+)/base transporters mediating bicarbonate absorption and pH(i) regulation in the OMCD. AB - The outer medullary collecting duct (OMCD) plays an important role in mediating transepithelial HCO transport [J(HCO(3)(-))] and urinary acidification. HCO absorption by type A intercalated cells in the OMCD inner stripe (OMCD(is)) segment is thought to by mediated by an apical vacuolar H(+)-ATPase and H(+)-K(+) ATPase coupled to a basolateral Cl(-)-HCO exchanger (AE1). Besides these Na(+) independent transporters, previous studies have shown that OMCD(is) type A intercalated cells have an apical electroneutral EIPA-sensitive, DIDS-insensitive Na(+)-HCO cotransporter (NBC3); a basolateral Na(+)/H(+) antiporter; and a basolateral Na(+)-K(+)-ATPase. In this study, we reexamined the Na(+) dependence of transepithelial Na(+) transport in the OMCD(is) and determined the role of apical NBC3 in intracellular (pH(i)) regulation in OMCD(is) type A intercalated cells. Control tubules absorbed HCO at a rate of approximately 13 pmol. min(-1). mm(-1). Lowering luminal Na(+) from 140 to 40 mM decreased [J(HCO(3)(-))] by approximately 15% without a change in transepithelial potential (V(te)). Furthermore, 50 microM EIPA (lumen) also decreased [J(HCO(3)(-))] by approximately 13% without a change in V(te). The effect of lowering luminal Na(+) and adding EIPA were not additive. These results demonstrate that [J(HCO(3)(-))] in the OMCD(is) is in part Na(+) dependent. In separate experiments, the pH(i) recovery rate after an NH prepulse was monitored in single type A intercalated cells with confocal fluorescence microscopy. The pH(i) recovery rate was approximately 0.21 pH/min in Na(+)-containing solutions and decreased to approximately 0.16 pH/min with EIPA (50 microM, lumen). In tubules perfused/bathed without Na(+), luminal Na(+) addition resulted in a pH(i) recovery rate of approximately 0.36 pH/min, whereas the Na(+)-independent recovery rate was approximately 0.16 pH/min. EIPA (50 microM, lumen) decreased the Na(+)-dependent pH(i) recovery rate to approximately 0.07 pH/min. The Na(+) independent recovery rate was decreased to approximately 0.06 pH/min by bafilomycin (10 nM, lumen) and to approximately 0.10 pH/min using Schering 28080 (10 microM, lumen). These findings indicate that NBC3 contributes to pH(i) regulation in OMCD(is) type A intercalated cells and plays only a minor role in mediating [J(HCO(3)(-))] in the OMCD(is). PMID- 12372787 TI - Mild hyperuricemia induces glomerular hypertension in normal rats. AB - Mildly hyperuricemic rats develop renin-dependent hypertension and interstitial renal disease. Hyperuricemia might also induce changes in glomerular hemodynamics. Micropuncture experiments under deep anesthesia were performed in Sprague-Dawley rats fed a low-salt diet (LS group), fed a low-salt diet and treated with oxonic acid (OA/LS group), and fed a low-salt diet and treated with oxonic acid + allopurinol (OA/LS/AP group) for 5 wk. The OA/LS group developed hyperuricemia and hypertension compared with the LS group: 3.1 +/- 0.2 vs. 1.1 +/ 0.2 mg/dl (P < 0.01) and 143 +/- 4 vs. 126 +/- 2 mmHg (P < 0.01). Hyperuricemic rats developed increased glomerular capillary pressure compared with the LS rats: 56.7 +/- 1.2 vs. 51.9 +/- 1.4 mmHg (P < 0.05). Pre- and postglomerular resistances were not increased. Histology showed afferent arteriolar thickening with increased alpha-smooth muscle actin staining of the media. Allopurinol prevented hyperuricemia (1.14 +/- 0.2 mg/dl), systemic (121.8 +/- 2.8 mmHg) and glomerular hypertension (50.1 +/- 0.8 mmHg), and arteriolopathy in oxonic acid treated rats. Linear regression analysis showed that glomerular capillary pressure and arteriolar thickening correlated positively with serum uric acid and systolic blood pressure. Glomerular hypertension may be partially mediated by an abnormal vascular response to systemic hypertension due to arteriolopathy of the afferent arteriole. PMID- 12372788 TI - SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes. AB - The apical- and basolateral-specific distribution of target soluble N ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H(+)-ATPase in alpha- but not beta-intercalated cells, suggesting its involvement in H(+)-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability. PMID- 12372789 TI - Decreased renal heme oxygenase-1 expression contributes to decreased renal function during cirrhosis. AB - Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme, catalyzing the oxidative cleavage of heme molecules to biliverdin, carbon monoxide, and iron. The present study was designed to investigate the role of HO 1 in the pathogenesis of renal dysfunction during cirrhosis. Biliary cirrhosis was induced in rats by common bile duct ligation (CBDL). Animals were studied 2 and 5 wk after surgery. In kidney from CBDL rats, HO-1 protein expression increased slightly at 2 wk but was abolished at 5 wk. In addition, we confirmed histologically that HO-1 expression was suppressed in renal tubules and interlobular arterioles in 5-wk-old CBDL rats. Conversely, HO-1 expression in liver was strongly increased. Consistent with the development of cirrhosis and renal dysfunction mean arterial pressure (MAP), glomerular filtration rate (GFR), and renal blood flow (RBF) were decreased in CBDL rats compared with sham operated controls. In sham rats, treatment with the selective HO inhibitor zinc protoporphyrin markedly decreased GFR and RBF to values similar to those measured in CBDL rats without decreasing MAP. In conclusion, decreased renal HO-1 expression contributes to deteriorated renal function and hemodynamics during cirrhosis. This finding provides a novel mechanism for the pathophysiology of renal dysfunction during cirrhosis. PMID- 12372790 TI - Overload proteinuria is followed by salt-sensitive hypertension caused by renal infiltration of immune cells. AB - Recent evidence suggests that salt-sensitive hypertension develops as a consequence of renal infiltration with immunocompetent cells. We investigated whether proteinuria, which is known to induce interstitial nephritis, causes salt sensitive hypertension. Female Lewis rats received 2 g of BSA intraperitoneally daily for 2 wk. After protein overload (PO), 6 wk of a high-salt diet induced hypertension [systolic blood pressure (SBP) = 156 +/- 11.8 mmHg], whereas rats that remained on a normal-salt diet and control rats (without PO) on a high-salt diet were normotensive. Administration of mycophenolate mofetil (20 mg. kg(-1). day(-1)) during PO resulted in prevention of proteinuria-related interstitial nephritis, reduction of renal angiotensin II-positive cells and oxidative stress (superoxide-positive cells and renal malondialdehyde content), and resistance to the hypertensive effect of the high-salt diet (SBP = 129 +/- 12.2 mmHg). The present studies support the participation of renal inflammatory infiltrate in the pathogenesis of salt-sensitive hypertension and provide a direct link between two risk factors of progressive renal damage: proteinuria and hypertension. PMID- 12372791 TI - Angiotensin II clamp prevents the second step in renal apical NHE3 internalization during acute hypertension. AB - Acute hypertension inhibits proximal tubule (PT) sodium reabsorption. The resultant increase in NaCl delivery to the macula densa suppresses renin release. We tested whether the sustained pressure-induced inhibition of PT sodium reabsorption requires a renin-mediated decrease in ANG II levels. Plasma ANG II concentration of anesthesized Sprague-Dawley rats was clamped by simultaneous infusion of the ANG I-converting enzyme inhibitor captopril (12 microg/min) and ANG II (20 ng. kg(-1). min(-1)). Blood pressure was increased 50 mmHg for 20 min by arterial constriction +/- ANG II clamp or by sham operation. This acute hypertension increased urine output and endogenous Li(+) clearance, and these responses were blunted 40-50% in ANG II clamped rats. Acute hypertension provoked a rapid redistribution of Na(+)/H(+) exchanger isoform 3 (NHE3) out of apical brush-border membranes (21 +/- 4% decrease of total NHE3 abundance) to endosomal/lysosomal membranes (16 +/- 6% increase of total). In ANG II-clamped rats, acute hypertension also provoked disappearance of NHE3 from the apical membranes (27 +/- 2% decrease of total), but NHE3 was shifted to membranes enriched in intermicrovillar cleft and dense apical tubules (step 1) rather than endosomal/lysosomal membranes (step 2). This difference was independently confirmed by confocal analysis. In contrast, the pressure-induced redistribution of Na(+)-P(i) cotransporter type 2 was not altered by ANG II clamp. We conclude that the responses to acute hypertension, including diuresis and redistribution of PT NHE3 into intracellular membranes, require a responsive renin-angiotensin system and that the responses may be induced by the sustained increase in NaCl delivery to the macula densa during acute hypertension. PMID- 12372792 TI - Gene expression profiling reveals role for EGF-family ligands in mesangial cell proliferation. AB - Control of mesangial cell growth and matrix accumulation is critical for normal development of the glomerular tuft and progression of glomerular injury, but the genes that control mesangial cell growth are not well understood. We used high density oligonucleotide microarrays to analyze gene expression in well differentiated human mesangial cells treated with serum to stimulate proliferation. Parallel measurement of >12,000 genes and expressed sequence tags identified 5,806 mRNA transcripts in quiescent, unstimulated cells and 609 genes significantly induced or repressed by serum. Functional classification of serum regulated genes revealed many genes not directly related to cell cycle progression that, instead, might control renal hemodynamics and glomerular filtration or cause tissue injury, leukocyte exudation, matrix accumulation, and fibrosis. Hierarchical cluster analysis defined sets of coregulated genes with similar functions and identified networks of proinflammatory genes with similar expression patterns. Pathway analysis of the gene expression profile suggested an autocrine role in mesangial cell proliferation for three growth factors in the epidermal growth factor (EGF) family: heparin-binding EGF-like growth factor, amphiregulin, and epiregulin. A functional role for EGF receptor (EGFR) activation was confirmed by blocking serum-induced proliferation with an EGFR selective kinase inhibitor and a specific EGFR-neutralizing antibody. Taken together, these results suggest a role for EGFR signaling in control of mesangial cell growth in response to serum. PMID- 12372793 TI - A rat kidney tubule suspension for the study of vasopressin-induced shuttling of AQP2 water channels. AB - AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP. PMID- 12372794 TI - Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli. AB - The interaction of weak electromagnetic fields (EMF) with living cells is a most important but still unresolved biophysical problem. For this interaction, thermal and other types of noise appear to cause severe restrictions in the action of weak signals on relevant components of the cell. A recently presented general concept of regulation of ion and substrate pathways through microvilli provides a possible theoretical basis for the comprehension of physiological effects of even extremely low magnetic fields. The actin-based core of microfilaments in microvilli is proposed to represent a cellular interaction site for magnetic fields. Both the central role of F-actin in Ca2+ signaling and its polyelectrolyte nature eliciting specific ion conduction properties render the microvillar actin filament bundle an ideal interaction site for magnetic and electric fields. Ion channels at the tip of microvilli are connected with the cytoplasm by a bundle of microfilaments forming a diffusion barrier system. Because of its polyelectrolyte nature, the microfilament core of microvilli allows Ca2+ entry into the cytoplasm via nonlinear cable-like cation conduction through arrays of condensed ion clouds. The interaction of ion clouds with periodically applied EMFs and field-induced cation pumping through the cascade of potential barriers on the F-actin polyelectrolyte follows well-known physical principles of ion-magnetic field (MF) interaction and signal discrimination as described by the stochastic resonance and Brownian motor hypotheses. The proposed interaction mechanism is in accord with our present knowledge about Ca2+ signaling as the biological main target of MFs and the postulated extreme sensitivity for coherent excitation by very low field energies within specific amplitude and frequency windows. Microvillar F-actin bundles shielded by a lipid membrane appear to function like electronic integration devices for signal-to noise enhancement; the influence of coherent signals on cation transduction is amplified, whereas that of random noise is reduced. PMID- 12372795 TI - PARs in the stars: proteinase-activated receptors and astrocyte function. Focus on "Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways". PMID- 12372796 TI - Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways. AB - Protease-activated receptors (PARs), newly identified members of G protein coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/(betagamma-subunits) phosphatidylinositol 3-kinase branch, and 2) the G(q)-PLC-(InsP(3) receptor)/Ca2+ -PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation. PMID- 12372797 TI - Inhibition of DNA replication by fish oil-treated cytoplasm is counteracted by fish oil-treated nuclear extract. AB - We have recently noted that cells treated with fish oil and n-3-fatty acids show slower DNA replication rates than cells treated with a control emulsion or corn oil only. However, it is not clearly understood how such an effect is induced. Fish oil and its metabolites are known to have several modulating effects on signal transduction pathways. Alternatively, they may influence DNA replication by interacting directly with nuclear components. To investigate this problem in greater detail, we have studied the kinetics of DNA synthesis in a cell-free system derived from HeLa cells. Nuclei and cytosolic extract were isolated from cells synchronized in early S phase after treatment with control emulsion, corn oil, or fish oil, respectively. The nuclei were reconstituted with cytosolic extract and a reaction mixture containing bromodeoxyuridine (BrdU) triphosphate to label newly synthesized DNA. The rate of DNA synthesis was measured by bivariate DNA/BrdU analysis and flow cytometry. We show that fish oil-treated cytosol inhibits the elongation of newly synthesized DNA by ~80% in control nuclei. However, nuclei treated with fish oil escape this inhibitory effect. We also show that addition of nuclear extract from fish oil-treated cells reverses the inhibitory effect seen in the reconstitution system of control nuclei and fish oil-treated cytosol. These results indicate that polyunsaturated fatty acids can modulate DNA synthesis through cytosolic as well as soluble nuclear factors. PMID- 12372798 TI - Altered expression of skeletal muscle myosin isoforms in cancer cachexia. AB - Cachexia is commonly seen in cancer and is characterized by severe muscle wasting, but little is known about the effect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression of myosin isoforms toward increased fast (type II) isoform expression. We injected mice with murine C-26 adenocarcinoma cells, a tumor cell line that has been shown to cause muscle wasting. Mice were killed 21 days after tumor injection, and hindlimb muscles were removed. Myosin heavy chain (MHC) and myosin light chain (MLC) content was determined in muscle homogenates by SDS-PAGE. Body weight was significantly lower in tumor-bearing (T) mice. There was a significant decrease in muscle mass in all three muscles tested compared with control, with the largest decrease occurring in the soleus. Although no type IIb MHC was detected in the soleus samples from control mice, type IIb comprised 19% of the total MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. These data are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia. PMID- 12372799 TI - Regulation of arterial tone by smooth muscle myosin type II. AB - The initiation of contractile force in arterial smooth muscle (SM) is believed to be regulated by the intracellular Ca2+ concentration and SM myosin type II phosphorylation. We tested the hypothesis that SM myosin type II operates as a molecular motor protein in electromechanical, but not in protein kinase C (PKC) induced, contraction of small resistance-sized cerebral arteries. We utilized a SM type II myosin heavy chain (MHC) knockout mouse model and measured arterial wall Ca2+ concentration ([Ca2+](i)) and the diameter of pressurized cerebral arteries (30-100 microm) by means of digital fluorescence video imaging. Intravasal pressure elevation caused a graded [Ca2+](i) increase and constricted cerebral arteries of neonatal wild-type mice by 20-30%. In contrast, intravasal pressure elevation caused a graded increase of [Ca2+](i) without constriction in (-/-) MHC-deficient arteries. KCl (60 mM) induced a further [Ca2+](i) increase but failed to induce vasoconstriction of (-/-) MHC-deficient cerebral arteries. Activation of PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM) induced a strong, sustained constriction of (-/-) MHC-deficient cerebral arteries without changing [Ca2+](i). These results demonstrate a major role for SM type II myosin in the development of myogenic tone and Ca2+ -dependent constriction of resistance-sized cerebral arteries. In contrast, the sustained contractile response did not depend on myosin and intracellular Ca2+ but instead depended on PKC. We suggest that SM myosin type II operates as a molecular motor protein in the development of myogenic tone but not in pharmacomechanical coupling by PKC in cerebral arteries. Thus PKC-dependent phosphorylation of cytoskeletal proteins may be responsible for sustained contraction in vascular SM. PMID- 12372800 TI - Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells. AB - Protein kinase C (PKC) plays an important role in activating store-operated Ca2+ channels (SOC) in human mesangial cells (MC). The present study was performed to determine the specific isoform(s) of conventional PKC involved in activating SOC in MC. Fura 2 fluorescence ratiometry showed that the thapsigargin-induced Ca2+ entry (equivalent to SOC) was significantly inhibited by 1 microM Go-6976 (a specific PKCalpha and betaI inhibitor) and PKCalpha antisense treatment (2.5 nM for 24-48 h). However, LY-379196 (PKCbeta inhibitor) and 2,2',3,3',4,4' hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether (HBDDE; PKCalpha and gamma inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single channel analysis in the cell-attached configuration revealed that Go-6976 and PKCalpha antisense significantly depressed thapsigargin-induced activation of SOC. However, LY-379196 and HBDDE did not affect the SOC responses. In inside-out patches, application of purified PKCalpha or betaI, but not betaII or gamma, significantly rescued SOC from postexcision rundown. Western blot analysis revealed that thapsigargin evoked a decrease in cytosolic expression with a corresponding increase in membrane expression of PKCalpha and gamma. However, the translocation from cytosol to membranes was not detected for PKCbetaI or betaII. These results suggest that PKCalpha participates in the intracellular signaling pathway for activating SOC upon release of intracellular stores of Ca2+. PMID- 12372801 TI - Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues. AB - Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles. PMID- 12372802 TI - Role of abnormal neutral endopeptidase-like activities in Hyp mouse bone cells in renal phosphate transport. AB - We investigated whether the absence of Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) in the Hyp mouse affects the expression and activity of neprilysin (NEP) and of endothelin-converting enzyme like endopeptidase (ECEL1/DINE) in bone marrow stromal cells (BMSC) and osteoblasts (Ob). Total NEP-like activity was higher in Ob than in BMSC regardless of genotype, and Hyp cells showed higher activities than normal. Conditioned media (CM) from Hyp BMSC and Ob inhibited inorganic phosphate (P(i)) uptake by mouse proximal tubule cells, and incubating Hyp Ob with phosphoramidon prevented the production of the inhibitor of renal P(i) uptake. A linear relationship was observed between the NEP-like activity of Hyp and normal cells and the inhibition of P(i) uptake. NEP and ECEL1/DINE mRNA levels were higher in Hyp cells than in normal cells, and in situ hybridization of ECEL1/DINE confirmed higher levels of expression in the Hyp mouse than in normal cells. In conclusion, we observed a correlation between the inhibition of P(i) uptake by CM from Hyp cells and elevated NEP-like activities. PMID- 12372803 TI - Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride. AB - Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl(-) transport. Reductions in either cell volume or cytosolic Cl(-) concentration ([Cl(-)](i)) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl(-)](i) was evaluated in model duck red blood cells. Replacement of [Cl(-)](i) with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl(-)](i) at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl(-)](i) is associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl(-)](i) accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl(-)] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl(-)]. The major effect of Cl(-) on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein. PMID- 12372804 TI - Termination of immediate-early gene expression after stimulation by parathyroid hormone or isoproterenol. AB - cAMP/PKA signaling transiently stimulates mRNA expression of immediate-early genes, including IL-6 and c-fos. We confirmed that these mRNAs are transiently stimulated by parathyroid hormone (PTH) in ROS 17/2.8 osteoblastic cells. Consistent with the role for cAMP/PKA signaling in this response, PTH induces transient cAMP elevation, PKA activation, and cAMP-responsive element-binding protein (CREB) phosphorylation. Our goal was to determine whether termination of immediate-early gene expression is due to receptor desensitization or cAMP degradation. The approaches used were 1) inhibition of PTH receptor desensitization with G protein-coupled receptor kinase 2 (GRK2) antisense oligonucleotides or antisense plasmids, 2) sustained activation of adenyl cyclase with forskolin, and 3) inhibition of cAMP degradation with 3-isobutyl-1 methylxanthine. These experiments show that mechanisms downstream of receptor desensitization and cAMP degradation are primarily responsible for termination of PKA activity, CREB phosphorylation, and immediate-early gene expression. Similar conclusions were also obtained in response to PTH in a second osteoblastic cell line (MC3T3-E1) and in response to isoproterenol in NIH3T3 fibroblasts. This conclusion may therefore reflect a general mechanism for termination of immediate early gene expression after induction by cAMP/PKA. PMID- 12372805 TI - Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action. AB - To determine the mechanism of fatty acid modulation of rabbit pulmonary artery large-conductance Ca2+ -activated K+ (BK(Ca)) channel activity, we studied effects of fatty acids and other lipids on channel activity in excised patches with patch-clamp techniques. The structural features of the fatty acid required to increase BK(Ca) channel activity (or average number of open channels, NP(o)) were identified to be the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which have a sufficiently long alkyl chain (C >or= 8), produced a decrease in NP(o). Neutral and short-chain lipids did not alter NP(o). Screening of membrane surface charge with high-ionic-strength bathing solutions (330 mM K+ or 130 mM K+, 300 mM Na+) did not alter the modulation of the BK(Ca) channel NP(o) by fatty acids and other charged lipids, indicating that channel modulation is unlikely to be due to an alteration of the membrane electric field or the attraction of local counterions to the channel. Fatty acids and other negatively charged lipids were able to modulate BK(Ca) channel activity in bathing solutions containing 0 mM Ca2+, 20 mM EGTA, suggesting that calcium is not required for this modulation. Together, these results indicate that modulation of BK(Ca) channels by fatty acids and other charged lipids most likely occurs by their direct interaction with the channel protein itself or with some other channel-associated component. PMID- 12372806 TI - Glutamate 172, essential for modulation of L247T alpha7 ACh receptors by Ca2+, lines the extracellular vestibule. AB - Neuronal alpha7 nicotinic ACh receptors (nAChRs) are permeable to and modulated by Ca2+, Ba2+, and Sr2+. These permeant divalent cations interact with slowly desensitizing L247T alpha7 nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro-beta-erythroidine (DHbetaE), and increase agonist-independent activity. Mutation of glutamate 172 (E172) to glutamine or cysteine eliminated these effects of permeant divalent cations. 2 (Trimethylammonium)ethyl methanethiosulfonate (MTSET), a cysteine-modifying reagent directed at water-accessible thiols, inhibited ACh-evoked currents of E172C/L247T alpha7 nAChRs by >90%, demonstrating that E172 was accessible to permeant ions. The data are consistent with a model of alpha7 receptors, derived from the crystal structure of the ACh binding protein (AChBP) from Lymnaea stagnalis, in which E172 projects toward the lumen of the extracellular vestibule. The observations that E172 was essential for divalent cation modulation of L247T alpha7 nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity. PMID- 12372807 TI - Essential role of Ca2+ -dependent phospholipase A2 in estradiol-induced lysosome activation. AB - The mechanism of lysosome activation by 17beta-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was completely prevented by preincubating the cells with the Ca2+ chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+ -dependent PLA2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+ -independent PLA2, (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2 one (BEL). The Ca2+ ionophore A-23187 yielded similar effects on [Ca2+](i) and lysosomes. Exposure to estradiol also resulted in cPLA2 translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+ -dependent mechanism involving activation of Ca2+ -dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects. PMID- 12372808 TI - Lyn- and ERK-mediated vs. Ca2+ -mediated neutrophil O responses with thermal injury. AB - We evaluated the dependency of neutrophil O production on PTK-Lyn and MAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn was assessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessed by Western blot analysis. O production was measured by isoluminol-enhanced luminometry. Imaging technique was employed to measure neutrophil [Ca2+](i) in individual cells. Thermal injury caused marked upregulation of Lyn and ERK1/2 accompanying enhanced neutrophil O production. Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478) before burn injury caused complete inhibition of the respective kinase activation. Both AG556 and AG1478 produced an ~66% inhibition in O production. Treatment with diltiazem (DZ) produced an ~37% inhibition of O production without affecting Lyn or ERK1/2 activation with burn injury. Ca2+ mobilization was upregulated with burn injury but not affected by treatment of burn rats with AG556. Unlike the partial inhibition of burn-induced O production by AG556, AG1478, or DZ, platelet-activating factor antagonist (PAFa) treatment of burn rats produced near complete inhibition of O production. PAFa treatment also blocked activation of Lyn. The findings suggest that the near complete inhibition of O production by PAFa was a result of blockade of PTK as well as Ca2+ signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result of potentiation of Ca2+ -linked and -independent signaling triggered by inflammatory agents such as PAF. PMID- 12372809 TI - ENaC plays a role in regulated antibody secretion by hybridomas. AB - Hybridomas are fused immortal lymphocytes that typically secrete monoclonal antibodies to a known antigen. Hybridomas express two ionic conductances that have properties consistent with epithelial sodium channel (ENaC) and CFTR. Both ion channels are expressed by lymphocytes. Both of these channels are known to play a role in epithelial cell physiology. However, the physiological role of these channels in lymphocytes is unclear. We tested the hypothesis that ENaC plays a role in the process of regulated antibody secretion. We have been able to demonstrate that hybridomas can be provoked to acutely secrete monoclonal antibodies by a variety of agonists. Concurrently, we were able to show that these same agonists activate amiloride-sensitive sodium currents in whole cell clamped hybridomas. Inhibition of ENaC by amiloride inhibited the acute provoked antibody secretion, thereby linking ENaC to the process of acute antibody secretion. Interestingly, the concentration of amiloride necessary to completely inhibit the provoked secretion was approximately an order of magnitude higher than the concentration necessary to inhibit all of the transmembrane current. However, because amiloride is a weak base, the equilibrium concentration necessary to produce partial inhibition was precisely in accord with the K(i) for amiloride and ENaC, indicating that the inhibition was intracellular. PMID- 12372810 TI - Inhibition of apical Cl-/OH- exchange activity in Caco-2 cells by phorbol esters is mediated by PKCepsilon. AB - The present studies were undertaken to examine the possible regulation of apical membrane Cl-/OH- exchanger in Caco-2 cells by protein kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, on OH- gradient-driven 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed. The results demonstrated that PMA decreased apical Cl-/OH- exchanger activity via phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of PKCepsilon. The data consistent with these conclusions are as follows: 1) short-term treatment of cells for 1-2 h with PMA (100 nM) significantly decreased Cl-/OH- exchange activity compared with control (4alpha-PMA); 2) pretreatment of cells with specific PKC inhibitors chelerythrine chloride, calphostin C, and GF-109203X completely blocked the inhibition of Cl-/OH- exchange activity by PMA; 3) specific inhibitors for PKCepsilon (Ro-318220) but not PKCalpha (Go-6976) significantly blocked the PMA-mediated inhibition; 4) specific PI3-kinase inhibitors wortmannin and LY-294002 significantly attenuated the inhibitory effect of PMA; and 5) PI3-kinase activators IRS-1 peptide and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] mimicked the effects of PMA. These findings provide the first evidence for PKCepsilon-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells and indicate the involvement of the PI3-kinase-mediated pathways in the regulation of Cl- absorption in intestinal epithelial cells. PMID- 12372811 TI - On the mechanism of thrombin-induced angiogenesis: involvement of alphavbeta3 integrin. AB - Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of alphavbeta3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with alphavbeta3 as demonstrated by direct binding of alphavbeta3 protein to immobilized thrombin. This interaction of thrombin with alphavbeta3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to alphavbeta3 or a specific peptide antagonist to alphavbeta3 can abolish all these alphavbeta3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to alphavbeta3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi. PMID- 12372812 TI - Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes. AB - To examine effects of cytosolic Na+, K+, and Cs+ on the voltage dependence of the Na+-K+ pump, we measured Na+-K+ pump current (Ip) of ventricular myocytes voltage clamped at potentials (Vm) from 100 to +60 mV. Superfusates were designed to eliminate voltage dependence at extracellular pump sites. The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip) of 80 mM and a K+ concentration from 0 to 80 mM or with solutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When [Na]pip was 80 mM, K+ in pipette solutions had a voltage-dependent inhibitory effect on Ip and induced a negative slope of the Ip-Vm relationship. Cs+ in pipette solutions had an effect on Ip qualitatively similar to that of K+. Increases in Ip with increases in [Na]pip were voltage dependent. The dielectric coefficient derived from [Na]pip-Ip relationships at the different test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+ free. PMID- 12372813 TI - The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II. AB - COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 +/- 1 mM H+ equivalents/min was inhibited 53 +/- 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl 4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 +/ 4% without effect on DRA transport activity (105 +/- 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII. PMID- 12372814 TI - Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells. AB - Evidence indicates that stretch of the uterus imposed by the growing fetus contributes to the onset of labor. Previously we have shown that mechanically stretching rat myometrial smooth muscle cells (SMCs) induces c-fos expression. To investigate this stretch-induced signaling, we examined the involvement of the mitogen-activated protein kinase (MAPK) family. We show that stretching rat myometrial SMCs induces a rapid and transient phosphorylation (activation) of MAPKs: extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. The use of selective inhibitors for the ERK pathway (PD 98059 and U-0126), p38 (SB-203580), and JNK pathway (curcumin) demonstrated that activation of all three MAPK signaling pathways was necessary for optimal stretch induced c-fos expression. We also demonstrate that upstream tyrosine kinase activity is involved in the mechanotransduction pathway leading to stretch induced MAPK activation and c-fos mRNA expression. To further examine the role of MAPKs in vivo, we used a unilaterally pregnant rat model. MAPKs (ERK and p38) are expressed in the pregnant rat myometrium with maximal ERK and p38 phosphorylation occurring in the 24 h immediately preceding labor. Importantly, the rise in MAPK phosphorylation was confined to the gravid horn and was absent in the empty uterine horn, suggesting that mechanical strain imposed by the growing fetus controls MAPK activation in the myometrium. Collectively, this data indicate that mechanical stretch modulates MAPK activity in the myometrium leading to c-fos expression. PMID- 12372815 TI - Interplay between integrins and FLK-1 in shear stress-induced signaling. AB - Blood flow can modulate vascular cell functions. We studied interactions between integrins and Flk-1 in transducing the mechanical shear stress due to flow. This application of a step shear stress caused Flk-1. Casitas B-lineage lymphoma (Cbl) activation (Flk-1. Cbl association, tyrosine phosphorylation of the Cbl-bound Flk 1, and tyrosine phosphorylation of Cbl) in bovine aortic endothelial cells (BAECs). The activation of integrins by plating BAECs on vitronectin or fibronectin also induced this Flk-1. Cbl activation. The shear-induced Flk-1. Cbl activation was blocked by inhibitory antibodies for alphavbeta3- or beta1 integrin, suggesting that it is mediated by integrins. Inhibition of Flk-1 by SU1498 also abolished this shear-induced Flk-1. Cbl activation. In contrast to the requirement of integrins for Flk-1. Cbl activation, the Flk-1 blocker SU1498 had no detectable effect on the shear-induced integrin activation, suggesting that integrins and Flk-1 play sequential roles in the signal transduction hierarchy induced by shear stress. Integrins are essential for the mechanical activation of Flk-1 by shear stress but not for the chemical activation of Flk-1 by VEGF. PMID- 12372816 TI - Opposing effects of PKCalpha and PKCepsilon on basolateral membrane dynamics in intestinal epithelia. AB - PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCepsilon followed by late activation of the conventional isoform PKCalpha in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCepsilon was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCalpha was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCalpha on basolateral endocytosis. Inhibition of PKC by the selective conventional PKC inhibitor Go-6976 (5 microM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCepsilon selective agonist carbachol (100 microM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Go-6850 but not by Go-6976, implicating PKCepsilon as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 microM) induced early activation of PKC when added simultaneously with PMA. This early activation of PKCalpha blocked the ability of PMA to remodel basolateral F actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCalpha stabilizes F-actin and thereby opposes the effect of PKCepsilon on membrane remodeling in T84 cells. PMID- 12372817 TI - Mechanical stimulation improves tissue-engineered human skeletal muscle. AB - Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue. PMID- 12372818 TI - Integration of the non-genomic and genomic actions of estrogen. Membrane initiated signaling by steroid to transcription and cell biology. AB - Estrogen binds to receptors that translocate to the plasma membrane and to the nucleus. The rapid, non-genomic actions of this sex steroid are attributed to membrane action, while gene transcription occurs through nuclear receptor function. However, gene transcription can also result from estrogen signaling initiated at the membrane, but the relative importance of this mechanism is not known. In vascular endothelial cells (EC), estradiol (E(2)) activates several kinase cascades, including phosphatidylinositol 3-phosphate (PI3K)/Akt, a signaling pathway that impacts EC biology. We determined here by DNA microarray that 40-min exposure to E(2) significantly increased 250 genes in EC, up regulation that was substantially prevented by the PI3K inhibitor, LY294002. This coincided with maximum E(2)-induced PI3K activity at 15-30 min. An important vascular gene strongly up-regulated by E(2) in our array produces cyclooxygenase 2 (Cox-2). In cultured EC, E(2) induced both Cox-2 gene expression and new Cox-2 protein synthesis by 40 and 60 min, respectively, and rapidly stimulated the secretion of prostaglandins PGI(2) and PGE(2). The up-regulation of gene expression reflected transcriptional transactivation, shown using Cox-2 promoter/luciferase reporters in the EC. Soluble inhibitors or dominant negative constructs for PI3K and Akt prevented all these actions of E(2). Functionally, EC migration was induced by the sex steroid, and this was significantly reversed by NS-398, a Cox-2 inhibitor. Gene transcription and cell biological effects of estrogen emanate from rapid and specific signaling, integrating cell surface and nuclear actions of this steroid. PMID- 12372819 TI - Unusual proteolytic activation of pro-hepatocyte growth factor by plasma kallikrein and coagulation factor XIa. AB - Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N terminal sequencing they were identified as the normal cleavage site Arg(494) Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494) Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis. PMID- 12372820 TI - Autocrine activation of the hepatocyte growth factor receptor/met tyrosine kinase induces tumor cell motility by regulating pseudopodial protrusion. AB - The multiple beta-actin rich pseudopodial protrusions of the invasive variant of Moloney sarcoma virus (MSV)-transformed epithelial MDCK cells (MSV-MDCK-INV) are strongly labeled for phosphotyrosine. Increased tyrosine phosphorylation among a number of proteins was detected in MSV-MDCK-INV cells relative to untransformed and MSV-transformed MDCK cells, especially for the hepatocyte growth factor receptor (HGF-R), otherwise known as c-met proto-oncogene. Cell surface expression of HGF-R was similar in the three cell lines, indicating that HGF-R is constitutively phosphorylated in MSV-MDCK-INV cells. Both the tyrosine kinase inhibitor herbimycin A and the HGFalpha antibody abolished HGF-R phosphorylation, induced retraction of pseudopodial protrusions, and promoted the establishment of cell-cell contacts as well as the apparition of numerous stabilizing stress fibers in MSV-MDCK-INV cells. Furthermore, anti-HGFalpha antibody abolished cell motility among MSV-MDCK-INV cells. Conditioned medium from MSV-MDCK-INV cells induced MDCK cell scattering, indicating that HGF is secreted by MSV-MDCK-INV cells. HGF titration followed by a subsequent washout of the antibodies led to renewed pseudopodial protrusion and cellular movement. We therefore show that activation of the tyrosine kinase activity of HGF-R/Met via an autocrine HGF loop is directly responsible for pseudopodial protrusion, thereby explaining the motile and invasive potential of this model epithelium-derived tumor cell line. PMID- 12372821 TI - Hypoxia and beta 2-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells. AB - Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of beta(2)-agonists. To investigate the mechanism of beta(2)-agonist mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O(2) for 24 h. Hypoxia reduced transepithelial sodium current and amiloride-sensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially beta and gamma) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of beta-and gamma ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxia-induced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that beta(2)-agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells. PMID- 12372822 TI - Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein. AB - Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion ( Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl containing buffer, the transition of the F198S variant from a normal alpha helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc) like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein. PMID- 12372830 TI - Structure-activity relationships of linear and cyclic peptides containing the NGR tumor-homing motif. AB - Cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have proven useful for delivering various anti-tumor compounds and viral particles to tumor vessels. We have investigated the role of cyclic constraints on the structure and tumor-homing properties of NGR peptides using tumor necrosis factor-alpha (TNF) derivatives containing disulfide-bridged (CNGRC-TNF) and linear (GNGRG-TNF) NGR domains. Experiments carried out in animal models showed that both GNGRG and CNGRC can target TNF to tumors. However, the anti-tumor activity of CNGRC-TNF was >10-fold higher than that of GNGRG-TNF. Molecular dynamic simulation of cyclic CNGRC showed the presence of a bend geometry involving residues Gly(3)-Arg(4). Molecular dynamic simulation of the same peptide without disulfide constraints showed that the most populated and thermodynamically favored configuration is characterized by the presence of a beta-turn involving residues Gly(3)-Arg(4) and hydrogen bonding interactions between the backbone atoms of Asn(2) and Cys(5). These results suggest that the NGR motif has a strong propensity to form beta turn in linear peptides and may explain the finding that GNGRG peptide can target TNF to tumors, albeit to a lower extent than CNGRC. The disulfide bridge constraint is critical for stabilizing the bent conformation and for increasing the tumor targeting efficiency. PMID- 12372831 TI - Structural evidence of functional divergence in human alkaline phosphatases. AB - The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes, i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-structure of PLAP(1) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family. PMID- 12372832 TI - Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein (StAR). AB - The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P 450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria. PMID- 12372833 TI - An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation. AB - Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeled Neisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein- (LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOS(agg)) with an apparent M(r) >or= 2 x 10(7) were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOS(agg) with LBP and sCD14 promoted LOS(agg) disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M(r) approximately 60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOS(agg):protein aggregates formed by the interaction of LOS(agg) with either LBP or sCD14 alone revealed that the sequence of LOS-protein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS:sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOS(agg) with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOS(agg). Activation of HUVEC by LOS(agg), as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HUVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOS(agg) or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOS(agg). PMID- 12372834 TI - Epithelial innate immunity. A novel antimicrobial peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans. AB - The gut epithelium is an essential interface in insects that transmit parasites. We investigated the role that local innate immunity might have on vector competence, taking Stomoxys calcitrans as a model. S. calcitrans is sympatric with tsetse flies, feeds on many of the same vertebrate hosts, and is thus regularly exposed to the trypanosomes that cause African sleeping sickness and nagana. Despite this, S. calcitrans is not a cyclical vector of these trypanosomes. Trypanosomes develop exclusively in the lumen of digestive organs, and so epithelial immune mechanisms, and in particular antimicrobial peptides (AMPs), may be the prime determinants of the fate of an infection. To investigate why S. calcitrans is not a cyclical vector of trypanosomes, we have looked in its midgut for AMPs with trypanolytic activity. We have identified a new AMP of 42 amino acids, which we named stomoxyn, constitutively expressed and secreted exclusively in the anterior midgut of S. calcitrans. It displays an amphipathic helical structure and exhibits a broad activity spectrum affecting the growth of microorganisms. Interestingly, this AMP exhibits trypanolytic activity to Trypanosoma brucei rhodesiense. We argue that stomoxyn may help to explain why S. calcitrans is not a vector of trypanosomes causing African sleeping sickness and nagana. PMID- 12372836 TI - In vivo interactions between gene products involved in the final stages of molybdenum cofactor biosynthesis in Escherichia coli. AB - The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin. Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli. Numerous interactions among these proteins have been identified. Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA. Probing the effects of various mo. mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions. These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins. PMID- 12372835 TI - Endocytosed transferrin receptors recycle via distinct dynamin and phosphatidylinositol 3-kinase-dependent pathways. AB - Recycling of endocytosed membrane proteins involves passage through early endosomes and recycling endosomes. Previously, we demonstrated a role for clathrin-coated vesicles in transferrin receptor recycling. These clathrin-coated vesicles are formed from recycling endosomes in a process that was inhibited in dynamin-1(G273D)-overexpressing cells. Here we show a second transferrin recycling pathway, which requires phosphatidylinositol 3-kinase activity. Two unrelated phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, retained endocytosed transferrin in early endosomes but did not affect transfer through recycling endosomes. The inhibitory effects of LY294002 and dynamin 1(G273D) on transferrin recycling were additive. In combination with brefeldin A, a drug that prevents the formation of clathrin-coated buds at recycling endosomes, LY294002 inhibited transferrin recycling synergistically. Collectively, these data indicate two distinct recycling pathways. One pathway involves transfer from early endosomes to recycling endosomes, from where clathrin/dynamin-coated vesicles provide for further transport, whereas the other route bypasses recycling endosomes and requires phosphatidylinositol 3-kinase activity. PMID- 12372837 TI - Phosphorylation of the catalytic subunit of protein kinase A. Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1. AB - The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase. It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation. To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit. Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1. Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194). Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus. To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies. The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA. PMID- 12372838 TI - The antibiotic activity of N-pentylpantothenamide results from its conversion to ethyldethia-coenzyme a, a coenzyme a antimetabolite. AB - Pantothenic acid (vitamin B(5)) is the natural precursor of coenzyme A (CoA), an essential cofactor in all organisms. The pantothenic acid antimetabolite N pentylpantothenamide inhibits the growth of Escherichia coli with a minimum inhibitory concentration of 2 microm. In this study, we examine the mechanism of this inhibition. Using the last five enzymes of the CoA biosynthetic pathway in E. coli we demonstrate that N-pentylpantothenamide does not inhibit the CoA biosynthetic enzymes but instead acts as an alternative substrate, forming the CoA analog ethyldethia-CoA. We show that N-pentylpantothenamide is converted to ethyldethia-CoA 10.5 times faster than CoA is biosynthesized from pantothenic acid, demonstrating that ethyldethia-CoA biosynthesis can effectively compete with CoA biosynthesis in the cell. We conclude that the mechanism of toxicity of N-pentylpantothenamide is most likely due to its biosynthetic conversion to the CoA analog ethyldethia-CoA, which may act as an inhibitor of CoA- and acetyl-CoA utilizing enzymes. PMID- 12372839 TI - An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells. AB - 1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H(2)O(2). To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1 cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death. PMID- 12372840 TI - The viral oncogene human papillomavirus E7 deregulates transcriptional silencing by Brm-related gene 1 via molecular interactions. AB - BRG-1, a component of the human SWI/SNF complex, either activates or represses cellular promoters by modulating chromatin structure via the formation of a multiple polypeptide complex. Human papillomavirus E7 binds and destabilizes pRb, resulting in the blockage of G(1) arrest in the cell cycle. We show here that the high-risk human papillomavirus E7 protein group binds BRG-1 and modulates repression of the c-fos promoter mediated by this protein. In addition, both wild type and Rb binding-defective E7 proteins abolish flat cell formation by BRG-1 in SW13 cells, whereas E7 COOH-terminal mutants do not affect this process. BRG-1 triggered repression of the c-fos promoter is sensitive to trichostatin A. We further establish that BRG-1 contains an activation domain and a trichostatin A sensitive repression domain. These results collectively suggest that the viral oncogene E7 targets both pRb and BRG-1 via protein-protein interactions, resulting in the deregulation of host cell cycle control. PMID- 12372841 TI - The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion. AB - ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion. PMID- 12372842 TI - Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. AB - In both type 1 and type 2 diabetes, the late diabetic complications in nerve, vascular endothelium, and kidney arise from chronic elevations of glucose and possibly other metabolites including free fatty acids (FFA). Recent evidence suggests that common stress-activated signaling pathways such as nuclear factor kappaB, p38 MAPK, and NH2-terminal Jun kinases/stress-activated protein kinases underlie the development of these late diabetic complications. In addition, in type 2 diabetes, there is evidence that the activation of these same stress pathways by glucose and possibly FFA leads to both insulin resistance and impaired insulin secretion. Thus, we propose a unifying hypothesis whereby hyperglycemia and FFA-induced activation of the nuclear factor-kappaB, p38 MAPK, and NH2-terminal Jun kinases/stress-activated protein kinases stress pathways, along with the activation of the advanced glycosylation end-products/receptor for advanced glycosylation end-products, protein kinase C, and sorbitol stress pathways, plays a key role in causing late complications in type 1 and type 2 diabetes, along with insulin resistance and impaired insulin secretion in type 2 diabetes. Studies with antioxidants such as vitamin E, alpha-lipoic acid, and N acetylcysteine suggest that new strategies may become available to treat these conditions. PMID- 12372843 TI - Growth hormone receptor antagonists: discovery, development, and use in patients with acromegaly. AB - An understanding of the events that occur during GH receptor (GHR) signaling has facilitated the development of a GHR antagonist (pegvisomant) for use in humans. This molecule has been designed to compete with native GH for the GHR and to prevent its proper or functional dimerization-a process that is critical for GH signal transduction and IGF-I synthesis and secretion. Clinical trials in patients with acromegaly show GHR blockade to be an exciting new mode of therapy for this condition, and pegvisomant may have a therapeutic role in diseases, such as diabetes and malignancy, in which abnormalities of the GH/IGF-I axis have been observed. This review charts the discovery and development of GHR antagonists and details the experience gained in patients with acromegaly. PMID- 12372844 TI - Soluble metalloendopeptidases and neuroendocrine signaling. AB - Peptidases play a vital and often highly specific role in the physiological and pathological generation and termination of peptide hormone signals. The thermolysin-like family of metalloendopeptidases involved in the extracellular processing of neuroendocrine and cardiovascular peptides are of particular significance, reflecting both their specificity for particular peptide substrates and their utility as therapeutic targets. Although the functions of the membrane bound members of this family, such as angiotensin-converting enzyme and neutral endopeptidase, are well established, a role for the predominantly soluble family members in peptide metabolism is only just emerging. This review will focus on the biochemistry, cell biology, and physiology of the soluble metalloendopeptidases EC 3.4.24.15 (thimet oligopeptidase) and EC 3.4.24.16 (neurolysin), as well as presenting evidence that both peptidases play an important role in such diverse functions as reproduction, nociception, and cardiovascular homeostasis. PMID- 12372846 TI - Estrogen modulation of endothelial nitric oxide synthase. AB - Over the past decade, clinical and basic research has demonstrated that estrogen has a dramatic impact on the response to vascular injury and the development of atherosclerosis. Further work has indicated that this is at least partially mediated by an enhancement in nitric oxide (NO) production by the endothelial isoform of NO synthase (eNOS) due to increases in both eNOS expression and level of activation. The effects on eNOS abundance are primarily mediated at the level of gene transcription, and they are dependent on estrogen receptors (ERs), which classically serve as transcription factors, but they are independent of estrogen response element action. Estrogen also has potent nongenomic effects on eNOS activity mediated by a subpopulation of ERalpha localized to caveolae in endothelial cells, where they are coupled to eNOS in a functional signaling module. These observations, which emphasize dependence on cell surface-associated receptors, provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae. Estrogen binding to ERalpha on the SRFC in caveolae leads to G(alphai) activation, which mediates downstream events. The downstream signaling includes activation of tyrosine kinase-MAPK and Akt/protein kinase B signaling, stimulation of heat shock protein 90 binding to eNOS, and perturbation of the local calcium environment, leading to eNOS phosphorylation and calmodulin-mediated eNOS stimulation. These unique genomic and nongenomic processes are critical to the vasoprotective and atheroprotective characteristics of estrogen. In addition, they serve as excellent paradigms for further elucidation of novel mechanisms of steroid hormone action. PMID- 12372848 TI - The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism. AB - The nuclear pregnane X receptor (PXR; NR1I2) is an important component of the body's adaptive defense mechanism against toxic substances including foreign chemicals (xenobiotics). PXR is activated by a large number of endogenous and exogenous chemicals including steroids, antibiotics, antimycotics, bile acids, and the herbal antidepressant St. John's wort. Elucidation of the three dimensional structure of the PXR ligand binding domain revealed that it has a large, spherical ligand binding cavity that allows it to interact with a wide range of hydrophobic chemicals. Thus, unlike other nuclear receptors that interact selectively with their physiological ligands, PXR serves as a generalized sensor of hydrophobic toxins. PXR binds as a heterodimer with the 9 cis retinoic acid receptor (NR2B) to DNA response elements in the regulatory regions of cytochrome P450 3A monooxygenase genes and a number of other genes involved in the metabolism and elimination of xenobiotics from the body. Although PXR evolved to protect the body, its activation by a variety of prescription drugs represents the molecular basis for an important class of harmful drug-drug interactions. Thus, assays that detect PXR activity will be useful in developing safer prescription drugs. PMID- 12372852 TI - Phosphorylation-independent desensitization of G protein-coupled receptors? AB - G protein-coupled receptors (GPCRs) are involved in a multitude of signaling processes and respond to a wide range of ligands. The activity of GPCRs is subject to three principal modes of regulation: desensitization, trafficking, and down-regulation. Desensitization is defined as a loss in the responsiveness of a signaling system. The generally established paradigm for GPCR desensitization involves receptor phosphorylation by GPCR kinases (GRKs), initiated by agonist induced conformational changes in the receptor or by kinases activated by specific signaling pathways. GRKs have several interaction domains and may be able to contribute to receptor desensitization through mechanisms that do not involve the kinase activity of GRK. Pao and Benovic discuss some of these interactions and their relevance for the regulation of GPCR signaling. PMID- 12372849 TI - Sulfonation and molecular action. AB - The sulfonation of endogenous molecules is a pervasive biological phenomenon that is not always easily understood, and although it is increasingly recognized as a function of fundamental importance, there remain areas in which significant cognizance is still lacking or at most minimal. This is particularly true in the field of endocrinology, in which the sulfoconjugation of hormones is a widespread occurrence that is only partially, if at all, appreciated. In the realm of steroid/sterol sulfoconjugation, the discovery of a novel gene that utilizes an alternative exon 1 to encode for two sulfotransferase isoforms, one of which sulfonates cholesterol and the other pregnenolone, has been an important advance. This is significant because cholesterol sulfate plays a crucial role in physiological systems such as keratinocyte differentiation and development of the skin barrier, and pregnenolone sulfate is now acknowledged as an important neurosteroid. The sulfonation of thyroglobulin and thyroid hormones has been extensively investigated and, although this transformation is better understood, there remain areas of incomplete comprehension. The sulfonation of catecholamines is a prevalent modification that has been extensively studied but, unfortunately, remains poorly understood. The sulfonation of pituitary glycoprotein hormones, especially LH and TSH, does not affect binding to their cognate receptors; however, sulfonation does play an important role in their plasma clearance, which indirectly has a significant effect on biological activity. On the other hand, the sulfonation of distinct neuroendocrine peptides does have a profound influence on receptor binding and, thus, a direct effect on biological activity. The sulfonation of specific extracellular structures plays an essential role in the binding and signaling of a large family of extracellular growth factors. In summary, sulfonation is a ubiquitous posttranslational modification of hormones and extracellular components that can lead to dramatic structural changes in affected molecules, the biological significance of which is now beginning to be appreciated. PMID- 12372853 TI - In-gel kinase assay as a method to identify kinase substrates. AB - Phosphorylation of proteins by kinases is central to many cellular processes, including signal transduction. Thus, assays to identify or characterize kinases are a key tool for research in this area. Kinase substrates can be incorporated into polyacrylamide gels and used to characterize kinase activity in mixed samples. This methodology can be adapted for the identification of novel kinase substrates or kinase-kinases that participate in the regulation of cell signaling. Here, I review the rationale and principles of an in-gel kinase assay. This strategy relies on co-polymerization of a substrate within the gel matrix, followed by detection of enzymatic activity in situ. The following Protocol provides a detailed method for performing the in-gel kinase assay and discusses the uses of the assay to evaluate kinase activity in the context of proliferation, differentiation, and survival pathways. PMID- 12372855 TI - Constituents of the pollen of Crocus sativus L. and their tyrosinase inhibitory activity. AB - Five new naturally occurring monoterpenoids, crocusatins-A (1), -B (2a), -C (3), D (4a) -E (5), a new lactate, sodium (2S)-(O-hydroxyphenyl)lactate (6), and eighteen known compounds were isolated and characterized from the pollen of Crocus sativus L. The tyrosinase inhibitory activities of these compounds were also discussed. PMID- 12372856 TI - Further studies on synthesis of the 12,13-seco norditerpenoid alkaloids. AB - After a series of optimization for the reaction conditions (reagents, reaction temperature, etc.), treatment of the sulfonates 4, 8, 13 and 15 with 8% NaOH (room temperature, 24 h) via a semipinacol rearrangement afforded the corresponding C-nor compounds 5, 9, 12 and 16, as the major of a pair of epimer at C-16, to an excellent extent, in 95%, 92%, 100% and 90% yield, respectively. The 12,13-seco compounds 21 and 22 (23) were obtained in 20% and 60% yield, respectively, by treating 5 with Br(2)-glacial HOAc (room temperature, 24 h). Treatment of the C-nor compounds 5 or 6, 16 or 17, and 28 from 10 with SOCl(2) anhydrous benzene (room temperature, overnight) afforded the 12,13-seco compounds 24, 26 and 30 in 70% or 100%, 40% and 66% yield, respectively. When treatment of the C-nor compound 29 from 9 under same conditions gave the 12,13-seco products 30, 31 and 32 in 33%, 26% and 20% yield. When treating 21 or 24, and 26 with 5% KOH in EtOH afforded the 12,13-seco compounds 25 and 27 quantitatively, respectively. The compound 31 converted to 30 quantitatively by treatment with Na(2)CO(3) in MeOH. All of the new compounds were isolated and fully characterized. PMID- 12372857 TI - Synthesis and structure of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic Acid. AB - The first synthesis of (alphaS,betaS)-beta-hydroxy-alpha-[(methoxycarbonyl)amino] 4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7 butanoic acid methyl ester [(alphaS,betaS)-11] has been achieved by OsO(4) oxidation of [S-(E)]-4-[4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7 yl]-2-[(methoxycarbonyl)amino]-3-butenoic acid methyl ester (13) followed by successive gamma-deoxygenation through the cyclocarbonates, separation from the (alphaS,betaR)-isomer by means of flash chromatography, and deprotection. On the other hand, the minor nucleoside of rat liver tRNA(Phe) was isolated on a scale of 100 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P(1), followed by reverse-phase column chromatography, complete digestion with nuclease P(1)/alkaline phosphatase, and reverse-phase HPLC. Comparison of this nucleoside with the synthetic one has unambiguously established its structure to be (alphaS,betaS)-11. PMID- 12372858 TI - Potential bile acid metabolites. 25. Synthesis and chemical properties of stereoisomeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24 oic acids. AB - Epimeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids and some related compounds were synthesized from chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), respectively. The key reaction involved one-step remote oxyfunctionalization of unactivated methine carbons at C-17 of CDCA and at C-14 of UDCA as their methyl ester-peracetate derivatives with dimethyldioxirane (DMDO). After dehydration of the resulting 17alpha- and 14alpha hydroxy derivatives with POCl(3) or conc. H(2)SO(4), the respective Delta(16)- and Delta(14)-unsaturated products were subjected to hydration via hydroboration followed by oxidation to yield the 3,7,16- and 3,7,15-triketones, respectively. Stereoselective reduction of the respective triketones with tert-butylamine borane complex afforded the epimeric 3alpha,7alpha,16- or 3alpha,7alpha,15 trihydroxy derivatives exclusively. A facile formation of the corresponding epsilon-lactones between the side chain carboxyl group at C-24 and the 16alpha- (or 16beta-) hydroxyl group in bile acids is also clarified. PMID- 12372859 TI - Irradiating or autoclaving chitosan/polyol solutions: effect on thermogelling chitosan-beta-glycerophosphate systems. AB - The effects of steam sterilization and gamma-irradiation on chitosan and thermogelling chitosan-beta-glycerophosphate (GP) solutions containing polyol additives were investigated. The selected polyols were triethylene glycol, glycerol, sorbitol, glucose and poly(ethylene glycol) (PEG). They were incorporated to chitosan solutions prior to sterilization in a proportion ranging from 1 to 5% (w/v). The solutions were characterized with respect to their viscosity, thermogelling properties, compressive stress relaxation behavior and chitosan degradation. All polyols reduced the autoclaving-induced viscosity loss and had a positive impact on the solution thermogelling properties and compressive performance of the gels. Steam sterilization in the presence of glucose resulted in a substantial increase in the solution viscosity and gel strength. This was associated with a strong discoloration suggesting chemical alteration of the system. PEG was the most effective agent in preventing hydrolytic degradation of chitosan chains. Gamma-irradiation strongly decreased the chitosan solution viscosity regardless of the presence of additives, even when sterilization was carried out at -80 degrees C. Moreover, the thermogelling properties were dramatically altered, and thus, gamma-irradiation would not be an appropriate method to sterilize chitosan solutions. In conclusion, polyols are potentially useful additive to maximise the viscoelastic and mechanical properties of chitosan-GP after steam sterilization. PMID- 12372860 TI - Characteristics of hyaluronate-hydroxyethyl acrylate blend gel and release of cationic amphiphilic solutes. AB - Hyaluronate-hydroxyethyl acrylate blend gel (HA-PHEA) were prepared to modify the brittleness of hyaluronate gel (HA) and the characteristics of HA-PHEA gel were compared with those of HA and polyhydroxyethyl acrylate (PHEA) gels. These gels were high in water content and transparent. HA-PHEA gel was improved in viscoelastic properties due to the elasticity and the high affinity with water of PHEA, and the drying-swelling cycles became reversible. The effective charge densities theta of the gels estimated from membrane potentials were -0.002, 0.008 and 0 mol dm(-3) for HA-PHEA, HA and PHEA gels. Effects of electro- static and nonelectrostatic interactions on absorptions and releases were studied using sodium benzoate (NaBA) as an anionic solute, and methylene blue (MB), chlorpromazine (CPHCl) and benzethonium chloride (BZTCl) as cationic solutes, in which CPHCl and BZTCl are cationic amphiphilic solutes. The releases of MB, CPHCl and BZTCl from HA-PHEA and HA gels were suppressed comparing with those of NaBA. By adding salts, the releases of MB and CPHCl were enhanced but those of BZTCl were suppressed due to enhancement of the intra- and intermicelle formation. In the releases of the cationic solutes from HA-PHEA gel, electrostatic and nonelectrostatic interactions with HA were found to play important roles. Behaviors of the releases from HA-PHEA gel were found to possess the features of HA gel. PMID- 12372861 TI - Studies on non-thiazolidinedione antidiabetic agents. 1. Discovery of novel oxyiminoacetic acid derivatives. AB - A novel series of oxyiminoacetic acid derivatives were synthesized in an effort to develop a potent antidiabetic agent, which does not contain the 2,4 thiazolidinedione moiety. These compounds were evaluated for glucose and lipid lowering effects in genetically obese and diabetic KKA(y) mice. Several of the compounds showed strong antidiabetic activity, including functional potency at peroxisome proliferator-activated receptor (PPAR)-gamma. (Z)-2-[4-[(5-Methyl-2 phenyl-1,3-oxazol-4-yl)methoxy]benzyloxyimino]-2-(4-phenoxyphenyl)acetic acid (25) significantly reduced plasma glucose (33%, p<0.01) and plasma triglycelide levels (43%, p<0.01) even at a dosage of 0.001% in diet. Pharmacokinetic analyses of 25 are also reported. PMID- 12372862 TI - Phytochemical study on American plants I. Two new phenol glucosides, together with known biflavones and diterpene, from leaves of Juniperus occidentalis Hook. AB - Two new phenol glucosides termed juniperosides I (1) and II (2) were isolated, together with known two biflavones, cupressuflavone and amentoflavone and a diterpene, 3beta-hydroxy sandaracopimaric acid, from leaves of Juniperus occidentalis HOOK. (Cupressaceae) collected in Oregon, U.S.A., and their structures were established as (1S)- and (1R)-1-(2'-hydroxy-6' methylphenyl)ethanol 2'-O-beta-D-glucopyranosides (1, 2), respectively, on the basis of spectral, chemical, and synthetic evidence. The glycosides 1 and 2, as well as the corresponding aglycones 1a and 2a, are apparently novel types of naturally occurring compounds; to our knowledge, isolation of these types of natural phenol derivatives has only rarely been reported from the vegetable kingdom. PMID- 12372863 TI - Optical resolution by preferential crystallization of (RS)-2-benzoylamino-2 benzyl-3-hydroxypropanoic acid and its use in synthesizing optically active 2 amino-2-methyl-3-phenylpropanoic acid. AB - To synthesize optically active 2-amino-2-methyl-3-phenylpropanoic acid (1), (RS) 2-benzoylamino-2-benzyl-3-hydroxypropanoic acid [(RS)-2] was first optically resolved using cinchonidine as a resolving agent to yield optically pure (S)- and (R)-2 in yields of about 70%, based on half of the starting amount of (RS)-2. Next, the racemic structure of (RS)-2 was examined based on melting point, solubility, IR spectrum, and binary and ternary phase diagrams, with the aim of optical resolution by preferential crystallization of (RS)-2. Results indicated that the (RS)-2 exists as a conglomerate at room temperature, although it forms a racemic compound at the melting point. The optical resolution by preferential crystallization yielded (S)- and (R)-2 with optical purities of about 90%, which were fully purified by recrystallization. After O-tosylation of (S)- and (R)-2, reduction by zinc powder and sodium iodide gave (R)- and (S)-1, respectively. PMID- 12372864 TI - New isoflavones and flavanol from Iris potaninii. AB - Two new isoflavones, 6, 3', 4'-trimethoxy-7, 8, 5'-trihydroxyisoflavone (1), 7, 4'-dimethoxy-8, 3', 5'-trihydroxy-6-O-beta-D-glucopyranosylisoflavone (2), and 5, 3, 3'-trihydroxy-7, 4'-dimethoxyflavanone (3) have been isolated from the underground parts of Iris potaninii along with known isoflavones (4-8) and iriflophenone (9). The structures of the new compounds were determined using NMR and mass spectroscopic methods. PMID- 12372865 TI - Enhancement effects of double-chained cationic surfactants of n dimethyldialkylammoniums on permeability of salicylate through guinea pig dorsal skin. AB - We examined the enhancement effects of the double-chained cationic surfactants of n-dimethyldialkylammoniums (CH(3))(2)N(+)(C(n)H(2n+1))(2) on the permeation of anionic salicylate through excised guinea pig dorsal skin at pH 7.4. Among them, n-dimethyldidecylammonium (2C10), which seemed to form micelles, had dose dependent enhancement effects at concentrations of more than 0.1 mM, and about a ninety-fold increase in the permeability coefficient of salicylate was observed at 2 mM. The enhancement effect of 2C10 was larger than those of single-chained cationic surfactants of n-alkyltrimethylammoniums. n-Dimethyldilaurylammonium (2C12), which seemed to form bilayer vesicles, induced about a twenty five-fold increase in the permeability coefficient. The enhancement effects of n dimethyldialkylammoniums decreased with the increase in their alkyl chain lengths. In contrast, only slight stimulation by these cationic surfactants was observed for silicon rubber membrane permeation of salicylate. Analysis of the retention of the salicylate in the skin suggested that the double-chained cationic surfactants-induced increase in the transfer of salicylate to the skin is the main reason for the marked stimulation of the skin permeation. PMID- 12372866 TI - Reverse-phase HPLC separation of D-amygdalin and neoamygdalin and optimum conditions for inhibition of racemization of amygdalin. AB - In boiling aqueous solution, D-amygdalin usually begins to convert into neoamygdalin in 3 min and more than 30% of the initial D-amygdalin is found as neoamygdalin after 30 min. In this report, we establish methods for simple HPLC analysis and the inhibition of D-amygdalin conversion. D-Amygdalin and its conversion product, neoamygdalin, were clearly separated on reverse-phase column chromatography by an optimized eluent of 10 mM sodium phosphate buffer (pH 3.8) containing 6% acetonitrile. Linearity for analyzing D-amygdalin and neoamygdalin was observed in the range from 0.05 to 0.5 mM. The detection limits for D amygdalin and neoamygdalin were ca. 5 microM per injected amount. We found that D amygdalin conversion was completely inhibited by adding 0.05% citric acid to the aqueous solution before boiling. To prevent the loss of pharmaceutical potency of Tonin, we applied this method to measure the conversion rate of D-amygdalin. We confirmed that D-amygdalin conversion in Tonin is effectively inhibited by acidic boiling solution with 0.1% citric acid. PMID- 12372867 TI - Synthesis and absolute configuration of a new 3,4-dihydro-beta-carboline-type alkaloid, 3,4-dehydro-5(S)-5-carboxystrictosidine, isolated from Peruvian Una de Gato (Uncaria tomentosa). AB - The structure including the absolute configuration of a new glucoalkaloid, 3,4 dehydro-5(S)-5-carboxystrictosidine, isolated from Peruvian Una de Gato (Cat's Claw, original plant: Uncaria tomentosa), was confirmed by synthesis starting from secologanin and L-tryptophan. PMID- 12372868 TI - Synthesis of series of 1- and 3-differently substituted xanthines from imidazoles. AB - A new and general method is described for the synthesis, in three steps and in good overall yields, of tetrasubstituted xanthines from an easily prepared imidazole precursor. The method is especially useful for the preparation in standardized conditions of series of xanthines combining a broad variety of primary or secondary alkyl, benzyl or aryl groups at N1 and of alkyl or arylmethyl groups at N3, that are not readily available by other methods. PMID- 12372869 TI - Lupane-triterpene glycosides from the leaves of Acanthopanax gracilistylus. AB - A novel lupane-triterpene glycoside, called wujiapioside B (1), was isolated from the leaves of Acanthopanax gracilistylus (Araliaceae) together with three known lupane-triterpene glycosides, acankoreoside C (2), acantrifoside A (3) and 3 epibetulinic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1- >6)-beta-D-glucopyranosyl ester (4). Based on spectroscopic data, the chemical structure of 1 was determined as 3alpha,23-dihydroxy-lup-20(29)-en-28-oic acid 28 O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D glucopyranosyl ester. Compounds 2-3 were obtained for the first time from this plant and compound 4 has not been isolated from Acanthopanax genus yet. PMID- 12372870 TI - Isolation and structure of monomethylated GM3-type ganglioside molecular species from the starfish Luidia maculata. AB - Two monomethylated GM(3)-Type ganglioside molecular species, 1 and 2, have been obtained from the polar lipid fraction of the chloroform/methanol extract of the starfish Luidia maculata. The structures of these gangliosides have been determined on the basis of chemical and spectroscopic evidence as 1-O-[8-O-methyl (N-acetyl-alpha-D-neuraminosyl)-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta-D glucopyranosyl]-ceramide (1) and 1-O-[8-O-methyl-(N-glycolyl-alpha-D neuraminosyl)-(2-->3)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranosyl] ceramide (2). The ceramide moieties were composed of heterogeneous unsubstituted fatty acid, 2-hydroxy fatty acid, sphingosine and phytosphingosine units. Compound 1, designated as LMG-3, represents new ganglioside molecular species. Compound 2 was a known ganglioside molecular species. PMID- 12372871 TI - New bibenzyl cannabinoid from the New Zealand liverwort Radula marginata. AB - The ether extract of the New Zealand liverwort Radula marginata afforded a new cannabinoid type bibenzyl compound named perrottetinenic acid, and two new bibenzyls, together with a known cannabinoid, perrottetinene. Their structures were established by two dimensional (2D) NMR spectral data. The structure of perrottetinenic acid was a similar to that of Delta(1)-tetrahydrocannabinol, a known hallucinogen. Cannabinoid type bibenzyls have been isolated from liverwort Radula perrottetii, though have not previously been reported from the liverwort R. marginata. PMID- 12372872 TI - A new pyrroloquinazoline alkaloid from Linaria vulgaris. AB - A new alkaloid, 1,2,3,9-tetrahydropyrrolo(2,1-b)quinazolin-1-carboxylic acid (1), together with eight known compounds, 7-hydroxy vasicine (2), benzyl alcohol beta D-(2'-O-beta-xylopyranosyl)glucopyranoside (3), benzyl alcohol O-beta-D glucopyranoside (4), benzyl alcohol O-beta-D-primveroside (5), 3,5-dimethyl-4 hydroxy benzaldehyde (6), gluco-syringic acid (7), syringin (8), and liriodendrin (9), were isolated from the plants of Linaria vulgaris. Their structures were established by spectroscopic methods. PMID- 12372873 TI - An iridoid glucoside dimer and a non-glycosidic iridoid from the leaves of Lasianthus wallichii. AB - A new iridoid glucoside dimer (1) and a non-glycosidic iridoid (2) was isolated together with the known compounds, asperuloside (3), paederoside (4), daphylloside (5), citroside A (6) and benzyl 6-O-alpha-L-rhamnopyranosyl-beta-D glucopyranoside (7), from the leaves of Lasianthus wallichii. The structures of the new compounds were elucidated by spectroscopic and chemical evidence. PMID- 12372874 TI - A new method for synthesis of 7-deoxytaxane analogues by hydrogenation of delta(6,7)-taxane derivatives. AB - A new method for the synthesis of 7-deoxytaxane analogues has been established through hydrogenation of Delta(6,7)-taxane derivatives. Among several catalysts examined, Pd-C was found to be a most effective catalyst for the preparation of target compound. PMID- 12372875 TI - Novel [D-Arg2]dermorphin(1-4) analogs with mu-opioid receptor antagonist activity. AB - Ten Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) analogs in which specific amino acid side chains are shifted to the N(alpha)-position were synthesized, and the binding to these analogs to the mu receptor and their in vitro biological properties were evaluated. Some analogs in which a N,N-bis(p-hydroxybenzyl)-Gly residue was substituted for Tyr(1) exhibited mu receptor antagonist activities (pA(2)) between 5.3 and 6.1. Of these analogs, [N,N-bis(p-hydroxybenzyl)-Gly(1)]YRFB was found to be the most potent specific antagonist for the mu-opioid receptor. PMID- 12372876 TI - Synthesis of optically active organoantimony compounds having an (S)-alpha methylbenzyldimethylamine group and its evaluation for asymmetric reaction. AB - Novel, enantiomerically pure organoantimony compounds having a C-chiral amine moiety, (S)-(alpha-methyl-2-di-p-tolylstibanobenzyl)dimethylamine [AMSb] (2) and its (eta(6)-arene)chromium complex [AMSb-Cr(CO)(3)] (4), were prepared from common (S)-(alpha-methylbenzyl)dimethylamine (1) via its ortho-lithiated intermediates in short steps. The catalytic activity and enantioselectivity of the ligands 2 and 4 for asymmetric reaction are evaluated, and the optically active (eta(6)-arene)chromium complex 4 has been shown to be an effective ligand for rhodium-catalyzed asymmetric hydrosilylation of ketones. PMID- 12372877 TI - A practical procedure for the synthesis of esonarimod, (R,S)-2-acetylthiomethyl-4 (4-methylphenyl)-4-oxobutanoic acid, an antirheumatic agent (part 1). AB - An efficient and practical procedure for the synthesis of esonarimod, (R,S)-2 acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (1), a new antirheumatic drug, has been developed. The intermediate, 2-methylene-4-(4-methylphenyl)-4 oxobutanoic acid (2), was prepared by Friedel-Crafts acylation of toluene with itaconic anhydride (3) in the presence of aluminum trichloride and nitrobenzene in 63% yield without silica gel column purification. Compound 1 was prepared by Michael addition of 2 with thioacetic acid (4) in 74% yield. Overall, 1 was obtained in 47% yield from 3. The structures and synthetic mechanisms of by products (five compounds) of 2 were also clarified. PMID- 12372878 TI - New megastigmane glycoside and aromadendrane derivative from the aerial part of Piper elongatum. AB - A new megastigmane glycoside, called pipeloside A, and a new aromadendrane type sesquiterpenoid, pipelol A, were isolated from the MeOH extract of the aerial part of Piper elongatum VAHL. along with a known megastigmane glycoside, byzantionoside B. The structures of these compounds were elucidated on the basis of spectroscopic data and chemical evidence. PMID- 12372879 TI - Antioxidant ortho-benzoyloxyphenyl acetic acid ester, vaccihein A, from the fruit of rabbiteye blueberry (Vaccinium ashei). AB - A new ortho-benzoyloxyphenyl acetic acid ester, called vaccihein A (1), was isolated from the fruit of rabbiteye blueberry (Vaccinium ashei). The chemical structure was determined on the basis of spectroscopic data. Compound 1 had antioxidative activity using the ferric thiocyanate method. In addition, 1 showed a scavenging effect on the stable free radical 1,1-diphenyl-2-picrylhydrazyl. PMID- 12372880 TI - Synthesis and activity of a metabolite of (S)-6-amino-5-(6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxamido)-3-methyl-1-phenyl-2,4-(1H,3H)-pyrimidinedione (CX-659S). AB - CX-659S (1) [(S)-6-amino-5-(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxamido)-3 methyl-1-phenyl-2,4-(1H,3H)-pyrimidinedione], has been developed as a new type anti-inflammatory agent for the treatment of dermatitis. The structure of a major metabolite of CX-659S was determined as (S)-6-amino-5-[2-hydroxy-2-methyl-4 (2,4,5-trimethyl-3,6-dioxo-1,4-cyclohexadienyl)butanamide]-3-methyl-1-phenyl-2,4 (1H,3H)-pyrimidinedione (2) by direct comparison with the synthesized authentic compound. The anti-inflammatory activity of 2 was equipotent with that of 1 on the contact hypersensitivity reaction (CHR) induced by picryl chloride (PC) in mice, suggesting that compound 2 contributes, at least in part, to the anti inflammatory activity of CX-659S. PMID- 12372881 TI - Biomimetic reduction of nimesulide with NaBH4 catalyzed by metalloporphyrins. AB - The biomimetic reduction of anti-inflammatory drug, nimesulide (1) with sodium borohydride catalyzed by 5,10,15,20-tetraarylporphyrinatoiron(III) chlorides [TAPFe(III)Cl] has been studied in organic solvents under anaerobic and aerobic conditions. PMID- 12372882 TI - Local treatment of penile squamous cell carcinoma. AB - Treatment of primary tumour represents one of the main issues in the management of squamous cell carcinoma of the penis. Radical surgery assures the best results in terms of oncological radicality but causes important anatomical and functional limitations and a significant quality of life compromise. It is possible to suggest a penile-sparing treatment in the presence of small size and low stage tumours. The local recurrence rates seem higher than radical surgery and the functional and aesthetic results are not excellent in all cases. Moreover, radiotherapy seems to have a negligible percentage of local complications. PMID- 12372883 TI - p21 and p53 Immunostaining and survival following systemic chemotherapy for urothelial cancer. AB - INTRODUCTION: Induction of apoptosis and regulation of cell cycle checkpoints are important mechanisms of chemotherapy-induced cell death. The intact p53 tumor suppressor gene is required for efficient activation of apoptosis. The WAF1/p21 gene is transcriptionally activated by p53 and mediates p53-dependent G1 arrest following DNA damage. Therefore, p53 and p21 expression might be related to urothelial tumor response to cytotoxic therapy. METHODS: In a retrospective study, archival tumor specimens from 60 patients treated with cisplatinum-based systemic chemotherapy for locally advanced and/or metastatic urothelial cancer were immunohistochemically stained for p53 and p21. Response to chemotherapy and overall survival were correlated with the results of immunohistochemistry. RESULTS: Thirty-five tumors (58%) of the 60 specimens showed p53 accumulation, and 25 (42%) expressed detectable p21. No association between p53 accumulation and expression of p21 was observed. Correlation with complete and partial remissions following inductive chemotherapy (n = 39) demonstrated that patients with intact p53 responded significantly better (70 vs. 31%, p < 0.05). However, no difference in overall survival was observed with regard to p53 immunostaining (median 12 and 17 months for p53-positive and p53-negative tumors, respectively). The p21 expression was related neither to response nor to overall survival following inductive chemotherapy. In patients receiving adjuvant chemotherapy after cystectomy (n = 21), the outcome was correlated with the immunohistochemistry results. While the survival times for p53-negative patients (60 months) and p53-positive patients (23 months) did not translate into a significant difference, the median overall survival for patients with p21 positive or p21-negative tumors (60 vs. 21 months) was significantly different (p < 0.005). CONCLUSIONS: The short survival of patients with metastatic bladder cancer may conceal putative differences between different prognostic groups in smaller trials. In contrast, p21 immunohistochemistry appears to be of prognostic value in patients receiving systemic adjuvant chemotherapy for locally advanced bladder cancer. The observations made in this retrospective study in a limited number of patients warrant further investigation on the correlation between G1/S checkpoint regulatory genes and adjuvant chemotherapy in larger prospective studies. PMID- 12372884 TI - Is incidental appendectomy necessary during radical cystectomy? AB - OBJECTIVE: To find out the incidence of acute appendicitis leading to acute abdominal pain and necessitating appendectomy in the follow-up of patients after radical cystectomy and urinary diversions. METHODS: A prospective 160 consecutive radical cystectomy patients with urinary diversion in whom appendectomy was not done between January 1991 and June 2001 were reviewed for the incidence of acute appendicitis. Ages ranged between 26 and 73 years. There were 143 males and 17 females. 120 patients had ileal conduit, 20 sigmoid neobladder, 5 continent urinary diversion, and 15 ureterosigmoidostomy as urinary diversion. Each patients was followed up regularly till death or last follow-up. The follow-up ranged between 4 months and 10 years (mean 6 years). RESULTS: Intestinal obstruction (11%) and acute pyelonephritis (16%) were the most common causes of acute abdominal pain. The remaining causes include sigmoid neobladder perforation (0.6%), parastomal hernia (0.6%), urinary retention due to mucus (1.8%) and renal colic (1.8%). In all patients, diagnosis was easily made and they were managed accordingly. None of the patients had acute appendicitis requiring appendectomy on follow-up. CONCLUSION: Incidental appendectomy is not required during radical cystectomy as the risk of subsequent appendicitis is extremely low. PMID- 12372885 TI - Digital fluorographic video-urodynamics in the long-term morphofunctional evaluation of Alcini's Ileocecourethrostomy and ileal reservoir. AB - INTRODUCTION: The authors present the functional long-term follow-up by means of digital fluorographic video-urodynamics (DFVUDM) of two different surgical urinary diversions. MATERIALS AND METHODS: 64 of 101 patients submitted to radical cystectomy from 1983 to 1999 for infiltrating bladder cancer, were diverted by means of an Alcini's ileocecourethrostomy (ICUS+T), and the remaining 37 patients by means of an ileal reservoir (IR). All of those orthotopically diverted patients were submitted to an accurate follow-up which included DFVUDM 1, 3, 6, and 9 years after the surgical procedure (mean follow-up 51 +/- 42 months). RESULTS: All the evaluated patients showed a neobladder with good function during both the filling and the voiding phases. In 88.8% of the DFVUDM examinations, it was possible to find a residual peristaltic activity of the neobladder walls. Such a residual peristaltic activity caused urinary leakage during the examination in 11.1% of cases, while vesico-ureteral reflux was detected in 12.5%. The patients voided by relaxing the perineal floor and/or by contracting the abdominal muscles: the emptying of the reservoir was often excellent with average residual urine of 28.5 ml. None of the patients needed clean intermittent catheterization. EMG evaluation of the pelvic floor in some of patients showed a peculiar EMG pattern characterized by an insufficient voluntary control of the perineal musculature with a slight increase of EMG activity during bladder filling. Moreover, an insufficient relaxation of the pelvic floor muscles in the beginning of and during the micturition has been seen. This particular EMG pattern was present in 22.22% of all patients included in this study while it was particularly high (81.81%) in patients with leakage. CONCLUSION: DFVUDM evaluation represents a highly sophisticated tool which allows an accurate long term morphofunctional evaluation of the urinary diverted patients. In this study, it is shown that the functional results of the two studied surgical procedures, namely Alcini's ICUS+T and IR, are quite similar, demonstrating that the taeniotomies on the cecal tract may have almost the same functional effects of detubularization. Although DFVUDM revealed imperfect functional performances in some patients, the quality of life of diverted patients in our series seems to be satisfactory. PMID- 12372886 TI - Expression of p16 and cyclin D1 in bladder cancer and correlation in cancer progression. AB - INTRODUCTION: Gene p16 encodes a cyclin-dependent kinase inhibitor which functions to regulate cyclin D1, cell cycle progression and malignancies. The relationship between p16 and cyclin D1 is thought to alter bladder cancer formation and tumor progression. We aimed to investigate the expression of p16 and cyclin D1 genes in order to evaluate their clinical significance in bladder cancer. MATERIALS AND METHODS: Tissue samples from 67 patients with transitional cell carcinoma were examined with an immunohistochemical stain for the expression of p16 and cyclin D1 genes. The expression rate was compared to 12 normal urinary bladder mucosa samples obtained from transurethral surgery from noncancer patients. RESULTS: The results revealed significant differences between normal bladder mucosa (100%) and cancer tissue (40.3%) for the positive staining of p16 protein (p < 0.001), while positive staining for the cyclin D1 protein in the patient group (67.2%) was significantly higher (p = 0.003) than that in the control group (16.7%). With the progression of tumor grade and clinical staging the positive rate of p16 gene expression increased, whereas, that of cyclin D1 decreased. Expression of the p16 gene in the non-invasive group was greater than that in the invasive group and a lower expression rate of the cyclin D1 gene in the non-invasive group compared to the invasive group. CONCLUSIONS: The results revealed that expression of the p16 gene is inversely proportional to the expression of the cyclin D1 gene. Therefore, abnormal expression of the p16 and cyclin D1 genes play important roles in tumorigenesis and tumor progression. PMID- 12372887 TI - An evaluation of quality of life in patients who underwent orthotopic bladder replacement after cystectomy: comparison of ileal neobladder versus colon neobladder. AB - OBJECTIVE: The objective of this study was to determine whether the quality of life (QOL) in patients who underwent orthotopic bladder replacement after radical cystectomy was affected by the intestinal segment used for the creation of a neobladder. MATERIALS AND METHODS: A total of 52 patients who underwent radical cystectomy for bladder cancer were included in this study; i.e., 24 patients with an ileal neobladder and 28 patients with a sigmoid neobladder. QOL was evaluated using the SF-36 health-related QOL survey and a questionnaire designed to evaluate the continent status. RESULTS: The mean follow-up periods for patients with an ileal and a sigmoid neobladder was 40.2 and 43.1 months, respectively. The SF-36 survey revealed that patients with colon neobladder had a significantly higher score for role-emotional functioning than those with ileal neobladder, while there was no significant difference in the remaining seven scores between patients with ileal and colon neobladders; however, general health and social functioning in patients with both types of neobladder appeared to be significantly lower than those in the general population in the United States. The results of the questionnaire analyzing the continent status were also similar between these two groups, including the desire to urinate, the incidence of both day- and nighttime urinary leakage, the frequency of pad exchange, and the concern of urine odor. CONCLUSIONS: Six of the eight scales concerning health related QOL were favorable with both patients with ileal and colon neobladders, and the health-related QOL in orthotopic neobladder patients except for role emotional functioning was not affected by the segment of the intestine used for neobladder construction. Moreover, no significant differences were observed in the QOL associated with continent status between these two groups. Therefore, patients with both types of orthotopic neobladder were generally satisfied with their health-related as well as disease-specific QOL. PMID- 12372888 TI - Microstaging of pT1 transitional cell carcinoma of the bladder. Does it really differentiate two populations with different prognoses? (pT1 subcategory). AB - INTRODUCTION: Our objective was to evaluate the feasibility and value of microstaging in pT1 transitional cell carcinoma (TCC) of the bladder in a well defined group of patients treated with transurethral resection (TUR) only. MATERIALS AND METHODS: The clinical records of 152 patients who underwent TUR for the treatment of primary superficial TCC of the bladder between 1983 and 1997 were reviewed. Patients with primary carcinoma in situ and who received adjuvant intravesical treatments were excluded from study. We subclassified the pT1 tumors into two groups according to muscularis mucosae (MM) invasion (pT1 and pT1b). The recurrence and progression rate of cancers was analyzed according to the stage, grade, multiplicity and tumor size. Mean follow-up was 68 months. Estimation of the cumulative distribution of the disease-free interval in separate groups was calculated according to the Kaplan-Meier method. Multivariate analysis of the data was performed by using Cox regression method. A value of p < 0.05 was taken to be statistically significant with odds ratios. RESULTS: Of the 152 patients, tumor stage was pTa in 62 (40.8%) patients and pT1 in 90 (59.2%) patients. Among those pT1 tumors, MM was identified in 50 (55.5%) of cases (pT1a = 34, pT1b = 16). In the remaining 40 (44.5%) patients, MM could not be assessed. Kaplan-Meier analysis revealed that recurrence and progression were statistically significant for stage, multiplicity and grade of tumor. However, multivariate analysis revealed that stage was the only prognostic factor for recurrence and progression (p = 0.0001). CONCLUSION: The present study underscores the fact that pT1b tumors have a distinct natural history. If initial conservative treatment is selected, the patients must be followed very cautiously. PMID- 12372889 TI - Genomic and functional investigations of mutations of the SLC3A1 gene in cystinuria. AB - BACKGROUND: Cystinuria is the second most frequent autosomal recessively inherited disorder in Europe, and it is based on a disturbance of the transepithelial transport of cystine and amino acids in the proximal renal tubule as well as in the intestinum. From the point of view of the urologist, patients suffering from cystine stones represent an important population because they develop a great number of recurrences which necessitate frequent stone removal. METHODS/RESULTS: Advances in the field of molecular genetics have rendered it possible to correlate genotype and phenotype of these patients. In the candidate gene, the SLC3A1 gene, 25 mutations in patients suffering from cystinuria have been described so far. Investigations in our patients (n = 15) as well as results obtained by other study groups have revealed an average detection rate of approximately 50%. The low rate as well as in-depth physiological analyses of the individual mutations indicate that the SLC3A1 is a subunit of a heteromeric complex. This supposition is supported by the structure of the transporter as well as by biochemical analyses of the urine of cystinuric patients and their relatives. Investigations on other genomic segments and candidate genes localized there are being performed. However, it has already appeared that cystinuria is not based on alterations of one single gene, but that a variety of factors combine in the development of this disease. CONCLUSIONS: To be able to offer a molecular genetic diagnosis for patients suffering from cystinuria, the search for mutations of the SLC3A1 gene is being expanded and a screening for other candidate genes set up which are designed to early recognize the risk factors of cystinuria as well as to be able to initiate early therapy. PMID- 12372890 TI - Chemolitholysis and lithotripsy of infectious urinary stones - an in vitro study. AB - OBJECTIVES: This study was performed to look for an improvement of therapeutic strategies with regard to the treatment of infectious urinary stones using artificial stones made of struvite and apatite ('Bon(n) stones') which are comparable to their natural counterparts. MATERIALS AND METHODS: Using an experimental arrangement simulating the physiological conditions in the upper urinary tract, the efficacy of artificial urine (pH 5.7), Suby G solution (pH 3.6), mixtures of artificial urine with Suby G (pH 3.9 and pH 4.1) in dissolving artificial struvite and apatite stones (Bon(n) stones) was investigated. The dissolution of natural infectious urinary stones was also measured. Additionally, investigations on shock-wave lithotripsy (SWL) combined with initial chemolytic treatment of the stones were performed. RESULTS: The efficacy of Suby G solution in dissolving artificial stones was demonstrated. Direct comparison of chemolysis of natural and artificial stones showed no statistical difference between infectious urinary stones and Bon(n) stones of the same material. The investigations on SWL showed a significant improvement on stone comminution, especially of artificial apatite stones after initial chemolytic treatment with Suby G. CONCLUSION: New basics to improve dissolution of infectious urinary stones have been developed by performing standardized in vitro investigations. Local chemolysis with Suby G is an effective tool in the treatment of infectious stone disease. SWL can be improved by varying the physical properties of infectious stones through initial treatment with Suby G solution. PMID- 12372891 TI - Isolation of human leukocyte antigen (HLA)-associated peptide(s) in the absence of HLA-restricted specific cytolytic T lymphocytes. AB - BACKGROUND/METHODS: In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection. RESULTS: The results showed that: (1) Transfection of the class I negative Fen cell line with normal beta-microglobulin (beta(2)-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the beta(2)-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700-3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 x 10(6) of positive Fen cells contained about 200 microg total protein of which about 0.10 microg was class I and about 2 ng was class I associated peptides. CONCLUSIONS: These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients. PMID- 12372892 TI - Comparison of long-term results according to the primary mode of management and type of injury for posterior urethral injuries. AB - BACKGROUND: We retrospectively reviewed the records of patients with traumatic posterior urethral injuries, analyzed postoperative findings to compare the results of the primary mode of management, and evaluated whether the Colapinto and McCallum classification system was valuable for predicting the complications. METHODS: 55 patients with traumatic posterior urethral injuries were included in the study. A total of 35 patients underwent immediate realignment over a Foley catheter including direct Foley catheter insertion (group 1) and 20 underwent initial suprapubic tube placement followed by delayed urethroplasty (group 2). Urethral injuries were interpreted using the Colapinto and McCallum classification based on the retrograde urethrographies. RESULTS: 44 (80.0%) of the patients were classified to type III in both groups (group 1, 29 [82.9%]; group 2, 15 [75.0%]). Of group 1, mild, moderate and severe urethral strictures developed in 7 (20.0%), 8 (22.9%) and 6 (17.1%), respectively, and developed in 6 (30.0%), 2 (10.0%) and 5 (25.0%), respectively, of group 2. Six (17.1%) and 2 (20.0%) had decreased potency and 4 (11.5%) and 1 (5.0%) were impotent in group 1 and 2, respectively. Of group 1, incontinence developed in 3 patients but 1 did not need treatment, and developed in 2 (10.0%) but 1 (5.0%) did not need treatment of group 2. The score test for trend demonstrated that there were no significant differences of these results. CONCLUSIONS: Our findings suggest that complications in patients with posterior urethral injuries are not related to the primary mode of management. Because most injuries are type III, the evolution toward the classification system is needed. PMID- 12372893 TI - Ultrastructural basis for the efficiency of an ileal orthotopic neobladder 27 years after surgery. AB - The morphological and functional basis of the excellent clinical outcome of ileal orthotopic neobladders are largely unknown. Only long-term follow-up studies will provide an adequate answer to this unsettled question. We have studied a patient who underwent this type of surgery over 27 years ago. Besides an important secretive adaptation we have found, at the ultrastructural level, that the monolayered epithelium does not show signs of true metaplasia and that changes had occurred in the intercellular junctions, namely that desmosomes are significantly increased. Although limited to a single case, these features, if confirmed by further observations, suggest a working hypothesis for the understanding of the definitive phenotypic adaptation of the ileal epithelium to the new aggressive environment. PMID- 12372894 TI - Fungating scrotal mass. Rare clinical presentation of testicular tumor. AB - We present a case of large fungating scrotal mass in a 18-year-old male. Investigations revealed non-seminomatous germ cell tumor with retroperitoneal nodes. Systemic chemotherapy was followed by excision of the mass. This rare presentation is reported here. PMID- 12372895 TI - Pelvic lipomatosis in a child. AB - We report a 10-year-old boy with pelvic lipomatosis causing chronic urinary retention. CT scan features, therapy and review of the literature are discussed. PMID- 12372896 TI - Primary mucosa-associated lymphoid tissue lymphoma in the renal pelvis. AB - A primary low-grade lymphoma of mucosa-associated lymphoid tissue arising from the renal pelvis is extremely rare. We present a 77-year-old man with this disease which was difficult to diagnose preoperatively. PMID- 12372897 TI - Primary adenocarcinoma arising from a paraurethral cyst in a female patient. AB - A very rare case of primary adenocarcinoma arising from a paraurethral cyst in a 63-year-old woman is reported. Initially she was diagnosed as having a simple paraurethral cyst because of absent communication with the urethra. The resected paraurethral cyst was histologically associated with adenocarcinoma. We also performed chemotherapy composed of methotrexate, vinblastine, Adriamycin and cisplatin because of lymph node metastasis. Our treatment, however, was not effective and the patient died of systemic metastases. PMID- 12372898 TI - Acute lumbago and sciatica as first symptoms of focal xanthogranulomatous pyelonephritis. AB - Xanthogranulomatous pyelonephritis (XGP) is a rare inflammatory disease of the kidney, presenting in a diffuse or focal form. The preoperative diagnosis of XGP is made only in 10% of the cases because neither the clinical nor the radiological presentation are specific and could be confused with renal tumors, thus deserving the name of 'great imitator'. We report a case of focal XGP in a middle-aged man presenting with acute lumbago and sciatica, an unusual clinical presentation. PMID- 12372899 TI - Giant seminal vesicle stones. Report of two cases. AB - Seminal vesicle stones are rare conditions and only a few cases have been reported in the literature. Here we present 2 cases with single and multiple seminal vesicle calculi. PMID- 12372900 TI - N-tosyl-L-phenylalanyl-chloromethyl ketone eliminates the increase in caspase-3 and bcl-2 caused by brain injury in the newborn rat. AB - N-Tosyl-L-phenylalanyl-chloromethyl ketone (TPCK) is neuroprotective in rat pups. We measured bcl-2, Bax and caspase-3 to determine the mechanisms. Seven-day-old rats had the right carotid artery ligated and were subjected to 2.5 h of 8% oxygen. Ten mg/kg of PTCK or vehicle was given intraperitoneally 15 min prior to hypoxia. At 24 h after hypoxia the brains were removed. Bcl-2 in the hippocampus increased from 0.149 +/- (SE) 0.023 in the shams to 0.289 +/- 0.037 with injury and vehicle (p < 0.05 vs. shams), which was reduced to 0.177 +/- 0.030 by TPCK ( p < 0.05 vs. vehicle). Bcl-2 in the cortex increased from 0.180 +/- 0.037 in the shams to 0.655 +/- 0.078 with injury and vehicle (p < 0.01 vs. shams), which was reduced to 0.354 +/- 0.035 by TPCK (p < 0.01 vs. vehicle). Bax, measured only in the mitochondrial enriched fraction of the cortex, was unchanged. Caspase-3 activity increased with injury to 245 +/- 38% of baseline in the hippocampus (p < 0.01) and to 261 +/- 69% in the cortex (p < 0.01). Treatment with TPCK reduced this to 132 +/- 16% in the hippocampus (p < 0.01 vs. vehicle) and 140 +/- 14% in the cortex (p < 0.05 vs. vehicle). In this experiment TPCK reduces bcl-2 and caspase-3 concentration in animals who have been shown in our previous studies to be protected by TPCK from hypoxic ischemic brain injury. This is consistent with the hypothesis that TPCK produces neuroprotection by blocking the apoptotic cascade between bcl-2 and caspase-3. PMID- 12372901 TI - Effect of riluzole on cytosolic Ca2+ increase in human osteosarcoma cells. AB - In human osteosarcoma MG63 cells, the effect of the neuroprotective drug riluzole on the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura 2. Riluzole (50-500 micromol/l) caused a rapid and sustained plateau increase in [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 150 micromol/l). The riluzole-induced rise in [Ca(2+)](i) was prevented by 58 and 20% by extracellular Ca(2+) removal and nifedipine, respectively, but was not changed by La(3+) and verapamil. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic increase in [Ca(2+)](i), after which the increasing effect of riluzole on [Ca(2+)](i) was attenuated by 84%; also, pretreatment with riluzole abolished the thapsigargin-induced [Ca(2+)](i) increase. U73122, an inhibitor of phospholipase C, abrogated the ATP (but not riluzole)-induced rise in [Ca(2+)](i). A low concentration (6 micromol/l) of riluzole selectively potentiated the bradykinin (but not ATP and histamine) induced increase in [Ca(2+)](i). These results suggest that riluzole rapidly increases [Ca(2+)](i) by stimulating both the extracellular Ca(2+) influx via a nifedipine-sensitive pathway and intracellular Ca(2+) release from the ER via an as yet unidentified mechanism(s). PMID- 12372902 TI - Coordination of histamine H3 receptor antagonists with human adrenal cytochrome P450 enzymes. AB - Optical difference spectroscopy was used to identify and quantify human adrenal microsomal and mitochondrial cytochrome P450 enzyme interactions with the histamine H(3) receptor antagonists thioperamide, clobenpropit and ciproxifan. Addition of these structurally diverse imidazole H(3) receptor antagonists to cytochrome-P450-containing human adrenal microsomal and mitochondrial preparations resulted in concentration-dependent type II optical difference spectra. Respective spectral dissociation constants (K(S)) for the drug interactions with human adrenal microsomal and mitochondrial cytochrome P450 were 1.5 and 1.6 micromol/l for thioperamide, 3.1 and 0.28 micromol/l for clobenpropit and 0.10 and 0.11 micromol/l for ciproxifan. The three compounds demonstrated a similar activity profile in cytochrome-P450-containing bovine adrenal microsomal and mitochondrial preparations. Findings indicate direct coordination of these imidazole-containing H(3) receptor antagonists with the heme moiety of human adrenal cytochrome P450 isozymes. PMID- 12372903 TI - Excitatory amino acid-elicited tonic convulsions in mice and N-methyl-D-aspartate receptor activation: role of Ca(2+) influx and involvement of intracellular Ca(2+)-dependent biochemical processes. AB - Intravenously administered nimodipine (an L-type Ca(2+) antagonist) as well as dizocilpine (an N-methyl-D-aspartate--NMDA--antagonist) showed a wide spectrum of anticonvulsant activity in intracerebroventricular mouse models for excessive activation of excitatory amino acid receptors. The duration of Bay k-8644 (L-type Ca(2+) agonist; intracerebroventricular administration) caused seizures was significantly reduced by intravenously administered nimodipine. Intracisternal administration of Bay k-8644 lowered the convulsion threshold of an intracerebroventricular injection of NMDA. Intracisternal administration of omega conotoxin GVIA (N-type Ca(2+) antagonist) only tended to inhibit the NMDA-induced tonic convulsions. Intracisternal administration of staurosporine (a protein kinase C inhibitor) or calmidazolium (a calmodulin antagonist) was effective in inhibiting the NMDA-induced tonic convulsions. Calmidazolium, unlike staurosporine, produced side effects at a dose showing its anticonvulsant activity. From these results, it is suggested that excessive activation of excitatory amino acid receptors results in tonic convulsions by virtue of a massive increase of Ca(2+) influx mainly through NMDA receptor channels, and at least in part through L-type Ca(2+) channels, and in subsequent activation of protein kinase C and possibly calmodulin. PMID- 12372904 TI - Anti-emetic activity of the novel nonpeptide tachykinin NK1 receptor antagonist ezlopitant (CJ-11,974) against acute and delayed cisplatin-induced emesis in the ferret. AB - The anti-emetic effects of a novel tachykinin NK(1) receptor antagonist, ezlopitant ((2S,3S-cis)-2-diphenylmethyl)- N-[(2-methoxy, 5 isopropylphenyl)methyl]-1-azabicyclo- [2.2.2]octan-3-amine), were investigated in ferrets. Ezlopitant inhibited [(3)H]substance P ([(3)H]SP) binding to the human, guinea pig, ferret and gerbil NK(1) receptors (K(i) = 0.2, 0.9. 0.6 and 0.5 nmol/l, respectively), but had no affinity to NK(2) and NK(3) receptors up to 1 micromol/l. Ezlopitant also inhibited SP-induced contraction of guinea pig trachea with a pA(2) value of 7.8, but had no effects on the baseline tension and maximum contractile response. In ferrets, ezlopitant, either orally (0.03-3 mg/kg) or subcutaneously (0.3-3 mg/kg), prevented acute retching and vomiting responses induced by intraperitoneal injection of cisplatin (10 mg/kg). In addition, repeated subcutaneous injection of ezlopitant significantly inhibited delayed retching and vomiting responses that occurred in ferrets treated with the lower dose of cisplatin (5 mg/kg, i.p.). Ezlopitant (0.1-1 mg/kg, s.c.) also produced a dose-dependent inhibition of hindpaw tapping induced by intracerebroventricular injection of [Sar(9),Met(O(2))(11)]SP in gerbils, which is known to be mediated by NK(1) receptors in the brain. These findings indicate that ezlopitant is a potent and selective NK(1) receptor antagonist, and that it inhibits both acute and delayed emetic reactions induced by cisplatin in ferrets via acting on NK(1) receptors in the central nervous system. PMID- 12372905 TI - Migratory responses of polymorphonuclear leukocytes to kinin peptides. AB - The present study examines the influence of kinins on the migratory capacity of human polymorphonuclear leukocytes under in vitro conditions using the Boyden chamber technique. By means of checkerboard analysis the migration of neutrophils induced by bradykinin could be characterized as true chemotaxis. The stimulation of human neutrophils with bradykinin, with the nonpeptide B(2) receptor agonist FR190997 as well as with des-Arg(9)-bradykinin and des-Arg(10)-kallidin results in a concentration-dependent migration. Pretreatment of the neutrophils with the B(2) receptor antagonist HOE-140 (icatibant) inhibited the bradykinin-induced migration but not that induced by B(1) receptor agonists, whereas the B(1 )receptor antagonist des-Arg(10)HOE-140 abolished the migration elicited by des Arg(9)-bradykinin or des-Arg(10)-kallidin but not that evoked by bradykinin. Pretreatment of the neutrophils with the leukotriene B(4) (LTB(4)) antagonist ZK158252 inhibited the LTB(4)-induced chemotaxis as well as the chemotaxis produced by bradykinin and des-Arg(10)-kallidin. An involvement of interleukin 1beta and of the chemokine IL-8 in the bradykinin-induced migration in vitro could be excluded during the migration time of the neutrophils. In conclusion, the present study provides pharmacological evidence showing that B(1) and B(2) kinin receptors are involved in the migration of human neutrophils in vitro, that LTB(4) participates in the downstream pathway and that the B(1) kinin receptor seems to be expressed already under physiological conditions. PMID- 12372906 TI - Michael adducts of palmitoylascorbic acid: effects on the oxidative burst of neutrophils and the production of tumor necrosis factor-alpha in monocytes. AB - The effects of Michael adducts of 6-O-palmitoyl-L-ascorbic acid (compounds 1-4) on the phosphorylation-dependent response of stimulated monocytes and neutrophils was investigated. The pyranosyl derivative 3 increased the production of tumor necrosis factor-alpha in human monocytes stimulated with lipopolysaccharide (LPS). Compound 3 also enhanced the release of tumor necrosis factor-alpha from nonstimulated monocytes. Michael adducts 1-4 inhibited the formation of reactive oxygen species in fMLP-stimulated human neutrophils as measured by luminol chemiluminescence. Treatment with 6-O-palmitoyl-L-ascorbic acid (compound 5) also led to a decreased luminescence response of neutrophils. Results are discussed with respect to the inhibitory activity of Michael adducts of ascorbic acids towards protein phosphatases PP1/PP2A. PMID- 12372907 TI - Effects of morphine on oedema and tissue concentration of nerve growth factor in experimental inflammation of the rat paw. AB - Injection of carrageenan (1 mg) into the rat hind paw caused a time-dependent increase in paw volume that was maximal 3 h after injection. At this time, the concentration of nerve growth factor (NGF) in the skin of the inflamed paw was more than twofold higher than in the contralateral, non-inflamed paw. Treatment of rats with indomethacin reduced inflammatory oedema by 57%, morphine treatment attenuated oedema by 62%. While indomethacin had no statistically significant effect on the concentration of NGF in the skin of inflamed paws, morphine attenuated the NGF response by 24.2% in a naloxone reversible manner. These data suggest that drug-induced inhibition of inflammatory oedema is not predictive of its effect on an inflammation-induced rise in tissue NGF. Furthermore, our results confirm and extend previous observations suggesting an anti-inflammatory activity of morphine. PMID- 12372909 TI - Vertebral artery dissection in children: a comprehensive review. AB - Vertebral artery dissection (VAD) has been infrequently recognized in children. The authors have reviewed 68 reported cases of VAD in children in the existing literature. An association between routine types of neck movement in sports and the evolution of VAD was recognized in half of the reported cases. Boys outnumbered girls by a ratio of 6.6 to 1, in contrast to adults, for whom the male to female ratio is approximately equal (1.3 to 1). Neck pain, one of the hallmark symptoms of VAD in adults, was infrequently noted in this young population (12%). Most children presented with various combinations of symptoms and signs, including ataxia (53%), headache (38%) and vomiting (34%). Eye signs or symptoms were noted in 72% of patients, and paresis/paralysis of one or more extremity occurred in 54%. Angiography was the method most frequently used to diagnose VAD (63/68; 93%). Magnetic resonance angiography (MRA) revealed pathognomonic signs of VAD in only 3 out of 13 patients evaluated (23%). In this series of 68 patients, 48 reports failed to indicate whether or not a cervical X ray was performed, but in the 20 patients for whom such information was recorded, half had skeletal abnormalities in the occipital/atlas/axis region. The most common treatments were antiplatelet therapy (n = 15) and anticoagulation with (n = 8) or without (n = 7) supplemental antiplatelet therapy. Asymptomatic recovery occurred in 12 of the 15 patients (80%) who received antiplatelet therapy compared with 4 of the 15 patients (27%) who received anticoagulation therapy with or without antiplatelet therapy. There is a very high incidence of associated cervical anomalies in children with VAD. Further studies are required to determine if noninvasive examinations such as magnetic resonance imaging, ultrasonography, computed tomography angiography and MRA could replace angiography as the modality of choice in establishing the diagnosis of VAD in children. The role of different therapies for children presenting with symptoms related to VAD is unclear. PMID- 12372910 TI - Shunt dependency in shunted arachnoid cyst: a reason to avoid shunting. AB - Cystoperitoneal (CP) shunting is minimally invasive and achieves a high rate of resolution on neuroimaging. However, in the absence of definite symptoms, shunting should be reconsidered, because some patients can experience shunt dependency after CP shunting. In this study, the risk of shunt dependency in patients with arachnoid cysts treated with CP shunting and the management of these patients are described. Eight patients (7 boys and 1 girl) were diagnosed as shunt dependent following CP shunting. At the time of the first operation (mean age at first shunting 6.1 years, range 1-11 years), a causal relationship between symptoms and the arachnoid cyst was evident in only 2 cases. Clinical manifestations, neuroimaging (computed tomography and/or magnetic resonance imaging) and intracranial pressure (ICP) data were reviewed retrospectively. The mean age of the patients at the time of shunt dependency was 9.8 years (range 6 13 years), and the mean time between the first shunt operation and shunt dependency was 41 months (range 17-80 months). Although neuroimaging demonstrated a collapsed cyst and small ventricles in most patients, ICP monitoring revealed significant intracranial hypertension. The release of shunt ligation, revision or additional shunting, such as ventriculoperitoneal shunting or lumboperitoneal shunting, resulted in the complete resolution of symptoms except in one patient who lost vision. This study shows that shunt dependency after CP shunting is a real problem and requires more attention. ICP monitoring can demonstrate the presence of intracranial hypertension when clinical and radiological analyses do not. PMID- 12372911 TI - The effects of protein, red blood cells and whole blood on PS valve function. AB - We tested the performance of low- and medium-pressure PS flow control valves as they were perfused with (1) solutions with varying concentrations of protein, (2) solutions with varying numbers of red blood cells (RBC) or (3) solutions with varying concentrations of whole blood. Perfusion was performed with a peristaltic pump at a constant rate and each trial lasted 2 weeks or until valve failure. Mean valve pressures were measured and recorded electronically, and opening and closing pressures were obtained at baseline and at the end of the perfusion period or upon valve failure. Any buildup of material within the valve was noted and recorded. Our findings were as follows: (1) protein levels have little practical effect on valve function; (2) moderate numbers of RBC cause increased variability in valve function while large numbers of RBC uniformly lead to valve failure; (3) prolonged perfusion with solutions of dilute whole blood is poorly tolerated, and (4) valve failure is preceded by a period of increased variability in perfusion pressure. PMID- 12372912 TI - Timing of shunt surgery in childhood tuberculous meningitis with hydrocephalus. AB - Hydrocephalus is a common complication of tuberculous meningitis (TBM) in children. The aims of this study are to review our experience of hydrocephalus in childhood TBM and to evaluate the effect of the timing of ventriculoperitoneal shunting (VPS) on the final outcome. In this study, 156 patients with TBM and hydrocephalus were reviewed retrospectively between 1990 and 2000. Patients' ages ranged from 6 months to 15 years, with a mean age of 4.1 years. There were 85 boys, and the male-to-female ratio was 1.19:1.0. Sixty-two percent of the children were younger than 6 years old. VPS was performed 2 days after the diagnosis in 100 patients, and in the remaining 56 patients, 3 weeks after the diagnosis. The average follow-up period was 8.5 months. Good recovery or minor sequelae was seen in 82 patients (52.6%), and 51 died (12.3%). The timing of the VPS procedure and cerebral complications had an effect on the final outcome. Early VPS gave a better outcome in mild and moderate hydrocephalus (p = 0.040). This study has shown that early surgical procedure for mild/moderate hydrocephalus has a positive effect on the morbidity and mortality of hydrocephalus in childhood TBM (p = 0.014, p = 0.040, respectively). In severe hydrocephalus, there was a tendency for early shunting to have a positive effect on morbidity, although this did not reach statistical significance. PMID- 12372913 TI - Asymmetry of tonsillar ectopia in Chiari I malformation. AB - There has been almost no evaluation or discussion of the symmetry of tonsillar ectopia in the medical literature. We measured the degree of left and right tonsillar herniation in 42 pediatric patients with a symptomatic Chiari I malformation and made clinical/radiological correlations. 18% of all patients with tonsillar asymmetry had clinical symptoms or physical findings referable to the inequality of their hindbrain herniation. In addition, 95% of patients with a coexisting syringomyelia had a right cerebellar tonsillar herniation greater than the left. We hypothesize that slight differences in posterior cranial fossa morphology may physically allow for greater unilateral herniation of the left or right cerebellar tonsil, which may be manifested in a patient's clinical presentation. PMID- 12372914 TI - L-asparaginase-induced reversible posterior leukoencephalopathy syndrome in a child with acute lymphoblastic leukemia. AB - Reversible posterior leukoencephalopathy syndrome (RPLS) is being increasingly described with various etiologies even in the absence of hypertension. We present an 11-year-old patient with acute lymphoblastic leukemia who presented with seizures while on treatment with L-asparaginase. MRI showed bilaterally symmetrical nonenhancing occipital lesions characteristic of RPLS. L-Asparaginase induced RPLS is a rare cause of neurological symptoms in patients on induction chemotherapy. PMID- 12372915 TI - Torsion of a lumbar nerve root schwannoma. AB - We report a very rare case of a lumbar nerve root schwannoma presenting with torsion and infarction. The patient was a 16-year-old male presenting with severe low back pain and urinary retention following an aggressive game of hockey. Subsequent MRI of the lumbar spine revealed a nonenhancing lumbar intradural lesion at the level of L3. The patient was taken to the operating room where he underwent a bilateral L2 and L3 laminectomy and gross total resection of an intradural nerve root tumor, which appeared to have undergone torsion and infarction. Subsequent histopathological examination of the surgical specimen verified the diagnosis of infarcted schwannoma. This is a unique case of lumbar nerve root schwannoma, with atypical MRI findings, presenting with infarction due to torsion of the involved nerve root. PMID- 12372916 TI - Craniopharyngioma involving the infrasellar region: a case report and review of the literature. AB - Craniopharyngiomas are most commonly located in the intradural suprasellar region. However, ectopic craniopharyngiomas can originate in the infrasellar region. The authors present the case of an 8-year-old boy diagnosed with a large cystic craniopharyngioma involving the nasal cavities and maxillary, ethmoid and sphenoid sinuses, in addition to the sella turcica. The primary symptoms of this patient were nasal obstruction, unilateral decreased visual acuity and exophthalmos. He subsequently underwent combined radical resection of the tumor by means of an endonasal transsphenoidal approach followed by radiation therapy. During the surgery, the dura mater of the sellar floor was found to be intact. The neuroradiological and surgical findings suggested that this tumor originated in the extradural infrasellar region and invaded the sella turcica. PMID- 12372917 TI - Dolichoodontoid in a pediatric patient. AB - Dolichoodontoid is defined as hypertrophy of the apical portion of the odontoid process of the axis. We report the first case of a dolichoodontoid process in a pediatric patient with other congenital anomalies. This rare entity has had three previous citations in the literature. Our patient presented with scoliosis in which subsequent MRI demonstrated a Chiari I malformation, a small thoracic syrinx and a dolichoodontoid process. The diagnosis of this malformation should be entertained in cases of craniocervical anomalies. PMID- 12372918 TI - Congenital hemiparesis and seizures secondary to perinatal occlusion of the middle cerebral artery. PMID- 12372919 TI - The synchrony of arterial and CSF pulsations is not due to resonance. PMID- 12372921 TI - Self-management of oral anticoagulants with a whole blood prothrombin-time monitor in elderly patients with atrial fibrillation. AB - The efficacy of oral anticoagulants (OAC) in reducing the incidence of stroke in elderly patients with atrial fibrillation (AF) has been well documented. The intensity of OAC therapy and deviations in the prothrombin time (PT) are the strongest risk factor for bleeding complications in elderly patients. The aim of this study was to evaluate a more rigorous regulation of OAC by the use of a portable whole blood PT-monitor (CoaguChek) in elderly patients with AF (age 65 80 years). The study group consisted of 20 patients, of whom 17 were evaluable, which were trained to use to CoaguChek monitor and adjust their anticoagulant dose for 12 months. The control group, 20 patients matched for age, gender and the duration of OAC treatment, were tested in an anticoagulant clinic and their OAC dose was adjusted by a physician. To validate the PT-monitor results, the patients performed a total of 129 simultaneous venous blood PT tests at various time points. The correlation coefficient R(2) was 0.707 indicating the accuracy of the CoaguChek results. The self-managed patients perform more frequent measurements 46 +/- 8.9 vs. 15.7 +/- 3.1 PT tests per patient. They demonstrated a within the therapeutic range INR in 80.5% of the tests (95% confidence interval, 76.5-84.1%) as compared to 72.4% (95% confidence interval, 68.5-76.5%) in the control group (p = 0.057). The median value for all CoaguChek International Normalized Ratio (INR) recordings was within therapeutic range in the self-management group as well as in the control group. There were fewer INR results below or above the therapeutic range in the study group. None of the patients had hemorrhagic or thrombotic events during the study. Overall, the study group expressed high satisfaction from using the home monitor. We conclude that home PT monitoring and self-management of OAC are feasible in a motivated population of elderly patients with atrial fibrillation and are probably cost effective. PMID- 12372922 TI - Effect of low-molecular-weight heparin on potassium homeostasis. AB - BACKGROUND: Low-molecular-weight heparins (LMWHs) are being preferred to unfractionated heparin (UFH) because of their superior convenience and a comparable or slightly better toxicity profile. Whether LMWH has an inhibitory effect on aldosterone that causes hyperkalemia is yet uncertain. METHODS: Twenty eight patients (all male; mean age: 70 years, range 52-87 years) placed on LMWH therapy (40 mg subcutaneously every 12 h) for deep venous thrombosis prophylaxis after an operation were included in the study. Transtubular potassium concentration gradient (TTKG) was calculated 1 day prior to LMWH therapy and again after 4 days of treatment. Of the 28 patients enrolled in the study, we were able to calculate the TTKG in only 19 patients: 9 had a urinary osmolarity (either before or after LMWH therapy) less than the serum osmolarity, making the TTKG calculation unreliable. The Wilcoxon signed-rank test was used to analyze differences in the median serum potassium levels and TTKG before and after LMWH therapy. RESULTS: All patients had adequate renal function (creatinine clearance >90 ml/min). Mean (+/- SD) serum potassium concentration before LMWH was 4.25 (+/ 0.40) mmol/dl. It increased to 4.35 (+/- 0.41) mmol/dl after initiating LMWH therapy (p = 0.09). Similarly, the mean (+/- SD) TKKG calculated was 5.52 (+/- 2.33) before and 5.97 (+/- 3.06) after 4 days of LMWH (p = 0.54). CONCLUSIONS: Unlike UFH, LMWH (Lovenox in doses used for postoperative prophylaxis against deep venous thrombosis does not seem to have a significant effect on potassium homeostasis. PMID- 12372923 TI - Treatment with vitamin K antagonists: frequency of indications and appropriateness of continuation. AB - To prevent venous and arterial thrombosis vitamin K antagonists (VKA) are the treatment of choice for many indications. It is important to balance the benefits and the potential hazards of this treatment. An effective way to prevent unnecessary bleeding during VKA treatment is to stop treatment when the indication is no longer present. In this study, we analyze the distribution of indications in a randomly selected group of 250 patients starting VKA treatment at the Amsterdam Thrombosis Service. The proportion of patients still treated after one year of follow-up was also investigated. The distribution of the indications among patients starting VKA therapy was approximately 50% for venous thromboembolism treatment and prophylaxis, and approximately 50% for prophylaxis of arterial thrombosis. After one year of follow-up, 164 (65.6%) of the 250 patients had stopped VKA therapy. Reasons for stopping included: no indication for continuing VKA treatment, e.g. end of treatment, prophylaxis or restoration of sinus rhythm (137 patients); death (17) and other reasons (10). Six (2.4%; 95% CI: 0.9-5.1%) patients had a questionable indication for long-term treatment, and 9 (3.6%, 95% CI: 1.6-6.7%) patients had no clear indication for continued VKA treatment. We conclude that in the setting of the Amsterdam Thrombosis Service, only a small proportion of patients is treated with long-term VKA therapy without a valid indication after 1 year. PMID- 12372924 TI - Thromboplastin Bilbao: reproducibility and sensitivity of a Spanish thromboplastin. AB - Prothrombin time (PT) is the control test for oral anticoagulant therapy as well as the screening test for defects of the extrinsic pathway of coagulation. Its responsiveness to decreased extrinsic clotting factors depends on the source and type of tissue factor thromboplastin extract. In 1994, a rabbit brain thromboplastin - Thromboplastin Bilbao (TBi) - was introduced as a replacement for a human brain preparation used since 1983, with the aim of establishing a national standard. The purpose of this study was to check the reproducibility, the inter-assay/intra-assay accuracy and the stability of this reagent under temperature changes and over time. A method modified from Frei et al. [World Health Organisation Regional Publications, Eastern Mediterranean Series, Alexandria, 1995] was used for the preparation of thromboplastin extract. Thirty five batches of human TBi were prepared from 1983 to 1988, while from 1993 to 1999 13 batches of rabbit TBi were produced. The inter-assay reproducibility of rabbit TBi exhibited a coefficient of variation (CV) of 1.07-1.57% for normal plasma and of 1.25-2.56% for anticoagulated plasma. The intra-assay CV was 0.06 1.30% for normal plasma and 1.23-2.66% for anticoagulated plasma. The stability of the reagent to temperature changes and time was also estimated, with similar results for the two thromboplastins. As a result of the Oral Anticoagulant Treatment Quality Assessment Scheme in the Basque Country, an in-house rabbit thromboplastin with good sensitivity and reproducibility was developed. PMID- 12372925 TI - Effect of an ionic compared to a non-ionic X-ray contrast agent on platelets and coagulation during diagnostic cardiac catheterisation. AB - The aim of the present study was to evaluate the effects of ionic (ioxaglate) and non-ionic (iopromide) contrast media on haemostatic parameters ex vivo. In 40 patients undergoing coronary angiography, platelet function (platelet reactivity and serotonin concentration) and coagulation markers [thrombin-antithrombin III complexes, prothrombin fragments (F1+2) and the D-dimers] were measured. The use of an ionic X-ray contrast agent (XCA) (ioxaglate) in diagnostic cardiac catheterisation angiography is associated with lower thrombin generation and lower activation of the platelet system than when a non-ionic XCA is employed (iopromide). The results thus confirm the results of various in vitro studies and animal investigations. PMID- 12372926 TI - Hemostasis and fibrinolysis factors in first-degree relatives of patients with Type 2 diabetes without hypertension. AB - First-degree relatives of type 2 diabetic patients with or without a family history of hypertension are at increased risk for cardiovascular diseases. The aim of this study was to verify some possible hemostatic alterations in first degree relatives of type 2 diabetic, normotensive and hypertensive patients. In 78 non-diabetic, normotensive first-degree relatives of type 2 diabetic patients (47 without a family history of hypertension and 31 with a family history of hypertension) and in 36 normoglycemic, normotensive subjects with no family history of hypertension, we evaluated plasma levels of fasting glucose and insulin, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), D-dimer (DD) and prothrombin fragment 1 + 2 (F1+2). Insulin resistance, calculated by the HOMA model, and plasma levels of t-PA and PAI-1 were significantly higher in relatives of diabetics compared to controls. As far as the thrombin activation indexes are concerned, we detected a significant increase in DD and F1+2 in relatives of diabetics with hypertension compared to other study subjects. In conclusion, our data indicate that familial predisposition may influence the hemostatic system in first-degree relatives of diabetic and/or hypertensive patients. PMID- 12372927 TI - Prospective assessment of a nomogram for the initiation of oral anticoagulation therapy for outpatient treatment of venous thromboembolism. AB - Venous thromboembolism is a common medical problem. Recently, the emphasis has been on a switch to outpatient low molecular weight heparin therapy. Previous warfarin nomograms have been developed only for inpatients. We prospectively assessed a warfarin initiation nomogram in 105 consecutive outpatients; the nomogram requires International Normalized Ratio (INR) testing on only days 3, 5, and 8. Eighty-three percent had a therapeutic INR by day 5 and 98% by day 8. There were no major bleeds and only 6 instances of INR >4.5. This outpatient warfarin nomogram appears to be safe and efficacious in obtaining timely therapeutic levels of warfarin and deserves further study. PMID- 12372928 TI - Low molecular weight heparin for the prevention of deep venous thrombosis: a suitable monitoring in elderly patients? AB - Monitoring of anti-Xa activity (aXa) levels is not routinely required in patients receiving enoxaparine at prophylactic dosages, since aXa is supposed to stay below the manufacturer's recommended range in patients treated for venous thrombosis (0.5-1 IU/ml). In order to aXa in elderly subjects receiving prophylactic enoxaparin, 68 consecutive patients (mean age 82.5 +/- 10.7 years) hospitalized in a medical department receiving 4000 IU enoxaparin daily subcutaneously for the prevention of venous thromboembolic disease were studied. After the first injection of enoxaparin, the aXa of 57.4% patients was superior to 0.5 IU/ml while 69.4% had an aXa higher than 0.5 after 8.4 +/- 1.2 days. A negative relationship between aXa and body weight and a trend towards a positive correlation between aXa and age but not with creatinine clearance were noted. Our findings question the opportunity to monitor aXa in elderly patients receiving 4000 IU enoxaparin as antithrombotic prophylaxis. PMID- 12372929 TI - Abnormally short activated partial thromboplastin times are related to elevated plasma levels of TAT, F1+2, D-dimer and FVIII:C. AB - Abnormally short activated partial thromboplastin times (APTTs) are associated with an increased risk of thrombotic disorders. We have examined the status of coagulation activity in subjects with short APTTs. In addition, the presence of the thrombotic risk factors G1691A-factor V, G20210A-prothrombin gene mutation and factor VIII coagulant activity (FVIII:C) was determined. Plasma levels of TAT, F1+2, D-dimer and FVIII:C were markedly higher in subjects with short APTTs compared with subjects with normal APTTs. APTTs were inversely related to TAT, F1+2, D-dimer and FVIII:C levels. The prevalence of G1691A-factor V and G20210A prothrombin gene mutation between the group with short APTTs and the control group was not significantly different. Hence, these gene polymorphisms do not contribute to the increased risk of thrombosis associated with short APTTs. In conclusion, short APTTs are indicative of marked coagulation activity and elevated FVIII:C levels. Elevated FVIII:C levels may play a pathogenic role in the increased risk of thrombosis associated with abnormally short APTTs. PMID- 12372930 TI - Quantification of antithrombin isoform proportions in plasma samples of healthy subjects, sepsis patients, and in antithrombin concentrates. AB - Antithrombin (AT) circulates in plasma in two isoforms, AT-alpha (90-95%) and AT beta (5-10%). AT isoform proportions were measured in plasma samples of 17 healthy subjects and 26 posttraumatic or postoperative septic patients, as well as in 4 commercially available AT concentrates. Total AT was immune-purified from plasma and concentrates. Micellar electrokinetic chromatography was used to analytically separate and quantify the isoforms. Compared with plasma samples of healthy donors, septic plasmas revealed significantly reduced AT activity (p < 0.001) and beta-isoform content (p < 0.05). AT-beta correlated inversely with urea and creatinine serum concentrations (p < 0.01), indicating a relationship between better renal function and higher beta-isoform content. beta-Isoform neither correlated with age, gender, and 28-day mortality, nor with plasma concentrations of various inflammatory and organ function parameters. The commercial AT concentrate, which is equivalent to the current WHO standard, had an AT-beta content close to that found in plasma of healthy subjects. The availability of this novel quantitative AT isoform assay allows, for the first time, a closer look at the role of AT isoforms in hemostasis and sepsis pathophysiology. PMID- 12372931 TI - Vasopressin, hypercalciuria and aquaporin--the key elements for impaired renal water handling in astronauts? PMID- 12372932 TI - Cellular interactions in the pathogenesis of human proliferative glomerulonephritis. The role of beta-2 integrin-expressing leukocytes. PMID- 12372933 TI - Gene expression analysis in microdissected renal tissue. Current challenges and strategies. AB - The architecture and compartmentalization of the kidney has stimulated the development of an array of microtechniques to study the functional differences between the distinct nephron segments. With the vast amounts of genomic sequence data now available, the groundwork has been laid for a comprehensive characterization of the molecular pathways defining the differences in nephron function. With the development of sensitive gene expression techniques the tools for a comprehensive molecular analysis of specific renal microenvironments have been provided: Quantitative RT-PCR technologies now allow the analysis of specific mRNAs from as little as single microdissected renal cells. A more global view of gene expression regulation is a logical development from the application of large scale profiling techniques. In this review, we will discuss the power and pitfalls of these approaches, including their potential for the functional characterization of nephron heterogeneity and diagnostic application in renal disease. PMID- 12372934 TI - Impairment of vascular responses to reactive hyperemia and nitric oxide in chronic renal failure. AB - BACKGROUND: Cardiovascular events are the leading cause of morbidity and mortality in patients with end-stage renal disease. The role of endothelial dysfunction, an early marker of arteriosclerosis, in patients with chronic renal failure (CRF) before the initiation of maintenance hemodialysis (HD), and the factors affecting endothelial dysfunction in the setting of chronic renal failure remain poorly understood. METHODS: We evaluated endothelial function by measuring flow-mediated vasodilation (%FMD) during reactive hyperemia in healthy individuals (HCS) and patients with chronic renal failure with (HD) or without (ND) hemodialysis. Nonspecific endothelium-independent vasodilation (%NTG) was measured after the administration of sublingual glyceryl trinitrate spray (0.3 mg). Factors affecting %FMD and %NTG were also tested. RESULTS: In ND and HD, plasma homocysteine, cysteine and stable NO metabolite (NO(-)(3)) concentrations were significantly elevated. In ND and HD, reactive hyperemia as well as %NTG and %FMD were attenuated to a similar degree. On multivariate regression analysis, NO(-)(3) concentration was directly correlated with both %FMD and %NTG, while the glutathione (GSH) concentration correlated with only %NTG. CONCLUSIONS: Our findings indicate that chronic renal failure before the initiation of maintenance hemodialysis impairs endothelial function and/or the response to NO, which is accompanied by the attenuated reactive hyperemia. Furthermore, the impairment might be related to the decreased synthesis or the dissipation of NO. PMID- 12372935 TI - Fenofibrate increases creatininemia by increasing metabolic production of creatinine. AB - Fenofibrate is a potent hypolipemic agent, widely used in patients with renal insufficiency in whom dyslipidemia is frequent. A moderate reversible increase in creatinine plasma levels is an established side effect of fenofibrate therapy, which mechanism remains unknown. We have previously reported that in 13 patients with normal renal function or moderate renal insufficiency, two weeks of fenofibrate therapy increased creatininemia without any changes in renal plasma flow and glomerular filtration rate [1]. In 13 additional patients, muscular enzymes (AST, GPT, CPK, LDH) and myoglobin were measured before and after 2 weeks on fenofibrate, and the values of creatininemia obtained by the Jaffe technique and HPLC were compared. CPK and AST activity and plasma myoglobin increased in 2 patients with fenofibrate, but muscular enzymes remained unchanged in the population as a whole, and were not correlated to the changes in creatininemia. The changes in creatininemia induced by fenofibrate measured by the Jaffe technique were strongly correlated to those measured by HPLC (r(2) = 0.675, p = 0.0006). Analysis of the pooled data of the two arms of the study showed in 26 patients that two weeks of fenofibrate therapy efficiently reduced total cholesterol and triglycerides plasma levels, and raised creatininemia from 139 +/ 8 to 160 +/- 10 micromol/l (p < 0.0001), but confirmed that creatininuria also increased to the extent that creatinine clearance remained unchanged (68 +/- 6 vs. 67 +/- 6 ml/min, n.s.). It is concluded that the increase in creatininemia induced by fenofibrate in renal patients does not reflect an impairment of renal function, nor an alteration of tubular creatinine secretion, and is not falsely increased by a dosage interference. Fenofibrate-induced increase of daily creatinine production is neither readily explained by accelerated muscular cell lysis. It is proposed that fenofibrate increases the metabolic production rate of creatinine. PMID- 12372936 TI - B7-1 (CD80) and B7-2 (CD 86) expression in human tubular epithelial cells in vivo and in vitro. AB - BACKGROUND: Tubulointerstitial inflammation with infiltration of mononuclear cells plays an important role in acute allograft rejection and in the progression of renal diseases. We therefore investigated in vivo the expression of the costimulatory molecules B7-1 and B7-2 on proximal tubular epithelial cells (PTEC) under normal and pathologic conditions and analyzed the regulation and functional role of these molecules after cytokine and CD40 activation in vitro. METHODS: Immunohistological staining for B7-1 and B7-2 on cryostat sections of core needle biopsies from patients with different renal diseases was examined. Patients were divided into three groups: group A: diffuse interstitial inflammation; group B: minor interstitial inflammation; group C: no interstitial inflammation. In addition, the expression of B7-1 and B7-2 protein and mRNA of cultured PTEC that had been stimulated with cytokine-combinations in absence or presence of a stimulatory anti-CD40 antibody was investigated by means of FACS analysis and RT PCR. The functional role was analyzed in MKLCs with cytokine and anti-CD40 prestimulated PTEC by measuring IFN-gamma and IL-2 expression in absence or presence of CTLA4-Ig by ELISA. RESULTS: Group A patients showed intense tubular staining for B7-1 and B7-2, group B patients showed mild staining, whereas in group C patients B7-1 and B7-2 staining was negative or only weakly positive. In vitro, the presence of B7-1 and B7-2 on PTEC was increased after stimulation with combinations of IL-1alpha, IL-4, IFN-gamma or IL-13 instead of IL-4 and CD40 activation. B7-1 and B7-2 mRNA could be detected in PTEC as well. In MKLCs only cytokine and anti-CD40 prestimulated PTEC were able to stimulate IFN-gamma and IL 2 production by purified T cells, which could be blocked dose-dependently by CTLA4-Ig. CONCLUSIONS: This study clearly shows that B7-1 and B7-2 can be induced on PTEC in vivo and in vitro. After B7-1 and B7-2 induction, PTEC costimulate CD28 on T lymphocytes resulting in cytokine production. This might be of relevance in allograft rejection and in various kidney diseases. PMID- 12372937 TI - Wall shear stress assessment in the common carotid artery of end-stage renal failure patients. AB - Under physiological circumstances in the common carotid artery (CCA), mean wall shear stress (WSS), defined as mean wall shear rate (WSR) times local whole blood viscosity (WBV), is maintained at approximately 1.5 Pa. In patients with end stage renal failure (ESRF) whole blood viscosity is low and it is not unlikely that mean WSS is lower in these patients than in control subjects. Moreover, hemodialysis causes an acute increase in blood viscosity with possible effects on WSS. In this study WSS in the CCA was determined with the Shear Rate Estimating System, an apparatus based on ultrasound, in ESRF patients (n = 13) and in presumed healthy age- and sex-matched control subjects (n = 13). Prior to hemodialysis, mean WSS (0.67 +/- 0.23 Pa) was significantly lower (p < 0.05) in patients with ESRF, due to both a lower WBV (2.80 +/- 0.52 mPa.s) and mean WSR (271 +/- 109 s(-1)), than in the control subjects (mean WSS: 1.24 +/- 0.20 Pa; WBV: 3.20 +/- 0.29 mPa.s; WSR: 387 +/- 51 s(-1)). Hemodialysis induced an increase in WBV (up to 3.71 +/- 1.54 mPa.s, p < 0.01), but mean WSS did not change significantly due to a reciprocal decrease in mean wall shear rate. These findings demonstrate that WSS is lower in hemodialysis patients than in control subjects, and that mean WSS is maintained at this low level despite an acute change in blood viscosity. PMID- 12372938 TI - Renal in situ hybridization studies of extracellular matrix related molecules in type 1 diabetes mellitus. AB - BACKGROUND/AIM: Progressive expansion of mesangial matrix and glomerular basement membrane thickening represent alterations in the balance between synthesis and degradation of glomerular extracellular matrix (ECM) protein and are hallmarks of diabetic nephropathy. In order to elucidate the basis for this imbalance between the synthesis and the degradation of ECM in renal tissues from patients of type 1 diabetes mellitus (type 1D) with diabetic nephropathy (DN), we examined the expression of alpha1 chain of type IV collagen (IV-C), matrix metalloproteinase-2 and -3 (MMP-2, MMP-3), tissue inhibitor of metalloproteinase-1 (TIMP-1) and beta actin mRNA using a high-resolution in situ hybridization with digoxigenin-labeled oligonucleotide. METHODS: Patients were divided into two groups based on both of degree of mesangial expansion using electron microscopic point counting morphometric methods and duration of type 1D: 7 'fast-track' patients were selected for their very rapid development of DN structural changes and 8 'slow track' patients for their very slow development of DN structural changes. Seven normal human kidney (NHK) tissues were used as controls. RESULTS: Positive cells for each mRNA were observed in glomerular resident cells, including glomerular mesangial, epithelial and endothelial cells and cells of Bowman's capsule. The percentage of glomerular cells positive for IV-C, MMP-2 and MMP-3 mRNA was significantly greater in the 'slow-track' vs. 'fast-track' patients. No significant differences in percentage positive cells was seen for beta-actin mRNA. Furthermore, to elucidate the total number of positive cells per glomerulus for each mRNA, we estimated total cell number of glomerulus using morphometric techniques on light microscopy tissues. The total cell number per glomerulus was significantly greater in 'fast-track' than that in 'slow-track' patients and NHK. The total number of positive cells per glomerulus for MMP-2 in NHK was significantly greater than that in 'slow-track' and 'fast-track' patients. CONCLUSIONS: Thus, IV-C, MMP-2, MMP-3 and TIMP-1 mRNA are expressed in resident glomerular cells in renal tissues from NHK and type 1D. Glomerular alterations in these in situ mRNA expressions sufficient to explain ECM accumulation and DN risk were not uncovered. These largely negative results could be due to methodologic quantitative imprecision or could indicate that post-translational differences account for ECM imbalance in DN. However, these studies make it clear that unraveling the nature of the ECM production/removal imbalance in DN will require careful consideration of alterations in glomerular cell number. PMID- 12372939 TI - Familial juvenile gouty nephropathy: exclusion of 16p12 from the candidate locus. AB - BACKGROUND/AIMS: Familial juvenile gouty nephropathy (FJGN, MIM 162000) is an autosomal-dominant renal disease characterized by underexcretion-type hyperuricemia, gouty arthritis, and progressive renal disease at younger ages. We analyzed the localization of the responsible gene for FJGN concerning the chromosomal region of 16p12 using parametric linkage analysis in our FJGN. METHODS: The affected members of this family were accompanied with polyuria due to nephrogenic diabetes insipidus and without hypertension. Fifteen samples were collected from 9 affected and 6 nonaffected members of the family. By using microsatellite markers mainly focused on the short arm of chromosome 16, two point and multipoint linkage analyses were carried out. RESULTS: All of the 2 point logarithm of odds (LOD) scores were typically negative and all of the multipoint LOD scores were less than -3.0 in our FJGN family. CONCLUSIONS: The results suggested that the localization of the responsible gene to 16p12 can be excluded in our FJGN family. This finding means that the responsible gene for FJGN is not common. PMID- 12372940 TI - Potassium-lowering effect of mineralocorticoid therapy in patients undergoing hemodialysis. AB - The present study was conducted to examine potassium lowering effect of exogenous mineralocorticoid (fludrocortisone acetate; FCA) administration to the patients with chronic renal failure undergoing hemodialysis. Fifteen patients on hemodialysis receiving FCA with its dosage gradually increased from 0 to 0.20 mg/day were observed for five successive 4-week periods. The serum potassium concentration was significantly decreased after FCA administration concomitant with the decrease of the salivary sodium to potassium ratio. Such decrease in serum potassium concentration was more significant in patients with <150 pg/ml of plasma aldosterone concentration (PAC) (low PAC group) than in those with >/=150 pg/ml of PAC (high PAC group). 0.05 mg of FCA was sufficient to lower serum potassium in low PAC group, while 0.15 mg of FCA was required for high PAC group. FCA administration did not affect serum sodium, chloride and bicarbonate concentrations. Body weight and blood pressure were not increased during the experimental periods. There were no significant changes in plasma level of glucose, insulin, epinephrine and norepinephrine. These results suggested that FCA could be effective to treat hyperkalemia without any adverse effects in patients undergoing hemodialysis. PMID- 12372941 TI - Can haemodialysis-induced hypotension be predicted? AB - BACKGROUND: During haemodialysis (HD) ultrafiltration may affect the central blood volume to an extent that blood pressure decreases. Thoracic electrical impedance (TI) is applied to monitor the central blood volume and we evaluated if it can be used to predict HD-induced hypotension. METHODS: In 12 hypotensive prone (H) and 13 non-hypotensive prone (N) patients, blood pressure and heart rate were recorded during one dialysis session every 30 min, while TI, thoracic intracellular water (Th(ICW)) and total body impedance (TBI) were followed every 10 min. Hypotension was defined as a decrease in systolic blood pressure (SAP) >/=30 mm Hg or a SAP < 90 mm Hg. RESULTS: All 12 H patients developed hypotension after 190 +/- 10 min (mean +/- SE) as SAP decreased 35 +/- 5 mm Hg, while the 13 N patients maintained blood pressure. TBI increased in all patients and the increase was similar (60 +/- 5 and 56 +/- 6 Omega in H and N patients, respectively). In N patients TI did not change significantly for the first 2 h of HD, while it became elevated by 2.8 +/- 0.6 Omega (1.5 kHz) and 2.3 +/- 0.7 Omega (100 kHz) by the end of the dialysis. In H patients, the increase in TI took place at the onset of HD to reach higher values (by 7.0 +/- 0.5 Omega at 1.5 kHz and 5.9 +/- 0.5 Omega at 100 kHz). Th(ICW) was changed only in H patients (decreased by 7.9 +/- 2.1 Siemens (S) 10(-4), p < 0.05), while HR increased (9 +/ 2 beats/min) in 8 of 12 H patients, while it decreased in 1 patient (by 9 beats/min). CONCLUSIONS: The results suggest that in HD patients hypotension is elicited by a reduction in the central blood volume that affects heart rate and the distribution of red cells within the body. To prevent HD-induced hypotention, the ultrafiltration rate could be reduced when an increase in thoracic impedance approaches 5 Omega, or when an index of intracellular water decreases by 6 10( 4). PMID- 12372942 TI - Standard heparin versus low-molecular-weight heparin. A medium-term comparison in hemodialysis. AB - BACKGROUND: To compare standard heparin (SH) and low molecular weight heparin (LMWH) in terms of anticoagulation, platelet activation and lipid metabolism, we selected 54 patients who had been on 4-hour hemodialysis three times weekly for at least 12 months, without bleeding disorders or dyslipidemic diseases. 28 were on hemodialysis with Polysulfone low-flux, 26 were on hemodiafiltration with Polysulfone high-flux. All patients underwent EPO. METHODS: During the first 18 months, we administered SH 1,500 IU on starting dialysis and 1,500 +/- 500 IU in continuous intradialytic infusion per session. In the following 18 months, we administered LMWH 64.6 IU/kg on starting dialysis in a single arterious bolus. We assessed aPTT, anti-factor Xa activity, TAT and FPA, beta-TG and PF4. Blood samples were taken monthly at times 0, 30, 60, 180 and 240 min, as well as 1, 4 and 20 h after dialysis end. Predialysis cholesterol, HDL, LDL, triglycerides and lipoprotein(a) were checked monthly. RESULTS: During both LMWH and SH sessions no clotting or major bleeding complications were observed. APTT with LMWH was lower than that found with SH (p < 0.001); aFXa using LMWH was higher than when using SH (p < 0.001); TAT and FPA were lower in LMWH sessions (p < 0.01) than in SH sessions. We also detected lower beta-TG (p < 0.05) and PF4 levels (p < 0.05) using LMWH than using SH. As regards lipids, we only observed a significant decrease in triglycerides after 18 months of LMWH treatment. CONCLUSIONS: Routine use of LMWH during hemodialysis affords a safe and effective alternative to SH, and causes reduced platelet activation. PMID- 12372943 TI - Endotoxin-free dialysate improves response to erythropoietin in hemodialysis patients. AB - BACKGROUND/AIMS: Inflammatory process induced by endotoxin is one of the causes of resistance to recombinant human erythropoietin (rHuEPO) in hemodialysis patients. Thus dialysate contaminated with endotoxin may diminish response to rHuEPO. We investigated whether dose of rHuEPO could be reduced with endotoxin free ultrafiltered dialysate. METHODS: Twenty-seven chronic hemodialysis patients receiving rHuEPO were studied. The patients did not have known causes of anemia other than chronic renal failure. An endotoxin-cut polyethylene ultrafilter was installed into the dialysate fluid circuit. Hematocrit and dose of rHuEPO were monitored before and after installation. Dose of rHuEPO was adjusted to keep hematocrit at about 30%. Endotoxin concentration of dialysate was measured by commercial limulus test (Endospecy. RESULTS: After installation of ultrafilter, dialysate endotoxin concentration decreased from >100 to <1.0 endotoxin units/liter (EU/l). Dose of rHuEPO decreased from 90.0 U/kg/week (median) to 57.3 U/kg/week (p < 0.05) and hematocrit increased from 30.3% (median) to 32.2% (p = 0.03) after 5 months of installation of ultrafilter. The running cost of the ultrafilter corresponded to only 4% of the cost of spared rHuEPO. CONCLUSIONS: Ultrafiltered endotoxin-free dialysate caused significant reduction in dose of rHuEPO to keep target hematocrit level. Endotoxin-cut ultrafilter was beneficial to hemodialysis patients in medical and in economical aspects. PMID- 12372944 TI - Blood volume control by biofeedback and dialysis-induced symptomatology. A short term clinical study. AB - In earlier studies, a reduction in intradialytic procedures was observed in patients with severe intradialytic hypotension symptomatology by the use of blood volume controlled biofeedback systems. However, few data are present on the use of biofeedback-controlled treatments in patients experiencing minor intradialytic symptoms. In the present study, 157 standard and 158 biofeedback-controlled treatments were compared during a 2-month period in 16 hemodialysis patients. Both the percentage of hypotensive episodes (6.3 +/- 11.3 vs. 15.8 +/- 18.3%; p < 0.05) as well as other intradialytic symptoms (cramps, nausea, headache, abdominal pain) (11.0 +/- 12.8 vs. 18.1 +/- 16.9%; p < 0.05) were significantly less during biofeedback-controlled treatments compared to standard dialysis treatments, despite a similar decline in relative blood volume (8.8 +/- 3.5 vs. 8.3 +/- 3.1%; p = n.s.). Interdialytic weight gain and intradialytic rise in plasma sodium levels were comparable. Concluding, in this short-term preliminary study, blood volume controlled biofeedback improved dialysis tolerance also in patients with minor intradialytic symptomatology. PMID- 12372945 TI - Increased circulating levels of natriuretic peptides predict future cardiac event in patients with chronic hemodialysis. AB - BACKGROUND/AIMS: Cardiovascular events are the major determinant of the prognosis in patients with chronic hemodialysis. The present study was designed to investigate whether increased plasma levels of atrial or brain natriuretic peptides (ANP or BNP) predict future cardiac events in such patients. METHODS: Fifty-three patients undergoing chronic hemodialysis without clinical symptoms suggestive of cardiac disorders were enrolled and their blood was sampled for ANP and BNP measurements. Electrocardiograms demonstrated left ventricular hypertrophy in 28 patients but no other abnormal findings. We followed them up for 11.3 +/- 0.2 months. The endpoint was cardiac events. RESULTS: Cardiac events occurred in 13 patients (CE group). Both ANP and BNP levels were higher in CE group than in patients without cardiac events (ANP: 118 +/- 21 vs. 56 +/- 5 pg/ml, BNP: 769 +/- 204 vs. 193 +/- 25 pg/ml, respectively). Receiver operating characteristics curve revealed that the cut-off levels of ANP and BNP were 58 and 390 pg/ml, respectively. Using the Kaplan-Meier method, the incidence of cardiac events was significantly greater in patients with higher levels of ANP (50.0 vs. 0.0%) or BNP (72.7 vs. 11.9%) than in those with lower levels of the peptides. CONCLUSIONS: Elevated levels of ANP or BNP indicate an increased risk of cardiac events and these peptides are clinically useful to predict cardiac events in patients with hemodialysis. PMID- 12372946 TI - Maintenance hemodialysis and circulating ionized magnesium. AB - BACKGROUND: Circulating magnesium exists in the bound and in the free ionized form, that is biologically active. In kidney disease the relationship between ionized and total circulating magnesium is often altered. Little information is available on the influence of hemodialysis on the relationship between ionized and total circulating magnesium in end-stage kidney disease. METHODS: Plasma total and ionized magnesium and the plasma ionized magnesium fraction were assessed before and after hemodialysis (dialysate magnesium content 0.75 mmol/l) in 46 patients with end-stage kidney disease and in a control group of 25 healthy subjects. RESULTS: In patients plasma total (from 1.19 [1.05-1.33] to 1.10 [1.02 1.16] mmol/l; median and interquartile range) and ionized (from 0.71 [0.66-0.78] to 0.65 [0.63-0.69] mmol/l) magnesium significantly decreased during dialysis (control subjects: 0.82 [0.80-0.92], respectively, 0.57 [0.54-0.59] mmol/l). The plasma ionized magnesium fraction was significantly lower in patients both before (0.61 [0.58-0.64)] and after (0.60 [0.56-0.62]) hemodialysis than in controls (0.68 [0.65-0.70]). CONCLUSIONS: The study demonstrates a tendency towards a reduced circulating ionized magnesium fraction in end-stage kidney disease that is not corrected by hemodialysis. PMID- 12372947 TI - The synthesis by fine-needle aspiration biopsy cultures of IL-7, IL-16 and IL-18 is significantly associated with acute rejection in kidney transplants. AB - BACKGROUND: T-cell activation, the key event in the development of acute allograft rejection, depends on co-stimulatory signals delivered by antigen presenting cells (APC). APC-derived cytokines may provide co-stimulation and modulate alloimmune reaction. We have studied cytokine synthesis by fine-needle aspiration biopsy (FNAB) culture and we found significant differences for interleukin (IL)-2, IL-6, IL-10, M-CSF and IL-1ra on comparing acute rejection versus stable kidney transplant patients. We report our findings on FNAB cultures synthesis of IL-7, IL-15, IL-16, IL-17, IL-18 and RANTES (regulated upon activation, normal T-cell expressed and secreted), all potential modulators of anti-graft reaction. PATIENTS AND METHODS: Kidney transplants (KTX) treated with CsA-AZA-Pred from the beginning, were divided into four groups. Group I: day 7 post-KTX, stable; II: day 7 post-KTX, 6.5 +/- 5.5 days before acute rejection; III: first day of acute rejection; IV: day 14 post-KTX, stable. Patients from I and IV remained rejection-free for the first 6 months, at least. All rejection episodes were confirmed by classical core renal biopsy. FNAB samples were cultured according to our published methodology and culture supernatants were collected at 48 h and analysed by ELISA for IL-7, IL-15, IL-16, IL-17, IL-18 and RANTES. RESULTS: Group III synthesized significantly higher amounts of IL-7, IL 16 and IL-18 than stable patients (groups I and IV). RANTES production did not show significant differences among the four groups. We did not find any trace of IL-15. CONCLUSIONS: IL-18 may play the activation role that has been attributed to IL-12 which previously, we did not find to correlate significantly with acute rejection in KTX. IL-16 seems to play an activation role rather than an inhibition of anti-graft reaction. We confirm that RANTES is not significantly associated with acute rejection in KTX. PMID- 12372948 TI - Expression in mouse kidney of membrane copper transporters Atp7a and Atp7b. AB - Copper is essential for activity of many enzymes, but is toxic in excess. Several copper proteins are required for copper homeostasis. ATP7A and ATP7B are genes encoding membrane copper transporters. ATP7A, defective in Menkes disease (MNK), is expressed in many tissues involved primarily in copper uptake from dietary sources. ATP7B, defective in Wilson disease (WND), is essential for copper excretion. Although MNK patients have a copper deficiency in most tissues, copper accumulates in proximal tubules in the kidney. WND patients also have copper accumulation in the proximal tubules. In some WND patients this copper accumulation may result in tubular dysfunction, resulting in the increased excretion of low molecular weight substances (e.g. amino acids and calcium). In mouse, we have demonstrated, by in situ hybridization, the expression pattern in the kidney of mouse orthologues, Atp7a and Atp7b, and have confirmed Atp7b expression by immunohistochemistry. Both Atp7a and Atp7b are expressed in glomeruli; however, Atp7b is also seen in the kidney medulla. This suggests that glomeruli are responsible for regulating copper levels in the filtrate. In WND patients, urinary copper levels are extremely high suggesting Atp7b in the loops of Henle may have a role in copper reabsorption. PMID- 12372949 TI - An oral adsorbent downregulates renal expression of genes that promote interstitial inflammation and fibrosis in diabetic rats. AB - BACKGROUND/AIMS: An oral adsorbent, AST-120, removes uremic toxins such as indoxyl sulfate, and delays the progression of renal failure. This study was designed to investigate the effects of AST-120 on the molecular basis of interstitial inflammation and fibrosis, using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus. METHODS: Four weeks after unilateral nephrectomy, the uninephrectomized OLETF (1/2NxOLETF) rats were divided into two groups: AST-120-administered and control 1/2NxOLETF rats. After the administration of AST-120 for 48 weeks, we examined the effects of AST 120 on renal functional, pathological, and gene expressional changes. RESULTS: The administration of AST-120 to the 1/2NxOLETF rats attenuated the progression of renal dysfunction, proteinuria, glomerular sclerosis, tubular injury, and interstitial inflammation and fibrosis. AST-120 significantly reduced renal expression of intercellular adhesion molecule (ICAM)-1, osteopontin, monocyte chemotactic protein (MCP)-1, and transforming growth factor (TGF)-beta1, as well as clusterin. All the five molecules were expressed mainly in tubular cells. AST 120 also decreased serum and urinary levels of indoxyl sulfate and the overload of indoxyl sulfate in tubular cells. CONCLUSIONS: AST-120 ameliorates tubulointerstitial injury by reducing renal expression of ICAM-1, osteopontin, MCP-1, TGF-beta1 and clusterin in 1/2NxOLETF rats. PMID- 12372950 TI - The herbal medicine Sairei-to inhibits proliferation of rat mesangial cells. AB - BACKGROUND: The herbal medicine Sairei-to is efficacious in renal diseases where mesangial proliferation is a key event. We examined whether Sairei-to inhibits proliferation of cultured rat mesangial cells and investigated its mechanism of action. METHODS: The effect of Sairei-to on [(3)H]-thymidine incorporation stimulated by serum was assessed. Cell cycle was analyzed by flowcytometry. Extracellular signal-regulated kinase (ERK) activity was determined by immunecomplex kinase assay. Tyrosine phosphorylation of cellular proteins, and phosphorylation of ERK and Raf-1 were analyzed by immunoblot. Cyclic AMP was measured by radioimmunoassay. RESULTS: Incubation of mesangial cells for 18 h with water-soluble but not insoluble fraction of Sairei-to inhibited serum stimulated [(3)H]-thymidine incorporation. In subsequent experiments, water soluble fraction, at a dose required for a half-maximal response (2 mg/ml), was used. Sairei-to inhibited S-phase entry stimulated by serum. Serum-induced tyrosine phosphorylation of p44 and p42 ERK was inhibited by Sairei-to, but that of other cellular proteins was not affected. Suppression of serum-stimulated ERK activation by Sairei-to was confirmed by immunecomplex kinase assay. Activation of Raf-1, an upstream activator of ERK, was also attenuated by Sairei-to. Incubation of cells with Sairei-to significantly increased the generation of cAMP. CONCLUSIONS: Sairei-to inhibits serum-induced DNA synthesis of rat mesangial cells by suppressing Raf-1/ERK cascade probably via cAMP. PMID- 12372951 TI - Heme oxygenase-1 localization in the rat nephron. AB - BACKGROUND/AIMS: Renal tubules undergo oxidative injury in various nephropathies. It is unknown whether tubular cells possess mechanisms to attenuate this form of injury. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, may provide such a mechanism by reducing levels of free heme, a prooxidant molecule, and by limiting activity of heme-containing prooxidant enzymes. Determination of the distribution of HO-1 in the nephron may identify those segments where HO-1 can afford protection against oxidative injury. METHODS: Rats were injected subcutaneously with two different inducers of HO-1: Stannous chloride and cobalt protoporphyrin. At completion of injections, frozen sections of kidneys were stained for HO-1 using a biotin-conjugated monoclonal anti-HO-1 antibody. To identify the origin of tubules staining positive for HO-1, Tetragonolobus purpureas (TP)-derived lectin and Arachnis hypogaea (AH)-derived lectin were applied to sequential sections of the kidney cortex. RESULTS: In rats injected with either HO-1 inducer, HO-1 was immunolocalized in tubules but not in glomeruli. Staining of sequential sections with TP-derived lectin, which binds mainly to proximal tubular cells, was negative in the tubules that stained positive for HO-1. Staining of sequential sections with AH-derived lectin, which binds mainly to distal and collecting tubular cells, was positive in those tubules that were also positive for HO-1. CONCLUSIONS: In kidneys of rats injected with inducers of HO-1, distal and collecting tubular cells were identified as the main segments of the nephron that express HO-1. We suggest that the distal nephron, by expressing HO-1, may be less vulnerable to oxidative injury. PMID- 12372952 TI - In vitro effects of Habu snake venom on cultured mesangial cells. AB - BACKGROUND: Habu snake venom (HSV)-induced glomerulonephritis is a unique model showing a progressive course of mesangial proliferation. To elucidate the in vitro effects of HSV, we examined whether HSV itself could have direct effects on the cultured mesangial cells, such as cell proliferation and activation of chemokine gene expression. METHODS: The incorporation of 5-[(125)I]iodo-2' deoxyuridine was measured with a gamma-counter, and gene expressions of growth factors, chemokines and cytokines were evaluated by a real time quantitative PCR. RESULTS: We demonstrated that excessive or continuous HSV stimulation decreased a mesangial cell viability. However, adequate and temporary HSV stimulation induced proliferation of mesangial cells in vitro along with a significant elevation of monocyte chemoattractant protein-1 (MCP-1) mRNA levels. In addition to these in vitro results, we showed that MCP-1 mRNA levels increased in renal cortices of glomerulonephritis induced by HSV. Immunohistochemistry also showed a positive staining for MCP-1 in the marginal area of glomerulus with mesangiolysis. CONCLUSIONS: These data suggest that HSV itself may elicit direct biological effects on mesangial cells which may participate in pathophysiology of glomerulonephritis induced by HSV. PMID- 12372953 TI - MPO-ANCA-associated small vessel vasculitis presenting as fever of unknown origin. Report of one case. AB - Microscopic polyangiitis (MPA) is an antineutrophil cytoplasmic antibody (ANCA) associated small vessel vasculitis which can present with various clinical manifestations, for which the mainstay of treatment is systemic corticosteroids and immunosuppressants. We report a case of a 54-year-old female admitted to the hospital because of fever during the last month, leukocytosis and elevated erythrocyte sedimentation rate. Persistence of elevated serum creatinine levels and accompanying hematuria led us to perform a renal biopsy, and MPA was diagnosed on the basis of light and immunofluorescence microscopy. Remission was induced with oral corticosteroids and cyclophosphamide therapy in conjunction with plasmapheresis (PF). The objective of this report was to assess the role of PF in the treatment of MPA and report on its utility in patients with MPA who are not responding to standard therapy or who require unacceptably high doses of steroids or immunosuppressants. In a patient presenting with fever of unknown origin, microscopic polyangiitis should also be considered in the differential diagnosis. PMID- 12372954 TI - Acute interstitial nephritis due to nicergoline (Sermion). AB - We report a case of acute interstitial nephritis (AIN) due to nicergoline (Sermion). A 50-year-old patient admitted to our hospital for fever and acute renal failure. Before admission, he had been taking nicergoline and bendazac lysine due to retinal vein occlusion at ophthalmologic department. Thereafter, he experienced intermittent fever and skin rash. On admission, clinical symptoms (i.e. arthralgia and fever) and laboratory findings (i.e. eosinophilia and renal failure) suggested AIN, and which was confirmed by pathologic findings on renal biopsy. A lymphocyte transformation test demonstrated a positive result against nicergoline. Treatment was consisted of withdrawal of nicergoline and intravenous methylprednisolone, and his renal function was completely recovered. To our knowledge, this is the first report of nicergoline-associated AIN. PMID- 12372955 TI - Nephrotic syndrome with portal, splenic and renal vein thrombosis. A case report. AB - BACKGROUND: Thromboembolism is known as a major complication of nephrotic syndrome, but only 4 cases of portal vein thrombosis have been reported as a complication of nephrotic syndrome. All of these 4 cases had acute symptoms, and 3 of 4 were in relapsing phase of nephrotic syndrome when thrombi were found. We describe here a case of 51-year-old woman with fresh nephrotic syndrome that was asymptomatically complicated by portal, splenic and renal vein thrombosis. CONCLUSIONS: In the presence of fresh nephrotic syndrome of minimal change, asymptomatic and widely distributed, including portal vein, thrombus formation occurred. If the clinical course shows resistance to therapy, we must consider the complication of venous thrombosis. Anticoagulant therapy with heparin and warfarin was effective and all thrombi disappeared without any other complications. PMID- 12372956 TI - Salvage of cyclosporine A-induced oxidative stress and renal dysfunction by carvedilol. AB - BACKGROUND: Cyclosporine A (CsA) is the first-line immunosuppressant employed for the management of solid organ transplantation and autoimmune diseases. Nephrotoxicity is the major limitation of CsA use. Recent evidence suggests that reactive oxygen species (ROS) play an important role in mediating CsA nephrotoxicity. The present study was designed to investigate effects of carvedilol, a third-generation beta-blocker with potent free radical-scavenging activity on CsA-induced oxidative stress and resultant renal dysfunction in a rat model of chronic CsA nephrotoxicity. METHODS: Carvedilol (2.0 and 4.0 mg/kg i.p.) and propranolol (10 mg/kg i.p.) were administered to separate group of animals 24 h before and concurrently with CsA (20 mg/kg s.c.) for 21 days. Renal function was assessed by estimating plasma creatinine, blood urea nitrogen (BUN), creatinine and urea clearance. Tissue lipid peroxidation was measured as thiobarbituric acid-reacting substances (TBARS). Renal morphological alterations were assessed by histopathological examination of hematoxylin-eosin, PAS and Masson's trichrome stained sections of the kidneys. RESULTS: CsA (20 mg/kg s.c) administration for 21 days produced elevated levels of TBARS and deteriorated renal function as assessed by increased plasma creatinine, BUN and decreased creatinine and urea clearance as compared to vehicle-treated rats. The kidneys of CsA-treated rats showed severe striped interstitial fibrosis, arteriolopathy, glomerular basement thickening, tubular vacuolization and hyaline casts. Propranolol neither decreased TBARS nor improved the renal dysfunction and morphological changes induced by CsA. Both doses of carvedilol markedly reduced elevated levels of TBARS, whereas the higher dose of carvedilol significantly attenuated renal dysfunction and morphological changes in CsA-treated rats. CONCLUSIONS: These data clearly indicate the renoprotective potential of carvedilol in CsA-induced nephrotoxicity and suggest a significant contribution of its antilipoperoxidative property in this beneficial effect. PMID- 12372957 TI - Tranilast slows the progression of advanced diabetic nephropathy. AB - BACKGROUND: Tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, suppresses collagen synthesis by various cells, including macrophages and fibroblasts, by interfering with the actions of transforming growth factor-beta 1. We investigated the effect of tranilast on progression of diabetic nephropathy (DN), since this process is associated with accumulation of collagens in the glomerulus and interstitium. METHODS: Tranilast (100 mg, 3 times daily) was administered to 9 outpatients with advanced DN who were receiving an angiotensin-converting enzyme inhibitor or an angiotensin II receptor antagonist and who exhibited a progressive decline in renal function. The decline in renal function before and during tranilast treatment was evaluated for each patient on the basis of the slope in reciprocal serum creatinine (1/S(Cr)) over time. Urinary type IV collagen (U-IV.C) and protein (U-P) excretions were measured just before commencement of tranilast treatment and every 2 months during the treatment. RESULTS: One male patient dropped out soon after commencement of tranilast treatment due to development of lung cancer, and hemodialysis was introduced in one female patient 6 months after the start of treatment. In the 8 patients who did not drop out, 1/S(Cr) was significantly less steep during tranilast treatment than before treatment (-0.00748 +/- 0.00700 vs. -0.01348 +/- 0.00636 dl/mg/month, respectively; p = 0.0374). U-IV.C and U-P tended to decrease with time, although the decrease was statistically insignificant. CONCLUSIONS: Our data suggest that tranilast treatment may suppress accumulation of collagens in renal tissue and may be therapeutically useful for reducing the progression of advanced DN. PMID- 12372958 TI - Can immunosuppressive therapy be useful in IgA nephropathy when the 'Point of No Return' has already been exceeded? AB - Immunosuppressive treatment of IgA nephropathy (IgAN) has been a controversial issue since many years, mainly because of skepticism on awaited results and fear of possible side effects. Some authors proposed the existence of a 'point of no return', after which the worsening in renal function becomes inexorable and treatment ineffective. Indeed, the decision to treat these patients is easily followed by disappointment due to lack of favorable results. We report the case of a 24-year-old woman with a diagnosis of IgAN with advanced sclerosis and chronic renal failure. After treatment with a 6-month steroid course, she experienced a long-lasting stabilization of renal function (serum creatinine) and decrease in proteinuria (from 2.9 to 0.46 g/24 h) that still persisted at the end of follow-up (48 months). Analysis of this case and review of the literature suggest that immunosuppressive treatment could delay the beginning of renal replacement therapy in the advanced phase of IgAN. However, the results of long term, randomized, controlled, adequately sized trials are awaited. PMID- 12372960 TI - Urinary complement factor H in renal disease. AB - BACKGROUND: Complement factor H (hCFH) plays a key inhibitory role in the control of the alternative complement pathway. We examined whether urinary hCFH (U-hCFH) levels is useful as an indirect indicator of renal damage. METHODS: Urine samples were obtained from 104 patients with renal disease. Urine was collected with 10 mM EDTA and U-hCFH levels were measured using the BTA TRAK Assay Kit. RESULTS: In the 62 patients with nephritis, the levels of U-hCFH were elevated (range 15 52,198 U/ml) over the normal range (0-14 U/ml). U-hCFH levels of patients with chronic renal failure, lupus nephritis, membranoproliferative glomerulonephritis, focal glomerulosclerosis were higher than that of IgA nephropathy patients (p < 0.05). In the patients with minimal change disease, showed high levels of U-hCFH during the nephrotic syndrome. U-hCFH was correlated significantly with urinary protein and urinary N-acetyl-beta-D-glucosaminidase. CONCLUSIONS: We demonstrated that U-hCFH was detected in the urine of nephritis patients. PMID- 12372959 TI - Significant elevations in serum mannose-binding lectin levels in patients with chronic renal failure. AB - BACKGROUND/AIMS: Mannose-binding lectin (MBL), a liver-derived C-type serum lectin, activates the complement cascade through the lectin pathway. Since the complement system contributes to the host defense against infections and mediates inflammatory processes including atherosclerosis, and since chronic renal failure (CRF) patients are prone to the development of infectious complications and cardiovascular disease, we focused on serum MBL levels in CRF patients who were either uremic, or who were receiving hemodialysis treatment. METHODS: MBL levels were measured in the sera of subjects with CRF before they began dialysis treatment (pre-HD patients; n = 23) and in the sera of subjects who were receiving maintenance hemodialysis (HD patients; n = 178), by ELISA using polyclonal anti-rabbit IgG and a monoclonal antibody directed against MBL (3E7). METHODS: Mean levels (+/- SD) of serum MBL were significantly higher in pre-HD subjects (4.343 +/- 2.533 microg/ml, p < 0.05) and in HD subjects (8.897 +/- 4.920 microg/ml, p < 0.05), than in healthy controls (1.452 +/- 0.692 microg/ml). Levels were also significantly higher in HD subjects than in pre-HD subjects (p < 0.05). CONCLUSIONS: Elevated serum MBL levels in patients with CRF might have significantly influence pathologic conditions such as alterations of the immune system and acceleration of atherogenesis. PMID- 12372961 TI - Renal amyloidosis secondary to tuberculosis of cecum. AB - Renal amyloidosis can occur as a primary or secondary, systemic or localized disorder. It is defined as a chronic infiltrative disorder characterized by impaired organ function caused by extracellular insoluble protein fibrils. Although colonic tuberculosis is not uncommon, the occurrence of reactive renal amyloidosis in such patients is not as prevalent. We report a single case of renal amyloidosis in a patient with tuberculosis of the cecum who presented with nephrotic syndrome. PMID- 12372962 TI - Hypokalemia due to Fanconi syndrome in a patient with obstructive jaundice. AB - There are limited data regarding tubular dysfunction in patients with obstructive jaundice. Here, we present a patient aged 79 years with protracted obstructive jaundice due to choledocholithiasis who developed hypokalemia with inappropriate kaliuria as a part of a non-acidotic generalized proximal tubular dysfunction, which included renal phosphate, uric acid and glucose wasting. PMID- 12372963 TI - Validation of simple indices to assess insulin sensitivity and pancreatic Beta cell function in patients with renal dysfunction. AB - BACKGROUND/AIMS: Insulin resistance and hyperinsulinemia has been reported in patients with chronic renal failure. However, usefulness and validation of new indices for assessment to insulin sensitivity and pancreatic beta-cell function were unknown. METHODS: We evaluated insulin sensitivity and pancreatic beta-cell function in 61 normal glucose tolerant (NGT) and 60 diabetic (DM) subjects; both groups were subdivided as normal renal function (NRF; C(cr) >/= 70 ml/min) and impaired renal function (IRF; C(cr) <70 ml/min). Insulin sensitivity were assessed by homeostasis model assessment of insulin resistance (HOMA-IR) and plasma glucose and insulin concentrations obtained at fasting or during a 75-gram oral glucose tolerance test (insulin sensitivity index), and pancreatic beta-cell function were assessed by insulinogenic index, first-phase insulin secretion index, and area under the response curve for plasma insulin (insulin-AUC(0-180)). RESULTS: There was no evidence of insulin resistance in NGT-IRF group. No differences in both insulinogenic index and first-phase insulin secretion index between NGT-NRF and NGT-IRF, but these were significantly decreased in DM-NRF and DM-IRF. There were inverse linear correlations between the insulin sensitivity index and insulin-AUC(0-180) in patients with NGT and DM, respectively. These correlations were similarly robust in NRF subjects and IRF subjects. CONCLUSIONS: New indices for assessment of insulin sensitivity and pancreatic beta-cell function calculated from plasma glucose and plasma insulin concentrations after OGTT are applicable for clinical use even in patients with renal dysfunction. PMID- 12372964 TI - New trends in the treatment of scleroderma renal crisis. AB - The main pathological changes observed in scleroderma kidney are edema and proliferation of intimal cells, glomerular changes with thickening and obliteration of arteries leading to decreased renal perfusion and increased renin release. Angiotensin converting enzyme inhibitors are the cornerstone in the treatment of patients with scleroderma renal crisis. Statins are used in the prevention of primary and secondary cardiovascular events. These drugs control cell proliferation and may prevent the injury observed in scleroderma kidney. PMID- 12372965 TI - Treatment with vasodilators and crude extract of Ganoderma lucidum suppresses proteinuria in nephrosis with focal segmental glomerulosclerosis. PMID- 12372966 TI - Leptin correlates with some hemostatic parameters in CAPD patients. AB - Hyperleptinemia is also common in chronic renal failure, particularly in CAPD. On the other hand, cardiovascular events related to thrombosis are a predominant cause of death and account also for an important morbidity in uremic patients. Treatment with recombinant human erythropoietin (rHuEPO) may shift the precarious hemostatic balance towards thrombosis. Therefore, the aim of the study was to assess the relationships between leptin and platelet aggregation and some hemostatic parameters in CAPD patients treated with rHuEPO. The study was performed on 15 patients maintained on CAPD given rHuEPO and 13 subjects without rHuEPO therapy served as a control group. Platelet aggregation was studied in both platelet-rich plasma (PRP) and in the whole blood. Tissue factor (TF) and tissue factor pathway inhibitor (TFPI) (antigens and activities), von Willebrand factor, trombomodulin, protein C, thrombin activatable fibrinolysis inhibitor (TAFI) and leptin (serum and dialysate) were assayed by using commercially available kits. Patients in both groups studied did not differ significantly with respect to age, BMI, duration of renal replacement therapy, and other hematological and hemostatic parameters studied as well as leptin serum and dialysate leptin. In CAPD patients treated with rHuEPO serum and dialysate leptin significantly correlated with tissue factor pathway inhibitor, protein C, thrombomodulin, ristocetin-induced platelet aggregation in the whole blood and PRP. In CAPD patients not treated with rHuEPO the significant correlations were observed between serum and dialysate leptin and protein C. Positive correlations between platelet aggregation and leptinemia in CAPD patients might indicate that hyperleptinemia could be associated with the cardiovascular disease in dialyzed patients. Leptin might contribute at least in part to the thrombotic complications observed in CAPD patients. PMID- 12372967 TI - Acute hydrothorax in CAPD. Eearly thoracoscopic (VATS) intervention allows return to peritoneal dialysis. AB - BACKGROUND/AIMS: Hydrothorax is a complication of continuous ambulatory peritoneal dialysis (CAPD) occurring due to pleuroperitoneal fistulae. Several treatments exist with no consensus as to best management. We report on the largest series of video-assisted thoracoscopic surgery (VATS) treated pleuroperitoneal fistulae yet available. METHODS: Between 1995 and 2000, we treated 6 CAPD patients for acute right hydrothoraces using VATS. Data pertaining to size and sterility of hydrothoraces, presence of diaphragmatic defects, surgical procedures performed, morbidity and return to CAPD were obtained. RESULTS: Hydrothoraces were drained in all patients and there were no significant growths on subsequent culture. Fistulae were directly identified and closed in three patients. In the remaining patients, endoclips were placed across the base of small diaphragmatic blebs (the presumed site of communication). Parietal pleurectomy was performed uneventfully in all patients. There was no morbidity, all patients returned to haemodialysis and there have been no recurrences. CONCLUSIONS: Pleuroperitoneal fistulae produce symptomatic hydrothoraces in CAPD patients. A variety of approaches to the problem have been described. This is the largest series of VATS available and shows the usefulness of this approach in both closing the defect and producing an effective pleurectomy to prevent recurrence. PMID- 12372968 TI - Hypersensitivity to paraoxybenzoic Acid esters (parabens) in a dialysis patient. AB - A 24-year-old woman undergoing chronic dialysis therapy who had been diagnosed as having 'heparin allergy' presented acute allergic reaction after the intracatheter injection of a heparin agent. Further investigation revealed that mild eosinophilia had persisted for more than 3 years before this incidence despite avoiding the use of heparin agents. The leukocyte migration inhibition test (LMIT) showed that heparin agent A which was used for the patient showed positive reaction while heparin agent B which was not used for the patient showed a negative reaction. Paraoxybenzoic acid esters (parabens) are contained in heparin agent A but not in heparin agent B. Parabens showed positive reactions on LMIT. Parabens are the most common preservatives in drugs, foods, and cosmetics and they sometimes induce allergic reactions through percutaneous and possibly ingestive sensitization. However, they cause more severe allergic reactions when used intravenously. We concluded that the allergen in this patient was not heparin but parabens. Hypereosinophilia of unknown origin often occurs in dialysis patients. Such patients may be hypersensitive to parabens. PMID- 12372969 TI - A rare combination of sites of involvement by Mycobacterium intracellulare in a hemodialysis patient: multifocal synovitis, spondylitis, and multiple skin lesions. AB - PURPOSE: Atypical mycobacterial infection is a rare but serious hazard in immunocompromised patients including those undergoing maintenance hemodialysis and immunosuppressive therapy. Recognition of unusual involvement patterns is important. METHODS: We describe an extremely rare combination of complications caused by such an organism in a patient with end-stage renal disease: spinal osteolysis and multiple skin lesions associated with synovitis. RESULTS: The patient had received a renal allograft 18 years previously but developed infection with Mycobacterium avium-M. intracellulare complex including dermatologic manifestations, spondylitis, and synovitis involving the wrist and lateral malleolus after initiation of hemodialysis when the transplanted kidney failed. An empirical antibiotic regimen failed to alleviate skin lesions or fevers, or to lower an elevated C-reactive protein concentration, until the patient's dose of methylprednisolone was increased to treat mild adrenal insufficiency. The increase resulted in rapid resolution of skin lesions. A compression fracture 6 months later was attributed to spondylitis caused by the same organism. CONCLUSIONS: We suspect that spondylitis represented the primary focus of M. intracellulare infection. PMID- 12372970 TI - Time course of serum prolactin and sex hormones following successful renal transplantation. AB - BACKGROUND: Chronic renal failure is commonly associated with disturbances in hypothalamic-pituitary-gonadal function. METHODS: The gonadotrophins, prolactin and estradiol or testosterone levels were measured immediately before renal transplantation, at discharge from the transplantation unit (19 +/- 8 days after Tx) and 6 months after transplantation in 21 patients, 7 females and 14 males, age range 21-60 years. RESULTS: The mean prolactin level was high during uremia and decreased rapidly after transplantation, from 441 to 167 mU/l in males and from 1,057 to 521 mU/l in females. Hypergonadotrophism was seen in most uremic patients, with the mean LH and FSH levels of 14.2 and 6.0 U/l in males and 14.7 and 4.0 U/l in females, respectively. A temporary change to hypogonadotrophic hypogonadism took place 2-3 weeks after transplantation and was followed by normalization of the hypothalamic-gonadal function. The levels of circulating sex steroids were suppressed when the patients were discharged from the transplantation unit but returned to the normal range at 6 months. CONCLUSIONS: We conclude that renal transplantation corrects the hyperprolactinemia induced by uremia and is followed by rapid onset of restoration of the hypothalamic pituitary-gonadal axis. PMID- 12372971 TI - Immunohistochemical staining for proliferation antigen as a predictor of chronic graft dysfunction and renal graft loss. AB - BACKGROUND: Assessment of proliferation rate (PR%) using monoclonal antibody for Ki-67 antigen has recently been found to have prognostic importance in lung and cardiac allografts. We ascertained whether the same might be true for renal allografts. METHODS: Newly cut sections from 20 archival paraffin blocks of renal allograft biopsy material showing acute cellular rejection and/or acute tubular necrosis (ATN) and absence of other pathology were stained using MIB-1 antibody and were further double-stained with anti-CD3, anti-CD20 or anti-CD68 antibodies. Counts of staining of mononuclear interstitial cells were correlated with clinical and pathological data. RESULTS: Mean PR% was significantly greater than that in control renal allografts (13.23 +/- 1.94 vs. 2.84 +/- 1.66, p < 0.01). PR% of cases with ATN and no or borderline rejection was significantly lower than that of the remaining cases with acute rejection pathology (6.68 +/- 1.15 vs. 14.31 +/- 1.62, p < 0.05). However, PR% was neither correlated to histological rejection grade nor to long-term graft outcome. Double labelling failed to identify the cell type of most infiltrating MIB-1 positive cells. CONCLUSIONS: Positive MIB-1 staining helps to identify the presence of rejection but does not appear to predict prognosis or correlate with the Banff classification of rejection pathology. PMID- 12372972 TI - Severe alloimmune hemolytic anemia after renal transplantation. AB - Alloimmune hemolytic anemia is a rare complication following allogeneic organ transplantation. Despite that some other drugs have also been reported, in the majority of cases this complication has been associated with cyclosporine therapy. We here present a case of severe alloimmune hemolytic anemia due to ABO minor incompatibility after renal transplantation in a patient treated with tacrolimus. PMID- 12372973 TI - Acute uric acid nephropathy by overdosage of benziodarone in a renal transplant recipient. PMID- 12372974 TI - Brain atrophy in uremia. Research of the causes and future perspectives. PMID- 12372975 TI - Renal infarction in a hyperhomocysteinemic patient. PMID- 12372976 TI - Cerivastatin induces rhabdomyolysis and acute renal failure. PMID- 12372977 TI - End-stage renal disease in mayer-rokitansky-kuster-hauser syndrome. PMID- 12372978 TI - Preexisting essential hypertension accelerates the development of diabetic renal lesions in early stage nephropathy. PMID- 12372979 TI - Pharmacokinetics of tramadol in a hemodialysis patient. PMID- 12372980 TI - Role of submandibular salivary glands in LPS-induced lung inflammation in rats. AB - OBJECTIVE: Literature data suggest that rodent salivary glands can exert a neuroimmunomodulatory influence on distant inflammatory events. The release of regulatory factors by salivary glands appears to be influenced by time-dependent factors. In this paper we examined this possibility directly by studying the role of submandibular salivary glands in the temporal profile of lypopolysaccharide (LPS)-induced lung inflammation in rats. METHODS: The submandibular glands were removed (SMGx) or not (sham) and, 4 days later, the animals received an intravenous LPS injection (Salmonella abortus equi, 1 mg/kg). Cells in peripheral blood and in bronchoalveolar and bone marrow lavages were quantified after 90 min, 1, 3 and 5 days. Tumor necrosis factor (TNF) activity and corticosterone concentrations in serum were also determined. Baseline values were determined in a group of naive rats. RESULTS: One day after the LPS injection, neutrophil counts in lungs and blood in both animal groups were elevated, but the SMGx rats presented a significantly lower response in comparison to the sham-operated controls. Five days after LPS treatment, however, SMGx rats had higher neutrophil counts in the lungs than did sham animals, but numbers of blood neutrophils were equal. Ninety minutes after LPS injection, a peak of serum TNF activity was detected in both groups compared with naive levels. At this time point, TNF activity was about 135% higher in the serum of the SMGx group than in controls. Corticosterone levels of sham-operated controls rose only on the 5th day after LPS, whereas SMGx rats had significant peaks of corticosterone both on the 1st and the 5th day, but not on the 3rd day. CONCLUSION: Our data indicate that submandibular glands have a dual effect on inflammatory pulmonary response by differentially modulating the profile of lung neutrophil influx. PMID- 12372981 TI - Autonomic regulation of experimental autoimmune encephalomyelitis: the role of interferon-gamma. AB - OBJECTIVE: The effect of chemical sympathectomy on experimental autoimmune encephalomyelitis (EAE) was studied in interferon-gamma (IFN-gamma) knockout C57BL/6 mice. METHODS: Mice were immunized with myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide. Sympathectomy was achieved by subcutaneous administration of 6-hydroxydopamine. RESULTS: We showed previously that wild-type mice developed a mild form of EAE with complete recovery. Sympathectomy caused more serious disease and mice did not recover completely, indicating that the sympathetic nervous system (SNS) downmodulates the process of EAE. The clinical signs of disease were more serious in untreated IFN-gamma(-/-) mice than in wild type animals. Unexpectedly, sympathectomy resulted in suppression of active EAE in IFN-gamma(-/-) mice, implying that control of actively induced EAE by the SNS depends on INF-gamma and the integrity of the cytokine network. We also induced EAE by passive transfer of MOG(35-55)-reactive lymph node cells, and this disease was aggravated by sympathectomy in both wild-type and knockout animals. CONCLUSIONS: Our study supports the idea that the integrity of the whole cytokine network is necessary to maintain normal nervous-immune system interactions. PMID- 12372982 TI - Feedback between glial tumor necrosis factor-alpha and gp120 from HIV-infected cells helps maintain infection and destroy neurons. AB - An envelope glycoprotein, gp20, of the human immunodeficiency virus (HIV) interacts with host systems to promote HIV replication. gp120 is also involved in tissue-destructive positive feedback cycles that contribute to HIV-related but non-lymphocytic-, non-immunodeficiency-related tissue-destructive morbidity. Exposure to gp120 results in tumor necrosis factor-alpha (TNF) upregulation, particularly in cells of monocyte lineage. The resultant increased TNF in the microenvironment of the TNF-producing monocyte lineage cells results in increased occupancy of TNF receptors on nearby lymphocytes, monocytes or glia in which HIV does replicate. Such TNF binding increases HIV replication. Increased replication results in increased gp120 available to bind to monocyte lineage cells, further increasing or maintaining those cells' TNF production in the face of other TNF suppressive forces. A trophic environment (TNF) for HIV replication is thereby maintained. gp120 raises cAMP levels. Increased cAMP is inherently TNF suppressive. This is a moderating negative feedback element embedded within the larger positive feedback cycle. HIV does not effectively replicate in neurons yet many HIV infections show significant neuron loss. gp120 stimulates glia to synthesize TNF. Increased TNF stimulates HIV to replicate in the cells present in which HIV is able to replicate. TNF also damages nearby neurons. The resultant increased gp120 would further stimulate glia, and the stimulated glia's TNF would damage local neurons. Damaged neurons make factors that activate glia to upregulate TNF synthesis. These feedback cycles centering on gp120 and TNF contribute to HIV pathophysiology, neuron loss and maintenance of infection. PMID- 12372983 TI - Immunological variables mediate cognitive dysfunction in gulf war veterans but not civilians with chronic fatigue syndrome. AB - We explored the relationship between a set of immunological variables and a set of cognitive and functional status measures and a diagnosis of chronic fatigue syndrome (CFS) in civilians and veterans using various regression and factor analytic methods. Our approach emphasized the extraction of a few distinct factors in order to limit statistical problems associated with doing large numbers of multiple comparisons. This approach led to our finding cytokine data grouping into type 1 and type 2 clusters. A type 2 cluster plus a T and B cell factor predicted CFS caseness for Gulf War veterans but not for civilians with CFS. When a cognitive variable, reaction time, was added into the model, both immunological factors lost statistical significance; this indicates that the cognitive variable reaction time moderated the effects of the immunological factors in predicting patient status. We did a similar analysis on the roles of the immunological and cognitive variables in functional status using SF-36 data. Higher levels of these same two immunological factors predicted poorer general health as well as poorer physical and social functioning in Gulf War veterans but not in civilians with CFS. When the reaction time factor was added, only the lymphocyte factor remained significant. This implies that lymphocytes are directly related to functional status in Gulf War veterans with CFS, but the Th2 factor produces its effect on functional status via changes in cognitive abilities. PMID- 12372984 TI - Alteration of locus coeruleus neuronal activity by interleukin-1 and the involvement of endogenous corticotropin-releasing hormone. AB - Activity of the locus coeruleus (LC), which is the source of most of the norepinephrine in the brain, may participate in effects of the cytokine interleukin (IL)-1. This report describes the influence of IL-1 beta on the electrophysiological single-unit activity of LC neurons. When microinjected into the LC, human recombinant IL-1 beta (50 pg to 5 ng) increased the activity of LC neurons, predominantly by increasing 'burst' firing, which occurs in response to a sensory stimulus. At the higher doses and/or with longer time delays after injection, the spontaneous depolarization rate was also increased. This excitation (1). did not occur if IL-1 beta was microinjected nearby but outside of the LC and (2). could be reversed by administration of IL-1 receptor antagonist (IL-1 RA). In contrast to excitatory effects, microinjection of a very low dose of IL-1 beta (5 pg) into the LC inhibited LC activity, and this change could also be blocked by IL-1 RA. In view of earlier findings that (1). LC electrophysiological activity could be inhibited by microinjection of corticotropin-releasing hormone (CRH) into the LC region and (2). IL-1 beta in the brain stimulates the release of CRH, the hypothesis was tested that the inhibition of LC activity produced by the low dose of IL-1 was mediated by CRH. Microinfusion of the CRH receptor antagonist alpha-helical CRH(9-41) blocked the inhibition of LC activity otherwise produced by 5 pg of IL-1 beta, thus indicating that IL-1 beta also influences the activity of LC neurons via CRH. Finally, microinjection of IL-1 RA alone was found to decrease LC activity, raising the possibility that LC neurons are under the influence of tonic excitation by IL-1 in the brain. In summary, the findings described here show that the activity of LC neurons can be influenced by IL-1 beta through stimulation of IL-1 beta receptors. The potential involvement of IL-1 beta in stress responses by means of this cytokine influencing the activity of LC neurons is discussed. PMID- 12372985 TI - Thermal response to zymosan: the differential role of complement. AB - OBJECTIVES: This study was designed to determine whether the complement (C) system may be involved in the febrile response to zymosan (Zym), a glycan derived from yeast cell walls. METHODS: Cobra venom factor (CVF) at 100 U/animal or its vehicle, pyrogen-free saline (PFS), was injected intravenously (i.v.) into guinea pigs to deplete serum C. Eighteen hours later, a low or high dose of Zym or its vehicle, PFS, was administered i.v. or intraperitoneally (i.p.) to these animals. The core temperature (T(c)) was measured continuously by thermocouples. Serum C levels were determined by sheep erythrocyte hemolytic assay. RESULTS: Zym at 1 mg/kg caused a 1 degrees C T(c) rise that was not significantly affected by CVF pretreatment. However, CVF-induced hypocomplementation converted the T(c) fall ( approximately 1.2 degrees C) produced by 100 mg/kg of Zym i.p. into a 1 degrees C T(c) rise. Similarly, CVF pretreatment did not affect the T(c) rise caused by 0.5 mg/kg of Zym i.v., but converted the T(c) fall induced by 25 mg/kg i.v. into a 1 degrees C T(c) rise. A separate experiment showed that 25, but not 0.5 mg/kg of Zym i.v., decreased serum C by 34% in 15 min; C did not recover over the next 6 h. A second i.v. injection of 25 mg Zym/kg 210 min later, when the T(c) had recovered but the serum C had not, yielded a smaller and briefer T(c) fall. CONCLUSION: These results suggest that Zym is inherently pyrogenic, but this effect is manifested only when the dose of zymosan is too small to activate C or when C has been reduced by prior activation. PMID- 12372986 TI - How mitochondrial damage affects cell function. AB - The pathophysiology of mitochondrial DNA (mtDNA) diseases is caused by increased cell death and dysfunction due to the accumulation of mutations to mtDNA. While the disruption of oxidative phosphorylation is central to mtDNA diseases, many other factors, such as Ca(2+) dyshomeostasis, increased oxidative stress and defective turnover of mitochondrial proteins, may also contribute. The relative importance of these processes in causing cell dysfunction and death is uncertain. It is also unclear whether these damaging processes lead to the disease phenotype through affecting cell function, increasing cell death or a combination of both. These uncertainties limit our understanding of mtDNA disease pathophysiology and our ability to develop rational therapies. Here, we outline how the accumulation of mtDNA mutations can lead to cell dysfunction by altering oxidative phosphorylation, Ca(2+) homeostasis, oxidative stress and protein turnover and discuss how these processes affect cell function and susceptibility to cell death. A better understanding of these processes will eventually clarify why particular mtDNA mutations cause defined syndromes in some cases but not in others and why the same mutation can lead to different phenotypes. PMID- 12372987 TI - On the release of cytochrome c from mitochondria during cell death signaling. AB - Mitochondria play key roles in apoptosis, a central step being the release of cytochrome c (cyt c) across the outer mitochondrial membrane into the cytoplasm. We review this process in terms of the influences that induce mitochondria to release cyt c, the possible mechanisms of such release and the downstream consequences for caspase activation. The contributions of members of the Bcl-2 family in regulating mitochondrial activities relevant to apoptotic signaling are considered. Antiapoptotic members, such as Bcl-2 itself, are antagonistic to other family members, which prominently include Bax amongst a host of other proapoptotic proteins homologous to Bcl-2. Focus is placed on technical methods of determining cyt c release, which encompass cell fractionation, biochemistry, immunochemistry and confocal microscopy [including observations of release in real time using cyt c-green fluorescent protein (GFP) fusion proteins]. The advantages and potential pitfalls of the various approaches are discussed, with some emphasis on the use of cyt c-GFP fusions and the determination of the characteristics of the putative outer membrane pore through which cyt c and other mitochondrial proteins with proapoptotic functions may pass. The richness of this field relating to mitochondria and cell death is brought out by consideration of studies carried out in mammalian and yeast cells. PMID- 12372988 TI - Critical role of mitochondrial reactive oxygen species formation in visible laser irradiation-induced apoptosis in rat brain astrocytes (RBA-1). AB - Laser irradiation-induced phototoxicity has been intensively applied in clinical photodynamic therapy for the treatment of a variety of tumors. However, the precise laser damage sites as well as the underlying mechanisms at the subcellular level are unknown. Using a mitochondrial fluorescent marker, MitoTracker Green, severe mitochondrial swelling was noted in laser-irradiated rat brain astrocytes. Nucleus condensation and fragmentation revealed by propidium iodide nucleic acid staining indicated that laser-irradiated cells died from apoptosis. Using an intracellular reactive oxygen species (ROS) fluorescent dye, 2',7'-dichlorofluorescin diacetate, heterogeneous distribution of ROS inside astrocytes was observed after laser irradiation. The level of ROS in the mitochondrial compartment was found to be higher than in other parts of the cell. With another ROS fluorescent dye, dihydrorhodamine-123, and time-lapse laser scanning confocal microscopy, a substantial increase in mitochondrial ROS (mROS) was visualized in visible laser-irradiated astrocytes. The antioxidants melatonin and vitamin E largely attenuated laser irradiation-induced mROS formation and prevented apoptosis. Cyclosporin A (CsA), a mitochondrial permeability transition (MPT) blocker, did not prevent visible laser irradiation-induced mROS formation and apoptosis. In conclusion, mROS formation contributes significantly to visible laser irradiation-induced apoptosis via an MPT-independent pathway. PMID- 12372989 TI - Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence. AB - Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations (< or = 180 microM) of hydrogen peroxide (H(2)O(2)). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 microM H(2)O(2). Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide or m chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less rho(o) cells was also significantly increased by exposure to low concentrations (e.g. 180 microM) of H(2)O(2). These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria. PMID- 12372990 TI - Clinical phenotype, prognosis and mitochondrial DNA mutation load in mitochondrial encephalomyopathies. AB - We studied 42 individuals, including 8 patients with either complete or partial syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS), 8 patients with either complete or partial syndrome of myoclonic epilepsy with ragged-red fibers (MERRF) and 26 maternal family members who carried either the A3243G or A8344G mutation of mitochondrial DNA (mtDNA). Clinical manifestations and prognosis were followed up in the patients harboring the A3243G or A8344G mutation. The relationship between clinical features and proportions of mutant mtDNAs in muscle biopsies, blood cells and/or hair follicles was studied. In the 8 regularly followed patients with the A3243G mutation, 4 died within 1 month to 7 years due to status epilepticus and/or recurrent stroke-like episodes. Two patients developed marked mental deterioration and 2 remained stationary. All of the patients harboring the A8344G mutation were stable or deteriorated slightly, except for 1 patient who died due to brain herniation after putaminal hemorrhage. The A3243G and A8344G mtDNA mutations were heteroplasmic in the muscle biopsies, blood cells and hair follicles of both the probands and their maternal family members. The mean proportion of A3243G mutant mtDNA in the muscle biopsies of the patients with MELAS syndrome (68.5 +/- 21.3%, range 33-92%) was significantly higher than that of the asymptomatic family members (37.1 +/- 12.6%, range 0-51%). The average proportions of A8344G mutant mtDNA in the muscle biopsies (90.1 +/- 3.9%, range 89-95%) and hair follicles (93.9 +/- 6.4%, range 84-99%) of the patients with MERRF syndrome were also significantly higher than those of the asymptomatic family members (muscle: 40.3 +/- 39.5%, range 1-80%; hair follicles: 51.0 +/- 44.5%, range 0.1-82%). We concluded that measurement of the proportion of mutant mtDNA in muscle biopsies may provide useful information in the identification of symptomatic patients with mitochondrial encephalomyopathies. For patients with the A3243G mutation, the prognosis was related to status epilepticus and the number of recurrent stroke-like episodes and was much worse than for patients with the A8344G mutation of mtDNA, who had stable or slowly deteriorating clinical courses. PMID- 12372991 TI - Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. AB - The restriction endonuclease SmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F(0)F(1)-ATPase. Our ultimate goal is to apply SmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed the SmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the SmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targeted EcoRI induced a decrease in cytochrome c oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases. PMID- 12372992 TI - Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat. AB - We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0-0.8 Hz) in systemic arterial pressure signals. LPS also elicited progressive hypotension, and death ensued within 4 h. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (Complexes I + III) and cytochrome c oxidase (Complex IV) in the RVLM during all three phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complexes II + III) remained unaltered. We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat. PMID- 12372993 TI - Mitochondrial DNA mutations and oxidative damage in skeletal muscle of patients with chronic uremia. AB - Abundant evidence has been gathered to suggest that mitochondrial DNA (mtDNA) sustains many more mutations and greater oxidative damage than does nuclear DNA in human tissues. Uremic patients are subject to a state of enhanced oxidative stress due to excess production of oxidants and a defective antioxidant defense system. This study was conducted to investigate mtDNA mutations and oxidative damage in skeletal muscle of patients with chronic uremia. Results showed that large-scale deletions between nucleotide position (np) 7,900 and 16,300 of mtDNA occurred at a high frequency in muscle of uremic patients. Among them, the 4,977 bp deletion (mtDNA(4977)) was the most frequent and most abundant large-scale mtDNA deletion in uremic skeletal muscle. The proportion of mtDNA(4977) was found to correlate positively with the level of 8-hydroxy 2'-deoxyguanosine (8-OHdG) in the total DNA of skeletal muscle (r = 0.62, p < 0.05). Using long-range PCR and DNA sequencing, we identified and characterized multiple deletions of mtDNA in skeletal muscle of 16 of 19 uremic patients examined. The 8,041-bp deletion, which occurred between np 8035 and 16,075, was flanked by a 5-bp direct repeat of 5'-CCCAT-3'. Some of the deletions were found in more than 1 patient. On the other hand, we found that the mean 8-OHdG/10(5 )dG ratio in the total cellular DNA of muscle of uremic patients was significantly higher than that of the controls (182.7 +/- 63.6 vs. 50.9 +/- 21.5, p = 0.05). In addition, the mean 8 OHdG/10(5 )dG ratio in muscle mtDNA of uremic patients was significantly higher than that in nuclear DNA (344.0 +/- 56.9 vs. 146.3 +/- 95.8, p = 0.001). Moreover, we found that the average content of lipid peroxides in mitochondrial membranes of skeletal muscle of uremic patients was significantly higher than that of age-matched healthy subjects (23.76 +/- 6.06 vs. 7.67 +/- 0.95 nmol/mg protein; p < 0.05). The average content of protein carbonyls in the mitochondrial membranes prepared from uremic skeletal muscles was significantly higher than that in normal controls (24.90 +/- 4.00 vs. 14.48 +/- 1.13 nmol/mg protein; p < 0.05). Taken together, these findings suggest that chronic uremia leads to mtDNA mutations together with enhanced oxidative damage to DNA, lipids, and proteins of mitochondria in skeletal muscle, which may contribute to the impairment of mitochondrial bioenergetic function and to skeletal myopathy commonly seen in uremic patients. PMID- 12372994 TI - Characterization and immunobiology of house dust mite allergens. AB - The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization. PMID- 12372995 TI - Role of adverse reactions to food in urticaria and exercise-induced anaphylaxis. AB - In urticaria, adverse reactions to food are only a frequent finding in the subset of patients with chronic continuous urticaria. Mostly these reactions are of pseudoallergic nature, directed against artificial additives as well as naturally occurring aromatic components. IgE-mediated allergic reactions are a rare cause in acute urticaria as well as in recurrent chronic urticaria. In other types of urticaria, e.g. physical urticaria, food plays hardly any role as an eliciting agent with the exception of ice-cold drinks in cold urticaria. By contrast, exercise-induced anaphylaxis is frequently food-dependent. Two subtypes are distinguished: unspecific food-dependent exercise-induced anaphylaxis (FDEIA), where the filling of the stomach independently of the kind of food ingested prior to exercise is responsible for the symptoms. In specific FDEIA, an IgE-mediated food allergy causes symptoms only in combination with exercise. In the latter group, wheat is an important allergen. PMID- 12372996 TI - Significance of carbohydrate epitopes in a latex allergen with beta-1,3-glucanase activity. AB - BACKGROUND: One of the latex allergens, Hev b 2, has beta-1,3-glucanase activity. The entire sequence of this allergen is already known. There is one potential N glycosylation site in this molecule ((27)Asn). Heterogeneous glycosylation of this Asn residue could be a source of the multiplicity of natural Hev b 2. Possible participation of the carbohydrate epitopes of latex beta-1,3-glucanase isoenzymes in their IgE-binding capacity and cross-reactivity was investigated in this study. METHODS: beta-1,3-Glucanase isoenzymes were separated based on their affinities for concanavalin A. IgE-binding capacity and cross-reactivity were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Sequence heterogeneity among the isoenzymes was probed by peptide mass mapping after lysyl endopeptidase digestion. To clarify the relation to Hev b 2, N terminal sequencing was performed on a fragmented peptide common to the separated isoenzymes. RESULTS: Basic beta-1,3-glucanase was subdivided into two glycosylated isoenzymes (GI and GII) and one non-glycosylated isoenzyme (GIII). IgE antibodies in latex-positive sera chiefly recognized the glycosylated isoenzymes. Inhibition ELISA supported the significance of the carbohydrate epitopes for the IgE recognition and cross-reactivity. However, non-glycosylated GIII, as well as GI and GII, produced positive results in a skin prick test. The three beta-1,3-glucanase isoenzymes shared a partial sequence in common with Hev b 2. CONCLUSIONS: Our results suggest that the carbohydrate epitopes in Hev b 2 homologues are relevant to an in vitro diagnosis of latex allergy and the accompanying cross-reactivity. Carbohydrate epitopes do not necessarily provoke allergic symptoms. Therefore, the actual allergenicity of Hev b 2 and its homologues should be carefully evaluated not only by in vitro IgE tests but also by in vivo tests. PMID- 12372997 TI - Molecular basis of arthropod cross-reactivity: IgE-binding cross-reactive epitopes of shrimp, house dust mite and cockroach tropomyosins. AB - BACKGROUND: Shrimp may cross-react with other crustaceans and mollusks and nonedible arthropods such as insects (cockroach and chironomids), arachnids (house dust mites) and even nematodes. Since the muscle protein tropomyosin has been implicated as a possible cross-reacting allergen, this study characterized the IgE-binding epitopes in shrimp tropomyosin, Pen a 1, that cross-react with other allergenic invertebrate tropomyosins in house dust mites (Der p 10, Der f 10) and cockroaches (Per a 7). Pen a 1-reactive sera from shrimp-allergic subjects were used to evaluate the effect on IgE binding of different amino acid substitutions in Pen a 1 epitopes based on homologous sequences in Per a 7 and Der p 10/Der f 10. METHODS: Peptides were synthesized spanning the length of Pen a 1 IgE-binding epitopes and amino acid substitutions were performed based on homologous amino acid sequences from Per a 7 and Der p 10/Der f 10. RESULTS: 7/8 individually recognized Pen a 1 epitopes (2, 3a, 3b, 4, 5a, 5b and 5c) had an identical amino acid sequence with lobster allergen, Hom a 1, 4/8 (3a, 3b, 4 and 5a) with Der p 10 and Der f 10, and 5/8 (2, 3a, 3b, 4 and 5a) with Per a 7. In addition, even homologous regions of other arthropod tropomyosins that differ in one or more amino acids from the sequences of Pen a 1 epitopes are still recognized by shrimp-allergic IgE antibodies. In total, shrimp-allergic sera recognize 6/8 peptides homologous to Pen a 1 epitopes in Per a 7, 7/8 in Der p 10/Der f 10, and 7/8 epitopes in Hom a 1. CONCLUSIONS: The IgE recognition by shrimp-allergic individuals of identified and/or similar amino acid sequences homologous to Pen a 1 epitopes in mite, cockroach and lobster tropomyosins are the basis of the in vitro cross-reactivity among invertebrate species. Based on amino acid sequence similarity and epitope reactivity, lobster tropomyosin has the strongest and cockroach the least cross-reactivity with shrimp. The clinical relevance of these cross-reactivities in developing allergic reactions to different arthropods needs to be determined. PMID- 12372998 TI - Pepsin-resistant 16-kD buckwheat protein is associated with immediate hypersensitivity reaction in patients with buckwheat allergy. AB - BACKGROUND: Buckwheat is becoming popular in many countries as a health food and the incidence of buckwheat allergy is increasing in Asia. The ingestion of small amounts sometimes provokes an anaphylactic reaction. However, it remains controversial which is the major allergen responsible for such reactions. METHODS: The patients whose sera are positive for buckwheat-specific IgE antibody measured by the CAP system fluorescein-enzyme immunoassay (CAP-FEIA) were classified into two subgroups depending on the history of immediate hypersensitivity reactions (IHR). Major buckwheat allergens were identified with immunoblotting, ELISA and N-terminal amino acid sequencing. Various treatments such as pepsin digestion were added to characterize the proteins. RESULTS: We found that the 24-kD protein that had previously been reported to be a major allergen reacted to IgE antibodies present in sera from almost all subjects (19/20) regardless of symptoms. On the other hand, 16- and 19-kD proteins were bound with IgE antibodies present in sera from 9 of the 10 patients with IHR including 8 patients with anaphylaxis but not in sera from buckwheat-specific IgE positive subjects without IHR. After pepsin treatment, the 16-kD protein but not the 19- and 24-kD proteins remained undigested and preserved the capacity of IgE binding. This pepsin-resistant 16-kD protein had no homology with the 24-kD protein by the N-terminal amino acid sequencing. CONCLUSIONS: The 16-kD buckwheat protein was resistant to pepsin digestion and appeared to be responsible for IHR including anaphylaxis, while the pepsin-sensitive 24-kD protein was responsible for CAP-FEIA but not IHR. PMID- 12372999 TI - High-density oligonucleotide array analysis of mRNA transcripts in peripheral blood cells of severe atopic dermatitis patients. AB - BACKGROUND: There are few laboratory tests for evaluating atopic dermatitis (AD) with the exception of IgE levels or the eosinophil count. We attempted to identify new diagnostic markers by screening the genome-wide expression of transcripts in peripheral blood mononuclear cells (PBMC). METHODS: For this study, we enrolled 7 nonatopic healthy volunteers, 5 AD patients who responded well to treatment and 6 who responded poorly. We compared genome-wide transcript levels in PBMC derived from patients with severe AD and healthy volunteers using high-density oligonucleotide arrays (GeneChip, Affymetrix). After the first screening with GeneChip, we employed real-time quantitative PCR to confirm differential expression levels. RESULTS: Screening with GeneChip showed that the levels of a total of 92 transcripts increased at least 3-fold in one population compared to another. After further evaluation of these genes with real-time quantitative PCR, the levels of 4 transcripts were confirmed to be significantly different in PBMC from AD patients compared to controls, namely IFN-gamma, TRAIL (TNF-related apoptosis-inducing ligand), ISGF-3 (STAT1) and defensin-1. With the exception of IFN-gamma, none of these genes has previously been implicated in AD pathology. CONCLUSION: These 4 transcripts in PBMC are expected to be useful markers for evaluating AD. PMID- 12373000 TI - A functional study on CysLT(1) receptors in human eosinophils. AB - BACKGROUND: The cysteinyl leukotrienes (CysLTs) mediate their biological actions through two receptors: CysLT(1) receptor and CysLT(2) receptor. OBJECTIVE: This study was undertaken to examine the direct effects of CysLTs on eosinophils, such as chemotaxis and degranulation, focusing on CysLT(1). METHODS: Eosinophils were isolated from venous blood from normal volunteers who had no history of allergy (purity >99%). They were subjected to reverse transcription-PCR analysis and flow cytometric analysis for CysLT(1). Binding assays were performed with [(3)H]LTD(4). Purified eosinophils loaded with Fura-2 acetoxymethyl ester were stimulated with CysLTs, and Ca(2+) influx was measured. Eosinophil migration in response to CysLTs was measured using a 96-well multiwell Boyden chamber. Eosinophils were treated with LTD(4) at 10(-6) M for 60 min followed by incubation for 4 h at 37 degrees C in the presence or absence of IL-5 and eosinophil-derived neurotoxin (EDN) release was evaluated. RESULTS: The expression of the mRNA and protein of CysLT(1) on eosinophils and [(3)H]LTD(4) specific binding to eosinophils were observed. Neither Th1 cytokine (IFN-gamma) nor Th2 cytokines (IL-4 or IL-5) affected CysLT(1) expression in eosinophils. CysLTs induced an increase in intracellular free Ca(2+) in eosinophils via CysLT(1), as suggested by the efficient inhibition by a CysLT(1) antagonist, pranlukast, in addition to the rank order of potency being LTD(4), LTC(4) and LTE(4). LTD(4) stimulated eosinophils to migrate at 10(-6) M via CysLT(1). LTE(4) also induced significant eosinophil migration at 10(-6) M. LTD(4) enhanced EDN release induced by IL-5 via CysLT(1). CONCLUSION: CysLTs induce migration and enhance degranulation in eosinophils via CysLT(1). Accordingly, interaction of CysLTs and CysLT(1) on eosinophils has the potential to play a prominent role in the pathophysiology of asthma. PMID- 12373001 TI - Mast cell-derived VEGF enhances the passage of IgE FE-3 through the rat aortic endothelial cell monolayer. AB - BACKGROUND: It is well known that the IgE-mediated allergic reaction in various extravascular tissues is induced by the mutual interaction of IgE, target cells (mast cells, MCs) and allergens. However, so far little has been known about the detailed mechanism whereby IgE in the circulating blood is transferred to the extravascular tissue. To examine whether or not MCs are involved in the permeability of IgE across rat aortic endothelial cells (RAECs) in vitro, we determined the permeability constant (PC) of dinitrophenyl-specific rat IgE (IgE FE-3) using a dual chamber system. METHODS: Isolated RAECs obtained by a primary explant technique were seeded on a collagen-coated membrane in the upper chamber. MCs were collected from the peritoneal cavity of Wistar rats and suspended in the lower chamber. The time-dependent changes in concentration of IgE FE-3 (IgE) in the upper and lower chambers were measured by IgE capture ELISA after addition of IgE (10 microg/ml) to the upper chamber. RESULTS: The cultured medium of IgE stimulated MCs significantly increased the PC of IgE (9.86 +/- 0.46 x 10(-6) cm/s), as compared to the value to which calcium ionophore A 23187-stimulated MCs increased the PC of IgE (7.97 +/- 0.21 x 10(-6) cm/s). The increase of PC by IgE stimulated MCs was most strongly inhibited by suramin (92.3% +/- 1.89), and was weakly inhibited by tranexamic acid, cimetidine and diphenhydramine. In addition, the PC of IgE was increased with the increase in the MC-derived vascular endothelial growth factor/permeability factor (VEGF). CONCLUSIONS: After IgE is transferred from the circulating blood to the extravascular tissue, it may bind to Fc epsilon RI of the MC and the other Fc epsilon RI-bearing cells. The MC is then activated through the interaction of IgE and Fc epsilon RI. Release of VEGF from the activated MC increases, and the VEGF enhances the permeability of IgE. PMID- 12373002 TI - Effect of intranasal administration of CV-11974, a type 1 angiotensin II receptor antagonist, on airway hyperresponsiveness and airway inflammation induced by antigen inhalation in guinea pigs. AB - BACKGROUND: Angiotensin II is a putative mediator in asthma, but the effect of topical administration of type 1 angiotensin II (AT1) receptor antagonists on allergic airway reactions is not known. OBJECTIVE: To investigate the effect of intranasal administration of CV-11974, an AT1 receptor antagonist, and of PD123319, a type 2 angiotensin II (AT2) receptor antagonist, on antigen-induced airway reactions in guinea pigs. METHODS: Thirty minutes after intranasal topical administration of CV-11974 (0.1 or 1.0 mg/ml) or PD123319 (10 mg/ml) into the airways, the animals were given an antigen challenge. Airway hyperresponsiveness and bronchoalveolar lavage fluid were analyzed 24 h after the antigen challenge. RESULTS: Although these compounds did not inhibit antigen-induced early-phase bronchoconstriction or late-phase airway eosinophilia, intranasal administration of CV-11974 (but not PD123319) inhibited antigen-induced airway hyperresponsiveness in a dose-dependent manner 24 h after the antigen challenge. CONCLUSION: Intranasal administration of an AT1 receptor antagonist reduces antigen-induced airway hyperresponsiveness. PMID- 12373004 TI - Growth and metabolic disorders in chronic paediatric diseases. Proceedings of a workshop. Cambridge, United Kingdom. December 10-11, 2001. PMID- 12373003 TI - Identification of sequential IgE-binding epitopes on bovine alpha(s2)-casein in cow's milk allergic patients. AB - BACKGROUND: Caseins are the major allergens responsible for cow's milk allergy (CMA). We have previously identified the IgE-binding epitopes of the major cow's milk (CM) proteins except for alpha(s2)-casein. METHODS: Overlapping decapeptides representing the entire length of alpha(s2)-casein were synthesized on a cellulose-derivatized membrane. Sera from 13 CM-allergic children, 4-15 years of age, with a median level of CM-specific IgE >100 kU/l (range 33.7 to > 100 kU/l) were used to identify IgE-binding epitopes. RESULTS: Four major and six minor sequential IgE-binding regions were identified on alpha(s2)-casein. The first major region is located in the middle of the protein at amino acids (AA) 83-100, and the other three major regions are located in the carboxy terminal portion of the protein at AA 143-158, 157-172 and 165-188. The minor IgE-binding regions were identified at AA 31-44, 43-56, 93-106, 105-114, 117-128, and 191-200. CONCLUSION: We identified 10 sequential IgE-binding regions on alpha(s2)-casein and performed the first crucial step in the development of immunotherapeutic interventions for CMA. PMID- 12373005 TI - Prepubertal growth in children with long-term parenteral nutrition. AB - In children who depend on long-term parenteral nutrition (PN), a major goal is to obtain optimal growth. The aim of this retrospective study was to analyze growth in children on long-term cyclic nocturnal home PN, over at least 8 years before puberty. Nine boys and 7 girls were studied. Their mean age at the time of study was 11 years with a mean PN duration of 10.5 (8.6-16.4) years. Diseases were short bowel syndrome (5), intractable diarrhea (4), chronic intestinal pseudo obstruction (4) and long segment Hirschsprung's disease (3). In each child, periods of at least 2 years were analyzed: either periods of regular growth (R: height gain >50th percentile), or slow growth (S: height gain < or =25th percentile). Results were expressed as mean +/- SD. Comparisons were performed using either Student's test for unpaired data or Wilcoxon's test for paired data. PN provided a mean of 224 +/- 80 mg nitrogen/kg/day and 43 +/- 14 kcal/kg/day equivalent to 50% of recommended supplies. At the time of study, the population presented with weight (W) = -0.7 +/- 0.8 SD and height (H) = -1.5 +/- 1.3 SD. The difference between W and expected W for H (W/H) was significant (p < 0.002). W/H ratio was 105 +/- 11%. For the total PN duration, weight gain was +0.2 +/- 1.5 SD and height loss was -0.75 +/- 1.4 SD. An excess weight gain occurred in parallel with the deflection of height gain. Of the 16 children, regular prepubertal growth was achieved in 4 only. The other 12 showed alternate periods of R and S. In 8 of them, 26.5 years of R and 33.5 years of S were compared, each child being his own control. PN nitrogen and energy supplies were significantly higher during R periods than during S periods. In the absence of any disease or treatment explaining the failure to thrive, inadequate PN supplies, especially in terms of nitrogen supply, are thought to be responsible for a negative nitrogen balance and slowed growth. In case of any deflection away from the individual growth curve, it is recommended to adjust the PN supply early, especially nitrogen supply. PMID- 12373006 TI - Fundamental mechanisms of growth failure in inflammatory bowel disease. AB - Growth failure is common in children with inflammatory bowel disease (IBD) and has been attributed chiefly to undernutrition. Liquid enteral feeding can reverse the calorie deficit and increase growth velocity. The inflammatory process per se may also directly inhibit linear growth. After institution of enteral nutrition, significant changes in serum growth factors and inflammatory indices have been observed before any changes in nutritional parameters [Bannerjee et al., Gastroenterology 2000;118:A526]. In rats with trinitrobenzenesulphonic acid (TNBS)-induced colitis, about 60% of the final growth impairment can be attributed to undernutrition, inflammation accounting for the remaining growth deficit. Young patients with Crohn's disease and growth failure have normal stimulated and spontaneous growth hormone (GH) secretion and reduced plasma concentrations of insulin-like growth factor-1 (IGF-I), suggesting a degree of GH resistance. Rats with TNBS colitis also have normal plasma GH and reduced IGF-I concentrations, mediated by a combination of undernutrition and active inflammation. Immunoneutralization of interleukin-6 (IL-6) increases hepatic IGF I mRNA expression, plasma concentrations of IGF-I and linear growth. In contrast, administration of anti-tumour necrosis factor-alpha antibodies (TNF-ab) had no effect on IGF-I in this model. TNFab did, however, increase linear growth, suggesting inhibitory effects of TNF-alpha on the growth axis by mechanisms other than reduction in IGF-I. Preliminary data suggests that TNF-alpha inhibits maturation of growth plate chondrocytes. We have identified IL-6 receptors on growth plate chondrocytes but to date have not identified the effect, if any, of IL-6 directly at the growth plate. PMID- 12373007 TI - Growth in paediatric Crohn's disease. AB - Growth failure (GF) is one of the major complications affecting children with inflammatory bowel disease. The faltering is temporary in 40-50% of cases and prolonged in 10-20% in Crohn's disease (CD). Such failure is rare in children with ulcerative colitis (5%). This complication is often associated with retarded bone development and delayed onset of sexual maturation. The delayed linear growth has a variety of causes including insufficient intake due to anorexia and the inflammatory process with increased energy and protein expenditure. Other factors are increased intestinal loss, secondary hypopituitarism and treatment with steroids. Therapeutic strategies of CD in children have changed this last decade by introducing new therapeutic agents such as topic steroids, immunosuppressors, anti-TNF (antibody and notably in children enteral nutrition which has shown its efficacy in inducing remissions of active CD, restoring nutritional status and stimulation of linear growth. The results of a recent prospective multicentric study over 2 years in 82 CD show that severe GF (-2 SD) is initially present in 15% (n = 12), among them 11 remain < -2SD after 2 years of follow-up. Six patients who were on the normal range initially increased their GF during the follow-up (< -2SD) (total 21% < -2SD (n = 17) at 2 years). At inclusion in this group there was no difference in growth velocity, used of steroids, enteral nutrition or severity of CD as compared to the group with no GF. It suggests that new treatment strategy should be developed in the future for this specific complication of paediatric CD. PMID- 12373008 TI - Nutrition and growth in cystic fibrosis. AB - Malnutrition is a common complication of chronic diseases in children and may lead to growth impairment (stunting). Malnutrition in cystic fibrosis (CF) results from increased energy expenditure, decreased energy intakes, malabsorption of ingested nutrients because of pancreatic insufficiency and chronic inflammation. Malnutrition and high levels of inflammatory cytokines affect IGF-1 production through interrelated mechanisms. Nutritional support was shown to improve both nutritional status and outcome in CF. However, some nutrients have a direct effect on the disease. n-3 fatty acids supplementation is able to correct lipid abnormalities resulting from a primary mechanism. Moreover, n-3 fatty acids have a direct effect on the inflammatory response, decreasing eicosanoid synthesis and modulating nuclear transcriptional factors nuclear factor kappaB and peroxisome proliferator-activated receptors gamma. Nutritional support may be considered part of the care of the CF patient together with antibiotics, pancreatic enzymes and physiotherapy, influencing significantly the evolution of the disease. PMID- 12373009 TI - Can glutamine and growth hormone promote protein anabolism in children with cystic fibrosis? AB - To determine whether recombinant human growth hormone (rhGH), glutamine (GLN) or a combination of both agents can enhance protein synthesis in cystic fibrosis (CF) patients, six 9.6 +/- 0.5-year-old prepubertal children (4 M, 2 F) with CF and stable lung disease with undernutrition (weight/height <50th percentile) or delayed growth (height <5th percentile) received stable isotope infusions, in the postabsorptive state and on 4 separate study days: (a) at baseline, and after a 4 week treatment with either, (b) oral GLN (0.7 g/kg/day), (c) rhGH (0.3 mg/kg/week, SC), or (d) both GLN and rhGH. Four-hour infusions of (13)C-leucine were used to assess leucine appearance rate (Ra, an index of protein breakdown), oxidation (Ox), and non-oxidative leucine disposal (NOLD, an index of protein synthesis). Results are expressed as changes (%) from baseline:We conclude that in children with CF: (1) due to high inter-subject variability, oral glutamine does not significantly enhance protein gain; (2) rhGH has significant anabolic effects which are mediated by stimulation of protein synthesis, and (3) glutamine does not enhance the effect of rhGH. PMID- 12373010 TI - Role of interleukin-6 in growth failure: an animal model. AB - Indirect evidence suggests a link between factors produced during the inflammatory response and stunted growth. The demonstration of this link was provided by the observation that mice transgenic for the inflammatory cytokine interleukin-6 (IL-6), expressing high circulating levels of IL-6 since birth, show a marked decrease in growth rate leading to adult mice 50-70% the size of wild-type littermates. The growth defect is completely abolished by neutralization of IL-6. In these mice the production of GH is normal, while circulating levels of IGF-I are markedly decreased. Administration of IL-6 to wild-type mice results in a marked decrease in IGF-I levels. These observations show that in vivo high levels of IL-6 are associated with low levels of IGF-I. However, IL-6 does not directly affect IGF-I production both in vitro and in vivo. In contrast, markedly decreased levels of IGFBP-3 are present in the IL-6 transgenic mice and administration of IL-6 to wild-type mice results in a marked decrease in IGFBP-3 levels. In these mice the decrease in IGFBP-3 levels is associated with impaired formation of the 150 kD ternary complex, even in the presence of normally functional ALS. As a consequence, IL-6 transgenic mice show increased clearance of circulating IGF-I, suggesting that IL-6 decreases IGF-I levels by increased clearance. Proteolytic degradation of IGFBP-3 occurs in the IL-6 transgenic mice, suggesting that the decrease in IGFBP-3 could be at least in part due to proteolysis. The abnormalities of the IGF-I system observed in the IL-6 transgenic mice are similar to those found in patients with systemic juvenile idiopathic arthritis, one of the chronic inflammatory diseases characterized by stunted growth and prominent production of IL-6. The IL-6 transgenic mice represent a faithful animal model of the growth impairment associated with chronic inflammation and may therefore provide information relevant to the understanding and treatment of this complication of inflammatory diseases. PMID- 12373011 TI - Treatment of growth failure in juvenile chronic arthritis. AB - We retrospectively assessed linear growth and final height in a group of 24 patients suffering from juvenile idiopathic arthritis (JIA) during childhood, receiving steroid therapy. In these patients, a significant loss of height (-2.7 +/- 1.5 SDS) occurred in the first years of the disease which was positively correlated with prednisone therapy duration. After remission of the disease and prednisone discontinuation, most of the patients (70%) had catch-up growth but 30% had a persistent loss of height. Their mean final height was strongly correlated with their mean height at the end of steroid therapy and was significantly different between the group of patients with catch-up growth (-1.5 +/- 1.6 SDS) and the group without catch-up growth (-3.6 +/- 1.2 SDS). This pattern of growth observed in JIA patients should help us to define strategies of GH treatment in these patients in order to improve their final height. We have previously reported the beneficial effects on growth and body composition of a 1 year GH treatment in a group of 14 growth-retarded patients suffering from juvenile idiopathic arthritis, receiving glucocorticoid therapy. These patients (n = 13) were treated again with GH at the same dosage (0.46 mg/kg/week) for another 3-year period. GH treatment markedly increased growth velocity in these patients, but had a minor effect on SDS height suggesting that these children will remain short at adult age. Using GH earlier in these patients during the course of their disease may prevent growth deterioration and metabolic complications induced by chronic inflammation and long-term steroid therapy. PMID- 12373012 TI - Mechanisms of steroid impairment of growth. AB - With child growth being multifactorial and the glucocorticoids (GC) having many target physiological and biochemical mechanisms, growth and the GC collide in several meeting points. Indirectly, GC have a general anti-anabolic and catabolic influences that include bone, cartilage and muscle proteins. The GC interfere with the GH-IGF-1 axis at the hypothalamic, pituitary and target organ levels, affecting hormone release, receptor abundance, signal transduction, gene transcription, pre-mRNA splicing and mRNA translation. GC disturb normal calcium balance at the intestine and kidney. Direct effects at the growth plate include the suppression of multiple gene expression, chondrocyte proliferation and matrix proteoglycan synthesis, sulfation, release and mineralization as well as the augmentation of hypertrophic cell apoptosis. At the tissues adjacent to the growth plate, GC enhance osteoclast and suppress osteoblast recruitment and function, they reduce muscle strength and disrupt the normal control of vascular invasion at the cartilage-bone interface. Growth damage from GC is maximal during the initial months of treatment and prevention is more effective than post factual therapy. To reduce these growth-retarding effects, the following measures, which are partly experimental, may be effective in a decreasing order: minimize GC dose and use an alternate-day treatment; utilize the oxazoline analog of prednisolone deflazacort, normalize calcium balance; employ hGH or IGF-1. PMID- 12373013 TI - Growth in disorders of adrenal hyperfunction. AB - Growth is disturbed by adrenal hypersecretion of androgens or cortisol. Androgen excess in virilizing adrenal tumours causes advanced growth and bone age. In 9 girls with virilizing tumours, mean heights at diagnosis and final heights were 1.23 +/- 0.42 and 1.3 +/- 0.37 SDS respectively. In poorly controlled CAH, excess androgens cause early epiphyseal fusion and adult short stature. Increased growth occurs only after 18 months of age, even in untreated CAH, i.e. hydrocortisone >10 mg/m(2)/day is not generally required and may suppress infantile growth, affecting childhood and adult height. Growth was studied in 19 patients, aged 6.4 17.8 years, with Cushing's disease (CD). At diagnosis, mean height SDS was -1.81 (1.2 to -4.17), 53% < -1.8 SDS, height velocity in 6 was 0.9-3.8 cm/year and mean BMI SDS 2.29 (0.7-5.06). From 1983 to 2001, CD was cured in 18 patients (61%) by transsphenoidal surgery (TSS) alone and 39% by TSS plus pituitary irradiation (RT). In 13 patients, growth hormone (GH) was assessed by ITT/glucagons at 1-108 months after cure. Four had severe GH deficiency (<9 mU/l), 7 subnormal (10-29 mU/l) and 2 normal (>30 mU/l) GH status. Subnormal GH was present in 7 subjects >2 years after TSS or RT cure. In 10 subjects, aged 12.9 +/- 3.4 years, growth after cure was studied for 9.1 +/- 5.0 years. Nine had no catch-up growth in the interval of 0.3-1.1 years after cure (mean HV 5.3 +/- 2.4 cm/year). All these had GH deficiency peak GH 0.5-20.9 mU/l, and received hGH 2.7 mg/m(2)/week, 3 with GnRHa. All 10 showed long-term catch-up growth with mean delta SDS at diagnosis (Ht SDS-target Ht SDS) -1.72 +/- 1.26 improving to -0.83 +/- 1.08 (p = 0.0005) at latest of final Ht. At diagnosis, virilization was present in 82% of 17 patients with CD. Mean SDS values of serum androstenedione, DHEA-S and testosterone were normal, i.e. 0.72 (-2.9 to 3.0), -0.8 (6.0 to 2.2), 0.7 (-7.9 to 9.5) respectively, whereas SHBG was reduced at -2.1 (-5.3 to 1.2), increasing free androgen levels. Bone age (BA) was delayed (mean 1.46 years) in 14/16 patients, suggesting cortisol excess contributed more then androgen effect to skeletal maturation. In conclusion, most paediatric patients with CD had subnormal linear growth with delayed BA. After cure by TSS or pituitary irradiation, GH deficiency was frequent and persisted for many years. Treatment with hGH induced significant long-term catch-up growth leading to reasonable final height. PMID- 12373014 TI - Mechanisms influencing bone metabolism in chronic illness. AB - Bone is permanently renewed by the coordinated actions of bone-resorbing osteoclasts and bone-forming osteoblasts, which model and remodel bone structure during growth and adult life. The origin of osteoblastic cells (osteoblasts, osteocytes and bone-lining cells) differs from that of osteoclasts, but both cell groups communicate with each other using cytokines and cell-cell contact as to optimally maintain bone homeostasis. This communication in many ways uses the same players as the communication between cells in the immune system. During acute life-threatening illness massive bone resorption is the rule, while bone formation is suppressed. During chronic illness, the balance between bone formation and bone resorption also shifts, frequently resulting in decreased bone mass and density. Several factors may contribute to the osteopenia that accompanies chronic illness, the most important being undernutrition and low body weight, inflammatory cytokines, disorders of the neuroendocrine axis (growth hormone/IGF-1 disturbances, thyroid and gonadal deficiency), immobilization, and the long-term use of glucocorticoids. Their combined effects not only influence the generation and activity of all bone cells involved, but probably also regulate their life span by apoptotic mechanisms. Osteopenia or even osteoporosis and bone fragility, and before puberty also decreased linear growth and lower peak bone mass are therefore frequent consequences of chronic illnesses. PMID- 12373015 TI - Influence of growth hormone on accretion of bone mass. AB - Growth hormone (GH) exerts important influences on bone metabolism during lifespan. During childhood, GH is a major determinant of acquisition of bone mass and in adult life, GH partly determines the rate of bone remodelling and therefore influences maintenance of bone mineral density (BMD). Insights into the importance of GH in these respects may be obtained by studies of BMD and indices of bone remodelling in GH deficiency (GHD) of adult-onset and childhood-onset. Adult-onset GHD, usually accompanied by other features of hypopituitarism, may be associated with osteopenia and an increased fracture risk. Postulated mechanisms include GHD and gonadal steroid deficiency of unknown duration; glucocorticoid and thyroxine replacement do not appear to exert a major role. GH replacement in adult-onset GHD results in an early increment in indices of bone remodelling which persists for up to 5 years; BMD increases by 0.5-1.0 SD in males and stabilizes in females over this time period. In adolescents with GHD who traditionally discontinue GH at completion of linear growth, BMD is substantially lower than peak bone mass for a young adult population. Studies addressing the effects of continuation of GH after achievement of final height are currently underway and will provide insights into the possible need to continue GH into adult life. Such studies may confirm a role for GH in promoting continued accrual of bone mass and thereby demonstrate that cessation of GH at achievement of final height, by limiting peak bone mass, may predispose to clinically significant osteoporosis in later life. In addition to the potential importance of GH for achievement of peak bone mass, there may be a superimposed accelerated loss of BMD with advancing age similar to the situation observed in adult-onset GHD. To date, this has been difficult to assess in adult GHD of childhood-onset because the relative contributions of low peak bone mass and increased loss of bone in later life could not be distinguished. PMID- 12373016 TI - Catch-up growth and endocrine changes in childhood celiac disease. Endocrine changes during catch-up growth. AB - Childhood celiac disease may lead to a failure of statural growth. After institution of a gluten-free diet most patients exhibit catch-up growth. Catch-up growth is a remarkable phenomenon characterized by a supranormal height velocity. One of the hypothetical mechanisms of catch-up growth is that an increased activity of the somatotrophic axis is involved. In order to provide further insight in the physiology of catch-up growth, auxological and endocrine changes were prospectively studied in 28 children with newly diagnosed celiac disease. The results demonstrate a malnutrition-like state of the somatotrophic axis at the time of diagnosis and a rapid recovery of this axis towards normal functioning after institution of the gluten-free diet. Although several correlations between these endocrine alterations and auxological parameters were detected, it is questionable whether the endocrine changes are the driving force behind catch-up growth. PMID- 12373017 TI - Growth and body composition in type 1 diabetes mellitus. AB - Over the last 50 years the prognosis for growth and pubertal development in children with type 1 diabetes mellitus (T1DM) has improved considerably. The early reports of Mauriac's syndrome were related not only to relative deficiency of insulin but also reduced caloric intake. Improved insulin delivery and liberalisation of caloric intake has resulted in improved growth, but subtle abnormalities persist. The frequently reported increased height at diagnosis may relate to prior hyperinsulinaemia and genetic background with respect to lDDM2 the insulin gene VNTR. Subsequent growth faltering is thought to be related to impairment of the GH/IGF-1 axis but children with T1DM are also more at risk of hypothyroidism and coeliac disease. At puberty, persisting abnormalities of the GH/IGF-1 axis and our inability to reverse these totally, even with intensified insulin therapy, contribute to the blunted pubertal growth in the girls but abnormal sex steroid concentrations may also be important. Intensification of insulin therapy may result in leptin resistance and excessive gains in fat mass, particularly in girls. Although it is likely that most children with T1DM will have normal final heights, this excessive weight gain in girls may lead to problems with compliance. Furthermore, hyperinsulinaemia in these subjects may also lead to ovarian hyperandrogenism, increased early risk of microvascular complications and long-term risk of cardiovascular disease. PMID- 12373018 TI - Growth and puberty and its management in thalassaemia. AB - The purpose of this review is to report the personal experience on growth and pubertal development in a large number of thalassaemic and ex-thalassaemic patients followed at the Pediatric and Adolescent Unit of Ferrara. Secondary amenorrhoea (SA), hypogonadism and short stature are the commonest endocrine and auxological complications. The anterior pituitary gland is particularly sensitive to free radical oxidative stresses and exposure to this. Magnetic resonance imaging (MRI) shows that even a modest amount of iron deposition within the anterior pituitary can interfere with its function. Other possible cause of hypogonadism in beta-thalassaemia major include liver disorders, chronic hypoxia, diabetes mellitus and zinc deficiency. The treatment of pubertal disorders consists of hormone replacement therapy with sex steroids. Successful induction of spermatogenesis and ovulation has been reported after hormonal stimulation with gonadotrophins. Height above the 10th centile was achieved in 50% of males and 64% of females. Eight prepubertal thalassaemic patients, 6 males and 2 females, ranging in age from 8.6 to 11.7 years, were treated with GH. After the first 12 months of GH treatment a significant increase of growth velocity was observed in 6 patients who doubled growth velocity before basal value (4 cm or more above the basal value), 2 patients had a partial response (2-4 cm above the basal value). In the following 3 years all thalassaemic patients had a partial response to the treatment with GH. These data indicate that despite somewhat reduced sensitivity to GH, compared to GH deficiency children, there is evidence indicating that thalassaemic patients may benefit from GH treatment. Sixty-eight thalassaemic patients (30 males and 38 females) who had successfully undergone bone marrow transplantation (BMT) during childhood were studied. Following BMT growth rate decelerated when compared to Tanner and Whitehouse standards. Twenty nine ex-thalassaemics reached final height. The patterns of growth during puberty was variable in ex-thalassaemic males, while in all but 3 ex-thalassaemic females we observed an improvement in the percentile of standing height. A gonadal dysfunction was found in 68% of ex-thalassaemic patients. Since the quality of life of these patients is an important aim, it is vital to monitor carefully the growth and pubertal development in order to detect abnormalities and to initiate appropriate and early treatment. PMID- 12373019 TI - Effects of chemotherapy on bone metabolism and skeletal growth. AB - In recent years there has been a significant increase in both acute and chronic toxicity associated with the more successful but now highly intensive chemotherapy (CT) regimens used to treat childhood cancers. The incidence of childhood cancers coincides with periods of rapid skeletal development. Consequently, short stature and osteoporosis are important long-term effects in adult survivors. Clinical data indicate that the effects of CT, including glucocorticoids, on final height are due to direct effects of these drugs on the skeleton. The multiple modes of action of CT drugs suggest a complex and diverse influence on chondrocytes, extracellular matrix and bone cells. However, only limited data demonstrate these direct effects on the proliferative capacity of growth plate chondrocytes and on key steps of endochondral ossification, the multistep process that determines rate and extent of long bone growth. Endochondral ossification requires coordinated maturation, proliferation and differentiation of growth plate chondrocytes leading to hypertrophic cells which eventually undergo apoptosis to leave a cartilaginous scaffold that is mineralized prior to the laying down of new bone. Disruption of the physiological cellular activity of growth plate chondrocytes and/or bone cells result in skeletal growth disturbances. Thus, CT drugs which disrupt normal cell division may manifest their effects on the growth plate as either a reduction in cell number and/or the loss of functional integrity of extracellular matrix. Histological and cell kinetic studies, using in vivo and in vitro models of long bone growth, are essential to increase our understanding of the cellular mechanisms involved and to finally determine how the individual growth potential might be maintained during treatment for childhood cancers. PMID- 12373020 TI - Growth and growth hormone status after a bone marrow transplant. AB - The three most common clinical situations which have given rise to diagnostic and therapeutic issues involve the child treated for: (1) a brain tumour or extracranial tumour with radiotherapy (XRT) which includes an XRT dose of > or =30 Gy to the hypothalamic-pituitary axis; (2) acute lymphoblastic leukaemia with a cranial XRT dose of 18-24 Gy, and (3) haematological malignancy or solid tumour requiring total body irradiation (dose 10-14 Gy) and BMT. The decision about the intent to treat and the timing of GH replacement needs to be taken in collaboration with the paediatric oncologist who will provide guidance about overall prognosis and the risk of relapse. After a dose of > or =30 Gy to the hypothalamic pituitary axis the risk of GH deficiency (GHD) 2 years later is very high (>50%) and therefore there is 'solid' epidemiological evidence, which predicts outcome. Therapeutically the choice is whether or not to offer GH replacement at 2 years in the presence of biochemical evidence of GHD but independent of auxology, or wait until the growth rate declines. Diagnostically the IGF-1 SDS is more useful than previously thought, particularly if XRT-induced GHD is severe; there may, however, be systematic discordancy between the GH responses to different pharmacological stimuli (ITT vs. arginine). For irradiated children in categories 2 and 3, greater emphasis is placed on auxology in determining the need for assessment of GH status. Early rather than very precocious puberty is a real issue and needs to be actively treated with a GnRH analogue if final height appears to be significantly compromised. PMID- 12373021 TI - [Breast diagnosis: new acquisitions and developments]. AB - The development and refinement of breast diagnosis have increased the significance of breast ultrasound, have led to the establishment of mammographic classification criteria (BI-RADS) and to the standardisation of the terminology used for reporting the results and thus its codification. Targeted stereotactic biopsies (Mammotome and SiteSelect) have become established methods of pretreatment diagnosis. PMID- 12373022 TI - [Advantages and limitations of breast ultrasound]. AB - Technological improvements in image quality have allowed to expand the indications for the use of breast ultrasound. This includes tumor differentiation, peroperative staging, follow-up after cancer treatment and interventional diagnosis. Up to now, only mammography has been useful for population-based screening. However, high-resolution and quality-controlled ultrasound can further improve early cancer detection. This is useful in high risk patients and women with dense breasts who are mammographically problematic. The population-wide use of this advanced indication for breast ultrasound depends on equipment quality and investigator experience. Up to now, there has been a lack of guidelines and regulations. The implementation of quality control is essential before high-quality and effective breast ultrasound can be generally offered. PMID- 12373023 TI - [Differential diagnosis of benign and malignant mammary lesions with special regard to the BI-RADS((R)) classification system in mammography]. AB - Mammography is the imaging method of choice for the diagnostic workup of breast tumors in screening programs as well as in symptomatic patients. Quality assurance is one of the main prerequisites for a successful performance in both fields. Quality assurance on the technical and film processing level can be easily achieved compared to reporting and outcome monitoring on the level of the physician performing mammography. This is especially due to the lack of standards of the written reports. This article reviews the Breast Imaging Reporting and Data System (BI-RADS((R))) developed and commercialized by the American College of Radiology, contains several examples of lesion classification and discusses the significance of the BI-RADS((R)) approach. PMID- 12373024 TI - [Stereotaxic breast biopsy techniques have become the standard of care for mammographically suspicious lesions]. AB - An optimal technique for the evaluation of nonpalpable, suspicious mammographic lesions should have a low technical failure rate, no false-negative results and should remove the lesion completely. Since most of these lesions are benign, the procedure should be carried out in an outpatient setting without general anesthesia. Cancer is missed in 2.6% of cases with excisional biopsy following needle localization. Furthermore, 50-83% of these patients undergo a second surgical intervention for definitive surgical treatment. In contrast, the rate of missed cancers is less than 0.7% following stereotaxic core or large-core biopsies. However, using these techniques, discordant results and histologic high risk lesions need to be recognized and reexcized. The cost-effectiveness of stereotaxic vacuum-assisted core biopsy has been demonstrated. Stereotaxic breast biopsy techniques such as vacuum-assisted core biopsy and large-core biopsy for suspicious mammographic lesions have low false-negative rates and result in few histologic underestimations. PMID- 12373025 TI - [Early experience with the advanced breast biopsy instrumentation system in a multicentre study]. AB - Early Experience with the Advanced Breast Biopsy Instrumentation System in a Multicentre Study In an Austrian multicentre trial between September 1998 and December 2001, 474 procedures were performed with the Advanced Breast Biopsy Instrumentation (ABBI), and 389 were entered in the protocol. For reasons of patient comfort, radiological accuracy and low complication rate, the stereotactic excision biopsy with the ABBI system is a useful alternative to 'open' biopsy of non-palpable breast lesions, although there are technical limitations. The question of the therapeutic option in breast cancer cannot be answered yet. PMID- 12373026 TI - [The evaluation of the second-look operation of patients with ovarian carcinoma and tubal carcinoma by means of a retrospective comparison study]. AB - INTRODUCTION: This investigation is a retrospective analysis to evaluate the influence of second-look surgery on the relapse-free and overall survival of patients with ovarian and tubal carcinomas. METHOD: For 208 patients with and without second-look operation out of 469 of the total collective, a matched analysis and a Cox regression model were established in the framework of a multivariate analysis. RESULTS: Second-look surgery in patients with ovarian cancer had no significant influence on the relapse-free and overall survival. The 10-year survival was equal in both groups: CONCLUSION: Second-look surgery cannot be justified on the basis of clinically noninvasive methods such as radiological findings with additional use of tumor markers. It should only be done in control clinical trials to evaluate new means of treatment. PMID- 12373027 TI - Medicine and the pharmaceutical industry. PMID- 12373028 TI - Therapy of Sneddon syndrome. AB - We report the case of a young woman with progressive cognitive decline and epilepsy. She showed ischemic cerebrovascular disease and proximal livedo racemosa. Antiphospholipid antibody (aPL) could not be detected and there were no microemboli on continuous transcranial Doppler ultrasonography monitoring. Histology of cerebral vessels showed intimal hyperplasia in small leptomeningeal venous vessels and micronecrosis of grey and white matter. We subsequently made the diagnosis of aPL-negative Sneddon Syndrome (SNS). Anticoagulation with warfarin could not be initiated because of a drug-resistant epilepsy with the risk of falls and subsequent bleeding; immunosuppression with steroids and azathioprine was ineffective, as was initial antiplatelet therapy with clopidogrel alone. However, when we intensified antiplatelet therapy by combining clopidogrel and ASS, a slowing of disease progression, as assessed by neuropsychological testing and magnetic resonance imaging, was noted on a follow up after 6 months. Therapeutic options in SNS in both aPL-positive and aPL negative patients with SNS are discussed. PMID- 12373029 TI - Complications following acute ischemic stroke. AB - The objective of this study was to assess typical early-onset complications following ischemic stroke in a large, hospital-based cohort to provide clinical data for future randomized trials and quality standards in clinical routine. 3,866 patients with acute ischemic stroke were prospectively documented in 14 Neurology Departments with an acute stroke unit. Within the first week after admission, increased intracranial pressure (7.6%) and recurrent cerebral ischemia (5.1%) were the most frequent neurological complications. Fever >38 degrees C (13.2%), severe arterial hypertension (7.5%) and pneumonia (7.4%) were the most frequent medical complications. Multivariate regression analysis yielded brain stem infarction and large-artery atherosclerosis as independent predictors for early recurrent ischemic stroke. This study provides representative data on onset and severity of early neurological and medical complications as well as possible predictors for early recurrent cerebral ischemia following acute ischemic stroke. PMID- 12373030 TI - Lateralization as a factor in the prognosis of middle cerebral artery territorial infarct. AB - The consequences of cerebral infarcts involving the left hemisphere differ from those of the right homologue areas. Data of 337 consecutive, unselected patients with acute ischaemic stroke in the territory of the right or left middle cerebral artery were analysed. Furthermore, lesion locations of 77 stroke patients with early death were compared with those of 315 patients followed for more than 28 days. Without any differences in stroke severity and in the volume of the lesions, the outcome of strokes of the right hemisphere was less favourable, and the case fatality rate was higher than in controls during a 10-year follow-up period. The rate of early death due to cardiogenic cause was relatively higher in the right hemispherical group. The asymmetry of the sympathetic nervous system, and the distinct psychosyndromes following the injury of the two hemispheres may underlie this difference in prognosis. PMID- 12373031 TI - Pre-attentive cognitive processing in epilepsy. A pilot study on the impact of epilepsy type and anti-epileptic treatment. AB - Patients suffering from epilepsy often complain of deficits in attentive cognition affecting, for instance, concentration, reaction time, memory and psychomotor speed. We were interested in the impact of epilepsy and its treatment on pre-attentive cognitive functions and enrolled 107 subjects (50 male, 57 female; mean age 26 years): 50 patients with partial epilepsy, 15 with primary generalised epilepsy and 42 healthy controls. We used a new computer-adapted neuropsychological test (called textest), measuring the pre-attentive visual processing speed. The subjects had to discriminate 5 different stimuli presented on a computer screen. As a primary result, we calculated the time the subject needed to detect 50% of the demonstrated stimuli correctly (t-50 time). We also applied the d2 test. For the textest results, no significant group differences between the different patient and healthy groups could be revealed, except for a significantly increased t-50 time in patients with polytherapy compared with the control group. Compared with healthy controls, significantly worse d2 test results were found in patients with epilepsy in general, a duration of disease >1 year, anti-epileptic drug therapy in general and in patients receiving polytherapy in particular (all p < 0.001). Our data suggest that polytherapy, but not monotherapy or the subtype of epilepsy, might have a slowing impact on pre attentive cognitive processing. PMID- 12373032 TI - Evidence for human leukocyte antigen-related susceptibility in idiopathic childhood ischemic stroke. AB - Stroke in children is a relatively uncommon condition and frequently associated with other diseases like cardiopathies, sickle cell disease and chronic smoking. In contrast to stroke in adults, it is rarely caused by atherosclerosis, hypertension or diabetes mellitus. Childhood stroke of unknown causes is called idiopathic stroke. The etiology of idiopathic stroke is unknown. However, several so-called idiopathic diseases develop on the basis of a genetic predisposition. As an approach to investigate this possibility in idiopathic childhood ischemic stroke, we studied the relationship between clinical and immunogenetic features in this disease. We demonstrate that the gene frequencies and relative risk of HLA-B51 were markedly increased in our patients compared with controls (p < 0.001). Thirteen of seventeen HLA-B51-positive patients had had a preceding respiratory infection, which was a higher proportion than in the control group (p < 0.05). In the patient group, the alleles HLA-DRB1*0802, -DRAI*0401 and DQBI*0402 were also significantly increased, defining the haplotype DRB1*0802 DRA1*0401-DQB1*0402 as a high-risk haplotype for idiopathic childhood ischemic stroke. Transient viral or bacterial infections, which involve vasculitis and vascular occlusion in the brain, can trigger idiopathic childhood ischemic stroke on the basis of an genetic predisposition. PMID- 12373033 TI - Proton magnetic resonance spectroscopy in dementia with Lewy bodies. AB - Proton magnetic resonance spectroscopy ((1)H-MRS) provides a non-invasive, in vivo insight into the brain metabolism, and has been successfully used in several neurological conditions. Our objective was to characterise the cerebral metabolic changes in dementia with Lewy bodies (DLB) patients using (1)H-MRS. Single Voxel (1)H-MRS was performed in 12 DLB patients with mild to moderate symptoms and 11 age-matched healthy controls. Volumes of interest (VOI) were selected, including white matter (WM) in the centrum semiovale and grey matter (GM) in the parasagittal parietal cortex. Main metabolic peaks corresponding to N acetylaspartate (NAA), glutamate/glutamine (Glx), choline-containing compounds (Cho), myo-inositol (Ins), and creatine plus phosphocreatine (Cr) were identified. These areas were measured and referred to that of the water. Metabolic ratios among the different peak areas were also calculated. In comparison with the control group, DLB patients showed significantly lower mean NAA/Cr, Glx1/Cr and Cho/Cr ratios in the WM, while their Ins/Cr and Ins/NAA ratios did not differ from those of the control group. In the GM, no significant differences were found between both groups. Correlations between age at onset, disease duration, Mini-Mental State Examination, the motor section of the Unified Parkinson's Disease Rating Scale, Hoehn and Yahr staging and metabolic ratios, both for WM and GM, were not significant in DLB patients. Our spectroscopy data show WM involvement, along with GM preservation, in DLB patients with early or intermediate stages. Hence, (1)H-MRS may provide adjunctive information in the ante-mortem diagnosis of DLB. PMID- 12373034 TI - Visual and spectral analysis of sleep EEG in acute hemispheric stroke. AB - BACKGROUND: Reports on the effects of focal hemispheric damage on sleep EEG are rare and contradictory. PATIENTS AND METHODS: Twenty patients (mean age +/- SD 53 +/- 14 years) with a first acute hemispheric stroke and no sleep apnea were studied. Stroke severity [National Institute of Health Stroke Scale (NIHSS)], volume (diffusion-weighted brain MRI), and short-term outcome (Rankin score) were assessed. Within the first 8 days after stroke onset, 1-3 sleep EEG recordings per patient were performed. Sleep scoring and spectral analysis were based on the central derivation of the healthy hemisphere. Data were compared with those of 10 age-matched and gender-matched hospitalized controls with no brain damage and no sleep apnea. RESULTS: Stroke patients had higher amounts of wakefulness after sleep onset (112 +/- 53 min vs. 60 +/- 38 min, p < 0.05) and a lower sleep efficiency (76 +/- 10% vs. 86 +/- 8%, p < 0.05) than controls. Time spent in slow wave sleep (SWS) and rapid eye movement (REM) sleep and total sleep time were lower in stroke patients, but differences were not significant. A positive correlation was found between the amount of SWS and stroke volume (r = 0.79). The slow-wave activity (SWA) ratio NREM sleep/wakefulness was lower in patients than in controls (p < 0.05), and correlated with NIHSS (r = -0.47). CONCLUSION: Acute hemispheric stroke is accompanied by alterations of sleep EEG over the healthy hemisphere that correlate with stroke volume and outcome. The increased SWA during wakefulness and SWS over the healthy hemisphere contralaterally to large strokes may reflect neuronal hypometabolism induced transhemispherically (diaschisis). PMID- 12373035 TI - Restoring migraine sufferers' ability to function normally: a comparison of rizatriptan and other triptans in randomized trials. AB - BACKGROUND: Many migraine patients are unable to function normally during a migraine attack. Assessments of treatment efficacy have tended to focus on migraine symptoms, rather than looking at functional impact. This study compared the efficacy of different oral triptans for restoring normal function in migraine sufferers. METHODS: Retrospective subgroup analysis of data from five randomized, placebo-controlled, double-blind clinical trials in which oral rizatriptan was directly compared with oral sumatriptan 100 mg (772 attacks), 50 mg (2,227 attacks), and 25 mg (1,182 attacks), naratriptan 2.5 mg (413 attacks), and zolmitriptan 2.5 mg (578 attacks) for the acute treatment of a moderate or severe migraine attack. Functional disability was evaluated by patients on a 4-grade scale ('normal', 'mild impairment', 'severe impairment', 'requires bedrest') at baseline and at 0.5, 1, 1.5, 2, 3 and 4 h after dosing. This analysis looked at the percentage of patients who had normal functional ability at 2 h, the last time point before escape medications were allowed, in the subgroup of patients who had some level of disability at baseline. RESULTS: Most patients in each trial and treatment group had some level of disability at baseline (range = 94 100%). At 2 h, more patients on rizatriptan 10 mg were able to function normally compared with sumatriptan 100 mg (39 vs. 32%, odds ratio = 1.4, p = 0.021), sumatriptan 50 mg (47 vs. 42%, odds ratio = 1.2, p = 0.033), sumatriptan 25 mg (48 vs. 36%, odds ratio = 1.7, p < 0.001), naratriptan 2.5 mg (39 vs. 22%, odds ratio = 2.5, p < 0.001), and zolmitriptan 2.5 mg (45 vs. 36%, odds ratio = 1.6, p = 0.008). CONCLUSION: In direct head-to-head comparative clinical trials, oral rizatriptan 10 mg enabled more migraine sufferers to function normally at 2 h after dosing than oral sumatriptan, naratriptan, and zolmitriptan. PMID- 12373036 TI - Lethal ischemic stroke after cisplatin-based chemotherapy for testicular carcinoma and cannabis inhalation. PMID- 12373037 TI - Zolpidem in restless legs syndrome. PMID- 12373038 TI - Wernicke's encephalopathy accompanied by multiple symptomatic cerebral hemorrhages during the recovery phase. PMID- 12373039 TI - Endocarditis due to anaerobic bacteria. AB - This review describes the microbiology, diagnosis and management of endocarditis due to anaerobic bacteria. Anaerobic bacteria are an uncommon but important cause of endocarditis. Most cases of anaerobic endocarditis are caused by anaerobic cocci, Propionibacterium acnes and Bacteroides fragilis group. Predisposing factors and signs and symptoms of endocarditis caused by anaerobic bacteria are similar to those seen in endocarditis with facultative anaerobic bacteria with the following exceptions: the gastrointestinal tract was the most common source for B. fragilis group endocarditis, the head and neck were the most common origin for Fusobacterium and Bacteroides spp., and the head and neck and genitourinary tract were the most common source for peptostreptococci. Complications with anaerobic endocarditis include valvular destruction, multiple mycotic aneurysms, aortic-ring abscess, aortitis, cardiogenic shock, dysrhythmias and septic shock. The mortality rate for patients with anaerobes endocarditis is 21-43%. Treatment of endocarditis involving anaerobic bacteria includes the use of antibiotic therapy effective against these organisms. PMID- 12373040 TI - Hormone replacement therapy does not affect plasma homocysteine in postmenopausal women with coronary artery disease. Free tissue factor pathway inhibitor antigen, a circulating anticoagulant, is related to plasma homocysteine. AB - OBJECTIVE: The objective was to evaluate the effect of hormone replacement therapy (HRT) on plasma homocysteine levels in postmenopausal women with coronary artery disease (CAD) and to investigate associations of homocysteine to other cardiovascular risk factors. METHODS: The women in this single-center, controlled, and randomized study were examined at baseline, and after 3 and 12 months, after they had been recruited consecutively from patients referred for investigational coronary angiography. All analyses were performed examiner blind. They were randomized to HRT consisting of transdermal application of continuous 17beta-estradiol with cyclic medroxyprogesterone acetate (MPA) tablets for 14 days every 3rd month, or to a control group. RESULTS: After 3 months of unopposed 17beta-estradiol, no significant effect on homocysteine was observed compared to the control group. The absolute decrease of 5% in median plasma homocysteine levels after 12-month HRT did not reach statistical significance. Plasma homocysteine seemed slightly higher in women with three- or four-vessel disease, but the difference was not significant. With increasing homocysteine levels, free tissue factor pathway inhibitor (TFPI) antigen increased, whereas E-selectin decreased. In women with diabetes or elevated blood glucose >6.0 mmol/l, plasma homocysteine was correlated to body mass index, C-peptide and insulin as well as age. CONCLUSION: Transdermal application of 17beta-estradiol and sequential MPA do not affect plasma homocysteine in women with established CAD. Plasma homocysteine is stable in women with CAD over time, and unless special intervention is undertaken, repetitive measurements are not necessary in this particular group of high-risk individuals. The circulating anticoagulant TEPI is related to plasma homocysteine. PMID- 12373041 TI - Clinical significance of the antibody against oxidized low-density lipoprotein in acute myocardial infarction. AB - To establish the clinical significance of the antibody against oxidized low density lipoprotein (anti-Ox-LDL) titer in patients with acute myocardial infarction (AMI), we measured the anti-Ox-LDL titer in 39 patients with AMI and 25 controls. In all AMI patients, the anti-Ox-LDL titer on admission was higher (p < 0.05) than the value in the controls. One month after admission, the titer decreased significantly (p < 0.001) reaching control levels. In patients who underwent thrombolytic therapy, the anti-Ox-LDL titer on admission was identical in patients with occluded infarct-related arteries (IRA) and patients with patent IRA during emergency coronary angiography. In patients who did not undergo thrombolytic therapy, the anti-Ox-LDL titer on admission was higher in patients with occluded IRA than in patients with patent IRA. An increased anti-Ox-LDL titer may be a risk factor for the onset of AMI. Spontaneous recanalization of the IRA may be associated with increased anti-Ox-LDL titers, while thrombolysis induced recanalization may be independent of it. PMID- 12373042 TI - Cardiac structural involvement in mucopolysaccharidoses. AB - Mucopolysaccharidoses (MPS) are lysosomal storage disorders due to impaired glycosaminoglycan degradation. Cardiac involvement is present in most patients with MPS although its clinical impact is still undetermined. Cardiovascular abnormalities were evaluated in 39 patients with MPS aged 4-22 years. Valvular lesions and different forms of cardiac involvement were detected. The most common lesion was thickening of the mitral valve with regurgitation or stenosis, regardless of the MPS type. Mitral valve thickening was observed in 23 patients, aortic valve thickening in 11 patients and congestive heart failure in only 1 patient with MPS III. The most severe changes were registered for MPS types I and II. Complete cardiological investigation should be routinely warranted in every patient inflicted with MPS. PMID- 12373043 TI - Oscillations in peripheral arterial tone in congestive heart failure patients: a new marker for Cheyne-Stokes breathing. AB - Cheyne-Stokes breathing (CSB), which is a prevalent finding in congestive heart failure (CHF) patients, has been shown to be of prognostic value. The oscillations in respiration were shown to be associated with oscillations in sympathetic nerve activation. We tested the hypothesis that the peripheral arterial tone (PAT) as measured by a novel finger plethysmograph can be used to detect CSB. Using a novel technique to measure the PAT, we monitored 10 patients with advanced CHF simultaneously with conventional polysomnographic recordings for either 1 or 2 nights. Records were scored for CSB during 3-min periods based on either respiratory effort and nasal-buccal airflow or on the PAT signal alone. The PAT sensitivity and specificity for the detection of periods containing CSB were 91 and 91% for the entire recording, 90.7 and 92.9% for non-REM sleep, 90.7 and 70% for REM sleep, and 73 and 97.3% for awake periods, respectively. PAT is a reliable marker of CSB in CHF patients. The novel finger plethysmograph can be used for screening and monitoring CSB. PMID- 12373044 TI - Isolated left ventricular noncompaction: an unclassified cardiomyopathy with severe prognosis in adults. AB - Noncompaction of the ventricular myocardium is a rare congenital cardiomyopathy, which appears to represent an arrest in intrauterine endomyocardial morphogenesis. It is diagnosed both in children and adults. Its common presentation involves heart failure symptoms, ventricular tachyarrhythmias and thromboembolic events, but the age of onset varies widely. The diagnosis is made by the combined appearance of numerous, excessively prominent trabeculations and multiple deep intertrabecular recesses perfused from the ventricular cavity, commonly involving the apical and midventricular segments of the left ventricle. Although the peculiar echocardiographic picture may possibly lead to the correct diagnosis, this condition may be often misdiagnosed or unrecognized since it is not widely known. PMID- 12373045 TI - Left ventricular diastolic dysfunction as an early manifestation of diabetic cardiomyopathy. AB - AIMS/HYPOTHESIS: Early determination of myocardial manifestations of diabetes mellitus is of major importance, since myocardial involvement considerably influences the prognosis of diabetic patients. The aim of this study was to investigate whether young patients with insulin-dependent diabetes mellitus and normal systolic left ventricular (LV) function already show a diastolic LV dysfunction and an increased risk of arrhythmias. METHODS: Echocardiography was performed in 87 patients suffering from type I diabetes mellitus, without known cardiac disease and in 87 controls. Patients with a known manifest cardiac disease or a long-term diabetic syndrome were excluded. Morphological parameters were determined using M-mode echocardiography. Doppler echocardiography was used to evaluate parameters of LV diastolic function. The risk of arrhythmia was assessed by means of electrocardiography, heart rate variability, and late potential analysis. RESULTS: The left atrial and ventricular dimensions and systolic functional parameters of all patients were normal. A diastolic dysfunction with a reduction in early diastolic filling, an increase in atrial filling, an extension of isovolumetric relaxation and deceleration time was documented in diabetic patients, as well as an increased number of supraventricular and ventricular premature beats. CONCLUSION: Even young patients with diabetes mellitus suffer from a diastolic dysfunction while systolic ventricular function is normal. Therefore, echocardiography with measurements of diastolic functional parameters appears to be a sensitive method for evaluating the manifestation and course of early diabetic cardiomyopathy. PMID- 12373046 TI - Correlation between ST-T-segment changes with markers of hemostasis in patients with acute coronary syndromes. AB - BACKGROUND: Disturbance of the hemostatic and the inflammatory system plays an important role in the pathophysiology of acute coronary syndromes (ACS). Their markers have been shown to predict further coronary events in patients with ACS. The prognostic value of the admission electrocardiogram (ECG), which is commonly used to evaluate ischemia, was studied previously. We investigated the correlation between serum markers of the hemostatic/inflammatory system and ECG changes in ACS. METHODS: A standard 12-lead ECG was obtained from 85 patients with ACS on admission (0d). Markers of the hemostatic and inflammatory system were measured on admission and after 2 days (2d). RESULTS: Patients with ST-T changes had higher fibrinogen and thrombin-antithrombin III complex (TAT) levels than patients without ECG alterations at both times (fibrinogen: 0d: 492 +/- 38 vs. 357 +/- 36 mg/dl, p < 0.01; 2d: 633 +/- 55 vs. 440 +/- 50 mg/dl, p < 0.02; TAT: 0d: 7.2 +/- 1.3 vs. 3.6 +/- 0.7 microg/l, p < 0.05; 2d: 5.3 +/- 0.9 vs. 3.2 +/- 0.5 microg/l, p < 0.05). Tissue-type plasminogen activator (TPA) was elevated in patients with ECG changes initially (10.1 +/- 0.6 vs. 7.2 +/- 0.7 ng/ml, p < 0.02). D-dimers, the acute-phase proteins C-reactive protein, serum amyloid A and the soluble adhesion molecules showed no significance. CONCLUSIONS: The data reveal a correlation between electrocardiographic changes and hemostasis in patients with ACS. The association of myocardial damage and a disturbed hemostatic system might stratify patients who are at high risk of suffering further coronary events. PMID- 12373047 TI - A new approach for transseptal catheterization in patients undergoing percutaneous balloon mitral valvuloplasty. AB - AIMS: To evaluate the safety and efficacy of a new approach for transseptal catheterization in patients undergoing percutaneous balloon mitral valvuloplasty (PBMV). METHODS: One hundred and two patients with rheumatic mitral stenosis were randomized into two groups. In the study group (RA approach), an imaginary horizontal line was drawn from the top end of the tricuspid valve under anteroposterior fluoroscopic view. The intersection of the horizontal line and the right edge of the corresponding thoracic vertebra was defined as the upper border of the Fossa ovalis. The atrial septum was punctured from a point 0.5 cm below the upper border of the Fossa ovalis. In the control group (LA approach), an imaginary horizontal line was drawn between the upper and middle third of the left atrium, and the intersection of this horizontal line and the right edge of the corresponding thoracic vertebra was used as an atrial septum puncture point. RESULTS: Atrial septum puncture succeeded in all patients in the study group and in 72.6% of the patients in the control group (p < 0.01). The average fluoroscopy times for transseptal catheterization in the study and the control groups were 2.0 +/- 0.5 and 3.0 +/- 1.0 min, respectively (p < 0.01). Transseptal catheterization was subsequently achieved using the RA approach in the 14 patients from the control group in whom the LA approach failed. CONCLUSIONS: The RA approach is a safe and effective means for transseptal catheterization in patients undergoing PBMV. PMID- 12373048 TI - Application of the vena contracta method for the calculation of the mitral valve area in mitral stenosis. AB - OBJECTIVES: The vena contracta is the narrowest region of the regurgitant or stenotic jet just downstream the orifice and reflects the size of that orifice. This study was performed to assess the accuracy of the vena contracta width (VCW) in evaluating the severity of mitral stenosis (MS) and to compare the mitral valve area (MVA) determined by VCW with MVAs obtained by other more traditional echocardiographic methods. METHODS: We studied 59 patients (43 females, 42 +/- 14 years) with MS. VCW was measured in the apical four chamber view by Doppler color flow mapping. The largest diameter of the VCW during diastole was measured for at least three cardiac cycles and averaged. MVA was calculated from the following equation: pir(2), where r = VCW/2. MVA was also determined by planimetry, the pressure half-time method, and by the Gorlin formula. RESULTS: In this study, the width of the vena contracta ranged from 0.89 to 1.73 cm (mean 1.30 +/- 0.21). MVA, calculated based on the VCW, ranged from 0.63 to 2.35 cm(2) (mean 1.36 +/- 0.41). MVA by VCW (1.36 +/- 0.41 cm(2)) showed good correlations with three comparative techniques: (1) the cross-sectional area by planimetry (1.35 +/- 0.36 cm(2), mean difference = 0.21 +/- 0.16 cm(2), y = 0.91x + 0.14, r = 0.79, SEE = 0.26 cm(2), p < 0.001); (2) the area derived from the Doppler pressure half-time (1.27 +/- 0.32 cm(2), mean difference = 0.22 +/- 0.19 cm(2), y = 0.97x + 0.13, r = 0.76, SEE = 0.27 cm(2), p < 0.001), and (3) the area derived from the Gorlin equation in the 18 patients who underwent catheterization (1.27 +/- 0.35 cm(2), mean difference = 0.19 +/- 0.16, y = 0.98x + 0.05, r = 0.81, SEE = 0.26 cm(2), p < 0.001). CONCLUSIONS: These findings suggest that Doppler color flow imaging of the MS jet in the vena contracta can provide an accurate estimation of MVA and appears to be potentially applicable in the assessment of the severity of MS. PMID- 12373049 TI - Myocardial perfusion abnormalities early (12-24 h) after coronary stenting or balloon angioplasty: implications regarding pathophysiology and late clinical outcome. AB - OBJECTIVE: We prospectively examined the prevalence of reversible perfusion defects on very early (12-24 h) thallium-201 single photon emission computed tomography (SPECT) scintigraphy after angiographically successful percutaneous coronary intervention (PCI) by stenting and/or stand-alone balloon angioplasty and the predictive value of these defects for late target lesion revascularization (TLR). PATIENTS AND METHODS: 83 consecutive patients undergoing PCI for 88 lesions (38 balloon angioplasties, 50 stents) underwent very early (12 24 h) SPECT thallium-201 scintigraphy at rest and following administration of 0.7 mg/kg intravenous dipyridamole after PCI. Univariate and multivariate clinical, procedural and scintigraphic correlates of target lesion revascularization during long-term follow-up were examined. RESULTS: Coronary stenting achieved a larger immediate post-PCI minimal luminal dimension (2.7 +/- 0.4 vs. 2.1 +/- 0.4 mm, p < 0.001) and less residual stenosis (4 +/- 12 vs. 19 +/- 11%, p < 0.001) than stand alone balloon angioplasty. Nonetheless, early reversible perfusion defects were similarly present in the territory supplied by 36% of stented lesions and 32% of lesions treated by balloon angioplasty (NS). Of 81 lesions (76 patients) available for long-term clinical follow-up, TLR was performed in 11% of the stent group and 14% of the balloon angioplasty group (NS). By multivariate logistic regression analysis, diabetes mellitus was the only predictor of late TLR (p < 0.05). The type of intervention (balloon or stent) predicted neither early perfusion defects nor late TLR. CONCLUSIONS: Early 201-thallium SPECT scintigraphy was abnormal in a third of patients treated by stand-alone balloon angioplasty or by stent placement. The very early SPECT scintigraphic findings did not differentiate between balloon and stent and did not predict late TLR. PMID- 12373050 TI - Burden of late repeat hospitalization in patients undergoing angioplasty or bypass surgery. A long-term (13 years) report from the Lady Davis Carmel Medical Center registry. AB - We investigated the incidence and determinants of early and late repeat hospitalization for cardiac causes in 378 patients following myocardial revascularization [199 coronary balloon angioplasty (PTCA), 179 coronary bypass surgery (CABG)] in a single cardiovascular center and followed for a median period of 13 years. Data were available for repeat rehospitalization in 91% and for mortality in all. Patients in the upper quartile for repeat hospitalization (>or=4 rehospitalizations) were defined as having multiple repeat hospitalizations. In the PTCA cohort, the rehospitalization rate was high (48%) in the first year, partly due to restenosis and to a group of patients who underwent planned repeat angiography, and then 15-26% annually. In the surgical cohort, annual repeat hospitalization was 8-12% during the first 4 years, but increased to a level similar to that in PTCA patients (19-26%) in the second half of the follow-up period. Independent predictors of multiple (>or=4) repeat hospitalizations included systemic hypertension (odds ratio 2.4, 95% CI 1.4-4.0), incomplete revascularization (odds ratio 2.0, 95% CI 1.1-3.4) and less extensive (<3 vessels) disease at the time of the index procedure (odds ratio 2.0, 95% CI 1.1-3.4). Predictors of repeat hospitalization were different from those of mortality (diabetes mellitus, 3-vessel disease). Late repeat hospitalizations after myocardial revascularization impose a considerable burden on the patient and the health care system, and represent an issue which should be better addressed. PMID- 12373051 TI - Factors predisposing to a nonadmission of patients with acute myocardial infarction. AB - Among patients presenting at the hospital with an acute myocardial infarction (AMI), about 2-6% are mistakenly discharged by emergency physicians. The relevance of diagnostic problems in the prehospital period of an AMI is unknown. We prospectively studied 421 patients seen by a primary care physician in the prehospital period of an AMI. Using a standardized interview, data were obtained to identify factors determining nonadmission. Of 421 AMI patients, 327 (77.7%) were directly admitted to hospital after examination by the physician, whereas 94 (22.3%) were not admitted. The median prehospital delay was 240 min in admitted and 2,200 min in nonadmitted patients. Using a stepwise logistic regression model, the following factors were identified as independent contributors to nonadmission: the patient not being much affected by the symptoms (2.48; 1.40 4.39), improvement of symptoms (2.59; 1.46-4.59), the patient not thinking to suffer an AMI (2.33; 1.28-4.17) and the patient being unable to imagine having a heart disease (1.93; 1.07-3.46). CONCLUSION: Nonadmission of AMI patients by health care professionals is a common problem. Several aspects of AMI presentation including the often limited intensity of symptoms and the variability of the clinical course may have to be re-emphasized by cardiologists. Taking a very careful history and being circumspect about the patient's interpretation of symptoms still are the keys to a correct diagnosis of AMI. PMID- 12373052 TI - Correlation between the electrocardiogram and regional wall motion abnormalities as detected by echocardiography in first inferior acute myocardial infarction. AB - We assessed the correlation between ST deviation in each of the six precordial leads and the presence of regional wall motion abnormalities (RWMA) as assessed by transthoracic echocardiography in 109 patients with first inferior acute myocardial infarction. ST depression in lead V1 and V2 was associated with higher incidence of RWMA of the mid-posterior segment (p < 0.02 for both leads). The specificity of ST segment depression in leads V1 and V2 for RWMA in mid-posterior segment was 87 and 57%, and the sensitivity 36 and 70%, respectively. Patients with ST depression in leads V2 or V3 had worse global RWMA score than patients without ST depression in these leads (p = 0.009 and p = 0.025, respectively). Patients with an ST elevation in lead V1, but not in leads V2 or V3, had a higher prevalence of right ventricular involvement (p < 0.0001). ST elevation in lead V5 was associated with more frequent involvement of the apical portion of the inferior wall (p < 0.02), with specificity of 88% and sensitivity of 33%. Global RWMA score was significantly worse for patients with ST elevation than for patients with isoelectric ST in lead V5 (p = 0.024). ST elevation in lead V6 was associated with RWMA in the mid-posterior segment (p < 0.006), with specificity of 91% and sensitivity of 33%, and worse global RWMA score (p = 0.022). PMID- 12373053 TI - Exercise test in women and men aged 75-77 years. AB - The purpose of this study was to investigate the physiological response to an exercise test in 75- to 77-year-old women and men. Out of a systematically chosen and representative sample of 1,245 persons from a population at the age of 70, 649 individuals remained available at the age of 75-77 years. An exercise test was performed in 335 participants (52%), 174 women and 161 men. 180 (28%) were excluded because of morbidity. 131 (20%) refused to perform an exercise test. Three persons had to be excluded because of missing data. The maximal heart rate was about 140 beats/min. Heart rates at different submaximal workloads were higher in females than in males. In males there was a significant nonlinear increase in heart rate at increasing work loads while in females this was not significant. The systolic blood pressure increased more in females than in males while working on the loads 30-50 W and 50-75 W. There was a significant nonlinear increase in systolic blood pressure in men without cardiovascular drugs during exercise on 30-75 W, but the corresponding increase was not significant in women. The highest average work load for the whole group measured during at least 4 min of near-maximal exercise on a bicycle was in women 48 W and in men 66 W. Forty four percent of the women and 22% of the men had a physical working capacity presumably interfering with their ability to perform activities of daily living. PMID- 12373054 TI - Turbulencies during hypothyroidism therapy. PMID- 12373055 TI - Possible role of estrogen receptor and apolipoprotein B-100 polymorphisms in coronary heart disease. PMID- 12373057 TI - Prinzmetal's variant angina and alterations in coronary flow velocity reserve. PMID- 12373056 TI - Myocardial calcification following septic shock. PMID- 12373058 TI - Does intermittent accessory pathway block during slow sinus rhythm always imply a low risk for rapid AV conduction of preexcited atrial fibrillation? PMID- 12373059 TI - Comparative anatomy of the paratympanic organ (vitali organ) in the middle ear of birds and non-avian vertebrates: focus on alligators, parakeets and armadillos. AB - The paratympanic organ (PTO) in the middle ear has been described in numerous bird species, but little is known about the distribution of this presumed lateral line remnant in other vertebrate classes. Here we provide evidence for a PTO in juvenile alligators, and make the first detailed description of its location and relation to ligaments in the reptilian middle ear. The alligator PTO measures about 450 micro m in diameter. The alligator PTO contains hair cells whose cilia extend into a mucous substance within the lumen. The PTO connects though a ligament to the ear drum, suggesting that pressure onto the tympanic membrane might induce fluid movement in the PTO. Labeling of innervating nerve fibers with the fluorescent dye, DiI, indicates that the alligator PTO is connected with the vestibular brainstem. Because all bird species examined possess a PTO except for owls and possibly parakeets, we verified the absence of a PTO in parakeets by examination of serial sections combined with GABA immunolabeling for potential hair cells. Bird species with significant upper beak movement lack a PTO, suggesting that PTO function is incompatible with upper beak movement. We also examined the middle ear of an armadillo, a mammal that has a very basal position within the eutherian phylogenetic tree. A small vesicle with ciliated cells was found, but did not label with a hair-cell specific marker, antibodies to myosin VIIa, and thus is not likely to represent a true PTO. Our evidence for a PTO in a non-avian species, the alligator, together with previous reports suggesting the presence of a PTO in some mammals, indicates that ancestral stem amniotes possessed a PTO, and that the PTO was not a de novo invention of birds. PMID- 12373060 TI - Distribution of NADPH-diaphorase/nitric oxide synthase in the brain of the caecilian Dermophis mexicanus (amphibia: gymnophiona): comparative aspects in amphibians. AB - The organization of nitrergic systems in the brains of anuran and urodele amphibians was recently studied and significant differences were noted between both amphibian orders. However, comparable data are not available for the third order of amphibians, the gymnophionans (caecilians). In the present study we have investigated the distribution of neuronal elements that express nitric oxide synthase (NOS) in the brain of the gymnophionan amphibian Dermophis mexicanus by means of immunohistochemistry with specific antibodies against NOS and enzyme histochemistry for NADPH-diaphorase. Both techniques yielded identical results and were equally suitable to demonstrate the nitrergic system. In addition, they were useful tools in the identification of cell groups and brain structures, otherwise indistinct in the brains of caecilians. The distribution of nitrergic structures observed in Dermophis conforms to the overall amphibian pattern but numerous distinct peculiarities were also noted. These included a dense innervation of the olfactory bulbs but a lack of reactivity in olfactory and vomeronasal fibers and glomeruli. A large population of nitrergic cells in the striatum and the presence of thalamic neurons, as well as the specific distribution of nitrergic cells in the isthmic region, are some of the differential features in the gymnophionan brain. Given the variability among species in the same class of vertebrates any discussion including amphibians should also include evidence for gymnophionans. PMID- 12373061 TI - Anterograde projections of the motorcortical tongue area in the saddle-back tamarin (Saguinus fuscicollis). AB - In the New World monkey Saguinus fuscicollis, the anterograde projections of the motorcortical tongue area were studied. Three animals were analyzed. In two, biotin dextran amine was used as tracer; in the third, Phaseolus vulgaris leucoagglutinin was used. Identification of the tongue area was carried out by electrical brain stimulation. Intracortical projections were found into the neighboring primary motor cortex, ventral premotor cortex, frontoopercular cortex, and primary and secondary somatosensory cortex. Projections also ended in the ventrolateral prefrontal cortex, orbital cortex, supplementary motor area, anterior cingulate cortex and agranular as well as granular insula. In addition, weaker labeling was found in the inferior and dorsal parietal cortex, and perirhinal and inferotemporal cortex. Subcortically, there was a heavy projection into the ventral putamen, a moderate projection into the caudate nucleus and claustrum, and a weak projection into the anterior, central and lateral amygdala. In the thalamus, terminal labeling was found in the nuclei ventralis posterior medialis, ventralis lateralis, reticularis, centralis lateralis, medialis dorsalis, pulvinaris oralis, centrum medianum, reuniens and suprageniculatus in an order of intensity. Subthalamically, weak projections could be traced into the zona incerta and lateral hypothalamus. In the midbrain, labeling was found in the deep layers of the colliculus superior, area praetectalis, dorsal reticular formation and, very sparsely, in the periaqueductal gray. In the lower brainstem, fibers ended in the griseum pontis, medial and lateral parabrachial nuclei, lateral pontine and medullary reticular formation, paramedian and dorsal reticular nuclei, solitary tract nucleus and principal as well as spinal trigeminal nuclei. No terminals were found in the hypoglossal nucleus itself; there were, however, terminals in the immediately bordering reticular formation. PMID- 12373062 TI - Variation in reproductive outcomes for captive male rhesus macaques (macaca mulatta) differing in CSF 5-hydroxyindoleacetic acid concentrations. AB - In rhesus macaque males, lower than average cerebrospinal fluid (CSF) concentrations of the principle metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA), have been linked to impulsivity, involvement in escalated aggression, failure to elicit consort relationships, production of fewer sperm plugs, and a relatively early age of mortality. Given these potential fitness costs, we performed two studies aimed at elucidating the effects of CSF 5-HIAA on reproduction. Study 1 retrospectively evaluated over a four-year period, the relative reproductive outcome for pairs of adult male rhesus macaques (n = 15) who lived in social groups and who differed in concentrations of CSF 5-HIAA. Study 2 examined the relationship between CSF 5-HIAA and sperm motility and density (n = 12), as a potential mechanism for maintaining variability in CSF 5 HIAA. For Study 1, an average measure from two CSF 5-HIAA samples was calculated for the two males who were present during the time when conception most likely took place (offspring birth date -165 +/- 14 days). Within-pair comparisons of CSF 5-HIAA concentrations between the sire and the non-successful male were drawn for each of the 72 offspring in the study. We found that while sires were typically the male with relatively higher CSF 5-HIAA within the pair, there were no absolute differences in CSF 5-HIAA between males who sired at least one offspring (sires) and those who failed to reproduce (non-sires). Furthermore, while absolute age was not predictive of reproductive outcome, sires with relatively high CSF 5-HIAA also tended to be also relatively older than their competitors. By contrast, for the males with relatively low CSF 5-HIAA who reproduced, sires were relatively younger than the non-sires. These differences in reproductive outcome for males differing in CSF 5-HIAA could not be explained by variability in sperm quantity or quality as we did not find evidence of a relationship between CSF 5-HIAA and either sperm measure. The results of this study suggest that as serotonergic function affects many aspects of behavior and survivorship, it might also be associated with reproductive outcome and different life-history strategies for males differing in concentrations of CSF 5-HIAA. PMID- 12373063 TI - Comparison of tracheal aspirate and bronchoalveolar lavage specimens from premature infants. AB - Lung fluid obtained by tracheal aspiration (TA) or bronchoalveolar lavage (BAL) has been used to study bronchopulmonary dysplasia (BPD). These two sample collection methods have seldom been compared. Paired BAL and TA specimens were collected 1, 3, 7 and 28 days after birth in 40 infants <34 weeks' gestation during a randomized, controlled trial of dexamethasone for BPD prophylaxis. Interleukin 8 (IL-8) and cell counts were measured. Compared to subjects without BPD, those who developed BPD or died by 28 days had elevated IL-8 in epithelial lining fluid on day 1 in both BAL specimens (20 ng/ml vs. 2 ng/ml) and TA specimens (101 ng/ml vs. 18 ng/ml). IL-8 levels (r = 0.55) and neutrophil proportions (r = 0.51) were moderately correlated between BAL and TA samples. TA specimens may be suitable substitutes for BAL samples in some studies of newborn lung fluid. PMID- 12373064 TI - Nitric oxide production and plasma cyclic guanosine monophosphate in premature infants with respiratory distress syndrome. AB - A low blood pressure is common in preterm infants with respiratory distress syndrome (RDS). A diminished vascular resistance appears to be an important cause. The endogenous production of nitric oxide (NO), a mediator of vascular smooth muscle relaxation, has been shown to be higher in infants with RDS than in those without. Infants with persistent pulmonary hypertension showed decreased endogenous NO levels as compared with controls. Severe RDS in preterm infants may be accompanied by persistent pulmonary hypertension. To elucidate the role of NO in RDS and low blood pressure, we determined the endogenous NO production in infants with and without RDS by measuring urinary nitrite and nitrate excretions and plasma cGMP levels. In consecutively admitted preterm infants (gestational age <32 weeks), urine samples for measurement of NO(2) and NO(3) and plasma samples for the determination of the cGMP concentrations were serially collected during the 1st week of life. Arterial blood pressure, therapy to support blood pressure, and additional relevant clinical data were registered simultaneously. 27 infants with and 39 without RDS were included. The urinary NO(x) levels increased in all patients and were not different between both groups. The plasma cGMP concentrations were higher in the RDS group on days 2, 3, 4, and 7 (p < 0.05). The severity of RDS was positively correlated with plasma cGMP (r = 0.50, p = 0.0001). Although the arterial blood pressure did not differ between the groups, more blood pressure support was needed in the RDS infants during the first 4 days (p < 0.05). A positive correlation was found between blood pressure support and plasma cGMP (r = 0.34, p < 0.0001). The endogenous NO production was not different in infants with and without RDS. Increased plasma cGMP levels in the RDS infants were associated with the severity of RDS and the intensity of antihypotensive treatment. The origin of cGMP in infants with RDS requires further research. PMID- 12373065 TI - Treating preterm infants at risk for chronic lung disease with dexamethasone leads to an impaired quality of general movements. AB - Mortality rates do not decline markedly after postnatal corticosteroid therapy and concern has been raised about its neurological sequelae. We studied 37 preterm infants with Prechtl's method for the qualitative assessment of general movements before, during and after dexamethasone therapy and found that the quality of general movements was impaired in 9 of 13 initially normal infants (p = 0.004, McNemar test). The quality of fidgety movements at 3 months was abnormal in the majority of the infants and correlated strongly with neurological abnormalities at 2 years (Spearman r = 0.785, p < 0.001). Prechtl's method may prove useful for the early neurological evaluation of alternative corticosteroid treatment strategies for the treatment of chronic lung disease. PMID- 12373066 TI - Natural history of serum immunoglobulin concentrations in low birth weight infants and association with respiratory tract infections. AB - Infections are a significant cause of morbidity and mortality among low birth weight (LBW) infants. THE AIMS OF OUR STUDY WERE: (1) to investigate whether serum antibody concentrations in 62 LBW infants (1,500-2,500 g) were normalized by 1 year of life, and (2) to determine the clinical relevance of humoral immaturity in these children during the 1st year compared to 20 appropriate-for gestational-age (AGA) term infants. At 1 year of life, immunoglobulin serum concentrations in LBW infants were comparable to those of the control group. The incidence of respiratory tract infections during the 1st year of life was not significantly different between LBW and AGA term infants. Interestingly, we demonstrated that LBW infants with a higher frequency of reported febrile upper respiratory tract infections had more elevated serum total IgG, IgG(1), IgG(3), total IgA, and IgA(1) concentrations. Thus, infants with a birth weight of 1,500 2,500 g do not appear to have an increased risk of respiratory tract infections compared to AGA term children during the 1st year of life. Furthermore, our data suggest that especially febrile infections induce higher serum immunoglobulin concentrations in LBW infants. PMID- 12373067 TI - Ureaplasma urealyticum induces apoptosis in human lung epithelial cells and macrophages. AB - Chronic lung disease (CLD) of prematurity remains a significant cause of morbidity among premature infants. It is a multifactorial disorder and characterized by an early increased number of neutrophils and alveolar macrophages, with later architectural epithelial and endothelial cell damage. Recently, apoptosis of type 2 pneumocytes in the lung of preterm neonates with acute and chronic lung disease has been examined and apoptosis of mesenchymal cells was detected in the chronic stage of bronchopulmonary dysplasia. Infection and inflammatory responses in the lungs play important roles. However, the contribution of Ureaplasma urealyticum to the development of CLD is debated. We found that U. urealyticum induced apoptosis in human type II lung epithelial cells (A549 cell line) and macrophages (derived from human monocytic cell line THP-1) by measuring the outer leaflets translocation of phosphatidylserine (flow cytometry analysis and fluorescence microscopy assessment), DNA fragmentation analysis, cell morphology changes such as diminution in cell volume, increased cytoplasmic staining, and nuclear pyknosis (hematoxylin and eosin staining) and viable counting (trypan blue exclusion). Anti-TNF-alpha monoclonal antibody partially protected the macrophages from undergoing apoptosis after infection with U. urealyticum. Our findings imply that U. urealyticum might be involved in impairing lung structure and host immune response during the development of CLD. PMID- 12373068 TI - Infants of mothers with HELLP syndrome compensate intrauterine growth retardation faster than unaffected premature infants: does HELLP change fetal programming? AB - OBJECT: To investigate the influence of HELLP (hemolysis, elevated liver enzymes and low platelet count) pregnancies on the postpartal course and further development of the neonate. METHODS: The postnatal course and further development up to 4 years of age of 43 infants after pregnancies complicated by HELLP syndrome were evaluated. 43 unexposed infants matched for gestational age and gender served as controls. RESULTS: Small-for-gestational age (SGA) neonates exhibiting hypoglycemia and hypoproteinemia during the first 4 weeks after birth were significantly more commonly observed in the HELLP group (p < 0.5). No other differences in the postpartal course or clinical outcome were detected. At the age of 4 years the gains in weight and length were significantly increased in the HELLP group (p < 0.01). CONCLUSION: The postnatal course of newborns after HELLP pregnancies is influenced by low energy stores. Fetal programming toward a more efficacious GH-IGF-1 pathway may explain the faster postnatal catch-up growth of premature SGA infants born to mothers with HELLP syndrome. PMID- 12373069 TI - Surfactant with SP-B and SP-C analogues improves lung function in surfactant deficient rats. AB - The use of mammalian lung surfactant extracts has sharply reduced mortality and morbidity from respiratory distress syndrome in premature infants. Synthesis of surfactant protein B and C (SP-B and SP-C) analogues may lead the way to a synthetic surfactant preparation. Dimeric SP-B(1-25) (dSP-B(1-25)) is based on the N-terminal domain of human SP-B and SP-Cfc is a modified human SP-C in which a single phenylalanine is substituted for a palmitoylated cysteine residue in the N-terminal segment (Phe-4 > Cys-4 variant). We tested the effects of synthetic surfactants with 1 or 2% dSP-B(1-25) and 1% SP-Cfc on lung function in surfactant deficient rats. Four experimental surfactant preparations were prepared by mixing 1% dSP-B(1-25), 2% dSP-B(1-25), 1% dSP-B(1-25) +1% SP-Cfc, and 2% dSP-B(1-25) +1% SP-Cfc with phospholipids (PL). PL and Survanta, a bovine lung extract, were controls. Groups of 8 rats were ventilated, lavaged until surfactant deficiency, and treated with 100 mg/kg surfactant. Arterial blood gas values and dynamic compliance were measured every 15 min and after 2 h of ventilation, the rats were killed and pressure-volume curves performed. Oxygenation improved quickly after instillation of surfactant with synthetic peptides and Survanta. Oxygenation and lung volumes were consistently higher in the 2% than in the 1% dSP-B(1-25) groups. Addition of 1% SP-Cfc to the synthetic surfactants further improved oxygenation and lung volume, but to a lesser extent than increasing the dSP-B(1 25) content from 1 to 2%. These data indicate that improvements in oxygenation and lung volume in lavaged rats are dependent on the concentration of dSP-B(1-25) in the surfactant preparation and that the presence of SP-Cfc has a relative minor effect on these parameters. PMID- 12373070 TI - Detrimental effects of nicotine and endotoxin in the newborn piglet brain during severe hypoxemia. AB - Hypoxia-ischemia is a major cause of perinatal brain damage, but evidence shows that brain injury also is associated with intrauterine infections and maternal smoking. The mechanisms are not known, and we therefore explored the effects of experimental inflammation or nicotine on perinatal brain metabolism and injury during severe hypoxemia. Twenty-eight 1-week-old piglets were anesthetized and instrumented with microdialysis probes in the striatum and brainstem. We studied three pretreatment groups: (1) 20 microg/kg i.v. nicotine (n = 9); (2) 1 microg/kg i.v. endotoxin from Escherichia coli (n = 11), or (3) control (n = 8). The piglets were subsequently exposed to 30 min of hypoxemia (6% O(2)). In order to minimize any ischemic component and increase survival, this was abrupted for 1 min if blood pressure fell to 30 mm Hg. During hypoxemia, both the pretreatment with endotoxin and nicotine induced higher levels of extracellular lactate and peak lactate/pyruvate ratio compared with controls (54.7 +/- 9.6 (p < 0.01) and 65.2 +/- 13.1 (p < 0.02) vs. 15.9 +/- 7.4, respectively), reflecting a deterioration of the metabolic status in these groups. The two pretreated groups reached significantly higher peak levels of extracellular glycerol (30.9 +/- 4.1 vs. 77.9 +/- 12.7 and 89.4 +/- 14.2 micromol/l, respectively, p = 0.01), indicating a higher level of cellular membrane disintegration or leakage. In addition, 3 endotoxin piglets and 4 nicotine piglets died during reoxygenation, while all controls survived (p = 0.13 and p < 0.04, respectively). Mortality was associated with a rise in extracellular glutamate at the end of hypoxemia/start reoxygenation (p = 0.02). These findings contribute in explaining how nicotine and inflammatory response to bacterial toxins could act as cofactors for hypoxic ischemic neurologic injury in the immature brain. PMID- 12373071 TI - Nitric oxide synthesis inhibition during cerebral hypoxemia and reoxygenation with 100% oxygen in newborn pigs. AB - The objective of our study was to evaluate the effects of N(sigma)-nitro-L arginine methyl ester (L-NAME), an inhibitor of the nitric oxide (NO) pathway, on cerebral microcirculation during hypoxemia and reoxygenation with 100% oxygen in newborn pigs. Twenty-two pigs were randomized to hypoxemia [inspired fraction of oxygen (FIO(2)) 0.08; 20 min] and reoxygenation (FIO(2) 1.0; 60 min) or normoxia. The hypoxemic animals were further randomized to receive either an intravenous bolus injection of 5 mg/kg L-NAME (n = 8) or a corresponding volume of isotonic saline (n = 8) 30 min before the onset of hypoxemia. The normoxemic group (n = 6) received the same pretreatment with L-NAME. Cerebral hemodynamics were assessed by laser Doppler flowmetry and intracranial pressure monitoring. The cerebral NO concentration was continuously measured using an electrochemical sensor. Pretreatment with L-NAME resulted in a more severe systemic hypotension and reduced cerebral microcirculation during the period of hypoxemia compared with the saline/hypoxemia group. NO synthesis inhibition during reoxygenation with 100% oxygen, however, blunted the increase in NO concentration (p < 0.05) without reduction of cerebral blood flow and cerebral perfusion pressure. In conclusion, in this newborn pig model, pretreatment with a bolus infusion of L-NAME induced severe hypotension and reduced cerebral microcirculation during hypoxemia. However, it appears to have no significant adverse effect on cerebral hemodynamics during the period of reoxygenation with 100% oxygen. This deleterious effect during hypoxemia limits the use of L-NAME as a preventive drug but suggests beneficial effects during reoxygenation with 100% oxygen. PMID- 12373072 TI - Exposure to the opioid antagonist naltrexone throughout gestation alters postnatal heart development. AB - The influence of endogenous opioid blockade by naltrexone during prenatal life on postnatal heart development was studied in rats. Pregnant Sprague-Dawley rats received daily injections of 50 mg/kg naltrexone (NTX) or saline throughout gestation; offspring were cross-fostered at birth to mothers not receiving NTX. In general, NTX-treated offspring weighed more than controls throughout preweaning life, whereas heart weights were often increased from age-matched controls up to 35 days. Biochemical analyses of nucleic acids and protein demonstrated that DNA and protein content were increased throughout development in NTX-treated animals relative to controls. Morphometric analyses revealed increases in total area of the heart and myocardial area in NTX-exposed rats relative to control levels. These data suggest that endogenous opioids function to regulate cardiac growth during the prenatal period, and that disruption of this process has long-term implication for cardiac biology. PMID- 12373074 TI - Hyperuricemia causes glomerular hypertrophy in the rat. AB - BACKGROUND/AIMS: Rats with mild hyperuricemia develop systemic hypertension, interstitial renal disease, afferent arteriolopathy, and increased renin expression [Mazzali et al.: Am J Physiol 2002;6:F991-F997]. We hypothesized that hyperuricemia might also induce glomerular changes. METHODS: We reviewed renal biopsies of rats previously made hyperuricemic for 7 weeks with the uricase inhibitor, oxonic acid. Controls included normal rats and oxonic acid-treated rats administered allopurinol, benziodarone, hydrochlorothiazide, or enalapril. Glomeruli were examined for size (computer image analysis) and structure (histology). An additional group of rats were administered oxonic acid or control diet for 6 months. RESULTS: Renal biopsies showed that hyperuricemic rats had a 30% increase in glomerular tuft area (p < 0.01); these changes were prevented by allopurinol and benziodarone. Control of blood pressure with hydrochlorothiazide did not prevent the development of glomerular hypertrophy, whereas enalapril partially reduced the glomerular hypertrophy. Prolonged hyperuricemia was associated with the development of microalbuminuria (p < 0.05) and glomerulosclerosis (22 vs. 10%, p < 0.05) compared to control rats. CONCLUSIONS: Hyperuricemic rats develop glomerular hypertrophy that can be prevented in part by ACE inhibitor therapy. Prolonged hyperuricemia is associated with the development of glomerulosclerosis in the rat. PMID- 12373075 TI - Effect of relaxin in two models of renal mass reduction. AB - BACKGROUND: Relaxin (Rlx), a 6-kD protein hormone, belongs to the insulin growth factor family. We have previously shown that Rlx reduces interstitial fibrosis in a model of chronic papillary necrosis. HYPOTHESIS: The purpose of this study was to extend these observations to a model of renal injury induced by mass reduction. MATERIAL AND METHODS: Renal mass was reduced by either infarction or surgical excision of both poles, with removal of the contralateral kidney. Two weeks later, creatinine clearance was done and animals from both groups implanted with osmotic pumps delivering either Rlx (2 microg/h) or vehicle (Veh). Treatment was continued for 4 weeks. The severity of the glomerular injury was quantified by planimetric measurements. Renal function was assessed by creatinine clearance and plasma creatinine. RESULTS: Rlx significantly decreased systolic blood pressure in animals with infarction. This was accompanied by a decrease in serum creatinine and a slight improvement in creatinine clearance. The severity of the glomerular lesion was reduced by Rlx (sclerosis index, Veh 1.16 +/- 0.13 vs. Rlx 0.74 +/- 0.16, p = 0.037). In the excision group the animals were normotensive. In this group, Rlx treatment was accompanied by a decrease in serum creatinine (Veh 1.01 +/- 0.03 vs. Rlx 0.81 +/- 0.05 mg/dl, p = 0.02) and an increase in GFR (Veh 0.90 +/- 0.14 vs. Rlx 1.33 +/- 0.11 ml/min, p = 0.03). The sclerosis index was also reduced. CONCLUSION: Rlx decreases renal injury by at least two mechanisms, one by lowering blood pressure as seen in the infarction model, the other independent of blood pressure as seen in the normotensive excision model where there was also a significant functional improvement. PMID- 12373076 TI - Protective effect of HMG-CoA reductase inhibitor on experimental renal ischemia reperfusion injury. AB - BACKGROUND: Increasing evidence supports an important role for inflammation in the pathogenesis of renal ischemia-reperfusion injury (IRI). Recently, HMG-CoA reductase inhibitors, 'statins', have demonstrated anti-inflammatory effects independent of cholesterol-lowering. HYPOTHESIS: We tested the hypothesis that a statin would improve outcome in a murine model of renal IRI. Upon finding a protective effect, we tested the hypothesis that the mechanisms by which statins protected in renal IRI was by reducing neutrophil and macrophage infiltration and upregulating the anti-inflammatory cytokine IL-6. METHODS: Cerivastatin at various dosing regimens was administered to NIH Swiss mice to evaluate the effects on renal IRI. Analysis of renal structure, function, neutrophil and macrophage infiltration, cytokine production, as well as mortality was performed in cerivastatin- and saline-treated groups. RESULTS: PRIMARY: Cerivastatin pretreatment for 3 days led to a significant improvement in renal function, tubular injury as well as survival after IRI compared to saline-treated mice. SECONDARY: Neutrophil and macrophage infiltration into kidney tissue was similar in both groups. IL-6 was markedly upregulated early in the kidneys of statin treated compared to saline-treated mice. CONCLUSION: These data demonstrate that a statin compound can improve the course of ischemic acute renal failure. Induction of protective molecules such as IL-6 may underlie this effect. PMID- 12373077 TI - Renal and intestinal handling of oxalate following oxalate loading in rats. AB - BACKGROUND: The enteric excretion of oxalate has been established in rats with chronic renal failure induced by 5/6 nephrectomy [Hatch et al.: Regulatory aspects of oxalate secretion in enteric oxalate elimination. JASN 1999;10:S324] and this response is mediated by angiotensin II receptor activation. However, the renal and intestinal handling of oxalate has not been evaluated for other common models of hyperoxaluria that simulate primary hyperoxaluria or oxalate stone disease. METHODS: We assessed the renal clearances of creatinine, oxalate and calcium in three rat models: chronic hyperoxaluria (CH), chronic hyperoxaluria with hyperoxalemia (CHH) and acute hyperoxaluria (AH), and evaluated the transepithelial transport of oxalate and chloride in large intestinal segments of these models and their sensitivity to angiotensin II antagonism. RESULTS: Hyperoxaluria alone (CH) was not associated with changes in colonic oxalate transport, whereas changes in net oxalate transport in distal colon from absorption to net secretion was observed in models with hyperoxalemia (CHH and AH). Angiotensin II receptor antagonism with losartan reduced net colonic oxalate secretion in AH but not CHH. CONCLUSIONS: Colonic secretion of oxalate is stimulated in rat models exhibiting hyperoxalemia suggesting a contribution of this extrarenal pathway to regulation of oxalate mass balance in clinical conditions manifesting hyperoxalemia. The transport avenues and regulatory mechanisms may not be identical to those observed during adaptive enteric oxalate secretion in chronic renal failure models. PMID- 12373078 TI - Delayed apoptosis post-cadmium injury in renal proximal tubule epithelial cells. AB - BACKGROUND: Accumulation of the widespread environmental toxin cadmium (Cd) in the kidney results initially in proximal tubule dysfunction. Exposure to Cd has been previously shown to induce apoptosis in LLC-PK (Lily Laboratory Culture, Porcine Kidney) cells, which are a model of proximal tubule epithelium. HYPOTHESIS: We postulated that modulation of the components of the apoptotic pathway triggered by Cd is amenable to therapeutic intervention. METHODS: We subjected confluent LLC-PK cells grown on two-compartment filters and on plastic to Cd (1-50 microM). Apoptosis and changes in components of the apoptotic pathway were measured by immunocytochemical and immunoblot analysis during the period of exposure and following Cd withdrawal. RESULTS: Insignificant apoptosis was seen during exposure to Cd and immediately after removal of this metal. Two waves of apoptosis were noted 6 and 48 h after the Cd was removed from the apical compartment. The apoptosis 48 h post-Cd exposure was accompanied by a decrease in cellular ATP levels and transepithelial resistance and preceded by an increase in p38 phosphorylation. Inhibition of p38 mitogen-activated protein kinase activity decreased the delayed apoptotic peak, without affecting the rate of recovery of the integrity of the renal epithelium. IGF-1 neither altered the delayed apoptosis nor facilitated the rate of recovery of the integrity of the renal epithelium. CONCLUSION: We demonstrate that following exposure to Cd, renal epithelial cells undergo significant apoptosis, which appears to involve p38 and is not amenable to IGF therapy. PMID- 12373079 TI - Nephrotic-like proteinuria in experimental diabetes. AB - AIMS/HYPOTHESIS: Streptozotocin (STZ) diabetic rats are characterized by the development of albuminuria. It is not known, however, whether the excess excretion of protein is primarily due to intact protein or protein fragments or whether it is specific for albumin or occurs for all high-molecular-weight plasma proteins. To test this we have measured the excretion rates and fractional clearances of [(14)C]albumin, [(3)H]immunoglobulin G and [(3)H]transferrin in diabetic rats. METHODS: The radiolabeled proteins were delivered to the circulation of conscious diabetic (STZ induced for 6 weeks) and control rats by ALZET osmotic pumps. The plasma level of the radiolabeled proteins reached steady state levels by day 7. Urine and plasma samples from day 7 were used to determine the excretion rates of the proteins by radioactivity and radioimmunoassay. RESULTS: When excretion rates were determined by radioactivity it was apparent that only the albumin excretion rate increased significantly with STZ diabetes to a value of 354 +/- 166 microg/min which agrees with proteinuria determined by Biuret assay of 299.9 +/- 52.4 microg/min. The major proportion of protein being excreted was in the form of protein fragments which are not detected by conventional immmunochemical assays. CONCLUSION: The previously unrecognized nephrotic-like levels of proteinuria in experimental diabetes appears to be associated with an albumin-specific mechanism responsible for the increase in albumin peptides in urine. There was significant lowering of plasma albumin concentration but plasma concentrations of transferrin and immunoglobulin G remained unchanged. There was also no significant appearance of intact protein in urine that is normally found in nephrotic states. PMID- 12373080 TI - Management of chronic kidney disease in an academic primary care clinic. AB - BACKGROUND: Three million people in the United States are estimated to have chronic kidney disease (CKD). Management of these CKD patients in the outpatient primary care clinic setting has not been well studied. HYPOTHESIS: Primary care management of CKD can be assessed and opportunities for improvement can be identified. METHODS: Management of CKD based on available published literature and guidelines was assessed in a single primary care site of an academic hospital with 23,000 annual visits and 8,300 patients. Charts of patients seen between October 1, 1997 and March 25, 1999 with an elevated SCr > or = 1.7 mg/dl on two separate measurements at least 6 months apart were reviewed for predefined indicators of CKD management. RESULTS: Assessment identified several aspects of CKD management to be suboptimal: control of blood pressure, use of angiotensin converting enzyme inhibitors or angiotensin receptor blockers, assessment of proteinuria, and renal consultation. Better management was found with respect to hemoglobin A1c measurement for diabetic patients. In general, CKD care was similar for diabetic and non-diabetic patients. CKD management was also similar regardless of level of creatinine clearance (> or = 50 vs. 50-30 vs. < or = 30 ml/min). CONCLUSION: CKD care can be measured in an outpatient academic primary care clinic and opportunities to improve were identified. PMID- 12373081 TI - Impact of burn size and initial serum albumin level on acute renal failure occurring in major burn. AB - BACKGROUND: Acute renal failure (ARF) is not a rare occurrence in severe burns and is an important complication leading to an increase in mortality. The severity of the burn is largely determined by the burn size, and severe burns are likely to cause enough loss of extracellular fluid and albumin from plasma volume to produce shock and hypoalbuminemia. HYPOTHESIS: We hypothesized that initial serum albumin level may be useful as an indicator of prognosis and severity of injury in burned patients. METHODS: The clinical characteristics of 147 adult patients with second- and third-degree burns covering 30% or more of their body surface area were analyzed retrospectively. Logistic regression was used to estimate the relative risks of ARF and mortality associated with the larger burn size and the lower serum albumin level at admission. RESULTS: Mean burned body surface was 60.0 +/- 21.8% (range 30-100%). Twenty-eight (19.0%) out of 147 patients experienced ARF, defined as a serum creatinine > or = 2 mg/dl, during the admission. The patients with ARF had larger burn size (79.5 +/- 15.4 vs. 55.3 +/- 20.5%, p < 0.0001) and lower serum albumin concentration at admission (1.92 +/- 0.66 vs. 2.48 +/- 0.82 g/dl, p < 0.0005) compared with those without ARF. All patients with ARF expired, whereas 29.4% (35/119) of the patients without ARF died. The burn size > or = 65% was associated with a risk of ARF that was 9.9 times and with a risk of death that was 14.2 times as high as that for the burn size <65%. The initial serum albumin level <2.5 g/dl was associated with a risk of death that was 2.7 times as high as that for the initial serum albumin level > or = 2.5 g/dl. CONCLUSIONS: When major burns are complicated by ARF, the mortality rate increases significantly. Burn size is an independent predictor of ARF occurring in major burns. Initially depressed serum albumin level is associated with an increase in mortality in the major burn patients. PMID- 12373082 TI - Cytogenetic and molecular genetic aspects of idiopathic myelofibrosis. AB - Idiopathic myelofibrosis is a chronic myeloproliferative disorder in which the characteristic fibroblast proliferation is thought to be a secondary phenomenon resulting from the inappropriate release of megakaryocyte- and/or monocyte derived growth factors, including PDGF, TGF-beta, bFGF and calmodulin. In contrast, the haematopoietic cells are clonal, although the underlying pathogenetic mechanisms remain essentially unknown. Cytogenetic studies have highlighted that 13q-, 20q-, +8 and abnormalities of chromosomes 1, 7 and 9 constitute more than 80% of the chromosomal changes. A third of idiopathic myelofibrosis cases have abnormal karyotypes at diagnosis, a figure that increases if follow-up analyses are performed. Evolution to more complex karyotypes may accompany clinical progression, with abnormalities increasing to around 90% following acute leukaemic transformation. Cytogenetic abnormalities have been associated with prognosis and to a lack of treatment response to androgens. Oncogene mutations are rare and include point mutations in N-RAS, c KIT and TP53. PMID- 12373083 TI - Polycythaemia vera: will new markers help us answer old questions? AB - The publication of the first molecular markers for polycythaemia vera (PV) has led to a renewed interest in finding the mutations leading to the development of this myeloproliferative disorder. A substantial amount of cell biological data on the PV stem cell, however, have been available for over two decades. This review will summarise, combine and integrate both fields of investigation. Based on this analysis, two alternative models for the development of myeloproliferative disorders are proposed. PMID- 12373084 TI - Functional studies on nine different haemoglobins with high oxygen affinity. AB - Nine carriers of beta-abnormal haemoglobins with increased oxygen affinity (Hb X) were examined. Their oxygen dissociation curves from whole blood were more or less left-shifted, with six of nine also characterized by biphasism. This refers to an 'inflection point' usually positioned at about 50-60% of Hb O(2) saturation, commonly believed to be a limit between oxygenation of the normal and abnormal components. In effect, the inflection does not always correspond to the Hb X level, which sometimes is much lower than 50% of the total Hb. Moreover, the upper half segment of the dissociation curve could not only be an expression of Hb A oxygenation, since it is always left-shifted. However, a high Hb A level is commonly believed to be the main compensatory factor of these subjects, but many indications suggest that often they have at least three, and not only two, main haemoglobin species: Hb A, Hb X plus hybrids of the type alpha(2)beta(A)beta(X). These would oxygenate after Hb X, but before Hb A. Finally, the interaction of 2,3-diphosphoglycerate with Hb X and/or hybrid tetramers must be altered, and the releasing of oxygen from both is more or less reduced. Unfortunately, it is difficult to demonstrate the presence of hybrids directly, i.e. with amino acid analysis of the abnormal beta-globin. PMID- 12373085 TI - Unusual myelodysplastic syndrome with the initial presentation mimicking idiopathic thrombocytopenic purpura. AB - Idiopathic thrombocytopenic purpura (ITP) and primary myelodysplastic syndrome (MDS) are hematological disorders that are frequently associated with thrombocytopenia, and both are heterogeneous disorders of uncertain etiology. Their diagnosis requires the exclusion of other hematological or immunological disorders whose diagnosis is usually not difficult. However, in some patients presenting with thrombocytopenia, the differential diagnosis is complex. We performed a retrospective study of 47 consecutive patients treated between 1990 and 2001; in 25 patients the initial diagnosis was ITP, in 22 it was MDS; we compared their backgrounds, laboratory data and clinical outcomes. Among the 25 ITP patients, there were 5 confusing cases. Following treatment, they presented with inexplicable refractory anemia and/or neutropenia. Cytopenia, the polyploidization pattern of megakaryocytes, and chromosomal aberrations were of diagnostic relevance in these patients' defective hematopoiesis. Their cytopenia progressed relatively slowly and none progressed to leukemic transformation. We suggest that these 5 patients should be classified into an unusual subtype of MDS with clinical characteristics resembling ITP. PMID- 12373086 TI - Effects of deferiprone on immune status and cytokine pattern in thalassaemia major. AB - OBJECTIVE: The present study was undertaken to evaluate the possible occurrence of immunological abnormalities in thalassaemia major patients treated with deferiprone (L1). METHODS: Longitudinal observational cohort study. RESULTS: The absolute number of CD8+ lymphocytes was high and the CD4/CD8 ratio low before L1 treatment; these parameters returned to normal after 3 months of L1 treatment. TNF-alpha, IL-2 and IL-2sRalpha were elevated before L1 treatment (11.83 +/- 1.75, 11.75 +/- 3.91, 1,409 +/- 621 pg/ml, respectively), while IL-6 was normal (2.58 +/- 0.79 pg/ml). After 12 months of treatment, IL-10 was higher than in previous periods, although always within the normal range. TNF-alpha, IL-2 and IL 2sRalpha returned to normal after 12, 6, and 3 months of L1 treatment, respectively. PMID- 12373087 TI - A rare, in-frame BCR-ABL fusion (e13a3) in a patient with an aggressive chronic myeloid leukaemia. AB - We have identified a rare BCR-ABL chimaeric gene with multiplex and nested RT-PCR in a patient with an unusually aggressive chronic myeloid leukaemia. cDNA sequencing showed an in-frame rearrangement with a breakpoint in BCR exon e13 (b2) and fusion with ABL exon 3 following skipping of the entire ABL exon a2. These data confirm the heterogeneity of breakpoints in BCR-ABL rearrangements. PMID- 12373088 TI - Warm-antibody autoimmune hemolytic anemia developing after thrombotic thrombocytopenic purpura. AB - Thrombotic thrombocytopenic purpura (TTP) and warm-antibody autoimmune hemolytic anemia (AIHA) are uncommon diseases. Although TTP has been increasingly described in association with autoimmune antibodies, there are very few reports of the association with autoimmune hematological conditions, including idiopathic thrombocytopenic purpura and AIHA. Here we describe a patient with classic manifestations of TTP, who was successfully treated with plasma exchange. A few weeks later, she developed warm-antibody AIHA, which responded promptly to prednisone. PMID- 12373089 TI - Molecular characterization of thalassemia intermedia associated with HPFH-6/beta thalassemia and HPFH-6/Hb E in Thai patients. AB - We report the molecular and hematological characterizations of thalassemia caused by interactions of the hereditary persistence of fetal hemoglobin (HPFH)-6 with beta-thalassemia in 2 Thai patients and the HPFH-6 with Hb E in another Thai patient. Marked hypochromic microcytosis, characteristics of thalassemia intermedia, were obvious in the former 2 cases but the latter had much milder clinical phenotype with normal Hb and a slightly reduced mean corpuscular volume (MCV) value. Hb analysis revealed no Hb A but Hb A(2)F patterns in the compound HPFH-6/beta-thalassemia patients and the EF pattern in the HPFH-6/Hb E patient. The (G)gamma-globin chain predominated in all cases. Globin gene analyses demonstrated that all patients carried the 101-kb HPFH-6 deletion in trans to the beta-thalassemia genes with the IVS1#5 G-C mutation and the G insertion between codons 8/9 and the beta(E)-gene, respectively. Hematologic data of the patients were compared to those of the HPFH-6 heterozygotes found in their family members and different genotype-phenotype interactions of this HPFH determinant in these Thai patients are illustrated. PMID- 12373090 TI - Successful use of recombinant factor VIIa for massive bleeding after caesarean section due to HELLP syndrome. PMID- 12373091 TI - Fatal hepatic veno-occlusive disease in a phase I study of mylotarg and troxatyl in patients with refractory acute myeloid leukemia or myelodysplastic syndrome. PMID- 12373092 TI - Inflammation and resistance to erythropoietin in hemodialysis patients. PMID- 12373094 TI - Microtubule reconfiguration during axogenesis. AB - When cultured on polylysine, rat sympathetic neurons extend modest lamellae which contain a mass of relatively short non-aligned microtubules. Microtubules display movements, but these movements do not result in any obvious alterations in the overall configuration of the array. Application of a mixture of growth factors called matrigel results in a rapid expansion of the lamellae followed by the outgrowth of axons. Microtubules undergo rapid behavioral changes that result in dramatic alterations in the microtubule array. Microtubules become significantly longer, and extend to the periphery of the lamellae where they invade newly forming axons. The microtubules align with one another and relative to the cell cortex, and draw together into bundles. Microtubules within a bundle move apart as well, particularly at the tips of developing axons. These observations demonstrate a complexity of microtubule behaviors, some of which can be explained by interactions with actin and/or by forces generated by molecular motor proteins. PMID- 12373095 TI - Histochemical localisation of FMRFamide-gated Na+ channels in Helisoma trivolvis and Helix aspersa neurones. AB - FMRFamide-gated Na+ channels of molluscan neurones belong to the ENa/Deg family of channels which have diverse functions. FMRFamide (Phe-Met-Arg-Phe-NH2) Na+ channels were detected electrophysiologically in specified neurones of Helix (Helix aspersa) and Helisoma (Helisoma trivolvis), and clones (FaNaCs) subsequently identified. We have now made a study to determine the distribution of mRNA for the clones HaFaNaC (Helix) and HtFaNaC (Helisoma) in the nervous systems of these species using standard in situ hybridization techniques. Immunohistochemical experiments were also made using an HtFaNaC antibody to detect the channel protein in Helisoma neurones. Many neurones in the central ganglia, including those which exhibit the FMRFamide Na+ current, stained for FaNaC-mRNA, suggesting a much wider distribution of the channel than was indicated by the earlier work. An immunoreactive response to the channel antibody was also observed in some Helisoma neurones, such as the giant dopamine neurone of the left pedal ganglion, also shown to possess HtFaNaC-mRNA and to exhibit the FMRFamide Na+ current. Taken together, these experiments suggest that the clones HaFaNaC and HtFaNaC are major, if not the only, subunits of the FMRFamide-gated Na+ channel detected electrophysiologically in the identified neurones of these species. However, fewer neurones in Helisoma reacted with the HtFaNaC-antibody than those which exhibited message for the channel. This discrepancy may be due to a difference in sensitivity of the two techniques, or because not all of the channel mRNA is normally expressed as a membrane protein. PMID- 12373096 TI - Localization of Triton-insoluble cAMP-dependent kinase type RIbeta in rat and mouse brain. AB - In eukaryotic cells, cAMP regulates many different cellular functions. Its effects are in most cases mediated by cAMP-dependent protein kinases. These consist of two regulatory and two catalytic subunits. In mammals, four different isoforms of cAMP-dependent protein kinases regulatory subunits have been characterized (RIalpha and beta, RIIalpha and beta). These four isoforms show a high level of homology and slightly different biochemical properties. In addition to biochemical properties, a different anatomical distribution of the regulatory isoforms may contribute to determine the specificity of diverse cAMP effects. By immunohistochemistry, the distribution of the detergent-insoluble fraction of RIbeta isoform has been examined in rat and mouse brain. Biochemical fractionation shows that a large fraction of both RIalpha and RIbeta isoforms is bound to the cytoskeleton. RIbeta labelling can be observed only in few locations: Purkinje cells, olfactory mitral cells, lateral thalamic neurons, superior olivary complex neurons. These cell populations are involved in the so called Purkinje cell degeneration. On the other hand, RIalpha aggregates have a more widespread distribution, in brain areas involved in visceroemotional control. At the subcellular level, these two subunits show a different pattern of labelling: in most cells a sharply defined clustered labelling is observed for RIalpha isoforms, while the RIbeta isoform presents a weaker, diffuse intracytoplasmic distribution. Competition experiments point to the presence of, as yet unidentified, different and selective anchoring proteins for the two similar RIalpha and beta isoforms. It is suggested that, as is the case for structural proteins, a different supramolecular organization of similar regulatory proteins may be crucial in order to fulfill different functions. PMID- 12373097 TI - Reorganization of actin during repair of hair bundle mechanoreceptors. AB - Hair bundle mechanoreceptors can be damaged by over-stimulation or by exposure to calcium-free buffers. Provided the trauma is slight, hair bundles recover, although the subcellular mechanisms for such recovery are poorly understood. Hair bundle mechanoreceptors on tentacles of sea anemones are especially resilient, recovering from severe trauma within several hours. During the recovery period, large protein complexes are secreted called "repair proteins" containing replacement linkages for those lost during trauma. In the present study, we find that recovery requires reorganization of the actin-based cytoskeleton in hair bundles. F-actin is first partially depolymerized and then repolymerized in hair bundles based on confocal microscopy. Furthermore, stereocilia show considerable motility during repair based on field emission scanning electron microscopy of hair bundles fixed at 1 min intervals after exposure to exogenously supplied repair protein complexes. Recovery of vibration sensitivity occurs at the organismal level within 8 min. Paradoxically, a full recovery of morphology of hair bundles requires approximately 45 min and a recovery of F-actin levels requires approximately 40 min. Similarly, a full recovery of mechanoelectric responses of hair cells requires approximately 45 min. Thus, it appears that the recovery of responsiveness at the organismal level precedes a full recovery of hair bundles. PMID- 12373098 TI - Detection of retrogradely transported WGA-HRP in axotomized adult hamster facial motoneurons occurs after initiation of the axon reaction. AB - We have previously shown that facial nerve transection at the stylomastoid foramen activates ribosomal RNA transcription in injured facial motoneurons (FMN) of the adult hamster within 30 minutes postoperative. The signal for the initiation of the nerve cell body response to injury in vertebrates is currently unknown. It has been hypothesized that the signal for initiating the injury response is dependent on retrograde transport, where the signal itself is either the loss of a repressor substance from the periphery or the loss of retrogradely transported target-derived factors. To examine if a retrograde transport-mediated signal would be sufficient to produce the rapid ribosomal effects observed in hamster FMN following injury, adult hamsters were subjected to right facial nerve axotomies, with the neuronal tracer wheat germ agglutinin horseradish peroxidase (WGA-HRP; M.W. 80,000) applied at the proximal stump of the transected nerve. At time points ranging from 0.5 to 24 hours postoperative (hpo), the animals were killed and brainstem sections containing bilateral facial nuclei processed for WGA-HRP label using the TMB method. The earliest time point at which WGA-HRP was detected in the axotomized facial nucleus occurred at 3 hpo. To eliminate molecular weight as a confounding factor, an additional retrograde transport study was performed using the smaller tracer, Fluoro-Gold (M.W. 532.59). Fluoro Gold was not detected until well after the 3 hpo time point. Thus, it appears that initiation of the axon reaction in hamster FMN is likely to be independent of the retrograde transport properties of the injured neuron. PMID- 12373099 TI - Neurofilamentous hypertrophy of intramedullary axonal arbors in intact spinal motoneurons undergoing peripheral sprouting. AB - An incomplete motor nerve injury or a partial loss of motoneurons leads to a partial denervation of skeletal muscle. As part of a compensatory response, the remaining intact motoneurons undergo peripheral sprouting and increase their motor unit size. Our knowledge about the responses in the more proximal parts of these sprouting motoneurons is sparse, however. We investigated the effects of an incomplete transection of the medial gastrocnemius (MG) nerve in the adult cat on the morphology of the intramedullary axon and axon collateral systems of the remaining intact MG motoneurons. At twelve weeks following the partial transection of the MG nerve, intracellular recording and labeling techniques were used to deposit horseradish peroxidase into single intact MG motoneurons for detailed morphological studies. The light microscopic appearance and caliber of the intramedullary stem motor axons of the intact MG motoneurons were indistinguishable from controls. The number and size of the intramedullary motoraxon collateral systems were also unchanged. However, frequent and marked hypertrophy of the distal portions of the motoraxon collaterals was encountered. Electron microscopic studies of the hypertrophied collaterals demonstrated abnormal accumulations of disorganized neurofilaments arranged in bundles or whorls. The morphological changes were indistinguishable from the neurofilamentous hypertrophy that has previously been reported in Wallerian degeneration, in experimental and human motor neuron disease and in some regenerating axonal processes of spinal motoneurons. We conclude that, neurofilamentous hypertrophy of the intramedullary arbors of motor axons may also be part of a reactive and non-degenerative response in intact motoneurons undergoing compensatory peripheral sprouting. PMID- 12373100 TI - Neurotrimin expression during cerebellar development suggests roles in axon fasciculation and synaptogenesis. AB - We investigated the temporal expression of the neural cell adhesion molecule, neurotrimin, in the rat cerebellum and the brainstem from birth to adulthood using immunoreactive labeling. A wave of expression accompanied the development of projection pathways extending from brainstem nuclei (pons/inferior olive) through the cerebellar peduncles into the arbor vitae and disappeared with myelination by P14. Immuno-EM revealed expression of neurotrimin on the surface of unmyelinated axons but not on astrocytes or oligodendroglia. With the development of the molecular and internal granular layers, intense labeling occurred on the surface of parallel fiber bundles, granule cells and mossy fibers. With synaptogenesis, each excitatory junction was labeled by the immunoreaction. By P21, neurotrimin reactivity decreased on the surfaces of neuronal somata, dendrites and axons but remained at excitatory synaptic contact sites in both the molecular and granular layers. The spatial-temporal expression pattern of neurotrimin suggests that this adhesion molecule plays a role in axonal fasciculation of specific cerebellar systems and may also be involved in the formation of excitatory synapses and their stabilization into adulthood. PMID- 12373142 TI - Testing for KIT (CD117) in gastrointestinal stromal tumors: another HercepTest? PMID- 12373143 TI - Immunohistochemical labeling of normal melanocytes. AB - Comparison of seven antibodies for the demonstration of normal melanocytes in formalin-fixed, paraffin-embedded surgical discard skin showed that the monoclonal antibody Mel-5 (clone TA99) directed against pigment associated antigen was the most sensitive. Quantitative data were obtained for the sensitivity of the antibodies NKI/beteb, S100, T311, Melan A (clone A103), c-kit, and Mel-5 in parallel sections of human skin. An anticytokeratin antibody (CK34betaE12) was also used to stain basal keratinocytes and provide a negative image of the melanocytes present. Optimal conditions for the use of Mel-5 in paraffin sections of skin are described. PMID- 12373146 TI - Detection of ganglion cells in the colonic plexuses by immunostaining for neuron specific marker NeuN: an aid for the diagnosis of Hirschsprung disease. AB - The majority of ganglion cells in the colonic plexuses can be easily and specifically identified by immunostaining for neuronal marker NeuN. The distance between the neighboring solitary ganglion cells or groups of ganglion cells varied from 0.18 to 4.0 mm, average 1.0 mm, in ganglionic segments of colons of patients with Hirschsprung disease, and from 0.3 to 6.3 mm, average 1.43 mm, in colons of pediatric patients with chronic constipation of various etiologies. No ganglion cells were detected in aganglionic colonic segments of patients with Hirschsprung disease by this method. PMID- 12373145 TI - Hepatocellular carcinoma and markers of apoptosis (bcl-2, bax, bcl-x): prognostic significance. AB - Patients with tumors expressing promoters of apoptosis (bax) versus inhibitors of apoptosis (bcl-2, bcl-x) may have increased survival. The purpose of this study was to determine the frequency of expression of apoptotic markers in hepatocellular carcinoma (HCC) and their relationship with prognosis. Seventy HCC were immunostained for bcl-2, bax, and bcl-x. Staining intensity in tumor cells was graded 0 to 3+. Follow-up data were available for mean survival (57 cases) and death rates (58 cases). These values and clinical parameters were related to prognosis. Staining frequency for bcl-2, bax, and bcl-x was 20%, 66%, and 60%, respectively. Immunostaining intensity of bax correlated with overall survival and death rates: of 57 patients, the 37% with 0 to 1+ intensity had a median survival of 6.6 months, the 63% with 2 to 3+ intensity had a median survival of 31.9 months (P = 0.05); 86% of 19 patients with 0 to 1+ intensity died, and 50% of 36 patients with 2 to 3+ intensity died (P < 0.05). Intensity of bcl-x staining tended to correlate with survival: of the 57 patients with 0 to 1+, 42% had a median survival of 32.7 months compared with 5.8 months in the 58% with 2 to 3+ intensity (P = 0.06). By multivariate analysis, this relationship held for bax (P = 0.011) and bcl-x (P = 0.048). There was no correlation between bcl-2 expression, stage, or gender and prognosis. Patients with bax-expressing HCC experience improved survival compared with those with no or low bax expression, in uni- and multivariate models. Patients with no or low bcl-x tended toward improved survival compared with patients with more bcl-x in their HCC. bcl-2 expression did not correlate with prognosis. PMID- 12373144 TI - The histiocytic marker PG-M1 is helpful in differentiating histiocytes and histiocytic tumors from melanomas. AB - Previous studies have shown that immunohistochemical stains for histiocytes are immunoreactive for melanomas. Accordingly, their value in differentiating histiocytes and histiocytic lesions from melanomas was questioned. PG-M1, the most specific histiocytic marker, was not evaluated in these studies. Our aims were to assess the reactivity of PG-M1 with a series of primary cutaneous and metastatic melanomas and to establish the potential usefulness of this antibody in the differentiation between histiocytes and histiocytic tumors and melanomas. PG-M1 staining was performed in 50 primary cutaneous and metastatic melanomas. For comparison, additional sections were stained with KP-1 and lysozyme (commonly used as histiocytic markers) and with S-100 and HMB-45 (commonly used as melanoma markers). The intensity (1+, 2+) and extent (1+ to 4+) were recorded semiquantitatively. PG-M1 stained weakly (1+) and focally (2+) only four cases of melanoma (8%). In contrast, histiocytes were strongly reactive for PG-M1 in all cases, being readily differentiated from melanoma cells including the positive cases. KP-1 stained melanoma cells in 44 cases (88%), lysozyme in 11 cases (22%), S-100 in 50 cases (100%), and HMB-45 in 48 cases (96%). No changes were found after restaining of selected KP-1 and lysozyme positive melanomas using an endogenous avidin/biotin blocking kit. PG-M1 is helpful in discriminating histiocytes and histiocytic lesions from melanoma cells. We recommend its inclusion in any antibody panel put together to distinguish between them. PMID- 12373147 TI - APC gene expression in gastric carcinoma: an immunohistochemical study. AB - Gastric carcinoma is one of the most common malignancies worldwide, particularly in Japan and China. Inactivation of the adenomatous polyposis coli ( ) gene, a tumor suppressor gene, has been shown to play a significant role in the development of colorectal carcinoma, and it has been suggested that it may play a role throughout the digestive tract, including the stomach. This study assesses gene expression in normal gastric mucosa and gastric adenocarcinoma using an antibody to the C-terminal region. One hundred twenty cases of gastric adenocarcinoma were examined from the files of Beaumont Hospital, Dublin, Ireland, and China Medical University, Shenyang, China. Ninety-one cases were informative. Of these, 78% revealed loss of staining. Loss of staining in adenocarcinoma showed no association with tumor type, tumor, stage or patient nationality. Loss of staining was also found in nine of 35 cases (26%) of intestinal metaplasia. In conclusion, loss of the gene, as determined by immunohistochemical staining, appears to be an early event in gastric carcinogenesis. Immunohistochemistry is a sensitive method for detection of this loss. PMID- 12373148 TI - Expression of E2F-4 gene in colorectal adenocarcinoma and corresponding covering mucosa: an immunohistochemistry, image analysis, and immunoblot study. AB - E2F-4 is a transcription factor involved in the transition of the cell from the resting state (G0/G1) to the proliferative stage (S). It has been associated with the p107 and p130 members of the Rb-family and it is responsible for many important growth suppressive functions. E2F-1, one member of the E2F family, has a similar structure to E2F-4; however, both have different mechanisms of action in regulating cell-cycle progression. Although E2F-4 acts mainly as a repressor in the early part of the cell cycle, E2F-1 has the ability to function as both an oncogene and a tumor suppressor gene. In an attempt to identify the role of E2F-4 as a potential mediator of cell proliferation, differentiation, tumorigenesis, and apoptosis in colorectal mucosa comparing with that of E2F-1, the authors examine 20 patients with human colon cancer and their corresponding histologically healthy mucosa by using immunohistochemical methods, computerized quantitative image analysis, and immunoblot analysis. Immunohistochemical studies were performed with formalin-fixed, paraffin-embedded sections stained with a monoclonal antibody against the E2F-4 protein. Apoptosis levels were determined by in situ assay. Positivity was scored by a Computerized Image Analyzer to detect the relative amount of the protein. Immunoblot analysis was performed on protein extracts from snap-frozen tissues of the same specimens. The results show that the expression of E2F-4 was greater in the tumor cells than in their corresponding benign epithelium as determined by immunohistochemical staining and image analysis. This was confirmed by semiquantitative IB analysis of the E2F-4 protein. The labeling index (LI) of E2F-4 in the tumors was inversely proportional to the LI of apoptotic cells. Within these cases, 12 cases showed a very high E2F-4 LI corresponding to low apoptosis LI. Three cases with relatively lower levels of E2F-4 LI were characterized with high apoptotic rates. These data suggest that E2F-4 gene overexpression plays a role in the development of colorectal tumors and appears to play a role in suppressing apoptosis. PMID- 12373149 TI - Prostatic adenocarcinoma with urothelial (transitional cell) carcinoma features. AB - Prostatic adenocarcinoma and urothelial carcinoma (transitional cell carcinoma) may coexist in the prostate. However, a carcinoma with mixed features has not been recognized. Four cases, three surgical pathology cases and one autopsy case of prostatic adenocarcinoma with urothelial carcinoma features, were retrospectively found in a urological pathology teaching file maintained from 1984 to 1993. Subsequently, 181 consecutive cases of radical prostatectomy from 1994 to 1999 were reviewed, and two prostatic adenocarcinoma areas with features of urothelial carcinoma were identified. Areas with urothelial carcinoma features were identified in the intraductal component of the carcinoma in five cases and in the invasive component in three cases. The intraductal carcinoma with urothelial carcinoma areas usually merged with regions of prostatic adenocarcinoma with a papillary or cribriform pattern. All prostatic adenocarcinomas having areas with urothelial carcinoma features were of high stage, and five of six cases had ductal features. The urothelial carcinoma component displayed a positive reactivity for thrombomodulin and negative or weaker reactivity for PAP and PSA than the prostatic adenocarcinoma component in the same tumor. Excluding the case noted at autopsy, all patients died of the disease within 3 years. Urothelial carcinoma features were usually associated with ductal carcinoma of high stage. Areas of prostatic adenocarcinoma with urothelial carcinoma features should be considered histopathologically as areas of mixed carcinoma of the prostate. Prostatic adenocarcinoma with areas of urothelial carcinoma features may pose a difficult differential diagnosis problem with urothelial carcinoma, especially with small biopsies with focal weak immunoreactivity for PAP, PSA, and thrombomodulin. PMID- 12373150 TI - C-erb B2 (Her2/neu) is neither overexpressed nor amplified in hepatic neoplasms. AB - The human c-erb B2 proto-oncogene (Her2/ ) encodes a 185-kD transmembrane putative growth factor receptor of the tyrosine kinase family. Overexpression or amplification of this oncoprotein/oncogene has been established in breast, ovarian, salivary gland, and gastric carcinomas and has been implicated in other neoplasms. Recently, overexpression of c-erb B2 has been demonstrated in hepatocellular carcinoma using enzyme-linked immunosorbent assay. Patients with hepatocellular carcinoma have a poor prognosis, and overexpression of c-erb B2 may have prognostic and treatment implications. The authors evaluated the expression and amplification of c-erb B2 in hepatic neoplasms utilizing routine immunohistochemistry and fluorescence in situ hybridization. Formalin-fixed paraffin-embedded tissue sections from 27 hepatocellular carcinomas and 7 hepatocellular adenomas were immunostained with anti-c-erb B2 utilizing a modified avidin-biotin technique following heat induced antigen retrieval. Ten sections from hepatocellular carcinomas were subjected to fluorescence in situ hybridization assay. Positive and negative controls stained appropriately. Slides were evaluated independently by two pathologists. None of the hepatocellular carcinomas or hepatocellular adenomas was immunoreactive with anti-c-erb B2. Adjacent cirrhotic liver parenchyma, present in 11 cases, was also uniformly negative. None of hepatocellular carcinomas showed any evidence of c-erb B2 amplification by fluorescence in situ hybridization. Immunoreactivity for c-erb B2 was not demonstrated in hepatocellular adenomas, cirrhotic livers, or hepatocellular carcinomas using routine immunohistochemical methods. C-erb B2 amplification was not demonstrated in hepatocellular carcinomas. Neither overexpression nor amplification of c-erb B2 (Her2/ ) can be regarded as a useful prognostic factor in hepatocellular carcinoma. PMID- 12373151 TI - Endometrial carcinoma cells are nonpermissive for CD44-erbB2 interactions. AB - The erbB2 receptor tyrosine kinase and the CD44 transmembrane glycoprotein interact with one another in numerous cell types. This interaction helps to maintain erbB2 activity that contributes to tumor progression. We investigated whether CD44 and erbB2 similarly interact in endometrial carcinomas in vitro and in situ. In contrast to other carcinomas, CD44 did not colocalize with erbB2 in any of the 51 cases of endometrial cancer analyzed. CD44 also did not coimmunoprecipitate or colocalize with erbB2 in two endometrial carcinoma cell lines. We propose that the lack of CD44-erbB2 interactions may reduce the contribution of erbB2 to endometrial carcinoma progression. PMID- 12373152 TI - Three-dimensional visualization of connexin 43 on the human cardiomyocytes. AB - Gap junctions created by a family of connexin proteins play an important role in the development of human heart. It has been previously shown that the abnormalities of right ventricular outflow tract are related to an altered level of expression of connexin 43. The right ventricular outflow tract narrowing, stenosis, or atresia of the main pulmonary artery and hypertrophy of the right ventricle are observed in tetralogy of Fallot. The aim of the current study was to determine the distribution of connexin 43 on the surface of human cardiomyocytes obtained during reparative surgery for tetralogy of Fallot. Connexin 43 distribution in these cells was compared with distribution of connexin 43 in cardiomyocytes obtained from patients without right ventricular outflow tract pathology. Cardiomyocytes isolated from tissue biopsy were cultured on collagen substratum, fixed with paraformaldehyde, and incubated with goat antihuman connexin 43 antibodies and secondary donkey antigoat antibodies conjugated with fluorescent indocarbocyanine. Z-series of optical sections were recorded using a laser scanning confocal microscope. Three-dimensional data stacks were visualized using volume-rendering techniques. Images of connexin 43 fluorescence revealed a pattern of three-dimensional distribution of connexin on the surface of an individual cardiomyocyte. Cardiomyocytes from tetralogy of Fallot and hearts with normal right ventricular outflow tract differ in the organization of connexin 43. Cardiomyocytes from tetralogy of Fallot hearts revealed disturbed distribution of connexin 43. The protein is located irregularly on the entire surface of the cell. In the controls, connexin 43 can be visualized within the intercalated disks only. These disturbances may influence heart maturation, cause hypertrophy of the right ventricle, and induce severe arrhythmias in children with tetralogy of Fallot. PMID- 12373153 TI - Immunolocalization of peripheral lymph node addressins in normal and neoplastic human thymuses. AB - Peripheral lymph node addressin is a specific L-selectin ligand of the high endothelial venules that plays an important role in lymphocyte homing to lymph nodes. Tissue selective migration of lymphocytes through this pathway to the thymus has also been proposed. In this work, peripheral lymph node addressin expression was investigated immunohistochemically with a monoclonal antibody, clone MECA-79, in formaldehyde-fixed, paraffin-embedded tissue sections of 5 normal neonatal thymuses, 25 thymomas, 3 thymic carcinomas, and 2 thymic lymphoid hyperplasias. In normal thymuses, peripheral lymph node addressin expression was found in the endothelium of corticomedullary and medullary vessels surrounded by perivascular space. In type B thymomas and thymic lymphoid hyperplasias, peripheral lymph node addressin was detected in the vessels with perivascular spaces, at the medullary differentiation areas, and in paralymphoid follicles, respectively. However, in type A thymomas and thymic carcinomas, MECA-79-positive vessels were restricted to the remnants of pre-existing thymic tissue, and they were absent from the neoplastic areas. These findings suggest that in normal and most neoplastic thymuses, peripheral lymph node addressin is expressed by regions of vascular endothelium corresponding to postcapillary venules that may serve as a pathway for homing of recirculating lymphocytes to the thymus. PMID- 12373154 TI - Immunohistochemical detection of immunoglobulin light chain expression in B-cell non-Hodgkin lymphomas using formalin-fixed, paraffin-embedded tissues and a heat induced epitope retrieval technique. AB - Definitive diagnosis of B-cell non-Hodgkin lymphomas often requires demonstration of B-cell monoclonality. Immunohistochemical detection of monotypic immunoglobulin light chain expression, and thereby B-cell monoclonality, may be accomplished readily using fresh cell suspensions or frozen tissue sections. However, immunohistochemical detection of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues is more difficult; with few exceptions, techniques suitable for formalin-fixed, paraffin-embedded tissues are not widely available. This report describes and validates a method for detecting immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues using a heat-induced epitope retrieval technique. This method was evaluated in a series of 113 cases of B-cell non-Hodgkin lymphoma, including 73 cases with correlative flow cytometric immunophenotyping data. Monotypic light chain expression was demonstrated in 91 (81%) of 113 cases, including several small core biopsy specimens with extremely limited tissue. Compared with the reference method (flow cytometric immunophenotyping), the specificity of the assay was 100%. Interobserver reproducibility was excellent, with 87% concordance between two independent observers categorizing cases as indeterminate, suggestive or diagnostic of kappa or lambda light chain restriction (Cohen kappa statistic: 0.81). In summary, the described method permits demonstration of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues in approximately 80% of cases of B-cell non-Hodgkin lymphoma with a high degree of specificity and excellent interobserver reproducibility. The assay is sufficiently robust for diagnostic use in small biopsies in which fresh tissue is unavailable. PMID- 12373155 TI - Superheating antigen retrieval. AB - Heat-induced antigen retrieval in a variety of solutions has been shown to enhance the immunoreactivity of a wide range of antigens in routine formalin fixed, paraffin-embedded tissues. Accurate time and temperature control is important for standardization and optimization of the procedure but is difficult to achieve. This study used a device to attain precise time and temperature control for antigen retrieval at 120 degrees C under 1.9 bar pressure. It compares the efficacy of this method with antigen retrieval in a conventional pressure cooker, by microwave heating at 98 degrees C, and ultrasound retrieval at 40 and 70 W for 40 and 100 seconds. Multitissue and multitumor blocks containing a spectrum of normal tissues and a variety of tumors, respectively, were used, and 42 routine diagnostic antibodies were applied with a standard peroxidase conjugated streptavidin technique. Sections in which antigen retrieval was not performed served as controls. The three heat-induced methods showed distinctly better immunostaining for all antigens compared with those obtained with ultrasound retrieval. The latter method did not produce consistent staining and intensity, and the extent of staining was only marginally better than sections not subjected to antigen retrieval. Superheating at 120 degrees C produced the best overall results with the exception of antibodies to cytokeratin clones Cam 5.2, AE1/3, and 34BE12 in which superheating resulted in slightly inferior immunostaining compared with heating in a pressure cooker and at 98 degrees C. PMID- 12373156 TI - Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues. AB - Formalin-fixed, paraffin-embedded tissue (PET) is an invaluable resource for retrospective molecular genetic studies, but the extraction of high-quality genomic DNA from the PET may be problematic. We report a simple method that significantly improves the ability to amplify DNA recovered from formalin-fixed PET. Based on the standard procedure of a commercially available DNA preparation kit, the QIAamp DNA mini kit or the HighPure DNA preparation kit, we developed this method by eliminating the xylene/ethanol extraction step and adding a heat treatment step. With this method, we have observed a five- to 10-fold increase in amplification efficiency of a fragment in a range of 90 to 386 base pairs. We also have obtained much higher amplification efficiencies for a multiplex polymerase chain reaction. PMID- 12373157 TI - Stereologic estimation of the number of neuroendocrine cells in normal human prostate detected by immunohistochemistry. AB - Neuroendocrine cells may play a role in both normal and pathologic conditions of the human prostate. It may be interesting to investigate 1) whether there are significant amounts of neuroendocrine cells in human adult normal prostate, and 2) whether the distribution of these cells shows regional differences. This study estimates both absolute and relative amounts of neuroendocrine cells immunostained for two neuronal markers (chromogranin A and protein gene product 9.5) and for serotonin in the three regions of human prostate, transition zone, central zone, and peripheral zone, using unbiased stereologic measurements. There was observed a predominance of neuroendocrine cells in the transition zone of the normal prostate. The neuroendocrine cells of this region may play a role in the genesis of benign prostate hyperplasia. The significant presence of neuroendocrine cells secreting neuropeptides in peripheral zone could be correlated with the induction of androgen-independent growth in prostate carcinogenesis. The wolffian origin attributed to the central zone can explain its poor population of neuroendocrine cells. PMID- 12373158 TI - Effect of tissue fixatives on the immunohistochemical expression of ABH blood group isoantigens. AB - Immunohistochemical analysis of ABH blood group isoantigens has been shown to be a useful ancillary technique for resolving problems associated with specimen mix ups in the daily practice of surgical pathology. However, the effects of different fixatives on the expression of these antigens in paraffin-embedded tissues are not known. Therefore, the effects of seven different fixatives on the immunohistochemical expression of ABH blood group isoantigens were studied in tissues from several organs. The following fixatives were used: acetone, 70% ethanol, B5, Bouin, Carnoy, methanol, and 10% formalin. After fixation for 6, 12, and 72 hours, the tissue blocks were embedded in paraffin, and immunohistochemistry was performed on 4 microm-thick tissue sections using monoclonal antibodies to blood group isoantigens (A, B, and H) and the avidin biotin detection method. Also, immunostaining was performed on step tissue sections with and without antigen retrieval using citrate buffer at pH 6.0. The expression of the blood group isoantigens was concordant with the blood group of the patient in all the cases studied, irrespective of the fixative and time of fixation. However, in the absence of antigen retrieval, the intensity of the staining reaction was diminished. These results showed that irrespective of the fixative used, immunohistochemical staining of paraffin-embedded tissue sections with ABH blood group antibodies is a rapid, reliable, and cost-effective method for sorting out interpretative problems of tissue contaminants (floaters) and specimen mix-ups in surgical pathology. PMID- 12373159 TI - Re: Ortiz-Hildago C, Torres JE. Cytokeratin-positive interstitial reticulum cells in Kikuchi-Fujimoto disease (Appl Immunohistochem Molecul Morphol 2002;10:194-5). PMID- 12373243 TI - Independent value of tissue harmonic echocardiography for risk stratification in patients with non-ST-segment elevation acute chest pain. AB - BACKGROUND: Clinical factors, electrocardiography, and cardiac troponins provide a satisfactory, although not ideal, means for risk-stratifying patients with non ST-segment elevation acute chest pain. Tissue harmonic echocardiography enables improved assessment of wall motion abnormalities compared with fundamental echocardiography and may be a useful adjunct for the detection of myocardial ischemia and infarction. We aimed to determine the value of tissue harmonic echocardiography in relation to electrocardiographic and biochemical factors for risk stratification of these patients. RESULTS: Eighty patients with non-ST segment elevation chest pain were studied using tissue harmonic echocardiography and troponin-T and -I. Fifty-five (69%) patients had abnormal electrocardiograms and 47 (59%) patients had abnormal echocardiograms. Thirteen patients (17%) had elevated troponin-T levels and 17 (21%) had elevated levels of troponin-I. Twelve patients (15%) had a myocardial infarction as the presenting event and, of the remaining 68 patients, 24 sustained an adverse cardiac event during the follow-up period (3 cardiac deaths, 4 nonfatal myocardial infarctions, 17 revascularization procedures). Troponin-T (98%), troponin-I (97%), and echocardiography (97%) all had similar negative predictive values for myocardial infarction as the presenting event, but troponin-T was the only independent predictor of this endpoint (relative risk 230, 95% CI 22-2427). An abnormal echocardiogram was the only independent predictor of subsequent events. The independent predictors of all events were age, troponin-T, and echocardiography. CONCLUSION: Tissue harmonic echocardiography provides independent information for risk stratification of patients with non-ST-segment elevation acute chest pain. PMID- 12373244 TI - Pitfalls of echocardiographic measurement in tissue harmonic imaging: in vitro and in vivo study. AB - BACKGROUND: Tissue harmonic imaging (THI) is a useful method for endocardial border detection by transthoracic echocardiography, especially in technically difficult patients, even though accuracy of this method in the echocardiographic measurement is unclear. The purpose of this study is to evaluate the accuracy of echocardiographic measurement by THI in vivo and in vitro. METHODS: In vitro, we measured wall thickness, dimension, and volume of the excised hearts by THI. In 11 patients, we assessed the comparative accuracy of THI and fundamental imaging (FI) in determination of left ventricular (LV) wall thickness, dimension, volume, and ejection fraction. RESULTS: In vitro, thickness measurements by THI overestimated true length, and both volume and dimension measurements by THI underestimated true values. In vivo, LV ejection fraction measurements obtained by THI exhibited excellent correlation and agreement with those obtained by FI. However, LV wall thickness determined by THI was significantly larger than that determined by FI, and the dimensions and volume of LV measured by THI were significantly smaller than those measured by FI. CONCLUSION: Although THI is an excellent imaging technique for determination of LV ejection fraction, echocardiographic measurement by THI underestimates LV dimensions and volume, and overestimates LV wall thickness. PMID- 12373245 TI - A validation study of aortic stroke volume using dynamic 4-dimensional color Doppler: an in vivo study. AB - OBJECTIVE: To explore the feasibility of directly quantifying transaortic stroke volume with a newly developed dynamic 3-dimensional (3D) color Doppler flow measurement technique, an in vivo experimental study was performed. BACKGROUND: Traditional methods for flow quantification require geometric assumptions about flow area and flow profiles. Accurate quantification of flow across the aortic valve is clinically important as a means of estimating cardiac output. METHODS: Eight open-chest sheep were scanned with apical epicardial placement of a 7 to 4 MHz multiplane transesophageal probe scanning parallel to aortic flow and running on an ATL HDI 5000 system. An electromagnetic flow meter implanted on the ascending aorta was used as reference. Thirty different hemodynamic conditions were studied after steady states were obtained in the animals by administration of blood, angiotensin, and sodium nitroprusside. Electrocardiogram-gated digital color 3D velocity data were acquired for each of the 30 steady states. The aortic stroke volumes were computed by temporal and spatial integration of flow areas and actual velocities across a projected surface perpendicular to the direction of flow, at a level just below the aortic valve. RESULTS: There was close correlation between the 3D color Doppler calculated aortic stroke volumes and the electromagnetic data (r = 0.91, y = 0.96x + 1.01, standard error of the estimate = 2.6 mL/beat). CONCLUSION: Our results showed that dynamic 3D color Doppler measurements obtained in an open-chest animals provide the basis for accurate, geometry-independent quantitative evaluation of the aortic flow. Therefore, 3D digital color Doppler flow computation could potentially represent an important method for noninvasively determining cardiac output in patients. PMID- 12373246 TI - Left atrial volume determination by three-dimensional echocardiography reconstruction: validation and application of a simplified technique. AB - Left atrial (LA) size assessment by anteroposterior dimension is limited in accuracy. Conventional 3-dimensional (3D) reconstruction has been validated, but the process is time-consuming and the 3D system is not widely available. We developed an algorithm to simplify 3D reconstruction of LA on the basis of 3 standard apical views, tested it in 44 hemodynamic stages of 8 open-chest dogs, and compared it with LA volumes assessed by conventional 3D reconstruction. Simplified 3D reconstruction provided an accurate LA volume measurement (y = 0.93x + 0.7, r = 0.95, standard error of the estimate [SEE] = 3.6) with more than 60% of time saved. Cubic equation of anteroposterior dimension and biplane modified Simpson's method were less accurate (for biplane modified Simpson's method, y = 0.8x + 2.6, r = 0.88, SEE = 5.0; for cubic equation of anteroposterior dimension, y = 0.65x + 2.6, r = 0.76, SEE = 8.2). Without the need for a 3D-imaging acquisition tool, simplified 3D reconstruction can be applied in the clinical setting for LA size quantitation with significant time saved. PMID- 12373247 TI - Left atrial inflow propagation rate: a new transesophageal echocardiographic index of preload. AB - We postulated that the rate of blood propagating into the left atrium from the left upper pulmonic vein would be a useful measure of pulmonary capillary wedge pressure (PCWP). In 23 adult patients who were critically ill (ie, study group) and receiving mechanical ventilation, color M-mode multiplane transesophageal echocardiography was used to measure left atrial inflow propagation rate (LAIF PR) as a potential index of PCWP measured by right heart catheterization. LAIF-PR was measured in systole and diastole as the slope of the color M-mode signal entering the left atrium from the left upper pulmonic vein. Correlation with PCWP was good for systolic (r = -0.847, P < .0001) and diastolic (r = -0.78, P < .0001) LAIF-PR. The reliability of univariate linear regression equations derived from the study group was tested in 29 subsequent patients (ie, testing group). Measured PCWP was accurately estimated within 5 mm Hg in 85% (22 of 26 patients) and 68% (17 of 25 patients) of the testing group by systolic and diastolic LAIF PR, respectively. Color M-mode transesophageal echocardiography-derived LAIF-PR, particularly in systole, is a promising new index to estimate PCWP in patients who are critically ill. PMID- 12373248 TI - Evaluation of left ventricular diastolic function from spectral and color M-mode Doppler in genetically altered mice. AB - Doppler indices of transmitral flow are commonly used to assess noninvasively left ventricular (LV) diastolic function in species larger than mice. The objective of our study was to characterize patterns of LV diastolic function in 2 genetically altered mouse models using Doppler- and color M-mode echocardiography. Phospholamban (PLB) reversibly inhibits the sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and is a key regulator of myocardial relaxation. Twelve-week-old PLB knockout mice (PLB/KO) were examined in parallel with age matched transgenic mice expressing a mutant form of PLB (PLB/N27A) that exhibited superinhibition of SERCA. Transmitral Doppler flow indexes, including isovolumic relaxation time, the ratio of peak early-to-late filling velocities, and deceleration time of peak early transmitral velocity indicate impaired diastolic filling in the PLB/N27A mutants, but improved LV diastolic function in the PLB/KO mice. In addition, a relatively load-independent parameter of LV relaxation measured by color M-mode Doppler, the propagation velocity of early flow into the LV cavity confirmed the observed differences. We conclude that transmitral filling patterns and color M-mode flow propagation velocity reflect changes in myocardial relaxation in mice with genetically altered levels of PLB and may be useful tools to characterize LV diastolic function in other mouse models of disease. PMID- 12373249 TI - Noninvasive assessment of coronary flow velocity and coronary flow velocity reserve in the right coronary artery by transthoracic Doppler echocardiography: comparison with intracoronary Doppler guidewire. AB - The aim of this study was to evaluate whether coronary flow velocity (CFV) and coronary flow velocity reserve (CFVR) in the posterior descending right coronary artery can be reliably measured by transthoracic Doppler echocardiography (TTDE). In 17 patients, CFV in the posterior descending right coronary artery was measured with TTDE at the time of Doppler guidewire examination. CFV was measured by both methods at baseline and under hyperemic conditions. TTDE data were obtained for 12 patients. CFV and CFVR by TTDE show a good correlation with those obtained by the Doppler guidewire method (average diastolic peak velocity: r = 0.98, y = 0.85x + 5.26; diastolic peak velocity: r = 0.97, y = 0.94x + 3.39; CFVR: r = 0.97, y = 0.87x + 0.56). CFV and CFVR in the posterior descending right coronary artery obtained noninvasively by TTDE accurately reflect these values obtained by the invasive Doppler guidewire method. PMID- 12373250 TI - Does contrast echocardiography with Optison induce myocardial necrosis in humans? AB - Myocardial contrast echocardiography is a promising diagnostic tool for detecting microvascular integrity. Multiple experimental laboratories have shown that diagnostic combined microbubble contrast and ultrasound exposure can cause vessel rupture and myocardial damage in laboratory animals. This study investigated the phenomenon of contrast ultrasonically induced myocardial damage in human beings. Twenty consecutive patients (mean age of 60 +/- 12 years, 14 men) underwent contrast echocardiography with intravenous Optison using a mechanical index of at least 1.4 (Vivid Five System (GE, Vingmed Ultrasound, Horton, Norway). Creatine kinase (CK), creatine kinase-isoenzyme MB (CK-MB); CK-MB mass, myoglobin, and troponin I were measured before and 2, 4, 8, and 24 hours after contrast echocardiography. There was no significant correlation concerning the response to contrast echocardiography for any pair of parameters at any time after the intervention. Only in 2 patients were there higher values for troponin I before and after contrast echocardiography without an increase of myoglobin, CK, or CK MB mass and activity. These values were therefore interpreted as false positive because of renal failure and severe heart failure. The use of contrast echocardiography is without demonstrated risk of myocardial damage even in patients with different cardiologic entities. PMID- 12373251 TI - Pulmonary venous systolic flow: influence of gravity on pulmonary venous flow velocities assessed in patients with atrial fibrillation. AB - The origin of the pulmonary venous (PV) systolic flow wave is still unclear and could be the atrial relaxation and systolic descent of the atrioventricular plane, which decrease atrial pressure (suction) or raised PV pressure. In atrial fibrillation (AF), loss of atrial contraction and relaxation significantly modifies the systolic PV flow wave. The effect of recumbent positional changes on PV, however, has not yet been characterized in AF. The purpose of this study was to evaluate the effect of positional changes on systolic PV flow in patients with AF studied by transesophageal echocardiography. The study group consisted of 45 patients with AF (34 patients with AF, alone, and 11 patients with mitral stenosis [MS]). To assess the influence of left atrial pressure, we included patients with MS and AF. Pulsed wave Doppler transesophageal echocardiography of the left and right upper PV were performed in the left lateral recumbent position in all patients and repeated records were obtained with the subject in the supine position in 25 (AF alone: n = 20, MS: n = 5) of 45 patients. In the left lateral recumbent position, the systolic PV flow velocity and systolic fraction of the left PV, which were recorded on the recumbent subject's lower side, were significantly increased compared with those of the right PV in both AF alone and MS with AF (33.9 +/- 10.8 vs 13.8 +/- 6.4 cm/s, 0.45 +/- 0.09 vs 0.20 +/- 0.10 in AF alone; 30.2 +/- 11.7 vs 14.6 +/- 6.0 cm/s, 0.43 +/- 0.12 vs 0.20 +/- 0.07 in MS, respectively, P < .01). By changing the position from the left lateral to the supine position, systolic PV flow velocity and systolic fraction of the left and right PV became the same (29.3 +/- 8.4 vs 27.9 +/- 8.4 cm/s, 0.39 +/- 0.09 vs 0.36 +/- 0.06 in AF alone, 23.5 +/- 8.8 vs 27.5 +/- 5.0 cm/s, 0.35 +/- 0.08 vs 0.35 +/- 0.09 in MS, respectively). These findings show that the PV volume (hydrostatic pressure) significantly modifies systolic PV flow wave in patients without atrial contraction and relaxation. We should take into consideration the body position on which PV flow is studied. PMID- 12373252 TI - Transcatheter closure of atrial septal defect and patent foramen ovale in adult patients using the Amplatzer occlusion device: no evidence for thrombus deposition with antiplatelet agents. AB - Transcatheter closure of atrial septal defect (ASD) and patent foramen ovale (PFO) using the Amplatzer septal occluder (AGA Medical, Minneapolis, Minn) is an alternative to surgical closure. There are only limited data on the thrombogenic potential of the device. Thirty-seven patients (14 men, 23 women) underwent device closure of their ASD (n = 21) or PFO (n = 16) at a mean age of 47 +/- 14 years (range, 18-72). The device was successfully deployed in all patients. Thirty-three of 37 patients received antiplatelet therapy with clopidogrel bisulfate and aspirin for a total of 6 months. Four patients in atrial fibrillation were also anticoagulated (international normalized ratio 2.0 to 3.0). No thrombus was detected in any patient on either side of the device by transthoracic and transesophageal echocardiography and there were no cases of symptomatic thromboembolism. Right-to-left interatrial shunting was diagnosed by contrast transesophageal echocardiography with the Valsalva's maneuver. At 1 month follow-up, minimal right-to-left shunting was detected in 6 patients (2 PFO, 4 ASD). Two patients (PFO) had minimal shunting at 1 month but not at 6 months. In 3 patients (ASD), inducible right-to-left shunting persisted at 6 months. In conclusion, our results obtained from a modest number of patients indicate that antiplatelet therapy is safe and effective in preventing thrombus formation on the septal occluder surface. PMID- 12373253 TI - Acute atypical type-A thoracic aortic dissection with intramural hematoma: the importance of patient symptoms and the transthoracic echocardiographic examination. AB - This report demonstrates how transthoracic echocardiography, in conjunction with the sonographer's attention to patient symptomatology, heightened the clinical suspicion of an atypical aortic dissection, leading to further investigation and confirmation. Although initially undiagnosed by computed tomography, a structure suggestive of aortic dissection was subsequently found by transthoracic echocardiography. Transesophageal echocardiography and a second computed tomography examination validated the transthoracic findings. An atypical type A aortic dissection with intramural hematoma was confirmed at operation. PMID- 12373254 TI - Long-term follow-up of acquired coronary artery fistula after septal myectomy for hypertrophic cardiomyopathy. AB - The long-term follow-up of acquired coronary artery to left ventricle fistula is unclear. We describe 2 cases of coronary artery to left ventricle fistula monitored for an average of 15 years with no clinical or echocardiographic deterioration. Medical therapy appears to be the appropriate treatment strategy in these patients. PMID- 12373255 TI - Rapid hemodynamic deterioration because of acute rupture of an aneurysm of the sinus of Valsalva: the importance of echocardiography in early diagnosis. AB - We report a patient with an acute rupture of an aneurysm of the right sinus of Valsalva of a bicuspid aortic valve into the right atrium. This congenital disorder is a result of a weak point in the aortic wall resulting from a localized interruption of the media. Because of rapid hemodynamic deterioration, early diagnosis is mandatory and often lifesaving. Echocardiography is the most reliable method to confirm the diagnosis. PMID- 12373256 TI - Myocardial contrast echocardiography: a series on contrast echocardiography, Article 5. PMID- 12373257 TI - Understanding people who smoke and how they change: a foundation for smoking cessation in primary care, part 1. AB - The purpose of this article is to develop an understanding of people who smoke and how they change as a foundation for the delivery of smoking cessation interventions in primary care. Central to our approach is the transtheoretical model of change (TMC). The TMC is an evidence-based model of behavior change that has been developed and tested during the past 2 decades by Prochaska and his colleagues in the context of smoking cessation. We use a review of the literature, in-depth interviews of people who successfully quit smoking, and our experience applying the TMC in the context of primary care and a smoking cessation clinic to explore the clinical work of smoking cessation. This article on smoking cessation will be presented in 2 issues. Part 1 describes the theoretical information known about smoking cessation: why smoking is a powerful behavior, the scientific background of the TMC, and the building-block constructs of the TMC. The first section of part 2 is a review of the Public Health Service clinical practice guideline, Treating Tobacco Use and Dependence, published in 2000. The second section of part 2 is a discussion of clinical assessments and strategies for working with smokers, which is grounded in the Public Health Service practice guideline, our understanding of people who smoke, and the TMC. Woven throughout are transcripts of interviews with 4 people in which they describe their smoking experiences and their pathways to cessation. PMID- 12373259 TI - An update on pollen and fungal spore aerobiology. AB - Changes in climate are altering pollen distribution. Predictive modeling can be used to forecast long- and short-term changes in pollen concentrations. Increasing evidence confirms the presence of pollen allergens on small, respirable particles in the air, explaining the occurrence of pollen-season increases in asthma. Like pollens, aboveground indoor fungal aerosols primarily reflect outdoor concentrations. Basement spore concentrations might be higher and reflective of local sources. Fungal presence in the indoor or outdoor air can be monitored on an area basis or with personal monitors. The samples can be analyzed by means of microscopy, culture, DNA probes, HPLC, or immunodetection. Total fungal biomass can be estimated on the basis of measurements of ergosterol or glucan in environmental samples. Unfortunately, there are no generally accepted standards for interpretation of fungal levels in indoor or outdoor air. At present, the best approach to indoor fungal control is moisture control in the indoor environment. This will essentially prevent fungal growth, except from extraordinary events. PMID- 12373260 TI - The role of transcription factors in allergic inflammation. AB - The induction of allergic inflammation and the expression of allergic disorders are dependent on the coordinated regulation of numerous genes. The products of these genes determine lymphocyte phenotype, immunologic responsiveness, eosinophil and mast cell development, activation, migration and life span, adhesion molecule expression, cytokine synthesis, cell-surface receptor display, and processes governing fibrosis and tissue repair. Although the expression of gene products involved in these processes is regulated at multiple levels (eg, transcription, mRNA processing, translation, phosphorylation, and degradation), transcription represents an essential and often the most important determinant of their contribution to cellular function. Signal-dependent and cell type-specific regulation of gene expression is generally achieved by means of combinatorial interactions between sequence-specific transcription factors that recruit chromatin remodeling machinery and general transcription factors to promoter and enhancer regions of RNA polymerase II-dependent genes. As targets of signal transduction pathways, transcription factors integrate the response of the cell to the myriad of inputs it receives. This integration can be accomplished by the effect of signaling cascades on the activation status or subcellular locus of transcription factors or by transcription factor dimerization induced by means of ligand binding. This review will identify the major families of transcription factors important in allergic mechanisms and discuss their interactions, their mechanisms of action, and their interrelated and competitive actions, as well as implications for therapy of allergic disorders. PMID- 12373261 TI - The future of allergy and clinical immunology lies in evaluation, treatment, and research on allergic disease. PMID- 12373262 TI - Recognition of clinical immunology as a distinct medical subspecialty: importance for the practice of allergy. AB - BACKGROUND: Although residents trained in accredited teaching programs in allergy and immunology are exposed to many areas of clinical immunology, the vast majority of these residents' subsequent practices are composed of caring for patients with allergic and asthmatic conditions. Except for rheumatologists, almost all other clinical immunologists appear to lack organized training programs, defined certification pathways, and clear career opportunities. OBJECTIVE: Recognition of clinical immunology as a distinct medical subspecialty with many areas of expertise will enhance the image of allergists and clinical immunologists, ensure subspecialty certification, and provide better career opportunities. METHODS: Documents, publications, and private opinions of individuals within professional allergy and clinical immunology organizations were evaluated for possible contribution to the subject content of this article. RESULTS: There is a need for defined residency programs, medical board certification, and professional organizations that speak for and provide postgraduate education for all clinical immunologists. Molecular and genetic discoveries are delineating the central role of fundamental immunology in all immune-mediated diseases and future therapy of allergic and immunologic diseases. CONCLUSIONS: Allergists of the 21st century should participate in the growing recognition of clinical immunology as an important medical subspecialty that can provide science-based therapies for allergic and immunologic disorders. The future practice of allergy depends largely on the molecular and genetic discoveries that serve to unite all practitioners of clinical immunology. Forging common alliances of education, certification, and career pathways with other clinical immunologists is the correct investment for a bright future for allergy. PMID- 12373263 TI - Imbalance between vascular endothelial growth factor and endostatin levels in induced sputum from asthmatic subjects. AB - BACKGROUND: Angiogenesis has recently attracted considerable attention as a component of airway remodeling in bronchial asthma. Vascular endothelial growth factor (VEGF) is highly expressed in asthmatic airways, and its contribution to airway remodeling has been reported. Although angiogenesis is regulated by a balance of angiogenic and antiangiogenic factors, the relative levels of antiangiogenic factors in asthmatic airways have not been evaluated. OBJECTIVE: We sought to determine whether an imbalance between angiogenic and antiangiogenic factors exists in asthmatic airways. METHODS: We simultaneously measured VEGF and endostatin levels and evaluated their correlation and balance in induced sputum from 18 steroid-naive asthmatic subjects and 11 healthy control subjects. After initial sputum induction, asthmatic subjects underwent 8 weeks of inhaled beclomethasone dipropionate (BDP; 800 microg/d) therapy, and sputum induction was then repeated. RESULTS: VEGF and endostatin levels in induced sputum were significantly higher in asthmatic subjects than in control subjects (P <.001). There was a significant correlation between VEGF and endostatin levels in both control subjects (r = 0.995, P <.001) and asthmatic subjects (r = 0.923, P <.001). Moreover, the VEGF/endostatin level ratio in asthmatic subjects was significantly higher than that in control subjects (P <.0001). After 8 weeks of inhaled BDP therapy, the VEGF level in induced sputum in asthmatic subjects was significantly decreased (P <.001), whereas the endostatin level was not. A correlation between VEGF and endostatin levels existed even after BDP therapy (r = 0.861, P <.001). Moreover, the VEGF/endostatin level ratio was significantly decreased to the same level as in the control subjects after BDP therapy (P <.0001). CONCLUSION: There was an imbalance between VEGF and endostatin levels in induced sputum from asthmatic subjects. This imbalance might play an important role in the pathogenesis of bronchial asthma through its effects on angiogenesis. PMID- 12373265 TI - Daycare centers and schools as sources of exposure to mites, cockroach, and endotoxin in the city of Sao Paulo, Brazil. AB - BACKGROUND: Public places, including schools, have been identified as sources of exposure to allergens derived from mites, cockroach, cat, and dog and to endotoxin. OBJECTIVES: The purposes of this study were to assess and compare exposure to allergens and endotoxin in 4 types of public child-care facilities in Brazil and to investigate whether the presence of children and the performance of cleaning procedures could have an influence on allergen and endotoxin levels. METHODS: We have analyzed dust from bedding, floors, chairs, and tables of daycare centers (DCs), preschools, kindergartens, and elementary schools (ESs). Major allergens from mites, cockroach, cat, and dog were quantitated by means of ELISA, and endotoxin content was determined by using the Limulus Amebocyte Lysate assay. RESULTS: Group 1 mite allergens were greater than 2 microg/g in 67% of DC and preschool samples and in 8.9% and 2.2% of kindergarten and ES samples, respectively. The presence of bedding in DCs and preschools accounted for increased levels of mite allergens in these settings. Levels of Bla g 1 were higher in ES floors compared with those found in DC and preschool floors. Low levels (<1 microg/g) of Fel d 1 e Can f 1 were found in most samples. Levels of endotoxin in DCs and preschools were 3 times higher than in ESs. CONCLUSIONS: DCs and schools in Brazil should be considered as important sources of exposure to dust mites and cockroach allergens and to endotoxin. Recommendations for mite allergen avoidance should include appropriate care of bedding in DCs and preschools. PMID- 12373264 TI - The cost-effectiveness of an inner-city asthma intervention for children. AB - BACKGROUND: Comprehensive management efforts to reduce asthma morbidity among children in urban areas with high levels of poverty and large minority populations have been inconclusive. The National Cooperative Inner-City Asthma Study (NCICAS) demonstrated improved symptom outcomes but did not evaluate cost effectiveness in this population. OBJECTIVE: We sought to examine the incremental cost-effectiveness of a comprehensive social worker-based education program and environmental control in children with asthma stratified by baseline level of asthma control. METHODS: We performed a prospective cost-effectiveness analysis alongside a randomized trial. A total of 1033 children and their families residing in 8 inner-city urban areas in the United States were enrolled in the NCICAS. Outcomes included symptom-free days, cost per symptom-free day gained, and annual costs of asthma morbidity compared by baseline symptom control, previous hospitalization, and previous unscheduled physician visits. RESULTS: The NCICAS intervention significantly reduced asthma symptoms. First-year intervention costs were 245 US dollars higher for the intervention children compared with those receiving usual care. There were no additional intervention related costs during the second year. When compared with usual care, the intervention improved outcomes at an average additional cost of 9.20 US dollars per symptom-free day gained (95% CI, -12.56 to 55.29 US dollars). The intervention was cost saving in 3 strata of children with increasing asthma severity. CONCLUSIONS: A multifaceted asthma intervention program reduced symptom days and was cost-effective for inner-city children with asthma. In children with more severe disease, the intervention was substantially more effective and reduced costs compared with that seen in control children. Organizations serving this population should consider this strategy as part of a comprehensive disease management program for asthma. PMID- 12373267 TI - Predictors of protocol adherence in a pediatric asthma clinical trial. AB - BACKGROUND: Declining protocol adherence can threaten the validity of a clinical trial. OBJECTIVE: We sought to explore patient and family factors important for protocol adherence in the 133 patients followed at one of the 8 Childhood Asthma Management Program (CAMP) clinical centers. Difficulties with timely return of diary cards (diary card problem), with keeping or frequently rescheduling appointments (appointment problem), and with commitment to all aspects of the trial (commitment problem) were tracked prospectively during the treatment phase of CAMP, which ranged from 20 to 40 months at the time of the analysis. METHODS: We performed a cross-sectional analysis. RESULTS: During the course of this investigation, no St Louis CAMP patients dropped out of the study, although signs of eroding participation were observed in 44% of patients. For this cross sectional analysis, the percentage of patients exhibiting protocol-adherence problems was greater the longer patients had been in the trial: 33.3% at 20 to 25 months, 39.5% at 26 to 30 months, 51.4% at 31 to 35 months, and 69.2% at 36 to 40 months (P <.01). The diary card problem was present in 22.2% of the patients enrolled in the trial for 20 to 25 months compared with 66.7% for patients enrolled for 36 to 40 months (P <.005). Appointment and commitment problems were present in smaller percentages of patients and did not change by time in the trial (P =.41 and.22, respectively). A logistic regression analysis of demographic characteristics indicated that age at randomization and time in the trial were significant factors: for every 2-year increase in age, a child was twice as likely to have a commitment problem (odds ratio [OR], 1.96; 95% CI, 1.50 2.57), and for each additional 5 months of participation in the study, a child was twice as likely to have a diary card problem (OR, 1.91; 95% CI, 1.76-2.07). There was no influence of family income, patient race, or patient sex on the occurrence of any of the 3 protocol-adherence problems. A similar analysis of psychologic characteristics of the child and family indicated (1) a 2-fold increase in the risk of a diary card problem with a 10% increase in the percentage of total commissions on the attention scale of the Gordon Diagnostic Study (OR, 2.18; 95% CI, 2.02-2.35), (2) a 2-fold decrease in the risk of an appointment problem with a 10-unit increase in the Child Manifest Anxiety Scale (OR, 0.46; 95% CI, 0.44-0.49), (3) a 2-fold decrease in risk of an appointment problem with a 10-unit increase in the cohesion subscale of the Family Environment Scale (OR, 0.58; 95% CI, 0.55-0.60), and (4) a 5-fold decrease in the risk of a commitment problem with a 10-unit increase in the Child Depression Index score (OR, 0.21; 95% CI, 0.18-0.24). CONCLUSIONS: Adherence and retention problems commonly occur in longer clinical trials. CAMP patients and families were selected in part on the basis of likelihood of being able to participate in the trial to enhance the conclusions of the trial. Despite this selection process, adherence problems were noted. Problems increased with duration of participation, increasing child age, and the presence of less family cohesion or attention problems in the child. In contrast, the presence of mild emotional distress (anxiety and depression) in the child was associated with fewer protocol adherence problems. Incorporating procedures that help anticipate and identify adherence problems early might improve continued participation in all aspects of a trial and even retention in long-term clinical trials. PMID- 12373268 TI - Rapid effect of inhaled fluticasone propionate on airway responsiveness to adenosine 5'-monophosphate in mild asthma. AB - Inhaled adenosine 5'-monophosphate (AMP) has an "indirect" bronchoconstrictive effect through mast cell degranulation and mediator release, whereas inhaled histamine has a "direct" effect on smooth muscle. Prolonged treatment with inhaled glucocorticosteroids attenuates airway responsiveness (AR) to AMP and histamine. We investigated the early effects of inhaled fluticasone propionate (FP) therapy on AR in 3 consecutive double-blind, randomized, placebo-controlled crossover studies in steroid-naive subjects with mild asthma. In one study, each of 12 subjects received FP 1000 microg or matched placebo for 7 inhalations at 12 hourly intervals; AR to AMP and FEV(1) were measured 2 hours after the 3rd and 7th inhalations. In a second study, each of 12 subjects received FP 100, 250, or 1000 microg or matched placebo for 3 inhalations at 12 hourly intervals; AR to AMP and FEV(1) were measured 2 hours after the 1st and 3rd inhalations. In a third study, each of 8 subjects received a single inhalation of FP 1000 microg or matched placebo; AR to histamine was measured 2 hours later. In the first study, FP 1000 microg significantly attenuated AR to AMP by 2.7 and 2.5 doubling doses after 3 and 7 inhalations, respectively (P < or =.0001). In the second study, FP 100, 250, and 1000 microg significantly attenuated AR to AMP by 1.9, 2.2, and 2.7 doubling doses, respectively, after 1 inhalation and by 2.4, 2.2, and 3.2 doubling doses, respectively, after 3 inhalations (P < or =.0001); a small but significant increase in FEV(1) (>0.15 L) was observed after 3 inhalations but not after 1 inhalation of FP irrespective of dose (P < or =.05). In the third study, a single inhalation of FP 1000 microg had no effect on AR to histamine. We have demonstrated a reduction in AR to AMP but not AR to histamine within 2 hours of a single inhalation of FP. This reflects a rapid, topical anti-inflammatory action of inhaled FP by a mechanism of action that remains unknown. PMID- 12373266 TI - Cockroach extract antigen increases bronchial airway epithelial permeability. AB - BACKGROUND: The bronchial epithelial cells of airways are subject to recurrent environmental injury throughout the life of an individual. Recently, a high incidence of asthma has been reported in inner-city children. The increased incidence of asthma in inner-city children is thought to be caused, in part, by frequent exposure to allergens of the common household pest the cockroach. OBJECTIVE: We sought to investigate whether cockroach extract antigen (CrAg) induces vascular permeability factor, also known as vascular endothelial growth factor (VEGF), and whether it increases permeability in bronchial airway epithelial cells (BAECs). METHODS: We estimated CrAg-induced VEGF release in BAECs by using an ELISA and VEGF mRNA expression by using an RT-PCR reaction. The influence of CrAg on BAEC barrier function was estimated by measuring electrical resistance with an electric cell substrate impedance-sensing system. RESULTS: Our results demonstrate that CrAg induces VEGF release in BAECs in a time-dependent manner. The VEGF induction was also confirmed by means of VEGF mRNA expression in CrAg-stimulated BAECs. CrAg decreased electrical resistance across BAEC monolayers. The maximum decrease in electrical resistance was noticed 6 hours after activation and reached a plateau thereafter. Neutralizing antibodies to VEGF significantly inhibited the decrease in BAEC electrical resistance caused by CrAg. CONCLUSIONS: These data suggest that CrAg induces VEGF release in BAECs and alters bronchial airway permeability. PMID- 12373269 TI - Persistence of viral RNA in 2 rat strains differing in susceptibility to postbronchiolitis airway dysfunction. AB - After viral bronchiolitis at an early age, a chronic asthma-like syndrome develops in BN, but not F344, rats. We hypothesized that the BN strain is less effective at clearing virus from the involved tissues. Weanling BN and F344 rats were inoculated with Sendai virus, and lung and peribronchial lymph nodes were harvested from each strain at 5 to 84 days after infection; control tissues were obtained from noninfected rats. Lung viral titers were similar for the 2 strains, with no infectious virus detectable by day 10. However, viral RNA was detected consistently by means of RT-PCR analyses in lungs and lymph nodes of both strains from days 10 to 27 and was still present at day 84 in some of the tissues from each strain. In contrast, there were strain-related differences in immune responses because IL-13 levels remained increased in the lung secretions of BN rats at 4 weeks after inoculation. Thus although Sendai virus could persist for at least 3 months after an acute infection in rats, this did not differ with strain. The persistent increase in IL-13 suggests instead that the strain-related variability in virus-associated airway pathology might be determined by the host response to infection rather than by the intensity or duration of infection. PMID- 12373270 TI - Hypersensitivity reactions after respiratory sensitization: effect of intranasal peptides containing T-cell epitopes. AB - BACKGROUND: The intranasal administration of peptides containing T-cell epitopes has been shown to inhibit T-cell and antibody responses of mice injected with allergen, but responses to respiratory sensitization might be regulated differently. OBJECTIVE: This study was designed to examine the effect of intranasal peptide on antigen-induced lung inflammatory responses and delayed hypersensitivity after sensitization by the respiratory mucosa or without sensitization. METHODS: Mice were treated with an intranasal tolerizing regimen of a peptide containing the major T-cell epitope of Der p 1. Delayed hypersensitivity and lung inflammation to challenge with Der p 1 was measured either without further treatment or after sensitization induced by means of the intranasal administration of Der p 1 with a mutated enterotoxin adjuvant. Lung inflammatory responses were examined by means of lavage and histologic section, and delayed hypersensitivity responses were measured on the basis of ear swelling. RESULTS: Delayed hypersensitivity reactions were induced in mice treated with intranasal peptide, and large reactions were found in mice given intranasal peptide and sensitized with intranasal Der p 1 and adjuvant. Mice pretreated with peptide and sensitized with Der p 1 had an increased lymphocytic infiltration after allergen-specific challenge, as measured by means of bronchoalveolar lavage and shown histologically. These hypersensitivity results are in contrast to previous data that show tolerance to injected antigen. CONCLUSIONS: Although the intranasal administration of a peptide containing a T cell epitope markedly inhibits responses to sensitization produced by the injection of allergen, the peptide induces immune responses and increases hypersensitivity to respiratory sensitization. PMID- 12373271 TI - Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients. AB - BACKGROUND: Among factors potentially involved in the increased prevalence of allergic diseases, modification of the intestinal bacteria flora or lack of bacterial stimulation during childhood has been proposed. Lactic acid bacteria (LAB) present in fermented foods or belonging to the natural intestinal microflora were shown to exert beneficial effects on human health. Recent reports have indicated their capacity to reduce allergic symptoms. OBJECTIVE: The purpose of this investigation was to determine the effect of LAB on the production of type 2 cytokines, which characterize allergic diseases. METHODS: PBMCs from patients allergic to house dust mite versus those from healthy donors were stimulated for 48 hours with the related Dermatophagoides pteronyssinus allergen or with a staphylococcal superantigen. The effect of LAB preincubation was assessed by measuring the type 2 cytokine production by means of specific ELISA. RESULTS: The tested gram-positive LAB were shown to inhibit the secretion of T(H)2 cytokines (IL-4 and IL-5). This effect was dose dependent and was observed irrespective of the LAB strain used. No significant inhibition was induced by the control, gram-negative Escherichia coli TG1. Interestingly, LAB reduced the T(H)2 cytokine production from allergic PBMCs specifically restimulated with the related allergen. The inhibition mechanism was shown to be dependent on antigen presenting cells (ie, monocytes) and on the involvement of IL-12 and IFN-gamma. CONCLUSION: The tested LAB strains were demonstrated to exhibit an anti-T(H)2 activity, and thus different strains of this family might be useful in the prevention of allergic diseases. PMID- 12373272 TI - T lymphocyte-mediated changes in airway smooth muscle responsiveness are attributed to induced autocrine release and actions of IL-5 and IL-1beta. AB - BACKGROUND: Bidirectional stimulatory cross-talk was recently found to exist between activated T cells and airway smooth muscle (ASM) cells, a process that involves coligation of specific cellular adhesion-costimulatory molecules that results in the induction of proasthmatic-like changes in ASM responsiveness. OBJECTIVE: The present study examined whether the cooperative intercellular signaling between activated T cells and ASM cells is coupled to the induced expression and actions of IL-5 and IL-1beta. METHODS: Agonist-induced constrictor and relaxant responses were examined in rabbit ASM segments exposed to resting and anti-CD3-activated T cells in the absence and presence of either an anti-IL-5 receptor mAb or the recombinant human IL-1 receptor antagonist. In addition, mRNA and protein expression of IL-5 and IL-1beta were assayed under control and anti CD3-stimulated conditions. RESULTS: Relative to inactive T cells, incubation of ASM tissues with anti-CD3-activated T cells induced proasthmatic-like changes in agonist-mediated ASM responsiveness. This T cell-induced perturbation in ASM responsiveness was ablated by pretreating the tissues with either an anti-IL-5 receptor mAb or IL-1 receptor antagonist. Moreover, exposure of ASM cells to anti CD3-activated T cells elicited an initial increased mRNA expression and release of IL-5, followed by an enhanced expression and release of IL-1beta, and the induced release of these cytokines was prevented in ASM cells that were pretreated with an anti-IL-5 receptor mAb. CONCLUSION: Collectively, these observations provide new evidence demonstrating that exposure of naive ASM cells to activated T cells induces the sequential release of IL-5 and IL-1beta from the ASM cells and that the latter cytokines act in an autocrine manner to elicit the proasthmatic phenotype of altered ASM responsiveness. PMID- 12373273 TI - Different effects of endotoxin versus mite and cat allergen exposure on T-cell differentiation in infants. AB - BACKGROUND: Early exposure to bacterial endotoxin has been proposed to protect against allergy development in children. Whether endotoxin is able to direct T cell differentiation into a predominance of type 1 immunity is still unresolved. OBJECTIVE: We sought to compare the effects of endotoxin and mite and cat allergens on T-cell differentiation in infants. METHODS: In a random population sample of 135 2-year-old children of an ongoing birth-cohort study, peripheral blood CD4+ and CD8+ T-cell subsets were defined by the expression of the chemokine receptors CCR5 and CCR3 as surrogate markers for type 1 and type 2 T cells, respectively. Endotoxin and mite and cat allergens were measured in house dust collected from the mother's mattress at the child's age of 3 months to assess early exposure. RESULTS: In the CD4+ T-cell subset, endotoxin levels were positively associated with high proportions of type 1 CCR5+ cells (odds ratio for fourth exposure quartile [OR(Q4)], 7.68; 95% CI, 1.35-43.75), whereas cat allergen levels were associated with increased proportions of type 2 CCR3+ cells (OR(Q4), 4.07; 95% CI, 1.05-15.85). In contrast to endotoxin, allergen levels were associated with CD8+ T cells, showing an inverse relationship between mite allergen concentrations and high proportions of CCR5+ or CCR3+ cells (CCR5+ cells: OR(Q4), 0.14; 95% CI, 0.03-0.74; CCR3+ cells: OR(Q4), 0.16; 95% CI, 0.03 0.89) and a positive association of cat allergen levels with increased proportions of CCR5+ cells (OR(Q4), 9.24, 95% CI, 1.61-53.10), as well as CCR3+ cells (OR(Q3), 6.64; 95% CI, 1.21-36.51). CONCLUSION: Our results indicate that endotoxin has the potential to promote the development of type 1 CD4+ T cells, whereas mite and cat allergens primarily modify the proportion of CD8+ cells of both types. PMID- 12373274 TI - Airway inflammation and functional changes after exposure to different concentrations of isocyanates. AB - BACKGROUND: Isocyanates are a common cause of occupational asthma (OA). OBJECTIVES: We sought (1) to examine whether asthmatic reactions to isocyanates could be induced at concentrations as low as 1 ppb in subjects with OA caused by isocyanates previously diagnosed in our center and (2) to compare the inflammatory and functional changes after exposure to 1 and 15 ppb of isocyanates with similar total doses (concentration of isocyanates x duration of exposure). METHODS: Specific inhalation challenges were performed in 12 asthmatic subjects with previously confirmed OA caused by isocyanates. Eight subjects were exposed to 1 ppb at 10% of the dose of isocyanates that induced an asthmatic reaction at the time of the diagnosis. Seven subjects were exposed to the same total dose of isocyanates by using concentrations of 1 and 15 ppb 1 month apart. RESULTS: Exposure to 1 ppb at 10% of the dose that had induced functional changes at the time of diagnosis induced an asthmatic reaction in 3 of 8 subjects. There was a significant correlation between the percentage of maximum decrease in FEV(1) after exposure to 1 ppb and the increase in sputum neutrophils (rho = 0.8). By keeping the total dose (concentration of isocyanates x duration of exposure) of isocyanates similar, 4 of 7 subjects experienced an asthmatic reaction after exposure to 1 ppb, whereas only one subject experienced an 18.5% decrease in FEV(1) after exposure to 15 ppb. CONCLUSION: Isocyanates can induce functional and inflammatory changes (mainly neutrophilic) at concentrations as low as 1 ppb. For the same total dose of exposure, low concentrations of isocyanates are as harmful as or even more harmful than higher concentrations for subjects with OA to isocyanates. PMID- 12373275 TI - Epinephrine dispensing patterns for an out-of-hospital population: a novel approach to studying the epidemiology of anaphylaxis. AB - BACKGROUND: The underlying rate of occurrence of anaphylaxis from all potential triggers in the general population is unknown. OBJECTIVE: We sought to obtain a perspective on the epidemiology of anaphylaxis in a defined general population by studying epinephrine dispensing patterns in an out-of-hospital setting. METHODS: Using an administrative claims pharmaceutical database, we analyzed dispensing data for all epinephrine formulations prescribed for out-of-hospital treatment over 5 consecutive fiscal years in a population of 1.15 million persons in the province of Manitoba, Canada. We identified the number and percentage of individuals in the general population for whom epinephrine was dispensed on one or more occasions, their age and sex, and the type of formulation dispensed. In addition to performing these analyses for the entire population of children, adults, and elderly individuals, we also performed analyses by 5-year age groupings; furthermore, for individuals younger than 5 years of age, we performed a detailed analysis by 6-month age groupings. RESULTS: During the 5-year period, 0.95% of this defined general population had injectable epinephrine dispensed for out-of-hospital treatment. There were substantial variations in epinephrine dispensing rates across subsets of the population, ranging from 1.44% for individuals younger than 17 years of age, to 0.9% for individuals 17 to 64 years of age (inclusive), to 0.32% for those age 65 years or older. In infancy, childhood, and early adolescence boys were more likely to have epinephrine dispensed than girls. Beginning at age 15 years and continuing into adulthood, girls and women were more likely to have epinephrine dispensed than boys and men. In the elderly there were no differences in dispensing patterns between the sexes. The highest epinephrine dispensing rate (5.3%) was found for boys age 12 to 17 months. CONCLUSIONS: Epinephrine dispensing data provide a novel and practical approach for studying and monitoring the epidemiology of anaphylaxis in a community. Using this approach, we provide evidence that anaphylaxis from all triggers peaks in early childhood and then gradually declines into old age. PMID- 12373276 TI - Analysis of the major epitope of the alpha2 chain of bovine type I collagen in children with bovine gelatin allergy. AB - BACKGROUND: Anaphylaxis to measles, mumps, and rubella vaccines has been reported. It has been found that most of these reactions to live vaccines are caused by type I allergy with the bovine gelatin present in the vaccines as an allergen. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. We previously reported that allergic reactions to gelatin are caused by the type I collagen alpha2 (alpha2[I]) chain. OBJECTIVE: To aid in the development of gelatin that has little or no allergenicity in human subjects, we investigated epitopes of bovine alpha2(I) chain with use of IgE in gelatin-sensitive children. METHODS: Serum samples were collected from 15 patients who had systemic allergic reactions to vaccines and high levels of specific IgE to bovine gelatin. Eleven overlapping recombinant proteins that cover bovine alpha2(I) were prepared with a bacterial expression vector. We examined IgE reactivity to these recombinant proteins by means of ELISA. Fifteen peptides covering a major reactive recombinant protein were synthesized. The IgE reacting epitope was identified by means of IgE-ELISA inhibition with these synthetic peptides and pooled serum from the patients. RESULTS: We found that of the 15 patients, 13 showed IgE reactivity to a recombinant protein (no. 3) spanning the central region of the collagenous domain ((418)Gly-(662)Pro). Furthermore, all 13 patients showed IgE reactivity to the 4-kd recombinant protein (no. 3a) spanning the region from (461)Pro to (500)Glu. In IgE-ELISA inhibition we found that a minimum IgE epitope of gelatin allergen was composed of the 10-amino-acid sequence (485)Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro(494). This sequence is not observed in the human type I collagen alpha1 and alpha2 chains, nor is it found in the bovine type I collagen alpha1 chain. CONCLUSIONS: We found that Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro is a major IgE epitope of the alpha2 chain of bovine type I collagen in patients with gelatin allergy. The degree of anaphylaxis to gelatin in vaccines might be reduced by digestion of this IgE-binding site in gelatin. PMID- 12373277 TI - IgG and IgA antibody levels to cow's milk are low at age 10 years in children born preterm. AB - BACKGROUND: Both innate and specific defenses of the preterm infant are even less developed than those of term infants, and the immune systems of preterm infants might be skewed differently at birth. Their immune responses to food antigens started early in life might therefore differ from those of term infants. OBJECTIVE: We sought to compare antibody levels to cow's milk, ovalbumin, and gliadin at age 10 years in children who had been born either preterm or at term. METHODS: IgG and IgA isotype antibodies to whole cow's milk, beta-lactoglobulin, alpha-casein, and ovalbumin, as well as IgG antibody levels to gliadin and to tetanus and diphtheria toxoids, were measured for a group of 62 children born preterm and 61 control subjects born at term. These children were studied at the same time for atopy. RESULTS: Children born preterm had markedly lower levels of antibodies to cow's milk and to its protein fractions (P <.0001 for IgA and IgG antibodies to cow's milk and alpha-casein and IgG beta-lactoglobulin antibodies). IgG gliadin antibodies were also significantly lower in the preterm group (P =.03), although the difference was not significant for IgG ovalbumin antibodies. In the preterm group both those born before gestational week 30 and those given cow's milk-based formula early (before day 50) had the lowest levels of cow's milk antibodies. In the preterm group atopy was associated with low levels of IgG cow's milk antibodies but with high levels of IgG ovalbumin antibodies. CONCLUSIONS: Early introduction of food antigens into the immature gastrointestinal tract of preterm infants might result in tolerance. The presence of less atopy in these children might also be a result of tolerance development. PMID- 12373278 TI - Chronic urticaria and angioedema as the first presentations of common variable immunodeficiency. PMID- 12373279 TI - Disappearance of aspirin intolerance with antiasthma treatment. PMID- 12373280 TI - Aspirin-induced urticaria and angioedema, but not bronchoconstriction, associated with cysteinyl leukotriene overproduction in 2 patients with asthma. PMID- 12373281 TI - Is dithiothreitol affecting cells and soluble mediators during sputum processing? A modified methodology to process sputum. PMID- 12373282 TI - Cohorts in economic comparison might not have been comparable. PMID- 12373283 TI - The PAT study's methods, asthma classification, and results are questionable. PMID- 12373284 TI - Can immunotherapy prevent progression to asthma in allergic individuals? PMID- 12373285 TI - Cooke and Vander Veer on heredity and sensitization. PMID- 12373287 TI - The role of O-linked protein glycosylation in beta-cell dysfunction. AB - Although only recently described, the pathway of O-linked protein glycosylation is already being implicated in diseases as diverse as cancer and Alzheimer's. Unlike the better known N-linked pathway, O-linked protein glycosylation is a dynamic and regulated event, much like tyrosine phosphorylation. During the process of O-glycosylation, the enzyme O-GlcNAc transferase (OGT) uses the substrate UDP-N-acetylglucosamine (UDP-GlcNAc) to attach a single O-linked N acetylglucosamine (O-GlcNAc) to nuclear and cytosolic proteins on serine or threonine residues. Conversely, the enzyme O-GlcNAc-selective N-acetyl-beta-D glucosaminidase (O-GlcNAcase) removes the O-GlcNAc, returning the protein to its baseline state until the cycle repeats itself. Although proving to be of interest in many different tissues, this pathway is especially important in pancreatic beta-cells. The beta-cell is unique in containing much more OGT than any other cell type. This enables beta-cells to respond to physiological increases in the glucose concentration by converting glucose to the OGT substrate UDP-GlcNAc, thereby dynamically coupling intracellular O-linked protein glycosylation to the extracellular glucose concentration. As a result, the beta-cell also appears to be especially susceptible to disruption of the O-glycosylation pathway. The diabetogenic agent streptozotocin (STZ), a UDP-GlcNAc analogue, causes beta-cell toxicity by irreversibly inhibiting O-GlcNAcase, while the diabetogenic agent alloxan (ALX), also a UDP-GlcNAc analog irreversibly inhibits OGT. This review will summarize what is currently known about beta-cell O-glycosylation and expand upon historical observations of chemically-induced beta-cell toxicity in animals to develop a model suggesting how beta-cell O-glycosylation is also involved in the development and progression of type 2 diabetes in humans. PMID- 12373289 TI - Protein expression of NF-kappaB in human colorectal adenocarcinoma. AB - NF-kappaB is a transcription factor believed to mediate a cellular survival response following the apoptotic stimulus TNF-alpha. To clarify the role of NF kappaB in colorectal cancer, we investigated the relationship of NF-kappaB expression by immunohistochemistry with clinicopathological variables, and other factors including apoptotic index, bcl-2 expression, p53 and K-ras mutations in 138 colorectal adenocarcinomas. Eighty-seven (63%) tumours showed cytoplasmic and 51 (37%) nuclear NF-kappaB expression. Nuclear NF-kappaB was correlated with mucinous carcinomas (p=0.006) and was more apparent in the invasive margin of some tumours. A trend was seen between nuclear NF-kappaB expression and K-ras mutation (p=0.15). However, NF-kappaB was not correlated with gender, age, tumour location, Dukes' stage, survival, apoptotic index, bcl-2 expression and p53 mutations. Conclusively, NF-kappaB might be activated in more aggressive colorectal tumour cells, since both tumour cells in the invasive margin and the mucinous tumour cells could represent more aggressive tumour cells. PMID- 12373288 TI - Differential regulation of extrinsic and intrinsic cell death pathways by protein kinase C. AB - The pathway of cell death depends on the apoptotic stimuli as well as on the cell type. In the present study, we have compared how extrinsic and intrinsic cell death pathways are regulated by the protein kinase C (PKC) signal transduction pathway in the same cell type. PDBu, an activator of PKC, potentiated cell death mediated by the DNA damaging agent cisplatin but it blocked tumor necrosis factor alpha (TNF)-induced cell death in HeLa cells. Conversely, rottlerin, an inhibitor of PKCdelta, decreased sensitivity of HeLa cells to cisplatin but it potentiated TNF-induced cell death. Although both TNF and cisplatin caused activation of caspases, PKC modulators had opposing effects on caspase activation. Rottlerin inhibited mitochondrial or intrinsic cell death pathway by inhibiting cisplatin induced processing of apical caspase-9 and its downstream caspases. In contrast, it potentiated receptor-initiated or extrinsic cell death pathway by enhancing activation of caspase-2 and/or caspase-8. These results suggest that PKC acts at distinct steps to regulate receptor-mediated and DNA damage-induced apoptosis. PMID- 12373290 TI - The correlation between CpG methylation and protein expression of P16 in oral squamous cell carcinomas. AB - We examined the P16 expression by immunohistochemical stain and detected the methylation by methylation specific polymerase chain reaction (MSP) in 48 primary oral squamous cell carcinoma (SCC) tissues. The results showed that 20/48 (41.7%) of cancerous tissues had CpG methylation around the promoter region, but 8/48 (17%) of the nearby non-cancerous tissues also had CpG methylation, around the promoter region. The results from immunohistochemical studies showed that reduced and heterogeneous expression of P16 were found in the tissues, which had CpG methylation around the promoter region. In conclusion, the methylation of P16 in oral SCC occurs in pre-cancerous and cancerous stage, which results in decreasing or abolishing the P16 expression, which is heterogeneous in the cancer cells. PMID- 12373291 TI - Diol- and triol-types of phytol induce apoptosis in lymphoid leukemia Molt 4B cells. AB - The exposure of human lymphoid leukemia Molt 4B cells to diol- and triol-types of phytol which were synthesized and identified by Mass, and 1H- and 13C-NMR, led to both growth inhibition and induction of programmed cell death (apoptosis). Morphological changes showing apoptotic bodies were observed in the Molt 4B cells treated with diol- and triol-types of phytol. The fragmentations of DNA by the diol- and triol-types of phytol to oligonucleosomal-sized fragments, that is a characteristic of apoptosis, were observed to be both concentration- and time dependent. These findings suggest that growth inhibition of Molt 4B cells by the diol- and triol-types of phytol results from the induction of apoptosis in the leukemic cells. PMID- 12373292 TI - New human embryo liver cell lines obtained by stabilization and immortalization enhance in vitro clonal growth of cordonal blood cells. AB - We developed two new cell lines derived from embryo liver and tested their inductive capacity on in vitro clonal growth of cordonal blood (CB) hematopoietic cells. One line was stabilized and named BAEP2-WILD (W), and the other one was immortalized by retroviral transduction with SV40 Large T antigen and called BAEP2-SV40. Southern blot analysis demonstrated the integration of the Large T antigen gene in the BAEP2-SV40 cell genome, but this line did not display the expected growth arrest at the non-permissive temperature of 39 degrees C. Immunocytochemistry showed that BAEP2-SV40 cell line was positive for several cytokeratins and stromal markers (vimentin, desmin and laminin), as well as for epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (Rs). In contrast, BAEP2-W evidenced positivity only for cytokeratin-7 and laminin, and low positivity to EGF, EGF-R, FGF and FGF-R. BAEP2-SV40 cell line, but not BAEP2-W, expressed interleukin (IL)-1, IL-6, granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor, stem cell factor and vascular-cell adhesion molecule-1 mRNAs, and secreted IL-6 and GM CSF. Taken together, these findings could suggest that BAEP2-W cell line possesses the phenotype of fetal hepatocytes, while BAEP2-SV40 cell line has that of stromal cells. The supernatants conditioned by both cell lines stimulated the clonal growth of CB hematopoietic cells cultured on semisolid media deprived of growth factors and cytokines, the inductive capacity of the BAEP2-SV40 cell line being markedly higher than that of its wild counterpart, conceivably due to its ability to produce cytokines. Our study indicates that these two new cell lines, and especially BAEP2-SV40 one could be used in co-culture systems as feeder layers for hematopoietic CB SC expansion in vitro. PMID- 12373293 TI - The possible intraspousal transmission of HCV in terms of lichen planus. AB - Lichen planus (LP), common mucocutaneous disorder, involves not only oral mucosa and skin but genitalia membrane. LP is frequently seen in patients with HCV infection. This study evaluated patients with HCV-associated oral lichen planus (OLP) for vulvar and vaginal LP involvement, and the possible intraspousal transmission of HCV. We examined a total of 24 female Japanese patients with OLP for genitalia LP: 14 OLP-HCV positive and 10 OLP-HCV negative. All subjects were evaluated for genital LP by a gynecologist. All 24 subjects and 10 of the husbands were tested for anti-HCV and serum HCV RNA. Vulvar LP was observed in 10 (41.7%) of 24 patients with OLP. Vulvar LP in 14 (OLP-HCV positive) and 10 patients (OLP-HCV negative) were observed in 42.9 and 40%, respectively. There were no significant differences (age, sites of OLP, blood transfusion, HCV infection, and degree of liver diseases) between the vulvar LP and non-vulvar LP patients. Two spouses of 10 married couples were shown to be infected with HCV. In one couple with HCV infection, the wife and husband had also erosive OLP, the wife had erosive vulvar LP. In conclusion, the majority of OLP patients suffered from genitalia LP in Japan. Clinicians should follow the OLP patients with sufficient attention to the presence of extraoral manifestations. These data may suggest the occurrence of intraspousal transmission of HCV through erosive vulvar LP. PMID- 12373294 TI - MICA gene polymorphisms in an Italian paediatric series of juvenile Behcet disease. AB - The objective of this study was to investigate MICA (major histocompatibility complex MHC class I chain-related genes) polymorphisms in an Italian series of patients with juvenile Behcet disease (jBD) and to compare these genetic findings with the high prevalence of inflammatory mucosal disease, which occurs in Western populations. Ten families which included at least 1 affected patient were studied. We genotyped 18 patients (13 children and 5 adults) affected with the complete or incomplete form of jBD comparing the results to those found in a population of 20 apparently healthy individuals. The MICA transmembrane polymorphism was analysed by PCR and polyacrylamide gel electrophoresis. HLA typing was assessed by SSP-PCR technique. Statistical analysis was performed using chi2 based methods. In our series the prevalence of gastrointestinal disease was high (41%). Seven of 10 patients were HLA-B51 positive. MICA A6 allele was present in 70% of probands as compared to 25% of an ethnically matched control population. On the other hand, MICA A5.1 was present in 20% of probands as compared to 60% in controls. Out of 5 A6 homozygotes, 2 probands and 2 affected relatives developed a severe gut inflammatory disease. The study of MICA gene polymorphisms disclosed an independent association with genetic risk for jBD. The combination of MICA A6 and HLA-B51 is the strongest genetic marker for this disease. Homozygous A6 patients seem to develop more severe mucosal gut involvement. This finding sheds light on the role of a receptor for MICA, named NKG2D, presented by natural killer cells, and CD8+, alphabetaT cells and gammadeltaT cells, usually localised in gut mucosa. PMID- 12373295 TI - Suppression of tumor growth through introduction of an antisense plasmid of macrophage migration inhibitory factor. AB - The potential role in cell growth of Macrophage migration inhibitory factor (MIF) has been studied, however, the mechanism of its anti-tumor effect is poorly understood. Antisense-MIF plasmids were directly injected into colon 26 tumors embedded in the back of mice. Furthermore, the role of MIF in the cell cycle was assessed with regard to retinoblastoma (Rb) protein and transcription factor E2F. Plasmids containing sense- and antisense-MIF genes were transfected into human colon cancer KM12SM cells in vitro. To examine the Rb protein-E2F pathway, plasmids containing each specific cis-acting enhancer for Rb protein and E2F with luciferase reporter genes, pRB-luc and pE2F-luc, respectively, were used. Antisense MIF treatment significantly reduced the tumor size. In vitro cell proliferation was significantly suppressed by the antisense treatment as examined by BrdU uptake. Transcriptions of Rb protein were 8.4x10(3) (RLU), 9.5x10(3) and 24.3x10(3) in the antisense MIF, PBK, and the sense MIF, respectively. As for E2F, transcription activities were 3.8x10(3), 3.6x10(3) and 7.7x10(3), respectively. These results indicate the possibility that MIF may promote tumor growth, in which the activation-inactivation mechanism of the Rb protein-E2F pathway could be profoundly involved. PMID- 12373296 TI - Plasma exchange therapy for the treatment of Escherichia coli O-157 associated hemolytic uremic syndrome. AB - Plasma exchange (PE) therapy was administered to three patients with Escherichia coli O-157 associated hemolytic uremic syndrome (HUS) in early phase. Following several PE treatments, all cases completely recovered without any apparent complications. The usefulness of PE therapy in removing microbial fragments and inflammatory cytokines was evaluated. The peptidoglycan (PG) level, interleukin-1 receptor antagonist (IL-1Ra) were higher in HUS patients starting PE therapy than in patients who had received several sessions of PE therapy. PE therapy was an effective early phase treatment for Escherichia coli O-157 associated HUS. PMID- 12373297 TI - Evaluation of telomerase in the development and progression of colon cancer. AB - Telomerase activity, a cardinal requirement for immortalization, is a crucial step in the development of cancer and has been studied in many kinds of malignant tumours for clinical diagnostic and/or prognostic utilities. Using a PCR-based TRAP assay, we investigated telomerase activity in 8 adenomatous polyps, 9 dysplastic polyps, and in 36 paired cancer-normal mucosa specimens, one liver and one spleen metastasis from patients resected for sporadic colorectal cancer. Telomerase was absent or very low in normal mucosa and in adenomatous polyps. Dysplastic polyps and adenocarcinoma samples showed telomerase activity, with higher levels in cancer tissues compared to dysplastic lesions. A high telomerase activity was shown to be associated with late-staged cancers and metastasis, providing arguments supporting the role of telomerase not only in the development but also in the progression of colorectal carcinoma. Moreover, telomerase evaluation may help to confirm the malignant transformation in polypoid colorectal lesions with different levels of dysplastic alterations. PMID- 12373298 TI - GnRH agonist inhibits human telomerase reverse transcriptase mRNA expression in endometrial cancer cells. AB - We investigated the relationship between the antiproliferative effect of GnRH agonist and telomerase activity using the endometrial cancer cell line HEC-1A. The subjects were 38 endometrial cancer, and 2 atypical endometrial hyperplasia patients. GnRH-R expression was detected using RT-PCR. HEC-1A cells were incubated with 10(-7)-10(-4) M GnRH agonist (leuprolide acetate), and cell proliferation was determined using MTT assay. The telomerase activity was detected by the TRAP assay and expression of human telomerase reverse transcriptase (hTERT) was assessed by RT-PCR. GnRH-R mRNA was detected at 94.7% (36/38) in endometrial cancer and in both of the atypical endometrial hyperplasia and in HEC-1A cells. Cell proliferation of HEC-1A showed significant inhibition at leuprolide acetate concentrations of 10(-6) M or higher compared with untreated control culture (p<0.05). The telomerase activity showed no marked difference compared with untreated culture. However, hTERT mRNA expression showed a decrease in the leuprolide-treated cells. It is suggested that the mechanism of the antitumor effect of GnRH agonist involved the inhibition of hTERT mRNA expression in the endometrial cancer cells. PMID- 12373299 TI - Pancreatic elastase IIIA and its variants are expressed in pancreatic carcinoma cells. AB - Ninety percent of pancreatic cancers are classified as ductal adenocarcinoma and are not known to secrete pancreatic elastases of the acinar enzymes. In the present study, we investigated the expression and the changes in elastase genes expressed in pancreatic ductal carcinoma cells. The expression of elastase gene family molecules in pancreatic cancer cells was analyzed by reverse transcription polymerase chain reaction (RT-PCR) method using primer sets for conservative active domains among pancreatic elastases (PEs) and neutrophil elastase (NE). The PCR products were subcloned, sequenced and analyzed. Three distinct products were isolated in pancreatic carcinoma cells using the RT-PCR analyses. The sequencing revealed that one was identical with the PE IIIA isoform, and the remaining two were alternatively splicing forms of the PE IIIA. Four of five pancreatic cell lines expressed these splicing variants in a cell-dependent manner. The present study is the first report to demonstrate the expression of PE IIIA and its splicing variants in pancreatic carcinoma cells might represent another infiltrative feature of the pancreatic ductal cell carcinoma. PMID- 12373300 TI - Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates. AB - We had shown previously that the novel, marine, anticancer compound dehydrothyrsiferol (DHT) does not modulate P-glycoprotein (P-gp) dependent drug efflux. Many chemotherapeutics with clinical impact are substrates for the structurally distant related membrane transport protein MRP1 (multidrug resistance-associated protein 1). Thus, we were interested in analysing the behaviour of DHT and control compounds in specific drug transport of MRP1 overexpressing cells. We established a fluorescence based drug efflux system for specific, functional detection of interference of a test compound in MRP1 mediated drug extrusion. Briefly, MRP1 overexpressing HL60/Adr cells were incubated to uptake and then efflux fluorescent 5(6)-carboxyfluorescein diacetate (CFDA), rhodamine 123 (Rh123), or 3,3-diethylocarbocyanine iodide (DiOC2), respectively. Changes in cell fluorescence intensity after coincubation with the compound of interest were determined by flow cytometry. MRP1 mediated efflux of CFDA was analysed in the presence of DHT, the known substrates genistein, probenecid, and the specific inhibitor MK-571. To exclude unknown P-gp related interference in drug transport, efflux of the fluorescent P-gp substrate DiOC2 and specific inhibition by cyclosporin A (CsA) were analysed. Cytotoxicity of DHT in resistant HL60/Adr cells was found to be even superior to that in the parental HL60 leukaemia cell line. Consequently, DHT did not interfere in MRP1 mediated drug transport. In contrast to DiOC2, rhodamine 123 was not specifically effluxed by P-gp but also by MRP1. Therefore, we propose the MRP1 specific CFDA efflux model as a screening and/or excluding system for MRP1 substrates. Together with previous data our results suggest DHT to be an interesting candidate for further investigation directed towards a drug development regimen. PMID- 12373301 TI - Sulfotransferase 1A2*2 is a risk factor for early-onset breast cancer. AB - The estrogen-signaling pathway plays an important role in the pathophysiology of breast cancer, and the sulfotransferase 1A (SULT1A) family has been found to be both downstream targets of tamoxifen and a risk factor of breast cancer. We have used PCR-RFLP and direct sequencing methods to determine SULT1A2 polymorphisms in 230 Taiwanese breast cancer patients. The results showed that the frequencies of SULT1A2*1 and SULT1A2*2 occurring were 94.8% and 5.2%, respectively. No SULT1A2*3 allele was found in these patients. In comparison with the frequency of healthy controls (96.0% and 4.0% for SULT1A2*1 and SULT1A2*2, respectively), the allelic frequencies of SULT1A2 polymorphisms in these patients were not statistically significant (p=0.398). However, the SULT1A2*2 allele seems to influence the age of onset among early-onset breast cancer patients (p=0.021, OR=2.74, 95%CI=1.13 6.65). PMID- 12373302 TI - Lipid-mediated gene transfection of intercellular adhesion molecule-1 suppresses the peritoneal metastasis of gastric carcinoma. AB - We have previously reported that a decreased expression level of ICAM-1 in cancer cells frequently led to the development of lymph node metastasis, and suggested that ICAM-1 gene transfection may inhibit lymph node metastasis. In the present study, we investigated whether ICAM-1 gene therapy for the peritoneal metastasis of gastric carcinoma is useful as a new immuno-gene therapy. ICAM-1 gene was transfected into a gastric cancer cell line, OCUM-2MD3 (2MD3), which has a high metastatic ability to the peritoneum. A transfectant cancer cell line, 2MD3/ICAM 1, had high ICAM-1 expression on the cell surface. The adhesion and cytotoxicity abilities of peripheral blood mononuclear cells were significantly increased against 2MD3/ICAM-1 cells in comparison with 2MD3 cells. Mice inoculated with 2MD3/ICAM-1 cells in the peritoneal cavity had a significantly better survival rate than those inoculated with 2MD3 cells (log-rank test, p<0.05). Histologic findings revealed that more mononuclear cells existed around the metastatic nodules in 2MD3/ICAM-1. Although gastric carcinoma frequently causes peritoneal metastasis, no useful therapy for the metastasis of gastric carcinoma has been developed. These findings revealed that ICAM-1 gene transfection to cancer cells could be a useful immuno-gene therapy for the peritoneal metastasis of gastric carcinoma. PMID- 12373303 TI - Detection of circulating prostate tumor cells: alternative spliced variant of PSM induced false-positive result. AB - RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA. PMID- 12373304 TI - Dietary bile acids inhibit potentially elemental diet-induced small intestinal atrophy in rats. AB - The mechanism responsible for elemental diet (ED)-induced small intestinal atrophy is still unknown. However, it is possible that bile acids in the gut lumen influence this process. The aim of this study was to evaluate the effects of oral bile acid administration during ED feeding. Specific pathogen-free male Sprague-Dawley rats, 10 weeks old, were fed an ED only, ED plus 0.1% (w/w) hyocholic acid, or ED plus 0.1% (w/w) hyodeoxycholic acid ad libitum for 4 weeks. The control rats were fed standard chow ad libitum for 4 weeks. After 4 weeks, the wet weight and whole length of the small intestine, and the mucosal diamine oxidase (DAO) and alkaline phosphatase (ALP) activities were measured. Microscopic histological observation was also performed. ED feeding induced atrophy and elevations in the mucosal DAO and ALP activities in the small intestine. Hyocholic acid and hyodeoxycholic acid administration both tended to inhibit these alterations. In conclusion, ED feeding induced atrophy and elevations in the mucosal DAO and ALP activities in the small intestine. Oral bile acid administration may prevent this atrophy and the elevations in mucosal DAO and ALP activities, which may lead to new therapeutic strategies in patients managed with ED. PMID- 12373305 TI - Clinicopathological analysis of liver abscess in Japan. AB - Currently, pyogenic liver abscess is not frequent, but it is a severe infectious disease. However a strategy for the effective treatment of liver abscess is not established. We analyzed 75 cases of liver abscess over an eight year period and evaluated their prognosis, any associated underlying disease, or the effect of percutaneous transhepatic abscess drainage (PTAD). For all 75 cases, laboratory data were analyzed and imaging studies were performed. Next, PTAD and antibiotic administration were started on these cases as first choice treatments. These treatments were continued until the laboratory data of the patient were restored to within the normal range. Those cases that were PTAD non-effective or required operation for underlying diseases, underwent operations. Of the total 75 cases, 63 survived after treatment and 12 cases died. Bacteria were detected in 50 cases and Klebsiella pneumoniae was detected in 31 of these 50 cases, but 25 out of 75 cases were negative. The biliary system was the main route of infection. PTAD was effective, especially in cases that were complicated with disseminated intravascular coagulation (DIC) or acute renal failure (ARF). PTAD is an effective treatment for liver abscess, it is especially useful in the restoration of severe general conditions as indicated by this study. PMID- 12373306 TI - Interleukin (IL)-4 and IL-17 synergistically stimulate IL-6 secretion in human colonic myofibroblasts. AB - There is increasing evidence that interleukin (IL)-4 can aid in Th1-type inflammatory responses in chronic colitis models. In this study, we evaluated the effects of IL-4 and/or IL-17 on IL-6 secretion in human colonic myofibroblasts. IL-6 secretion was determined by ELISA and Northern blotting. IL-6 secretion was rapidly induced by either IL-4 or IL-17. IL-17 induced IL-6 mRNA expression within 1 h after stimulation, and reached a maximum at 3 h. IL-6 mRNA induction by IL-4 occurred more rapidly. A maximum induction of IL-6 mRNA by IL-4 was observed at 1 h after stimulation, and this was rapidly decreased. The combination of IL-4 plus IL-17 greatly enhanced IL-6 secretion and mRNA expression. In conclusion, IL-4, in particular IL-4 plus IL-17, induced IL-6 secretion in human colonic myofibroblasts. Th2 immune responses might play an important role in the pathogenesis of gut inflammation. PMID- 12373307 TI - Prognostic significance of Epstein-Barr virus involvement in gastric carcinoma in Japan. AB - We examined the prognosis of 64 EBV-associated gastric carcinoma (EBV-GC) cases and 128 EBV-negative gastric carcinoma cases. EBV-GCs were identified by detecting EBV-encoded small RNA (EBER) using in situ hybridization assay of paraffin-embedded tissue. For each EBV-GC case, 2 EBER-negative cases (EBV negative cases) were selected, matching the EBV-GC case with respect to age, sex, tumor location, and depth of invasion. The average follow-up period was 70.9 months (SD=61.1) in EBV-GCs and 63.8 months (SD=59.7) in EBV-negative cases. Tumor-advanced stage determined by TNM classification of UICC, tumor location, and p53 over-expression were statistically significant prognostic factors. On the other hand, EBER expression was not related to the survival of patients. However, further analysis specific for intestinal and diffuse types of Lauren classification revealed that the association of EBER expression with prognosis was different in the two histological types. EBER expression was related to poor prognosis in intestinal-type carcinoma [hazard ratio (HR) =2.5, 95% confidence intervals (CI) =1.3-4.8] after adjusting for stage, p53 over-expression, and tumor location, whereas the diffuse-type EBV-GC had better prognosis (HR=0.4, 95% CI=0.2-0.9) even when lymphoepithelioma-like carcinomas were excluded. To examine the interactive prognostic effects between EBER expression and p53 over expression, the study subjects were divided into 4 groups on the basis of EBER expression and p53 over-expression. In intestinal-type tumors, the cases having both EBER expression and p53 over-expression showed the poorest prognosis (HR=10.0, 95% CI=3.3-30.4), and the cases with either EBER expression or p53 over expression had an intermediate prognosis. In diffuse-type tumor, only EBER was an important prognostic factor. These results give additional evidence implicating EBV in the natural history of EBV-GCs. PMID- 12373308 TI - Immortal phenotype of the esophageal epithelial cells in the process of immortalization. AB - To search for potential biomarkers used to monitor the process of immortalization, we investigated the relative level of telomerase activity and other immortal phenotypes in the SHEE esophageal epithelial cell line. This human fetal esophageal epithelial cell line, induced by human papilloma virus (HPV) 18 E6E7, was continually propagated over 100 passages. Fourteenth passage cells (SHEE14) were cultured in a flask with a serum-free medium and continually cultured to the 30th passage (SHEE30). Cells of SHEE14, SHEE20 and SHEE30 were examined according to cell morphology, cell cycle, apoptosis, contact-inhibition growth, anchorage- dependency, dose-dependency to epithelial growth factors (EGF), telomerase activity and tumorigenicity. The SHEE14 cells exhibited good differentiation with contact-inhibition and anchorage-dependent growth. The SHEE20 cells exhibited increase of senescent and apoptotic cells, and difficulty in propagation. The SHEE30 cells exhibited a higher proliferative index and some undifferentiated cells, with weakened contact-inhibition and anchorage-dependent growth. The telomerase was activated in cells of SHEE30, but not in SHEE14 and SHEE20 cells. The different response to dose-dependency to EGF was not statistically different in SHEE14 and SHEE30. Three groups of cells displayed lack of tumor formation in nude mice. Compared with SHEE14 and SHEE20, SHEE30 cells were of immortalized status with immortal phenotype, which consisted of telomerase activity, increase of cell proliferation, weakened contact-inhibition and anchorage-dependent growth, dose dependency to EGF and lack of tumor formation. From passage 14 to 30th passage, SHEE cells went through cellular senescence, apoptosis and immortalization. With a view toward diagnostic and biological aspects, telomerase activity is a crucial step and a cardinal requirement for immortalization. The telomerase activity and other immortal phenotypes are potential markers for monitoring the process of immortalization. PMID- 12373309 TI - Additional benefit of lamivudine treatment as a preventive therapy for hepatic encephalopathy in patients with decompensated liver cirrhosis associated with hepatitis B. AB - Lamivudine has previously been found to be effective not only in patients with compensated liver disease due to hepatitis B virus (HBV) but also in those with hepatic decompensation. However, long-term follow-up of patients with hepatic encephalopathy (HE) has not been previously reported. We describe a patient with recurrent HE associated with decompensated liver cirrhosis due to hepatitis B. After the initiation of treatment with lamivudine, manifestation of HE has not been observed for about 2 years and liver function has improved as well. This experience suggests that improved liver function using lamivudine may contribute to prevention from recurrence of HE. PMID- 12373310 TI - Clonality analysis of follicular lymphoma using laser capture microdissection method. AB - Whether a common and a single clone present, or not, among follicles of follicular lymphoma (FL) was examined in 12 cases with FL. Histologic grade was I in 6 cases, II in 3, and III in 3. DNA was selectively extracted from the neoplastic follicles of paraffin-embedded samples with use of laser capture microdissection method, and used for PCR-based analysis of rearrangement of immunoglobulin heavy chain variable region gene. Three different follicles in each case of FL were microdissected. Semi-nested PCR was performed using two sets of primers (Fr2A and Fr3A). In PCR with Fr2A primers, nine of 12 cases showed a common band among neoplastic follicles. The remaining three cases showed no PCR products. With Fr3A primers, eight of 12 cases showed a common band among follicles of the same case. The other four cases showed oligoclonal bands, among them presence of a common band was difficult to assess. Oligoclonal bands were more frequently observed in PCR with Fr3A than that with Fr2A and in grade I or II than in grade III cases. In total, 11 of 12 cases showed a common band in PCR with either Fr2A or Fr3A primers. In two cases, DNA extracted from whole section was amplified with both Fr2A and Fr3A or only Fr3A primers, showing smear or oligoclonal bands. These results showed the presence of a single clone of cells in neoplastic follicles of FL and the usefulness of PCR-based rearrangement analysis of immunoglobulin heavy chain gene combined with microdissection methods for differential diagnosis of FL from follicular hyperplasia. PMID- 12373311 TI - EGF and dextran-conjugated EGF induces differential phosphorylation of the EGF receptor. AB - Dextran-conjugated EGF (EGF-dextran) has a potential use for targeted radionuclide therapy of tumors that overexpress the epidermal growth factor receptor (EGFR). There are plans to treat both bladder carcinomas and malignant gliomas with local injections of radiolabeled EGF-dextran since these tumors often express high levels of EGFR. In this report we show that EGF and EGF dextran differentially activate the EGFR. In the human glioma cell line U-343, activation of the serine/threonine kinases Erk and Akt is identical upon stimulation with EGF or EGF-dextran. However, the effect on phospholipase Cgamma1 (PLCgamma1) phosphorylation differs. In cells stimulated with EGF-dextran, the PLCgamma1 phosphorylation is lower than in cells stimulated with EGF. This observation could be explained by the fact that the PLCgamma1 association sites in the EGFR, tyrosine residues 992 and 1173, were phosphorylated to a lower degree when the receptor was stimulated with EGF-dextran as compared to with EGF. PMID- 12373312 TI - Carrier analysis and prenatal diagnosis of haemophilia A in North India. AB - The feasibility of DNA diagnosis for haemophilia A in North India was evaluated using intragenic polymorphic DNA markers in factor VIII gene for linkage analysis as well as direct detection of inversion mutation in intron 22 of the gene. The informativity of RFLP (HindIII, BclI and XbaI) and STR (introns 13 and 22) markers for linkage analysis in factor VIII gene was determined in 100 normal individuals. The observed heterozygosity for RFLP markers HindIII, BclI and XbaI was 0.63, 0.60 and 0.48 while that of STR markers introns 13 and 22 were 0.60 and 0.40 respectively. Six and four alleles were identified for introns 13 and 22 and the most frequent allele was 13(CA)26 and 22(AG)n(GT)26 with an allele frequency of 0.53 and 0.62 respectively. The heterozygosities observed for RFLP markers was higher (>70%) than the STR markers (50%) in the affected families with haemophilia A. Inversion mutation was detected in 37% of severely affected patients. Based on present and previous studies from India, a strategy has been proposed to provide molecular diagnosis to a large number of undiagnosed cases of haemophilia A. PMID- 12373313 TI - Effects of experimental conditions on absorption of glycol ethers through human skin in vitro. AB - OBJECTIVES: To determine effects of experimental variables on the dermal absorption of 2-ethoxyethanol (EE), 2-butoxyethanol (BE) and 1-methoxy-2-propanol (M2P) through human skin in vitro. METHODS: Percutaneous absorption of EE, BE and M2P, in aqueous solution (3 mg ml(-1), 200 microl) or undiluted (10.5 microl), though full thickness or dermatomed human breast skin (0.64 cm(2) exposed area) was measured for 24 h using flow-through diffusion cells. Tissue culture medium was used as receptor fluid, with 2% (w/v) bovine serum albumin (BSA) or 2%-6% (w/v) polyethylene glycol 20 oleyl ether (PEG 20) added for some studies. Volatilised test compounds were trapped on charcoal filters placed above cells. RESULTS: In aqueous solution, steady-state flux of BE (544+/-64 nmol cm(-2) h( 1)) exceeded that of EE (143+/-19 nmol cm(-2) h(-1)) and M2P (48+/-6 nmol cm(-2) h(-1)). Reducing the dose volume to 100 microl decreased the steady-state flux of BE by about 55%, though the flux of EE was approximately doubled. Doubling the dose concentration of EE increased the flux by about eight-fold. Using full thickness skin increased tau of both EE and BE and reduced the steady-state flux of BE. Absorption rates of undiluted solvents in finite doses exceeded those measured with aqueous solutions, though the apparent permeability coefficient was higher with aqueous doses. Addition of BSA or PEG 20 to receptor fluid markedly increased absorption in both aqueous and undiluted doses. CONCLUSIONS: The dermal absorption potential of M2P from a liquid application was markedly lower than from EE or BE in all but infinite undiluted doses. The influence of receptor fluid on dermal absorption of glycol ethers could be relevant to prediction of absorption in vivo. PMID- 12373314 TI - Blood lead levels of primary-school children in Penghu County, Taiwan: distribution and influencing factors. AB - OBJECTIVES: The purpose of this study was to investigate the blood lead levels (BLLs) of primary-school children aged 7 to 12 in Penghu island and to determine the factors affecting their BLLs. METHODS: A total of 1,885 participants were recruited and BLLs were measured with a flameless atomic absorption spectrophotometer. A questionnaire was used to collect personal information. RESULTS: The results indicated that the mean BLL of primary-school children in Penghu was 6.0+/-2.4 microg/dl (1.0 approximately 29.3 microg/dl). The mean BLL of schoolboys ( n=1,046) was 6.3+/-2.6 microg/dl, with a maximum of 29.3 microg/dl, while the mean BLL of schoolgirls ( n=839) was 5.7+/-2.2 microg/dl, with a maximum of 23.4 microg/dl. Risk-factor analysis showed that personal characteristics (i.e., gender, frequency of milk consumption, grade levels) and geographic factors (i.e., levels of urbanization) significantly influence the BLLs. However, there was no significant impact on BLLs from drinking water, residential distance from a major road, and living close to lead-emitting sources. CONCLUSIONS: Geographical factors were highly associated with BLLs. The BLLs of the primary-school children living in the main Penghu island were lower than those in the other small islands. PMID- 12373315 TI - Assessment of exhaustion and related risk factors in employees in the manufacturing industry--a cross-sectional study. AB - OBJECTIVES: Vital exhaustion, a construct overlapping with burnout, is an independent risk factor for adverse health outcomes, including cardiovascular disease. We aimed to assess vital exhaustion in employees in the manufacturing industry and identify work characteristics associated with exhaustion. METHODS: Cross-sectional study. A stratified, representative random sample of employees from a manufacturing plant for airplane parts and assemblies was invited ( n=647), of whom 537 employees (83% accrual) volunteered to participate. Scores obtained by the nine-item Shortened Maastricht Exhaustion Questionnaire were compared with the mental and physical summary scales of the SF-12 General Health Survey and to a list of 20 health complaints. Pathogenic and salutogenic work characteristics were assessed by the self-reported 52-item, 13-subscale SALSA questionnaire. RESULTS: Vital exhaustion correlated with the mental summary score of the SF-12 and the number of health complaints and was positively associated with age. Exhaustion was not associated with gender, position (socio-economic status) or being on a wage (paid per completed item up to a contracted amount) or salary (payment as fixed monthly income). The instrument identified departments with high levels of exhaustion. Of the observed variance in exhaustion, 29% was explained by age, department, and five work characteristics: High levels of exhaustion (score >10) were related to excessive workload or qualitative demands (scoring in the highest quartile; OR(adj) 7.5; 95% CI 2.4-23), to adverse physical work conditions (OR(adj) 6.9; 95% CI 2.2-21), to adverse co-worker behavior (OR(adj) 4.8; 95% CI 1.4-16), to qualification potential (OR(adj) 0.32; 95% CI 0.11-0.97), and to social support by co-workers (OR(adj) 0.34; 95% CI 0.13 0.99), respectively. CONCLUSIONS: The nine-item instrument allows rapid screening of employees for self-reported levels of exhaustion. Besides physical work conditions and workload, absence or presence of social support by co-workers is strongly associated with exhaustion. PMID- 12373316 TI - Perceived odor and irritation of isopropanol: a comparison between naive controls and occupationally exposed workers. AB - OBJECTIVES: To assess sensory irritation levels from isopropanol (IPA) unconfounded by subjective evaluations of odor for comparison against the recommended exposure limits (400 ppm threshold limit value (TLV); American Conference of Governmental Industrial Hygienists). METHOD: The lateralization method was used to assess intra-nasal irritation thresholds for IPA, while odor detection thresholds were also measured. Thresholds for 1-butanol and phenyl ethyl alcohol (PEA) were obtained as positive and negative irritant controls. To compare potency and hedonic characteristics, subjects provided subjective ratings of odor, irritation and annoyance intensity for three concentrations of each chemical. Workers occupationally exposed to IPA ( n=26) were compared with previously unexposed controls ( n=26). RESULTS: The (geometric) mean odor detection threshold for IPA was slightly higher among exposed workers than controls (39 ppm vs. 11 ppm). Lateralization thresholds measuring intra-nasal irritation were elevated when compared with controls (6,083 ppm in exposed workers vs. 3,361 ppm in naive controls), with a significantly higher proportion of phlebotomists being unable to lateralize the maximum concentration regarded as safe, than controls. Calculations of the 6th percentile for lateralization thresholds revealed that 95% of the sample did not experience sensory irritation below 512 ppm. Thus, while odor detection thresholds were well below the current recommended exposure limits, the irritation thresholds were well above these values. The odor, irritation and annoyance from IPA was perceived, on average, as between weak and almost strong, from lowest to highest concentration. CONCLUSIONS: The results indicate that current exposure guidelines would be adequately protective of the acute adverse effect of nasal sensory irritation, as operationally defined by the intra-nasal lateralization threshold. Exposures to higher concentrations should perhaps be evaluated on the basis of existing knowledge about systemic, rather than local (e.g., irritation), toxic effects. IPA appears to be a weak sensory irritant and occupational exposure to IPA appears to elicit small changes in sensitivity that do not generalize to other odorants (e.g., PEA and 1-butanol) and are likely to be reversible. PMID- 12373317 TI - Are changes in mechanical exposure and musculoskeletal health good performance indicators for primary interventions? AB - OBJECTIVES: The purpose of this review is to present more insight into the effects of primary interventions on both mechanical exposure and musculoskeletal health and to determine whether these outcomes are good performance indicators of such interventions. METHODS: The literature was scrutinised for relevant references. Primary prevention was defined as any activity aimed at preventing the occurrence of musculoskeletal disorders in occupational populations. Primary outcome measures were mechanical exposure, musculoskeletal health, and sick leave due to musculoskeletal disorders. The impact of interventions was assessed by calculating the reduction in mechanical exposure and the preventable fraction (PF) of the musculoskeletal complaints. After selection, 40 studies were included for further analysis. RESULTS: In general, of the 40 included studies, 29 (73%) found a reduction in musculoskeletal symptoms (PF range 0.10-0.95). Eighteen out of 29 studies (62%) reported a statistically significant reduction in musculoskeletal disorders. In 12 of the 40 studies (30%) changes in both mechanical exposure and musculoskeletal health were used as performance indicators for the intervention. Of these studies nine (67%) showed a reduction in both mechanical exposure (range 14%-87% reduction) and musculoskeletal disorders or sick leave due to musculoskeletal disorders (PF range 0.15-0.92). From these nine it was seen that a reduction of at least 14% in mechanical exposure resulted in a concomitant reduction in musculoskeletal health. CONCLUSIONS: More quantitative information is needed to describe the relationship between mechanical exposure and musculoskeletal health as presented in the model. In this case it is recommended that in primary intervention studies not only changes in health outcomes be measured but also changes in mechanical exposure along the pathway of the intervention. In this way a better insight will be gained about the dose-response relationships between exposure to physical-load risk factors and work-related musculoskeletal disorders (WRMSD). More insight into these relationships will eventually lead to more efficient implementations of primary intervention strategies. PMID- 12373318 TI - Markers of insulin resistance in day and shift workers aged 30-59 years. AB - OBJECTIVES: To examine relationships between shift work and markers (metabolic abnormalities) of insulin resistance (IR). METHODS: A cross-sectional study of 2,824 day and 826 shift workers. All the subjects were male blue-collar workers aged 30-59 years. Four IR markers [(1) hypertension (systolic blood pressure >or=140 mmHg or diastolic blood pressure >or=90 mmHg or under treatment for hypertension); (2) hyperglycemia (fasting serum glucose >or=7.00 mmol/l or under treatment for diabetes); (3) hypertriglyceridemia (fasting serum triglyceride >or=1.70 mmol/l or under treatment for hyperlipidemia); (4) hypo-HDL cholesterolemia (fasting serum HDL-cholesterol <1.04 mmol/l)] were checked. Subsequently, IR syndrome, a cluster of IR markers, was expediently diagnosed by the number (N) of IR markers found in each worker. N>or=1, N>or=2 or N>or=3 was used as a cutpoint for the diagnosis. The prevalence of each IR marker and IR syndrome was compared between the two worker groups. Age, body mass index (kg/m(2)), job, drinking, smoking, and exercise were used as confounding factors. Job, work schedule and lifestyles were based on self-administered questionnaires. RESULTS: Hypertriglyceridemia (28.7% in day workers, 31.2% in shift workers) was most prevalent (4.6%), and hyperglycemia (5.4%) was least, in the four IR markers in both worker groups. Approximately half of the subjects had at least one IR marker in both groups (N>or=1; 46.0% and 46.6%, respectively). However, a full cluster of IR markers was rare in both groups (N=4; 0.2% and 0.1%, respectively). Prevalence of IR syndrome by any cutpoints increased with age in day workers, but shift workers aged 50 years or older had lower prevalence than shift workers younger than 50 years. In subjects younger than 50 years, all IR markers (except for hypo-HDL-cholesterolemia) and IR syndrome by any cutpoints were more prevalent in shift workers than in day workers. Contrariwise, in subjects aged 50 years or older, lower prevalence of all IR markers and IR syndrome in shift workers than in day workers was found. These results were not influenced by statistical adjustments for the confounding factors by multiple logistic regression analysis. CONCLUSIONS: Shift work may be associated with IR syndrome in workers younger than 50 years. These relations may be underestimated mainly by broad definition of shift work and healthy-worker effects. PMID- 12373319 TI - Relationship between blood lead levels and renal function in lead battery workers. AB - OBJECTIVES: The aim of this study was to investigate the correlation between blood lead (PbB) levels and renal function indices of blood-urea nitrogen (BUN), serum creatinine (SC) and uric acid (UA) among lead battery workers with exposure to lead. METHODS: A total of 229 workers of both genders from two lead battery factories were recruited in this cross-sectional study. The personal airborne and blood samples were collected on the same day. The airborne lead (PbA) and PbB levels, and individual renal function parameters were measured and statistically analyzed. RESULTS: A positive correlation between PbB levels and individual renal function index of BUN, SC, and UA was found ( P<0.01). The PbB levels and renal function indices showed significant difference between male and female workers. Based on a multiple regression model, an increment of 10 micro g/dl PbB produced an increase of 0.62 mg/dl BUN, after being adjusted for work duration and age, and an increase of 0.085 mg/dl UA, after being adjusted for gender and body weight. Workers with PbB 60 microg/dl showed a positive dose-effect relationship with significant difference in BUN ( P<0.001) and UA ( P<0.05), and the percentage of workers with BUN and UA over the reference value also showed an increasing trend. CONCLUSION: Blood-urea nitrogen and uric acid could be considered as suitable prognostic indicators of renal dysfunction in lead-exposed workers. Our results showed that PbB levels higher than 60 micro g/dl had increasing chances of inducing adverse renal effects. PMID- 12373320 TI - Intra-individual variation in the metabolism of inorganic arsenic. AB - OBJECTIVE: Inorganic arsenic is metabolized to methylarsonic acid (MMA) and dimethylarsinic acid (DMA), which are excreted in the urine. Previous studies have shown a marked inter-individual variation in the metabolism, but the within person variation is unknown. Therefore, we determined the variation in the relative amount of urinary arsenic metabolites, i.e., inorganic arsenic, MMA and DMA, over time in women living in an Andean village with elevated arsenic levels in drinking water. METHODS: For our investigation of the individual day-to-day variation, daily spot urine samples were collected (at the same time of the day) for 5 consecutive days by 15 women. For our investigation of the within-day variation, seven women collected all the urine voided over a 72-h period, each voided into a separate container. RESULTS: Repeated measures analysis of variance (ANOVA) revealed no significant day-to-day variation, either in the relative distribution of inorganic arsenic metabolites (inorganic arsenic, MMA and DMA), or in the concentration of the metabolites. However, the percentage of DMA was slightly higher (on average 5.0%; P=0.003) and the percentage of inorganic arsenic lower (on average 5.8%; P=0.001) during the morning/day (03:00-15:00) compared with the evening/night (15:00-03:00). No within-day variation in the percentage of MMA was observed. CONCLUSION: The arsenic methylation efficiency of an individual is remarkably stable over time. PMID- 12373322 TI - The risk of asthma among Finnish patients with farmer's lung. AB - OBJECTIVES: To assess the incidence and risk of asthma in patients with farmer's lung in comparison with farm workers without farmer's lung. METHODS: The details of farmers and animal-husbandry workers notified in 1988-1999 for farmer's lung ( n=1,272) or other occupational disease ( n=5,045) to the Finnish Register of Occupational Diseases were followed until 31 December 2000 through two national registries of individuals eligible for reimbursement of the cost of asthma medication and the Population Register Center. Incidence rates of asthma were calculated, and a log-linear model adjusted for age, gender and occupation was used to estimate relative risks of asthma among those with farmer's lung compared to those with other occupational disease. RESULTS: Of the patients with farmer's lung, 109 (8.6%) were diagnosed with asthma during the follow-up compared with 202 (4.0%) incident cases of asthma among those in the reference population. The crude relative risk of asthma was 2.1 (95% CI 1.6-2.6) among those with farmer's lung compared with the reference population. The age- and occupation-adjusted relative risk of asthma among patients with farmer's lung was 2.5 (1.8-3.5) in men and 1.4 (1.0-1.9) in women. The rate of asthma was especially high during the first 2 years after notification of farmer's lung. CONCLUSIONS: Patients with farmer's lung have an increased risk of developing asthma in comparison to farm workers in general. Most of the cases of asthma occur relatively shortly after the diagnosis of farmer's lung, which should be taken into account in medical follow-up of patients with farmer's lung. PMID- 12373321 TI - Health consequences of an intravenous injection of metallic mercury. AB - BACKGROUND: Mercury poisoning presents a variety of clinical pictures depending on chemical structure, the route of exposure, amount absorbed and individual factors. Thus, an injection of metallic mercury can be considered relatively harmless in contrast to inhalation of mercury vapor. Injection of elemental mercury is rare, and a total of only 78 cases have been reported in the literature over the period 1923-2000. CASE REPORT: We report a suicide attempt by intravenous injection of approximately 8 g metallic mercury. By X-ray examination widespread multiple mercury shadows were visible in the whole lung and also in the subcutaneous region of the cubital fossa, the small pelvis and the right hypogastrium. Mercury excretion after treatment with 2,3-dimercaptopropane-1 sulfonate (DMPS) was significantly higher than in occupationally exposed workers. CLINICAL SYMPTOMS: The patient showed symptoms typical of acute mercury intoxication, including gastroenteritis, ulceromembranous colitis and stomatitis mercuralis. No biochemical abnormalities in hepatic or renal function occurred, despite the persistence of metallic densities in the body. The patient's lung function was normal. The patient transitionally developed erethismus and tremor mercuralis. After 1 month of DMPS treatment, the mercury levels in blood were still high and the tremor was persistent. Three years after the suicide attempt the surgical removal of residual mercury in the left fossa cubitalis was performed. The extirpation of residual mercury was successful in cutting the mercury levels to almost half. After the operation the patient showed no symptoms of chronic mercury intoxication. CONCLUSIONS: Since only 1 mg of mercury per day could be removed with DMPS treatment, it can be calculated, that it would take about 8,000 daily treatments to remove a total of 8 g solely by DMPS. Although DMPS itself does not dissolve the metallic deposits, it may considerably reduce the blood level of mercury and may therefore mitigate clinical symptoms, albeit transitorily. We therefore recommend that in cases of symptomatic metallic mercury injections, where the mercury cannot be removed by surgery, the patient's condition should be managed by repeated long-term DMPS treatment in order to control blood mercury levels. PMID- 12373323 TI - Biomechanical strains on the hand-wrist system during grapevine pruning. AB - OBJECTIVES: In order to understand the high prevalence of musculoskeletal disorders of the hand among vineyard workers, we conducted a study to evaluate biomechanical strains on the hand-wrist system during grapevine pruning. METHODS: Surface electromyography (sEMG) activity of the right flexor digitorum muscle and wrist posture were analysed in six healthy vineyard workers using the same hand powered pruning shears during grapevine pruning. RESULTS: The biomechanical strains on the hand-wrist system were high during grapevine pruning. Mean sEMG activity during pruning was high [23.5% (standard error of the mean (SEM): 0.4) in the maximal voluntary handgrip contraction (MVC)], as was the mean cutting frequency per minute (38; range=24-48). Approximately 14% of cuts were performed with the wrist in extreme flexion/extension (F/E) (>60% of the maximum range). Numerous cuts required moderate (20%-40% of the maximum range) or extreme (>50% of the maximum range) ulnar deviation (17% and 12% of cuts, respectively). Approximately 18% of cuts required both high muscular activity (sEMG >15% MVC) and extreme ulnar/radial (U/R) deviation of the wrist (>50% of the maximum range). CONCLUSION: Pruning imposes high biomechanical strains on the hand-wrist system in view of the repetitiveness of the task. The magnitude of physical exposure during pruning explains the high prevalence of hand disorders among vineyard workers. The use of ergonomic pruning shears is advised to lower force exertion and to reduce the frequency of awkward wrist postures during pruning. PMID- 12373324 TI - Peteosthor - a medical disaster due to Radium-224A personal recollection. AB - Up to the end of World War II, there was no effective therapy against bone tuberculosis, and even today there is no treatment for ankylosing spondylitis. However, in the 1940s up to about 1956, radiotherapy with "Peteosthor" - a drug containing Thorium X ((224)Ra) as an effective compound - was introduced in Germany as a presumed cure and it maintained a central place in the treatment of these diseases. In 1948, I was entrusted to assess the new treatment. Animal studies and the clinical evaluation of the patients made me soon realise a number of severe adverse health effects which induced me to pronounce and subsequently repeat warnings against the intravenous administration of high doses of (224)Ra, especially because it was then administered predominantly to children and juveniles. As a consequence, this type of treatment was finally abandoned in 1956. But there remained a need to observe and document the resulting late health effects. Already in 1967, our initial follow-up study provided data for about 800 patients with exact information on the (224)Ra dose levels administered, administration schemes, and the resulting detrimental effects. A large number of bone sarcomas was the most severe consequence, but even today there is a broad spectrum of other grave health effects. A summary of the major scientific insights which have been achieved in the course of the still on-going epidemiological studies is part of this report. PMID- 12373325 TI - Therapy of ankylosing spondylitis with 224Ra-radium chloride: dosimetry and risk considerations. AB - Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease which reduces the quality of life and leads to disability in approximately one-third of the patients. The spectrum of therapeutic modalities is limited. The renaissance of the use of (224)Ra-radium chloride for AS treatment, however, gives rise to concern which should result in the reconsideration of (224)Ra dosimetry and in the discussion of the risks associated with this treatment. The present study introduces new dosimetric calculations for alpha and beta/gamma rays performed according to the model proposed by the International Commission on Radiological Protection (ICRP). After a treatment schedule of 10 intravenous injections, each with 1 MBq of (224)Ra, the absorbed doses were calculated to be highest on the bone surface of the patient (4.4 Gy) with a resulting effective dose of 2.5 Sv. PMID- 12373326 TI - Studies of strontium biokinetics in humans. Part 1: optimisation of intrinsic labelling of foodstuffs with stable isotopes of strontium. AB - The radioactive isotopes of strontium, mainly (90)Sr, which are common fission products, may significantly contribute to the internal exposure of the population in case of their accidental release into the environment and transfer to the food chain. For (90)Sr, the internal radiation dose is significantly dependent on the fractional absorption of the ingested activity (f(1)-value). Human data on the absorption of dietary strontium and of soluble forms of the element give values ranging from about 0.15 to 0.45 (up to 1.0) for adults. The International Commission on Radiological Protection (ICRP) has adopted f(1)-values of 0.6 for children of less than 1 year of age, 0.4 for children between 1 and 15 years and 0.3 for adolescents above 15 years of age. This study was aimed at investigating how far these values correspond to the actual uptake of strontium from contaminated foodstuffs. A methodology is presented that has been developed for preparing foodstuffs intrinsically labelled with stable isotopes and that will be used in tracer kinetic investigations. The results show that cress and salad can be adequately labelled, i.e. a strontium concentration of 1.36+/-0.47 g per kg of cress (wet weight) and of 0.29+/-0.04 g per kg of salad (wet weight) may be obtained within 15 days and 24 days, respectively. For the biokinetic investigations on humans, applying stable isotopes of Sr as tracers, about 0.1-1 mg strontium is required per volunteer, i.e. a few grams of the edible parts of the labelled material are sufficient. PMID- 12373327 TI - External exposure of the population living in areas of Russia contaminated due to the Chernobyl accident. AB - An updated version of external dose modeling is presented with reference to the population in Russian areas contaminated due to the Chernobyl accident. An earlier version has been modified by applying a study time interval with a starting point immediately after radionuclide deposition (rather than 4 years after the accident as applied earlier) and by introducing an estimate of individual dose distributions. New input data to the model are the nuclide specific composition of the deposit, additional data about migration of caesium in soil, time dependence of location factors and uncertainty distributions of all input parameters. Model results (i.e. effective dose-rates and accumulated effective doses) from external exposure for the rural and urban populations in contaminated areas of Russia during 100 years after the accident are presented. Radionuclide contributions to the dose during various time intervals after the accident have been estimated. The model has been validated by measurements of absorbed dose-rate in air during the first 30 days after the accident and by TLD measurements of individual external doses among inhabitants of contaminated rural settlements in the year 1993. Both the measurements and model show that the geometric mean of individual external doses is about 10% lower than the arithmetic mean and the upper bound of the 95% confidence range is larger by a factor of about 2. PMID- 12373328 TI - Thyroid cancer incidence among liquidators of the Chernobyl accident. Absence of dependence of radiation risks on external radiation dose. AB - The increase of thyroid cancer incidence rate among children living in the Chernobyl contaminated territories of Belarus, Russia and Ukraine has widely been accepted. Our current work deals with thyroid cancer incidence in the cohort of liquidators (99024 persons) living in 6 regions of Russia: North-West, Volgo Vyatsky, Central-Chernozemny, Povolzhsky, North-Caucasus and Urals. In the period 1986-1998, a total of 58 thyroid cancer cases were detected in this cohort. We found a statistically significant increase of the thyroid cancer incidence rate in liquidators as compared to the baseline (male population of Russia) level (SIR=4.33, 95% CI: 3.29; 5.60). It was demonstrated that there is no dependence of incidence rates due to external radiation exposure (ERR/Gy=-2.23, 95% CI: 4.67; 0.22). PMID- 12373329 TI - The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation 1-10 Gy. AB - Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence. Cell survival curves of irradiated CHO-K1 cells showed that treatment with nocodazole increases radiosensitivity as indicated by a decrease of the mean inactivation dose (D) from 4.446 to 4.376. Nocodazole treatment increased the initial radiation-induced DNA damage detected by the FADU assay from 7% to 13%. In repair defective xrs1 cells, the same conditions increased the radiosensitivity from 1.209 to 0.7836 and the initial DNA damage from 43% to 57%. Alterations to chromatin structure by hypertonic medium increased radiosensitivity in CHO-K1 cells from of 4.446 to 3.092 and the initial DNA damage from 7% to 15%. In xrs1 cells these conditions caused radiosensitivity to decrease from 1.209 to 1.609 and the initial DNA damage to decrease from 43% to 36%. Disruption of chromatin structure by hypertonic treatment was found to be time-dependent. A threefold increase of exposure time to hypertonic medium from 40 to 120 min increased the initial DNA damage in CHO-K1 cells from 7% to 18% but decreased initial DNA damage in xrs1 cells from 43% to 21%. Perturbation of chromatin structure with hypertonic treatment has been shown to increase the radiosensitivity and the initial DNA damage in repair-competent CHO-K1 cells and decrease the radiosensitivity and DNA damage in repair-defective xrs1 cells. Hypertonic treatment thus abolishes differences in chromatin structure between cell lines and differences in initial DNA damage. Radiosensitivity and initial DNA damage are correlated ( r(2)=0.92; p=0.0026) and this correlation also holds when chromatin compaction is altered. The experiments demonstrate that initial DNA damage and chromatin structure are major determinants of radiosensitivity. PMID- 12373330 TI - Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons. AB - The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons. PMID- 12373332 TI - Accounting for the depth distribution of 137Cs in on-line mobile gamma spectrometry through primary and forward-scattered photons. AB - Stationary and mobile field gamma spectrometry is a useful tool for rapid estimation of environmental radioactivity inventories on and in the ground. A weak point however, is that the depth distribution of the activity in the ground must be known in order to calculate the true activity per unit area or unit mass from an observed photon fluence rate. A promising method for converting incoming spectral data into both true activity content and depth distribution in real time is the peak-to-valley method, which is based on an analysis of the ratio between count rates from primary and forward-scattered photons. In this study the peak-to valley method was adapted to car-borne mobile gamma spectrometry, where the depth distribution of (137)Cs is fitted to a Lorenz function. Results from field experiments with a large HPGe detector, utilising point sources at different depths, are presented. It was found that the method can be useful for mobile measurements with a measuring time of 5-10 min for activity concentrations of about 100 kBq.m(-2) or higher, resulting in an uncertainty in the estimate of the true activity of about 50%. PMID- 12373331 TI - Influence of a low background radiation environment on biochemical and biological responses in V79 cells. AB - We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanita (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive. PMID- 12373335 TI - Apoptosis of human dermal endothelial cells as a potential side effect following therapeutic administration of UVA1 irradiation: preliminary results. AB - Apoptosis is a highly selective form of cell suicide with characteristic morphological and biochemical features. UVA1 phototherapy has been introduced into the treatment of many T cell-derived skin diseases. The aim of our pilot study was to assess apoptosis of endothelial cells in relation to time after irradiation with medium-dose UVA1 using four different staining techniques. With in situ nick end labelling (ISEL) and Hoechst 33342 staining we investigated DNA degradation during apoptosis and used M30 CytoDEATH to selectively stain the cytoplasm of apoptotic cells. Additionally, the expression of the tumour suppressor gene p53 was determined. ISEL and Hoechst 33342 revealed only a few positive endothelial cells 3 h after UVA1 irradiation. After 6 h almost all vessels were positively stained. By 12 h after irradiation this peak concentration had lowered again. The first p53-positive endothelial cells were seen 6 h after UVA1 irradiation and reached a maximum at 12 h after irradiation. Fibroblasts of the lower dermis were positively stained after 6 and 12 h. M30 positive endothelial cells were found from 3 to 12 hours after irradiation. ISEL and Hoechst 33342 staining clearly revealed UVA1-induced apoptotic cell elimination predominantly restricted to endothelial cells as a possible side effect of UVA1 irradiation. The induction of apoptosis was specifically verified by M30 immunostaining of early caspase cleavage. Whereas the p53-positive endothelial cells underwent programmed cell death as demonstrated by M30, ISEL and Hoechst 33342, some fibroblasts seemed to accumulate the p53 antibody, but this did not induce apoptotic cascades. PMID- 12373334 TI - Comparison of the distribution and numbers of antigen-presenting cells among T lymphocyte-mediated dermatoses: CD1a+, factor XIIIa+, and CD68+ cells in eczematous dermatitis, psoriasis, lichen planus and graft-versus-host disease. AB - Antigen-presenting cells (APCs) participate in the initiation of the inflammatory process in various immune-mediated dermatoses through the activation of antigen specific T lymphocytes. The skin contains several different subsets of APCs. To investigate the role of these APCs in T-cell immune-mediated inflammation, we examined the distribution and numbers of epidermal and dermal CD1a(+) dendritic cells (DCs), factor XIIIa(+) dermal DCs, and CD68(+) macrophages in five T-cell mediated inflammatory skin diseases. Immunohistochemistry of CD1a, factor XIIIa, and CD68 was performed using paraffin-embedded tissue obtained from a total of 51 patients with eczematous dermatitis (histologically spongiotic dermatitis), psoriasis, lichen planus, acute graft-versus-host disease (GVHD), and chronic GVHD. The numbers of positive cells for each staining were compared with those in site-matched normal skin control specimens from aged-matched subjects. In spongiotic dermatitis and lichen planus, the numbers of epidermal and dermal CD1a(+) cells and factor XIIIa(+) cells were significantly greater than in normal control skin, while in psoriasis only factor XIIIa(+) cells were significantly increased in number. Acute and chronic GVHD showed a reduced number of dermal CD1a(+) cells. Interestingly, factor XIIIa(+) cells were decreased in acute GVHD while they were increased in chronic GVHD. There was a significant reduction in epidermal CD1a(+) cells in acute GVHD, but not in chronic GVHD. The differences in the numbers of APCs in lesional skin appeared to reflect differences in the pathophysiology of these inflammatory skin diseases. PMID- 12373333 TI - Applicability of radiosurgery with heavy ion beams to inactivate specific organs in living organisms. AB - It was the aim of the study to test the applicability of radiosurgery in inactivating a specific organ through local irradiation with heavy ion beams. Silkworms were exposed to whole-body or local irradiation with carbon ion beams ((12)C(5+), 18.3 MeV/u, range=1.1 mm). After irradiation at the wandering stage, no significant differences were observed regarding either survival or cocoon quality between locally irradiated larvae and controls. Only localized effects were seen, such as the deletion of wings and functional disorders of the reproduction primordium, depending on both irradiation dose and site. This observation was not true for whole-body irradiated larvae. After local irradiation of the hemopoietic organs at the 4th instar premolting stage, the hemocyte densities were clearly reduced and the hemopoietic organ capacity was disrupted. The change in hemocyte densities was accompanied by changes of hemolymph components. These results show that radiosurgery utilizing heavy ion beams can destroy a specific organ or tissue in a living organism. PMID- 12373336 TI - U3 snoRNP associates with fibrillarin a component of the scleroderma clumpy nucleolar domain. AB - Serum from patients with scleroderma recognizes the clumpy autoantigen. The present studies addressed the issue as to whether the clumpy nucleolar autoantigen recognized by scleroderma serum is fibrillarin-U3 snoRNP. Clones encoding for clumpy autoantigen were immunodetected from a lambdagt11 HeLa cell random-primed library with the serum from a patient with diffuse scleroderma and autoautoantibodies against clumpy autoantigen. Sequences from the recombinant phages were amplified by PCR and subcloned into a pCRII vector. The DNA was sequenced by a dideoxy termination reaction. Ten lambdagt11 clumpy clones were detected by immunoscreening. One containing the glycine-rich and RNP2 fibrillarin domains was expressed in lysogenic bacteria. The recombinant proteins were used to elicit antibodies in rabbits, and these exhibited clumpy nucleolar reactivity. The recombinant fibrillarin tested by ELISA was recognized by the clumpy scleroderma serum from the majority of patients. In situ hybridization assays showed that the fibrillarin tagged by the elicited antibodies was colocalized with U3 snoRNP in the nucleolus in a clumpy manner and coprecipitated the U3 snoRNP. In conclusion, the fibrillarin-U3 snoRNP complex is the major component of the clumpy subcellular domain. Therefore these molecules constitute an important target of scleroderma autoantibodies. PMID- 12373337 TI - Ultrastructural changes in mice actively producing antibodies to desmoglein 3 parallel those in patients with pemphigus vulgaris. AB - Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by autoantibodies against the desmosomal cadherins, desmogleins 1 and 3 (Dsg1, Dsg3) of which Dsg3 plays a major pathogenic role. We have previously generated a novel active disease mouse model for PV, which was produced by the transfer of splenocytes from Dsg3(-/-) mice, immunized with recombinant mouse Dsg3, into Rag2(-/-)-immunodeficient mice that express Dsg3. In this study, we undertook a further analysis of these PV model mice using electron microscopy (EM). We compared the ultrastructure of the epithelia of PV model mice with that of Dsg3( /-) mice to highlight the mechanisms of blister formation in PV. These PV model mice showed the ultrastructural phenotype of PV, which is characterized by suprabasal acantholysis, rows of tombstone basal keratinocytes and half desmosomes. Additionally, patchy hair loss was observed in PV model mice as in Dsg3(-/-) mice, and the ultrastructure of the telogen hair follicles was indistinguishable between PV model mice and Dsg3(-/-) mice. These results demonstrate that anti-Dsg3 autoantibodies interfere with the cell-cell adhesion of keratinocytes in PV model mice. In conclusion, our model mice closely represent the disease phenotype of PV at the ultrastructural level and can therefore be utilized not only as a clinical disease model but also to study the molecular mechanisms involved in blister formation in PV. PMID- 12373338 TI - Transient production of stem cell factor in dermal cells but increasing expression of Kit receptor in mast cells during normal wound healing. AB - Mast cells are involved in inflammatory skin disorders and wound healing processes, but the mechanism behind mast cell activation is obscure. In this study, we stained the stem cell factor (SCF) and the Kit receptor in tryptase positive mast cells, since these molecules are essential for mast cell survival, growth, migration and activation. For this purpose, biopsies were taken from the edge of normally healing wounds of 12 patients undergoing skin transplantation on days 0, 1, 3, 7 and 14, and from chronic leg ulcers and psoriatic skin for comparison. In healing wounds, SCF-positive cells rapidly increased in number in the dermis peaking on day 1, but declined thereafter to their baseline values. The percentage of Kit-positive mast cells increased slowly but steadily reaching a maximum (73+/-22%, P=0.02) on day 14. In chronic ulcers, most of the mast cells were Kit-positive both in the wound bed and in the perilesional skin (87+/-9% and 86+/-13%, respectively). The number of SCF-positive cells was higher in the wound bed than in the dermis of perilesional skin. In the psoriatic skin of ten patients, lesional specimens showed significantly higher numbers of SCF-positive dermal cells as well as a higher percentage (88+/-12% vs 46+/-26%, P=0.004) of Kit-positive mast cells than nonlesional skin. In conclusion, our findings show that the expression of SCF increases rapidly in the early stages of wound healing but declines thereafter, whereas the expression of Kit in mast cells is induced slowly in healing wounds. In chronic wounds as well as in psoriatic lesions, both SCF and Kit are intensely expressed. Thus, it seems possible that SCF and Kit receptor interact, and this could lead to persistent mast cell activation and growth in chronic wounds and psoriasis, whereas only temporary mast cell activation is apparently needed in healing wounds. PMID- 12373339 TI - Immunolocalization of fibroblast growth factor receptors in normal and wounded human skin. AB - Fibroblast growth factors (FGFs) have been shown to play diverse roles in various tissues. To define their sites of action in normal human skin and during wound healing, we determined the protein expression of the four known fibroblast growth factor receptors (FGFRs) in normal and wounded human skin by immunohistochemistry. Four receptors (FGFR-1 to FGFR-4) showed distinct patterns of expression in normal skin. Expression of FGFR-1 was widespread in the epidermis, appendages, arrector pili muscles, blood vessels, and dermal fibroblasts. Intense expression of FGFR-2 and FGFR-4 was seen in the arrector pili muscles and smooth muscle cells of vessels. In the epidermis, the basal layer showed immunoreactivity for FGFR-2, whereas the suprabasal layers and the inner layers of hair follicles showed strong immunoreactivity for FGFR-3. In wounded skin, there was strong expression of FGFR-1 and FGFR-3, and moderate expression of FGFR-2 and FGFR-4 in the basal layer in newly forming epidermis. In granulation tissues, neocapillaries expressed all four FGFRs, fibroblasts/myofibroblasts expressed FGFR-1 and FGFR-3, and mononuclear inflammatory cells expressed FGFR-1 and FGFR-3. Our results suggest that the differences in the spatial patterns of FGFR expression in normal skin may generate functional diversity in response to FGFs and that in wounded skin, FGFs may function in wound healing via the induced FGFRs. PMID- 12373340 TI - Exact sampling formulas for multi-type Galton-Watson processes. AB - Exact formulas for the mean and variance of the proportion of different types in a fixed generation of a multi-type Galton-Watson process are derived. The formulas are given in terms of iterates of the probability generating function of the offspring distribution. It is also shown that the sequence of types backwards from a randomly sampled particle in a fixed generation is a non-homogeneous Markov chain where the transition probabilities can be given explicitly, again in terms of probability generating functions. Two biological applications are considered: mutations in mitochondrial DNA and the polymerase chain reaction. PMID- 12373341 TI - Monotone travelling fronts in an age-structured reaction-diffusion model of a single species. AB - We consider a partially coupled diffusive population model in which the state variables represent the densities of the immature and mature population of a single species. The equation for the mature population can be considered on its own, and is a delay differential equation with a delay-dependent coefficient. For the case when the immatures are immobile, we prove that travelling wavefront solutions exist connecting the zero solution of the equation for the matures with the delay-dependent positive equilibrium state. As a perturbation of this case we then consider the case of low immature diffusivity showing that the travelling front solutions continue to persist. Our findings are contrasted with recent studies of the delayed Fisher equation. Travelling fronts of the latter are known to lose monotonicity for sufficiently large delays. In contrast, travelling fronts of our equation appear to remain monotone for all values of the delay. PMID- 12373342 TI - A discrete, size-structured model of phytoplankton growth in the chemostat: introduction of inhomogeneous cell division size. AB - We introduce inhomogeneous, substrate dependent cell division in a time discrete, nonlinear matrix model of size-structured population growth in the chemostat, first introduced by Gage et al. [8] and later analysed by Smith [13]. We show that mass conservation is verified, and conclude that our system admits one non zero globally stable equilibrium, which we express explicitly. Then we run numerical simulations of the system, and compare the predictions of the model to data related to phytoplankton growth, whose obtention we discuss. We end with the identification of several parameters of the system. PMID- 12373343 TI - Investigating a simple model of cutaneous wound healing angiogenesis. AB - A simple model of wound healing angiogenesis is presented, and investigated using numerical and asymptotic techniques. The model captures many key qualitative features of the wound healing angiogenic response, such as the propagation of a structural unit into the wound centre. A detailed perturbative study is pursued, and is shown to capture all features of the model. This enables one to show that the level of the angiogenic response predicted by the model is governed to a good approximation by a small number of parameter groupings. Further investigation leads to predictions concerning how one should select between potential optimal means of stimulating cell proliferation in order to increase the level of the angiogenic response. PMID- 12373344 TI - Analysis of linear determinacy for spread in cooperative models. AB - The discrete-time recursion system u_[n+1]=Q[u_n] with u_n(x) a vector of population distributions of species and Q an operator which models the growth, interaction, and migration of the species is considered. Previously known results are extended so that one can treat the local invasion of an equilibrium of cooperating species by a new species or mutant. It is found that, in general, the resulting change in the equilibrium density of each species spreads at its own asymptotic speed, with the speed of the invader the slowest of the speeds. Conditions on Q are given which insure that all species spread at the same asymptotic speed, and that this speed agrees with the more easily calculated speed of a linearized problem for the invader alone. If this is true we say that the recursion has a single speed and is linearly determinate. The conditions are such that they can be verified for a class of reaction-diffusion models. PMID- 12373345 TI - Spreading speed and linear determinacy for two-species competition models. AB - One crucial measure of a species' invasiveness is the rate at which it spreads into a competitor's environment. A heuristic spread rate formula for a spatially explicit, two-species competition model relies on 'linear determinacy' which equates spread rate in the full nonlinear model with spread rate in the system linearized about the leading edge of the invasion. However, linear determinacy is not always valid for two-species competition; it has been shown numerically that the formula only works for certain values of model parameters when the model is diffusive Lotka-Volterra competition [2]. This paper derives a set of sufficient conditions for linear determinacy in spatially explicit two-species competition models. These conditions can be interpreted as requiring sufficiently large dispersal of the invader relative to dispersal of the out-competed resident and sufficiently weak interactions between the resident and the invader. When these conditions are not satisfied, spread rate may exceed linearly determined predictions. The mathematical methods rely on the application of results established in a companion paper [11]. PMID- 12373346 TI - Modeling alignment and movement of animals and cells. AB - Schools of fish and flocks of birds are examples for groups of individuals moving in a highly organized way. Individuals adapt their orientation and speed to that of their (nearest) neighbors. Adaptation of orientation can also be found on the cellular and subcellular level and is called alignment. A model for alignment and movement is derived on the basis of reaction transport equations in one space dimension. Existence of solutions is shown and long time behavior of the system is described. The effect of schooling on the risk of predation is investigated. Then the model is generalized to two space dimensions and compared to other models for alignment which do not incorporate individual movement in space. PMID- 12373347 TI - Determination of hammerhead ribozyme kinetic constants at high molar ratio ribozyme-substrate. AB - Hammerhead ribozymes provide valuable tools in the field of gene therapy due to their cleavage specificity and the broad range of RNA targets. A major prerequisite for the selection of suitable ribozymes for in vivo application is represented by in vitro determination of ribozyme cleavage kinetic constants. From the experimental cleavage data, kinetic constants are usually calculated under the assumption of rapid conversion of the substrate into the ribozyme substrate complex. However, this condition is often not satisfied for ribozymes carrying additional RNA stretches, due to cloning strategies or necessary for ribozyme expression in the cell. To overcome this problem, we propose a mathematical model which is able to calculate ribozyme kinetic constants in the case of non-rapid conversion of substrate into ribozyme-substrate complex. In addition, our system gives the opportunity to evaluate the nature of the S conversion into ES through the determination of a model parameter. The validity of the proposed model is restricted to the hypothesis of a ribozyme excess over the substrate at the beginning of the cleavage reaction and to the absence of any mass exchange with the external environment. PMID- 12373348 TI - Evaluating the prognosis of patients with myelodysplastic syndromes. AB - One of the hallmarks of myelodysplastic syndromes (MDS) is their prognostic heterogeneity which complicates decision making regarding treatment for individual patients. The French-American-British (FAB) classification provides significant prognostic information, but carries the disadvantage of arbitrary demarcation of subgroups and overemphasis of morphological findings. In addition, there is considerable variation in survival and risk of acute myeloblastic leukemia (AML) development even within defined FAB subgroups, particularly in patients with refractory anemia with ring sideroblasts (RARS) and chronic myelomonocytic leukemia (CMML). Over the last 2 decades, several research groups have tried to identify additional clinical, hematological, and cell biological parameters in order to more accurately predict the natural course of MDS. These investigations have clarified that the number and extent of peripheral blood cytopenias, the bone marrow blast count, and the cytogenetic pattern are the most powerful prognostic indicators in MDS. Recent efforts have been directed at constructing prognostic scoring systems. These scoring systems try to enhance the predictive power by combining several features of the disease, which have proved their independent prognostic weight on multivariate analysis. The International MDS Risk Analysis Workshop substantially advanced the prognostic categorization of MDS patients by proposing a new scoring system (International Prognosis Scoring System, IPSS) that can be successfully applied to risk assessment of newly diagnosed patients and will likely prove useful for the design and analysis of therapeutic trials in MDS. PMID- 12373349 TI - Myeloid antigen expression provides favorable outcome in patients with adult acute lymphoblastic leukemia: a single-center study. AB - Between July 1992 and July 2001, 81 patients with de novo adult acute lymphoblastic leukemia (ALL) treated according to the German Multicenter Study Group for Adult ALL (GMALL) 01/81 protocol were evaluated in order to analyze the effect of aberrant myeloid antigen expression on prognosis. We observed myeloid antigen aberrant expression in 21 of the adult ALL cases. We did not observe any effect of aberrant myeloid antigen expression on the time to achieve remission, relapse rate, and death rate. After 5 years of follow-up, cumulative disease-free survival of myeloid antigen (My) (+) and My (-) adult ALL patients was 67% and 43%, respectively. These data were not found to be statistically significant (P=0.29), but we did find a statistically significant difference in overall survivals between these two groups (85% vs 50%) (P=0.05). Twenty-nine patients died and the remaining 52 patients were followed for a median of 31 months. We could not find any special effect of the known prognostic factors on prediction of relapse in multivariate analysis. However, myeloid antigen expression was the most significant factor, which affected long-term survival in our patients (P=0.01). These data indicate that myeloid antigen expression is useful for predicting a favorable outcome of adult patients with ALL. PMID- 12373350 TI - Anthracycline-related toxicity requiring cardiac transplantation in long-term disease-free survivors with acute promyelocytic leukemia. AB - We describe three cases of acute promyelocytic leukemia (APL) with long-term disease-free survival who developed congestive heart failure (CHF) requiring cardiac transplantation. All three patients presented late-onset cardiotoxicity. Cardiac failure occurred progressively after 31-month, 32-month, and 14-month intervals, respectively, following completion of first anthracycline therapy. Cumulative anthracycline doses were 585 mg of daunorubicin and 64 mg of mitoxantrone in case 1, 1779 mg of daunorubicin in case 2, and 825 mg of daunorubicin in case 3. The questions relating to the pathogenesis of cardiac failure are discussed. We also discuss the prophylactic measures required for such treatment-related side effects. PMID- 12373351 TI - Evaluation of apoptosis induced in vitro by cladribine (2-CdA) combined with anthracyclines in lymphocytes from patients with B-cell chronic lymphocytic leukemia. AB - The aim of the study was to evaluate the effect of three anthracyclines [doxorubicin (DOX), mitoxantrone (MIT), and idarubicin (IDA)] on the rate of apoptosis triggered by 2-chlorodeoxyadenosine (2-CdA) in peripheral blood mononuclear cells isolated from 52 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL). The cells were cultured up to 48 h in the presence of drugs alone and in the following combinations: 2-CdA+DOX, 2-CdA+MIT, and 2 CdA+IDA. Apoptosis was assessed after 24 h and 48 h of incubation using annexin V/propidium iodide assay by flow cytometry. The apoptotic index (AI) was defined as a percentage of annexin V-positive B-CLL cells. Additionally, in some patients other hallmarks of apoptosis (activation of caspases, DNA fragmentation) were assessed in parallel for confirmation of apoptotic mode of induced cell death. All of the cytostatics induced apoptosis of B-CLL cells at a rate significantly higher than the index of spontaneous apoptosis occurring during 24 h and 48 h of cell culture. 2-CdA in combination with DOX significantly increased the percentage of annexin V-positive cells, particularly after 48 h of incubation, as compared with DOX used in monotherapy (median AI for 2-CdA+DOX=37.9%, median AI for DOX =13.8%, P=0.0011, and median AI for 2-CdA=22.1%, P=0.013). Combination of 2-CdA with MIT induced a similar effect, also more distinct after 48 h (median AI for 2-CdA+MIT=41.05%, median AI for MIT=16.3%, p=0.0012, and median AI for 2 CdA=22.1%, p=0.017). For both combinations median AI were similar to the sum of median AI for each drug when used alone. IDA in a concentration ten times lower (0.1 micro g/ml) than used before in acute leukemia cells produced high cytotoxic effects, masking the additive effect of combination with 2-CdA. Only at a dose of 25 ng/ml of IDA, significant differences in AI after 24 h and 48 h were detected between samples treated with 2-CdA+IDA (median 27.5% and 65.0%, respectively) and those incubated with IDA alone (median 10.5% and 33.4%; P=0.0004 and 0.0274, respectively). Similarly, there were significant differences between AI of cells treated with 2-CdA+IDA and 2-CdA alone (median 9.5% at 24 h and 23.5% at 48 h; P=0.0013 and 0.0207, respectively). In conclusion; these data indicate an additive cytotoxic effect on B-CLL cells of DOX, MIT, and IDA applied in vitro with 2-CdA; all of them induced apoptosis with similar efficacy. We suggest that further preclinical and clinical studies concerning combined use of 2-CdA with anthracyclines are desirable. High sensitivity of B-CLL cells to IDA suggests the possibility of lowering its dose in patients, especially when combined with 2 CdA. PMID- 12373352 TI - Hodgkin's disease of the nasopharynx: diagnostic and therapeutic approach with a review of the literature. AB - The lymphoid tissues of Waldeyer's ring, including the nasopharynx, are rarely involved in Hodgkin's disease (HD). Between March 1977 and July 2001, about 2150 patients affected by HD were observed in our institute; 7 of them (0.32%), all male patients, had HD of the nasopharynx. They had no symptoms and blood tests were normal. All patients were treated with chemotherapy and/or radiotherapy and achieved complete remission. At a median follow-up of 72 months, they are alive and in continuous complete remission. We conclude that Hodgkin's disease of the nasopharynx is a rare and predominantly male disease with a particularly favorable prognosis. Bone marrow biopsy could be avoided. We believe that two to four cycles of a chemotherapeutic regimen and involved field radiotherapy at an intermediate-high dosage (25-30 Gy) could be the first line treatment for these patients. PMID- 12373353 TI - Vertebral fractures in multiple myeloma: first results of assessment of fracture risk using dynamic contrast-enhanced magnetic resonance imaging. AB - The objective of this study was to evaluate dynamic contrast-enhanced magnetic resonance imaging (d-MRI) as a prognostic indicator of lumbar vertebral fractures in patients with multiple myeloma. d-MRI of the lumbar spine was performed in ten patients with multiple myeloma. A fast gradient echo sequence (turbo fast low angle shot, two-dimensional) was used, together with controlled bolus injection of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). The maximum increase in signal intensity [amplitude (A),arbitrary units (a.u.)] was assessed for each lumbar vertebra. About half a year later (median: 6.2 months) magnetic resonance imaging was repeated to detect new fractures. Amplitudes of vertebrae which fractured after the initial d-MRI were compared with amplitudes of vertebrae which did not fracture during follow-up. Six of ten patients (7 of 50 lumbar vertebrae) showed new fractures. Five patients fractured one vertebra each, whereas one patient had several vertebrae involved. The initial d-MRI showed significantly higher amplitudes (p<0.0001) in those vertebrae that subsequently fractured (A: 33.1+/-8.1 vs 16.7+/-4.2). On retrospective analysis, a cutoff level of 25 a.u. discriminated without overlap between vertebrae that fractured during follow-up and those which did not. The maximum increase in signal intensity (the amplitude) on d-MRI appears to be a prognostic marker capable of predicting vertebral fractures of the lumbar spine in patients with multiple myeloma. d-MRI may therefore be helpful in identifying patients who might benefit from localized radiation therapy or surgical intervention. PMID- 12373354 TI - Flt3 ligand and thrombopoietin serum levels during peripheral blood stem cell mobilization with chemotherapy and recombinant human glycosylated granulocyte colony-stimulating factor (rhu-G-CSF, lenograstim) and after high-dose chemotherapy. AB - The purpose of this investigation was to study thrombopoietin (TPO) and Flt3 ligand (FL) serum levels in the course of peripheral blood stem cell (PBSC) mobilization and high-dose chemotherapy (HDC) and to correlate the values with stem cell yield and engraftment. Thirty-nine patients were included. PBSC were mobilized by chemotherapy followed by two body surface area-dependent doses of glycosylated recombinant human granulocyte colony-stimulating factor (rhu-G-CSF, lenograstim). PBSC could be harvested in 35 patients and 30 received a total of 62 courses of HDC (1-3 per patient). Fifty-six were analyzed and TPO and FL serum levels were measured at the start of PBSC mobilization, at the first PBSC collection, on the day of PBSC infusion, and until engraftment. Mean baseline TPO and FL serum levels were 173 pg/ml and 192 pg/ml and increased to 493 and 323 pg/ml at the start of PBSC collection. Maximum values were 2279 pg/ml TPO after HDC 1 and 2375 pg/ml after HDC 2, while the mean maximum serum levels for FL were 1181 and 1236 pg/ml after HDC 1 and 2 and PBSC transfusion, respectively. FL serum levels at the start of PBSC mobilization correlated with the total yield of CD34+ cells (17.61+/-18.8x10(6)/kg body weight, r=0.81), while TPO serum levels on days 11-13 after PBSC infusion were inversely correlated with the amount of transfused CD34+61+62+ cells (r=-0.88 and -0.79 for HDC 1 and 2). There was no strong correlation between TPO or FL serum levels and WBC and platelet engraftment. In conclusion, chemotherapy followed by glycosylated rhu-G-CSF induced elevated serum levels of TPO and to a lower degree of FL at the start of PBSC collection. The maximum increase was 13.7-fold for TPO and 6.4-fold for FL after PBSC infusion indicating endogenous release which should be considered if the clinical use of these cytokines is intended in this setting. PMID- 12373355 TI - Candida arthritis in a patient with chronic myelogenous leukemia (CML) in blastic transformation, unresponsive to fluconazole, but treated effectively with liposomal amphotericin B. AB - Candida arthritis is quite rare and might be caused either by direct intra articular inoculation of Candida or secondary to hematogeneous seeding of Candida in immunocompromised hosts. Until now less than 50 cases of Candida arthritis have been reported in the literature. We report a case of Candida arthritis, which occurred in a patient with chronic myelogenous leukemia (CML) in blastic transformation. Aggressive chemotherapy and broad-spectrum antibiotics for a prolonged period for febrile neutropenia had been given to the patient. Arthritis of the left knee appeared during the recovery phase of leukopenia. Despite treatment with fluconazole, no clinical or microbiological improvement was obtained. Thus, administration of liposomal amphotericin B was started and after 3 days there was improvement. We can conclude that fluconazole might not be sufficient in some Candida arthritis cases and liposomal amphotericin B might be a good alternative in these resistant cases. PMID- 12373356 TI - Splenic actinomycotic abscess in a patient with acute myeloid leukemia. AB - Actinomycosis is a gram-positive anaerobic bacterium. Actinomyces organisms are important constituents of the normal flora of mucous membranes and are considered opportunistic pathogens. The three major clinical presentations of actinomycosis include the cervicofacial, thoracic, and abdominopelvic regions. Actinomycosis infection in patients with febrile neutropenia is uncommon and actinomycosis splenic involvement in acute leukemia patients is very rare. We describe a man with acute myeloid leukemia and splenic actinomycotic abscess that developed after chemotherapy following prolonged neutropenia. PMID- 12373357 TI - Eosinophilic pneumonia after administration of fludarabine for the treatment of non-Hodgkin's lymphoma. AB - Fludarabine is a purine analogue which is effective in the treatment of patients with low-grade non-Hodgkin's lymphoma, including chronic lymphocytic leukemia. While pulmonary toxicity due to cytotoxic drugs is increasingly diagnosed, only few cases of interstitial pneumonitis have been described following fludarabine administration. Here we report the first case in the literature of an acute eosinophilic pneumonia associated with peripheral blood eosinophilia after the administration of fludarabine monotherapy for stage IV follicular lymphoma. PMID- 12373358 TI - Fournier's gangrene after unrelated cord blood stem cell transplantation. AB - A 16-year-old boy with refractory acute myelogenous leukemia developed Fournier's gangrene as an early complication after two-antigen HLA-mismatched unrelated cord blood stem cell transplantation. On day 25 after the transplantation, he noted abrupt onset of penile swelling with miction pain. The penile inflammation rapidly extended posteriorly to involve the scrotum and perianal tissues, inferiorly to involve the thighs, and superiorly up the lower abdominal region within the next 36 h, and he died from sepsis on day 27. Fournier's gangrene presenting as a genitoperineal necrotizing fasciitis should be considered as a potential complication in umbilical-cord blood recipients in the cytopenic post transplant phase. PMID- 12373359 TI - Subdural hematoma in two hematopoietic stem cell transplant patients with post dural puncture headache and initially normal CT brain scan. AB - Subdural hematoma (SDH) is a rare complication in patients after lumbar puncture. We report two patients receiving hematopoietic stem cell transplantation (HSCT) who developed post-dural puncture headache (PDPH) and SDH following intrathecal methotrexate (MTX). Both patients initially had normal computed tomography (CT) scan findings at the onset of headache. The diagnosis was established only when a repeat CT brain scan was performed for deteriorating neurological signs coinciding with improving platelet counts. These cases demonstrate the importance of continued vigilance for the early recognition of this salvageable entity. A normal initial CT finding and platelet count do not exclude the occurrence of SDH. A repeat CT scan, or even magnetic resonance imaging (MRI), are indicated if the clinical suspicion remains strong. PMID- 12373360 TI - Inverse correlation of plasma leptin and soluble transferrin receptor levels in beta-thalassemia patients. AB - The aim of the study was to investigate the association of leptin with hematological parameters in beta-thalassemia patients in Greece. We measured plasma levels of soluble transferrin receptor (sTfR) and leptin by enzyme-linked immunosorbent assay (ELISA) in 40 beta-thalassemia patients (21 transfusion dependent and 19 not transfused or sporadically transfused), in 20 beta thalassemia carriers, and in 30 healthy individuals (HI). The percentage of reticulocytes (RET) was measured by the NE 9500 Sysmex automated method. Body mass index (BMI) was calculated by dividing body weight (kg) by square height (m). Endocrine measurements including sex hormones were also determined. sTfR concentrations were significantly higher in both transfusion-dependent (females 10.5+/-2.9, males 9.1+/-3.1) and non-transfusion-dependent patients (females 15.8+/-5.4, males 19.8+/-13.7) as compared to carriers (females 3.1+/-2.5, males 3.8+/-1.8) and to HI (females 1.5+/-1.2, males 2.5+/-2.1). Leptin levels were lower both in female and in male transfusion-dependent patients (0.5+/-0.3 and 1.2+/-1, respectively) and in non-transfused males (1.9+/-2) compared to carriers (females 7.9+/-2.7, males 13.1+/-9.1) and HI (females 14.6+/-6, males 7.5+/-3). There was a negative correlation between leptin and sTfR levels in transfused patients (R=-0.61, p<0.05). A stronger negative correlation (R=-0.7, p=0.006) was found in hypogonadic men and women with beta-thalassemia. These findings enhance previous results indicating that leptin may play some role in hematopoiesis and could associate the pathophysiology of thalassemic patients with the triggering effect of leptin in reproductive ability. PMID- 12373361 TI - F blast production correlates strongly with upregulation of inducible nitric oxide synthase in myelodysplastic syndromes. PMID- 12373362 TI - Role of visual input in nonlinear postural control system. AB - Stabilometry signals involve irregular and unpredictable components. The purpose of the present study was to investigate these signals with a nonlinear technique to examine how the complexity of the postural control system breaks down under altered visual conditions. We evaluated the dynamical similarities of the postural control system when the eyes were open or closed, or when there was optokinetic stimulation (OKS). A similarity index was calculated by the cross correlation integral between the two dynamics: eyes open and eyes closed, or eyes open with OKS. Using this technique, dynamical changes were not observed between eyes-open and eyes-closed conditions. This result suggests that the nonvision condition does not produce any striking effect on the postural control system; instead, the eyes-open condition causes a decrease in the stochastic activity of the postural control system, which may originate mainly from the stiffness of the musculoskeletal systems. In contrast, the visual input of OKS affected the dynamics of the postural control system in nearly half of the subjects (group 2) despite showing no significant differences between the eyes-open condition and the other conditions for area as the conventional parameter. However, the other half of the subjects (group 1) did not experience any influence of OKS on their postural dynamics, despite showing significant differences between eyes-open and the other conditions for all traditional parameters. From the results for group 2, we hypothesize that OKS may induce the striking effect on dynamics properties of the multilink network system involving visual and vestibular cortex related to self-motion perception, which acts to decrease the stochastic activity in order to correct disturbed posture. PMID- 12373363 TI - What and where in human audition: selective deficits following focal hemispheric lesions. AB - A sound that we hear in a natural setting allows us to identify the sound source and localize it in space. The two aspects can be disrupted independently as shown in a study of 15 patients with focal right-hemispheric lesions. Four patients were normal in sound recognition but severely impaired in sound localization, whereas three other patients had difficulties in recognizing sounds but localized them well. The lesions involved the inferior parietal and frontal cortices, and the superior temporal gyrus in patients with selective sound localization deficit; and the temporal pole and anterior part of the fusiform, inferior and middle temporal gyri in patients with selective recognition deficit. These results suggest separate cortical processing pathways for auditory recognition and localization. PMID- 12373364 TI - Heaviness perception. I. Constant involvement of haptically perceived size in weight discrimination. AB - With visual input blocked, subjects in this study utilized fingertips only to investigate the involvement of haptically perceived size in heaviness perception among humans. The objects used for testing consisted of three sets - copper (CP), aluminum (AL), and plastic (PL) - of ten cubes of various weights (0.05-0.98 N). All of the cubes were covered with a smooth vinyl material to eliminate any extraneous input concerning the actual composition. Screens enclosed the working space to eliminate any possible visual cues. Each comparison was between a pair of cubes of the same material to eliminate the effect of density. Fifteen subjects ( M=19.2, SD=0.68 years) attempted to judge differences in heaviness between the first and second cube in each trial that had been handed to them by the experimenter and were grasped between the thumb and the index finger. A total of 340 trials with 70 combinations of weight composed of 160 ascending trials (heavier), 160 descending trials (lighter), and 20 identical weight trials were pseudo-randomly presented to each subject for each material. Combinations of difference in weight and the number of trials were identical for all materials so that haptic size was regarded as the single independent factor. Accuracy of the subjects' responses for identical weight differences that resulted from placing a pair of cubes of the same combination was compared among the three materials. It was observed that a material like CP that had a lesser size effect facilitated significantly more accurate discrimination of the identical weight differences than PL with its greater size effect. This suggests that small changes in haptic size by the fingertips have a direct influence on heaviness perception when comparing objects of equal density. This finding, therefore, can be considered analogous to the size-weight illusion when comparing objects of unequal density. The findings of this study also suggest the constant involvement of haptic size in heaviness perception by humans along with the existence of a processing mechanism that integrates the factors of weight and haptic size in which heaviness increases either as weight increases or as size decreases, and vice versa. PMID- 12373365 TI - Heaviness perception. II. Contributions of object weight, haptic size, and density to the accurate perception of heaviness or lightness. AB - The present study investigated the contributions of object weight, haptic size, and density to the accurate perception of heaviness or lightness in the process of discriminating differences in weight between pairs of cubes with cue conflicts such as that resulting from the size-weight illusion. Fifteen subjects, with visual input blocked and relying on the input gained by grasping the cubes with only their fingertips, attempted to accurately discriminate possible differences in weight factor between the two respective cubes in each step of the trials. Three sets - one set each of copper (CP), aluminum (AL), and plastic (PL) - of seven cubes of various weight (0.10-0.74 N) were used. All of the cubes were covered with smooth, thin vinyl to eliminate possible input concerning density or material per se. Screens were strategically placed to eliminate any visual cues. One hundred and ninety-six trials with 37 combinations were pseudorandomly presented to subjects in the following conditions: PL versus AL, AL versus CP, and CP versus PL. Trials included 2 x 3 combinations on the basis of density (98 trials for higher and 98 for lower conditions) and weight (84 ascending trials for heavier, 28 for identical, and 84 descending for lighter conditions). The response for each trial given by each subject was regarded as correct when it accurately identified the weight relationship between the first and second cube. It was found that the subjects fairly accurately identified the weight relationship when density and weight both increased for the second cube (95.6% for given trials), and when density and weight both decreased (94.6%). The current results were markedly greater than those in the constant-density conditions obtained previously, suggesting that changes in density may be as much of an aid in the perception of heaviness and lightness as is weight. Whenever two cues conflicted directionally with each other, however, accuracy fell dramatically to 33.6% for lower density/ascending weight, and to 22.7% for higher density/descending weight. These results indicate the possibility of two different cues contributing to the perception of heaviness and lightness. Cue conflict such as the size-weight illusion naturally occurs when discriminating weight between objects. The present results, however, suggest that a person may perceive heaviness on the basis of the well-regulated relations between changes of density, size, and weight. The way in which these two cues are related through the haptic size is discussed. PMID- 12373366 TI - Linearity of canal-otolith interaction during eccentric rotation in humans. AB - During natural behavior, the head may simultaneously undergo rotation, transduced by the semicircular canals, and translation, transduced by the otolith organs. It has been demonstrated in monkey that the vestibulo-ocular reflexes (VORs) elicited by both endorgans (i.e., the angular and linear VORs, or AVOR and LVOR) sum linearly during combined rotation and translation, but this finding has proven more elusive in humans. To investigate the combined AVOR/LVOR response, six human subjects underwent yaw eccentric rotation at 3 Hz in darkness while displaced from the axis of rotation. Responses to on-center yaw rotation (AVOR alone) and interaural translation (LVOR alone) were also recorded. During eccentric rotation with the subject facing away from the axis of rotation (i.e., nose out), in which a yaw to the right occurs simultaneously with a translation to the right (i.e., translation in phase with rotation), the AVOR and LVOR acted synergistically. Responses were always out of phase with rotation, and became larger in magnitude as vergence increased. For nose-in eccentric rotation, during which translation is out of phase with rotation, the LVOR acted antagonistically to the AVOR. During near viewing, the LVOR often dominated the overall response when eccentricity was sufficiently large, producing eye movements that were in phase with the rotational stimuli. As vergence decreased, the LVOR influence diminished, eventually resulting in responses that were out of phase with rotation at lowest vergence. When the response to pure yaw rotation was vectorially removed from the responses to eccentric rotation, the results proved statistically indistinguishable from the LVOR recorded during interaural translation, suggesting that the ocular response to combined angular and linear motion reflects the linear combination of the AVOR and LVOR. PMID- 12373367 TI - Nitric oxide-containing pyramidal neurons of the subiculum innervate the CA1 area. AB - Neurons and axon terminals containing neuron-specific nitric oxide synthase (nNOS) were examined in the rat subiculum and CA1 area of Ammon's horn. In the subiculum, a large subpopulation of the pyramidal neurons and non-pyramidal cells are immunoreactive for nNOS, whereas in the neighbouring CA1 area of Ammon's horn only non-pyramidal neurons are labelled with the antibody against nNOS. In the pyramidal layer of the subiculum, nNOS-positive axon terminals form both asymmetric and symmetric synapses. In the adjacent CA1 area the nNOS-positive terminals that form symmetric synapses are found in all layers, whereas those terminals that form asymmetric synapses are only in strata radiatum and oriens, but not in stratum lacunosum-moleculare. In both the subiculum and CA1 area, labelled terminals make symmetric synapses only on dendritic shafts, whereas asymmetric synapses are exclusively on dendritic spines. Previous observations demonstrated that all nNOS-positive non-pyramidal cells are GABAergic local circuit neurons, which form exclusively symmetric synapses. We suggest that nNOS immunoreactive pyramidal cells of the subiculum may innervate neighbouring subicular pyramidal cells and, to a smaller extent, pyramidal cells of the adjacent CA1 area, forming a backward projection between the subicular and hippocampal principal neurons. PMID- 12373368 TI - The antisaccade task in a sample of 2,006 young men. I. Normal population characteristics. AB - A population of 2,075 young men aged 18-25 years selected from the conscripts of the Greek Air Force performed an antisaccade task as part of a prospective study for the identification of risk factors in the development of psychoses. The aim of this study, which is ongoing, is to follow this population and investigate the possible predictive value of oculomotor, cognitive, and psychometric factors for the development of psychosis and other psychiatric conditions. In this report we present data concerning the antisaccade task in this population. We measured performance indices, including the percentage of errors (PE), the latencies of different eye movement responses (latency for correct antisaccades, errors, corrections), and performance in perseveration-prone trials. These indices were also evaluated with respect to IQ (measured by the Raven progressive matrices test) and educational level. Mean PE was 23%, with 17% variance. This large variance is of particular importance whenever the detection of a putative deviant behavior is explored. As mean latency of the first eye movement decreased, the PE increased, as did the latency variance. While the negative correlation between percentage of error and mean latency is well established, the relationship of the latency variability of the first response to error production has not been studied before. Thus, optimal performance appears to require both an intermediate mean latency and a small variability. Furthermore, performance seems to be affected by IQ (the higher the IQ score, the lower the percentage of errors). This report offers an analysis of the interindividual variation in the performance of the antisaccade task and discusses some of the sources of this variation. PMID- 12373369 TI - The antisaccade task in a sample of 2,006 young males. II. Effects of task parameters. AB - Antisaccade performance was investigated in a sample of 2,006 young males as part of a large epidemiological study investigating psychosis proneness. This report summarizes the effects of task parameters on performance using a sample of 55,678 antisaccade trials collected from a subpopulation of 947 individuals. Neither the amplitude nor the latency of an error prosaccade in the antisaccade task was correlated with the latency of the ensuing corrective antisaccade that almost always followed an error. However, the latency of the corrective antisaccade decreased with increasing stimulus distance. Concerning the effects of specific task parameters, trials with stimuli closer to the central fixation point and trials preceded by shorter fixation intervals resulted in more errors and longer latencies for the antisaccades. Finally, there were learning and fatigue effects reflected mainly in the error rate, which was greater at the beginning and at the end of the 5-min task. We used a model to predict whether an error or a correct antisaccade would follow a particular trial. All task parameters were significant predictors of the trial outcome but their power was negligible. However, when modeled alone, response latency of the first movement predicted 40% of errors. In particular, the smaller this latency was, the higher the probability of an error. These findings are discussed in light of current hypotheses on antisaccade production mechanisms involving mainly the superior colliculus. PMID- 12373370 TI - Cortical binocularity in convergent strabismus after section of the optic chiasm. AB - In convergent strabismus (esotropia) the spatial asynchrony of the two eye inputs unbalances the interocular interactions, leading to the functional advantage of the nondeviated eye and the inhibition of the esotropic eye. It may be argued that the strabismic suppression, if it is the effect of inhibitory interactions between the eyes, could be removed by interrupting the interocular pathways at the optic chiasm. After chiasmatic section, each eye is connected only to the ipsilateral cortex through the uncrossed retinal projections and so the functionality of each eye's input is no longer interfered with by interocular mechanisms. In strabismic cats submitted, as adults, to section of the optic chiasm, we performed electrophysiological recordings from the striate cortex. Results show that in these animals: (1) the cortical responsiveness to the strabismic eye is strikingly higher than in esotropes with an intact optic chiasm; (2) the effectiveness of stimulation of the deviated eye is not different from that of the nondeviated eye in driving neurons of corresponding cortex; (3) surprisingly, a high degree of binocular activation is present in the cortex ipsilateral to the deviated eye, while in the cortex connected to the nondeviated eye the greatest majority of neurons are monocularly driven. Cortical binocularity depends on the corpus callosum, which conveys the input from the nondeviated eye to the opposite cortex (which receives the direct strabismic input), but not vice versa. The asymmetry of callosal transmission parallels the morphological asymmetry of callosal connections that occur in convergent strabismus. All together the findings indicate that the impaired effectiveness of esotropic input does not result from developmental deficit of the strabismic afferents but, rather, from inhibitory influences that are actively exerted through the interocular pathways. Strabismic suppression may be accomplished by the same interocular mechanisms underlying binocular rivalry. PMID- 12373371 TI - Effects of different types of light touch on postural sway. AB - When a standing person applies a light finger touch to an external stable object, postural sway is reduced. We tested a hypothesis that two factors related to touch can induce this effect, the presence of a stable reference point and the modulation of contact forces leading to tissue deformation. Force platform signals were analyzed while subjects stood quietly with or without additional light touch to an external object (contact forces under 1 N). The point of touch on the body was manipulated. We also investigated the effects of active touch vs fixation of a finger at a point in external space. The results show that touch to the head or neck can be more effective in reducing body sway than a finger touch. A larger reduction in sway was observed when the finger was fixed in a clip (the net forces between the clip and the point of its fixation to the stand were under 1 N) as compared to a free light touch to a pad. The subjects showed a reduction in postural sway while holding a load suspended using a pulley system; in this situation, contact with the load via the pulley provided modulation of contact forces but not a fixed reference point. This finding emphasizes the importance of such factors as stability of the contact point and modulation of contact forces, as compared to active touch or to an implicit task of stabilizing the kinematic chain. The system of postural stabilization can reduce postural sway, making use of either of two sources of sensory information associated with touch, one related to providing a fixed reference point in space, and the other related to transient force changes at the point of contact related to the sway. PMID- 12373372 TI - Investigations into the source of binocular input to the nucleus of the optic tract in an Australian marsupial, the wallaby Macropus eugenii. AB - Recordings from direction-selective neurons in the nucleus of the optic tract (NOT) of the marsupial wallaby, Macropus eugenii, show that 53% of cells are sensitive to visual stimulation of both eyes. Anatomical tracing studies using horseradish peroxidase reveal many retinal terminals in the contralateral NOT but very few in the ipsilateral nucleus. There was no convincing evidence of cortical inputs to the ipsilateral NOT despite large injections of tracer into the visual cortex. During visual stimulation in the visual field of the contralateral eye with moving patterns, the excitatory responses in the NOT generated by ipsiversive motion (right-to-left when recording from the left NOT) were usually larger than the inhibitory responses produced by contraversive motion. Conversely, during ipsilateral eye stimulation, the negative motion components to contraversive motion were usually larger than the positive components to ipsiversive motion. This response pattern resembles that observed in the NOT of the American opossum, Didelphis aurita, where binocularity appears to arise through a commissural subcortical pathway that connects the two nuclei and inverts the directional tuning of the transmitted signals. We propose that the lack of significant input from the ipsilateral eye and cortex in the wallaby suggests that binocularity must arise from another pathway, possibly a commissural route between the nuclei. As directional information appears not to be carried by the internucleus pathway in rats and cats, our results suggest that binocularity in the NOT arises from different sources in marsupials as compared to eutherians. PMID- 12373373 TI - Neck proprioception compensates for age-related deterioration of vestibular self motion perception. AB - Vestibular functions are known to show some deterioration with age. Vestibular deterioration is often thought to be compensated for by an increase in neck proprioceptive gain. We studied this presumed compensatory mechanism by measuring psychophysical responses to vestibular (horizontal canal), neck and combined stimuli in 50 healthy human subjects as a function of age (range 15-76 years). After passive horizontal rotations of head and/or trunk (torso) in complete darkness (dominant frequencies 0.05, 0.1, and 0.4 Hz), subjects readjusted a visual target to its remembered prerotational location in space. (1) Vestibular only stimulus(whole-body rotation); subjects' responses were shifted towards postrotatory body position, this only slightly at 0.4 Hz and pronounced at 0.1 and 0.05 Hz. These errors reflect the known physiological drop of vestibular gain at low rotational frequency. They exhibited a slight but significant increase with age. (2) Neck-only stimulus(trunk rotated, head stationary); the responses showed errors similar to those upon vestibular stimulation (with offset towards postrotatory trunk position) and this again slightly more with increasing age. (3) Vestibular-neck stimulus combinationduring head rotation on stationary trunk; the errors were close to zero, independent of stimulus frequency and the subjects' age. (4) Opposite stimulus combination(trunk rotated in the same direction as the head, but with double amplitude); the errors were clearly enhanced, essentially reflecting the sum of those with vestibular-only and neck only stimulation. Taken together, we find a parallel increase in neck- and vestibular-related errors with age, in seeming contrast to previous studies. We explain our and the previous findings by a vestibular-neck interaction model in which two different neck signals are involved. One neck signal is used, in combination with the vestibular signal, for estimating trunk-in-space rotation. It is internally shaped to always match the vestibular signal, so that these two signals cancel each other out when summed during head rotation on stationary trunk. Because of this matching, perceived trunk stationariness during head rotation on the stationary trunk is independent of vestibular deterioration (related to stimulus frequency, age, ototoxic medication, etc.). The other neck proprioceptive signal, coding head-on-trunk rotation, is superimposed on the estimate of trunk-in-space rotation, thereby yielding a notion of head-in-space. This neck signal remains essentially unchanged with vestibular deterioration. Generally, we hold that the transformation of the vestibular signal from the head down to the trunk proceeds further to include the hip and the legs as well as the haptically perceived body support surface; by this, subjects yield a notion of support kinematics in space. As a consequence, spatial orientation is impaired by chronic vestibular deterioration only to the extent that the body support is moving in space, while it is unimpaired (determined by proprioception alone) during body motion with respect to a stationary support. PMID- 12373374 TI - Movement-related potentials associated with self-paced, cued and imagined arm movements. AB - Self-paced movements, movement to a cue and imagined movement have all been reported to be preceded by a prolonged negativity on averaged electroencephalograph (EEG) recordings. Considerable evidence supports an important contribution from the supplementary motor area (SMA) to this potential and all three types of movement have been shown to be associated with SMA activation. This study was designed to compare the premovement component of these movement-related potentials (MRPs) in a group of subjects who performed each of these three types of movement. In addition, in view of the greater SMA activation in association with proximal arm movements, we studied movements at multiple joints in the right arm. All the potentials were largest at Cz. Self-paced movements were preceded by a negativity (mean onset 1.2 s prior to electromyographic activity) with two distinct phases - an early slow increase (early BP, Bereitschaftspotential) and a later, steeper phase (NS', negative slope). Proximal movements were associated with a larger peak amplitude (mean peak amplitude for shoulder 11.6 micro V, finger movement 9.0 micro V at Cz, n=14) due to a bigger NS' phase. Movements to a regular cue, but not to a randomly timed cue, were also preceded by a long duration negativity, but the NS' phase began earlier and was less distinct than for self-paced movements (mean peak amplitude for shoulder movement 9.1 micro V, finger 8.2 micro V at Cz, n=12). Imagining the movements to a regular cue was associated with a slow negativity, with no clear NS' phase (mean peak amplitude for shoulder movement 6.5 micro V, finger 6.2 micro V at Cz). Our results indicate that the MRPs prior to the three types of movement have distinct characteristics, most notably for the NS' phase. The MRP associated with movement to a regular cue may be analogous to the S2-related negativity of the contingent negative variation (CNV). We discuss the findings in the light of current evidence from functional imaging as to the cortical areas activated in similar movements. PMID- 12373376 TI - Columnar organization of mechanoreceptive neurons in the cortical taste area in the rat. AB - Mechanoreceptive neurons with or without taste responsiveness were recorded in the cortical taste area (CTA) of rats every 50 or 100 micro m along an electrode track made as perpendicular to the surface as possible. Three groups of mechanoreceptive neurons were recognized based on the adequate stimulus, i.e., low-threshold mechanoreceptive ( n=16), nociceptive-specific ( n=48), and wide dynamic range neurons ( n=392). Except for nine neurons, almost all had receptive fields (RFs) in the oral cavity ( n=447). They were categorized into three RF types: those with RFs only in the oral cavity (OC type; n=23), those with RFs both in the oral cavity and on the lip (OL type; n=44) and those with RFs in the oral cavity and on the external surface of the body (WB type; n=380), e.g., tail. Neurons with inhibitory RFs were often located in the infragranular layers. Several neurons with the same receptive features were sequentially recorded along the track, suggesting the presence of columnar organization. The diameter of the possible functional column was the largest (mean 113.85 micro m) for WB type neurons, but smaller in the other two types (mean 85 micro m for the OC type and 62.5 micro m for the OL type). Neurons were segregated according to the adequate stimulus within the column for WB type neurons. Taste-responsive mechanoreceptive neurons ( n=33) were recorded at 15 tracks, and two taste neurons were sequentially recorded in five cases, in three of which two successive neurons sharing the best stimulus were recorded. Taste neurons are possibly arranged in a column with a very small diameter within the large column of mechanoreceptive neurons. PMID- 12373375 TI - Short-term reduction of intracortical inhibition in the human motor cortex induced by repetitive transcranial magnetic stimulation. AB - Ten healthy subjects and two patients who had an electrode implanted into the cervical epidural space underwent repetitive transcranial magnetic stimulation (rTMS; 50 stimuli at 5 Hz at active motor threshold intensity) of the hand motor area. We evaluated intracortical inhibition before and after rTMS. In healthy subjects, we also evaluated threshold and amplitude of motor evoked potentials (MEPs), duration of cortical silent period and short-latency intracortical facilitation. rTMS led to a short-lasting reduction in the amount of intracortical inhibition in control subjects with a high interindividual variability. There was no significant effect on other measures of motor cortex excitability. Direct recordings of descending corticospinal volleys from the patients were consistent with the idea that the effect of rTMS on intracortical inhibition occurred at the cortical level. Since the level of intracortical inhibition can be influenced by drugs that act on GABAergic systems, this may mean that low-intensity repetitive magnetic stimulation at 5 Hz can selectively modify the excitability of GABAergic networks in the human motor cortex. PMID- 12373377 TI - The effects of muscle fatigue on rapid finger oscillations. AB - Two types of model for the control of rapid finger oscillations can be contrasted. According to the first type, any change of muscle characteristics induced by sustained isometric contractions should result in kinematic changes. According to the second type, kinematic characteristics should remain invariant while the timing of bursts of muscle activity exhibits compensatory adjustments. Following sustained isometric contractions of finger flexors and extensors, we observed essentially unchanged durations of the flexion and extension components of rapid finger oscillations, while the pattern of inter-burst intervals was modified so as to compensate the changed phasing of electromyographic bursts. These findings strongly suggest that even in extremely simple rapid finger oscillations motor control is configured to result in invariant kinematics. PMID- 12373379 TI - Polyurethane foams as solid chromogenic reagents for diffuse reflectance spectroscopy. AB - The chemical reactions of the functional groups in polyurethane foams (PUF) have been studied by use of diffuse reflectance spectroscopy and infrared spectroscopy. It was found that the functional groups are highly reactive towards diazotization by sodium nitrite, azo coupling with 4-nitrophenyldiazonium tetrafluoroborate, oxidation by active chlorine, and condensation with formaldehyde, resulting in the formation of intensely colored products. Heterogeneous chemical reactions of PUF with these compounds in aqueous solution proceed rapidly at room temperature and at low solute concentrations. PUF do not undergo degradation as a result of chemical interactions. The linear response of the Kubelka-Munk function to analyte concentration makes it possible to recommend PUF as solid chromogenic reagents for the determination of nitrite, nitrate, and 1- and 2-naphthols. PMID- 12373380 TI - Identification, characterization and determination of metal-binding proteins by liquid chromatography. A review. AB - The use of liquid chromatography in the separation and determination of metal binding proteins is reviewed. Advantages and drawbacks of different chromatographic techniques based on various principles: size exclusion, ion exchange (cationic and ionic), reversed phase and affinity, are presented and discussed. The topic "metal-binding proteins" is considered and presented from two different points of view. The first one regards metal speciation in biological samples (serum and blood). In metal speciation studies, the exact identity of the protein to which the metal is bound often remains unknown. The second point of view is that, frequently, the interest of analyzing metal-binding proteins is not related anymore to the metallic fraction of the protein, but to other chemical structures attached to the protein, such as carbohydrates, which indirectly determine how good the function of the protein is. In this review, special attention is paid to studies dealing with the glycosylation of transferrin, and with the glycated isoform of haemoglobin. PMID- 12373381 TI - Determination of the age of highly enriched uranium. AB - This paper describes the analytical methods (thermal ionization mass spectrometry, inductively coupled plasma mass spectrometry, and alpha spectrometry) that have been developed for determination of the age of uranium and discusses their advantages and limitations. With regard to potential application of the methods (e.g. Fissile Material Cut-off Treaty), the discussion focuses on highly enriched uranium, because this seems to be of highest strategic relevance. The different analytical methods were tested and validated by use of uranium reference materials of different (235)U isotope abundance and of known ages. The results show that thermal ionization mass spectrometry and alpha spectrometry are both very accurate and precise techniques for this application. Inductively coupled plasma mass spectrometry, on the other hand, although less precise, because of the different approach to the analytical problem, is still sufficiently accurate to be used as a rapid screening method. PMID- 12373382 TI - A simple, low-cost, remote fiber-optic micro volume fluorescence flowcell for capillary flow-injection analysis. AB - A small volume flowcell for fluorescence detection in capillary flow injection (CFI) analysis has been created by using a low cost, commercially available fluidic device. Fluorescence detection is achieved using an optical fiber to deliver excitation light to the sample flowing through the device and another optical fiber to collect fluorescence emission. The flowcell is a standard fluidic cross with a swept volume of 721 nL. Optical fibers were oriented at right angles using standard sleeves and ferrules to set their position near the cross intersection. Multiple excitation sources were used including a low power UV laser and blue and UV light emitting diodes (LED). The full emission spectrum detection limits, using the laser, for fluorescein and bovine serum albumin (BSA) were 0.30 ppb and 2.1 x 10(-4)% (w/w), respectively. Two fluidic crosses were used in series for multi-wavelength fluorescence excitation using fiber-optically coupled LED. PMID- 12373383 TI - Subcellular mass determination by 4He+ energy-loss micro-spectrometry. AB - The scanning transmission ion microscope (STIM) has been used to determine the intracellular mass of human cultured cells. A 4He+ microbeam of 2.0 MeV energy was chosen to obtain enhanced ion-energy-loss sensitivity through the micron thick freeze-dried cells. Local sample mass calculation, based on energy-loss conversion by use of appropriate matrix stopping powers, was performed by use of dedicated software. The method was validated with epoxy resin sections and polymer foil as analogues of biological samples in the range of (intra)cellular thickness, 150 to 3000 nm. STIM analysis resulted in less than 5% error in mass determination. 4He+ energy-loss micro-spectrometry was performed on freeze-dried human ovarian cancer cells, the mean areal mass obtained was 120 microg cm(-2) (200 microg cm(-2) in the nucleus and 250 microg cm(-2) in nucleoli). This method is particularly useful for mass normalization of X-ray fluorescence yields resulting from particle-induced X-ray emission microanalysis (micro-PIXE). When performed successively these two ion-beam micro-analytical methods enable the mapping of true element concentrations within single cells. PMID- 12373384 TI - Determination of proteins at nanogram levels by their quenching effect on the chemiluminescence reaction between luminol and hydrogen peroxide with manganese tetrasulfonatophthalocyanine as a new catalyst. AB - The manganese-tetrasulfonatophthalocyanine (MnTSPc) catalyzed luminol-hydrogen peroxide chemiluminescence (CL) systems can be quenched in the presence of proteins. A highly sensitive CL quenching method has been developed for the determination of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.1-20 microg/mL for human serum albumin (HSA), 0.2-20 microg/mL for human gamma-IgG, and 0.5-50 microg/mL for the bovine serum albumin (BSA) with the corresponding detection limits were 1.9 ng/mL, 2.7 ng/mL, and 3.4 ng/mL. The method has been applied to the analysis of total proteins in human serum samples and the results were in good agreement with clinical data provided. PMID- 12373385 TI - A chromoreactand for optical sensing of amphetamines. AB - A bisazo dye is presented that undergoes a reversible chemical reaction with amphetamine in thin layers of plasticised PVC and changes its colour from blue to red. The sensitivity of the dye in the polymer layer covers the range from 0.3 to 30 mmol L(-1) amphetamine with a limit of detection of 0.1 mmol L(-1). The maximum signal changes are observed at 630 nm making the dye compatible with cheap light sources and detectors. PMID- 12373386 TI - Suitability of hyperbranched polyester for sensoric applications--investigation with reflectometric interference spectroscopy. AB - Hyperbranched polyesters (HBP) with different end groups (P-OH, P-COOH, P-OAc) were prepared as thin films. Their surface properties were investigated using zeta potential and contact angle measurements. The differences in surface properties between P-OH and P-COOH, on the one hand, and P-OAc, on the other hand, predicted different behavior in sensoric applications. Therefore, the vapor of the homologous series of alcohols from methanol to pentanol was exposed to the thin films. Changes in thickness were observed with reflectometric interference spectroscopy (RIfS). First investigations in a current analytical problem for the detection and discrimination of refrigerants (freons) using P-OH as sensitive layer have been shown. Polydimethylsiloxane (PDMS) and poly(ether urethane) (PUT) were used as reference sensor materials for the RIfS measurements. PMID- 12373387 TI - Determination of hydrogen peroxide based on a metal dispersed sol-gel derived ceramic-graphite composite electrode. AB - A new copper dispersed ceramic-graphite composite electrode was fabricated by the initial mixing of copper nitrate and (3-mercaptopropyl)trimethoxy silane (MPS) followed by stirring with graphite powder. The combination of the metal catalysis and the advantages of the ceramic composite favored the electrocatalytic reduction of hydrogen peroxide (H2O2) at a reduced overpotential of -0.2 V with good sensitivity, stability and reproducibility. The sensor showed a good linear response to H2O2 in the range from 8.3 x 10(-6) M to 2.0 x 10(-3) M with a correlation coefficient of 0.9989 and the detection limit was 6.2 x 10(-6) M (S/N =3). PMID- 12373388 TI - Use of a copper electrode in alkaline medium as an amperometric sensor for sulphite in a flow-through configuration. AB - A flow injection analysis (FIA) method has been developed for the determination of sulphite in beverages. The method is based on the amperometric detection (0.60 V vs Ag/AgCl (sat. NaCl)) of the analyte at a copper surface in an alkaline medium (1 M NaOH solution) with a manifold that incorporates flow extraction of sulphite as SO2 through a PTFE membrane. Under optimal experimental conditions the peak current response increases linearly with sulphite concentration over the range from 1.0 to 5.0 mM. The repeatability of the electrode response in the FIA configuration was evaluated as 4% ( n =20), the limit of detection of the method was 0.04 mM (S/N =3) and the analytical frequency was 50 h(-1). Since ethanol is also electroactive and permeates through the PTFE membrane, a strategy involving in a first step measurements of only ethanol by manipulating the pH of the donor stream was employed for wine samples. Then, both ethanol and sulphite were measured at the copper electrode at 0.40 V vs Ag/AgCl (sat. NaCl) and the sulphite concentration was determined by difference. Results for 3 different beverage samples (alcoholic and non-alcoholic) showed excellent agreement with the ones obtained by using a recommended procedure for sulphite analysis. PMID- 12373389 TI - Development of a laser-induced cell lysis system. AB - A novel cell lysis system was developed that is based on laser-induced disruption of bacterial and yeast cells. It will find application as a rapid, efficient and clean sample preparation step in bioanalytical detection systems. Using E. coli as our model analyte, we optimized cell lysis with respect to optimal laser wavelength, lowest energy input requirements, RNA release from the cells, and potential protein damage. The optimized system was finally applied to the lysis of four additional microorganisms. All experiments were carried out with about 2000 cells per sample or less. Initially, lysis was determined by the detection of cell survival after laser treatment using standard microbiological techniques, (i.e., cells were grown on nutrient agar plates). Then, actual release of mRNA from the cells was proven. Wavelengths investigated ranged from 500 nm to 1550 nm. An average power of 100 mW for the lasers was shown to be sufficient to obtain cell lysis at wavelengths above 1000 nm, with optimal wavelengths between 1250 nm and 1550 nm. Since water absorbs energy at those wavelengths, it is assumed that laser exposure results in an instantaneous increase of the cell temperature, which causes rupture of the cell membrane. Second, damage to protein solutions treated under optimized laser-lysis conditions was also studied. Using a pure solution of horseradish peroxidase as a model protein, no loss in enzyme activity was observed. Thus, it was concluded that damage to intracellular proteins is unlikely. Third, RNA release was tested using an E. coli specific RNA biosensor. Release of RNA was not detected from untreated cells, but laser treated E. coli cells displayed significant RNA release due to laser-induced cell lysis. Finally, lysis of M. luteus, B. subtilis, B. cereus, and S. cerevisiae were investigated under optimized conditions. In all cases, laser-induced lysis of the cells was confirmed by determination of cell survival. Hence, laser induced cell lysis is an efficient procedure that can be used for sample preparation, without damage to macromolecules, in bioanalytical detection systems for microorganisms. Miniaturized lasers and miniaturized cell-lysis chambers will create a simple, field-usable cell lysis system and allow the application of laser-induced cell lysis in micro Total Analysis Systems. PMID- 12373390 TI - Determination of 6-oxo-morphinans, as the oximes, by difference circular dichroism spectroscopy. AB - A negative Cotton effect is observed in the circular dichroism (CD) spectra of 6 oxo-morphinans in the wavelength range of n-pi* electron transitions. 6-oxo Morphinans can be transformed into oxime derivatives with hydroxylamine and after oxime formation the CD spectra are significantly different. Oxime formation was monitored by CD and by HPLC. It was established that under the experimental conditions used oxime formation was complete within 90 min. The method suggested for the determination of 6-oxo-morphinans is based on the considerable differences between the ellipticities before and after the oxime formation. The ellipticity difference varies linearly with concentration in the range 2 x 10(-5) 5 x 10(-4) mol L(-1) for the three 6-oxo-morphinans examined (oxycodone, hydrocodone, and 14-hydroxycodeinone). For hydrocodone the dependence is also linear in a lower concentration range (5 x 10(-6)-10(-4) mol L(-1)). The new difference CD spectroscopic method can be applied to the selective determination of 6-oxo-morphinans in bulk and dosage forms. PMID- 12373391 TI - Determination of antitubercular drugs by micellar electrokinetic capillary chromatography (MEKC). AB - A method for the determination of isoniazid (ISO), pyrazinamide (PYR) and rifampicin (RIF) in pharmaceutical products, by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection is described. The influence of pH, concentration of surfactants, buffer and organic solvents, over the separation were studied as experimental variables. The optimal separation was carried out at 30 degrees C and 20 kV, using a 40 mM borate buffer and 100 mM sodium dodecylsulphate (SDS) adjusted to pH 8.5. Under these conditions, the analysis is accomplished in about 8 min. The method was applied to the determination of these compounds in different pharmaceuticals with good results when compared with a reference liquid chromatographic (LC) method. PMID- 12373393 TI - Porphyrin binding to DNA investigated by cyclodextrin supramolecular system. AB - The interactive mode of meso-tetrakis- (4- N-trimethylaminobenzyl) porphyrin (TAPP) with DNA has been investigated by the cyclodextrin (CD)-porphyrin supramolecular system. The binding of TAPP with DNA is inhibited by the anion CD derivative, sulfurbutyl-beta-cyclodextrin (SB-beta-CD); however, the neutral CD cannot influence the binding. As for the inclusion procedure of the CDs-TAPP system, the (1)H-NMR data suggests that the hydrophobic segment of TAPP enters into the cavity of CD, which means that the hydrophobic part of TAPP is not the binding site in the TAPP-DNA interaction. Therefore, the binding model cannot be the intercalation and is not related to the grooves of DNA. In addition, experimental data show that the charge attraction, which exists in the inclusion procedure of SB-beta-CD and TAPP hampers the binding of TAPP with DNA. The negative groups of SB-beta-CD compete with the phosphate groups of the DNA backbone, and have an attraction to the positive groups of TAPP. This competitive attraction supports the theory that the binding mode of TAPP with DNA is "electrostatic binding". Furthermore, there are two binding sites between one TAPP molecule and one DNA standard. We produce a possible binding structure (a suprahelical structure of DNA) for this TAPP-DNA complex. This structure is in good agreement with the literature. PMID- 12373392 TI - An international intercomparison exercise for the determination of purified microcystin-LR and microcystins in cyanobacterial field material. AB - The comparability of current microcystin analysis methods has been evaluated in an international intercomparison exercise. The focus was on the analysis of microcystins by high-performance liquid chromatography coupled with ultraviolet or photodiode-array detection (HPLC-PDA/UV), currently the most widespread method for microcystin analysis, but the exercise was open for other methods such as enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay (PPA) and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS).Thirty-one laboratories from 13 countries participated in the study. For a microcystin-LR (MC-LR) standard solution (S1) of undisclosed quantity, and for a field sample (S3) from a natural cyanobacterial bloom, repeatabilities between 4 and 15% and reproducibilities between 24 and 49% were obtained. No significant differences between single methods were found for S1 and S3, except for a significantly higher repeatability value of ELISA for S1. However, the analysis of microcystins in the field sample (S3) by HPLC-PDA/UV was significantly more variable than for the standard solution (S1). Both the extraction and the analysis of the microcystins appeared to contribute to this variability. It is concluded that standard MC-LR (S1) can be measured with adequate precision by all participating laboratories independently of the method used. With respect to the different methods used the results for the field sample can also be regarded as satisfactory, but clearly showed the need for improvement by standardisation between laboratories. Furthermore, quantification with in house standards compared to quantification using the supplied MC-LR standard indicated that routine microcystin analysis in laboratories may be also influenced by the variability of available standards, emphasising the need for the production of certified reference materials (CRM). PMID- 12373394 TI - First- and second-order multivariate calibration applied to biological samples: determination of anti-inflammatories in serum and urine. AB - First- and second-order multivariate calibration of fluorescence data have been compared as regards the determination of anti-inflammatories and metabolites in the biological fluids serum and urine. The simultaneous resolution of naproxen salicylic acid mixtures in serum and naproxen-salicylic acid-salicyluric acid mixtures in urine was accomplished and employed for a discussion of the relative advantages of the applied chemometric tools. The analysis of second-order fluorescence excitation-emission matrices was performed using iteratively reweighted generalized rank annihilation method (IRGRAM), parallel factor analysis (PARAFAC), and self-weighted alternating trilinear decomposition (SWATLD). The results were compared with first-order fluorescence emission data analyzed with partial least-squares regression (PLS). In all cases, the performance of the methods was improved through the formation of inclusion complexes of the analytes with beta-cyclodextrin. The concentration ranges in which the analytes could be determined were as follows: naproxen, 0-250 ng mL(-1) in serum and 0-200 ng mL(-1) in urine; salicylic acid, 0-500 ng mL(-1) in serum and 0-300 ng mL(-1) in urine, and salicyluric acid, 0-300 ng mL(-1) in urine. PMID- 12373395 TI - Complementary use of partial least-squares and artificial neural networks for the non-linear spectrophotometric analysis of pharmaceutical samples. AB - The complementary use of partial least-squares (PLS) multivariate calibration and artificial neural networks (ANNs) for the simultaneous spectrophotometric determination of three active components in a pharmaceutical formulation has been explored. The presence of non-linearities caused by chemical interactions was confirmed by a recently discussed methodology based on Mallows augmented partial residual plots. Ternary mixtures of chlorpheniramine, naphazoline and dexamethasone in a matrix of excipients have been resolved by using PLS for the two major analytes (chlorpheniramine and naphazoline) and ANNs for the minor one (dexamethasone). Notwithstanding the large number of constituents, their high degree of spectral overlap and the occurrence of non-linearities, rapid and simultaneous analysis has been achieved, with reasonably good accuracy and precision. No extraction procedures using non-aqueous solvents are required. PMID- 12373396 TI - Stability of total selenium and selenium species in lyophilised oysters and in their enzymatic extracts. AB - To obtain reliable information on speciation analysis it is necessary to previously evaluate the stability of the species in the sample of interest. Furthermore, in those cases in which sample treatment to extract the species is time-consuming, an evaluation of how to maintain species integrity in the extracts is paramount. Thus, the present paper reports the stability of total Se, SeMet and TMSe+ in freeze-dried oyster and in the enzymatic extracts stored in Pyrex and polyethylene containers at different temperatures (-18, 4 and 20 degrees C). Total selenium determinations and Se speciation were carried out by HG-AAS after acid digestion in a microwave oven and by on-line coupling of cation exchange HPLC-ICP-MS after enzymatic hydrolysis, respectively. The results obtained for the freeze-dried sample showed that total Se and the selenium species evaluated are stable for at least 12 months, under all the conditions tested. However, Se species in the enzymatic extracts are only stable for 10 days if stored at 4 degrees C in Pyrex containers. These results show that the extracts do not necessarily have to be analysed just after sample treatment. PMID- 12373397 TI - Determination of lead in biological samples by use of slurry sampling electrothermal atomic absorption spectrometry. AB - Slurry-sampling electrothermal atomic absorption spectrometry has been applied to the determination of lead in several biological samples (fish and marine algae). The slurries were prepared both by magnetic shaking and microwave-heating and the effect of instrument operating conditions and slurry preparation conditions on the signal were examined. In addition, results from slurry sampling were compared with those obtained by microwave-assisted acid digestion of the same samples and no significant differences were found between them when the analysis of variance (ANOVA) was applied. The between-batch precision of the slurry techniques employed was similar to that for the microwave-assisted digestion procedure; values were always below 6.7%, except for the Dicentrarchus labrax sample for which the value obtained was 9.5% when using slurry magnetic shaking and 7.6% when using the slurry microwave heating. The accuracy of the slurry methodology employed was also evaluated by analysis of two biological reference materials (NIST-1577b and IAEA-V10); percentage recoveries obtained were between 95.6 and 98.5% of the values certified for lead. PMID- 12373398 TI - Spectrophotometric determination of verapamil hydrochloride in drug formulations with chloramine-T as oxidant. AB - A new spectrophotometric method is described for the determination of verapamil hydrochloride, based on its oxidation with chloramine-T in hydrochloric acid medium. It produces a yellow colored compound with maximum absorbance at 425 nm. Beer's law was obeyed in the concentration range 0-340 micro g mL(-1) with molar absorptivity 2 x 10(3) L mol(-1) cm(-1) and RSD 0.3-0.82%. All variables were studied to optimize the reaction conditions. No interferences were observed from the common excipients present in the formulations. The method has been applied successfully to the determination of the drug in pharmaceutical preparations. Statistical comparison of the results with those from the reference method reveals excellent agreement and confirms that accuracy and precision are not significantly different. PMID- 12373400 TI - Determination of total phenols in environmental wastewater by flow-injection analysis with a biamperometric detector. AB - A flow injection (FI) method with a biamperometric detector, based on the biamperometry for an irreversible redox couple, is described for the determination of phenols in environmental wastewater. The method relies on coupling of the oxidation of phenols at one platinum-wire electrode with the reduction of MnO4- at another platinum wire electrode to enable biamperometric detection with an applied potential difference of 0 V. The linear dynamic range for the dependence of current on phenol concentration was from 1.0 x 10(-6) to 1.0 x 10(-4) mol L(-1), with a detection limit of 4.0 x 10(-7) mol L(-1) (signal to-noise ratio, S/N=3). In comparison with the 4-aminoantipyrine (4-AAP) standard method and the 3-methyl-2-benzothiazoline hydrazone (MBTH) method the proposed method can be used to detect many para-substituted phenols that do not react with 4-AAP and MBTH, and response factors are higher for most of the phenols tested. The method, which is simple, economic, and rapid (180 samples h(-1)), has been applied to the analysis of four wastewater samples. The results obtained were compared with those from 4-AAP method. The recoveries obtained by adding phenol standards to samples ranged from 94.3 to 105.2% with a standard deviation of 3.6%. PMID- 12373399 TI - Laser-induced fluorescence with an OPO system. Part I. Optimisation of the analytical system by use of experimental design methodology. Application to the direct quantification of traces of benzo[ a]pyrene. AB - This study deals with the optimisation and application of a method for direct analysis of trace pollutants in water by laser-induced fluorescence. The arrangement used consisted of an Nd:YAG Laser coupled with an optical parametric oscillator (LYOPO) and connected to a spectrophotometer and a high-sensitivity camera. Optimisation was achieved by developing an experimental design methodology to maximise the signal-to-noise ratio and reduce the limit of detection. The technique was then applied to the detection of benzo[ a]pyrene in water. The experimental results were evidence of its high sensitivity and time resolution potential. The detection limit for benzo[ a]pyrene was 0.7 ng L(-1) in drinking water and 4 ng L(-1) in raw water containing 1 mg L(-1) humic acids. PMID- 12373401 TI - Interlaboratory assessment of measurement precision and bias in the coulometric Karl Fischer determination of water. AB - The precision and bias of the coulometric Karl Fischer ASTM method D1533-00 have been assessed in a collaborative ASTM round robin program for a group of 34 laboratories. The test materials used in this study included water saturated 1 octanol (WSO), water saturated 1-butanol (WSB), and a series of new and used transformer oil samples. Fundamental systematic biases have been demonstrated in the accuracy of the measurement of water in the WSO, WSB, and transformer oil samples. The systematic bias in the measurement of the WSO and WSB standards indicates that for some laboratories either the instruments were not accurate or the quantity of the standard was not measured accurately. A second type of systematic bias consisted of measurement errors associated with the selection of the Karl Fischer solvent that was used with each instrument, and this was superimposed upon the error in the measurement of the water in the standards. Using the statistical calculation method ASTM D 6300 the repeatability and reproducibility for water in transformer oil were found to be 7 mg/kg and 14 mg/kg respectively. The method detection limit of water was 8 mg/kg oil. The method bias was estimated based on the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2890, WSO, since no suitable reference material for water in transformer oil was available for this study. PMID- 12373402 TI - Stability study of As(III), As(V), MMA and DMA by anion exchange chromatography and HG-AFS in wastewater samples. AB - The stability of arsenic species (arsenate [As(V)], monomethylarsonate [MMA], dimethylarsinate [DMA] and arsenite [As(III)]) in two types of urban wastewater samples (raw and treated) was evaluated. Water samples containing a mixture of the different arsenic species were stored in the absence of light at three different temperatures: +4 degrees C, +20 degrees C and +40 degrees C. At regular time intervals, arsenic species were determined by high performance liquid chromatography (HPLC)-hydride generation (HG)-atomic fluorescence spectrometry (AFS). The experimental conditions for the separation of arsenic species by HPLC and their determination by AFS were directly optimised from wastewater samples. As(III), As(V), MMA and DMA were separated on an anion exchange column using phosphate buffer (pH 6.0) as the mobile phase. Under these conditions the four arsenic species were separated in less than 10 min. The detection limits were 0.6, 0.9, 0.9 and 1.8 micro g L(-1) for As(III), DMA, MMA and As(V), respectively. As(V), MMA and DMA were found stable in the two types of urban wastewater samples over the 4-month period at the three different temperatures tested, while the concentration of As(III) in raw wastewater sample decreased after 2 weeks of storage. A greater stability of As(III) was found in the treated urban wastewater sample. As(III) remained unaltered in this matrix at pH 7.27 over the period studied, while at lower pH (1.6) losses of As(III) were detected after 1 month of storage. The results show that the decrease in As(III) concentration with time was accompanied by an increase in As(V) concentration. PMID- 12373403 TI - On-column complexation and simultaneous separation of vanadium(IV) and vanadium(V) by capillary electrophoresis with direct UV detection. AB - An on-column complexation method has been developed for the simultaneous determination of V(IV) and V(V). Vanadium species were chelated with aminopolycarboxylic acids to form anionic complexes which were separated by capillary zone electrophoresis (CZE) with direct UV detection. Ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentacetric acid (DTPA), nitrilotriacetic acid (NTA), and N-2-hydroxyethylethlendiaminetriacetric acid (HEDTA) were investigated as both ligand and running electrolyte. Of the ligands studied the complexes of EDTA with V(IV) and V(V) resulted in the highest selectivity and UV response. The conditions used for on-column complexation and separation, including pH, and electrolyte ligand concentration, were examined to achieve reasonable separation selectivity and detection sensitivity. The optimum separation of the anionic forms of V(IV) and V(V) was obtained by use of CZE with UV detection at 185 nm and an electrolyte containing 5 mmol L(-1) EDTA at pH 4.0. Linear calibration plots were obtained in the concentration range10-300 micro mol L(-1); detection limits were 3 micro mol L(-1) for V(IV) and 1 micro mol L(-1) for V(V). The proposed method was demonstrated for the determination of vanadium in groundwater spiked with V(IV) and V(V). PMID- 12373404 TI - Hydride generation for the direct determination of trace and ultra-trace level of arsenic and antimony in waters using derivative atomic absorption spectrometry. AB - A new method is developed for the direct determination of trace and ultra-trace level of arsenic and antimony in waters by hydride generation derivative atomic absorption spectrometry (DHGAAS). The signal model and fundamentals of DHGAAS are described. The effects of atomization temperature, argon flow rate, acidity and concentration of KBH(4)and KI were investigated and analytical conditions were optimized. The sensitivities for arsenic and antimony were increased 36.4 and 27.6 times better than those of conventional hydride generation atomic absorption spectrometry (HGAAS). For a 2 mV min(-1) sensitivity range setting, the characteristic concentration was 0.003 microg L(-1) for arsenic and 0.004 microg L(-1)for antimony, and the detection limits (3sigma) were 0.015 micro g L(-1) for arsenic and 0.020 microg L(-1) for antimony. The proposed method was applied to the determination of arsenic and antimony in several water samples with satisfactory results. PMID- 12373405 TI - Field preconcentration of cadmium from seawater by using a minicolumn packed with Amberlite XAD-4/4-(2-pyridylazo) resorcinol and its flow-injection-flame atomic absorption spectrometric determination at the ng L(-1) Level. AB - A flow injection analysis-flame atomic absorption spectrometric method for the determination of cadmium in seawater was developed with the aim of yielding a sensitive assay with a low detection limit. The method employs a field flow preconcentration technique involving a minicolumn containing Amberlite XAD-4 impregnated with the complexing agent 4-(2-pyridylazo) resorcinol. A Plackett Burman 2(7)x3/32 design for seven factors (sample pH, sample flow rate, eluent volume, eluent concentration, eluent flow rate, ethanol percentage in the eluent and minicolumn diameter) was carried out in order to find the significant variables affecting the field continuous preconcentration system (FCPS) and the flow injection elution manifold for cadmium determination in seawater samples by flame atomic absorption spectrometry. Cadmium can be preconcentrated with an enrichment factor of 1053 for a sample volume of 200 mL and a preconcentration time of 57 min. In these experimental conditions, the method provides a linear relationship between absorbance and cadmium concentration in the range from 22 1900 ng L(-1), with a detection limit (3SD) of 6 ng L(-1). The precision (expressed as relative standard deviation) for eleven independent determinations reached values of 8.9-0.8% in cadmium solutions of 50-700 ng L(-1). Analysis of certified reference materials (SLEW-3 and NASS-5) showed good agreement with the certified value. This procedure was applied to the determination of cadmium in seawater from Galicia (Spain). PMID- 12373406 TI - A high-efficiency annular diffusion scrubber for the collection of water-soluble trace atmospheric gases. AB - The design and construction details of an annular diffusion scrubber to be used as a quantitative gas sampler are described. A large-diameter inner tube (12 mm o.d.) wrapped with liquid channel interior of thin membrane (70 micro m thickness) provided high collection efficiency. With SO2 as the test gas, the performance data for a high-efficiency annular diffusion scrubber, coupled to an ion chromatograph, are presented. Quantitative collection and excellent reproducibility are observed at air sampling rates up to 2 L/min. The estimated detection limit is 4 pptv for a 20-L air sampling volume. PMID- 12373407 TI - Removal of trivalent chromium from tannery waste waters using bone charcoal. AB - The ability of bone charcoal to remove Cr(III) from aqueous solutions by adsorption has been investigated. The adsorbent used was first characterised and then the adsorption was studied as a function of time and amount of charcoal. Tests were carried out with synthetic solutions whose Cr concentrations (500 mg L(-1)) were similar to those found in some effluents of Moroccan tannery industries. Cr removal efficiencies higher than 90% were obtained at pH 3.5 using 3 g of bone charcoal and a stirring time of about 30 min. Results of Cr removal by all sieved fractions of bone charcoal had shown the same interesting capabilities for Cr(III) retention. The cross interference with other elements was also investigated. Pre-treatment of bone charcoal by nitric acid led to an increase in its specific surface area but induced a drastic reduction in its Cr elimination abilities. Adsorption tests were also carried out using calcinated bone charcoal. The results obtained showed a similar percentage of Cr retention to those found with untreated bone charcoal. On the other hand, a double treatment of bone charcoal with HCl and NaOH provided an enhancement of Cr(III) retention. The role played by the mineral fraction of the solid phase of bone was thus evidenced. PMID- 12373408 TI - Microwave-assisted extraction and mild saponification for determination of organochlorine pesticides in oyster samples. AB - A sample-preparation procedure (extraction and saponification) using microwave energy is proposed for determination of organochlorine pesticides in oyster samples. A Plackett-Burman factorial design has been used to optimize the microwave-assisted extraction and mild saponification on a freeze dried sample spiked with a mixture of aldrin, endrin, dieldrin, heptachlor, heptachorepoxide, isodrin, transnonachlor, p, p'-DDE, and p, p'-DDD. Six variables: solvent volume, extraction time, extraction temperature, amount of acetone (%) in the extractant solvent, amount of sample, and volume of NaOH solution were considered in the optimization process. The results show that the amount of sample is statistically significant for dieldrin, aldrin, p, p'-DDE, heptachlor, and transnonachlor and solvent volume for dieldrin, aldrin, and p, p'-DDE. The volume of NaOH solution is statistically significant for aldrin and p, p'-DDE only. Extraction temperature and extraction time seem to be the main factors determining the efficiency of extraction process for isodrin and p, p'-DDE, respectively. The optimized procedure was compared with conventional Soxhlet extraction. PMID- 12373409 TI - Enrichment of iron(III), cobalt(II), nickel(II), and copper(II) by solid-phase extraction with 1,8-dihydroxyanthraquinone anchored to silica gel before their determination by flame atomic absorption spectrometry. AB - A chelating matrix prepared by immobilizing 1,8-dihydroxyanthraquinone on silica gel modified with 3-aminopropyltriethoxysilane has been characterized by use of cross-polarization magic angle spinning (CPMAS) NMR, diffuse reflectance infrared Fourier transformation (DRIFT) spectroscopy, and thermogravimetric analysis and used to preconcentrate Fe(III), Co(II), Ni(II), and Cu(II) before their determination by flame atomic absorption spectrometry. The optimum pH ranges for quantitative sorption are 6.5-8.0, 6.0-7.0, 6.0-8.0, and 7.0-8.5 for Cu, Fe, Co, and Ni, respectively. All the metal ions can be desorbed with 2 mol L(-1) HCl or HNO3. The sorption capacity ( micromol g(-1) matrix) and preconcentration factor were 226.6, 250; 365.6, 300; 101.8, 150; and 109.0, 250 for Cu, Fe, Co, and Ni, respectively. The lowest concentration for quantitative recovery was 4.0, 3.3, 6.6, and 4.0 ng mL(-1), respectively for the four metal ions. The limits up to which electrolytes NaNO3, NaCl, NaBr, Na2SO4, and Na3PO4 and cations Ca(II) and Mg(II) can coexist with the four metal ions during their sorption without adverse effect are reported. The simultaneous enrichment and determination of all the four metals is possible if the total load of metal ions is less than the sorption capacity. Flame AAS was used to determine the metal ions in underground, tap, and river water samples (RSD90% of responding on the injection-lever. Availability of 0.01 mg/kg per injection cocaine resulted in approximately equal levels of responding on the food- and injection-levers. Presession IM cocaine injections dose-dependently increased responding on the injection-lever. CONCLUSIONS: Stable behavior can be maintained under concurrent FR schedules of food and cocaine presentation in monkeys, and the distribution of behavior on food- and injection-levers is dependent on the available dose of cocaine. PMID- 12373430 TI - Cocaine- and food-maintained responding under a multiple schedule in rhesus monkeys: environmental context and the effects of a dopamine antagonist. AB - RATIONALE: Environmental context has been shown to influence responding under multiple schedules of food reinforcement and to modify the behavioral effects of drugs. However, no systematic study has been conducted under conditions of cocaine self-administration. The hypothesis was that changes in the magnitude of food reinforcement would affect cocaine-maintained response rates and influence the behavioral potency of a dopamine antagonist to decrease cocaine self administration. OBJECTIVE: A multiple schedule was used to evaluate the effects of changes in the magnitude of food reinforcement on the self-administration cocaine dose-response curve and on the behavioral potency of a dopamine receptor antagonist to decrease food- and cocaine-maintained responding. METHODS: Rhesus monkeys (n=3) were trained to self-administer intravenous cocaine under a multiple fixed-interval (FI) 5-min schedule of food and cocaine presentation. Food (one or four pellets) was available in the first and third components and cocaine (saline, 0.01-0.3 mg/kg per injection) was available in the second and fourth components. After completion of cocaine dose-response curves, the effects of the dopamine D(2)/D(3) receptor antagonist 2,3-dimethoxy-N-(9-p-fluorobenzyl) azabicyclo[3.3.1]nonan-3beta-yl benzamide (MABN) were examined. RESULTS: Cocaine- and food-maintained responding varied as a function of dose and were characterized as inverted U-shaped functions; cocaine-maintained response rates were significantly influenced by the magnitude of food in the other component. The behavioral potency of MABN on food- and cocaine-maintained responding was not influenced by the magnitude of food reinforcement. CONCLUSIONS: These results suggest that cocaine self-administration under a multiple schedule with food reinforcement is influenced by the environmental context. These schedule interactions, however, did not alter the behavioral effects of a dopamine D(2)/D(3) receptor antagonist. PMID- 12373431 TI - Role of dose order in the development of tolerance to effects of cocaine on schedule-controlled behavior in pigeons. AB - RATIONALE: Tolerance to behavioral effects of cocaine can be produced by exposure to varying doses. The degree to which tolerance develops may depend on dose order. OBJECTIVE: To investigate the relationships between three sequences of doses of cocaine in a daily, variable-dosing regimen and the development of tolerance to effects on schedule-controlled behavior. METHODS: Twelve pigeons responded daily under a fixed-ratio 20 schedule of reinforcement, and performance was investigated under a range of doses of cocaine (0.3-10.0 mg/kg, i.m.) by administering the drug once every 7 days (acute effects). After determination of acute effects of cocaine, the drug was administered daily with dose varying from day to day. Dose order varied systematically across three groups of four pigeons; doses were delivered in ascending, descending, or "sawtooth" (ascending then descending) sequences. This variable-dosing regimen continued until drug effects were stable (at least 13 cycles through all doses). RESULTS: During the acute dosing regimen, response rates following small cocaine doses were similar to those under control conditions; following moderate-to-high doses, responding was diminished relative to control rates. During the variable-dosing regimen, tolerance to the rate-decreasing effects of cocaine was observed in all groups, regardless of the order in which the drug was delivered, and the magnitude of tolerance was similar across groups. Systematic differences in the rate of recovery from initial response decrements were observed across groups, with rate of recovery fastest under the ascending sequence. CONCLUSIONS: These results suggest that dose order under a variable-dosing regimen does not significantly affect the final attainment of tolerance, although it may contribute to the speed with which tolerance develops. PMID- 12373432 TI - Improved multiparameter models of drug effects on response rate under multiple variable interval schedules: evidence from rat studies. AB - RATIONALE: The functional analysis of the behavioral effects of drugs has lagged behind more biological research approaches in elucidating how drugs influence behavior. Part of the problem is the scarcity of mathematical models of responding under standardized behavioral procedures. A two-parameter model derived from the matching law to account for response rate as a function of reinforcement rate in single response alternative procedures provides a good description of responding under multi-component multiple variable interval (multVI) schedules of reinforcement, both in the absence of drugs and in the presence of a variety of drugs. OBJECTIVE: This paper explores how well the two parameter model and its exponentiated variant (three-parameter model) describe drug effects on responding under multVI schedules. METHODS: Data from seven published papers concerning the effects of drugs on multVI schedules are reanalyzed. RESULTS: The three-parameter model describes the effects of drugs on responding more accurately than does the two-parameter model. The three-parameter model also describes well the type of relation between response rates under drug and no-drug conditions that the rate dependency hypothesis is usually invoked to explain. CONCLUSIONS: The three-parameter model can be improved by adding a parameter to account for base response rate, that is, response rate when reinforcement rate is zero. The parameters of the best descriptive model could be used to devise a classification system for the behavioral effects of drugs under multVI schedules. The parameters should be conceptualized by relating the drug induced changes in parameter value to converging changes in measurements of brain function. This would connect the behavioral and the biological analyses of drug action to the benefit of both research fields. PMID- 12373433 TI - The generalized matching law as a predictor of choice between cocaine and food in rhesus monkeys. AB - RATIONALE: The generalized matching law predicts that the relative rate of behavior maintained by different reinforcers will match the relative rate of reinforcement. It has previously been shown that responding maintained by either food deliveries or cocaine injections under concurrent variable-interval (conc VI) schedules is well described by the generalized matching law. However, the generality of this conclusion to the choice between a drug and a non-drug reinforcer has not been well established. OBJECTIVE: The objective of the present study was to determine the extent to which the generalized matching law could account for choice between cocaine and food. METHODS: Four male rhesus monkeys (Macaca mulatta) lever pressed under various pairs of conc VI schedules with food and/or cocaine injection as the maintaining events. Two doses of cocaine (0.025 and 0.05 mg/kg per injection) were selected to provide information about reinforcer magnitude. RESULTS: As has been found in a context of choice between identical reinforcers, the generalized matching law accounted for most behavior. As in earlier studies with identical reinforcers, there was less responding apportioned to the alternative with the greater reinforcement frequency than predicted by the generalized matching law, i.e., undermatching was observed frequently. There was a tendency for more responding to be emitted toward the food alternative when the lower dose of cocaine was available and toward the drug alternative when the higher dose of cocaine was available. CONCLUSION: These results suggest that, as proposed by the generalized matching law, relative reinforcement rate is an important determinant of choice between a drug and a non drug reinforcer. PMID- 12373434 TI - Second-order schedules of drug self-administration in animals. AB - On a second-order schedule, a subject responds according to one schedule (the unit schedule) for a brief presentation of a stimulus such as a light. Responding by the subject on this unit schedule is then reinforced according to another schedule of reinforcement. Second-order schedules of drug injection allow the study of more complex behavioral sequences than do simple schedules and may more accurately reflect the human drug-abuse situation. Much of the early work in this area used primates as subjects and focused on the behavioral variables controlling responding. It was shown that long sequences of behavior could be maintained on second-order schedules with relatively infrequent injections of drug and that the second-order, brief-stimulus presentations were critical to the acquisition and maintenance of responding. Also, the continued presentation of the brief stimulus in extinction often led to prolonged extinction behavior. These studies clearly showed that environmental stimuli greatly influence drug self-administration behavior under second-order schedules. The focus of much of the more recent work with second-order schedules has been on the evaluation of pharmacological treatments for drug addiction, both as antagonist and substitution therapies. Both types of potential therapies have shown promise in these preclinical models of addictive behavior. The recent extension of second order self-administration studies to rats as subjects has facilitated the investigation of neural mechanisms involved in this behavior. While this use of second-order schedules is a relatively recent phenomenon, significant contributions have already been made in identifying neural mechanisms critical to second-order schedule drug self-administration. This active area of research holds great promise for delineating specific brain regions critical to different aspects of drug addiction. PMID- 12373435 TI - Observing responses maintained by stimuli associated with cocaine or remifentanil reinforcement in rhesus monkeys. AB - RATIONALE: The stimuli associated with drug reinforcement may be particularly relevant to drug abuse and relapse. OBJECTIVES: The study measured behavior maintained by conditioned reinforcing stimuli in an observing response procedure. METHODS: The experiment was conducted with rhesus monkeys in three stages: 1) discriminative control was established by reinforcing responding on one lever with either intravenous cocaine or remifentanil in the presence of one stimulus and extinguishing the response in the presence of another stimulus, 2) discriminative control was suspended by not presenting the stimuli, and 3) a final stage was implemented wherein the stimuli from the first stage were presented only when one or more responses were made on a second (observing) lever. RESULTS: Under FR1 conditions, observing responses were maintained at low rates, but increased markedly when the response requirement was increased. CONCLUSIONS: The procedure maintained observing responses quite well and may be useful to an analysis of conditioned reinforcement based on drug reinforcement. PMID- 12373436 TI - Functional imaging and neurochemical correlates of stimulant self-administration in primates. AB - RATIONALE: Recent advances in neuroimaging and in vivo neurochemistry have documented drug-induced functional changes in brain activity under physiologically relevant conditions. These approaches have significant strengths and important limitations that should be considered. OBJECTIVES: The present review describes current in vivo approaches to characterize drug effects as they relate to behavior, and highlights key contributions derived from each approach in the context of stimulant self-administration in primates. METHODS: Techniques relating in vitro neurochemistry to behavioral pharmacology are compared to several in vivo approaches, including microdialysis, positron emission tomography (PET) neuroimaging and functional magnetic resonance imaging (fMRI). RESULTS: In vitro neurochemical correlates of behavioral pharmacology established a significant relationship between dopamine and the reinforcing effects of abused stimulants. Subsequent in vivo microdialysis studies in behaving animals supported a critical role for nucleus accumbens dopamine in the reinforcing effects of stimulants. PET neuroimaging in monkeys and humans documented a close relationship between dopamine transporter (DAT) occupancy in vivo and the reinforcing effects of stimulants. The effectiveness of selective DAT inhibitors to reduce cocaine self-administration also was linked to DAT occupancy in vivo. Lastly, the measurement of cerebral blood flow and metabolism with PET and fMRI has begun to define the neuronal circuitry influenced by acute and chronic stimulant exposure. CONCLUSIONS: Collectively, in vivo neurochemistry and functional imaging have complemented in vitro approaches and have enhanced our current understanding of the neurobiology of abused stimulants. PMID- 12373437 TI - The 5-choice serial reaction time task: behavioural pharmacology and functional neurochemistry. AB - RATIONALE: The developmental history and application of the 5-choice serial reaction time task (5CSRTT) for measuring effects of drugs and other manipulations on attentional performance (and stimulus control) in rats is reviewed. OBJECTIVES: The 5CSRTT has been used for measuring effects of systemic drug treatments and also central manipulations such as neurochemical lesions on various aspects of attentional control, including sustained, selective and divided attention--and is relevant to the definition of neural systems of attention and applications to human disorders such as attention deficit/hyperactivity disorder (ADHD) and Alzheimer's disease. METHODS: The 5CSRTT is implemented in a specially designed operant chamber with multiple response locations ('nine-hole box') using food reinforcers to maintain performance on baseline sessions (about 100 trials) at criterion levels of accuracy and trials completed. The 5CSRTT can be used for measuring various aspects of attentional control over performance with its main measures of accuracy, premature responding, correct response latencies and latency to collect earned food pellets. RESULTS: The data reviewed include studies mainly of systemic and intra-cerebral effects of adrenoceptor, dopamine receptor, serotoninergic receptor and cholinergic receptor agents. These are compared with investigations of effects of selective chemical neurotoxins and excitotoxins applied to discrete parts of the forebrain, in order to define the neural and neurochemical substrates of attentional function. Furthermore, these results are integrated with findings from in vivo microdialysis in freely moving rats or metabolic studies. CONCLUSIONS: The monoaminergic and cholinergic systems appear to play separable roles in different aspects of performance controlled by the 5CSRTT, in neural systems centred on the prefrontal cortex, cingulate cortex and striatum. These conclusions are considered in the methodological and theoretical context of other psychopharmacological studies of attention in animals and humans. PMID- 12373438 TI - The effects of psychotomimetics and psychomotor stimulants on two schedules promoting response switching in the rat. AB - RATIONALE: Psychosis and psychotomimetic drugs result in a disorganisation of the structure of thought and behaviour. Normalising these is one of the objects of antipsychotic therapy, and methods for predicting such a therapeutic effect would be of value. OBJECTIVE: The effects of a number of psychotomimetic agents were examined on the way in which rats distributed responding over two response levers using two different procedures, to assay their effects on behavioural organisation. Previously, amphetamine has been found to increase response switching using these schedules. METHODS: In the first, the random reinforcement procedure, one of the two levers was selected at random as "correct", and responses on this lever were reinforced with food under a random ratio schedule. No signal was given to distinguish the levers. Responding could also result in the food tray being illuminated, but no food pellet was delivered ("no-food" event). Responses on the second lever ("incorrect") had no programmed consequences. After each food delivery or "no-food" event the levers designated as "correct" and "incorrect" were reassigned at random, and the rat had to open the food tray to restart the schedule. In the second procedure, the rats were required to make 21 responses before a switch between the two levers resulted in food delivery [Fixed Ratio (FR) 21-switch]. The responses making up the FR could be distributed freely between the two levers. RESULTS: Phencyclidine (PCP), scopolamine, caffeine and ethanol increased switching under the random reinforcement procedure, but (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and atropine did not. PCP, caffeine, lysergic acid diethylamide (LSD) and atropine increased switching under the FR21-switch procedure, but ethanol did not. The increases in switching produced by PCP, LSD and the anticholinergics were accompanied by marked reductions in response rate, whereas those produced by amphetamine and caffeine were not. The effects of amphetamine, and PCP were strongly dependent on the baseline probability of switching, those of atropine and caffeine moderately so, and those of LSD and ethanol only weakly so. CONCLUSIONS: Of the agents tested, psychomotor stimulants appear to produce the most selective increases in switching. The procedures described here may be useful for assaying the disorganisation of behaviour produced by other psychotomimetics and may have value in the detection of novel antipsychotic drugs. PMID- 12373439 TI - Effect of pentobarbital, d-amphetamine, and nicotine on two models of sustained attention in pigeons. AB - RATIONALE: The animal literature examining the effects of drugs of abuse on sustained attention has provided conflicting results. One reason for these inconsistencies could be the different type of tasks used to measure sustained attention. OBJECTIVE: Acute effects of pentobarbital (0.3, 1, 3, 5.6, 10, and 13 mg/kg), d-amphetamine (0.03, 0.1, 0.3, 1, 3, and 5.6 mg/kg), and nicotine (0.03, 0.1, 0.3, 1, and 3 mg/kg) were compared in two models of sustained attention. METHODS: Dose-response curves were compared in eight male, white Carneaux pigeons trained under a continuous-trial attention procedure and in six male, white Carneaux pigeons trained under a discrete-trial attention procedure. Both procedures required subjects to respond to a variable and brief signal presentation (signal was presented on average every 6.5 s). RESULTS: Under the continuous-trial procedure, pentobarbital decreased hits as well as increased the number of false alarms and misses at doses that did not impair the ability of the animals to respond. d-Amphetamine and nicotine dose dependently decreased hits and increased misses at doses that did not impair rates of responding. However, neither psychomotor stimulant caused a significant increase in false alarms. Under the discrete-trial procedure, pentobarbital, d-amphetamine, and nicotine decreased hits and correct rejections and increased misses and errors of omission. For the most part, these drug effects occurred at doses that increased the latencies of the animals to respond. When comparing drug effects between the continuous- and discrete-trial procedures, a difference in the false alarm rate was observed. CONCLUSIONS: The present study shows that the continuous-trial procedure was able to detect differences between drug classes that were not apparent under the discrete-trial procedure. Although the lack of a true measure of a false alarm rate continues to be a problem with the continuous-trial procedure, it may be an important procedure for studying the effects of pharmacological agents. PMID- 12373440 TI - Separate and combined effects of marijuana and alcohol on mood, equilibrium and simulated driving. AB - RATIONALE: Marijuana and alcohol, when used separately and in combination, contribute to automobile accidents and failed sobriety tests of standing balance. However, the extent to which the drugs have additive effects on both of these measures is unknown. OBJECTIVES: This study was designed to compare directly the separate and combined effects of marijuana and alcohol on simulated emergency braking and dynamic posturography. METHODS: Twelve healthy subjects who regularly used both marijuana and alcohol completed nine test sessions in a counterbalanced within-subject design. Subjects drank a beverage (0, 0.25, or 0.5 g/kg alcohol) then smoked a cigarette (0, 1.75, or 3.33% THC). Testing began 2 min after smoking and was conducted within the ascending limb of the blood alcohol curve. RESULTS: The 0.5 g/kg alcohol dose significantly increased brake latency without affecting body sway. In contrast, the 3.3% THC dose increased body sway but did not affect brake latency. There were no additive drug effects on mood or behavior. CONCLUSIONS: Although field sobriety tests are often used to determine driving impairment, these results suggest that impaired balance following marijuana use may not coincide with slowed reaction time. Conversely, braking impairment from low doses of alcohol may not be revealed by tests of balance. PMID- 12373441 TI - Determinants of the behavioral effects of opioids and their antagonists: contributions of the Laboratory of Psychobiology. AB - RATIONALE: The behavioral pharmacology of opioids has been influenced significantly by the research and writings of Drs. Peter B. Dews, Roger T. Kelleher, and William H. Morse, their colleagues, and their students. OBJECTIVE: Their conceptual and methodological approach to the topic is reviewed briefly, and three areas of research are described to provide an empirical perspective. RESULTS: The objective of determining the general effects of opioids on behavior is described; the effects of opioids on schedule-controlled behavior and punished behavior are described and compared to non-opioids. The differential effects of opioid antagonists on responding reinforced by different stimuli are also presented. CONCLUSION: The conceptual and methodological approach taken by the group, as well as their discoveries in the behavioral pharmacology of opioids, will continue to exert a positive influence on the field. PMID- 12373442 TI - Kappa opioid antagonist effects of the novel kappa antagonist 5' guanidinonaltrindole (GNTI) in an assay of schedule-controlled behavior in rhesus monkeys. AB - RATIONALE: Opioid receptors are divided into three types: kappa, mu, and delta receptors. Receptor-selective antagonists are useful experimental tools for evaluation of opioid receptor-mediated processes. 5'-Guanidinonaltrindole (GNTI) was recently developed as a novel kappa-selective antagonist. OBJECTIVES: To evaluate the potency, time course, and selectivity of GNTI's opioid antagonist effects in rhesus monkeys in an assay of schedule-controlled responding. METHODS: Five rhesus monkeys were trained to respond under a fixed ratio 30 schedule of food reinforcement. The rate-decreasing effects of the kappa agonists U50,488 and U69,593, the mu agonist morphine, and the delta agonist SNC80 were examined alone and after pretreatment with GNTI (0.1 and 1.0 mg/kg i.m.; 1 h to 14 days). RESULTS: U50,488, U69,593, morphine, and SNC80 dose-dependently decreased response rates in this procedure. GNTI produced a dose- and time-dependent antagonism of the rate-decreasing effects of U50,488. The kappa antagonist effects of GNTI had a slow onset and a long duration of action, and peak antagonist effects were observed after 24 h. A higher dose of 3.2 mg/kg GNTI eliminated responding in one monkey and was not studied further. The antagonist effects of GNTI were kappa selective, because 1.0 mg/kg GNTI also antagonized the effects of U69,593, but not those of morphine or SNC80. CONCLUSIONS: These results suggest that GNTI is a potent and selective kappa antagonist with a slow onset and long duration of action in rhesus monkeys. Relative to the prototype kappa antagonist nor-binaltorphimine, GNTI may have some advantages as a tool for the study of kappa receptor-mediated processes. PMID- 12373443 TI - Antinociceptive effects of the selective delta opioid agonist SNC80 alone and in combination with mu opioids in the squirrel monkey titration procedure. AB - RATIONALE: The nonpeptidic compound SNC80 [(+)-4[(alphaR)-alpha-((2S, 5R)-4-allyl 2, 5,-dimethyl-l-piperazinyl)-3-methoxybenzyl]- N, N-diethylbenzamide], has a high degree of selectivity for delta opioid receptors. Moreover, compounds with delta opioid activity have been shown to enhance the effects of mu agonists under certain conditions. OBJECTIVES: The present study examined the effects of SNC80 alone and in combination with the mu opioid agonists, morphine, butorphanol, and buprenorphine to determine whether SNC80 would enhance their antinociceptive effects. METHODS: In the squirrel monkey shock titration procedure increasing levels of shock are delivered to the monkey's tail in incremental steps and responses on a lever decrease shock intensity. The level at which monkeys maintain the shock (median shock level, MSL) and rate of responding (RR) are examined. RESULTS: SNC80 alone did not consistently alter responding under the titration procedure; however, morphine, butorphanol, and buprenorphine increased MSL without decreasing RR markedly. SNC80 (0.1-3.0 mg/kg) enhanced the effects of single doses of morphine, butorphanol, and buprenorphine that either did not increase or produced very small increases in MSL when administered alone. Interestingly, SNC80 enhanced the effects of morphine, butorphanol, and buprenorphine on MSL without decreasing RR. CONCLUSIONS: SNC80 does not produce antinociceptive effects in the squirrel monkey titration procedure but can enhance the effects of selected doses of morphine, butorphanol, and buprenorphine on MSL without decreasing RR. These data suggest that SNC80-induced enhancement of the antinociceptive effects of mu opioids is dependent on dose, time, and method of administration and is not the result of sedation or motor dysfunction. PMID- 12373444 TI - Platelet-activating factor antagonists decrease the inflammatory nociceptive response in rats. AB - RATIONALE: Platelet-activating factor (PAF) is a membrane-derived phospholipid mediator that has biological effects on a variety of cells and tissues. A variety of stimuli, including those producing inflammation, promote the synthesis and release of PAF from various cell types. Evidence suggests that PAF exerts cellular actions through a plasma membrane receptor as well as via intracellular (microsomal) PAF binding sites. OBJECTIVE: The present study was designed to: 1) investigate the role of PAF in a model of inflammatory nociception in rats (i.e. the formalin test), and 2) localize PAF's site(s) of action in nociception. To do this, we assessed the effect of administering two PAF antagonists (BN 52021 and BN 50730, which are selective for cell surface and intracellular PAF binding sites, respectively) on formalin-induced nociceptive responses. METHODS: Forty minutes prior to formalin injection into the rat hindpaw, male Sprague-Dawley rats received systemic injections of BN 52021 (10, 1, or 0.1 mg/kg), BN 50730 (10, 1, or 0.1 mg/kg), or vehicle (45% 2-hydroxypropyl-beta-cyclodextrin in distilled water, HBC) and the effects of the drugs on nociceptive behavioral responses were measured. RESULTS: Rats receiving systemic BN 52021 or BN 50730 displayed a significant reduction of nociceptive responses in the late, but not early, phase of formalin-induced nociception. CONCLUSIONS: These findings suggest a role for endogenous PAF in nociceptive transmission, especially for persistent pain such as that which occurs in the late phase of the formalin test. The findings also indicate that both intracellular and cell surface PAF binding sites are involved in nociceptive modulation in rats, and that PAF antagonists might be useful for treating some patients with acute or chronic pain. PMID- 12373445 TI - Social and neural determinants of aggressive behavior: pharmacotherapeutic targets at serotonin, dopamine and gamma-aminobutyric acid systems. AB - BACKGROUND AND RATIONALE: Aggressive outbursts that result in harm and injury present a major problem for the public health and criminal justice systems, but there are no adequate treatment options. Obstacles at the level of social policy, institutional regulation, and scientific strategy in developing animal models continue to impede the development of specific anti-aggressive agents for emergency and long-term treatments. OBJECTIVE: To be more relevant to the clinical situation, preclinical aggression research has begun to focus on the neurobiological determinants of escalated aggressive behavior that exceeds species-typical patterns. It is the goal of this review to examine novel pharmacological and molecular tools that target the neural mechanisms for different kinds of aggressive behavior more selectively than previously possible and to outline potential pharmacotherapeutic options. RESULTS AND CONCLUSIONS: (1) The preclinical focus on the behavioral characteristics and determinants of intense aggression promises to be most relevant to the clinical distinction between the proposed impulsive-reactive-hostile-affective subtypes of human aggression and the controlled-proactive-instrumental-predatory subtypes of aggression. The neural circuits for many types of human and animal aggression critically involve serotonin, dopamine and gamma-aminobutyric acid (GABA) and specific receptor subtypes. (2) The dynamic changes in frontal cortical serotonin that are triggered by engaging in aggressive behavior imply that serotonergic drug effects are largely determined by the functional state of the receptors at the time of drug treatment. Of the numerous 5-HT receptors currently identified, the 5-HT(1B) receptors offer a promising target for reducing impulsive aggressive behavior, particularly if the action can be limited to sites in the central nervous system. (3) Aggressive confrontations are salient stressors, both for the aggressor as well as the victim of aggression, that are accompanied by activation of the mesocorticolimbic but not the striatal dopamine system. Dopaminergic manipulations, particularly targeting the D(2) receptor family, can influence aggressive behavior in animals and human patients, suggesting that mesocorticolimbic dopamine may have important enabling or permissive functions. (4) GABA is critical in the neurochemical control of aggressive behavior as evidenced by studies that directly modify GABAergic neurotransmission and neurochemical studies that correlate GABA measurements with aggressive behavioral responses in several animal species. The GABA(A) receptor complex is a mechanism through which certain benzodiazepines and alcohol enhance and inhibit aggressive behaviors. Social and pharmacological experiences decisively determine the effects of GABAergic positive modulators on aggression. PMID- 12373446 TI - Aggressive behavior as a reinforcer in mice: activation by allopregnanolone. AB - RATIONALE: The neurobiological mechanisms that underlie the motivation to engage in an aggressive confrontation remain to be investigated. OBJECTIVE: The objective was to develop a method to differentiate pharmacologically the performance elements of aggressive behavior from behaviors that precede an aggressive encounter. METHODS AND RESULTS: Male CFW mice were housed as "residents" and trained to poke their nose in a hole in a panel placed into the home cage. After fulfilling a specific response requirement, an "intruder" male mouse was introduced for a brief aggressive encounter. In experiment I, the mice were maintained on a fixed ratio schedule of ten responses (FR10) and after stable responding, extinction and stimulus control were assessed by switching the active hole in an ABA design. In experiment II and III, the mice were maintained on a fixed interval schedule of 10 min (FI10 min) and responded with accelerating rates towards the end of the interval (mean index of curvature was 0.37). In experiment III, the mice were given the GABA(A) receptor positive modulator allopregnanolone (5.6-17 mg/kg or vehicle, IP), before responding on an FI10 min schedule reinforced by a 5-min aggressive encounter. Allopregnanolone had bitonic effects on FI responding and aggressive behavior. The low dose of allopregnanolone nearly doubled overall response rate without affecting the index of curvature, attack bites or sideways threats. The moderate dose increased attack behaviors by about 45% and had little effect on response rate and the index of curvature. In contrast, the higher dose decreased the index of curvature but had no effect on aggressive behavior or overall response rate. CONCLUSIONS: These data support previous demonstrations that certain GABA(A) positive modulators heighten aggressive behavior. Moreover, examining operant responding that is reinforced by the opportunity for aggression, it may be possible to dissociate pharmacological effects on the behaviors leading up to an aggressive encounter from their effects on specific aggressive acts. PMID- 12373447 TI - On serotonin and experimental anxiety. AB - BACKGROUND: This review describes the development of a research line on the role of serotonin (5-HT) in experimental anxiety that was initiated in 1969, in the laboratory founded by P.B. Dews, W.H. Morse and R.T. Kelleher at the Harvard Medical School, and has evolved until this date. RESULTS: Initially, it was found that two non-selective 5-HT receptor antagonists released punished responding in pigeons with a magnitude comparable to that of benzodiazepine anxiolytics. This result was one of the key evidences that led to the concept that 5-HT enhanced anxiety by acting both in the forebrain and in the periaqueductal gray matter (PAG). Further evidence supported this hypothesis regarding the forebrain, but results with electrical stimulation and intracerebral drug injection into the PAG indicated that 5-HT inhibited aversive behavior evoked from this area. As a result, it has been suggested that 5-HT has a dual role in the regulation of defense, namely enhancing learned responses to potential or distal threat through actions in the forebrain while inhibiting unconditioned responses to proximal threat by acting on the PAG. The former would be related to generalized anxiety and the latter to panic disorder. To test this hypothesis, a new animal model, named the elevated T-maze, has been designed. It consists of one arm enclosed by walls that is perpendicular to two open arms elevated from the floor. The same rat performs two tasks, namely inhibitory avoidance of the elevated open arms, representing conditioned anxiety and one-way escape from one of the open arms, representative of unconditioned fear. CONCLUSION: The differential effects of drugs acting on 5-HT observed in the two tasks of the ETM generally support the hypothesis under scrutiny. PMID- 12373448 TI - Relation between discriminative and reinforcing effects of midazolam, pentobarbital, chlordiazepoxide, zolpidem, and imidazenil in baboons. AB - RATIONALE: If a psychoactive drug shares discriminative effects with one that maintains self-administration, it is often inferred that the test drug is likely to be self-administered and to have abuse liability. This presumed predictive relationship has not been studied directly, however. OBJECTIVE: To determine at the level of the individual subject (1) whether a novel drug dose that shares discriminative effects with a reinforcing drug dose also will serve as a reinforcer, and (2) whether the results of generalization tests for drugs pharmacologically similar to the training drug predict whether the test drugs will or will not be self-administered. METHODS: Baboons were trained to discriminate midazolam (0.32 mg/kg, IV) from saline and also under a schedule of IV drug reinforcement. At the beginning of a period of self-administration, the first self-injection was followed 10 min later by a drug discrimination test session. The baboon then had the opportunity to self-administer the same dose 24 h/day (3-h timeout after each injection). A second drug discrimination test followed the last self-injection of the condition. RESULTS: Zolpidem and imidazenil shared discriminative effects with midazolam. Zolpidem was reinforcing in all baboons, but imidazenil was not. Chlordiazepoxide partially shared discriminative effects with midazolam, and the rate of self-administration was low. Pentobarbital did not share discriminative effects with midazolam, but was reinforcing. For all drugs, some doses did not share discriminative effects with midazolam but were reinforcing. Generalization gradients from tests after the last self-injection were similar to those after the first self-injection. CONCLUSIONS: The discriminative effect of a drug in relation to a training drug of the same pharmacological class is not isomorphic with its reinforcing effectiveness. PMID- 12373449 TI - Some contextual and historical determinants of the effects of chlordiazepoxide on punished responding of rats. AB - RATIONALE: A host of factors that modulate the increases produced by benzodiazepines on responding suppressed by punishment have been described. Nonetheless, the necessary and sufficient conditions for the anxiolytic-like activity in this animal model have not been fully delineated. OBJECTIVES: The present experiments sought to determine the necessity of the reinforcing event (food delivery), the role of the relationship of food delivery to the punishing stimulus, and the prevailing historical context of behavior in determining the effects of chlordiazepoxide (CDAP) from 1 to 17 mg/kg, i.p. on punished responding. METHODS: Male, Sprague-Dawley rats pressed a lever under a multiple schedule. In the presence of one stimulus, every 30th response produced food and, in the presence of an alternate stimulus, every 10th response produced food, a brief electric shock, or food plus shock. Additionally, the baseline schedule was manipulated to determine antecedent experience that may contribute to the efficacy of CDAP. RESULTS: Chlordiazepoxide generally produced little or no effect under the FR 30 schedule but increased response rates under the FR 10 schedule when responding produced either food plus shock (to 600% of control) or shock alone (300% of control) but not food alone. The increases produced when shock alone was delivered were eliminated when rats did not have a history of food plus shock pairings. In addition to increasing suppressed responding, CDAP also prevented the suppression in both punished and non-punished response rates that resulted from adding a food plus shock or shock alone contingency. CONCLUSIONS: Chlordiazepoxide and perhaps benzodiazepines in general have robust efficacy for both reducing response suppression and for preventing its occurrence. This efficacy is modulated by conditions present at the time of drug exposure and by the history of the organism with respect to response contingencies. PMID- 12373450 TI - Potential antidepressant properties of pramipexole detected in locomotor and operant behavioral investigations in mice. AB - RATIONALE: Pramipexole is a dopamine agonist which binds selectively to dopamine D(3) and D(2) receptors. There is evidence that, in addition to its beneficial effects in parkinsonism, this compound may also be of value in addressing symptomatology associated with depressive diseases. OBJECTIVES: The present study was aimed at investigating behavioral effects of pramipexole that may indicate possible antidepressant properties. METHODS: We measured how different doses of pramipexole influence nocturnal locomotion and operant responding after prolonged conditioning of a schedule of FI 2 min (FI2) in female NMRI mice. RESULTS: The present data indicate that active doses of pramipexole inhibit nocturnal locomotion during the first hour after administration and later elevate activity in mice. After prolonged FI conditioning some of the mice changed to a pattern of lowered responding prior to reinforcement ("low temporal control") whereas others maintained the typical high increase of responding prior to reinforcement ("high temporal control"). Additionally, low oral doses of pramipexole increase operant behavior in "low temporal control" mice prior to reinforcement leaving the pattern of operant responding of "high temporal control" mice unchanged. CONCLUSIONS: Based on other preclinical data and initial clinical results, we speculate that these effects may reflect anti-anhedonic/antidepressive properties of pramipexole. PMID- 12373451 TI - A repeated 28-day oral dose toxicity study of genistein in rats, based on the 'Enhanced OECD Test Guideline 407' for screening endocrine-disrupting chemicals. AB - In association with the international validation project to establish an OECD Enhanced Test Guideline 407, we performed a 28-day repeated-dose toxicity study of genistein, which is known as a phytoestrogen. Attention was paid to the sensitivity of certain additional parameters, such as histopathology observations and organ weights of endocrine related organs, sperm characteristics, serum hormone levels and estrous cycle, for detecting endocrine-related effects of endocrine-disrupting chemicals based on the existing TG 407. Seven-week-old Crj:CD(SD)IGS rats were assigned to one of four groups, each consisting of ten males and ten females, and genistein was administered once daily by gavage at doses of 0 (control), 120, 400 or 1000 mg/kg body weight per day. Male rats were killed on the day after the 28th administration. Female rats were killed on the day of the diestrus stage during the 4 days after the 28th administration. Endocrine-disrupting effects of genistein were detected in females by histopathology. The changes included vacuolation and mucinification of the vaginal epithelium in the 400 and 1000 mg/kg groups; however, the incidences of the lesion were very low. Although increased serum prolactin levels were recorded in the males of the 1000 mg/kg group, we could not determine whether this was indeed induced by genistein. General toxicological effects of genistein were detected in blood chemistry, such as increased triglycerides and total protein and a decreased albumin/globulin ratio, as well as increased liver weight and glycogen deposition in the periportal hepatocytes. Based on these results, the no observed-adverse-effect level (NOAEL) in the present study was estimated to be 120 mg/kg per day. In particular, endocrine-related effects were most sensitively detected by histopathology examination of sexual organs. However, the findings indicate that chemicals with weak endocrine-disrupting potential like genistein must be evaluated taking into consideration the results of other test systems. PMID- 12373452 TI - Simultaneous determination of styrene, toluene, and xylene metabolites in urine by gas chromatography/mass spectrometry. AB - Exposure to styrene, toluene, and xylene (STX) frequently occurs in various industrial settings leading to several adverse health effects. Therefore, the biological monitoring by determination of urinary mandelic acid (MA) and phenylglyoxylic acid (PGA), hippuric acid (HA), and 2-, 3-, and 4-methylhippuric acids (2-, 3-, and 4-MHAs), the metabolites of STX, is required or at least recommended in case of workers exposed by these agents. Considering the fact that co-exposure to STX frequently occurs, methods that have been described for the separate analysis of these compounds in urine samples cannot be used effectively for monitoring. Therefore, a reliable gas chromatographic/mass spectrometric (GC/MS) method was developed for the simultaneous identification and quantification of these metabolites. Following solid phase extraction of the urine samples, the extracts were silylated and analyzed by GC/MS using a HP-5MS capillary column. The method was evaluated for linearity, limits of detection and quantification, and specificity, as well as for precision, extraction efficiency, and stability at three different concentrations prepared in urine. The assay was linear up to 0.16 mg/ml for MA, and 0.32 mg/ml for PGA, HA, and 2-, 3- and 4 MHAs. The limits of detection and quantification of STX metabolites varied between 0.001 and 0.02 mg/ml, and from 0.01 to 0.04 mg/ml, respectively. The within-day and between-day precision, determined at low, medium, and high concentrations, ranged from 2 to 12% and 2 to 19%, respectively. The extraction efficiency was 70-80%. No degradation of the metabolites occurred in the urine samples under the possible working conditions. The method was applied for the analysis of the urine samples from exposed workers. The cost- and time effectiveness, the technical advantages and validity parameters of this GC/MS analysis make it suitable for biological monitoring of mixed exposure to styrene, toluene and xylene. PMID- 12373453 TI - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces Fas-dependent activation induced cell death in superantigen-primed T cells. AB - Immune response against a foreign antigen is characterized by a growth phase, in which antigen-specific T cells clonally expand, followed by a decline phase in which the activated T cells undergo apoptosis, a process termed activation induced cell death (AICD). In the current study, we have investigated the phase at which 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) acts to downregulate the antigen-specific T cell response. To this end, C57BL/6 +/+ mice were injected with staphylococcal enterotoxin A (SEA) into the footpads (10 micro g/footpad), and simultaneously treated with TCDD (10 or 50 micro g/kg intraperitoneally). At various time points, the draining lymph node (LN) cells were analyzed for SEA activated T cells. The data demonstrated that in C57BL/6 +/+ mice, TCDD treatment did not alter the growth phase but facilitated the decline phase of SEA-reactive T cells. TCDD caused a significant decrease in the percentage and absolute numbers of CD4(+) and CD8(+) SEA-responsive T cells expressing Vbeta3(+) and Vbeta11(+) but did not affect SEA-nonresponsive Vbeta8(+) T cells. Upon in vitro culture, TCDD-exposed SEA-immunized LN cells exhibited increased levels of apoptosis when compared with the vehicle controls. When Fas-deficient (C57BL/6 lpr/lpr) or Fas ligand defective (C57BL/6 gld/gld) mice were treated with TCDD, they failed to exhibit a decrease in percentage and cellularity of SEA-reactive T cells, thereby suggesting a role of Fas-Fas ligand interactions in the TCDD induced downregulation of SEA-reactive T cell response. The resistance to TCDD induced decrease in T cell responsiveness to SEA seen in Fas- and FasL-mutant mice was neither due to decreased aryl hydrocabon receptor (AhR) expression nor to altered T cell responsiveness to SEA. The current study demonstrates that TCDD does not prevent T cell activation, but prematurely induces Fas-based AICD, which may contribute to the deletion of antigen-primed T cells. PMID- 12373454 TI - Role of metabolites in MDMA (ecstasy)-induced nephrotoxicity: an in vitro study using rat and human renal proximal tubular cells. AB - The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and hepatotoxicity. However, its potential role in ecstasy-induced kidney toxicity has yet to be investigated. Thus, primary cultures of rat and human renal proximal tubular cells (PTCs) were used to investigate the cytotoxicity induced by MDMA and its metabolites methylenedioxyamphetamine (MDA), alpha-methyldopamine (alpha-MeDA), and the glutathione (GSH) conjugates 5-(glutathion- S-yl)-alpha MeDA and 2,5- bis(glutathion- S-yl)-alpha-MeDA. Cell viability was evaluated using the mitochondrial MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MDMA and MDA were not found to be toxic to either rat or human PTCs at any concentration tested (100-800 micro M). In contrast, 800 micro M alpha-MeDA caused 60% and 40% cell death in rat and human PTCs, respectively. Conjugation of alpha-MeDA with GSH resulted in the formation of even more potent nephrotoxicants. Thus, exposure of rat and human PTC monolayers to 400 micro M 5 (glutathion- S-yl)-alpha-MeDA caused approximately 80% and 70% cell death, respectively. 5-(Glutathion- S-yl)-alpha-MeDA (400 micro M) was more toxic than 2,5- bis(glutathion- S-yl)-alpha-MeDA to rat renal PTCs but equally potent in human renal PTCs. Pre-incubation of rat PTCs with either acivicin, an inhibitor of gamma-glutamyl transpeptidase (gamma-GT), or bestatin, an inhibitor of aminopeptidase M, resulted in increased toxicity of 5-(glutathion- S-yl)-alpha MeDA but had no effect on 2,5- bis(glutathion- S-yl)-alpha-MeDA-mediated cytotoxicity. The present data provide evidence that metabolism is required for the expression of MDMA-induced renal toxicity in vitro. In addition, metabolism of 5-(glutathion- S-yl)-alpha-MeDA by gamma-GT and aminopeptidase M to the corresponding cystein- S-yl-glycine and/or cystein- S-yl conjugates is likely to be associated with detoxication of this compound. Thus, it appears that toxicity induced by thioether metabolites of ecstasy at the apical membrane of renal proximal tubular cells is the result of extracellular events, presumably redox cycling. PMID- 12373455 TI - Equipotent cholinesterase reactivation in vitro by the nerve agent antidotes HI 6 dichloride and HI 6 dimethanesulfonate. AB - The well-documented efficacy of HI 6 dichloride in reactivating acetylcholinesterase (AChE) inhibited by nerve agents is curtailed by its poor water-solubility at temperatures below 10 degrees C. This drawback can be circumvented by using HI 6 dimethanesulfonate, which has been developed in our laboratory. Since investigations on the efficacy of this new entity are lacking, it has been proposed that some bridging experiments be performed, aimed at demonstrating reactivator equivalence in vitro. The reactivating properties of the two salts were compared on human erythrocyte AChE inhibited with paraoxon, sarin, cyclosarin and agent VX. The comparison was extended to cynomolgus erythrocytes exposed in vitro for sarin and VX. Finally, mouse diaphragm preparations were circumfused with sarin, cyclosarin and VX and reactivation by each of the HI 6 salts was examined in muscle homogenates. AChE activity in erythrocyte suspensions was monitored by a modified Ellman procedure, muscle AChE by a radiometric assay. In all models tested no differences between the HI 6 salts could be detected ( P=0.05). From these data, equipotency in AChE reactivation by the two HI 6 salts can be anticipated. PMID- 12373456 TI - Modification of the striatal dopaminergic neuron system by carbon monoxide exposure in free-moving rats, as determined by in vivo brain microdialysis. AB - Acute carbon monoxide (CO) intoxication in humans results in motor deficits, which resemble those in Parkinson's disease, suggesting possible disturbance of the central dopaminergic (DAergic) neuronal system by CO exposure. In the present study, therefore, we explored the effects of CO exposure on the DAergic neuronal system in the striatum of freely moving rats by means of in vivo brain microdialysis. Exposure of rats to CO (up to 0.3%) for 40 min caused an increase in extracellular dopamine (DA) levels and a decrease in extracellular levels of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum depending on the CO concentration. Reoxygenation following termination of the CO exposure resulted in a decline of DA to the control level and an overshoot in the recovery of DOPAC and HVA to levels higher than the control. A monoamine oxidase type A (MAO-A) inhibitor, clorgyline, significantly potentiated the CO-induced increase in DA and completely abolished the subsequent overshoot in the recovery of DOPAC and HVA. Tetrodotoxin, a Na(+) channel blocker, completely abolished both the CO-induced increase in DA and the overshoot of DOPAC and HVA. A DA uptake inhibitor, nomifensine, strongly potentiated the CO-induced increase in DA without affecting the subsequent overshoot of DOPAC and HVA. Clorgyline further potentiated the effect of nomifensine on the CO-induced increase in DA, although a slight overshoot of DOPAC and HVA appeared. These findings suggest that (1) CO exposure may stimulate Na(+)-dependent DA release in addition to suppressing DA metabolism, resulting in a marked increase in extracellular DA in rat striatum, and (2) CO withdrawal and subsequent reoxygenation may enhance the oxidative metabolism, preferentially mediated by MAO-A, of the increased extracellular DA. In the light of the neurotoxicity of DA per se and reactive substances, such as quinones and activated oxygen species, generated via DA oxidation, the significant modification of the striatal DAergic neuronal system by CO exposure might participate in the neurological outcome following acute CO intoxication. PMID- 12373457 TI - In ovo carcinogenicity assay (IOCA): evaluation of mannitol, caprolactam and nitrosoproline. AB - The in ovo carcinogenicity assay (IOCA) was used to examine whether the noncarcinogens epsilon-caprolactam (CAP), D-mannitol (MAN) and nitrosoproline (NPRO) induce toxicity and subsequently morphological changes in embryonic turkey livers compared with the carcinogen diethylnitrosamine (DEN). Various doses of the test compounds were injected into fertilized turkey or quail eggs prior to incubation. Embryonic livers were collected 3-4 days before hatching and processed for histology. The positive control DEN induced hepatocyte altered foci (HAF) and karyomegalic hepatocytes, whereas histological analysis of livers from embryos exposed to CAP, MAN and NPRO did not show such histological changes. The effects of the tested compounds on liver were further examined in hepatocytes cultured from exposed turkey and quail embryos. As observed in ovo, megalocytes as well as karyomegalic hepatocytes were present in hepatocyte cultures established from DEN-exposed turkey embryos, but not from embryos exposed to CAP, MAN or NPRO. It is concluded that CAP, MAN and NPRO do not induce histological changes in embryonic liver of the type produced by the carcinogen DEN, correlating with findings for these compounds in rodent studies. PMID- 12373458 TI - Let's open the door! PMID- 12373459 TI - Is there a role for plasmapheresis/plasma exchange therapy in septic shock, MODS, and thrombocytopenia-associated multiple organ failure? We still do not know--but perhaps we are closer. PMID- 12373460 TI - Intrinsic (or auto-) PEEP during controlled mechanical ventilation. PMID- 12373461 TI - Pressure ulcers in intensive care patients: a review of risks and prevention. AB - OBJECTIVE: Review of the literature concerning pressure ulcers in the intensive care setting. DATA SOURCE AND STUDY SELECTIONS: Computerized databases (Medline from 1980 until 1999 and CINAHL from 1982 until 1999). The indexing terms for article retrieval were: "pressure ulcers", "pressure sores", "decubitus", and "intensive care". Nineteen articles met the selection criteria, and seven more were found from the references of these articles. One thesis was also analyzed. RESULTS: Data on prevention, incidence, and costs of pressure ulcers in ICU patients are scarce. Overall there are no conclusive studies on the identification of pressure ulcer risk factors. None of the existing risk assessment scales was developed especially for use in ICU patients. It is highly questionable to what extent these scales can be used in this setting as they are not even reliable in "standard care". The following risk factors might play a role in pressure ulcer development: duration of surgery and number of operations, fecal incontinence and/or diarrhea, low preoperative protein and albumin concentrations, disturbed sensory perception, moisture of the skin, impaired circulation, use of inotropic drugs, diabetes mellitus, too unstable to turn, decreased mobility, and high APACHE II score. The number of patients per study ranged from 5 from 638. The definition of "pressure ulcer" varied widely between authors or was not mentioned. CONCLUSIONS: Meaningful comparison cannot be made between the various studies because of the use of different grading systems for pressure ulcers, different methods of data collection, different (or lack of) population characteristics, unreported preventive measures, and the use of different inclusion and exclusion criteria. There is a need for well-conducted studies covering all these aspects. PMID- 12373462 TI - A multicenter survey of visiting policies in French intensive care units. AB - OBJECTIVE: To determine the visiting policies of French intensive care units. DESIGN AND SETTING: Descriptive study in intensive care units. METHODS: A questionnaire on their official visiting policies was sent to 200 French ICUs. RESULTS: Ninety-five ICUs completed the questionnaire (47.5%). Ninety-two (97%) ICUs reported restricted visiting-hour policies, allowing visits at only one or several preassigned times. Mean total daily visiting time was 168 min (range 30 370). The number of visitors was restricted in 90 ICUs (95%). The type of visitors (immediate relatives only) was restricted in 57 (60%). Visiting was forbidden for children in 10 (11%), and 41 (44%) fixed an age limit for visiting. A gowning procedure was imposed on visitors in 78 (82%). Eighteen (19%) ICUs had no waiting room available, 35 (37%) used a special room for providing families with information in addition to the waiting room, 61 (64%) provided an information leaflet. A structured first meeting was organized in 68 (71%). A last structured family meeting at the ICU discharge was provided in 6 (6%). CONCLUSIONS: Responding ICUs provide homogeneously restrictive visiting policies concerning visiting hours, number and type of visitors. However, family reception cannot be reduced to some quantitative factors and depends on multiple other parameters such as the organization of family meetings and the use of an information leaflet. These results should be an interesting starting point to observe any change in mentalities and practices in the future. PMID- 12373463 TI - A multidisciplinary quality management system for the early treatment of severely injured patients: implementation and results in two trauma centers. AB - OBJECTIVE: The impact of a multidisciplinary quality management system (MQMS) on the early treatment of severely injured patients was tested. DESIGN AND SETTING: Prospective clinical study in two level 1 trauma centers. METHODS AND MATERIALS: MQMS comprised a protocol for documentation, 20 assessment criteria, and the judgement of data by a quality circle. After implementation in Munich (1st period, n=90; 2nd period, n=77) the validation took place in Essen (1st period, n=175; 2nd period, n=150). RESULTS: Improvements in diagnostics were shown by significant time savings in radiological diagnostics and before computed tomography in severe traumatic brain injury. In patients with hemorrhagic shock there was a reduction in time before transfusion (49 to 14 min in Munich; 31 to 22 min in Essen) and before emergency operation (74 to 43 min in Munich; 69 to 45 min in Essen). The time before craniotomy was reduced from 97 to 67 min in Munich. The incidence of delayed diagnosis of life-threatening lesions was diminished from 6% to 3% in Munich (not found in Essen). The TRISS technique showed a reduction in mortality in both hospitals in the second period (Munich: 15.4% TRISS vs. 9.1% observed mortality; Essen: 17.8% vs. 11.3%). CONCLUSIONS: MQMS improved early clinical treatment in severe injury with respect to therapeutic effectiveness and outcome. The effectiveness of the MQMS was shown at two different hospitals PMID- 12373464 TI - Assessment of former ICU patients' quality of life: comparison of different quality-of-life measures. AB - OBJECTIVE: To compare three different measures to assess quality of life (QOL) after an Intensive Care Unit (ICU) stay: a standardized telephone interview, a satisfaction scale, and the Sickness Impact Profile (SIP). DESIGN: Prospective study, evaluating QOL 6 months after ICU discharge. SETTING: Medical ICU of a Swiss tertiary-care university hospital. PATIENTS AND METHODS: Patients admitted to the ICU between July and November 1998 for more than 24 h were included. Six months after ICU discharge overall QOL and health-related QOL were evaluated. Of the 118 patients approached, 85 returned valid questionnaires. RESULTS: The majority of patients indicated good QOL 6 months after ICU stay on each measure. A correlation for both overall and health-related QOL was found between the SIP and the satisfaction scales, between SIP and the telephone interviews, between the telephone interviews and satisfaction scales. The correlation between rating by scale or telephone interview and SIP in patients with cardiovascular disease differed from patients with other diagnoses. CONCLUSIONS: For the global assessment of overall or health-related QOL after ICU stay, long questionnaires such as SIP may be replaced by a short, structured telephone interview or, better, by a satisfaction scale. Quantitative measures such as SIP may be needed for comparison of therapeutic interventions or specific functional or psychosocial aspects. PMID- 12373465 TI - Intermittent versus continuous renal replacement therapy for acute renal failure in intensive care units: results from a multicenter prospective epidemiological survey. AB - OBJECTIVES: To describe the current practice of hemodialysis in acute renal failure (ARF) and to estimate the impact of hemodialysis modality on patient outcome. DESIGN: Prospective multicenter observational study conducted from March 1996 to May 1997. SETTING: The 28 multidisciplinary ICUs in the Rhone-Alpes region in France. PATIENTS: The 587 patients who required hemodialysis. MEASUREMENTS AND RESULTS: Patients were followed until hospital discharge. Among the 587 patients 354 received continuous (CRRT) and 233 intermittent (IRRT) renal replacement therapy as first choice. CRRT patients had a higher number of organ dysfunctions on admission and at the time of ARF and higher SAPS II at time of ARF. Mortality was 79% in the CRRT group and 59% in the IRRT group. Logistic regression analysis showed decreased patient survival to be associated with SAPS II on admission, oliguria, admission from hospital or emergency room, number of days between admission and ARF, cardiac dysfunction at time of ARF, and ischemic ARF. No underlying disease or nonfatal disease, and absence of hepatic dysfunction were associated with an increase in patient survival. The type of renal replacement therapy was not significantly associated with outcome. CONCLUSIONS: Renal replacement therapy mode was not found to have any prognostic value. Randomized controlled trials should be undertaken to assess this important question. PMID- 12373466 TI - Citrate anticoagulation in continuous venovenous hemodiafiltration: a metabolic challenge. AB - OBJECTIVE: Feasibility and safety evaluation of regional citrate anticoagulation (RCA) versus systemic heparinization for continuous venovenous hemodiafiltration. DESIGN AND SETTING: Combined retrospective and prospective observational study performed in a secondary multidisciplinary intensive care unit of the Ospedale Civico Lugano Switzerland. PATIENTS AND INTERVENTIONS: Twelve hemodynamically unstable patients (median APACHE II score 26, interquartile range 22-29) in whom heparin was judged to be at least temporarily contraindicated. A switch from RCA (predilution setting; same iso-osmotic replacement and dialysis fluid) to heparinization or vice versa was recommended for the final evaluation; 56 dialyzers were used for RCA (1,400 h) and 39 for heparinization (1,271 h). MEASUREMENTS AND RESULTS: Median dialyzer life span was 24.2 h (interquartile range 17.4-42.3) for RCA and 42.5 h (20.6-69.1) for heparinization. Fluid control and dialysis quality were similar in the two groups and required no additional intervention. The risk of significant hypocalcemia and metabolic alkalosis was higher at the beginning of the RCA program and decreased with the further training of the staff. Seven bleeding episodes occurred with heparinization vs. three in RCA. CONCLUSIONS: RCA may be a safe and useful form of anticoagulation which is more expensive than heparinization but helps to minimize bleeding risk. The risk of metabolic complications is higher at the beginning of a new RCA program. For centers lacking experienced staff we suggest reserving this technique for patients with rapid clotting of the extracorporeal circuit if treated without anticoagulation. PMID- 12373467 TI - Effects of spontaneous breathing during airway pressure release ventilation on renal perfusion and function in patients with acute lung injury. AB - OBJECTIVE: Controlled mechanical ventilation can impair systemic and renal blood flow and function, which may be aggravated by respiratory acidosis. We hypothesized that partial ventilatory support using airway pressure release ventilation (APRV) with spontaneous breathing provides better cardiopulmonary and renal function than full ventilatory support using APRV without spontaneous breathing. DESIGN: Prospective randomized study. SETTING: Intensive care unit of a university hospital. PATIENTS: Twelve patients with acute lung injury (ALI). INTERVENTIONS: Airway pressure release ventilation with and without spontaneous breathing, maintaining either the same minute ventilation (V(E)) or the same airway pressure (Paw) limits. MEASUREMENTS: Systemic hemodynamics were estimated by double-indicator dilution, effective renal blood flow (ERBF) by para aminohippurate, and glomerular filtration rate (GFR) by inulin clearance. RESULTS: Compared to APRV with spontaneous breathing, cardiac index (CI) was decreased when the upper Paw limit was increased to provide the same V(E) (4.26+/ 1.21 l min(-1) m(-2)vs 3.72+/-0.99 l min(-1) m(-2); p<0.05) while CI was increased when Paw limits were held constant (4.91+/-1.41 l min(-1) m(-2); p<0.05). Effective renal blood flow and GFR were higher during APRV with spontaneous breathing (858+/-388 ml min(-1) m(-2) and 94+/-47 ml min(-1) m(-2)) than during APRV without spontaneous breathing and the same V(E) (714+/-236 ml min(-1) m(-2)and 82+/-35 ml min(-1) m(-2)) or the same Paw (675+/-287 ml min(-1) m(-2) and 80+/-41 ml min(-1) m(-2); p<0.05). Urine volume did not change. CONCLUSIONS: Spontaneous breathing during APRV was associated with better renal perfusion and function than APRV without spontaneous breathing applying either the same V(E) or the same Paw limits. Maintaining spontaneous breathing during ventilatory support may, therefore, be advantageous in preventing deterioration of renal function in patients with ALI. PMID- 12373468 TI - Plasmapheresis in severe sepsis and septic shock: a prospective, randomised, controlled trial. AB - OBJECTIVE: To determine the therapeutic efficacy and safety of plasmapheresis in the treatment of patients with severe sepsis and septic shock. DESIGN: Prospective, randomised, clinical trial with a planned, midstudy, interim analysis. SETTING: Intensive care unit in a university hospital in Archangels, Russia. PATIENTS: Consecutive patients with severe sepsis or septic shock. INTERVENTIONS: One hundred and six patients were randomised to receive either standard therapy or an add-on treatment with plasmapheresis. MEASUREMENTS AND RESULTS: The primary endpoint was 28-day survival. Septic shock was diagnosed in 57% of the plasmapheresis-treated patients and 54% of the control patients. Mean APACHE III score at entry was 56.4 in the plasmapheresis group and 53.5 in the control group. The 28-day, all-cause mortality rate was 33.3% (18/54) in the plasmapheresis group and 53.8% (28/52) in the control group. This represents a relative risk for fatal outcome in the plasmapheresis group of 0.61, an absolute risk reduction of 20.5% and a number of patients needed to treat of 4.9. Apart from six transient episodes of hypotension and one allergic reaction to fresh frozen plasma, no adverse reactions were attributable to the plasmapheresis treatment in this study. CONCLUSIONS: Plasmapheresis may be an important adjuvant to conventional treatment to reduce mortality in patients with severe sepsis or septic shock. Plasmapheresis is a safe procedure in the treatment of septic patients. A prospective randomised multicentre trial is warranted to confirm our results and to determine which subgroups of septic patients will benefit most from this treatment modality. PMID- 12373469 TI - Direct costs of severe sepsis in three German intensive care units based on retrospective electronic patient record analysis of resource use. AB - OBJECTIVE: To determine the direct costs of severe sepsis patients in German intensive care units (ICUs). DESIGN: Retrospective electronic data analysis. SETTING: Three adult intensive care units (surgical/medical) in three university hospitals in Germany. PATIENTS: 385 patients identified by standard definitions as suffering from severe sepsis. MEASUREMENTS AND RESULTS: A bottom-up approach was used to determine the direct ICU cost on actual resource use (medication, laboratory tests, microbiological analysis, disposables, and clinical procedures) for patients with severe sepsis. To determine the total direct costs, center specific personnel and basic bed ("hotel") costs were added to total resources consumed. Average hospital mortality of severely septic patients was 42.6%. Mean ICU length of stay (LOS) was 16.6 days. Survivors stayed on average 4 days longer than nonsurvivors. The mean direct ICU costs of care were 23,297+/-18,631 euros per patient and 1,318 euros per day. In comparison, average daily charges being paid for an ICU patient by the health care system in Germany are 851 euros (based on official statistics). Nonsurvivors were more expensive than survivors in total direct costs (25,446 vs. 21,984 euros) and in per day direct cost (1,649 vs. 1,162 euros). Medication makes up the largest part of the direct costs, followed by expenses for personnel. CONCLUSIONS. Patients with severe sepsis have a high ICU mortality rate and long ICU LOS and are substantially expensive to treat. Nonsurviving septic patients are more costly than survivors despite shorter ICU LOS. This is due to higher medication costs indicating increased efforts to keep patients alive. PMID- 12373470 TI - Inhibition of human neutrophil chemotaxis toward interleukin 8 with six clinical antithrombin concentrates in vitro. AB - OBJECTIVE: Antithrombin exerts direct effects on neutrophils by inhibiting chemokine-induced migration. This study examined the potency of different pharmaceutical antithrombin preparations in inhibiting neutrophil chemotaxis toward interleukin 8. METHODS: Cell migration was tested by the leading front assay in modified Boyden microchemotaxis chambers bearing nitrocellulose filters. Human neutrophils were incubated with six different antithrombin concentrates or an immunopurified antithrombin preparation at concentrations of 1 micro IUeth-5 IU/ml. RESULTS: All antithrombin concentrates irrespective of the pharmaceutical source deactivated neutrophil chemotaxis. At concentrations below 100 mIU/ml neutrophil chemotaxis toward interleukin 8 was decreased by the antithrombin preparations with varying potency, but at 1 mIU/ml no significant differences were observed. CONCLUSIONS: As the ability of antithrombin to deactivate neutrophil chemotaxis toward interleukin 8 shows differences depending on the source of commercial antithrombin, these results suggest that at equivalent WHO standard concentrations clinical antithrombin concentrates may differ in anti inflammatory potential. PMID- 12373471 TI - Effect of centre-, patient- and procedure-related factors on intensive care resource utilisation after cardiac surgery. AB - OBJECTIVE: To determine associations between intensive care resource utilisation and centre-, patient- and procedure-related factors. DESIGN: Prospective multicentre cohort study. SETTING: Twenty-one European intensive care units. PATIENTS AND PARTICIPANTS: Four thousand four hundred adult patients who had undergone cardiac or thoracic aortic surgery in 21 centres. INTERVENTION: None (observational study). MEASUREMENTS AND RESULTS: Primary outcomes were duration of artificial ventilation and intensive care unit (ICU) length of stay. Exposures were centres and patient- and procedure-related factors. Both outcomes varied fourfold among centres. Median time to extubation varied from 5 to 19 h and ICU length of stay varied from 22 to 91 h. Cox regression analysis was performed to adjust risks of prolonged ventilation and ICU length of stay for patient-, procedure- and centre-related factors. Patient- and procedure-related factors were the main risk factors among individual patients, accounting for nearly two thirds of the risk of prolonged ventilation and ICU length of stay. Centre related variation accounted for the remaining risk. CONCLUSIONS: In European ICUs resource utilisation is highly variable after cardiac surgery. Up to two thirds more patients could be treated with current ICU resources if the most efficient strategies and structures were applied across all centres. PMID- 12373472 TI - Prophylactic use of the phospodiesterase III inhibitor enoximone in elderly cardiac surgery patients: effect on hemodynamics, inflammation, and markers of organ function. AB - OBJECTIVE: Elderly patients appear prone to develop overwhelming post-bypass inflammation and organ dysfunction. We assessed the effect of prophylactic administration of the phosphodiesterase III inhibitor enoximone on inflammation and organ function. DESIGN: Prospective, blinded, randomized, placebo-controlled study. SETTING: Clinical investigations on a surgical intensive care unit. PATIENTS: 40 consecutive patients aged over 80 years undergoing first-time coronary artery bypass grafting. INTERVENTIONS: Enoximone was given to 20 patients after induction of anesthesia (initial bolus 0.5 mg/kg) followed by a continuous infusion of 2.5 micro g/kg per minute until the 2nd postoperative day. Control patients ( n=20) received saline solution. MEASUREMENTS AND RESULTS: Interleukins 6, 8, and 10 and soluble adhesion molecules were measured. Liver function was assessed by the monoethylglycine-xylidide test and by measuring alpha-glutathione S-transferase plasma levels; splanchnic perfusion by continuous gastric tonometry; renal function by measuring creatinine and alpha(1) microglobulin. Interleukins increased significantly more in controls than in the enoximone-pretreated patients. Soluble adhesion molecules were significantly more increased in controls. Liver function was more altered in controls than in the enoximone-pretreated patients. alpha(1)-Microglobulin increased significantly more in controls than in the enoximone group, indicating less tubular damage in the verum group. CONCLUSION: . Prophylactic use of enoximone in cardiac surgery patients aged over 80 years resulted in less post-bypass inflammation and improvement in markers of organ function than in the placebo group. The exact mechanisms by which enoximone exerts its beneficial effects in these patients remains to be elucidated. PMID- 12373473 TI - Evaluation of a noninvasive method for cardiac output measurement in critical care patients. AB - OBJECTIVE: Thermodilution (TD) is the gold standard to monitor cardiac output (CO) in critical care. However, there is concern about the safety of right ventricular catheterization. The CO(2) rebreathing technique allows noninvasive CO determination by means of the indirect Fick principle. Our objectives were: (a) to assess the accuracy of a new system of CO measurement using the CO(2) partial rebreathing method (PRCO); (b) to evaluate whether the PRCO itself may induce changes in CO. DESIGN AND SETTING: Prospective study in the intensive care department in a university-affiliated hospital. PATIENTS: Twenty-two mechanically ventilated critically ill patients. INTERVENTIONS: CO measured simultaneously by PRCO and TDCO. MEASUREMENTS AND RESULTS: PRCO and TDCO values were compared by concordance analysis. Stability of cardiac output during PRCO was evaluated by comparing the TDCO measurements before, during, and after the partial rebreathing period using analysis of variance. From a total of 79 valid sets of measurements, bias and precision was calculated at -0.18+/-1.39 l/min. The concordance analysis of lower and intermediate CO values (<7 l/min) yielded a bias and precision calculation of -0.07+/-0.91 l/min. No changes in hemodynamics were observed during the partial rebreathing period. CONCLUSIONS: The noninvasive partial CO(2) rebreathing technique may be an alternative method for CO determination in mechanically ventilated critically ill patients. The rebreathing maneuver alone does not induce changes in CO. PMID- 12373474 TI - Blood alcohol concentration for monitoring ethanol treatment to prevent alcohol withdrawal in the intensive care unit. AB - OBJECTIVE: Alcohol withdrawal syndrome (AWS) is a serious complication during postoperative treatment in chronic alcoholics. Despite prophylactic treatment, AWS occurs in at least 25% of these patients after elective surgery. An established protocol for the prevention of AWS is ethanol administration. The aim of this study was to evaluate possible differences in ethanol dose and levels between successfully treated patients and those who developed AWS. DESIGN: Prospective, observational study with retrospective post hoc analysis. SETTING: Intensive care unit (ICU). PATIENTS: Thirty-two alcohol-dependent patients undergoing elective or emergency surgery after trauma with postoperative admission to ICU. INTERVENTIONS: Continuous postoperative i.v. ethanol substitution. MEASUREMENTS AND RESULTS: Despite treatment, 13 patients developed AWS (failure group) and therapy was successful in the other 19 patients (success group). Major complications occurred more frequently in the failure group. The total dose of ethanol treatment and ethanol levels did not differ between the groups. Ethanol levels were determined in whole arterial blood (aBAC) and simultaneously taken in venous blood (vBAC), urine (UAC) and exhaled air (EAC). The following bias and precision, compared with aBAC, were found: vBAC less than UAC less than EAC. CONCLUSIONS: There is a high failure rate for i.v. ethanol prophylaxis. None of the methods to determine alcohol concentration were sufficient to monitor suitable ethanol treatment. It therefore seems to be more useful to titrate the individual dose for each patient by closer monitoring of the clinical status, adding additional therapy to counteract AWS if higher ethanol doses are required. PMID- 12373475 TI - Early versus delayed surfactant administration in extremely premature neonates with respiratory distress syndrome ventilated by high-frequency oscillatory ventilation. AB - OBJECTIVE: To determine whether early surfactant administration is superior to selective delayed treatment in terms of improving survival and/or reducing chronic lung disease in extremely premature neonates with respiratory distress syndrome (RDS) treated by high-frequency oscillatory ventilation (HFOV). DESIGN: Prospective randomized clinical trial. SETTING: Tertiary neonatal intensive care unit (NICU) in the Perinatology Center of Prague. PATIENTS: Forty-three extremely premature infants who needed artificial ventilation within 3 h after delivery. INTERVENTIONS: Patients were randomly assigned to either early ( n=21) or delayed (n=22) administration of surfactant. All were ventilated by HFOV as the primary mode of ventilation using the high volume strategy aimed at optimizing lung volume. Curosurf at a dose of 100 mg/kg was given as a single bolus via the endotracheal tube within 1 min immediately after intubation in the early group (EARL), or during HFOV only when defined criteria were reached in the delayed (DEL) group. MEASUREMENTS AND RESULTS: No differences were noted in demographic data between the two groups. Fewer infants randomized to the EARL group required oxygen use or died at 36 weeks (combined outcome 29% vs 64%, p=0.021), and there was a lower incidence of any intraventricular hemorrhage in this group (43 vs 82%, p=0.008). CONCLUSIONS: When compared to delayed dosing, early administration of surfactant followed by HFOV facilitates and accelerates respiratory stabilization during the acute phase of severe RDS, may reduce the incidence of chronic lung disease or death and may positively influence the incidence of severe intracranial pathology in extremely premature infants with primary surfactant insufficiency. PMID- 12373476 TI - Norepinephrine-induced hyperglycemia does not increase cortical lactate in brain injured rats. AB - OBJECTIVE: Hyperglycemia aggravates ischemic brain damage. Since catecholamines increase hepatic gluconeogenesis, resulting in hyperglycemia, we investigated whether norepinephrine and dopamine elevate arterial blood glucose, thereby increasing pericontusional cortical glucose and lactate concentrations and brain edema in brain-injured rats. DESIGN: Prospective, randomized, controlled animal study. SUBJECTS: Male Sprague Dawley rats. INTERVENTIONS: Physiological saline solution, norepinephrine, or dopamine were infused intravenously for 90 min beginning 4.5 h after inducing a focal cortical contusion. Blood glucose, lactate, and pericontusional cortical extracellular glucose and lactate were determined before, during and up to 60 min after the infusion period. Thereafter brains were removed to assess hemispheric water content. MEASUREMENTS AND RESULTS: Continuous norepinephrine and dopamine infusion significantly increased pericontusional glucose concentrations, being mostly sustained by norepinephrine (NaCl: 1.3+/-0.2, dopamine: 2.7+/-0.2, norepinephrine: 4.8+/-1.1 mM). While arterial blood glucose was only significantly elevated in norepinephrine-treated rats from 8.6+/-0.6 to 12.6+/-1.6 mM, the extracellular to blood glucose ratio was significantly increased in dopamine- and norepinephrine-treated rats (0.28+/ 0.01 and 0.38+/-0.05 vs. 0.17+/-0.01). Plasma and pericontusional lactate remained unchanged, and brain edema was similar in all groups. CONCLUSIONS: Norepinephrine and dopamine significantly increased pericontusional glucose concentrations which did not elevate extracellular lactate and aggravate underlying posttraumatic edema formation. In addition to possibly increased facilitated endothelial glucose transport, the elevated extracellular to blood glucose ratio suggests a passive concentration- and pressure- dependent entry via a damaged blood-brain barrier. This might contribute to the observed reversible increase in extracellular glucose. PMID- 12373477 TI - A standardisation of the ventilator terminology is needed. PMID- 12373478 TI - Acute lactic acidosis with Wernicke's encephalopathy due to acute thiamine deficiency. PMID- 12373479 TI - Cryptococcal meningitis in a patient with systemic immunosuppression 13 years after liver transplantation. PMID- 12373480 TI - Disseminated histoplasmosis in Switzerland: an unexpected cause of septic shock and multiple organ dysfunction. PMID- 12373481 TI - Development and use of palivizumab (Synagis): a passive immunoprophylactic agent for RSV. AB - Palivizumab (Synagis; Abbott Laboratories), a humanized, monoclonal antibody, prevents lower respiratory tract infection by respiratory syncytial virus (RSV). RSV causes significant morbidity and mortality in young children worldwide and is particularly severe in pre-term infants, children with cardiopulmonary disease, and the immunosuppressed population. The first such genetically engineered agent to be used effectively against a human infectious disease, palivizumab significantly reduces the number of hospitalizations caused by RSV in high-risk infants. This article reviews the preclinical development and clinical experience of palivizumab. PMID- 12373482 TI - Cefcapene inactivates chromosome-encoded class C beta-lactamases. AB - The stability of cefcapene and cefpodoxime, oral antibacterial cephalosporins, toward different classes of beta-lactamases was evaluated. For the class A beta lactamases, TEM-1, SHV-1, and NMC-A, only the steady-state kinetic parameter ( k(cat)/ Km) values were calculated (3100 - 1.1 x 10(7) M(-1) x s(-1)), because these enzymes have very high Km values for cefpodoxime and cefotaxime. As for class B beta-lactamases L1, IMP-1, and CcrA, in general, similar k(cat)/ Km values were obtained. However, regarding class C beta-lactamases from Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, and Citrobacter freundii, we found major differences in stability between the two compounds. Cefpodoxime acted as a good substrate for the class C beta-lactamases, except for the enzyme from E. cloacae; its k(cat) and Km values were successfully calculated ( k(cat)/ Km, 1.8 x 10(5) - 1.2 x 10(7) M(-1) x s(-1)). On the other hand, cefcapene acted as a poor substrate or an inactivator for class C beta lactamases; its k(2)/ K value was successfully calculated (8.7 x 10(5) - 7.0 x 10(6) M(-1) x s(-1)). In addition, k(3) values were determined for beta lactamases from P. aeruginosa (2.3 x 10(-2) x s(-1)) and C. freundii (2.1 x 10( 1) x s(-1)). Even though these values could be calculated, transient inactivation as an enzyme reactivation reaction for all these enzymes was observed. These findings suggest the potential of cephem compounds as inhibitors of class C beta lactamases. PMID- 12373483 TI - Properties of extended-spectrum beta-lactamases constructed by site-directed mutagenesis. AB - Plasmids carrying three types of TEM-type extended-spectrum beta-lactamase (ESBL) genes, encoding TEM-3, TEM-5, and TEM-9, respectively, were constructed by site directed mutagenesis. ESBL producers were prepared by transformation of Escherichia coli JM109 with a plasmid carrying one gene of either the three TEM types, an SHV-type, or a Toho-1 group gene. This strategy with the same vector and host strain can exclude the contribution of other factors to susceptibility, and is useful in Japan, where few TEM-type ESBL producers have been isolated. In vitro antibacterial activities of 23 beta-lactam antibiotics were tested against the ESBL producers by the agar dilution method, and the results were compared. The minimum inhibitory concentrations (MICs) of penicillins tested were more than 32 micro g/ml against both the parental RTEM and ESBL producers, but they were substantially decreased by a combination with beta-lactamase inhibitors. Compared with the MICs against the ESBL-nonproducing host strain, the MICs of the cephalosporins tested for the ESBL producers were increased more than eight times in most cases and in several cases soared to more than 2048 times against a Toho 1 ESBL producer. On the other hand, the MICs of carbapenem, cephamycin, and penem antibiotics were generally comparable to those against the host strain, and were increased by 32 times at most. Kinetic analysis revealed that extended-spectrum cephalosporins were hydrolyzed only slightly to moderately by the TEM-type ESBLs, while carbapenems and a cephamycin were scarcely hydrolyzed, and rather inhibited or inactivated the mutant enzymes. PMID- 12373484 TI - Role of fosfomycin in a synergistic combination with ofloxacin against Pseudomonas aeruginosa growing in a biofilm. AB - We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa biofilms of four clinical isolates with different susceptibilities to ofloxacin. A clear synergistic effect was detected in all four strains in accordance with their susceptibilities to ofloxacin. To clarify the mechanism of this synergistic action, changes in cellular accumulation of ofloxacin into fosfomycin-pretreated cells and morphological changes in cells treated with fosfomycin, ofloxacin, or fosfomycin plus ofloxacin were investigated. Pretreatment with fosfomycin significantly enhanced cellular uptake of labeled or unlabeled ofloxacin in biofilm cells as well as in floating cells. The accumulation of ofloxacin into fosfomycin-pretreated biofilm cells was further enhanced by treating cells simultaneously with ofloxacin and fosfomycin. Morphological studies using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM) demonstrated that fosfomycin induced dramatic changes in cell shape and the outer membrane structure responsible for the altered membrane permeability of both surface and embedded biofilm cells. The resulting increased accumulation of ofloxacin in multilayers of biofilm cells was correlated with the kinetics of biofilm cell eradication, and this synergistic killing effect was confirmed by a combined study using SEM, TEM, and CLSM. PMID- 12373485 TI - Chlorpromazine has intracellular killing activity against phagocytosed Staphylococcus aureus at clinical concentrations. AB - Chlorpromazine (CPZ) has in vitro antimicrobial activity against Staphylococcus aureus at concentrations that greatly exceed those achieved clinically. It is concentrated by tissues that are rich in macrophages and it is active against phagocytosed mycobacteria when the concentration in the medium is compatible with that achieved clinically. In this report we show that nontoxic concentrations of CPZ below clinical levels have killing activity against S. aureus phagocytosed by human monocyte-derived macrophages that have nominal killing activity against these bacteria. Little or no resistance to the antimicrobial activity of this compound is anticipated to result because of its large number of cellular targets. Therefore, CPZ may have a role in the management of intracellular staphylococcal infections that normally require the use of antibiotics whose potential toxicity exceeds that associated with short-term management with CPZ. PMID- 12373486 TI - Analysis of physical and laboratory findings in nontyphoidal salmonellosis. AB - Nontyphoidal salmonellosis has a wide variety of clinical presentations. With the aim of describing the detailed clinical presentations of gastroenteritis caused by nontyphoidal Salmonella spp., findings for 126 patients (1-94 years of age; 37.0 years on average) were analyzed. Nontyphoidal salmonellosis is prevalent from April to October in Akita, when the mean atmospheric temperature exceeds 10 degrees C. On physical examination, 3 patients had rebound tenderness and muscle guarding on their abdominal wall; 1 of these patients underwent surgery for associated acute appendicitis. Elderly patients tended to be more seriously affected, resulting in severe dehydration. Elevation of the serum C-reactive protein (CRP) level correlated well with a decline in the platelet count. Although nontyphoidal salmonellosis is a common cause of acute gastroenteritis, thorough investigation and meticulous care are required so that conditions requiring surgical treatment or those that are potentially fatal are not overlooked. PMID- 12373487 TI - Use of antifungal agents in febrile patients nonresponsive to antibacterial treatment: the current status in surgical and critical care patients in Japan. AB - Disseminated candidiasis is difficult to diagnose and treat, and often becomes life-threatening in critically ill patients. However, guidelines or consensus views regarding the management of disseminated candidiasis do not exist in Japan. To develop feasible guidelines in Japan, we studied the current status of antifungal treatment in critically ill patients in Japan. From April 1999 to January 2000, critically ill patients from either surgical wards or critical care/intensive care units of 26 teaching hospitals were studied, using a prospective enrollment protocol. Patient enrollment criteria included persistent fever(> or =38 degrees C) for 3 days or more despite antibacterial agent administration. Data were entered at each institution and managed centrally using Clinware software. Of the 200 patients in the study, 68 (34%) received antifungal agents. Factors associated with antifungal treatment in the 68 patients included: central venous or pulmonary artery catheter, re-surgery, carbapenem or cephem administration, prolonged antibacterial agent administration, and high body temperature. Proven or probable disseminated candidiasis was presumed or diagnosed by culture in 34 patients, and by clinical signs and/or serological tests in 34 patients. Fungi isolated from blood included Candida albicans (57%), C. tropicalis (14%), C. parapsilosis (7%), C. glabrata (7%), and others (14%). Treatment patterns were as follows: 65 patients (96%) were treated with fluconazole (60 as monotherapy and 5 with amphotericin B); 2 patients, with miconazole; and 1 patient, with amphotericin B. Excluding 6 unevaluable cases, antifungal treatment was efficacious in 75% (21/28) of the patients with positive fungal culture and in 68% (23/34) of patients diagnosed by other means. PMID- 12373488 TI - A prospective study of hemodialysis access-related bacterial infections. AB - The objective of this study was to describe hemodialysis vascular-access related infections that occurred in hemodialysis patients over an 18-month period. The study is a prospective descriptive analysis of incidence infection rates in a hemodialysis unit in a tertiary-care medical center. Prospective surveillance for hemodialysis vascular access-related infection was performed for all patients undergoing hemodialysis from November 1999 through April 2001 at King Fahd Hospital of King Faisal University, Al-Khobar, Saudi Arabia. The total number of dialysis sessions was calculated. The type of vascular access was noted. Cultures were obtained and all infections were recorded and infection rates were calculated. There were 9627 hemodialysis sessions (5437 via permanent fistulae or grafts, 2409 via temporary central catheters, and 1781 via permanent tunneled catheters) during the 18-month study period. We identified a total of 109 infections, for a rate of 11.32/1000 dialysis sessions (ds). Of the 109, 23 involved permanent fistulae or grafts (4.23/1000 ds); 18 involved permanent tunneled central catheter infections (10.1/1000 ds); and 68 involved temporary catheter infections (28.23/1000 ds). There were 38 bloodstream infections (3.95/1000 ds) and 34 episodes of clinical sepsis (3.53/1000 ds). Seventy-one vascular access infections without bacteremia were identified (7.38/1000 ds), including 16 permanent-fistulae or graft infections (2.94/1000 ds), 7 permanent tunneled central catheter infections (3.93/1000 ds), and 48 temporary-catheter infections (19.92/1000 ds). Staphylococcal organisms were responsible for 77% of the infections, with Staphylococcus epidermidis being the strain most commonly implicated. Gram-negative organisms were responsible for 23% of the infections. In conclusion, infection rates were highest in hemodialysis patients with temporary vascular access, compared with rates in those with permanent arteriovenous fistulae and synthetic grafts. Most of the bacterial organisms isolated from the vascular access sites were gram-positive cocci, with S. epidermidis accounting for 50% of the organisms. The rate of infection with gram negative bacilli was higher than in other reports. Our greater dependence on central venous catheters, due to local factors, coupled with the immune compromising comorbid conditions of our patients, may be contributory to the pattern of infection reported. Delays in the creation of vascular grafts for hemodialysis access should be avoided. PMID- 12373489 TI - Bacteremic and leukopenic pneumococcal pneumonia: successful treatment with antibiotics, pulse steroid, and continuous hemodiafiltration. AB - We describe a case of bacteremic, leukopenic pneumococcal pneumonia with respiratory failure, accompanied by diabetic ketoacidosis and hypothermia. Pulmonary leukostasis may play a role in the pathogenesis of the acute respiratory distress syndrome (ARDS) in pneumococcal pneumonia. The patient recovered with mechanical ventilation, intravenous antibiotics, pulse-steroid therapy, and continuous hemodiafiltration (CHDF). In particular, administration of steroid and the use of CHDF may improve the status of pulmonary leukostasis in leukopenic pneumococcal infection. PMID- 12373490 TI - Successful treatment with faropenem and clarithromycin of pulmonary Mycobacterium abscessus infection. AB - Mycobacterium abscessus accounts for 80% of rapidly growing mycobacterial pulmonary infections and can be lethal. Treatment is difficult because of the paucity of effective drugs. We describe a patient with pulmonary M. abscessus infection who was treated with a regimen that included faropenem, a novel oral penem, and clarithromycin. He showed favorable responses to the treatment for more than 12 months. In vitro, faropenem had considerable inhibitory activities against 56 strains of rapidly growing mycobacteria, including M. peregrinum, M. chelonae, M. fortuitum, and M. abscessus (stated in order of increasing minimal inhibitory concentrations). Thus, faropenem has the potential to be used as an adjunctive drug with clarithromycin for the treatment of infection with rapidly growing mycobacteria, including M. abscessus. PMID- 12373491 TI - Staphylococcal scalded-skin syndrome in an adult due to methicillin-resistant Staphylococcus aureus. AB - We report a case of a 71-year-old man with staphylococcal scalded-skin syndrome (SSSS). The patient, with a chronic history of diabetes mellitus, was admitted to our hospital with lumbago, and a diagnosis of renal-cell carcinoma with bone metastasis was made. In hospital he had sudden onset of high fever and erythema, followed by the formation of flaccid bullae and exfoliation, with a positive Nikolsky sign. Methicillin-resistant Staphylococcus aureus (MRSA), producing exfoliative toxin B, was isolated from blood and bile cultures, and Aeromonas hydrophila was isolated from bile culture. Skin biopsy specimen showed a cleavage of the epidermis at the level of the granular layer. The patient was diagnosed as having SSSS and cholecystitis, and was treated with intravenous antibiotics and percutaneous transhepatic gallbladder drainage, which led to recovery. SSSS in adults is usually associated with immunosuppression. A. hydrophila is recognized as an opportunistic pathogen. SSSS should be considered in the differential diagnosis of immunocompromised adult patients with sudden onset of high fever and erythema. PMID- 12373492 TI - A patient with adult Still's disease with an increased Chlamydia pneumoniae antibody titer. AB - Adult Still's disease is an important differential diagnosis of pyretic disease and it does not necessarily appear to be a distinct disease entity. The etiology of adult Still's disease is not yet known. However, it has been considered that adult Still's disease may be triggered by certain infections, such as the Coxsackie, parvo B19, rubella, mumps, Epstein-Barr, and cytomegalo virus, as well as mycoplasma, toxoplasma, and so on. Recently, we experienced a patient with adult Still's disease with an increased Chlamydia pneumoniae antibody titer. The titer decreased slowly after the beginning of steroid therapy, associated with improvement of clinical symptoms. In this report we mention the relationship between the pathogenesis of adult Still's disease and a high titer of Chlamydia pneumoniae antibody. PMID- 12373493 TI - Fulminant Japanese spotted fever definitively diagnosed by the polymerase chain reaction method. AB - A 72-year-old man was admitted to the emergency ward in our hospital on July 20, 2001, because of consciousness disturbance, fever, generalized skin eruption, and severe general weakness beginning 7 days previously. Physical examination on admission revealed marked systemic cyanosis, erythema, and purpura. Laboratory findings indicated disseminated intravascular coagulation (DIC) and multiorgan failure (platelet count, 0.9 x 10(4)/micro l; fibrin degradation product, 110 micro g/ml; C-reactive protein, 22.6 mg/dl). Soluble interleukin 2-receptor (sIL 2R) was markedly increased to 14 710 U/ml. Blood gas analysis demonstrated severe metabolic acidosis. He was diagnosed with multiorgan failure due to DIC. Administration of heparin and sodium bicarbonate was started immediately, but respiratory failure was exacerbated and systemic spasm caused by encephalitis was noted. Although he was supported by an artificial ventilator, deterioration of metabolic acidosis occurred, and the blood pressure decreased to less than 60 mm Hg. He died 5.5 h after admission. The serological test showed no positive antibody titers against Orientia tsutsugamushi, Rickettsia japonica, or Rickettsia typhi. However, a specific DNA band derived from R. japonica was detected by the polymerase chain reaction (PCR) method using a primer from a blood clot. Therefore, he was definitively diagnosed as having Japanese spotted fever. The PCR method may be markedly useful for establishing a definitive diagnosis of Japanese spotted fever during the critical stage. PMID- 12373494 TI - Time interval between the onset of type A influenza and consultation at the outpatient clinic in a community hospital: 1999-2000 epidemic. AB - We investigated the proportion of patients with laboratory-confirmed type A influenza who visited an outpatient clinic and who were suitable for receiving treatment with anti-influenza viral agents. Between December 1999 and March 2000, in a community hospital, 40 patients were diagnosed as having type A influenza by specific antigen detection ( n = 39) and reverse transcriptase-polymerase chain reaction ( n = 1). These patients with laboratory-confirmed type A influenza were enrolled in the study. We investigated the time interval between the onset of illness and visit to the outpatient clinic at the community hospital. The results indicated that 57.5% of the patients with type A influenza visited the hospital within 1 day of the onset of illness, and 77.5% visited the hospital within 2 days. The body temperature (mean +/- SD) during the initial consultation was 38.9 +/- 0.8 degrees C ( n = 40). Seventeen of the 40 patients (42.5%) were hospitalized. In conclusion, in the majority of patients, the time from onset of symptoms to consultation was appropriate for treatment with anti-influenza viral agents. A rapid antigen-detection assay, such as Directigen Flu A, is useful for early diagnosis and allows for early treatment with anti-influenza viral agents. PMID- 12373495 TI - Respiratory tract committee, protocol composition committee, clinical trial committee, Japanese society of chemotherapy. PMID- 12373496 TI - Delayed occurrence of cytomegalovirus disease in organ transplant recipients receiving antiviral prophylaxis: are we winning the battle only to lose the war? PMID- 12373497 TI - Activity of quinolones against gram-positive cocci: mechanisms of drug action and bacterial resistance. AB - The quinolones are a potent class of antimicrobial agents that target two essential enzymes of bacterial cells: DNA gyrase and topoisomerase IV. Resistance is mediated chiefly through stepwise mutations in the genes that encode these enzymes, leading to alterations of the target site. These mutations occur in an area called the "quinolone resistance determining region". In gram-positive organisms, mutations occur more often in topoisomerase IV than in DNA gyrase. This target preference appears to depend upon two factors: the species of organism and the selecting drug. Resistance can be enhanced by a decrease in intracellular drug concentration, which is mediated through efflux pumps. The newer generation of fluoroquinolones and non-fluorinated quinolones exhibits enhanced activity against gram-positive organisms compared to the older members of this drug class, although development of resistance to these drugs has been demonstrated in vitro. This review gives a chronological perspective of the literature on the action of DNA gyrase and topoisomerase IV and the mechanisms of resistance to quinolones in staphylococci, streptococci and enterococci. PMID- 12373498 TI - Characterisation of coagulase-negative staphylococci isolated from blood infections: incidence, susceptibility to glycopeptides, and molecular epidemiology. AB - The purpose of this study was to determine incidence of coagulase-negative staphylococci (CNS) bacteraemia and to characterise the epidemiology of isolates with reduced susceptibility to glycopeptides. CNS isolates from bloodstream infections were collected and characterised by determination of the species, analysis of antibiotic susceptibility, and restriction fragment length polymorphism using pulsed-field gel electrophoresis. The medical records of patients with positive cultures and the trends in glycopeptide use were reviewed to determine the effect of previous antibiotic treatment on the susceptibility profile of these organisms. The incidence of bacteraemia caused by CNS was 0.26 per 100 patients or 0.36 per 1,000 days of hospitalisation. According to genomic fingerprinting typing, 41 (67.2%) cases of bacteraemia were caused by a unique strain of CNS and 20 were caused by several strains. Nineteen of the 61 cases of bacteraemia studied were caused by an isolate with decreased susceptibility to teicoplanin. Genomic DNA analysis of the 90 CNS isolates recovered from the 61 cases of bacteraemia generated 50 unique profiles (1 isolate per major PFGE pattern) and 13 multiple profiles (several isolates per major PFGE pattern). Neither decreased susceptibility of an isolate to teicoplanin nor hospital acquisition was associated with a multiple profile. There was a significant correlation between the incidence of bacteraemia caused by CNS with decreased susceptibility to teicoplanin and glycopeptide use at the unit level but not in individual patients. Cross-transmission did not play an important role in the dissemination of CNS with decreased susceptibility to teicoplanin, thus strains probably become resistant as a result of antibiotic pressure. Prudent use of glycopeptides is necessary to minimise the spread of resistance to these agents. PMID- 12373499 TI - Egg-based media for delayed processing of nasopharyngeal swabs in colonization studies of Streptococcus pneumoniae. AB - This report describes the development and evaluation of a selective egg-based medium for ambient-temperature storage and transport of nasopharyngeal (NP) swabs in colonization studies of Streptococcus pneumoniae. Egg-thioglycolate-antibiotic (ETA) medium is based on Dorset's egg medium but made with thioglycolate broth and the addition of gentamicin (3 mg/l), nalidixic acid (15 mg/l), and amphotericin B (2.5 mg/l). Laboratory studies were conducted using mock swabs from 34 NP samples with known colony counts of pneumococci, which had previously been frozen in STGG (skim milk, tryptone, glucose, glycerol) broth. ETA facilitated better recovery of pneumococci than did either Stuart's or Amies' transport media. In a field study of 117 children, NP swabs were placed in ETA, after being vortexed in STGG and direct-plated for colony counts. Of 52 swabs that were culture positive for pneumococci, all 52 grew pneumococci after 4 days of storage in ETA, and 49 isolates were recovered after 7 days. Transport media such as Stuart's and Amies' require the processing of swabs within about 24 h, and storage in STGG broth requires freezing at -70 degrees C. ETA should be a useful addition to the storage media available for use in epidemiological studies of pneumococcal colonization, especially in situations where prompt processing, rapid transport, or low-temperature storage are not possible. PMID- 12373500 TI - Clinical and laboratory characteristics of infective endocarditis when associated with spondylodiscitis. AB - Spondylodiscitis is rarely observed in association with infective endocarditis (IE). In the study presented here, 92 cases of definite IE were examined. Spondylodiscitis was present in 14 (15%) cases. The mean age of patients with spondylodiscitis was 69.1+/-13.6 years (range, 33-87 years). The male-to-female ratio was 8:6. Predisposing heart disease was found in nine (64.3%) cases. Back pain was reported in all cases. Spondylodiscitis was diagnosed before endocarditis in all cases. The infection affected the lumbar spine in 10 (71%) cases. A bacterium was isolated in all cases: group D Streptococcus ( n=5; 35.7%), coagulase-negative Staphylococcus ( n=4; 28.6%), and others ( n=5). Endocarditis affected predominantly the aortic valve (43%). The outcome was favourable in 12 cases. No differences in clinical features, evolution of disease, or laboratory values were found between IE patients with and IE patients without spondylodiscitis. Spondylodiscitis does not appear to worsen prognosis of IE, although the need for cardiac valve replacement seems to be more frequent in IE patients with spondylodiscitis. IE should be included in the differential diagnosis in patients with infectious spondylodiscitis and risk factors for endocarditis. In such patients, echocardiography should be performed routinely. PMID- 12373501 TI - Vibrio cholerae bacteremia in a neutropenic patient with non-small-cell lung carcinoma. AB - Vibrio cholerae was isolated from the blood cultures of a neutropenic patient treated with chemotherapy for non-small-cell lung cancer. Attempts to isolate Vibrio spp. from a rectal swab and stool were unsuccessful. Piperacillin/tazobactam treatment resulted in eradication of the microorganism from the patient's blood. Although Vibrio spp. have occasionally been the source of infection in immunocompromised patients, this report describes the first case of non-0:1 Vibrio cholerae bacteremia in a neutropenic patient with a solid tumour. PMID- 12373502 TI - Prospective assessment of a new polymerase chain reaction target (STEVOR) for imported Plasmodium falciparum malaria. AB - The diagnostic value of a polymerase chain reaction (PCR)-based method for amplifying a new target of repeated genes (STEVOR) in Plasmodium falciparum was prospectively assessed on samples from 210 febrile patients returning from areas endemic for malaria. This method is capable of detecting 0.01 parasites in one microliter of blood. Plasmodium falciparum STEVOR PCR confirmed the results of the thin- and thick-film direct examination method but identified Plasmodium falciparum in four patients in whom direct examination was inconclusive at the species level. Moreover, PCR was positive in two patients with a negative direct examination. Thus, Plasmodium falciparum STEVOR PCR had 100% sensitivity and specificity and could be used in selected parasitology laboratories when expert advice is required. PMID- 12373503 TI - Unusual presentation of leishmaniasis as an adrenal cystic mass. AB - An unusual presentation of leishmaniasis that occurred in an Italian immunocompetent woman is described. The patient had a long history of coagulopathy due to factor VIII deficiency and pain in the right lumbar region. Computed axial tomography demonstrated a cystic mass in the right adrenal gland. Histological examination of the surgically removed cyst showed the presence of histiocytes containing Leishmania amastigotes. Serodiagnosis for leishmaniasis performed through immunofluorescent antibody testing and the rK39 enzyme immunoassay was positive, whereas a bone marrow aspirate did not reveal any parasite. The patient was not treated for leishmaniasis and recovered well after surgery. Serological testing was still positive 2 years after surgery, but clinical follow-up did not reveal the signs typical of visceral leishmaniasis. PMID- 12373504 TI - Effect of highly active antiretroviral therapy on hospitalization characteristics of HIV-infected patients. AB - In order to determine the impact that highly active antiretroviral therapy (HAART) has on inpatients with HIV infection, HIV-infected patients hospitalized from 1994 to 1999 at the Department of Internal Medicine, University of Basel, Switzerland, were investigated. During the observation period, 578 HIV-related hospitalizations occurred, and 502 charts from 262 different patients were available for evaluation. Analyses showed significant reductions in hospital mortality (from 13.2% to 6.5%) and length of stay for HIV-related admissions (from 16 to 11 days) in the post-HAART period, and the percentage of AIDS-related admissions decreased from 54.5% to 47.6%. However, the admission of HIV-infected patients to the intensive care unit increased from 6.3% to 11.8%, which could indicate that treating physicians have greater confidence in the outcome of HIV infected patients due to better therapeutic options. Since the advent of HAART, the yearly number of admissions related to HIV dropped by 49% and HAART was administered often during hospital stay. By the end of the study period, death due to multiple HIV-associated diseases and wasting had disappeared. PMID- 12373505 TI - Predictive value of immunologic parameters and HIV RNA in relation to humoral pneumococcal polysaccharide vaccine responses in patients with HIV infection. AB - In the study presented here immunologic markers and HIV RNA were related to specific antibody responses in 50 HIV-infected patients who had moderate immunodeficiency (median CD4+, 295) and were vaccinated with a pneumococcal polysaccharide vaccine. Low responses were associated with low IgG2 or high IgM levels ( P=0.01) and good responses with high IgG4 ( P=0.05) or IgG2 ( P=0.07) or low beta(2) microglobulin ( P=0.04) levels. A combination of IgG2 levels >1.0 g/l and IgM <1.6 g/l at baseline significantly predicted a twofold or better response in logistic regression analysis ( P=0.025). Neither CD4+ lymphocyte counts nor HIV RNA levels were predictive, but it should be noted that good antibody responses were not restricted to patients with high CD4+ cell counts or low HIV RNA levels. PMID- 12373506 TI - Disseminated infection caused by slow-growing Mycobacterium lentiflavum. PMID- 12373507 TI - Evaluation of wider system for direct identification and antimicrobial susceptibility testing of gram-negative bacilli from positive blood culture bottles. PMID- 12373508 TI - Antimicrobial therapy for anthrax. PMID- 12373510 TI - Amino acids in neurobiology: neuroprotective and neurotoxic aspects of amino acids involved in neurotransmission and neuromodulation -- general introduction. PMID- 12373512 TI - Co-activation of the phosphatidylinositol-3-kinase/Akt signaling pathway by N methyl-D-aspartate and TrkB receptors in cerebellar granule cell neurons. AB - Neuroprotective concentrations of N-methyl-D-aspartate (NMDA) promote survival of cerebellar granule cell neurons against glutamate excitotoxicity through a TrkB receptor-mediated brain-derived neurotrophic factor (BDNF) autocrine loop. However, the intracellular signaling pathway(s) are not clear. Our results show that PI-3 kinase/Akt is activated by either NMDA or BDNF displaying differential kinetics. BDNF and NMDA increased Akt phosphorylation within 5 minutes but maximal activation by NMDA was observed at 3 hours. Akt phosphorylation was completely blocked by the PI-3 kinase inhibitor LY294002. NMDA-mediated activation of Akt was completely blocked by MK-801 and partially blocked by the TrkB receptor inhibitor, K252a, indicating the requirement of TrkB receptors for maximal activation by NMDA. In contrast, BDNF-induced Akt phosphorylation was abolished by K252a, but not by the addition of MK-801. Therefore, the PI-3 kinase/Akt pathway is co-activated by NMDA and TrkB receptors. The kinetics of BDNF and NMDA-mediated activation of PI-3 kinase/Akt suggests that they have different roles in intraneuronal time-related events. PMID- 12373513 TI - Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures. AB - Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 microM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells. However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death. We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis. We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treatment by 71%, 92% and 91%, respectively. The inhibition of PC synthesis was accompanied by a decrease in the incorporation of [(3)H]Cho into phosphocholine and by an increase of the intracellular content of free [(3)H]Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity. However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extended after the end of treatment. These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity. PMID- 12373514 TI - Expression of cell-cycle-related proteins and excitoxicity. AB - Previous work from our laboratory has suggested the functional contribution of p53 to the cascade of events triggered by excitatory amino acids and leading to cell death in primary neurons. Here we show that this paradigm can be extended to cortical neurons treated with NMDA. We found that exposure of the cells to either 300 microM or 2 mM NMDA induced an enhancement of p53 protein levels which was already significant at 60 min after the lesion, while very low staining of the protein was observed in untreated cells. The effect was time- and concentration dependent, reaching the maximal induction at 3 h. NMDA treatment also resulted in an increase of gadd45 protein levels which was evident in both treatment at 3 h, the time when p53 was maximally induced. Our data give further evidence suggesting that a repertoire of events typical of proliferating cells is activated in degenerating neurons. PMID- 12373516 TI - Domoic acid neurotoxicity in hippocampal slice cultures. AB - The neurotoxicity of domoic acid was studied in 2-3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1-100 microM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3 oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC(50) values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC(50) value of 6 microM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 microM NBQX against 3 and 10 microM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 microM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 microM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD. PMID- 12373515 TI - Nefopam, an analogue of orphenadrine, protects against both NMDA receptor dependent and independent veratridine-induced neurotoxicity. AB - Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol. PMID- 12373517 TI - Comparison of the in vitro and in vivo neurotoxicity of three new sources of kainic acid. AB - Historically, all commercially available kainic acid has been derived from a single biological source using a consistent method of extraction and purification. That source became unavailable in 1995. Recently, three new commercial suppliers of kainic acid have made the product available, but the source of the material and the purification processes used differ. Our objective was to systematically compare the response produced by each of these new sources of kainic acid using three established neurobiological techniques: neuronal cell culture, hippocampal slice electrophysiology, and whole animal behavioural toxicity. Results in all three systems indicated no overall differences between the three formulations, although studies in both cerebellar neuron cultures and whole animal toxicity testing in mice, revealed some significant differences that may imply subtle differences in receptor selectivity and/or potency. We conclude that all three sources of kainic acid are viable alternatives to traditional kainate but they may not be identical. Until further information becomes available researchers may want to avoid using the three formulations interchangeably, and take note of the source of kainic acid when evaluating literature describing results from other laboratories. PMID- 12373519 TI - Neuroprotective and neurotoxic roles of levodopa (L-DOPA) in neurodegenerative disorders relating to Parkinson's disease. AB - Despite its being the most efficacious drug for symptom reversal in Parkinson's disease (PD), there is concern that chronic levodopa (L-DOPA) treatment may be detrimental. In this paper we review the potential for L-DOPA to 1). autoxidize from a catechol to a quinone, and 2). generate other reactive oxygen species (ROS). Overt toxicity and neuroprotective effects of L-DOPA, both in vivo and in vitro, are described in the context of whether L-DOPA may accelerate or delay progression of human Parkinson's disease. PMID- 12373520 TI - L-DOPA: from a biologically inactive amino acid to a successful therapeutic agent. AB - The article traces the development of research on the naturally occurring amino acid L-3,4-dihydroxyphenylalanine (L-dopa), from the first synthesis of its D,L racemate in 1911, and the isolation of its L-isomer from seedling of Vicia faba beans to the amino acid's successful application, from 1961 onward, as the most efficacious drug treatment of Parkinson's disease (PD). Upon its isolation from legumes in 1913, L-dopa was declared to be biologically inactive. However, two early pharmacological studies, published in 1927 and 1930 respectively, proved (in the rabbit) that D,L-dopa exerted significant effects on glucose metabolism (causing marked hyperglycemia) and on arterial blood pressure. Interest in L dopa's biological activity increased considerably following the discovery, in 1938, of the enzyme L-dopa decarboxylase and the demonstration that in the animal and human body L-dopa was enzymatically converted to dopamine (DA), the first biologically active amine in the biosynthetic chain of tissue catecholamines. This prompted, in the 1940s, many studies, both in animals and in humans, especially concerned with the vasopressor potential of L-dopa/DA. In the 1950s, the focus of L-dopa research shifted to its potential for replenishing the experimentally depleted (by insulin or reserpine) peripheral and brain catecholamine stores and the concomitant restoration of normal function. During that period, of special interest were the observations that L-dopa reversed the reserpine-induced state of "tranquilisation" and that its decarboxylation product DA occurred in high amounts in animal and human brain, with a preferential localization in the basal ganglia. These observations set the stage for the beginning of DA studies in PD brain. In 1960, the severe brain DA deficit, confined to patients with PD was discovered, and a year later L-dopa's strong therapeutic effect in patients with PD was demonstrated. In 1967, the chronic high-dose oral L-dopa regimen was successfully introduced into clinical practice. Despite some initial doubts about L-dopa's mechanism of action in PD, it is now generally recognized that L-dopa use in PD is a classic example of a brain neurotransmitter replacement therapy. However, the DA replacement potential of L dopa may not be its sole action of interest, as suggested by recent evidence that L-dopa may also have its own biological activity in the CNS, independent of DA. PMID- 12373522 TI - Modeling Alzheimer's disease and other proteopathies in vivo: is seeding the key? AB - Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of beta-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5-6 months prior to the usual onset of beta-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1). Premature deposition of beta-amyloid in eight month-old, beta-amyloid precursor protein ( betaAPP) transgenic mice (Kane et al., 2000); 2). augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3). evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4). tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of beta-amyloid in vivo by AD brain extracts suggests pathogenic similarities between beta-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. PMID- 12373521 TI - Restorative effects of glutamate antagonists in experimental parkinsonism. AB - Several compounds with antagonistic actions on N-methyl-D-aspartate (NMDA) receptors were tested for an antiakinesic action in hypoactive MPTP-treated C57 BL/6 mice rendered tolerant to the motor activity enhancing effects of the 20 mg/kg, s.c., dose of L-Dopa; each compound was administered 60 min before the administration of the dopamine precursor. The classes of compounds studied included the noncompetitive NMDA antagonists, memantine, amantadine and MK-801, the competitive NMDA antagonist, CGP 40116, the anticonvulsive and putative anticonvulsive agents, lamotrigine and FCE 26743, with a partial glutamatergic antagonistic action. All six compounds elevated locomotor, rearing and total activity counts of L-Dopa-tolerant mice in co-administration with L-Dopa in dose specific or dose-dependent manners but only memantine and MK-801 affected motor activity in the control mice, that also received chronic L-Dopa treatment. Thus, the restorative actions of those compounds in suprathreshold L-Dopa-tolerant MPTP treated mice subjected to "wearing-off" of L-Dopa efficacy were assessed in a series of experiments. Within each class of potentially therapeutic agents a differential restorative efficacy of the motor activity-stimulating effects of hypoactive MPTP mice was obtained, confirming the putative antiparkinsonian applications of compounds with glutamate antagonistic actions. PMID- 12373523 TI - Submolecular adventures of brain tyrosine: what are we searching for now? AB - This overview summarizes recent findings on the role of tyrosyl radical (TyrO(*)) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO(*) in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO(*) and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO(*), ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis. Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3 NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO(*). Nitration/denitration processes can be pathological, but they also may play: 1). a signal transduction role, because nitration of tyrosine residues through TyrO(*) formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2). a role of "blocker" for radical-radical reactions (scavenging of NO(*), NO(*)(2) and CO(3)(*-) by TyrO(*)); 3). a role of limiting factors for peroxynitrite formation, by lowering O(2)(*-) formation, which is strongly linked to the pathogenesis of neural diseases. It is still not known if tyrosine oxidation/nitration via TyrO(*) formation is 1). a footprint of generalized stress and neuronal disorders, or 2). an important part of O(2)(*-) and NO(*) metabolism, or 3). merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO(*) may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points. PMID- 12373525 TI - Transcription factors involved in the pathogenesis of L-DOPA-induced dyskinesia in a rat model of Parkinson's disease. AB - L-DOPA-induced dyskinesia (abnormal involuntary movements) is one of the most debilitating complications of chronic L-DOPA pharmacotherapy in Parkinson's disease. It is generally agreed that dyskinesia arises as a consequence of pulsatile dopamine-receptor stimulation in the brain, causing downstream changes in genes and proteins. Advance in our understanding of such changes is critically dependent on the availability of suitable animal models. We have introduced a new method to classify and rate L-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA) lesioned rats. This method allows us to dissect the molecular correlates of a dyskinetic motor response to L-DOPA in this species. One of the most prominent molecular changes underlying the development of dyskinesia in the rat consists in the striatal induction of prodynorphin gene expression by L-DOPA. This effect is mediated by FosB-related transcription factors of 32-37 kDa, which are co-induced with prodynophin in striatal neurons of the "direct pathway". Both AIM development and the associated upregulation of prodynorphin mRNA by L-DOPA are significantly inhibited by the intrastriatal infusion of fosB antisense. Antisense-mediated knockdown of CREB (cyclic AMP response-element binding proteins) has however no effect. Our results identify fosB as a potential target for adjunctive antiparkinsonian therapies. PMID- 12373526 TI - Functional alteration by NMDA antagonist: effects of L-Dopa, neuroleptics drug and postnatal administration. AB - Antiakinsic effects of the uncompetitive NMDA antagonists, memantine, amantadine and MK-801, and competitive antagonists, CGP 40116, alone or in co-administration with acute subthreshold dose of L-Dopa (5 mg/kg) in MPTP-treated mice, functional alterations induced by acute MK-801 in combinations with neuroleptic compounds or behavioural deficits following postnatal administration of MK-801 were investigated. Memantine and amantadine injected 60 min before the subthreshold dose of L-Dopa (5 mg/kg), induced antiakinesic actions in hypokinesic MPTP treated mice. Concurrently, higher doses of memantine and MK-801 caused dyskinesic changes, reducing further rearing (10 and 30 mg/kg) and locomotor (30 mg/kg) behaviour of the MPTP mice; MK-801 elevated locomotion (0.1 mg/kg) but reduced rearing (0.3 mg/kg). In control, saline-treated mice, memantine (3, 10 and 30 mg/kg) and MK-801 (0.1 and 0.3 mg/kg) increased locomotor behaviour but decreased rearing behaviour. In rats, MK-801 induced marked increases in locomotor activity and disruptions of circular swim maze acquisition that were to greater or lesser extents blocked or potentiated by neuroleptic compounds: SCH 23390 (0.005 and 0.05 mg/kg) and clozapine (5.0 and 10.0 mg/kg) dose-dependently antagonised MK-801 (0.3 mg/kg) induced locomotor activity whereas raclopride (0.1 mg/kg) and haloperidol (0.1 mg/kg) attenuated it dose-specifically. Amperozide (0.5 mg/kg) attenuated the MK-801 effect but potentiated it at the 2.0 mg/kg dose. In the circular swim maze, raclopride (0.01 mg/kg) and SCH 23390 (0.05 mg/kg) improved the acquisitive performance of rats administered MK-801 (0.03 mg/kg) acutely whereas clozapine (10.0 mg/kg) and amperozide (2.0 mg/kg) deteriorated the performance of MK-801-treated rats. Postnatal administration of MK-801 (0.05 mg/kg, day 11 after birth) induced severe functional alterations in adult mice. At 70 days of age, MK-801 mice showed an initial hypoactivity followed by marked hyperactivity in the motor activity test chambers. These mice showed deficits in habituation, a nonassociative form of learning. Their hyperactivity in the test chambers was reversed by a low dose of d-amphetamine (0.25 mg/kg). Taken together, these findings display a wide range of acute/long term functional alterations induced by NMDA antagonists, particularly MK-801, associated with animal models of brain disorders. PMID- 12373527 TI - Glutamate-mediated striatal dysregulation and the pathogenesis of motor response complications in Parkinson's disease. AB - Chronically administered levodopa to Parkinson's disease (PD) patients ultimately produces alterations in motor response. Similarly, in 6-hydroxydopamine lesioned hemi-parkinsonian rats, chronic twice-daily administration of levodopa progressively shortens the duration of contralateral turning, an index of, the wearing-off fluctuations that occur in parkinsonian patients. The pathogenesis of these response alterations involves, in part, upregulation of corticostriatal glutamatergic synaptic transmission. Changes involving kinase and phosphatase signaling pathways within striatal dopaminoceptive medium-spiny neurons now appear to contribute to increased synaptic efficacy of glutamatergic receptors in these neurons. Glutamate-mediated striatal sensitization subsequently modifies basal ganglia output in ways that favor the appearance of parkinsonian motor complications. At the molecular level, transcriptional activation of striatal CREB and cdk5 may contribute to the persistent expression of these levodopa induced response alterations. Conceivably, a safer and more effective therapy for PD can be provided by drugs that target signaling proteins within striatal spiny neurons or those that interact extracellularly with non-dopaminergic receptors such as AMPA and NMDA, adenosine, adrenergic, opioid, and serotonergic. PMID- 12373528 TI - Recognition and treatment of response fluctuations in Parkinson's disease: review article. AB - Patients with Parkinson's disease (PD) by definition benefit from treatment with the dopamine precursor levodopa. However, after 5 years of therapy 50% of patients experience motor response complications (MRC's): the benefit from each dose becomes shorter (wearing-off fluctuations), more unpredictable (on-off fluctuations) and associated with involuntary movements (dyskinesias). In addition these patients suffer from fluctuations in motor function that are inherent to the disease itself. Recent findings have lead to the suggestion that hyperfunction of NMDA receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of MRC's. In PD patients blockade of striatal glutamate receptors with several NMDA-antagonists improve MRC's. With progression of PD the severity and complexity of MRC's magnify, obfuscating their pattern and their relation to the medication cycle. Only through detailed history taking and patient education will the physician be able to clarify the situation and establish a rational, targeted approach to the treatment of patients with advanced PD complicated by motor fluctuations and dyskinesias. PMID- 12373529 TI - Glutamatergic mechanisms in different disease states: overview and therapeutical implications -- an introduction. AB - Glutamate is the most widely distributed excitatory transmitter in the central nervous system (CNS). It is acting via large - and still growing - families of receptors: NMDA-, AMPA-, kainate-, and metabotropic receptors. Glutamate has been implicated in a large number of CNS disorders, and it is hoped that novel glutamate receptor ligands offer new therapeutic possibilities in disease states such as chronic pain, stroke, epilepsy, depression, drug addiction and dependence or Parkinson's disease. While an extensive preclinical literature exists showing potential beneficial effects of NMDA-, AMPA-, kainate- and metabotropic receptor ligands, only NMDA receptor antagonists have been characterized clinically to any appreciable degree. In these trials it has been shown that while several compounds are therapeutically active, they also produce serious side effects at therapeutic doses. Current interest largely centers on the development of receptor subtype-selective compounds, namely compounds selective for receptors containing the NR2B subunit. Preclinical findings and the first clinical results are encouraging, and it may be that such subunit-selective compounds may have a sufficiently wide therapeutic window to be safe for human use. PMID- 12373530 TI - Modulation of glutamate receptors: strategies for the development of novel antidepressants. AB - On a biochemical level, conventional antidepressants have been shown to modulate synaptic levels of biogenic amines (i.e., serotonin, norepinephrine, and dopamine), most often by interfering with reuptake processes or inhibiting metabolism. Strategies directed at modulating glutamatergic transmission may overcome the principal limitations (i.e., delayed onset and low efficacy) that appear to be inherent to these conventional agents. In this brief overview, I summarize two glutamate-based approaches to develop novel antidepressants. These distinct and (on a cellular level) seemingly diametric strategies may converge on intracellular pathways that are also impacted upon by chronic treatment with biogenic amine based agents. PMID- 12373531 TI - Cystine/glutamate exchange serves as the source for extracellular glutamate: modifications by repeated cocaine administration. AB - Repeated administration of cocaine lowers the basal extracellular levels of glutamate in the nucleus accumbens as measured by microdialysis. The studies presented reveal that this long-term neuroadaptation elicited by repeated cocaine results from a decrease in the activity of cystine/glutamate exchange. PMID- 12373532 TI - The effects of NMDA receptor antagonists on acute morphine antinociception in mice. AB - Antagonists of the N-methyl- d-aspartate (NMDA) receptor complex inhibit the development of tolerance to antinociceptive effects of morphine and upon acute administration, influence morphine antinociceptive activity. The analysis of numerous studies investigating acute interaction between NMDA receptor antagonists and morphine in mice indicate a variety of procedural differences and reveal that these compounds may potentiate, attenuate and produce no effect on morphine antinociception. The conditions responsible for such conflicting experimental outcome of acute interaction remain unclear. It appears that the effects of NMDA receptor antagonists on morphine tolerance are not causally related to their acute effects on morphine antinociception. PMID- 12373533 TI - Novel approaches to targeting glutamate receptors for the treatment of chronic pain: review article. AB - Glutamatergic mechanisms are implicated in acute and chronic pain, and there is a great diversity of glutamate receptors that can be used as targets for novel analgesics. Some approaches, e.g. NMDA receptor antagonism, have been validated clinically, however, the central side-effects have remained the main problem with most compounds. Recently, some novel approaches have been explored as new compounds targeting some modulatory sites at the NMDA receptor (glycine(B) and NR2B-subtype selective antagonists), as well as kainate and metabotropic glutamate receptors, have been discovered. Many of these compounds have demonstrated efficacy in animal models of chronic pain, and some of them appear to have a reduced side-effect liability compared to clinically tested NMDA antagonists. These recent advances are reviewed in the present work. PMID- 12373534 TI - Formalin-induced changes of NMDA receptor subunit expression in the spinal cord of the rat. AB - Using RT-PCR, the present study investigated the effects of formalin administration on mRNA expression coding for NMDA receptor (NR) subunits and splice variants in rat lumbar spinal cord. Subsequent to formalin injection (5%; subcutaneously) into the hind paw of Sprague-Dawley rats, the animals exhibited the typical biphasic behavioural pain response. Spinal cord (L3-6) was prepared six hours after formalin injection. In controls, NR1-b predominated over NR1-a, and NR1-2 and NR1-4 exceeded over NR1-1 and NR1-3, respectively. Regarding the NR2 subunit expression in controls, NR2B exhibited the highest expression, followed by decreasing proportions of NR2C, NR2A, and NR2D. Formalin treatment did not affect NR1 splice variant expression but significantly increased and decreased the proportion of NR2A and NR2C, respectively. In summary, the present data demonstrate adaptive changes in the NR subunit expression pattern in rat spinal cord due to formalin injection. PMID- 12373536 TI - Localization and physiological roles of metabotropic glutamate receptors in the direct and indirect pathways of the basal ganglia. AB - Our current understanding of the circuitry of the basal ganglia, and the pathophysiology of Parkinson's disease has led to major breakthroughs in the treatment of this debilitating movement disorder. Unfortunately, there are significant problems with the currently available pharmacological therapies that focus on dopamine replacement or dopaminergic agonists. Because of this, much effort has been focused on developing novel targets for the treatment of Parkinson's disease. The metabotropic glutamate receptors are a family of G protein coupled receptors activated by glutamate. These receptors are differentially distributed throughout the basal ganglia in a manner suggesting that they may provide novel targets for the treatment of movement disorders. In this review we summarize anatomical and physiological data from our work and the work of other laboratories describing the distribution and physiological roles of metabotropic glutamate receptors in the basal ganglia with emphasis on possible therapeutic targets. PMID- 12373538 TI - The role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons: review article. AB - Degeneration of dopaminergic nigrostriatal neurons is a primary cause of Parkinson's disease. Oxidative stress, excitotoxicity and mitochondrial failure are thought to be key mechanisms responsible for degeneration of dopaminergic cells. We found that the selective antagonist of the mGluR5 subtype MPEP in a dose of 5 mg/kg diminished basal and veratridine (100 microM)-stimulated dopamine release in rat striatum in an in vivo model of microdialysis. In contrast, MPEP given intrastriatally in a high concentration (500 microM) enhanced the striatal extracellular concentration of dopamine. DCG-IV (100 microM), a non-selective agonist of group II mGluRs, inhibited the veratridine-stimulated striatal dopamine release. In an animal model of neuroxicity in vivo, methamphetamine (5 x 10 mg/kg, injected at 2 h intervals) produced deficits in the striatal content of dopamine and its metabolites DOPAC and HVA 72 h after the treatment. MPEP (5 x 5 mg/kg) given before each methamphetamine injection reversed the decrease in the striatal content of dopamine and diminished the methamphetamine-induced dopamine outflow from nigrostriatal terminals. It is concluded that the MPEP-produced blockade of mGluR5 situated on dopaminergic cells, or the suppression of glutamate release in the subthalamic nucleus or substantia nigra pars reticulata may directly and indirectly cause a decrease in striatal dopamine release. However, inhibitory effect of DCG-IV on dopamine release can be induced by attenuation of excitatory input from corticostriatal terminals by activation of mGluR2/3. Regulation of dopamine carriers by MPEP, an antagonist of group I mGluRs may be responsible for the reversal of toxicity induced by methamphetamine. PMID- 12373537 TI - The role of striatal metabotropic glutamate receptors in Parkinson's disease. AB - The primary cause of Parkinson's disease is a loss of dopamine in the corpus striatum. It has been postulated that this effect leads to disinhibition of the striopallidal pathway and secondarily, to a functional shift towards glutamatergic stimulation. The aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mGluRs) in the striatum may alleviate parkinsonian-like symptoms in rats. The non-competitive antagonist of receptor subtype 5 (mGluR5), MPEP (1.0-10 mg/kg ip), or the agonist of group II mGluRs, LY354,740 (5-10 mg/kg ip), reduced haloperidol-induced muscle rigidity and catalepsy. Intrastriatal injections of the mGluR1 antagonist, (RS) AIDA (7.5-15 microg/0.5 microl), but not of the agonist of group II mGluRs, 2R,4R-APDC (7.5-15 microg/0.5 microl), inhibited the muscle rigidity induced by haloperidol. In order to search for an influence of mGluRs on the striopallidal pathway, the effect of MPEP or of the agonist of group II mGluRs, DCG-IV, on the proenkephalin (PENK) mRNA expression in the dorso lateral striatum was examined by an in situ hybridization. Repeated MPEP (6 x 10 mg/kg ip) administration did not influence PENK expression in naive rats, but diminished that increased by haloperidol. In contrast, repeated DCG-IV (3 x 1 nmol/4 microl icv) injections enhanced both the control and the haloperidol increased levels of PENK expression. The obtained results suggest that blockade of group I mGluRs, or stimulation of group II mGluRs may be important to ameliorate parkinsonian symptoms. Striatal mGluRs may contribute to at least some of these effects. PMID- 12373539 TI - In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article. AB - There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain. We have characterized novel subtype selective mGlu(5) receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu(5) receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. PMID- 12373541 TI - Neurobiology of the CNS injury and repair: New roles of amino acids, growth factors and neuropeptides -- introduction. PMID- 12373540 TI - Antidepressant-like effect of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist in the olfactory bulbectomized rats. AB - Using the olfactory bulbectomy model of depression, we examined the antidepressant-like activity of 2-methyl-6-(phenylethynyl)-pyridine (MPEP) in rats. Bulbectomized rats required a significantly greater number of trials to acquire the response similar to sham-operated controls in the passive avoidance model. Both the prolonged (but not acute) treatment with MPEP and with antidepressant drug-desipramine restored the learning deficit. The results indicate that the prolonged blockade of mGlu5 receptors exerts antidepressant like effects in rats. PMID- 12373542 TI - Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage. AB - Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo. PMID- 12373543 TI - Repeated topical application of growth hormone attenuates blood-spinal cord barrier permeability and edema formation following spinal cord injury: an experimental study in the rat using Evans blue, ([125])I-sodium and lanthanum tracers. AB - The neuroprotective efficacy of growth hormone on a focal spinal cord trauma induced alteration in the blood-spinal cord barrier (BSCB) and edema formation was examined in a rat model. Under Equithesin anaesthesia, one segment laminectomy was done over the T10-11 segments. Spinal cord injury was produced by making an incision into the right dorsal horn of the T10-11 segments (2 mm deep and 4 mm long). The animals were allowed to survive 5 h after injury. Highly purified rat growth hormone [rGH, 25 microl of a 1microg/ml solution) was applied over 10 sec topically on the exposed surface of the spinal cord 30 min before injury. The identical doses of the rGH were repeated 0 min, 30 min, 60 min, 120 min, 180 min and 240 min following injury. Saline (0.9% NaCl) treated traumatised rats at identical intervals served as controls. Traumatised rats treated with saline exhibited marked edema formation and extravasation of Evans blue and ([125])Iodine tracers in the spinal cord. At the ultrastructural level, perivascular edema and exudation of lanthanum across the endothelial cells was quite frequent in the spinal cord. Pretreatment with rGH significantly attenuated the edema formation and the extravasation of tracers in the spinal cord. In these rats, perivascular edema and infiltration of lanthanum across the endothelial cells was not much evident. These observations show that the rGH has the capacity to reduce the early manifestations of microvascular permeability disturbances and edema formation following trauma and further suggest a possible therapeutic potential of the hormone for the treatment of spinal cord injuries. PMID- 12373544 TI - Role of VEGF in an experimental model of cortical micronecrosis. AB - Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke. After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis. PMID- 12373545 TI - Nitric oxide synthase inhibitors influence dynorphin A (1-17) immunoreactivity in the rat brain following hyperthermia. AB - The possibility that nitric oxide synthase (NOS) inhibitors influence dynorphin immunoreactivity following hyperthermia was examined in a rat model using a pharmacological approach. Previous reports from our laboratory show that hyperthermia induces an upregulation of NOS in several brain regions that seems to be instrumental in causing cell injury. Recent reports suggest that nitric oxide (NO) can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. Since dynorphin is neurotoxic in different animal models of brain or spinal cord injury, it may be that the peptide will contribute to the cell injury in hyperthermia. The present investigation was carried out to determine whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of NOS will influence the peptide distribution in the brain following heat stress. Rats subjected to hyperthermia at 38 degrees C for 4 h in a biological oxygen demand incubator (BOD) resulted in a marked upregulation of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. Pretreatment of rats with two potent NOS inhibitors, L-NAME (30 mg/kg/day, i.p. for 7 days) or L-NMMA (35 mg/kg/day, i.p. for 7 days) significantly attenuated the dynorphin immunoreactivity in the brain. These drugs were also able to reduce hyperthermia induced blood-brain barrier (BBB) permeability, brain edema formation and cell injury. Taken together, our results suggest that (i). hyperthermia has the capacity to upregulate dynorphin immunoreactivity in the brain, (ii). inhibition of NOS considerably attenuates the dynorphin immunoreaction following heat stress and (iii). upregulation of dynorphin is somehow contributing to hyperthermia induced brain damage, not reported earlier. PMID- 12373546 TI - A new antioxidant compound H-290/51 modulates glutamate and GABA immunoreactivity in the rat spinal cord following trauma. AB - The involvement of the excitatory amino acid glutamate and the inhibitory amino acid gamma-amino butyric acid (GABA) in the pathophysiology of spinal cord injury is not known in details. This investigation is focused on the role of glutamate and GABA in a rat model of spinal cord trauma using immunohistochemistry. Spinal cord injury produced by a longitudinal incision of the right dorsal horn of the T10-11 segments resulted in profound edema and cell damage in the adjacent T9 segment at 5 h. Pretreatment with H-290/51 (50 mg/kg, p.o.), a potent antioxidant compound, effectively reduced the blood-spinal cord barrier (BSCB) permeability, edema formation and cell injury following trauma. At this time, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity along with a distinct decrease in GABA immunostaining in the T9 segment. These changes in glutamate and GABA immunoreactivity in traumatised rats were considerably attenuated by pretreatment with H-290/51. These results suggest that (i). oxidative stress contributes to alterations in glutamate and GABA in spinal cord injury, (ii). glutamate and GABA are important factors in the breakdown of the BSCB, edema formation and cell changes, and (iii). the antioxidant compound H 290/51 has a potential therapeutic value in the treatment of spinal cord injuries. PMID- 12373547 TI - Topical application of dynorphin A (1-17) antiserum attenuates trauma induced alterations in spinal cord evoked potentials, microvascular permeability disturbances, edema formation and cell injury: an experimental study in the rat using electrophysiological and morphological approaches. AB - Dynorphin is a neuropeptide that is present in high quantities in the dorsal horn of the spinal cord. The peptide is actively involved in pain processing pathways. However, its involvement in spinal cord injury is not well known. Alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. Infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. Antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improve motor recovery, reduce edema and cell changes. However, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. Spinal cord evoked potentials (SCEP) are good indicator of spinal cord pathology following trauma. Therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in SCEP were examined in our rat model. In addition, spinal cord edema formation, microvascular permeability disturbances and cell injury were also investigated. Our results show that topical application of dynorphin antiserum (1 : 200) two min before injury markedly attenuated the SCEP changes immediately after injury. In the antiserum treated animals, a significant reduction in the microvascular permeability, edema formation and cell injury was observed in the traumatised spinal cord. These observations suggest that (i). dynorphin is involved in the altered bioelectrical activity of the spinal cord following trauma, (ii). the peptide actively participates in the pathophysiological processes of cell injury in the spinal cord trauma, and (iii). the dynorphin antiserum has potential therapeutic value for the treatment of spinal cord injuries. PMID- 12373548 TI - Role of cholecystokinin-A and cholecystokinin-B receptors in anxiety. AB - Evidence from several laboratories indicates that the anxiogenic effects of cholecystokinin (CCK) are mediated by CCKB receptors. However, it has been reported that CCKA receptors have been found in brain and CCKA antagonists have anxiolytic properties. The aim of this work was to study whether CCKA receptors are also involved in the modulation of anxiety. Anxiogenic effects were observed in the elevated plus maze in rats when pure CCKB receptor agonists (CCK-4 and CCK 8 non-sulfated) or CCK-8S, a CCKB/CCKA agonist, were injected into the lateral ventricle. In contrast, CCK-33, a CCKA agonist or CCK-(1-21) and CCK-(26-29) were ineffective. Furthermore, the anxiogenic effects of CCK-8S were prevented by blocking CCKB but not CCKA receptors. Finally, CCK-33 injected into the postero medial nucleus accumbens failed to affect the anxiety level of the rats. These results indicate that CCKA receptors are not involved in anxiety, as measured by the paradigms used in this work. PMID- 12373550 TI - Molecular imaging of perfusion disturbances in glaucoma. AB - Ocular ischemia resulting from perfusion disturbances may play a major role in initiation of glaucoma. Possibly secondary to ischemia autoimmunogenic events are activated in glaucoma patients with increased prevalence of systemic autoimmune diseases. The determination of potential molecular markers in blood leukocytes could be useful for early noninvasive diagnostics of glaucoma. Our study using subtractive hybridization showed altered gene expression in leukocytes of glaucoma patients in comparison to age and sex matched healthy subjects. Subtracted genes encoding lymphocyte IgE receptor (Fc epsilon RII/CD23), T cell specific tyrosine kinase, thromboxan A2 receptor, alkaline phosphatase and Na(+)/K(+)-ATPase are differentially expressed in circulating leukocytes of glaucoma patients. These genes show expression profiles characteristic for adherent leukocytes which could be an important contributor to blood-brain barrier breakdown which has been found in glaucoma patients. PMID- 12373551 TI - Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro. AB - Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta. PMID- 12373552 TI - Perfusion and molecular diffusion-weighted MR imaging of the brain: in vivo assessment of tissue alteration in cerebral ischemia. AB - The combined use of perfusion imaging (PI) and diffusion-weighted imaging (DWI) is opening a new window into the processes that occur during the first hours of ischemia. DWI detects changes in molecular diffusion associated with cytotoxic edema. PI characterizes the degree of regional hypoperfusion. Regions showing mismatches between DWI and PI, i.e. hypoperfused areas with normal diffusion behavior are considered potentially salvageable. We present results of 11 patients with an occlusion of the middle cerebral artery stem and spontaneous stroke evolution. Whereas the infarct was clearly visible on initial DWI and PI, surrounding tissue at risk of infarction was marked in all patients by an increased blood volume and transit time, but only in a subgroup (n = 3) where alteration were more pronounced this tissue at risk was progressively infarcted. These human DWI and PI data show alterations in the area of tissue at risk which correlates with infarct progression. PMID- 12373553 TI - In-vivo proton MR-spectroscopy of the human brain: assessment of N acetylaspartate (NAA) reduction as a marker for neurodegeneration. AB - Proton magnetic resonance spectroscopy ((1)H-MRS) is a non-invasive method to investigate changes in brain metabolite composition in different cerebral diseases. We performed proton spectroscopy in patients with dementia of the Alzheimer's type (AD) and in patients with motor neuron disease (MND) with the aim to detect the specific metabolic pattern for these neurodegenerative disorders. In the MND group we found a significant reduction of NAA/tCr metabolite ratios in the motor cortex, which correlates with the disease severity and the clinical lateralization of neurological symptoms and further decreases in the time course of the disease. In AD patients a reduction of NAA/tCr was observed in the medial temporal lobe. Since NAA is exclusively expressed in neurons as shown by immunohistochemical studies, reduced NAA levels suggest neuronal loss or dysfunction in the observed regions. The observed regional metabolic alterations reflect the neuronal basis of the characteristic neurological symptoms in AD (dementia) and MND (muscular palsy) and mirrors the disease progress over time. PMID- 12373554 TI - Effects of cytoskeletal modifications on Ca2+ influx after cerebral ischemia. AB - The fungal toxin cytochalasin D as well as endogenous gelsolin depolymerize filamentous actin which may induce dynamic uncoupling of membrane ion channels. In vitro application of cytochalasin D reduced NMDA-induced [(3)H]noradrenaline release from mouse brain neocortical slices by 38%. In gsn deficient neocortical synaptosomes [Ca(2+)](i) increase in response to K(+) (30 mM) depolarization was 33% higher than in wild-type. After transient focal cerebral ischemia K(+) induced [Ca(2+)](i) increase in neocortical synaptosomes was 56% lower than in synaptosomes prepared from the non-ischemic contralateral hemisphere. After in vivo pretreatment with cytochalasin D 10 min before MCA occlusion K(+)-induced [Ca(2+)](i) increase in synaptosomes in vitro prepared 1 h after reperfusion from the ischemic hemisphere was only 25% lower than in contralateral synaptosomes, while cytochalasin D pretreatment in vivo did not reduce K(+)-induced [Ca(2+)](i) increase in vitro. Hence, presynaptic Ca(2+) influx and subsequently neuronal vulnerability are attenuated by increased and are aggravated by decreased F-actin depolymerization. PMID- 12373555 TI - Early molecular events in the development of the diabetic cardiomyopathy. AB - Oxidative damage to DNA has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (DM). This study evaluates possible molecular mechanisms for early events in the development of DM-induced cardiomyopathy. METHODS: To analyze the mechanism of overexpression of p21(WAF1/CIP1) and inhibition of cyclin D(1) expression in cardiomyocytes of diabetic rats we examined the methylation status of these genes by MS-PCR and assessed the possibility of epigenetic control of their expression. RESULTS: We found that the steady-state expression of both genes is influenced by their methylation status, as an epigenetic event, of their 5'-flanking regions upon development of diabetes. CONCLUSIONS: Oxidative damage contributes to the development of cardiomyopathy via p53-dependent activation of cardiac cell death. This pathway includes de novomethylation of the P53-inducible p21(WAF1/CIP1)-gene encoding a protein which binds to and inhibits a broad range of cyclin-cyclin dependent kinase complexes. PMID- 12373556 TI - Effects of dietary deficiency of selective amino acids on the function of the cornea and lens in rats. AB - Effects of dietary deficiencies of tryptophan and methionin on the transparency of cornea and lens were investigated in young rats (Brown-Norway, BN; Sprague Dawley, SD) over 3 months. Transparency of the cornea and lens were evaluated in weekly intervals using a photo-slitlamp microscope. After sacrifice and lens fresh weight determination the lenses were prepared for histopathology. Methionin deficiency had no effect on the parameters investigated. Tryptophan deficiency caused severe loss of body weight in both strains, with additional loss of hair in SD rats. These developed corneal neovascularisations and cataracts. BN rats showed an enhanced zone of discontinuity in the lens. Diet intermission arrested the pathological processes in the eye which restarted when feeding the diet again. This observation is supported by lens fresh weight data. DNA staining evidenced that tryptophan deficiency arrested lens fiber maturation in both strains but stimulated corneal neovascularisation only in SD rats. PMID- 12373557 TI - Stereospecific inhibition of monoamine uptake transporters by meta hydroxyephedrine isomers. AB - Meta-hydroxyephedrine (HED) comprises four stereoisomers consisting of two enantiomeric pairs related to ephedrine and pseudoephedrine. HED is transported into adrenergic neurons and radiolabeled HED has been employed in positron emission tomography (PET) to image adrenergic neurons in vivo. To extend structure-activity analyses of binding sites within monoamine transporters and to determine which stereoisomer displayed the best selectivity for PET imaging applications, we tested the HED compounds for their abilities to inhibit [(3)H]neurotransmitter uptake into platelets, transfected cells, and chromaffin vesicles. We hypothesized that the HED compounds would be most potent at the norepinephrine transporter (NET) compared to the serotonin or dopamine transporters and that the 1R diastereomers would be more effective than 1S diastereomers. Supporting the hypotheses, all stereoisomers were most potent at the NET and the 1R,2S stereoisomer was the most potent inhibitor overall. However, the 1S,2R isomer may be preferred for PET applications because of better selectivity among the transporters and reduced neuronal recycling. PMID- 12373558 TI - The effects of iron deficiency and iron and zinc supplementation on rat hippocampus ferritin. AB - Iron deficiency (ID), the most prevalent nutritional disorder in the world, impairs cognition in early development. The involvement of hippocampus in cognition has prompted investigation into distribution of the iron storage protein ferritin (FER) in rat hippocampus. (a) In normal rats, FER positive cells appeared first in lateral CA3 and hilus of dentate gyrus and then spread over the entire mossy fiber (MF) system. No such spread was observed in CA1 field. (b) Nutritional iron deficiency retarded development of FER in the MF system. No change in FER was observed in CA1 field. (c) Zinc distribution can be altered by iron deficiency. Thus, the effect of zinc added to iron supplementation was tested in iron-deficient rats. Significant FER recovery was observed only in hippocampal MF of rats receiving both zinc and iron. It is apparent that for accelerating recovery of hippocampal function in iron deficiency, both zinc and iron are required. PMID- 12373559 TI - Deep brain stimulation of the subthalamic nucleus versus levodopa challenge in Parkinson's disease: measuring the on- and off-conditions with FDG-PET. AB - In order to compare the effects of high-frequency stimulation of the subthalamic nucleus (STN-DBS) and a levodopa-challenge on cerebral metabolic activity, we conducted PET scans with [(18)F]2-fluoro-2-deoxyglucose (FDG) in the drug- and stimulation- on- and off-condition in a single patient suffering from advanced PD. Our data revealed evidence for improved thalamocortical processing released from inhibition by overactive basal ganglia output nuclei in both on-conditions. While levodopa also led to a reduction of lentiform hyperactivity, effective STN stimulation seemed to interfere with distinct cerebellar and limbic circuits. PMID- 12373560 TI - New perspectives on neurochemical effects of amantadine in the brain of parkinsonian patients: a PET - [(11)C]raclopride study. AB - Amantadine, is a non competitive NMDA receptors antagonist that has been proved beneficial in Parkinson's disease. However its mechanism of action at therapeutic doses is still under discussion. Aim of this study was to evaluate the effect of repeated administration of amantadine on striatal dopaminergic system by measuring [(11)C]raclopride binding to striatal D(2) dopamine receptors, in patients with moderate idiopathic Parkinson's disease. Eight patients completed the study undergoing a PET scan, before and after 10-14 days treatment with Amantadine (200 mg/day). Patients were on treatment with L-DOPA, which was suspended 1 night before each PET scans, and free from dopaminergic agonists, anticholinergic and antidepressants. Amantadine treatment significantly increased [(11)C-]Raclopride binding (caudate: 10% p = 0.04; putamen 11% p = 0.01). A slight reduction (-7.3%, p = 0.062) of UPDRS total scores was also observed. The increased availability of striatal D(2) receptors, is likely to be caused by drug induced modification of receptors expression. This hypothesis is consistent with previous experiments, indicating an increase in striatal D(2) receptors in rats treated with amantadine or other non competitive NMDA antagonists and suggests that the neo-synthesis of D(2) receptors may represent a reinforcing mechanism of drug efficacy. PMID- 12373561 TI - Local application of L- threo-hydroxyaspartate and malonate in rats in vivo induces rigidity and damages neurons of the substantia nigra, pars compacta. AB - In order to study neuronal death in Parkinson's disease, neurons of the substantia nigra, pars compacta in rats were exposed to elevated levels of glutamate and decreased levels of energy in vivo and consequences for behavior and neuronal morphology were studied. Thus, repeated local injections (9x) of the glutamate uptake inhibitor L- threo-hydroxyaspartate (L-THA; 833 microM in 0.3 microl) in the presence or absence of the succinate dehydrogenase inhibitor malonate (25 mM in 0.3 microl) were applied during three weeks. 24 h after injection, rigidity and catalepsy were measured, as well as, at the end of the three week period, locomotion, rearing and exploratory behavior. Thereafter, the cytoarchitecture of the substantia nigra, pars compacta of the brains of these rats was described. The L-THA plus malonate injected rats did not differ in their behavior from carrier injected rats, except for rigidity: their scores were higher than that of carrier and L-THA injected rats (P < 0.05), while L-THA injected rats did not differ from carrier injected controls. Observations on cresyl violet sections revealed, that, although many neurons with a shrunken nucleolus and faintly stained cytoplasm were present in both L-THA and L-THA plus malonate treated rats, the ventral edge of the substantia nigra, pars compacta containing modified cells was longer in L-THA plus malonate than in L-THA injected rats (P < 0.05). This indicates, that a minimum amount of damage to neurons in the ventral part of the substantia nigra, pars compacta might be required for the expression of rigidity. PMID- 12373562 TI - Naloxone reduces levodopa-induced dyskinesias and apomorphine-induced rotations in primate models of parkinsonism. AB - Using in situ hybridization, it was found that subchronic treatment with levodopa/benserazide increased preproenkephalin-A and preproenkephalin-B mRNAs in the dopamine-depleted striatum. In order to examine whether dysfunction of the endogenous opioid system may underlie the development of levodopa-induced dyskinesias, the effect of naloxone, an opioid antagonist, on dyskinesias was investigated in two models of parkinsonism in the common marmoset. MPTP-treated monkeys were administered a daily oral dose of levodopa/benserazide which relieved the parkinsonian symptoms but induced severe and reproducible dyskinetic movements. Naloxone (0.1, 0.2 or 0.5 mg/kg) was given subcutaneously (s.c.) during peak-dose dyskinesia, which reduced the dyskinesias significantly using the highest dose, normalized the motor activity, but did not modify the antiparkinson effect. Unilaterally 6-OHDA -lesioned marmosets received apomorphine s.c., which caused a contralateral turning behavior that could be reduced up to 35 percent by concomitant administration of naloxone. Taken together the present results suggest a possible role for the endogenous opioid system in the pathogenesis of levodopa-induced dyskinesia in primates. PMID- 12373563 TI - Brain chemistry reflects dual states of pain and anxiety in chronic low back pain. AB - The neurobiology of the interaction between pain and anxiety is unknown. The present study examined interrelationships between: regional brain chemistry (as identified by in vivo proton magnetic resonance spectroscopy [(1)H-MRS] in dorsolateral prefrontal cortex [DLPFC], orbitofrontal cortex [OFC], cingulate and thalamus), pain (as measured by short form of the McGill Pain Questionnaire [SF MPQ]), and anxiety (measured by the State-Trait Anxiety Inventory) in chronic low back pain (CLBP) patients, and contrasted to the relationship between brain chemistry and anxiety in sex and age-matched normal subjects. The results show that brain chemistry depends on a 3-way interaction of brain regions examined, subject groups (normal vs. CLBP), and anxiety levels (high vs. low). The concentration of N-Acetyl aspartate (the largest peak in (1)H-MRS) in OFC could distinguish between anxiety levels and between subject groups. Chemical perceptual relationships were analyzed by calculating correlations between regional chemicals and perceptual measures of pain and anxiety. To isolate pain from anxiety, these maps were subdivided based on anxiety and, in the CLBP patients along anxiety-more-related vs. anxiety-less-related pain descriptors and along sensory vs. affective pain descriptors. There was a precise relationship between perception and brain chemistry. The chemical-perceptual network best related to pain in CLBP patients was comprised of the DLPFC and OFC; the chemical anxiety network was best related to the OFC chemistry in normals and to all four regions studied in CLBP patients; and the cingulate was best related to the affective component of pain. We conclude that the chemical-perceptual mapping differentiates between closely related perceptual states of pain and anxiety in chronic pain and provides a brain regional-chemical-perceptual description of the long-term reorganization that occurs with chronic pain. PMID- 12373564 TI - Glycogen storage disease types 1 and 2: recent developments, management and outcome. Proceedings of an international symposium. Fulda, Germany, November 2000. PMID- 12373565 TI - Historical highlights and unsolved problems in glycogen storage disease type 1. AB - Thirty-three years after Von Gierke described the first patient with glycogen storage disease type 1 (GSD1) in 1929, the Coris detected glucose-6-phosphatase (G6Pase) deficiency. The first mutation of this enzyme was identified 41 years later and subsequently the gene was mapped to chromosome 17q21, its enzyme topology defined, a nine transmembrane helical model suggested, an enzyme deficient knockout mouse created and by infusing an adenoviral vector associated murine G6Pase gene, correction of the clinical and laboratory abnormalities was observed. A similar successful gene transfer has been performed in enzyme deficient canine puppies. To explain the function of the G6Pase complex, a multicomponent translocase catalytic model has been proposed in which different transporters shuttle glucose-6-phosphate (G6P), inorganic phosphate (Pi) and glucose across the microsomal membrane. It was suggested that GSD1b patients suffered from a G6P transporter (G6PT) defect and the first mutation in the G6PT gene subsequently recognised. The gene mapped to chromosome 11q23 and its structural organisation was defined which showed a close functional linkage between G6PT and hydrolysis. Nordlie identified the first patient with Pi transport deficiency (GSD1c). However putative GSD1c and 1d patients based on kinetic studies were found to harbour mutations in the G6PT gene so that GSD1 patients are presently divided into 1a and non-1a. G6PT deficient patients suffer from numerical and functional leucocyte defects. A mRNA leucocyte G6PT deficiency has been suggested to account for the glucose phosphorylation and subsequent calcium sequestration defects observed in theses cells. Inflammatory bowel disease which occurs frequently in GSD non-1a patients has been related to their leucocyte abnormalities. Dietary management of GSD1 patients, designed to maintain a normal blood glucose level can be achieved during the night by nocturnal gastric infusions of glucose-containing solution or by the administration of uncooked cornstarch around the clock or by a combination of both. Both therapeutic modalities, if conducted in a meticulous manner, have a major impact on the quality of life, prevention of complications and subsequent prognosis. Open questions relate to the source of endogenous glucose production in GSD1 patients which increases as a function of age from 50% to 100% of normal, concomitant with an improvement in the patients fasting tolerance. Several complications, the nature of which is incompletely understood, tend to occur after the first decade: Liver adenomata with a small risk of transforming into hepatoma, progressive renal disease, which may be related to the hyperlipidaemia observed in this disease, often leading to end stage renal failure, osteopenia apparently based on high bone turnover, growth retardation and delayed puberty. CONCLUSION: this review highlights the present knowledge of glycogen storage disease type 1 and subtypes, discussing unsolved questions, which reflect the limitation of our knowledge in the understanding of this intriguing group of diseases. PMID- 12373566 TI - Glycogen storage disease type I: diagnosis and phenotype/genotype correlation. AB - Glycogen storage disease type Ia (GSD Ia) is caused by mutations in the G6PC gene encoding the phosphatase of the microsomal glucose-6-phosphatase system. GSD Ia is characterized by hepatomegaly, hypoglycemia, lactic acidemia, hyperuricemia, hyperlipidemia and short stature. Other forms of GSD I (GSD I non-a) are characterized by the additional symptom of frequent infections caused by neutropenia and neutrophil dysfunction. GSD I non-a is caused by mutations in a gene encoding glucose-6-phosphatase translocase (G6PT1). We report on the molecular genetic analyses of G6PC and G6PT1 in 130 GSD Ia patients and 15 GSD I non-a patients, respectively, and provide an overview of the current literature pertaining to the molecular genetics of GSD I. Among the GSD Ia patients, 34 different mutations were identified, two of which have not been described before (A65P; F177C). Seventeen different mutations were detected in the GSD I non-a patients. True common mutations were identified neither in GSD Ia nor in GSD I non-a patients. CONCLUSION: Glycogen storage disease type Ia and and type I non-a are genetically heterogenous disorders. For the diagnosis of the various forms of glycogen storage disease type I, molecular genetic analyses are reliable and convenient alternatives to the enzyme assays in liver biopsy specimens. Some genotype-phenotype correlations exist, for example, homozygosity for one G6PC mutation, G188R, seems to be associated with a glycogen storage disease type I non-a phenotype and homozygosity for the 727G>T mutation may be associated with a milder phenotype but an increased risk for hepatocellular carcinoma. PMID- 12373567 TI - Glycogen storage disease type I: diagnosis, management, clinical course and outcome. Results of the European Study on Glycogen Storage Disease Type I (ESGSD I). AB - Glycogen storage disease type I (GSD I) is a relatively rare metabolic disease and therefore, no metabolic centre has experience of large numbers of patients. To document outcome, to develop guidelines about (long-term) management and follow-up, and to develop therapeutic strategies, the collaborative European Study on GSD I (ESGSD I) was initiated. This paper is a descriptive analysis of data obtained from the retrospective part of the ESGSD I. Included were 231 GSD Ia and 57 GSD Ib patients. Median age of data collection was 10.4 years (range 0.4-45.4 years) for Ia and 7.1 years (0.4-30.6 years) for Ib patients. Data on dietary treatment, pharmacological treatment, and outcome including mental development, hyperlipidaemia and its complications, hyperuricaemia and its complications, bleeding tendency, anaemia, osteopenia, hepatomegaly, liver adenomas and carcinomas, progressive renal disease, height and adult height, pubertal development and bone maturation, school type, employment, and pregnancies are presented. Data on neutropenia, neutrophil dysfunction, infections, inflammatory bowel disease, and the use of granulocyte colony stimulating factor are presented elsewhere (Visser et al. 2000, J Pediatr 137:187 191; Visser et al. 2002, Eur J Pediatr DOI 10.1007/s00431-002-1010-0). CONCLUSION: there is still wide variation in methods of dietary and pharmacological treatment of glycogen storage disease type I. Intensive dietary treatment will improve, but not correct completely, clinical and biochemical status and fewer patients will die as a direct consequence of acute metabolic derangement. With ageing, more and more complications will develop of which progressive renal disease and the complications related to liver adenomas are likely to be two major causes of morbidity and mortality. PMID- 12373568 TI - Effect of continuous glucose therapy with uncooked cornstarch on the long-term clinical course of type 1a glycogen storage disease. AB - To evaluate the effects of uncooked cornstarch (UCS) on metabolic control, growth, and complications of pubertal and postpubertal subjects with type 1a glycogen storage disease, we studied 26 subjects (16 males), mean age 20.8+/-5.1 years, in whom continuous glucose therapy with cornstarch began at 6.8+/-4.3 years. At the time of this analysis, subjects had received cornstarch for 14.1+/ 3.5 years. Metabolic control was determined with subjects receiving their usual home dietary regimens: 4.1+/-1.3 doses of UCS in the day (9.7+/-2.6 g/h) and 2.0+/-0.4 doses at night (11.7+/-2.2 g/h). Mean height standard deviation score (SDS) was -1.2+/-1.3, significantly less than the mean target height of -0.2+/ 1.1 ( P<0.01). Mean weight SDS was 0.5+/-1.9 and body mass index SDS was 0.7+/ 1.0. Of all subjects, 50% had at least one focal hepatic lesion consistent with an adenoma. Urinary albumin excretion was increased (>20 micro g/min) in 31% of subjects; two subjects had clinical albuminuria (>300 mg per 24 h), but none has progressed to chronic renal insufficiency. Of 26 subjects, 13 (50%) had anemia. All of the complications were associated with evidence of suboptimal metabolic control, whereas subjects with no evidence of any long-term complications had near normal blood lactate and total CO(2) concentrations. CONCLUSION: The achievement of optimal biochemical control of glycogen storage disease type 1a continues to be a challenge, but is attainable by meticulous adherence to an individualized dietary regimen based on the results of periodic metabolic evaluation and home blood glucose monitoring. Minimizing the metabolic abnormalities of the disease may decrease the risk of long-term complications. PMID- 12373569 TI - Type I glycogen storage disease: favourable outcome on a strict management regimen avoiding increased lactate production during childhood and adolescence. AB - Our objective was to evaluate the long-term effects of dietary therapy of type I glycogen storage disease which avoids increased lactate production during childhood and adolescence. In order to suppress hepatic glucose and increased lactate production consistently day and night, the treatment regimen included nocturnal intragastric feeding of glucose polymer during childhood and adolescence. The aim was to keep the blood glucose concentration in the "high normal range" (4.3-5.5 mmol/l) and the lactate concentration in urine in the normal range (<0.06 mol/mol creatinine). The amounts of dietary carbohydrate required decreased in an age-related manner from 11.9+/-1.3 mg/kg body weight per min by day and 6.9+/-0.9 mg/kg body weight per min by night at 1 year of age to 5.2+/-1.0 and 2.9+/-1.2 mg/kg body weight per min, respectively, at the age of 16 years. In 15 infants, therapy started at 5.8+/-3.2 months of age and induced catch up growth over 1-2 years by which time the mean height SDS increased from 1.02+/-0.91 to -0.19+/-1.07. In the well controlled patients, further growth continued within that range. From 12 years of age, mean height SDS was in line with the respective mean SDS of mid-parental target height. The plasma lipid concentrations were markedly reduced, but were not brought into the normal range. So far, no adolescent showed liver adenoma or renal damage. Four patients with poor metabolic control due to poor compliance with treatment (frequently subnormal plasma glucose concentrations, severe hypoglycaemia, and increased urinary lactate excretion) showed retardation of growth and bone maturation. CONCLUSION: avoiding increased lactate production by keeping the blood glucose concentration permanently in the "high normal range" seems to be crucial for growth according to the genetic potential. PMID- 12373570 TI - Glycogen storage disease type I: pathophysiology of liver adenomas. AB - Of the many complications associated with glycogen storage disease type I, hepatic tumours cause great concern because of their malignant potential and the current difficulties in monitoring them. Hepatic adenomas occur in 22%-75% of affected adults, according to the population studied, and from those reported in the literature are thought to have an approximately 10% risk of undergoing malignant transformation. Their aetiology is unclear, but they occur generally in post-pubertal patients, and can be either single or multiple. This article discusses theories of their aetiology, methods of detection and monitoring, and treatment options. CONCLUSION: the incidence of liver tumours in younger adults seems less than in older ones, suggesting that better dietary treatment, and thus improved metabolic control, may be protective. Surgery (partial hepatectomy or orthotopic liver transplantation) is the definitive therapy for these tumours, but the timing of this intervention is difficult to determine and it is not without its own hazards. PMID- 12373571 TI - The muscle-bone relationship: methods and management - perspectives in glycogen storage disease. AB - Currently bone development is commonly presented as a process leading to the 'accumulation of peak bone mass'. Consequently, the usual approach to a suspected bone disorder in a child is to address the question are this child's bones as heavy as those of a healthy child of the same sex and age? However, from a functional perspective the aim of bone development should not be make bones as heavy as possible but to make them as stable as necessary. A functionally oriented approach should address two different questions: how strong are the bones? are they as strong as they need to be? It is clear that the bone has to be strong enough to withstand the mechanical forces to which it is exposed. CONCLUSION: since the main forces applied to bones are due to muscle action, the strength of a bone should be related to the force of the muscles attached to it. PMID- 12373572 TI - Glycogen storage disease type I: indications for liver and/or kidney transplantation. AB - Even though significant progress has been achieved in the management of patients with glycogen storage disease type I, hepatic (mainly adenomas) and renal (proteinuria, renal failure) complications may still develop. Orthotopic liver transplantation has been reported in less than 20 patients, and, in most cases, its indications were multiple hepatic adenomas, sometimes combined with poor metabolic control and/or growth retardation. Even though short-term outcome seems to be favourable, long-term complications have been reported in several cases. Thus it appears that improved metabolic control has to be attempted before performing liver transplantation in such patients. As for renal transplantation, it has been performed in patients with terminal renal failure. It is hoped that improving long-term metabolic control will prevent renal involvement from evolving to terminal renal failure. Finally, combined liver and kidney transplantation may be indicated in a few patients. CONCLUSION: organ (liver/kidney) transplantation in glycogen storage disease type I may be advantageous when long-term metabolic control has been attempted. Nevertheless, post-transplantat long-term complications may still develop. PMID- 12373573 TI - Adenovirus-mediated gene therapy in a mouse model of glycogen storage disease type 1a. AB - Glycogen storage disease type 1a (GSD-1a), characterized by growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia, and renal dysfunction, is caused by deficiencies in glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. Over the last 20 years, dietary therapies have greatly improved the prognosis of GSD-1a patients. However, the underlying pathological process remains uncorrected and the efficacy of dietary treatment is frequently limited by poor compliance. Therefore, long-term complications still develop in adult patients. To develop future therapeutic approaches for GSD-1a, we have generated G6Pase-deficient (G6Pase(-/-)) mice that mimic the pathophysiology of human GSD-1a patients. To evaluate the feasibility of gene replacement therapy for this disorder, we have infused recombinant adenovirus containing murine G6Pase gene (Ad-mG6Pase) into G6Pase(-/-) mice. While only 15% of G6Pase(-/-) mice under glucose therapy survived weaning, a 100% survival rate was achieved when G6Pase(-/-) mice were infused with Ad-mG6Pase and 90% of which lived to 3 months of age. Hepatic G6Pase activity in Ad-mG6Pase infused mice was restored to 19% of that in G6Pase(+/+) mice at 7 through 14 post infusion days. Ad-mG6Pase infusion also greatly improved growth of G6Pase(-/-) mice and normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles. Further, liver and kidney enlargement were less pronounced with near normal levels of glycogen depositions in both organs. CONCLUSION: our data demonstrate that a single administration of a recombinant adenovirus vector can alleviate the clinical manifestations of glycogen storage disease type 1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy. PMID- 12373574 TI - Is glycogen storage disease 1a associated with atherosclerosis? AB - Deficiency of microsomal glucose-6-phosphatase in liver and kidney leads to glycogen storage disease type 1a (GSD 1a). Notwithstanding intensive dietary therapy, moderate to severe dyslipidaemia and microalbuminuria, both known atherosclerotic risk factors, remain present. Although more patients reach adult age, no information is still available about accelerated atherosclerosis. The aim of our study was to investigate whether GSD 1a was associated with premature atherosclerosis. In nine adolescent patients (mean age 22.7+/-3.4 years) and nine matched healthy control subjects, lipid profile, blood pressure, ankle-brachial indices, aortic distensibility and intima-media thickness (IMT) of the carotid and femoral arteries were determined. As expected, lipid profiles were significantly unfavourable in the patient group compared with the control group. No differences were found in blood pressure, ankle-brachial indices and aortic distensibility between both groups. IMT segments were comparable in both groups, with even thinner segments in the patient group. In different multivariate models, GSD 1a remained an independent predictor for a thinner IMT (R(2)=0.90; beta=-0.69; P=0.018). CONCLUSION: glycogen storage disease type 1a is not associated with premature atherosclerosis, despite the existence of longstanding dyslipidaemia and microalbuminuria. PMID- 12373575 TI - Disturbed lipid metabolism in glycogen storage disease type 1. AB - Glycogen storage disease type 1 (GSD1) is an inborn error of metabolism caused by deficiency of glucose-6-phosphatase, the enzyme catalysing the conversion of glucose-6-phosphate (G6P) to glucose. GSD1 is associated with severe hyperlipidaemia and hepatic steatosis. The underlying mechanisms responsible for these abnormalities in lipid metabolism are only partly known. This review summarises data available on hyperlipidaemia and steatosis in GSD1 and postulates new hypotheses for unresolved issues. Evidence indicates that lipid clearance from the blood compartment is decreased in GSD1. Furthermore, in two GSD1a patients synthesis of palmitate, an indicator of de novo lipogenesis, and cholesterol were found to be increased 40-fold and 7-fold, respectively. Elevated hepatic G6P levels may play a regulatory role in lipid synthesis via activation of transcription of lipogenic genes. In addition, accelerated glycolysis will supply acetyl-CoA molecules required for lipogenesis. It is as yet unclear whether hepatic secretion of lipids in the form of very low density lipoprotein triglycerides (VLDL-TG) is altered in GSD1 patients: we recently found unaffected VLDL-TG secretion rates in an acute animal model of GSD1b. Hepatic steatosis, which is invariably present in GSD1 is probably mainly caused by an increased free fatty acid flux from adipose tissue to the liver and, to a limited extent, by increased de novo lipogenesis. CONCLUSION: future studies, using novel stable isotope methodologies, are warranted to further clarify the disturbances in lipid and lipoprotein metabolism in glycogen storage disease type 1 and the role of glucose-6-phosphate herein. PMID- 12373576 TI - Radical trapping in glycogen storage disease 1a. AB - Oxidative mechanisms involving lipid peroxidation in the subendothelium of the arterial vessel wall play a key role in atherogenesis. Despite severe hyperlipidaemia, patients with glycogen storage disease type 1a (GSD1a) do not develop premature atherosclerosis. Therefore, we analysed parameters of antioxidative defence and oxidative stress in plasma and serum of patients with GSD1a ( n=17) and compared them with those of patients with type 1 diabetes mellitus ( n=17), familial hypercholesterolaemia ( n=18) and healthy controls ( n=20). We measured the total radical trapping ability parameter (TRAP), single plasma antioxidants (sulfhydryl-groups, uric acid, vitamin C, alpha-tocopherol, coenzyme-Q10), markers of lipid peroxidation, lipoprotein (a) and homocysteine. Patients with GSD1a showed an elevated TRAP ( P<0.01) compared to the three other groups. This can mainly be attributed to elevated uric acid levels ( P<0.05 versus control). Lipoprotein (a) was significantly lower in the GSD1a group compared to the three other groups ( P<0.05). CONCLUSION: Patients with glycogen storage disease type 1a show an increased antioxidative defence in plasma which may protect them against lipid peroxidation and thus against premature atherosclerosis. Our finding of low lipoprotein(a) levels in this small group of patients warrants further investigation in a greater number of patients before assessing its role in atherogenesis in glycogen storage disease type 1a. PMID- 12373577 TI - Clinical symptoms and neutropenia: the balance of neutrophil development, functional activity, and cell death. AB - Neutrophilic granulocytes form the major type of leukocytes with counts ranging from about 1500-5000 cells/ micro l of blood under normal conditions. Neutrophils protect our body against bacterial and fungal infections. For this purpose, these cells are equipped with a machinery to sense the site of an infection and, upon local extravasation, rapidly move towards the site with invading micro-organisms, to ingest and kill them. As will be described, for proper functioning of this line of defence, a number of prerequisites have to be fulfilled. The quantitative defects are diagnosed more often and easier than the mere qualitative phagocytic defects. Nonetheless, functional defects may accompany neutropenia. These functional defects are seen in severe congenital neutropenia of which the gene defect has recently been elucidated, as well as in the more complex and syndromal forms of neutropenia such as Shwachman syndrome or the metabolic disease glycogen storage disease type 1b (non-a). The background of functional neutrophil defects is briefly reviewed. PMID- 12373578 TI - Granulocyte colony-stimulating factor in glycogen storage disease type 1b. Results of the European Study on Glycogen Storage Disease Type 1. AB - Patients with glycogen storage disease type 1b (GSD-1b) have neutropenia and neutrophil dysfunction that predispose to frequent infections and inflammatory bowel disease (IBD), for which granulocyte colony-stimulating factor (GCSF) is given. To investigate the use and the value of GCSF treatment in GSD-1b, a retrospective registry of GSD-1 patients born between 1960 and 1995 in 12 European countries was established. Included were 57 GSD-1b patients. Unglycosylated GCSF was given to 18 patients, median age of starting therapy was 8 years, longest duration of therapy 7 years. Dose varied between 2-10 micro g/kg, with a frequency from daily to twice per week. Neutropenia (defined as an absolute neutrophil count <0.5 x 10(9)/l) was found in 49 patients. In untreated patients, a significant decrease of haemoglobin, platelet counts and leucocyte counts with increasing age ( P<0.032, P<0.04 and P<0.001 respectively) was noted, whereas neutrophil counts remained low but stable with increasing age. In nine patients who were treated longer than 1 year, median neutrophil counts increased significantly and simultaneously median leucocyte counts and platelet counts decreased significantly. In all patients treated, the number and severity of infections decreased and the severity of IBD improved subjectively. The most serious complication of GCSF treatment was marked splenomegaly (four patients). CONCLUSION: in this retrospective study a significant haematological effect was documented and a subjective improvement of infections and inflammatory bowel disease. In view of the uncertainty, prospective controlled trials seem warranted to clarify the indication for the use of granulocyte colony-stimulating factor in this disease. PMID- 12373579 TI - Association of glycogen storage disease 1b and Crohn disease: results of a North American survey. AB - Glycogen storage disease (GSD)-1b has been associated with neutropenia and abnormalities of neutrophil function. Many individuals with GSD-1b manifest chronic gastrointestinal inflammation. Our study was performed to precisely establish the type, frequency, and spectrum of gastrointestinal disease in patients with GSD-lb. We established a medical database of 36 individuals affected with GSD-1b, utilizing patients at our center, disease registries, and direct contacts with North American referral centers specializing in genetic disorders. Records were reviewed, focusing on documentation of gastrointestinal involvement. Gastrointestinal symptoms or manifestations were present in approximately 75% of the patients, including chronic abdominal pain and diarrhea, perianal fistula or abscess, and oral aphthous ulceration. Of patients identified, 28% had documented inflammatory bowel disease (IBD) and an additional 22% of patients had symptoms highly suggestive of IBD, but had not undergone comprehensive diagnostic evaluation. Gastrointestinal involvement was found to be indistinguishable from idiopathic Crohn disease (CD) by detailed review of clinical, radiologic, endoscopic and histopathologic findings. Many patients had significant improvement or resolution of gastrointestinal disease in response to G-CSF treatment. The expression of CD in patients with GSD-1b may be delayed or prevented by G-CSF treatment. CONCLUSION: a strong association exists between glycogen storage disease type 1b and inflammatory bowel disease. A high index of suspicion for Crohn disease should be applied in evaluating patients with glycogen storage disease type 1b and intestinal symptoms. These results provide further support for the hypothesis that some forms of inflammatory bowel disease may result from impaired mucosal innate immunity. Additional investigations into the intestinal disease in glycogen storage disease type 1b may be directly relevant to the etiology and treatment of idiopathic Crohn disease. PMID- 12373580 TI - Severe pulmonary arterial hypertension in type 1 glycogen storage disease. AB - Pulmonary arterial hypertension is characterised by the presence of pulmonary hypertension (mean pulmonary artery pressure >25 mmHg at rest or >30 mmHg during exercise ) and normal pulmonary wedge pressure (<12 mmHg). Several risk factors for pulmonary arterial hypertension have been described. In the absence of any factor or condition suspected to play a causal or facilitating role in the process, pulmonary hypertension is "unexplained" (primary pulmonary hypertension, PPH). PPH is a rare condition, with an estimated incidence of 2 per million people. Recent genetic studies have identified mutations in the bone morphogenetic protein receptor-II (BMPR-II) gene, a receptor member of the transforming growth factor-beta family, in a majority of familial cases of PPH. Interestingly, 25% of patients displaying sporadic PPH may also have mutations in the BMPR-II gene, emphasising the relevance of genetic susceptibility for this severe condition. Other molecular and biochemical processes behind the complex vascular changes associated with pulmonary arterial hypertension are currently investigated. Type 1a glycogen storage disease caused by a deficiency of glucose 6-phosphatase has an estimated incidence of 1 per 100000 with a few reported cases of unexplained severe pulmonary hypertension. The occurrence of pulmonary arterial hypertension in type 1a glycogen storage disease could be due to vasoconstrictive amines such as serotonin, a pulmonary vasoconstrictor and growth factor for vascular smooth muscle cells stored in platelets. PMID- 12373581 TI - Contraception and pregnancy in women affected by glycogen storage diseases. AB - During the last decades, better understanding of specific enzymatic deficiencies has led to improved dietary management of children suffering from glycogen storage disease (GSD). Normal growth and development of infants can be achieved by a diet of regular meals supplemented by glucose and cornstarch during the night, and by monitoring glucose blood levels. This has resulted in an increase in the number of patients reaching adulthood and reproduction age. Therefore, developing a strategy for an optimal management of contraception and pregnancy is crucial for young women affected by GSD. Contraception has to be adapted to the specific metabolic requirements of women with GSD. Hormonal contraception is classically based on the combination of various synthetic progestogens and ethinyloestradiol. Ethinyloestradiol should be avoided because of a link with hepatic adenomas and is contraindicated in patients with hypertriglyceridaemia and hypercholesterolaemia. Blockade of ovulation can be achieved using high doses of progestogen alone, administered from the 5th to the 25th day of the cycle. Another scheme of hormonal contraception is based on daily administration of low doses of progestogen, which usually acts on local parameters of fertility, and can also achieve blockade of ovulation for the most recent compound proposed. Mechanical contraception using intra-uterine device is controversial for nulliparous patients. Benefits and side-effects of these different proposals are discussed. During pregnancy, the maternal nutritional state is important and a healthy maternal response to feeding and fasting is modified to ensure a constant supply of glucose for the developing fetus. Women with GSD are at risk of frequent hypoglycaemia. Only a few cases of successful pregnancies have been reported for patients with GSD. The outcomes using different approaches of dietary and obstetric management are discussed. CONCLUSION: in the future, multidisciplinary management is necessary to improve obstetric outcome of pregnancy in females affected with glycogen storage disease. PMID- 12373582 TI - More questions: 10 years later from glycogen storage disease patient support groups in Europe. AB - In 1990 the first Fulda Workshop on Glycogen Storage Disease (GSD) type I was held in November. Eight adult representatives from Patient groups in the UK, USA, Germany and the Netherlands were invited to come and set up an information table of posters, leaflets etc. We were also asked to present a short list of questions that can occur to parents after the initial shock of diagnosis and treatment of GSD has been made. These "Questions of Parents" were presented on the final day. Ten years later, patient representatives from Europe were invited to present "More Questions: 10 Years Later". On both occasions the questions centred around six broad areas: (1). treatments, (2). specific problems, (3). family planning, (4). long-term effects of having GSD type Ia and Ib, (5). research and (6). general questions. CONCLUSION: As representatives of GSD support groups, we hope that firm decisions can be agreed for common dietary and pharmacological treatment and follow-up procedures within the boundaries of cultural differences and financial circumstances. We anticipate that if there is a third Fulda workshop in 2010, the answer to the question "Is there a common set of protocols and guidelines among doctors and hospitals as to the correct treatment for glycogen storage disease type I?" will be a firm "Yes". PMID- 12373583 TI - Enzyme therapy for Pompe disease: from science to industrial enterprise. AB - Pompe disease or glycogen storage disease type II (OMIM 232300) is a metabolic myopathy with a broad clinical spectrum. Generalised muscle weakness combined with cardiomegaly presents within the first 3 months after birth, if the lysosomal alpha-glucosidase (AGLU) deficiency is complete. Residual enzyme activity prevents cardiac involvement and delays onset of muscle weakness. Enzyme therapy, by intravenous administration of acid AGLU, aims to supplement the missing enzyme activity. At the SHS symposium on Glycogen Storage Diseases Type I and II, in Fulda, two interim accounts were given of studies on the efficacy of enzyme therapy for Pompe disease; one with recombinant human acid AGLU produced in Chinese hamster ovary cells and the other with the same enzyme produced in the milk of transgenic rabbits. CONCLUSION: this review focuses on the latter study, discusses the scientific, technological and commercial aspects of the enterprise, and addresses the prospects and challenges of enzyme therapy for Pompe disease. PMID- 12373584 TI - Guidelines for management of glycogen storage disease type I - European Study on Glycogen Storage Disease Type I (ESGSD I). AB - Life-expectancy in glycogen storage disease type I (GSD I) has improved considerably. Its relative rarity implies that no metabolic centre has experience of large series of patients and experience with long-term management and follow up at each centre is limited. There is wide variation in methods of dietary and pharmacological treatment. Based on the data of the European Study on Glycogen Storage Disease Type I, discussions within this study group, discussions with the participants of the international SHS-symposium 'Glycogen Storage Disease Type I and II: Recent Developments, Management and Outcome' (Fulda, Germany; 22-25th November 2000) and on data from the literature, guidelines are presented concerning: (1). diagnosis, prenatal diagnosis and carrier detection; (2). (biomedical) targets; (3). recommendations for dietary treatment; (4). recommendations for pharmacological treatment; (5). metabolic derangement/intercurrent infections/emergency treatment/preparation elective surgery; and (6). management of complications (directly) related to metabolic disturbances and complications which may develop with ageing and their follow-up. CONCLUSION: In this paper guidelines for the management of GSD I are presented. PMID- 12373585 TI - Consensus guidelines for management of glycogen storage disease type 1b - European Study on Glycogen Storage Disease Type 1. AB - Life expectancy in glycogen storage disease type 1 (GSD-1) has improved considerably. Its relative rarity implies that no metabolic centre has experience of large series of patients and therefore experience with long-term management and follow-up at each centre is limited. There is wide variation in methods of dietary and pharmacological treatment. Based on data from the European Study on Glycogen Storage Disease Type 1, discussions within this study group together with those at the International SHS Symposium 'Glycogen Storage Disease Type I and II: Recent Developments, Management and Outcome', Fulda, Germany (2000) and on data from the literature, a series of guidelines were drawn up. CONCLUSION: the following guidelines for the management of patients with glycogen storage disease type 1b are in addition to those general guidelines for glycogen storage disease type 1 and address specific problems related to neutropenia and neutrophil dysfunction. PMID- 12373586 TI - In situ screening for genes expressed preferentially in secondary mesenchyme cells of sea urchin embryos. AB - In sea urchin embryos, four types of non-skeletogenic mesodermal cells are derived from secondary mesenchyme cells (SMCs). Although determining the complete lineage of SMCs is currently a high-priority goal, specific markers for each type of SMC-derived cell in Hemicentrotus pulcherrimus are unavailable. To identify genes preferentially expressed in the various types of SMC-derived cells, we constructed a cDNA library of the archenteron isolated from late gastrulae. Both the 5' and 3' ends of 1,050 cDNAs randomly selected from 7,500 picked clones were sequenced. Based on the sequence at the 3' end, the cDNAs were grouped into 671 independent clusters. Among these, 605 clusters were analysed by whole-mount in situ hybridisation; 28% (170 clusters) exhibited differential expression patterns, while 24% were ubiquitously expressed and 48% did not show any staining. Of 170 clusters showing differential expression patterns, 33 clusters were differentially expressed in SMC-derived cells. From these clusters, several genes were obtained that were specifically or predominantly expressed in each type of SMCs, including coelomic pouch cells in which specific expression patterns have not been reported previously, and hence will be useful for lineage studies. Furthermore, in situ hybridisation revealed the existence of a new type or subpopulation of SMCs distributed sparsely in the blastocoel. PMID- 12373587 TI - Identification and developmental expression of new biomineralization proteins in the sea urchin Strongylocentrotus purpuratus. AB - The endoskeleton of the sea urchin larva is a network of calcareous rods secreted by primary mesenchyme cells (PMCs). In this study, we identified seven new biomineralization-related proteins through an analysis of a large database of gene products expressed by PMCs. The proteins include three new spicule matrix proteins (SpSM29, SpSM32, and SpC-lectin), two proteins related to the PMC specific cell surface glycoprotein MSP130 (MSP130-related-1 and -2), and two novel proteins (SpP16 and SpP19). The genes encoding these proteins are expressed specifically by cells of the large micromere-PMC lineage and are activated zygotically beginning at the blastula stage, prior to PMC ingression. Several of the mRNAs show regulated patterns of expression within the PMC syncytium that correlate with the pattern of skeletal rod growth. This work identifies new proteins that may regulate the process of biomineralization in this tractable model system. PMID- 12373588 TI - Fgf genes in the basal chordate Ciona intestinalis. AB - In vertebrates, a number of fibroblast growth factors (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/16/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs. PMID- 12373589 TI - HrNodal, the ascidian nodal-related gene, is expressed in the left side of the epidermis, and lies upstream of HrPitx. AB - The nodal-related genes are well known for their fundamental roles during vertebrate development, including mesoderm induction, neural induction, and left right axis formation, as several nodal-related genes show left-sided expression in mesodermal lineages. We have isolated the first non-vertebrate nodal-related gene, HrNodal, from the ascidian Halocynthia roretzi. During the late cleavage and gastrula stages, HrNodal is transiently and bilaterally expressed in several different cell lineages. Expression at the tailbud stage is observed asymmetrically in the left side, but unexpectedly only in the epidermis of the embryo. We also demonstrate the relationship of HrNodal with HrPitx, a Halocynthia homologue of the Pitx2 gene. HrNodal overexpression results in the disturbance of left-sided HrPitx expression. Our results demonstrate that left right specification during ascidian embryogenesis involves the HrNodal gene, and that the left-sidedness of the expression is evolutionarily conserved throughout the chordate clade. PMID- 12373590 TI - Novel G-protein-coupled receptor gene expressed specifically in the entire neural tube of the ascidian Ciona intestinalis. AB - We have cloned a newly identified gene, designated CiNut, C iona i ntestinalis neural-tube-specific gene. CiNut shows weak similarity to known neural receptors such as adrenergic receptors. Moreover, seven transmembrane domains are predicted based on its amino acid sequence. Zygotic expression of CiNut starts at the gastrula stage, and is restricted to the entire neural tube in the neurula- and the tailbud-stage embryos. CiNut is thus thought to be a novel G-protein-coupled receptor important for neural tube formation, and should provide a useful tool for the analysis of the molecular mechanism of neural tube formation. PMID- 12373591 TI - Ancestry and diversity of BEL1-like homeobox genes revealed by gymnosperm ( Gnetum gnemon) homologs. AB - BEL1-like homeobox genes encode plant-specific transcription factors, at least some of which are important for ovule development. Here we report MELBEL1 MELBEL4,the first BEL1-like genes from a non-flowering plant, the gymnosperm Gnetum gnemon. Our analyses suggest that there was already at least one BEL1-like gene present in the most recent common ancestor of extant seed plants about 300 million years ago. Multiple sequence alignments revealed that since this time, not only the DNA-binding homeodomain, but also a protein-protein interaction domain upstream of the homeodomain, termed the BEL domain, has been highly conserved. Sequence comparison of the BEL domain with upstream domains that have been conserved in other TALE homeodomain proteins, i.e. MEIS, KNOX, and PBC, revealed only weak sequence similarity. However, since homology has been shown for MEIS, KNOX, and PBC domains and since KNOX and BEL domains directly interact in vivo, it appears likely that the BEL domain was also derived from an ancestral upstream (MEINOX) domain. PMID- 12373592 TI - First-line chemotherapy for ovarian cancer - the controversy continues. PMID- 12373593 TI - First-line treatment for advanced ovarian cancer: paclitaxel, platinum and the evidence. AB - Four large randomised trials of paclitaxel in combination with platinum against a platinum-based control treatment have now been published in full, representing around 88% (3588 out of 4057) of patients randomised into the eight known trials of this question. There is substantial heterogeneity in the results of these four trials. Four main explanations for this heterogeneity have been proposed: differences in the extent and timing of 'crossover' to taxanes in the control groups; differences in the types of patient included; differences in the effectiveness of the research regimens used; differences in the effectiveness of the control regimens used. In this study we examine whether any of these explanations is consistent with the pattern of results seen in these trials. Each explanation suggests that a particular characteristic of each trial was responsible for the results observed. For each explanation the trials were split into groups according to that characteristic, in order to partition the total heterogeneity into that seen 'within' and 'between' groups of trials. If a particular explanation was consistent with the pattern of results, we would expect to see relatively little heterogeneity within each group of trial results viewed in this way, with most of the heterogeneity being between groups which are dissimilar with respect to the key characteristic. Heterogeneity 'within' and 'between' groups was formally compared using the F-ratio. If any explanation appeared to be consistent with the results of the trials, it was considered whether the explanation was also consistent with other evidence available about these regimens. Only one explanation appeared to be consistent with the pattern of results seen in these trials, and that was differences in effectiveness of the control arms used in these trials. This suggests that the very positive results in favour of paclitaxel/cisplatin seen in two of the trials may have been due to the use of a suboptimal control arm. There is no direct evidence about the relative effectiveness of the control arms used in these trials, but indirect evidence is consistent with the conclusion that the cyclophosphamide/cisplatin regimen used in two of the trials may be less effective than the control regimens used in the other trials. Specific concerns about the choice of a cyclophosphamide/cisplatin control arm in the first of these trials to report were raised before the results of the other trials were known, i.e. before any heterogeneity had been observed. Further investigation of this question would be useful. In the meantime, given all of the randomised evidence on the efficacy and toxicity associated with the regimens used in these trials, we conclude that single agent carboplatin is a safe and effective first-line treatment for women with advanced ovarian cancer. PMID- 12373594 TI - Platinum drugs in the treatment of non-small-cell lung cancer. AB - The use of chemotherapy is considered standard therapy in patients with locally advanced non-small-cell lung cancer that cannot be treated with radiotherapy and in those with metastatic non-small-cell lung cancer and good performance status. This approach is also accepted in patients with earlier stage disease, when combined with radiotherapy in those with non-resectable locally advanced disease, or in the preoperative setting. Randomised clinical studies and meta-analyses of the literature have confirmed the beneficial survival effect of platinum-based chemotherapy. Cisplatin and carboplatin have been successfully used with other drugs in a wide variety of well-established two-drug combinations while three drug combinations are still under investigation. Cisplatin and carboplatin use is limited by toxicity and inherent resistance. These considerations have prompted research into new platinum agents, such as the trinuclear platinum agent BBR3464, the platinum complex ZD0473 and oxaliplatin. These compounds could be developed in combination with agents such as paclitaxel, gemcitabine or vinorelbine in patients with advanced and/or refractory solid tumours. PMID- 12373595 TI - BAG-1 expression and function in human cancer. AB - BAG-1 is a multifunctional protein that interacts with a wide range of target molecules to regulate apoptosis, proliferation, transcription, metastasis and motility. Interaction with chaperone molecules may mediate many of the effects of BAG-1. The pathways regulated by BAG-1 play key roles in the development and progression of cancer and determining response to therapy, and there has been considerable interest in determining the clinical significance of BAG-1 expression in malignant cells. There is an emerging picture that BAG-1 expression is frequently altered in a range of human cancers relative to normal cells and a recent report suggests the exciting possibility that BAG-1 expression may have clinical utility as a prognostic marker in early breast cancer. However, other studies of BAG-1 expression in breast cancer and other cancer types have yielded differing results. It is important to view these findings in the context of current knowledge of BAG-1 expression and function. This review summarises recent progress in understanding the clinical significance of BAG-1 expression in cancer in light of our understanding of BAG-1 function. PMID- 12373596 TI - Treatment of disseminated ocular melanoma with sequential fotemustine, interferon alpha, and interleukin 2. AB - Malignant melanoma of the uvea is remarkable for purely haematogenous dissemination and its tendency to metastasise to the liver. Although the liver is involved in up to 95% of patients, 50% of these also develop extrahepatic metastases, most often in the lungs, bone, skin, and brain. The only effective treatments reported to date relied on hepatic arterial chemoembolisation or perfusion. The objective of this study was to establish a therapy protocol addressing patients with both sole liver involvement and systemic disease. Forty eight patients with metastatic ocular melanoma received fotemustine 100 mg m(-2) either as 60-min infusion into the hepatic artery or as 15-min infusion via a peripheral vein, depending on the metastatic sites involved, i.e., restriction to the liver or hepatic together with extrahepatic disease. For the first treatment cycle this infusion was repeated after one week. For all cycles, subsequent to a three week resting period, patients received an immunotherapy consisting of subcutaneous interleukin 2 and interferon alpha(2). Although objective responses were more frequent within the cohort receiving intraarterial fotemustine (21.7 vs 8%), this difference did not translate into a significant benefit in overall survival, i.e., 369 and 349 days, respectively. Of note, this overall survival is much longer than that repeatedly reported for stage IV uveal melanoma not treated with fotemustine, suggesting a therapeutic activity of this cytostatic drug even after systemic administration. PMID- 12373597 TI - Phase I study of docetaxel plus ifosfamide in patients with advanced cancer. AB - The aim of this study was to determine the maximum tolerated dose of a fixed dose of docetaxel when combined with continuous infusion ifosfamide, with and without G-CSF support, in the treatment of advanced cancer, and to evaluate anti-tumour activity of this combination. Thirty-one patients with advanced malignancies were treated with docetaxel 75 mg/m(2) intravenously on days 1, and ifosfamide at increasing dose levels from 1500 mg/m(2)/day to 2750 mg/m(2)/day as a continuous infusion from day 1-3, every 3 weeks. A total of 107 cycles of treatment were administered. Without G-CSF support dose-limiting toxicity of grade 4 neutropenia greater than 5 days duration occurred at dose level 1. With the addition of G-CSF the maximum tolerated dose was docetaxel 75 mg/m(2) on day 1 and ifosfamide 2750 mg/m(2)/day on days 1-3. Dose limiting toxicity (DLT) included ifosfamide-induced encephalopathy, febrile neutropenia and grade three mucositis. Three complete responses and 3 partial responses were seen. This combination of docetaxel and infusional ifosfamide is feasible and effective. The recommended dose for future phase II studies is docetaxel 75 mg/m(2) on day 1 and ifosfamide 2500 mg/m(2)/day continuous infusion on days 1-3. PMID- 12373598 TI - Irinotecan, cisplatin and mitomycin in inoperable gastro-oesophageal and pancreatic cancers - a new active regimen. AB - Irinotecan, mitomycin and cisplatin all demonstrate activity in gastro oesophageal cancers. This novel combination was administered to outpatients with previously untreated inoperable gastro-oesophageal or pancreatic cancer, in a 28 day cycle. A total of 26 out of 31 patients with gastro-oesophageal cancer and 12 out of 14 patients with pancreatic cancer have been treated with this combination, and were evaluable for response. The overall response rates for patients with gastro-oesophageal cancer was 42%, with a median survival of 9.5 months. In patients with pancreatic cancer, the overall response rate was 42% with a median survival of 8 months. There was a statistically significant increase in survival between those patients who achieved a stable disease response and those who achieved either a partial response or complete response. The toxicity profiles for both cancers were virtually identical. There were five treatment-related deaths, and a high admission rate (42%). Thus irinotecan, mitomycin and cisplatin is a new combination with activity in inoperable upper gastro-oesophageal cancers, but with a high toxicity profile. Future developments include reducing the dose of irinotecan and number of cycles of therapy to four. PMID- 12373599 TI - Describing randomisation: patients' and the public's preferences compared with clinicians' practice. AB - Explaining the concept of randomisation in simple terms to patients during the discussion of randomised clinical trials can be a difficult task for many health care professionals. We report the results of a questionnaire-based survey, using seven descriptions of randomisation taken from Corbett's study. We examined the preferences of the general public and patients towards the descriptions and compared the results with the clinicians' choice. Participants in the survey were 341 lay people without cancer, 200 patients with cancer and 200 oncologists from cancer centres throughout the UK. It was difficult to identify 'the best' way to describe the process of randomisation. The two most favoured statements for patients and members of the public included a very explicit statement that mentioned 'a computer', 'chance' and 'not the doctor's or patient's decision' and a succinct statement that played down the role of 'chance'. Clinicians chose neither of these statements as closely resembling their own practice. Patients and members of the public most disliked the statement 'a computer will perform the equivalent of tossing a coin to allocate you to one of two methods of treatment'. This analogy used by 26% of oncologists, was viewed as trivialising and upsetting in the context of determining treatment for life threatening disease. PMID- 12373600 TI - Binding of TGF-beta1 latency-associated peptide (LAP) to alpha(v)beta6 integrin modulates behaviour of squamous carcinoma cells. AB - The integrin alpha(v)beta6 is not detectable on normal keratinocytes in vivo but expression is increased significantly in oral squamous cell carcinoma where this heterodimer has been shown to play a role in cell migration, invasion and protease expression. Although regarded initially as a fibronectin receptor, alpha(v)beta6 may bind to arginine-glycine-aspartic acid sequences in other matrix molecules including tenascin and vitronectin. Interestingly, alpha(v)beta6 has also been shown to have high affinity for the TGF-beta1 latency associated peptide and to participate in the activation of the TGF-beta1 latent complex. Since TGF-beta1 is present in squamous carcinomas, it is possible that latency associated peptide may modulate malignant keratinocyte behaviour independently from the classical TGF-beta signalling pathways through its interaction with integrins. We show here that when latency associated peptide is immobilised onto a surface, it acts as an alpha(v)beta6-specific ligand for oral squamous carcinoma cells promoting adhesion and haptotactic migration in addition to alpha(v)beta6-dependent increase in pro-MMP-9 expression. In contrast, even very low concentrations of soluble latency associated peptide (0.1 microg ml(-1)) inhibited alpha(v)beta6-dependent adhesion, migration and invasion. Thus alpha(v)beta6-dependent processes of oral squamous cell carcinoma, is likely to be modulated, not only by the local concentration of latency associated peptide in the stroma, but also whether it is immobilised in the matrix or released as a soluble protein. PMID- 12373601 TI - Role of biological markers in the clinical outcome of colon cancer. AB - We investigated a number of biological markers, evaluated under strict intralaboratory quality control conditions, in terms of their role in predicting clinical outcome of patients with colon cancer treated with 5-FU-containing regimens. Colon cancer tissue from 263 patients enrolled onto two randomised clinical trials were studied for their cytofluorimetrically determined DNA content and their immunohistochemically evaluated microvessel density, vascular endothelial growth factor expression, thymidylate synthase expression and tumour lymphocyte infiltration. Disease-free survival and overall survival of patients were analysed as a function of the different variables. At a median follow up of 57 months, age, gender and Dukes' stage showed an impact on disease-free survival, whereas no biological marker emerged as an indicator of better or worse disease-free survival. Only histological grade and Dukes' stage were found to influence overall survival. The different biological variables, studied with particular attention for determination reliability, proved to have no impact on the clinical outcome of patients with colon cancer. Therefore, other markers must be identified to complement clinico-pathological variables in the management of this disease. PMID- 12373602 TI - The association of breast mitogens with mammographic densities. AB - Radiologically dense breast tissue (mammographic density) is strongly associated with risk of breast cancer, but the biological basis for this association is unknown. In this study we have examined the association of circulating levels of hormones and growth factors with mammographic density. A total of 382 subjects, 193 premenopausal and 189 postmenopausal, without previous breast cancer or current hormone use, were selected in each of five categories of breast density from mammography units. Risk factor information, anthropometric measures, and blood samples were obtained, and oestradiol, progesterone, sex hormone binding globulin, growth hormone, insulin-like growth factor-I and its principal binding protein, and prolactin measured. Mammograms were digitised and measured using a computer-assisted method. After adjustment for other risk factors, we found in premenopausal women that serum insulin-like growth factor-I levels, and in postmenopausal women, serum levels of prolactin, were both significantly and positively associated with per cent density. Total oestradiol and progesterone levels were unrelated to per cent density in both groups. In postmenopausal women, free oestradiol (negatively), and sex hormone binding globulin (positively), were significantly related to per cent density. These data show an association between blood levels of breast mitogens and mammographic density, and suggest a biological basis for the associated risk of breast cancer. PMID- 12373603 TI - survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. AB - Oesophageal squamous cell carcinoma is one of the most malignant tumours. To identify patients with a high risk of recurrence of oesophageal squamous cell carcinoma, we investigated the prognostic significance of survivin mRNA expression in oesophageal squamous cell carcinoma, which has recently been reported to be a good marker for unfavourable prognosis in various tumours. Tumours and non-cancerous epitheliums adjacent to tumours were obtained by surgical resection from 57 patients with oesophageal squamous cell carcinoma. Expression levels of survivin and glyceraldehyde-3-phosphate dehydrogenase mRNA were analysed quantitatively by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The survivin/glyceraldehyde-3-phosphate dehydrogenase ratios of tumours were higher than those of non-cancerous tissues (P=0.0003). Tumour survivin/glyceraldehyde-3-phosphate dehydrogenase ratio did not correlate with histologic type, lymph node metastasis, and stage of tumours. In 53 surviving patients, the 5-year survival rate of 17 patients with high survivin mRNA expressed oesophageal squamous cell carcinoma (14.1%) was significantly poorer than that of 36 with low survivin mRNA expressed oesophageal squamous cell carcinoma (46.8%, P=0.0018). In these patients, tumour-survivin mRNA expression was recognised as a good marker of cancer recurrence independently from tumour stage. These findings indicate that survivin mRNA expression in oesophageal squamous cell carcinoma may be a good biomarker for identifying patients with high risk of cancer recurrence. PMID- 12373604 TI - BRCA2 gene mutations in families with aggregations of breast and stomach cancers. AB - Stomach cancer ranks second to lung cancer in the global cancer burden. It is estimated that 25% of families meeting the criteria for hereditary diffuse gastric carcinoma (HDCG) will have germline mutations in the E-cadherin gene. Evidence suggests that stomach cancer might also be a malignant manifestation of other inherited predispositions to disease. Recently, it has been reported that the incidence of stomach cancer is significantly increased in BRCA2 gene mutation carriers. We analysed by direct sequencing the BRCA2 gene in 29 breast cancer patients derived from 29 families with an aggregation of at least one female breast cancer diagnosed before the age of 50 years and one male stomach cancer diagnosed before the age of 55 years. In all but one of these families at least one additional relative was also affected by a malignant tumour. We identified three frameshift mutations and three sequence variants - potentially missense mutations, in six unrelated patients representing 20.7% (six out of 29) of the families investigated. Our results confirm that BRCA2 gene mutations are also associated with familial aggregations of not only breast but also of stomach cancer. In comparison to the number of cancers expected in the study population compared to the general population there is an over-representation of several cancers with significant confidence intervals to suggest that the associations are real and not a selection artefact. PMID- 12373605 TI - Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. AB - Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer. PMID- 12373606 TI - Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells. AB - One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5' splice site (5'ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5' free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5'ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells. PMID- 12373607 TI - HPC2/ELAC2 polymorphisms and prostate cancer risk: analysis by age of onset of disease. AB - The candidate prostate cancer susceptibility gene HPC2/ELAC2 has two common coding polymorphisms: (Ser-->Leu 217) and (Ala-->Thr 541). The Thr541 variant in the HPC2/ELAC2 gene has previously been reported to be at an increased frequency in prostate cancer cases. To evaluate this hypothesis we genotyped 432 prostate cancer patients (including 262 patients diagnosed 55 years (OR=1.27, 95% CI 0.59-2.74). We conclude that any association between the Thr541 variant and prostate cancer is likely to be weak. PMID- 12373608 TI - Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells. AB - To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF 7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes. PMID- 12373609 TI - Inhibition of proliferation and induction of differentiation of glioma cells with Datura stramonium agglutinin. AB - We found that a lectin, Datura stramonium agglutinin, induced irreversible differentiation in C6 glioma cells. The differentiated cells had long processes, a low rate of proliferation and a high content of glial fibrillary acidic protein. When the medium was replaced with Datura stramonium agglutinin-free medium after 1 h, cell proliferation continued to be inhibited. Experiments with several other lectins indicated that both recognition of linear N acetyllactosamine repeats and recognition of multiantennary units of cell-surface glycans were required for the inhibition of C6 proliferation. Proliferation of four human glial tumour cells was also inhibited by Datura stramonium agglutinin. Further, these differentiated human glial tumour cells had long processes and a high content of glial fibrillary acidic protein similar to differentiated C6 glioma cells. Taken together, these observations suggest that Datura stramonium agglutinin may be useful as a new therapy for treating glioma without side effects. PMID- 12373611 TI - The association between nutritional status and handgrip strength in older Rwandan refugees. AB - OBJECTIVES: To investigate the association between nutritional status and handgrip strength in older Rwandan refugees. DESIGN: Cross-sectional study. SETTING: Rwandan refugee camp located in Karagwe district in the north-west of Tanzania. The study was carried out in the post-emergency phase. The response rate was 85%. SUBJECTS: A total of 413 men and 415 women aged 50-92 y participated in the study. METHODS: Weight, height, mid-upper-arm circumference (MUAC) and triceps skinfold were obtained using standard techniques. For people with visible kyphosis, height was estimated from armspan using regression equations developed from non-kyphotic subjects within the sample. Handgrip was measured using a mechanical handgrip dynamometer. Information regarding physical activity and health status was obtained by interview and clinical screening. RESULTS: Handgrip strength (kg) was significantly higher in men than in women (30.3+/-6.7 vs 22.3+/-5.1), and significantly lower in each older age group in both sexes. Handgrip strength was positively correlated to BMI (body mass index) and AMA (arm muscle area). The relative risk of impaired handgrip strength in individuals with poor nutritional status (BMI<18.5 kg/m(2)) compared with those of adequate nutritional status was 1.75. After controlling for potential confounders (sex, age and height), BMI remained a significant contributor to the variation in handgrip strength. CONCLUSION: Poor nutritional status is associated with poor handgrip strength independent of sex, age and height, in this refugee population. This may indicate that underweight older people are likely to have more difficulties in functioning independently in the community. Research is needed to investigate if improving nutritional status can lead to better functional ability. SPONSORSHIP: Department for International Development (UK) and HelpAge International. PMID- 12373610 TI - A novel form of constitutively active farnesylated Akt1 prevents mammary epithelial cells from anoikis and suppresses chemotherapy-induced apoptosis. AB - Protein kinase B/Akt has been described as a central mediator of anti-apoptotic signals transduced by the PI3 kinase. Although the role of Akt in the suppression of apoptosis is well elucidated, a potential function of Akt in tumorigenesis and chemoresistance is less intensively documented. In this study, we describe the construction of a novel form of constitutively active Akt1, which relies on the deletion of its pleckstrin homology domain and the insertion of a C-terminal farnesylation sequence. Stable cell lines were generated with MCF10A mammary epithelial cells and A549 human NSCLC cells expressing constitutively active Akt1. Enigneered MCF10A cells were rendered resistant towards apoptosis resulting from loss of cellular substrate attachment (anoikis). We investigated the chemosensitivity of A549 cells expressing farnesylated Akt vs control cells. A profoundly decreased sensitivity towards Mitoxantrone and cisplatin was observed in cells expressing farnesylated Akt. No significant difference in sensitivity however was observed upon treatment with cell cycle specific chemotherapeutic agents like paclitaxel. Our data suggest, that Akt is a central mediator in the suppression of anoikis and modulation of chemotherapy-induced apoptosis. Therefore it represents a promising target for small molecule inhibitors to shift the apoptotic threshold in cancer cells after treatment with standard chemotherapy. PMID- 12373612 TI - Longitudinal investigation of energy expenditure in infants with cystic fibrosis. AB - OBJECTIVE: To determine when energy expenditure becomes elevated in infants with cystic fibrosis (CF). DESIGN: Longitudinal studies of total energy expenditure (TEE) using doubly labeled water were conducted in infants identified with CF by newborn screening through the first year of life. SETTING: Hospital and community based studies in Denver, Colorado, USA and Cambridge, UK. RESULTS: Eight of the 12 infants enrolled had begun enzyme therapy but were clinically asymptomatic. Four of the 12 infants were heterozygous for the delta F508 mutation, however no difference was seen in TEE from the remaining homozygous infants. TEE was compared to control cohorts at 2, 6 and 12 months of age. There was no difference from the control groups in TEE/kg fat free mass (FFM)/day at 2 months. However, by 6 months of age TEE/kg FFM/day in infants with CF exceeded that of age-matched controls by 25% (P<0.001). This elevation in TEE continued at 12 months of age exceeding that of controls by 30% (P<0.05). CONCLUSIONS: These results indicate that infants with CF have increased energy needs by 6 months of age and that early diagnosis alone does not prevent the development of increased caloric requirements. These findings emphasize the need for close nutritional monitoring to prevent suboptimal growth during infancy in this population. SPONSORSHIP: This research was supported by grant number 5 MO1 RR00069, General Clinical Research Centers Program, National Center for Research Resources, NIH. PMID- 12373614 TI - Phloem fortification in rye bread elevates serum enterolactone level. AB - OBJECTIVE: To analyse the lignan content of phloem powder enriched rye bread and to study the dose-response relationship of the effect of dietary plant lignans derived from phloem on intestinal production of enterolactone by measuring enterolactone concentration in serum. DESIGN: A randomized double-blind supplementation trial. SUBJECTS: Seventy-five non-smoking men recruited by newspaper advertisements. INTERVENTION: Subjects were randomized to three study groups receiving either rye bread high in phloem (HP, 14% of rye flour substituted with phloem powder), rye bread low in phloem (LP, 7% of rye flour substituted with phloem powder) or placebo rye bread. Participants consumed 70 g of study bread daily for 4 weeks and provided serum samples for enterolactone analysis at baseline and at the end of the intervention. RESULTS: There was a significant increase in serum enterolactone concentration in the LP and HP groups compared with the placebo group (P=0.009 and P=0.003, respectively). Considerable interindividual differences were observed in the response to dietary lignans within the study groups. CONCLUSIONS: Our results indicate that plant lignans attached to insoluble fibre layer in phloem can be further metabolized and converted to enterolactone presumably by the bacteria present in the colon. Phloem powder is useful source of lignans for functional foods aimed to elevate serum enterolactone levels. SPONSORSHIP: Phloem powder and the study breads were provided by Finnpettu Oy and Linkosuo Oy, respectively. The clinical study work was sponsored in part by Oy Jurilab Ltd. PMID- 12373613 TI - Determinants of weight and length of Indonesian neonates. AB - OBJECTIVE: To investigate the determinants of neonatal weight and length. DESIGN: From 16-20 week of pregnancy, 366 mothers of the neonates had participated in the community-based study to investigate the effect of weekly supplementation during pregnancy with iron and vitamin A on infant growth. Women from five villages were allocated randomly to receive two tablets each containing 60 mg iron as ferrous sulphate and 250 micro g folic acid (n=121) or two tablets each containing 2400 RE vitamin A in addition to the same amount of ferrous sulphate and folic acid (n=122). A third ('daily') group (n=123) participating in the national iron supplementation programme was recruited from four neighbouring villages. RESULTS: Neonatal weight and length did not differ between the two weekly groups and between the weekly iron group and the 'daily' group. Iron and vitamin A status during pregnancy did not influence neonatal weight and length significantly. Boys were 100 g heavier and 0.53 cm longer than girls (P<0.05). First born neonates were lighter (P<0.01) and tended to be shorter (P=0.070) than neonates of higher birth order. Maternal age and education as well as other socioeconomic determinants were not associated with neonatal weight and length. Neonatal weight was 32% explained by gestational age, maternal weight, postnatal measurement, gender and parity, while neonatal length was 28% explained by gestational age, maternal weight, postnatal measurement, gender and maternal height. CONCLUSIONS: Gestational age, maternal weight at second trimester and infant gender were the main predictors of neonatal weight and length. SPONSORSHIP: The study was supported by The Netherlands Organization for Scientific Research-Netherlands Foundation for the Advancement of Tropical Research (NWO-WOTRO; WV 93-280) and Neys-van Hoogstraten Foundation (IN 114), The Netherlands, and German Agency for Technical Cooperation (GTZ)/South East Asian Ministers of Education Organization (SEAMEO), Indonesia. PMID- 12373615 TI - An investigation of orotic acid levels in the breastmilk of smoking and non smoking mothers. AB - AIM: In this study; orotic acid levels in the milk of smoking and non-smoking mothers were investigated by high-performance liquid chromatography (HPLC). RESULTS: It was found that the amount of orotic acid in the milk of smoking mothers (3.92+/-0.20 micro g/ml) was higher than that of non-smoking mothers (1.66+/-0.15 micro g/ml). Orotic acids levels in the milk of smoking mothers in comparison with non-smokers were found to be statistically significant (P<0.005). CONCLUSION: Smoking may have increased the orotic acid levels by affecting pyrimidine biosynthesis pathway. PMID- 12373616 TI - Daidzein and genistein content of cereals. AB - OBJECTIVE: To analyse 75 cereals and three soy flours commonly eaten in Europe for the phytoestrogens daidzein and genistein. DESIGN: The phytoestrogens daidzein and genistein were extracted from dried foods, and the two isoflavones quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O glucosyl-4'5-dihydroxyisoflavone) as internal standards. SETTING: Foods from the Cambridge UK area were purchased, prepared for eating, which included cooking if necessary, and freeze dried. Three stock soy flours were also analysed. RESULTS: Eighteen of the foods assayed contained trace or no detectable daidzein or genistein. The soy flours were rich sources, containing 1639-2117 mg/kg. The concentration of the two isoflavones in the remaining foods ranged from 33 to 11,873 micro g/kg. CONCLUSION: These analyses will supply useful information to investigators determining the intake of phytoestrogens in cereal products in order to relate intakes to potential biological activities. SPONSORSHIP: This work was supported by the United Kingdom Medical Research Council, Ministry of Agriculture Fisheries and Food (contract FS2034) and the United States of America Army (contract DAMD 17-97-1-7028). PMID- 12373617 TI - Serum leptin in disabled and non-disabled children in an Indian slum population. AB - OBJECTIVE: To assess the concentration of serum leptin in a population of malnourished children and to compare the leptin levels of disabled and non disabled children in this population. DESIGN: Case-control study. SUBJECTS: Eighty-one children, comprising 41 children with mixed disabilities and 40 non disabled controls, were selected from 425 children involved in a case-control study assessing the nutritional status of children with disabilities in an Indian slum population. METHODS: Leptin was measured in the serum samples and was compared with anthropometry (weight-for-age Z-scores (WAZ), height-for-age Z scores (HAZ), weight-for-height Z-scores (WHZ), body mass index (BMI), mid-upper arm circumference (MUAC), sub-scapular skinfold thickness and triceps skinfold thickness) and serum acute phase proteins. RESULTS: The children were very malnourished with WAZ=-2.07 (s.d. 1.15), HAZ=-2.15 (s.d. 1.85) and WHZ=-1.07 (s.d. 0.83). Leptin was extremely low in both the disabled (1.44 ng/ml; 95% confidence interval, CI, 1.23-1.69) and the non-disabled (1.19 ng/ml; 95% CI 1.04 1.35) children. There were no differences between the disabled and non-disabled groups as a whole but 15 children with neurological disabilities had significantly higher (P<0.05) serum leptin (1.65 ng/ml; 95% CI 1.29-2.06) than the non-disabled children. Girls (1.55 ng/ml; 95% CI 1.29-1.87) had significantly higher concentrations of leptin than boys (1.11 ng/ml; 95% CI 1.02-1.22; P=0.002). Leptin did not correlate with any biochemical or anthropometric measures. CONCLUSIONS: In this population, where malnutrition was common, serum leptin levels were very low and did not correlate with anthropometry. SPONSORSHIP: UK Department for International Development; Virgin Airways through the Great Ormond Street Hospital Trustees. PMID- 12373618 TI - A comparison of international references for the assessment of child and adolescent overweight and obesity in different populations. AB - OBJECTIVE: To compare different references assessing child and adolescent overweight and obesity in different populations. DESIGN: Comparison cross sectional study. SETTING: The United States, Russia, China. SUBJECTS: A total of 6108 American, 6883 Russian and 3014 Chinese children aged 6-18 y. INVESTIGATION: Using nationwide survey data from the USA (NHANES III, 1988-1994), Russia (1992), and China (1991), we compared three references: (1) the International Obesity Task Force (IOTF) reference, sex-age-specific body mass index (BMI) cut-offs that correspond to BMIs of 25 for overweight and 30 for obesity at age 18; (2) the World Health Organization (WHO) reference--BMI 85th percentiles for overweight in adolescents (10-19 y) and weight-for-height Z-scores for obesity in children under 10; (3) a USA reference--BMI 85th and 95th percentiles to classify overweight and obesity, respectively. RESULTS: Using the IOTF reference and 85th BMI percentiles, overweight prevalence was 6.4 and 6.5% in China, 15.7 and 15.0% in Russia, and 25.5 and 24.4% in the USA, respectively. Notable differences existed for several ages. Kappa (=0.84-0.98) indicated an excellent agreement between the two references in general, although they varied by sex-age groupings and countries. Overweight prevalence was twice as high in children (6-9 y) than in adolescents (10-18 y) in China and Russia, but was similar in the USA. Estimates of obesity prevalence using these three references varied substantially. CONCLUSIONS: The references examined produce similar estimates of overall overweight prevalence but different estimates for obesity. One should be cautious when comparing results based on different references. SPONSORSHIP: University of Illinois and University of North Carolina. PMID- 12373619 TI - Nutritional habits in the Mediterranean Basin. The macronutrient composition of diet and its relation with the traditional Mediterranean diet. Multi-centre study of the Mediterranean Group for the Study of Diabetes (MGSD). AB - OBJECTIVE: To compare the nutritional habits among six Mediterranean countries and also with the various official recommendations and the 'Mediterranean diet' as originally described. DESIGN: Cross-sectional study. SETTINGS: Three centres in Greece, two in Italy and one in Algeria, Bulgaria, Egypt and Yugoslavia. SUBJECTS: Randomly selected non-diabetic subjects from the general population, of age 35-60, not on diet for at least 3 months before the study. INTERVENTIONS: A dietary questionnaire validated against the 3-Day Diet Diary was used. Demographic data were collected and anthropometrical measurements done. RESULTS: All results were age adjusted. Energy intake varied in men, from 1825 kcal/day in Italy-Rome to 3322 kcal/day in Bulgaria and in women, from 1561 kcal/day in Italy Rome to 2550 kcal/day in Algeria. Protein contribution (%) to the energy intake varied little, ranging from 13.4% in Greece to 18.5% in Italy-Rome, while fat ranged from 25.3% in Egypt to 40.2% in Bulgaria and carbohydrates from 41.5% in Bulgaria to 58.6% in Egypt. Fibre intake, g/1000 kcal, ranged from 6.8 in Bulgaria to 13.3 in Egypt and the ratio of plant to animal fat from 1.2 in Bulgaria to 2.8 in Greece. The proportion of subjects following the WHO and the Diabetes and Nutrition Study Group (DNSG) of the EASD recommendations for carbohydrates, fat and protein ranged from 4.2% in Bulgaria to 75.7% in Egypt. Comparison with the Mediterranean diet, as defined in the seven Country Study, showed significant differences especially for fruit, 123-377 vs 464 g/day of the Mediterranean diet, meat, 72-193 vs 35 g/day, cheese, 15-79 vs 13 g/day, bread, 126-367 vs 380 g/day. CONCLUSIONS: (a) Dietary habits of the 'normal' population vary greatly among the Mediterranean countries studied. (b) Egypt is closest to the DNSG recommendations. (c) Significant differences from the originally described Mediterranean diet are documented in most Mediterranean countries, showing a Westernization of the dietary habits. PMID- 12373620 TI - The nutrition transition in Spain: a European Mediterranean country. AB - BACKGROUND: Mediterranean diets are felt to be healthful diets linked with reduced mortality from diet-related noncommunicable diseases. OBJECTIVE: To examine trends in diet, activity, obesity and diet-related noncommunicable diseases for Spain and compare these with other European countries, particularly those from the Mediterranean area. DESIGN: A combination of large-scale primary and secondary nationally representative data analysis are used. DATA: Nationally representative data on household food consumption, physical activity, adult obesity, and cause of death are combined with regionally representative adolescent obesity data, obtained in the last four decades. Comparative diet and obesity data come from nationally representative comparable data, obtained during the same period. RESULTS: The Spanish diet has shifted toward a very high level of fat intake, high fruit and dairy intake and moderate vegetable intake. Dairy and fruit intakes were the highest in Europe, as was the proportion of energy from fat, when we compared with the available data. Adult overweight and obesity trends show a marked increase in the past decade to levels as high as Italy and far above France. Overweight for children aged 6-7 is above that of even the USA, while adolescent overweight levels are among the highest in the world. Cardiovascular disease mortality is low, as with Italy and France, and the cancer mortality rate is lower than Italy and France. CONCLUSIONS: We have observed that, in Spain, relatively high obesity prevalences and dairy intake levels are related to much lower levels of cardiovascular disease and cancer mortality than are found in other European countries. This unique Spanish dietary and obesity pattern should be further explored in order to clarify the causal links. SUPPORT: The National Institutes of Health (NIH; R01-HD30880 and R01-HD38700). PMID- 12373621 TI - Validation of the Italian food composition database of the European institute of oncology. AB - OBJECTIVE: To compare nutrient intakes obtained by chemical analysis of food composite or duplicate portion of diets with those obtained by weighed record method using the database of the European Institute of Oncology (EIO). SETTING: Nutrition Section, Department of Internal Medicine, University of Perugia, Italy. SUBJECTS: Fifteen subjects aged 40-59 y in 1960 (41 observations in three seasons), twenty-six subjects in 1965, and only nine remaining subjects in 1970 and 1991 were examined in Crevalcore. In Montegiorgio sixteen subjects aged 40-59 y in 1960 (39 observations in three seasons), thirty-two in 1965, twenty in 1970 and nine in 1991 were assessed. Forty-four subjects in Gubbio area (Biscina, Belvedere and Scritto; 21 males, 23 females; age 56.2+/-14.4 y) were evaluated in 1993 and 1994. METHODS: For dietary appraisal the individual weighed record method was used for 7, 3 or 2 days. Equivalent food composites were made up from local foodstuffs and the duplicate portions were chemically analysed for total nitrogen, fat, saturated and polyunsaturated fatty acids, carbohydrates, retinol, beta-carotene, thiamin and riboflavin. RESULTS: In Crevalcore, a significant difference for protein intake was found between analysis and calculation with EIO database in 1965 and 1991 (P<0.05). Fat intake was significant different for EIO database compared to analysis in 1965 survey (P<0.05), but not for other years. In Montegiorgio, there was a significant difference for protein intake between analysis and calculation with EIO database in 1970 and 1991 (both P<0.001). EIO database showed a significant difference in regard to analysis for fat intake in 1960 IV, 1965, 1970 and 1991 (P<0.05). In both areas there was a significant difference between analysis and EIO database for starch and fibre, but not for polyunsaturated fatty acids and soluble carbohydrates (all P<0.05). In Gubbio area, a significant difference was found between analysis and calculation with EIO database for fat, retinol, beta-carotene and riboflavin intakes (all P<0.05). CONCLUSIONS: According to previous and present studies food composition tables and databases, such as the EIO database, cannot be considered a reliable method to determine nutrient intakes, particularly for some vitamins. PMID- 12373622 TI - Family resemblance in breakfast energy intake: the Stanislas Family Study. AB - BACKGROUND: There seems to be a consensus that family influences on dietary habits are important. However, no data relative to breakfast have been published yet. OBJECTIVE: To investigate whether and how breakfast energy intake aggregates within French families. DESIGN: A total of 398 families of the Stanislas Family Study who filled in a 3 day food consumption diary were selected. Absolute and relative breakfast energy intakes (BEI in kcal/day and RBEI in percentage of daily intake, respectively) were both studied. RESULTS: By using a variance component analysis, no genetic influence was shown in family aggregation of both BEI and RBEI. Intra-generation common environmental contribution to total phenotypic variance of BEI and RBEI was higher than inter-generation; both were increased with frequency of sharing breakfast. Furthermore frequency of sharing breakfast contributed to increase family resemblance in breakfast energy intake, particularly in offspring for BEI and RBEI, and in spouses for RBEI. Smoking habits, alcohol consumption, BMI or physical activity were related to family resemblance, but after adjustment on each factor degrees of resemblance were almost unchanged. CONCLUSION: General findings of this study were that family aggregation in breakfast absolute and relative energy intakes was significant within Stanislas families. Family resemblance depended on inter- and intra generation components and was modified by the number of shared breakfasts. Our study confirmed that familial habits act on family resemblance in both absolute and relative breakfast energy intakes, so that family should be a favorite unit for health and diet promotion programs. SPONSORSHIP: Kellogg's PA, France. PMID- 12373623 TI - Effect of blackcurrant-, cranberry- and plum juice consumption on risk factors associated with kidney stone formation. AB - OBJECTIVE: To evaluate the influence of plum-, cranberry- and blackcurrant juice on urinary stone risk factors. DESIGN: Investigations were carried out in 12 healthy male subjects aged 18-38 y. All subjects received a standardized diet formulated according to the dietary recommendations of the German Society of Nutrition. The subjects provided 24 h urine collections in a control, three loading phases. In each loading phase a neutral mineral water was substituted for 330 ml of the particular juice. RESULTS: Cranberry juice decreased the urinary pH, whereas the excretion of oxalic acid and the relative supersaturation for uric acid were increased. Blackcurrant juice increased the urinary pH and the excretion of citric acid. The excretion of oxalic acid was increased too. All changes were statistically significant. The plum juice had no significant effect on the urinary composition. CONCLUSION: It is concluded that blackcurrant juice could support the treatment and metaphylaxis of uric acid stone disease because of its alkalizing effect. Since cranberry juice acidifies urine it could be useful in the treatment of brushite and struvite stones as well as urinary tract infection. SPONSORSHIP: Funded by our own Division respectively the University. PMID- 12373624 TI - Extra virgin olive oil phenols and markers of oxidation in Greek smokers: a randomized cross-over study. AB - OBJECTIVE: To examine the effect of a low phenol olive oil and high phenol olive oil on markers of oxidation and plasma susceptibility to oxidation in normolipaemic smokers. DESIGN: Randomized single-blind cross-over trial with two intervention periods. SETTING: The Medical School and University Hospital of the University of Crete, Heraklion, Crete, Greece. SUBJECTS: Twenty-five healthy males and females completed the study. INTERVENTIONS: Each intervention was of three weeks duration and intervention periods were separated by a two week washout. Seventy grams of extra virgin olive oil was supplied to each subject per day in the intervention periods. The olive oils supplied differed in their phenol content by 18.6 mg/day. Two fasting venous blood samples were taken at the end of each intervention period. RESULTS: The markers of antioxidant capacity measured in fasting plasma samples (total plasma resistance to oxidation, concentrations of protein carbonyl as a marker of protein oxidation, malondialdehyde and lipid hydroperoxides as markers of lipid oxidation and the ferric reducing ability of plasma) did not differ significantly between the low and high phenol olive oil diets. CONCLUSIONS: No effect of olive oil phenols on markers of oxidation in smokers was detected. It may be that the natural concentrations of phenols in olive oil are too low to produce an effect in the post-absorptive phase. Possible reasons for period effects and interactions between diet and administration period need attention to aid further cross-over trials of this kind. SPONSORSHIP: Unilever Research Vlaardingen, The Netherlands. PMID- 12373625 TI - Height, weight and haemoglobin status of 6 to 59-month-old Kazakh children living in Kzyl-Orda region, Kazakhstan. AB - OBJECTIVE: To estimate the prevalence of stunting, wasting and anaemia among children aged 6-59 months in the Kzyl-Orda region of Kazakhstan, and to determine the association between childhood height and haemoglobin concentration and a range of environmental and biological factors. DESIGN: A cross-sectional study using a randomly selected sample. The mothers of children were interviewed, and finger-prick blood samples and anthropometric measurements were collected on both mothers and their children. Associations between haemoglobin (Hb) concentration, anthropometric measurements and questionnaire data were evaluated by multivariate analysis. SETTING: Health centres in Kazalinsk, Djalagash and Zhanakorgan districts of Kzyl-Orda region, Kazakhstan. SUBJECTS: Two-thousand and twenty-four children aged between 6 and 59 months born to 1501 mothers who were randomly selected from health centre records. RESULTS: The overall prevalence of stunting (<-2.0 Z-scores height for age), wasting (<-2.0 Z-scores weight for height) and anaemia (Hb<11.0 g/dl) in the study children was found to be 15.8, 0.8 and 50.1%, respectively. However, analysis demonstrated considerable variation by age, with the second year of life showing the highest prevalence of both stunting and anaemia. Both childhood height and haemoglobin concentration were found to be significantly associated with a range of environmental and maternal variables. CONCLUSIONS: This study demonstrates that the prevalence of both stunting and anaemia among Kazakh children in the Kzyl-Orda region is considerable, and similar to that of other Central Asian children. These findings highlight Central Asia as a region with levels of childhood nutritional status that are of concern. SPONSORSHIP: Funding was provided by the United States Agency for International Development, Office of Nutrition, the United Kingdom Department for International Development, and the Polden-Puckham Trust. PMID- 12373626 TI - Effects of moderate weight loss on anginal symptoms and indices of coagulation and fibrinolysis in overweight patients with angina pectoris. AB - OBJECTIVE: To evaluate the effects of moderate weight loss, in overweight patients with angina, on plasma coagulation, fibrinolytic indicies and pain frequency. DESIGN: Single-stranded 12-week dietary intervention, an individualised eating plan with quantitative advice delivered by a dietitian. Target weight loss of 0.5 kg per week. SETTING: Outpatient research clinic. SUBJECTS: Fifty-four volunteers with angina pectoris were recruited. Five subjects withdrew, so 27 males, 22 females, mean body mass index (BMI) 29.3 (s.d. 4.3) kg/m(2) and age 60.3 (s.d. 6.5) y completed the intervention. MEASUREMENTS: Body weight and frequency of anginal pain. Plasma fibrinogen, red cell aggregation (RCA), viscosity, factor VII activity, plasminogen activator inhibitor (PAI) activity, tissue plasminogen activator antigen (t-PA), plasma cholesterol, triglyceride and insulin. RESULTS: After the 12-week dietary intervention period, mean body weight fell by 3.5 (s.d. 2.6) kg or 4.3% (P=0.0001), range -11.7 to +1.7 kg. Mean angina frequency fell by 1.8 (s.d. 3.6) from 3.2 to 1.4 episodes/week (P=0.009) and plasma cholesterol by 0.4 (s.d. 0.7) from 6.3 to 5.9 mmol/l (P=0.0001). HDL cholesterol and triglyceride were unchanged. Of the coagulation and fibrinolytic factors, factor VII activity and RCA were significantly reduced by 5 (s.d. 20), IU/dl (P=0.04) and 1.3 (s.d. 1.3) arbitrary units (P=0.014), respectively. CONCLUSIONS: A conventional dietetic intervention, resulting in 4% weight loss, offers the potential to reduce atherosclerotic and thrombotic risk, and to reduce pain frequency, in angina patients. Given the importance of this result in a public health context, these results indicate that this may be a fruitful area for future nutrition research. PMID- 12373627 TI - Does fat intake explain fatness in healthy children? PMID- 12373628 TI - History of the endocrine effects of licorice. AB - The history of licorice as an officinal plant dates back thousands of years, and licorice is still appreciated as a medicinal root. Many of its endocrine properties can be derived from observations of Authors of the ancient world, when hormones were not known. Inappropriate use of licorice can produce pseudoaldosteronism, by inactivating 11beta-hydroxysteroiod-dehydrogenase and by binding to mineralocorticoid receptors. Licorice possesses many other therapeutic properties as to potentiate the action of cortisol, to reduce testosterone synthesis, especially in women, to exert an estrogen-like activity and to reduce body fat mass. The chronological development of research on these effects is described. PMID- 12373629 TI - Primary hyperaldosteronism. AB - Primary hyperaldosteronism (PHA) is regarded as a rare disease with prevalence rates of 0.5 to 2% within the hypertensive population. Recent studies using more detailed screening procedures in small hypertensive cohorts have suggested that PHA may be more common than previously thought (3-18%). Since a validated and cost-effective routine screening protocol for this entity is not established, many clinicians are reluctant to consider PHA as an underlying cause for a patient's high blood pressure. The insufficient perception of PHA may have fatal consequences since most patients are curable by an operation and missing the diagnosis often leads to significant and irreversible end-organ damage. This review focuses on the diagnosis of PHA and gives a rational and cost-effective flow chart for routine screening and differential diagnosis of PHA in hypertensive patients. PMID- 12373630 TI - Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man. AB - Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID- 12373631 TI - Familial ACTH-independent Cushing's syndrome with bilateral macronodular adrenal hyperplasia clinically affecting only female family members. AB - Primary adrenal hyperplasia, which may occur as a familial disorder, is a rare cause of ACTH-independent Cushing's syndrome. In most of these cases the underlying pathology is primary adrenocortical micronodular dysplasia. Very few cases of familial Cushing's syndrome due to primary macronodular adrenal hyperplasia have been described. We report a family with seven affected family members. The pedigree indicates an autosomal dominantly inherited disorder. Interestingly only female family members developed the clinically apparent syndrome. The only available obligatory male gene carrier failed to adequately suppress his plasma cortisol level on overnight dexamethasone suppression test. His adrenal glands showed nodular enlargement on abdominal computed tomographic imaging. Screening of the MEN 1 gene and genetic analysis of the hot spot regions of the GNAS 1 (codons 201 and 227) and GNAI 2 (codons 179 and 205) genes did not show any mutations in the constitutional DNA or the adrenal tissue DNA of the index patient. In conclusion, this family is the largest kindred reported in the literature with ACTH-independent Cushing's syndrome due to autosomal dominant inherited macronodular adrenocortical hyperplasia. Four currently alive and affected family members in two generations and further careful observation of the yet unaffected members of the third available generation might offer the opportunity to identify the still unknown gene defect in the future. PMID- 12373632 TI - Glucocorticoid metabolism and the Metabolic Syndrome: associations in an elderly cohort. AB - OBJECTIVE: The phenotype of the Metabolic Syndrome (hypertension, insulin resistance and hyperlipidaemia) bears similarities to Cushing's Syndrome, in which the cause of these features is elevated cortisol production. We have investigated relationships between glucocorticoid production and features of the Metabolic Syndrome in a cohort of elderly subjects. DESIGN: A cross-sectional analysis was carried out of a subset of a birthweight cohort from Sheffield. METHODS: 92 men and 40 women (aged 69-75 y) representative of the original cohort were investigated. Features of the Metabolic Syndrome (blood pressure, BMI, waist hip ratio, fasting glucose, insulin and triglycerides) were recorded and urinary glucocorticoid metabolites were measured by gas chromatography mass spectrometry. RESULTS: Total glucocorticoid metabolites were correlated with the overall phenotype of the Metabolic Syndrome (P = 0.002), whereas specific pathways of metabolism (activity of 11 beta-hydroxysteroid dehydrogenases and A-ring reductases) did not show significant associations. Specifically total glucocorticoid production increased with increasing systolic blood pressure (r = 0.21, P = 0.013), fasting glucose (r = 0.19, P = 0.02) and insulin (r = 0.23, P = 0.025). Glucocorticoid production was greater with increasing abdominal girth (r = 0.19, P = 0.033), but there was no association with enhanced metabolism via a specific pathway. Within this cohort, birthweight was not associated with total glucocorticoid metabolites. However, decreasing birthweight (P = 0.022), increasing obesity (P = 0.026) and increasing total glucocorticoid production (P = 0.009) were all independent predictors of fasting glucose. CONCLUSIONS: These data support the concept that cortisol production is enhanced in the Metabolic Syndrome, although they did not confirm the recent evidence that increased cortisol secretion is predicted by low birthweight. PMID- 12373633 TI - Insulin resistance in patients with the mitochondrial tRNA(Leu(UUR)) gene mutation at position 3243. AB - The point mutation at position 3243 of the tRNA Leu(UUR) of the mitochondrial DNA is associated with mitochondrial encephalomyopathy, lactic acidosis and strokes (MELAS) as well as with mitochondrial diabetes and deafness (MIDD). A defect in insulin secretion has been found in most of these patients. However, there have been controversial findings to which extent insulin resistance contributes to pathogenesis. The aim of the present investigation was to study the insulin sensitivity index (SI), insulin secretion (AIR(Glucose)) and glucose effectiveness (Sg) in patients with the 3243-mutation. MATERIAL AND METHODS: 7 patients of a large pedigree (some of the members who were not investigated had MELAS) and 3 siblings of another family in whom the 3243-mutation had been detected, as well as 23 non-related, healthy control subjects underwent a modified intravenous glucose tolerance test (Bergman's minimal model). In addition, a screening of islet cell antibodies (ICA) was performed. RESULTS: All patients except for one with known diabetes mellitus revealed normal glucose tolerance. There was no difference between patients and controls for SI, AIR(Glucose) or Sg. However, when looking at the individual results, there were 4 closely related members of the large family with very poor insulin sensitivity. The other 2 patients of this pedigree were more distantly related and extremely insulin sensitive. The siblings of the other family revealed normal or even a very good insulin sensitivity. In one patient, ICA were detected. CONCLUSIONS: The 3243-mutation does not seem to be causative for insulin resistance in our patients. Whether nuclear genes are involved and indirectly influence the expression of the 3243-mutation or, more likely, directly lead to impaired insulin sensitivity in some of our patients cannot be answered by our data. It remains open whether there is a difference in the pathogenesis of diabetes between patients with MIDD and those with MELAS. PMID- 12373634 TI - Increased intraabdominal adipose tissue mass in fructose fed rats: correction by metformin. AB - Summary. The aim of the present study was to investigate the effect of metformin on insulin sensitivity, adipose tissue mass and sympathetic nervous system (SNS) activity in fructose fed rats. Male Sprague-Dawley rats were fed for six weeks either on a standard diet (C group) or on a high-fructose diet (F group, 10% in drinking water). In each group, half of the animals received metformin in drinking water for the last 4 weeks (500 mg/kg x day, C+M and F+M). Hyperinsulinemic-euglycemic clamps (6 mU insulin/kg.min) were performed on awake unrestrained rats to test insulin resistance. Six-week fructose diet induced a reproducible insulin resistance (31.1 +/- 1.9 C vs 22.5 +/- 3.2 mg glucose/kg.min F, p<0.05). Metformin treatment prevented insulin resistance (31.1 +/- 1.9 C vs 30,2 +/- 1.8 mg glucose/kg x min F+M, ns). To measure SNS activity, rats received, ten minutes before sacrifice, an i.p. injection of NSD (m hydroxybenzylhydrazine, inhibitor of DOPA decarboxylase, 100 mg/kg). DOPA accumulation was used as an index of SNS activity and measured in superior cervical, coeliac ganglias, retroperitoneal and epidydimal adipose tissues. SNS activity was increased in F group only in coeliac ganglia (16.8 +/- 1.1 C vs 22.6 +/- 2.2 ng DOPA/ganglia, F group, p<0.05) and not in superior cervical ganglia (8.4 +/- 0.7 C vs 8.6 +/- 0.7 ng DOPA/ganglia, F group, ns). Metformin had no effect on SNS activity in coeliac ganglia of control animals (15.9 +/- 1.7 C+M vs 16.8 +/- 1.1 ng DOPA/coeliac ganglia C, ns) but prevented the increase in SNS activity in fructose fed animals (22.6 +/- 2.2 F vs 16.3 +/- 2.8 ng DOPA/coeliac ganglia F + M). In fructose fed rats, metformin significantly increased sympathetic activity in retroperitoneal white adipose tissue (RPWAT) resulting in a marked decrease in depot mass but had no effect on epidydimal WAT. In conclusion, our results demonstrate that fructose diet caused a selective increase of SNS activity in coeliac ganglia. Metformin increased SNS activity in RPWAT resulting in a significant reduction in RPWAT mass, lowered SNS activity in coeliac ganglia to control values and restore whole body insulin sensitivity. PMID- 12373635 TI - Construction and characterization of a conditionally active construct of the insulin-regulated forkhead transcription factor FKHR. AB - Summary. Insulin is known to inhibit glucose-6-phosphatase gene expression through PI 3-kinase/PKB mediated phosphorylation and inactivation of the forkhead transcription factor FKHR, which is a potent transactivator of the glucose-6 phosphatase gene. To study the function and regulation of the transcription factor FKHR in hepatic cells, we constructed a hydroxytamoxifen-inducible version of FKHR by fusing a part of the hormone binding domain of the estrogen receptor (ER) to the C-terminus of FKHR (FKHR-ER). In HepG2-cells transiently transfected with plasmids encoding the FKHR-ER fusion protein and a glucose-6-phosphatase reporter construct, hydroxytamoxifen induced a marked induction of glucose-6 phosphatase promoter activity, whereas no effect was observed in control cells. We next generated a H4IIEC3 rat hepatoma cell line stably expressing both FKHR-ER and a glucose-6-phosphatase promoter-based reporter construct. After 2h stimulation with hydroxytamoxifen, the promoter activity was stimulated 3-5 fold, and continued to increase up to 100-fold after 15 h. The response was half maximal at 0.5 microM hydroxytamoxifen. Insulin (1 nM) decreased the hydroxytamoxifen induced promoter activity by about 70% of the maximal response. This cell system can be used for (1) the identification of FKHR dependent genes and for (2) high throughput screening (HTS) of agents affecting the activity of FKHR and its regulation by insulin. Abbreviations used: FKHR, forkhead in rhabdomyosarcoma; G6Pase, glucose-6-phosphatase; PKB, protein kinase B; PI 3 kinase, phosphatidyl-inositol 3-kinase; IRU, insulin-responsive unit; Tx, 4 hydroxytamoxifen, ER, estrogen receptor; HBD, hormone binding domain PMID- 12373636 TI - [Increasing significance of antidepressants in deliberate self-poisoning]. AB - BACKGROUND AND OBJECTIVE: Antidepressant drugs are frequently used in deliberate self-poisoning resulting in a major risk for the patients due to their cardiac and central-nervous toxicity. In the present study the cases of intoxications consulting our Poison Center should be analysed illustrating recent results and trends about self-poisoning with antidepressants. PATIENTS AND METHODS: During the study period from 1995 to 2001 35 394 inquiries concerning deliberate self poisoning were registered in our Poison Center. The substance used, age and gender of the patient as well as the degree of the observed symptoms were documented. Thereby, antidepressant drugs were grouped in tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRI) and other antidepressants. RESULTS: The use of antidepressants in deliberate self-poisoning continuously increased during the study period from 17.3 % to 22.9 % with SSRI and other antidepressants being observed more frequently as compared to TCA. Antidepressant drugs were mainly used from female patients and in the age group between 35 and 54 years. Antidepressant drugs caused severe intoxications and deaths more frequently as the remainder substances with TCA showing higher rates of complications as compared to SSRI and other antidepressants. CONCLUSIONS: In recent years, an increasing importance of antidepressant drugs in deliberate self poisoning was determined particularly concerning female and middle-aged patients. Due to the changing prescribing patterns larger numbers of intoxications with SSRI and other antidepressants were observed representing an advantage with respect to the reduced rate of complications known for these substances as compared to TCA. Nevertheless, the averagely more severe symptoms present in the three groups of antidepressants in comparison to the remainder drug overdoses demonstrated the need for hospitalization and monitoring of intoxications with antidepressants. PMID- 12373637 TI - [A scissors-effect in career development of female and male medical doctors]. AB - BACKGROUND AND OBJECTIVE: Women are equally interested in studying medicine as men, and there is an equal proportion of female and male graduates in medicine. Women's occupational careers in medicine, however, are on the average less successful than men's. Whereas there are already many cross-sectional studies on this issue the present paper reports data of a prospective longitudinal study. PARTICIPANTS AND METHODS: Participants were 139 female graduates and 172 male medical graduates. These were questioned three times, after their second "Staatsexamen", one and a half years later during their practical medical training time, and again one and a half years later within their specialized training. The questionnaires tapped performance data, occupational self-efficacy, attitudes, goals, work satisfaction, experience of work situation, occupational development and private development. RESULTS: There were no gender differences in grades, study duration, occupational self-efficacy and goals immediately after the exam. Experience of the work situation as well as work satisfaction did not differ, either. However, at time three female medical doctors were less often full-time employed than their male colleagues. This pertained not only to mothers but also to childless women. Full-time employed women often were singles without a partner. There were also gender differences in the expectations how occupation and family should be combined. Psychologically, women experienced a decrease in occupational self-efficacy, whereas men experienced an increase. Both equivocal female gender-role expectations and unfavorable organizational conditions of medical training are important for this development. CONCLUSION: The compatibility between work and family must be seen as a societal task. Otherwise many well-trained female medicine doctors will quit their jobs and as a consequence there will be a lack of doctors in the future. PMID- 12373638 TI - [Esophageal pseudodivertikulosis]. AB - HISTORY AND CLINICAL FINDINGS: A 56-year-old man presented himself at our polyclinic with the symptoms of dyspnoea at rest and exhaustion. The case history revealed an alcoholic liver cirrhosis (Child B) and recurrent heart burn as a sign of a gastro-esophageal reflux disease. The examination showed jaundice and enlargement of the liver as pathological features. INVESTIGATIONS: At gastroscopy multiple openings of pseudodiverticula and a high-grade inflammatory reaction of the esophageal mucosa was found, indicating pseudodiverticulosis of the esophagus. After staining with lugol-solution and directed biopsy of unstained areas there was no sign of malignancy in the histopathological report. TREATMENT: The candida esophagitis and gastroesophageal reflux disease were treated with antimycotic and proton-pump-inhibiting drugs after which the patient had no more complains. Regular gastroscopic controls are planned. CONCLUSION: Pseudodiverticulosis of the esophagus is a very rare disease which arises from ductal dilatation of the mucosal glands during chronic inflammation of the esophagus. In most cases the pseudodiverticulosis is an accidental finding without symptoms. Risks can arise from the most frequent complications like development of inflammatory strictures, motility disorders and transformation to malignancy. Therefore it is necessary to perform regular inspection of the esophagus by endoscopy. PMID- 12373639 TI - [Polyneuropathy--Case report]. PMID- 12373640 TI - [Polyneuropathy--diagnostic]. PMID- 12373642 TI - [Polyneuropathy--quiz for certification]. PMID- 12373641 TI - [Polyneuropathy--treatment]. PMID- 12373644 TI - [Bolus fibrinolysis in acute myocardial infarction]. PMID- 12373645 TI - [How is catecholamine therapy carried out in heart failure?]. PMID- 12373646 TI - [Does vasectomy increase risk of prostate carcinoma?]. PMID- 12373649 TI - [6th Refresher course on epilepsy. Santander, Spain, 21-22 February 2002]. PMID- 12373647 TI - A major susceptibility locus for systemic lupus erythemathosus maps to chromosome 1q31. AB - A set of 87 multicase families with systemic lupus erythemathosus (SLE) from European (Iceland, Sweden, England, Norway, Italy, and Greece) and recently admixed (Mexico, Colombia, and the United States) populations were genotyped and analyzed for 62 microsatellite markers on chromosome 1. By parametric two-point linkage analysis, six regions (1p36, 1p21, 1q23, 1q25, 1q31, and 1q43) were identified that have LOD scores of Z>or=1.50, with different contributions, depending on the population of origin of the families (European or admixed American). All of the regions have been described previously and have therefore been confirmed in this analysis. The locus at 1q31 showed a significant three point LOD score of Z=3.79 and was contributed by families from all populations, with several markers and under the same parametric model. Analysis of a known mutation in the CD45 gene did not support the role that this mutation plays in disease. We conclude that the locus at 1q31 contains a major susceptibility gene, important to SLE in general populations. PMID- 12373648 TI - Mutation in human desmoplakin domain binding to plakoglobin causes a dominant form of arrhythmogenic right ventricular cardiomyopathy. AB - Arrhythmogenic right ventricular cardiomyopathy (ARVD/C) is a genetically heterogeneous disease characterized by progressive degeneration of the right ventricular myocardium and increased risk of sudden death. Here, we report on a genome scan in one Italian family in which the disease appeared unlinked to any of the six different ARVD loci reported so far; we identify a mutation (S299R) in exon 7 of desmoplakin (DSP), which modifies a putative phosphorylation site in the N-terminal domain binding plakoglobin. It is interesting that a nonsense DSP mutation was reported elsewhere in the literature, inherited as a recessive trait and causing a biventricular dilative cardiomyopathy associated with palmoplantar keratoderma and woolly hairs. Therefore, different DSP mutations might produce different clinical phenotypes, with different modes of inheritance. PMID- 12373650 TI - [Congenital errors of metabolism with epileptic seizures during the first years of life]. AB - OBJECTIVE: In this paper we review the main aetiologies of metabolic origin which cause epilepsy in children aged between 1 and 10 years. DEVELOPMENT: There are many aetiological causes of convulsive seizures. Seizures and epilepsies due to congenital errors of metabolism are a minority but should be known. Their identification is not easy although at present there is a wide range of diagnostic methods to confirm the diagnosis when this is suspected. In this review we have only considered the congenital errors of metabolism which start between the ages of 12 months and 10 years, with convulsive seizures. We have divided the conditions into two subgroups depending on whether epilepsy was one of the main symptoms or only part of a set of neurological symptoms and signs. Finally we establish the possible diagnoses of congenital errors of metabolism with seizures occurring in this age group. CONCLUSIONS: In neuropaediatrics the paediatrician and epileptolgist must be aware of congenital errors of metabolism as being responsible for epilepsy, especially in cases of drug resistant epilepsy or when accompanied by other systemic features, neurological deterioration or unexplained biochemical alterations. PMID- 12373651 TI - [Transient cognitive disorder from sub clinical paroxysmal EEG activity]. AB - INTRODUCTION: Transient cognitive disorder (TCD) defines the existence of a decrease in reaction time that coincides with an epileptiform EEG discharge, without any simultaneous manifestation of a classical epileptic seizure. Aims. To analyse the relation between episodes of TCD and the neurolopsychological manifestations in infancy that condition a high percentage of neuropaediatric visits to the surgery. At the same time we analyse the relation between the interictal paroxysmal disorders of patients with childhood benign partial idiopathic epilepsy with centrotemporal spikes (BIE CS) and the neurolopsychological manifestations that are frequently detected in such patients. PATIENTS AND METHODS: Two groups of patients were studied. Group A: 23 children who sought medical attention because of different neurolopsychological disorders (language retardation, hyperactivity, lack of attention, retarded academic achievement, behavioural disorders, bad social interaction); gender: 16 males and 7 females; age interval: from 2 years and 10 months to 11 years and 1 month (average age: 6 years and 8 months). Group B: 10 patients who were BIE CS carriers, two of which evolved toward atypical BIE; gender: 5 males and 5 females; age interval: from 3 years and 3 months to 9 years and 9 months (average age: 7 years and 4 months). Both groups were submitted to a clinical examination protocol involving neurological, EEG, child psychiatric and psychological aspects. RESULTS: In group A, sub clinical paroxysmal EEG discharges were seen in three cases, two of which corresponded to a lack of attention disorder with hyperactivity, and the third had a generalised growth disorder. In group B we detected a high percentage of perceptive and psychomotor disorders, without the existence of differences between those who displayed an irritative focus in the right or in the left hemisphere, although the alteration in the level of language was greater in the latter. Likewise, in a large percentage of cases (80%) the evaluation of the level of personality revealed obvious anxiety traits, which were related with suffering from seizures. CONCLUSIONS: Sufficient evidence has been found to demonstrate the existence of the possible relation between different neuropsychological disorders and epileptic EEG discharges, although revealing it in daily clinical practice requires a thorough diagnostic protocol and an accurate neuropsychological examination under video EEG monitoring, the positive results of which are considered to be decisive in evaluating the possibility of pharmacological treatment. PMID- 12373652 TI - [Clinical, neuro-radiological and prognostic aspects of post-encephalitic catastrophic epilepsies]. AB - OBJECTIVE: To determine the prevalence of encephalitis and meningo encephalitis as the causative agents of catastrophic epilepsies (CE) and the incidence of post encephalitic CE, when catastrophic epilepsy is defined as often refractory to treatment and always associated with psychoneurological deterioration. PATIENTS AND METHODS: The prevalence of central nervous system (CNS) infections in determining West s syndrome (WS), Lennox Gastaut syndrome (LGS) and HHE syndrome (HHES) was detected in the large series published since 1980 in which the cause was stated. The incidence of CE in the course of meningoencephalitis was deduced from three studies done in the Virgen del Roc o Hospital: study 1 of 1,221 children admitted to hospital with the diagnosis of meningo encephalitis; study 2 of 55 cases of tuberculous meningitis; study 3 of 30 cases of encephalitis. RESULTS: CNS infections causing CE are responsible for from 3 to 11% of all WS, 3 to 8.2% of all LGS and 19% of the HHES with a catastrophic course. The commonest causes are infection due to cytomegalovirus and toxoplasmosis during the prenatal stage and the purulent meningitis, tuberculous meningitis and herpetic encephalitis during the neonatal and postnatal periods. The evidence of CE in meningo encephalitis varies according to the germ, age and severity of the aggression. CNS infections during the neonatal period in 3% of cases cause CE. In babies, newborn and subsequently, tuberculous meningitis (12.7%), measles meningo encephalitis (22%) and herpetic encephalitis (50%) lead to refractory epileptic seizures and very severe psychoneurological deterioration. CONCLUSIONS: 1. Encephalitis and meningo encephalitis are commoner than usually thought as a cause of CE. 2. They cause 3 11% of the WS, 3 8% of the LGS and 19% of the HHES. 3. The incidence of CE in the course of meningo encephalitis varies according to the germ involved and the severity of the aggression. 4. CE are very frequent during the course of herpetic encephalitis, measles meningo encephalitis and tuberculous meningo encephalitis. The latter two are becoming much less common. 5. The prognosis is extremely serious PMID- 12373653 TI - [Preventive and therapeutic attitude in post-traumatic epileptic seizures]. AB - INTRODUCTION AND DEVELOPMENT: Due to the vast number of different circumstances surrounding them, the frequency with which post traumatic epileptic seizures occur varies greatly from study to study. Immediate and early epileptic seizures, within a week of the traumatism having taken place, are usually of little importance as regards the risk of post traumatic seizures. The most important factors governing the presentation of post traumatic seizures have to do with the seriousness of the injury, the extension of the brain tissue that is affected and the penetrating nature of the brain traumatism. CONCLUSION: Although antiepileptic medication significantly reduces the risk of early seizures from occurring, a review of well designed clinical trials has found no evidence that these drugs reduce the morbidity and mortality associated with head injuries, or the appearance of late seizures. PMID- 12373654 TI - [Problems of diagnosis and treatment in frontal epilepsies]. AB - OBJECTIVE: In this paper we review the anatomy, clinical features, problems of diagnosis and alternative treatment of the frontal epilepsies. DEVELOPMENT: A knowledge of the functional anatomy of the frontal lobe, the largest in the brain, is essential to understanding the varied features of the epileptic seizures arising in it. Unlike seizures arising in the temporal lobe in which the anatomicoclinical correlation is clearer since the rhinencephalum and especially the amygdala are almost always involved, in frontal seizures it is very difficult to systematize the relationship between the clinical signs and the organization of the discharge since there is wide cortico subcortical propagation, both homolateral and contralateral. There are therefore various types of frontal seizures which we define from a clinical point of view. They are the ones causing the greatest problems of differential diagnosis with epileptic pseudo seizures. A continuous video EEG recording is often necessary to differentiate them. Resonance imaging has meant a major advance in making an aetiological diagnosis of this type of seizure. It can show cortical dysplasia, heterotopia and small tumours or vascular malformations which are not visualized by other means. We give a brief description of the newly discovered genetic frontal epilepsies. Finally we review the different types of treatment indicated for them. CONCLUSIONS: Between 20% and 30% of all partial epilepsies start in the frontal lobe, and they form 30% of all surgical operations. The correct diagnosis of frontal seizures is still a challenge for the neurosurgeon. Advances in neurophysiology, neuro radiology and genetics have been, and still are, very important in better understanding of the disorder. PMID- 12373655 TI - [Epilepsy and sleep as seen on video encephalographic recordings]. AB - INTRODUCTION: The relation between epilepsy and sleep has been known for some time. Seizures are often not observed by the examiner and it is necessary to make prolonged recordings during sleep, both slow or no REM sleep and paradoxical or REM sleep. Objective. To show the way in which a video recording may be made of a patient s seizures, together with an electroencephalogram recorded on a suitable disk and both sets of data be synchronised and studied as often as necessary. PATIENTS AND METHODS: We describe some of the epileptic seizures, recorded by this technique, during sleep. At the same time we show other paroxystic non epileptic episodes occurring during sleep which may be needed to be ruled out of the differential diagnosis. CONCLUSIONS: We show that as a general rule the basic activity of paroxystic disorders seen on an electroencephalogram occurs during slow sleep phases and particularly during their early stages. These studies are especially relevant in children and in neonates a prolonged recording is essential PMID- 12373656 TI - [Melatonin and epilepsy]. AB - OBJECTIVE: This review has been prepared in response to the increasing interest shown in understanding the part played by melatonin in the body, which has led to the search for new uses of it in cerebral disorders, such as sleep disorders including insomnia, irritability, depression, behaviour disorders and even the treatment of autism, since sleep disorders also occur in this condition. We pay particular attention to studies involving epilepsy. DEVELOPMENT: We show that interest in melatonin is rapidly increasing and new discoveries are being made of the part it plays in the biological regulation of circadian rhythm, sleep, mood, ageing, tumour growth and reproduction. Perhaps these processes between them have led to its use in the treatment of many current problems such as neuroprotection, migraine and the control of epileptic seizures. CONCLUSIONS: It has been shown that in both children and adults melatonin is of low toxicity and may be used in high risk persons. In this paper we make a careful analysis of recent publications in the medical literature dealing with the use of melatonin in the control of epileptic seizures and discuss its advantages and disadvantages. However, as with other types of treatment, further study, both experimental and otherwise, is necessary for confirmation. PMID- 12373657 TI - [Evidence based treatment of epilepsy]. AB - OBJECTIVE: Evidence based medicine is becoming popular in all fields of medicine. Through systematic reviews, critical evaluation, and statistical strategies such as meta analysis it aids to take decisions in clinical practice. We revise the information on the treatment of epilepsy found in the main sources of evidence based medicine. DEVELOPMENT: After commenting some basic concepts such as systematic review, meta analysis, odds ratio, relative risk and numbers needed to treat, we describe the main primary, secondary and tertiary drug information sources with emphasis on sources of evidence based medicine. Some representative examples are given about the information on treatment of epilepsy found in the main sources of evidence based medicine such as TRIP, DARE, Cochrane Library, clinical practice guidelines, Clinical Evidence or Bandolier. Most clinical trials analyze the efficacy and tolerability of add on new antiepileptic drugs in partial refractory epilepsy. However, we found few trials on efficacy and tolerability of these drugs in monotherapy, in newly diagnosed partial epilepsy, on other types of epilepsy or in children. There are also few trials comparing the new antiepileptic drugs between them or in relation to the old ones. CONCLUSION: Treatment of epilepsy is yet an art more than a science because clinical practice decisions depends on therapeutic habits and clinical expertise more than on the results emerging from randomized and controlled clinical trials. Large comparative trials are needed but relevant criteria of efficacy and validated procedures to evaluate quality of life, tolerability or cognitive function outcomes should be used in these trials. PMID- 12373658 TI - [Characteristics and indications of lamotrigine]. AB - OBJECTIVE: In this paper we make an extensive review of the most recent and important studies which have been published on lamotrigine, a phenyltriazinic compound unrelated to the other known anti epileptic drugs, which is available on the market in many countries and is currently considered to be a first line anti epileptic drug. DEVELOPMENT: It is known that its anti epileptic effect is mainly due to blocking the voltage sensitive sodium channels most effectively in depolarized cells, resulting in the presynaptic inhibition of excessive release of excitatory amino acids, particularly glutamate and aspartate. In this review, we fully discuss their different mechanisms of action and compare various experimental animal studies with the use of these drugs in humans and the results obtained in everyday clinical practice. Amongst the metabolic aspects we analyze the reasons why children are more prone to develop enzyme induction than adults are. It has been shown that in infancy patients who take inductors and inhibitors show figures for the half life of lamotrigine which are between those of patients who take inductors only and those taking inhibitors only, causing a so called mixed effect . Adverse effects include skin eruptions which may lead to withdrawal of the drug. However, it should be remembered that the proportion of persons with this side effect is much reduced when the correct dosage is used, and the sliding scale of dosage starts with sufficiently low doses. CONCLUSIONS: After extensive analysis of the results, we point out the new possibilities now available for the treatment of other disorders besides epilepsy. We show the positive aspects of this treatment, its use in epileptic seizures and interactions in disorders besides epilepsy now that the mechanism of action is better understood. PMID- 12373659 TI - [Characteristics and indications of gabapentin]. AB - Gabapentin is a drug that shares a similar structure to that of GABA, although its mechanism of action cannot be explained solely by a direct gaba mimetic effect. It is well absorbed when administered orally and displays linear kinetics up to doses of 1,800 mg/day. It is been found to be effective both in added therapy and in mono therapy, and is particularly useful in special populations like the elderly and children, as well as patients suffering from liver diseases. Its safety profile and the absence of interactions make it a suitable drug for the treatment of recently diagnosed epilepsies, both in mono therapy and in bi therapy. PMID- 12373660 TI - [Characteristics and indications of topiramate]. AB - AIM: To evaluate the efficiency of topiramate (TPM), an antiepileptic medication (AEM) which possesses multiple mechanisms of action and good pharmacokinetics, in the different types of childhood epilepsy and to make an appraisal of its value in migraines, bipolar disorder, eating disorders and neuropathic pain, according to studies that have been published. To do so, we have made use of an analysis of the literature, together with a multi centre study conducted in Spain and personal casuistry. METHOD: We consider the percentage of seizure free patients and of patients who responded (reduction of 50% or above in the frequency of the seizures) in childhood epilepsy, partial epilepsy, generalized tonic clonic seizures, absence seizures, tonic seizures, patients with diverse types of seizures, juvenile myoclonic epilepsy, Lennox Gastaut syndrome, falling sickness and GTCS, West s syndrome and Dravet s syndrome. With monotherapy, in partial epilepsy, between 39 and 54% of patients treated were seizure free. TPM has also proved to be efficient in experiments with animals, as a neuroprotector, and in clinical trials, in type I bipolar disorder, eating disorders, neuropathic pain and migraine. CONCLUSIONS: TPM is an AEM offering a wide therapeutic spectrum that has proved to be efficient in clinical trials, expansion phases and observational studies, as an associated drug in partial epilepsy, generalized epilepsy, Lennox Gastaut syndrome, West s syndrome and Dravet s syndrome. It has proved to be more efficient in monotherapy, in partial epilepsy, as a first line AEM. TPM has also proved to be useful in mood disorders, eating disorders, neuropathic pain and tremor in observational studies, although this efficiency has not been backed up by clinical trials. In migraine and in clinical trials TPM has shown its efficiency. Its neuroprotective effect opens up new therapeutic perspectives. PMID- 12373661 TI - [Characteristics and indications of tiagabine]. AB - INTRODUCTION: Tiagabine (TGB) is an anti epileptic drug whose mechanism of action is due to a reduction in the neurone and astrocyte uptake of gamma aminobutyric acid (GABA), causing its concentration at the synapse to be increased. DEVELOPMENT: We analyze the most usual pharmacokinetic and pharmacodynamic characteristics of TGB, considering current therapeutic indications showing its increased use in seizures and partial epileptic syndromes. We also assess the adverse effects described, with special reference to the results obtained by the Spanish group investigating TGB, in a large number of patients with a wide range of ages. Finally we review the occurrence of status epilepticus induced by using TGB. CONCLUSIONS: It is defined as a gabaergic drug which is well tolerated and causes no visual field reduction. It may be used in epilepsies and epileptic syndromes which can be treated with it. PMID- 12373662 TI - [Characteristics and indications of oxcarbazepine]. AB - OBJECTIVE: To review the major studies published concerning the pharmacokinetic characteristics, mechanism of action, clinical efficacy and adverse effects of oxcarbazepine (OXC). DEVELOPMENT: OXC is a ketoderivative of carbamazepine (CBZ), with a similar mechanism of action, possibly widening the voltage dependent potassium channels. Its pharmacokinetic characteristics are much better than those CBZ but the frequency and intensity of interactions is much less. In several double blind trials, using the drug in monotherapy in previously untreated patients, similar efficacy was found after OXC, phenytoin, valproate and CBZ but the fewest adverse effects were seen after OXC. CONCLUSIONS: OXC is an antiepileptic drug with better pharmacokinetic properties than CBZ and similar clinical efficacy, but better tolerated, so it may therefore be expected to replace this classical antiepileptic drug for use in monotherapy and polytherapy of both children and adults in all types of partial seizures. PMID- 12373663 TI - [Characteristics and indications of levetiracetam]. AB - OBJECTIVE: To describe the data contained in the most important studies published on the pharmacokinetic and pharmacodynamic properties of levetiracetam, and also the main clinical trials carried out using this new antiepileptic drug. DEVELOPMENT: Derived from piracetam, but with very different properties, levetiracetam is ineffective in the usual models of seizures induced in experimental animals, although it acts in models of prolonged activation, audiogenous seizures and absences and has a novel mode of action. It has pharmacokinetic properties which are nearer to that of the ideal anti epileptic drug. In clinical trials done in adults with partial epilepsies use of 1,000 to 4,000 mg of levetiracetam was significantly more effective than a placebo, and the drug was very well tolerated. CONCLUSIONS: Levetiracetam is the newest antiepileptic drug to appear on the market. Its pharmacodynamic and pharmacokinetic characteristics are excellent. It has currently been approved for use in the polytherapy of patients with partial seizures aged over 16 years. Several studies indicate that its therapeutic spectrum is probably wider, particularly in generalized seizures such as the myoclonias, absences and seizures induced by light stimulation. Thus the indications for levetiracetam may become clear over the next few years. PMID- 12373664 TI - [Monitoring serum levels of new antiepileptics]. AB - AIM: Therapeutic monitoring of old antiepileptic drugs has been useful in improving their use in clinical practice. The new antiepileptic drugs have been developed with the idea that monitoring their serum levels was going to be unnecessary. We review the characteristics of the new antiepileptic drugs that can be relevant to their being monitored and their possible uses. DEVELOPMENT: After discussion of the evolution of the therapeutic monitoring of antiepileptic drugs in general, we take a more detailed look at the requirements needed for it to be useful, such as the indications, the procedure and a correct interpretation of the results. We point out the reasons why monitoring the new antiepileptic drugs can be worthwhile and we examine the characteristics of felbamate, gabapentin, lamotrigine, oxcarbazepine, tiagabine, topiramate, vigabatrin and zonisamide which may be relevant in their monitoring. These include the type of kinetics, the factors that have an influence on the relationship between dosage and serum levels, the concentration/dose ratio, data on the relationship between serum levels and effects, the factors that can influence this relationship, as well as the characteristics of sampling. CONCLUSION: The new antiepileptic drugs present a wide interindividual and intraindividual variability which leads us to believe that some of they may be suitable candidates for therapeutic monitoring, but at present no target ranges have been clearly defined for any of them. Therefore, routine monitoring cannot be recommended, but it may be useful to establish an individual reference level that allows control over compliance and dosage readjustment in the presence of factors that alter their pharmacokinetics. Specific prospective studies are needed to establish target ranges that allow to individualize dosage in the absence of clinical criteria and to resolve doubts about the efficacy and toxicity of these drugs. Quicker and simpler assays that make monitoring easier are also needed. PMID- 12373665 TI - [Teratogenic effects of epilepsy and anti epileptic drugs]. AB - OBJECTIVE: In this paper we review the main studies on teratogenicity related to epilepsy and especially use of anti epileptic drugs (AED), with special emphasis on recently acquired knowledge regarding the new AED. DEVELOPMENT: When considering the teratogenic effects of epilepsy and the anti epileptic drugs it should be remembered that there are a series of premises and considerations which undoubtedly play an important part in causing possible damage to the foetus. These factors include changes caused during pregnancy, the passage of drugs across the placenta barrier , malformations occurring in the children and relations of women with epilepsy and finally the effect of seizures on the foetus. We then review the mechanisms of the teratogenicity of the classical AED and the new AED. Little is known about the adverse effects of the new AED, and many are used together as polytherapy. Multicenter studies involving large numbers of participants are therefore necessary to obtain results which can be extrapolated to the whole population. Unfortunately, at present, this is not yet so and there are no clear recommendations for their use during pregnancy. The EURAP was designed for this reason. It is one of the multicenter studies being carried out in Europe at present with many Spanish specialists participating. CONCLUSIONS: Multicenter studies with many participants are necessary to obtain reliable data on the teratogenicity of the various AED, used as monotherapy and bitherapy, particularly regarding the new AED. We conclude by considering measures to try to reduce, as far as possible, the teratogenic effects in pregnant women. PMID- 12373666 TI - [Indications and results of nonpharmacological treatments of epilepsies: vagal stimulation, ketogenic diet and gamma rays]. AB - OBJECTIVES: In this paper we review alternative non pharmacological treatments for patients with epilepsy, both focal and generalized, which are resistant to the pharmacological treatment normally used. DEVELOPMENT: Vagal nerve stimulation (VNS) is a recently used palliative technique whose mechanism is not clearly understood. We analyze the clinical trials reported to date and the main indications and contra indications. Although the ketogenic diet (KD) has been used since the 1920s, recently there has been renewed interest in using it. Several papers have been published describing its use in children with epilepsy which was difficult to control. The complex metabolic and endocrine aspects of this type of diet make it difficult to select patients who may benefit from it. Gamma knife surgery is a new technique which has been discussed in this paper since it has been recently used in cases of refractory epilepsy, especially temporal medial epilepsy and hypothalamic hamartomas. CONCLUSIONS: VNS and KD are alternative treatments which may be used in patients whose condition cannot be satisfactorily controlled by pharmacological treatment and are not candidates for the surgery of epilepsy. Gamma knife surgery is a surgical technique which has recently been introduced for the treatment of these patients. PMID- 12373667 TI - [Society, law and epilepsy]. AB - INTRODUCTION: Epilepsy is an important problem from a medical, social and legal point of view. Proof of this is the fact that it constitutes the second most commonly alleged cause for absolution of responsibility in Spain, according to the jurisprudence from the Supreme Court (1976 1995). METHOD: Throughout history it has been classified as a magical, supernatural disease and has been studied within psychiatry as an endogenous psychosis. It has therefore been considered a form of madness, which has led to court decisions that have taken this concept into account. The supposed dangerousness of suffers from epilepsy must be the exception, and their supposed epileptic characters and personalities that drive them to commit atrocious murders are no longer of any relevance. The problems stemming from epileptic seizures are to be seen in civil, penal, military, canonical and labour law, very often in an exclusive fashion. CONCLUSION: We think it is worthwhile reviewing these concepts with a view to their undergoing a later modification, which would lead to the full integration of these patients and to their being considered as suffering from a neurological illness. PMID- 12373668 TI - [Bilateral hemifacial spasm: eight personal case reports]. AB - INTRODUCTION: Hemifacial spasms consist in tonic clonic, involuntary, asymmetrical and asynchronous contractions in the territory innerved by the facial nerve. Several different causes may give rise to this disorder, the most frequent of which are vascular abnormalities in the cerebellopontine angle. Its clinical features and electrophysiological studies are commonly used in diagnosis and its etiological diagnosis is most frequently performed by means of magnetic resonance imaging. Symptoms are treated using local injections of Botulinum toxin Type A in the affected muscles. AIMS: To review our experience in the handling of this pathological condition and to determine the results of employing Botulinum toxin. PATIENTS AND METHODS: We describe the cases of bilateral hemifacial spasms that have been diagnosed in the Virgen Macarena Hospital in Seville and La Fe in Valencia since 1980, as well as the follow up after treatment with Botulinum toxin. RESULTS: We describe eight cases of this pathological condition in which patients were treated with Botulinum toxin, and in all cases there was an improvement in the symptoms. CONCLUSIONS: Treatment with Botulinum toxin is considered to be satisfactory and provides a marked improvement in the patients quality of life. PMID- 12373669 TI - [Postictal paralysis during video-EEG monitoring studies]. AB - OBJECTIVE: To know the frequency of Todd s paralysis during the video EEG monitoring studies, to investigate in its pathophysiology, and to confirm its value to localise the epileptic focus. PATIENTS AND METHODS: We reviewed 114 monitoring studies, in 102 patients. RESULTS: Sixty patients had epileptic seizures. An obvious paresis was noted in four seizures of two patients (3 and 1, respectively). Both patients had frontal epilepsy. During the paralysis, in the first patient the EEG showed ictal discharges on the contralateral centrotemporal area. In the second patient, the EEG demonstrated slow waves in the contralateral frontal region. The ictal onset was contralateral to the paresis in all cases. No patient with pseudoseizures had paralysis. CONCLUSIONS: Postconvulsive paralysis are not frequent in video EEG monitoring studies. However, if present it points out to a contralateral seizure onset. In our series it happened in patients with frontal seizures. The EEG may help to clarify if it correspond to a true postictal phenomenon or to a ictal paralysis. PMID- 12373670 TI - [Management of multiple cerebral cavernomatosis]. AB - INTRODUCTION: Cavernous angiomas are angiographically occult vascular malformations that are present in 0.4% of people, and represent 5 13% of all cerebrovascular malformations. They can be alone or multiple, and sporadic or familial. The presence of multiple lesions is more frequent in familial cavernomatosis. OBJECTIVES: Improve our knowledge of the natural history of multiple cavernomatosis in order to improve our diagnostic and therapeutic management of this entity. PATIENTS AND METHODS: We have retrospectively reviewed 18 cases of multiple cerebral cavernomatosis; 4 of them belonged to the same family and 2 belonged to another family. Number, size, characteristics and evolution of the lesions, symptoms, treatment and clinical outcome have been analysed during a follow up period longer than 5 years. RESULTS: 31.5% of the cavernous angiomas reviewed by our department were multiple (at least three lesions). During the 5 year follow up period only four (4/18) patients underwent surgical treatment. 50% of patients suffered at least one hemorrhagic event with clinical impairment, and the most frequent manifestations were headache, focal deficit and seizures. The hemorrhagic rate per lesion per year was under 1%, for the more than 200 lesions and the low frequency of hemorrhagic events with clinical impairment in the time. CONCLUSIONS: Surgical treatment must be considered in patients with accessible lesions that have produced symptoms several or progressive symptoms. The non surgical patients should be followed with yearly MRI. When more than one first degree relative has a cavernous malformation or familial antecedent with cerebral hemorrhage or epilepsy, serial follow up monitoring consisting of physical examinations and MRI should be suggested to family members. PMID- 12373671 TI - [Characteristic times in sleep-waking electroencephalograms]. AB - INTRODUCTION AND AIMS: EEG signals emerge from the collective behaviour of large neuronal aggregates and betrays the information processed by neocortex. This electrophysiological collective activity varies with the brain function. Thus, one can ask whether there exists any indication of that neuronal activity in the EEG record. In this work this question is considered in the particular case of sleep/awake EEG s. To this aim, the concept of lacunarity is proposed as a tool to analyse the texture of the EEG samples. From the resulting lacunarity profiles an index is defined which represents a characteristic time of each phase. PATIENTS AND METHODS: The samples analysed corresponds to 30 seconds epochs from polysomnographic night records. Essential details for the computation of lacunarity patterns and the propounded index are given. The mean values of characteristic times (in seconds) are: 0.43 (phase I); 0.73 (phase II); 1.12 (phase III/IV); 0.65 (REM); 0.12 (relaxed wakefulness). RESULTS AND CONCLUSIONS: With this criterium, the awake state is clearly distinguished from phase I and REM sleep, whereas REM sleep comes out to be similar to phase II. Finally, statistical analysis of results suggests the possibility to interpret this index as a complementary tool for reading polysomnographies as well as its application to different physiological or pathological situations of EEG records. PMID- 12373672 TI - [Database application for information on post-surgical evolution after functional neurosurgery]. AB - INTRODUCTION: A series of quantitative scales have been established internationally to evaluate the functional state of patients affected by movement disorders, such as Parkinson s disease. The values of these parameters offered by each patient, measured at different moments during his or her illness, allow us to conduct studies into their evolution as well as perform statistical studies about the casuistics. AIM. To provide a tool that enables us to study this vast amount of material in an efficient, sure and, above all, automated manner. Materials and methods. We selected the most interesting variables from the international protocols. We also designed and developed a database application for use under the Windows environment using Delphi 3.0 language and compiler and Structured Query Language. RESULTS: We designed, developed and validated a database system so as to be able to handle automatically the information on the clinical evolution of patients who had undergone functional neurosurgery. This system not only enables us to collect all relevant pre and post surgical information but also allows fast searches and selection, data processing using descriptive statistical techniques and the exportation of the data in a standard format. The system, which also allows final double blind clinical evaluation of each patient to be performed, has been used successfully in the Movement Disorders Clinic at the CIREN for over three years. CONCLUSIONS: This system allows for a considerable saving in the amount of time and effort needed for the post surgical evolution of patients, while also increasing the reliability of the results obtained. PMID- 12373673 TI - [Angelman syndrome: physical characteristics and behavioural phenotype in 37 patients with confirmed genetic diagnosis]. AB - INTRODUCTION: Angelman syndrome (AS) is characterised by mental retardation, ataxic gait, epilepsy, absence of language and a special series of physical traits behavioural phenotype. Its incidence is estimated as one in every 20,000 individuals. On the basis of discoveries made in molecular biology, patients can be classified as belonging to five types: deletion, paternal uniparental disomy (UPD), imprinting defects, mutation of the UBE3A ubiquitin protein ligase gene and unidentified mechanism (15% 20% of patients). Some studies report significant correlations between the phenotype and the genetic cause. PATIENTS AND METHODS: We reviewed, retrospectively, 37 patients suffering from AS with a positive genetic study and who had been controlled for at least two years in the Neurological Service at the Hospital Sant Joan de D u. Data was collected on physical characteristics, behavioural phenotype, type of communication, sleep disorders and the medication they needed, as well as epilepsy, start age, types of seizures, medication, schooling and social integration. RESULTS: 87% of cases were due to de novo deletion, 8% were caused by UPD, and 5% had their origins in imprinting defects. The average age of diagnosis was 6.5 years. The sleep disorders present in 48% of the patients required medication in 67% of cases, and 95% presented epilepsy. The most frequent seizures were myoclonic, tonic clonic and atonic. The electroencephalogram (EEG) was the characteristic found in the AS in 68%. The most effective treatment was afforded by valproate and clonazepam. CONCLUSIONS: As regards the phenotype, no differences were found according to the genetic alteration. The most effective treatment for the sleep disorders was melatonin. Epilepsy was an almost constant finding in our series, as was cognitive affectation. Lastly, it must be pointed out that educational and socio occupational integration is difficult for patients suffering from AS. PMID- 12373674 TI - [Bilateral hand dystonia secondary to a bilateral opercular syndrome or Foix Chavany-Marie syndrome]. AB - INTRODUCTION: The main clinical feature of the opercular syndrome (Foix Chavany Marie) is the automatic voluntary dissociation of the facio glosso pharyngeal movements (that is, the alteration of voluntary motility with preservation of authomatic movements). Less frequently, it is presented with movement disorders as dystonia. CASE REPORT: We report a male patient aged 40 years who developed a biopercular syndrome of vascular etiology (confirmed by neuroimaging), in the context or a clinical picture of global hipoxemia, for which the most outstanding clinical manifestation was the presence of dystonic posturing. CONCLUSION: Although dystonia is usually related with damage or dysfunction of the basal ganglia or thalamus, in some case it can be caused by lesions in other locations, such as in some patients with biopercular syndrome as in the present case PMID- 12373675 TI - [Central diabetes insipidus: a case report]. AB - INTRODUCTION: Diabetes insipidus (DI) is a syndrome characterised by polyuria which is almost always associated with polydipsia. The most frequent cause is central DI, which is the result of an inadequate secretion of the diuretic hormone, and diagnosis involves differentiating it from other causes of polyuria and polydipsia. CASE REPORT: The authors report the clinical case of a previously healthy 4 year old girl, who, in December 1998, was found to have intense polydipsia accompanied by polyuria. Behavioural treatment was begun as an answer to what was thought to be psychogenic polydipsia, although results were unsatisfactory and the patient was brought to the Paediatric Nephrology Service at Hospital Maria Pia in June 1999. CONCLUSION: A clinical study, which included the water restriction test and concentration tests with desmopressin, enabled us to diagnose central DI. In spite of the results from a cranial NMR scan being normal, follow up time is still too short to classify the aetiology as idiopathic. The girl is asymptomatic under treatment with intranasal desmopressin. The favourable evolution in this case highlights the need to act in a thorough manner in the study of situations involving polyuria/polydipsia. PMID- 12373676 TI - [Presentation of distal saccular aneurysm as a subdural haematoma]. AB - INTRODUCTION: Intracranial aneurysms are one of the most frequent vascular diseases. Nevertheless, saccular aneurysms that are not due to an inflammatory aetiology, which are located in the peripheral segment of the posterior circulation, are extremely rare. They are most frequently located in the thickest arterial branches within the region of the anterior brain circulation, as is the case of the complex made up of the anterior cerebral artery posterior communicating artery, middle cerebral artery and posterior communicating artery. No clinical manifestations are produced in many of these aneurysms, and their rupture and the subsequent development of a subarachnoid haemorrhage is the cause of the most intense neurological damage, which on occasions can lead to fatal consequences. CASE REPORT: We report the case of a patient who was a carrier of distal aneurysm, located in the posterior region of the brain circulation, and also the neuroradiological findings, the form of clinical presentation and surgical treatment carried out, which allowed us to identify and close the afferent vessel and the resection of the aneurysmatic sac. CONCLUSION: From the presentation of the symptoms of this patient in the form of a subarachnoid haemorrhage, accompanied by a subdural haematoma, it could be inferred that these clinical and imagenological findings point to the rupture of a distal aneurysm. Application of the stereotactic approach would be one of the first choice treatments for aneurysms in the distal region if we bear in mind the characteristics of the afferent vessel, the size of the neck and the morphology of the sac PMID- 12373677 TI - [Septo-optic dysplasia]. AB - INTRODUCTION: Septo optic syndrome, described by De Morsier in 1956, consists in the hypoplasia of one or both optic nerves, mid line brain malformations and hypothalamohypophysial dysfunction, which is inconstant. It is an infrequent, but treatable, cause of hepatic and neurological damage, and it is important to obtain an early diagnosis and to begin hormone replacement therapy. CASE REPORT: We report the clinical case of a female baby who was diagnosed early on as suffering from septo?optic dysplasia, after discovery of the existence of cholestatic jaundice. In our case the three components of the syndrome were present: hypothalamohypophysial dysfunction, bilateral hypoplasia of the optic nerves and brain malformations with dysplasia of the transparent septum. All this gives rise to complex clinical features and the predominance of hypernatraemic dehydration secondary to insipid diabetes, nystagmus and serious psychomotor retardation. Our patient died, as in other cases reported in the literature, from an episode of sudden death. DISCUSSION: Despite the importance of an early diagnosis of this disorder, it is usually late. Most children who present hypopituitarism traits in the neonatal period are not diagnosed at that time, with the subsequent risk of death or brain damage. Some clinical findings, which appear early on and can provide clues which aid us to reach a diagnosis, are the appearance of episodes of hypoglycaemia in the neonatal period, the existence of micropenis and cryptorchidism with hypoplasic testes, jaundice or the appearance of clinical manifestations of insipid diabetes. Later on nystagmus and neurological symptoms may appear. The final diagnosis is performed through the use of neuroimaging techniques (CT or MRI) and hormonal studies. PMID- 12373678 TI - [Spinocerebellar ataxia type 8: the case of a Spanish family]. AB - INTRODUCTION: Dominant autosomic ataxias include a group of neurodegenerative diseases characterized by the abnormal expansion of triplets. CASE REPORT: Male aged 33, with expansion of the SCA 8 gene (100 repetitions), who presented a clinical picture compatible with a pancerebellar syndrome. The patient had been diagnosed 11 years earlier as suffering from previously of histiocytosis X. A clinico genetic study was conducted on the patient and several members of his family (parents and two sisters). Both sisters and the father were found to be carriers of the expansion (110 and 150 repetitions, respectively), and are currently asymptomatic. RESULTS AND DISCUSSION: There is no relation between the number of repetitions and the age of onset of the disease. The normal interval in our population oscillates between 16 37 repetitions, and the pathological interval has not been well determined. There may be a relation between the SCA 8 form and histiocytosis X. PMID- 12373679 TI - [Visual pathways: a set of time-saving strategies]. AB - INTRODUCTION: The visual pathways in our brain perform a practically impossible task: they are able to identify objects in tenths of a second in spite of having to process millions of data items. Studies being conducted in basic Neuroscience are beginning to discover the tricks that make this process possible. METHOD: These studies are based on two main pillars. First, we have an increasingly more detailed knowledge of the anatomy of the visual pathway. Second, the analyses of the receptive fields of the visual cells are becoming more and more sophisticated (each cell in the visual pathway responds to specific stimuli located within a tiny area of the space we call receptive field). Recent studies, including those conducted in my own laboratory, are beginning to reveal the pathways that give rise to different types of receptive fields and their possible function. CONCLUSION: The study of the different types of receptive fields provides us with important lessons about how images are processed. This paper offers a brief review of the current concepts that attempt to explain why such a complex task as sight gives the impression of being so simple. PMID- 12373680 TI - [Inflammatory mechanisms, arteriosclerosis and ischemic stroke: clinical data and perspectives]. AB - OBJECTIVE: The atherosclerosis is the most common cause of death and disability in developed countries by causing ischemic cardiopathic and stroke. The ischemic atherotrombotic stroke is the most frequent form of the last one. In this sense we review herein the mechanisms underlying the artherosclerotic process. DEVELOPMENT: It is understood as an inflammatory disease, by taking into account the widely accepted hypothesis by Ross: it was firstly stated in structural terms, as macrophages and T/B linfocities were present in the arterial wall from the first stages of the disease (fatty streak) to the last and complicated ones. The starting point is a functional endothelial damage, secondary to mechanical or vascular risk factors and called response to injury hypothesis . The next step is an inflammatory cascade that involves humoral (citokines, growth factors) and cellular (increased quimiotaxis, adherece and infiltration of inflamatory cells) mechanisms. They interact among them, outbalanced and in a progresssive way that leads to the final fibroproliferative response. Every stage has his own inflammatory components and interactive pathways. The following elements are outstanding in this process: 1) Adhesion molecules, including E selectin, ICAM 1 and VCAM 1, that are increased locally in the plaques and as circulating elements; plaquetary receptors of the type IIb/IIIa are integrins wich belong to the same family; 2) Citokines with either proinflammatory activity like IL 1, the TNF a and linfocitary ligands like the CD 40, or with antiinflammatory activity like the gamma interpheron; 3) Growth factors, with plaquetary (PDGF) and fibroblastic (FGF) variants as the cornerstone; 4) Markers of systemic inflammation, overall plasma C reactive protein and fibrinogen, that predict the risk of stroke and cardiovascular death; IL 6, complement, thrombin and heat shock proteins (HSP) would act in a similar but less conclusive way. CONCLUSIONS: The evidences of the pivotal role of the inflammation in the stroke allow to develop therapeutical strategies to prevent the disease: fostering natural antiinflamatory mechanisms, or inhibiting inflammatory elements by selective (monoclonal antibodies) or non selective (IIb/IIIa receptors, antiinflammatory drugs) pathways are distinctily glimpsed, ongoing or fully developed. PMID- 12373681 TI - [Neuroimaging and cerebral palsy]. AB - INTRODUCTION: A high percentage of subjects with cerebral palsy (CP) present brain injuries, which are revealed by neuroimaging techniques. On the whole the pattern of brain damage is heterogeneous. DEVELOPMENT: We review the studies that have described the brain damage in CP using structural and functional neuroimaging techniques. Brain damage is considered according to the type of CP and taking the gestational age into account. CONCLUSIONS: According to structural neuroimaging studies carried out in spastic diplegia, the brain pattern differs with the gestational age. In early subjects with spastic diplegia it is the periventricular white matter that is mainly affected. In spastic quadriplegia, cortico subcortical lesions and hypoplasia of the corpus callosum are also observed. Unilateral lesions predominate in the case of hemiplegia. Hemiplegic subjects may also present damage to the white matter, cortico subcortical lesions and congenital brain malformations. In these subjects, some of the injury patterns observed seem to be related with the clinical features they display. Dyskinetic CP is characterised by the absence of lesions and alteration of the basal ganglia and the thalamus. Very few studies have been conducted that take the different types of CP into account in comparing the findings of structural and functional neuroimaging. PMID- 12373682 TI - [Neurological complications of cardiac catheterization]. AB - AIMS: To describe the neurological complications of cardiac catheterization, together with its risk factors and pathogenic mechanisms. METHOD: Over the past few years there has been a marked increase in the number of interventions involving cardiac catheterizations. For this very reason, we can expect a proportional rise in the number of complications. The incidence of neurological pathologies secondary to heart interventions oscillates between 0.01 and 0.4% of procedures performed. The most frequent clinical pictures are cerebrovascular disease, neuro ophthalmological syndromes and peripheral neuropathies, due to damage done to the median, femoral and lateral femoral cutaneous nerves, and to the lumbar plexus. The most usual mechanisms are cerebral ischemia originated by embolisms and direct compression of the peripheral nerves. Factors increasing the likelihood of complications are old age, the presence of classic vascular risk factors and, probably, the patient s being female. More risk is involved in mitral and aortic valvuloplasties and non elective revascularization procedures. The personal experience of the operator and the overall activity of the department of haemodynamics where the physician works are factors that are very closely linked to the incidence of complications. CONCLUSIONS: Knowledge about neurological illness secondary to cardiac catheterization and its mechanisms of production may allow us to identify higher risk patients, to develop protocols to prevent it and to apply early therapeutic measures. PMID- 12373683 TI - [Influence of factors not related with HIV infection on neuropsychological performance of HIV-seropositive patients]. AB - INTRODUCTION: The presence of neuropsychological impairment related to HIV infection has generated abundant literature whose results are disparate. Keeping in mind that it has been suggested that neuropsychological impairment could be associated to the presence of certain factors that would coexist with the own HIV, it is necessary to determine factors which contribute to transform a seropositive into a subject more neuropsychologically vulnerable. DEVELOPMENT: In this work we present a revision of those factors related with HIV infection that can influence neuropsychological performance of the patients as the antecedents of neurological and psychiatric pathology, depressed mood, drugs abuse, and cognitive reserve. CONCLUSIONS: The study of the influence of these factors can not only contribute to clarify the controversy on the presence of neuropsychological deficits, but also to understand why some seropositive patients are more neuropsychologically vulnerable than others, and ultimately to better understand the neuropsychological implications derived of HIV infection. PMID- 12373684 TI - [Patient aged 45 with refractory epilepsy of the temporal lobe since early childhood]. AB - INTRODUCTION: Temporal lobe epilepsy is the most frequent of the epilepsies related with localization and one of the most refractory to pharmacological treatment. Temporal lobectomy curbs seizures in many of these patients, which improves their quality of life. CASE REPORT: Patient aged 45 who, during early infancy, started to suffer simple partial seizures that later went on to become generalised. These were well under control by adolescence but at 23 they became complex partial seizures that were resistant to different antiepileptic drugs. The patient was submitted to a complete presurgical evaluation and a left anterior temporal lobectomy was performed at the age of 44. After surgery he evolved favourably. The final diagnosis was that he was suffering from mesial temporal sclerosis, associated with a subcortical neuronal heterotopy of the parahippocampal region. We discuss the semiology and the aetiology with regard to this patient and in a general sense, and we also define the foundations upholding the decision to perform surgery, the areas that make up the epileptogenic zone, and the neurophysiological and neuropsychological tests, and the structural and functional neuroimaging that are used to measure those areas. Likewise, the different techniques that can be used in resection of the temporal lobe are analysed. We also set out an etiopathogenic hypothesis according to the histopathological results and comment on a number of related general aspects. CONCLUSIONS: Progress in physiopathological knowledge, the development of diagnostic and surgical techniques, and its high efficiency and low morbidity have consolidated temporal lobectomy as a radical form of treatment for temporal lobe epilepsy that should be performed as early as possible once resistance to medication has been observed. PMID- 12373685 TI - [Odontoid process (dens axis)]. PMID- 12373687 TI - [Reply. Childhood epilepsy caused by Rolandic discharges: diagnosis using magnetoencephalography]. PMID- 12373688 TI - [Neurological care of pregnant women with epilepsy]. PMID- 12373690 TI - Cyclooxygenase-2 inhibition potentiates morphine antinociception at the spinal level in a postoperative pain model. AB - BACKGROUND AND OBJECTIVES: After peripheral inflammatory stimuli, spinal cord cyclooyxgenase-2 (COX-2) mRNA and protein levels increase, whereas COX-1 is unchanged. In animal models of inflammatory pain, intrathecal COX-2 selective inhibitors suppress hyperalgesia. However, the role of spinal COX-2 inhibition in postoperative pain is not well elucidated. This study investigates whether a water-soluble COX-2 selective inhibitor, L-745337, can modify allodynic responses in a rat model of postoperative pain. METHODS: Allodynia was induced in the left plantar hindpaw by surgical incision. Animals then received intrathecal (0-80 micro g) or subcutaneous (0-30 mg/kg) L-745337 coadministered with intrathecal morphine (0-2 nmol). Reduction of mechanical allodynia (increased withdrawal threshold) was quantified with calibrated von Frey hairs. RESULTS: L-745337 alone, whether intrathecal or systemic, had no effect on withdrawal threshold. When intrathecal L-745337 at doses of 40 to 80 micro g was combined with a subthreshold dose (0.5 nmol) of morphine, withdrawal thresholds were increased in a dose-dependent manner. Adding 80 micro g L-745337 to 1 nmol morphine produced an antiallodynic effect greater than that of morphine at twice the dose. Subcutaneous L-745337, up to 30 mg/kg combined with intrathecal morphine resulted in the same antiallodynic response as morphine alone. CONCLUSION: These results suggest a spinal interaction of COX-2 inhibition with opiate analgesia may allow a reduction of postoperative pain with lower doses of opiate. PMID- 12373691 TI - "See one, do one, teach one, have one": a novel variation on regional anesthesia training. AB - INTRODUCTION: Is it possible to determine the number of nerve blocks needed for residents to become competent in regional anesthesia? Several studies have focused on this question, and the Residency Review Committee (RRC) for Anesthesiology has now defined a "minimum clinical experience" for some aspects of regional anesthesia training. In our experience, personally being a regional block recipient can also serve to enhance training. METHODS: Many residents at Virginia Mason Medical Center have received regional anesthetics as volunteers in research projects. We designed questionnaires to define their perceptions in both performing and receiving regional anesthesia. RESULTS: Twenty-one residents were recipients of a total of 72 regional anesthetic procedures. Many residents' comments focused on the discomfort of local anesthesia infiltration, the value of sedation, a better appreciation of the patients' perspectives, and improved preoperative consultation. Residents experiencing paresthesias were more likely to consider paresthesias as bad (P =.0098). CONCLUSION: The lessons learned from personally receiving a regional anesthetic are invaluable and can improve the quality of training, as well as the relationship between anesthesiologist and patient. PMID- 12373692 TI - Does local anesthetic stereoselectivity or structure predict myocardial depression in anesthetized canines? AB - BACKGROUND AND OBJECTIVES: It is unclear whether the susceptibility to myocardial depression from an accidental intravascular local anesthetic (LA) administration is associated with LA stereoselectivity or structure. By using direct left ventricular pressure monitoring and echocardiographic indices of contractile function in anesthetized, ventilated dogs, we compared the cardiac depressant effects of bupivacaine, ropivacaine, levobupivacaine, and lidocaine. METHODS: Open-chest dogs were randomized to receive escalating incremental infusions of the 4 local anesthetics until cardiovascular collapse. We assumed a concentration relationship for potency of 4:1 for lidocaine/bupivacaine, ropivacaine, and levobupivacaine. RESULTS: All LAs produced concentration-dependent increases in left ventricular end diastolic pressure (LVEDP) and decreases in dP/dtmax, ejection fraction % (EF), fractional shortening (%) (FS), and cardiac output (CO). When comparing the long-acting agents, the effect was least for ropivacaine. The effective concentration estimates for ropivacaine that produced 35% reductions in dP/dtmax and FS were 4.0 micro g/mL (95% confidence intervals [CI(95)]: 3.1 to 5.2 micro g/mL) and 3.0 micro g/mL (CI (95): 2.1 to 4.2 micro g/mL), respectively. The concentrations of levobupivacaine that produced these same end points of contractile dysfunction were significantly less: 2.4 micro g/mL (CI(95): 1.9 to 3.1 micro g/mL) and 1.3 micro g/mL (CI(95): 0.9 to 1.8 micro g/mL), respectively, and these were not different from bupivacaine. As expected, the concentrations of lidocaine that produced 35% reductions in dP/dtmax and FS were significantly greater than the longer acting agents; 8.0 micro g/mL (CI(95): 5.7 to 11.0 micro g/mL) and 5.5 micro g/mL (CI(95): 3.5 to 8.7 micro g/mL), respectively. CONCLUSIONS: This study suggests that smaller molecular size and possibly a piperidine-free structure as opposed to stereoselectivity may be the more important factor in reducing the risk of LA-induced myocardial depression. PMID- 12373693 TI - Epidural blockade suppresses lipolysis during major abdominal surgery. AB - BACKGROUND AND OBJECTIVES: The purpose of the study was to investigate the effect of thoracic epidural administration of local anesthetic, i.e., epidural block on perioperative lipolysis. METHODS: Fourteen patients undergoing elective colorectal surgery were randomly assigned to receive either general anesthesia combined with epidural block (EDA, n = 7) or general anesthesia alone (control, n = 7). The rates of glycerol appearance (R(a) glycerol), i.e., lipolysis, were assessed by the stable isotope tracer [1,1,2,3,3-(2)H(5)]glycerol before, during, and 2 hours after the operation. Plasma concentrations of metabolic substrates (glycerol, free fatty acids [FFA], lactate) and hormones (insulin, glucagon, cortisol) were also determined. RESULTS: In the EDA group, R(a) glycerol decreased to lower intra- and postoperative values than in the control group (P <.05). Perioperative plasma concentrations of glycerol, FFA, lactate, and insulin remained unaltered with both anesthetic techniques. Intraoperative plasma glucagon and cortisol concentrations were lower in the EDA group than in the control group (P <.05). CONCLUSIONS: Epidural block suppresses lipolysis during and 2 hours after major abdominal surgery without affecting plasma glycerol or FFA concentrations. PMID- 12373694 TI - Paravertebral somatic nerve block compared with peripheral nerve blocks for outpatient inguinal herniorrhaphy. AB - BACKGROUND: Inguinal herniorrhaphy (IH) is a common outpatient procedure, yet postoperative pain and anesthetic side effects remain a problem. Paravertebral somatic nerve blocks (PVB) have the potential to offer unilateral abdominal wall anesthesia and long-lasting pain relief with minimal side effects. We compared PVB with peripheral neural blocks for outpatient IH. METHODS: Forty-six patients scheduled for IH were entered into this prospective, single-blind study. All patients underwent a standardized general anesthetic. Patients were randomly assigned to receive a PVB (levels T10-L2) preoperatively (n = 24) or an intraoperative peripheral block (PB) by the surgeon (n = 22), using 0.5% ropivacaine (40 mL). Opioid use, verbal analog pain scores, and side effects were documented for 72 hours. RESULTS: The use of opioids during surgery was less for the PVB group 162 +/- 70 mg than the PB group, 210 +/- 60 (P =.02). Need for opioids in PACU was less for the PVB group (39%) than the PB group (61%) (P =.002). Time until first pain after discharge was not different between groups, 312 +/- 446 minutes (PB) and 425 +/- 384 minutes (PVB) (P =.12). Of the PVB patients, 29% used no opioids at all compared with 18% of PB patients (P =.12). Mean time until first oxycodone use was similar between groups, 303 +/- 469 minutes (PB) and 295 +/- 225 minutes (PVB) (P =.18). Oxycodone use was also similar; 35 +/- 34 mg (PVB) versus 49 +/- 42 mg (PB) (P =.30). More patients in the PB group (50%) required antiemetic treatment in the postanesthesia care unit than the PVB group (21%) (P <.001). Side effects were similar at all other measurements. CONCLUSIONS: This study shows that PVB provides analgesia equivalent to extensive peripheral nerve block for inguinal herniorrhaphy, offering an alternative method of postoperative pain management and perhaps fewer side effects. PMID- 12373695 TI - Gabapentin in postamputation phantom limb pain: a randomized, double-blind, placebo-controlled, cross-over study. AB - BACKGROUND AND OBJECTIVES: Severe phantom limb pain after surgical amputation affects 50% to 67% of patients and is difficult to treat. Gabapentin is effective in several syndromes of neuropathic pain. Therefore, we evaluated its analgesic efficacy in phantom limb pain. METHODS: Patients attending a multidisciplinary pain clinic with phantom limb pain were enrolled into this randomized, double blind, placebo-controlled, cross-over study. Other anticonvulsant therapy was discontinued. Each treatment was 6 weeks separated by a 1-week washout period. Codeine/paracetamol was allowed as rescue analgesia. The daily dose of gabapentin was titrated in increments of 300 mg to 2400 mg or the maximum tolerated dose. Patients were assessed at weekly intervals. The primary outcome measure was visual analog scale (VAS) pain intensity difference (PID) compared with baseline at the end of each treatment. Secondary measures were indices of sleep interference, depression (Hospital Anxiety and Depression [HAD] scale), and activities of daily living (Bartel Index). RESULTS: Nineteen eligible patients (mean age, 56 years; range, 24 to 68 years; 16 men) were randomized, of whom 14 completed both arms of the study. Both placebo and gabapentin treatments resulted in reduced VAS scores compared with baseline. PID was significantly greater than placebo for gabapentin therapy at the end of the treatment (3.2 +/- 2.1 v 1.6 +/- 0.7, P =.03). There were no significant differences between placebo and gabapentin therapy in terms of the number of tablets of rescue medication required, sleep interference, HAD scale, or Bartel Index. The medication was well tolerated with few reports of adverse effects. CONCLUSIONS: After 6 weeks, gabapentin monotherapy was better than placebo in relieving postamputation phantom limb pain. There were no significant differences in mood, sleep interference, or activities of daily living, but a type II error cannot be excluded for these variables. PMID- 12373696 TI - Effects of atracurium added to local anesthetics on akinesia in peribulbar block. AB - BACKGROUND AND OBJECTIVES: Peribulbar anesthesia (PBA) is widely used in cataract surgery, but the onset time of akinesia is not as rapid as with retrobulbar block. The aim of this study was to evaluate whether addition of low-dose atracurium to the local anesthetic mixture has any effects on akinesia in PBA. METHODS: Sixty adults undergoing cataract surgery were randomly allocated to receive either 8 mL of a lidocaine-bupivacaine mixture, plus 0.5 mL 0.9% NaCl (group I) or 8 mL of the same local anesthetic mixture plus 0.5 mL (5 mg) atracurium (group II). The level of akinesia was graded by a observer unaware of group assignment. The onset time and duration of akinesia were also recorded by an observer, again unaware of group assignment. RESULTS: The onset time of complete akinesia in group II was significantly shorter than that in group I (P <.05). In group I, 86% of patients had an akinesia score of 0 (complete akinesia) in the first 10 minutes. The rate of complete akinesia was 93% in group II in the same period. This difference was not significant. The success rate of complete akinesia was 93% in group I and 100% in group II at the end of the measurement interval. None of the group II patients required supplementary block, while 2 patients in group I received additional injections for inadequate akinesia. CONCLUSION: Atracurium added at a dose of 5 mg to a lidocaine-bupivacaine mixture for peribulbar block decreases the onset time of akinesia and provides better surgical conditions without obvious side-effects. PMID- 12373697 TI - Successful interscalene block with a nerve stimulator may also result after a pectoralis major motor response. AB - BACKGROUND AND OBJECTIVES: Interscalene block of the brachial plexus is a well established anesthetic and analgesia technique for shoulder surgery. The endpoint for successful block using the nerve stimulator has been described by previous authors as a bicep motor response (twitch) and recently by a deltoid motor response. This retrospective observational case study of regular clinical practice examined the efficacy of using the pectoralis major motor response as an endpoint for a successful block. METHODS: A total of 120 patients who were scheduled for elective ambulatory shoulder surgery were retrospectively studied. All interscalene blocks were performed with aid of a nerve stimulator. Patients were categorized into 3 groups of 40 patients. Group 1 (biceps twitch), group 2 (deltoid twitch), and group 3 (pectoralis major twitch) were compared on success of the block. This retrospective study was conducted by reviewing interscalene block data sheets from the last 40 patients consecutively receiving interscalene block from either a bicep, deltoid, or pectoralis major motor response. A successful block was defined by the inability of the patient to raise their arm against gravity 20 minutes after injection of the local anesthetic. RESULTS: Pectoralis major motor response as an endpoint for local anesthetic injection was examined. Of 40 patients studied in this group, 38/40 were judged successful. This was comparable to the success rate in biceps (38/40 successful) and deltoid groups (37/40 successful). CONCLUSIONS: This retrospective observational case study of regular clinical practice suggests that a pectoralis major motor response can be a satisfactory endpoint for interscalene block. PMID- 12373698 TI - Repeated failure of epidural analgesia: an association with epidural fat? AB - BACKGROUND AND OBJECTIVES: To report the case of a patient who experienced repeated failed epidural analgesia associated with an unusual amount of fat in the epidural space (epidural lipomatosis). CASE REPORT: A 44-year-old female presented for an elective small bowel resection. An L(1-2) epidural catheter was placed for postoperative analgesia. The patient gave no indication of having pain at the time of emergence from general anesthesia or in the first 2 hours in the recovery room. Assessment of the level of hypoesthesia to ice while the patient was comfortable in the recovery room suggested a functional epidural catheter (cephalad level of T(10)). Two hours after admission to the recovery room the patient began to complain of increasing pain. Another 6 mL 0.25% bupivacaine was administered via the catheter. The patient's pain decreased, but remained substantial, and there was minimal evidence of sensory block above the T(10) level. Subsequently, a T(10) epidural catheter was placed. Testing confirmed proper placement of the catheter in the epidural space at the T(10) level. A test dose of 5 mL 0.25% bupivacaine resulted in prompt and complete relief of the patient's pain. However, the level of hypoesthesia to ice did not exceed the T(10) level. Approximately 1 hour later the patient complained of increasing discomfort again. There was no evidence of any sensory block, and there was no response to a bolus of 8 mL 1% lidocaine. A thorough examination of the patient did not suggest any cause for the pain other than a malfunctioning epidural catheter. A third epidural catheter was placed at the T(8-9) level. This catheter was again confirmed to be in the epidural space with a test dose of 10 mL 0.5% bupivacaine. The level of hypoesthesia to ice was restricted to a narrow bilateral band from T(7)-T(9). Analgesia failed 2 hours later. The epidural catheter was removed and the patient's pain was subsequently managed with intravenous patient-controlled analgesia (PCA) morphine. A magnetic resonance imaging (MRI) scan revealed extensive epidural fat dorsal to the spinal cord from C(5)-C(7) and from T(3)-T(9). An imaging diagnosis of asymptomatic epidural lipomatosis was established. CONCLUSION: We have described a case of repeated failure of epidural analgesia in a patient with epidural lipomatosis. PMID- 12373699 TI - Introduction: Gaston Labat award 2002-John A.W. Wildsmith. PMID- 12373700 TI - No sceptic me, but the long day's task is not yet done: the 2002 Gaston Labat lecture. PMID- 12373701 TI - Safety of regional anesthesia in Eisenmenger's syndrome. AB - BACKGROUND AND OBJECTIVES: Eisenmenger's syndrome is characterized by right-to left or bidirectional shunting and pulmonary hypertension. Perioperative risk is high for noncardiac surgery, and many clinicians avoid regional anesthesia because of the potential deleterious hemodynamic effects. We determined perioperative mortality based on published reports describing anesthetic management in patients with Eisenmenger's syndrome. METHODS: A literature search identified 57 articles describing 103 anesthetics in patients with Eisenmenger's syndrome. An additional 21 anesthetics were identified in patients receiving regional anesthesia for labor. RESULTS: Overall perioperative mortality was 14%; patients receiving regional anesthesia had a mortality of 5%, whereas those receiving general anesthesia had a mortality of 18%. This trend favored the use of regional anesthesia but was not statistically significant. A better predictor of outcome was the nature of the surgery (and presumably the surgical disease). Patients requiring major surgery had mortality of 24%, whereas those requiring minor surgery had mortality of 5% (P <.05). Patients in labor receiving regional anesthesia had a mortality rate of 24%, and most of these occurred several hours after delivery. CONCLUSIONS: This review of anesthesia and surgery in patients with Eisenmenger's syndrome reveals that most deaths probably occurred as a result of the surgical procedure and disease and not anesthesia. Although perioperative and peripartum mortalities are high, many anesthetic agents and techniques have been used with success. PMID- 12373702 TI - Mechanisms for pain caused by incisions. PMID- 12373703 TI - Computed tomography images of entrapped epidural catheter. AB - OBJECTIVE: Knotting and looping of catheters in the epidural space occur rarely. Visualization of a catheter by radiograph or fluoroscopy is not always possible and often inaccurate in locating the knot and/or the loop with precision. We report the case of an entrapped lumbar epidural catheter. Computed tomography (CT) clearly showed a knotted and looped catheter. CASE REPORT: A 27-year-old woman underwent epidural analgesia during labor. The epidural catheter was inserted 7 cm into the epidural space. After unsuccessful attempts at removing the catheter, a CT scan was performed, and it showed a catheter knot in the epidural space as well as a loop within the interlaminar ligamentum flavum between L3 and L4. This explained why attempts to remove the catheter by manual traction failed. Surgical removal of the catheter was subsequently performed. CONCLUSIONS: CT is useful in showing an entrapped epidural catheter and the mechanisms of entrapment. Surgery should be considered when gentle traction fails to retrieve the catheter. CT allows the clinician to localize the catheter with accuracy, thus facilitating surgical follow-up. PMID- 12373704 TI - Edward Tuohy: the man, his needle, and its place in obstetric analgesia. AB - The introduction of a needle designed by Ralph Huber and Edward Tuohy made continuous epidural anesthesia for labor possible. Neither the needle nor the regional anesthetic technique evolved in a vacuum; both were the culmination of a range of ideas developed by individuals around the world. PMID- 12373705 TI - Analgesic effects of ketamine ointment in patients with complex regional pain syndrome type 1. AB - OBJECTIVE: Ketamine hydrochloride (KET), an agent used for general anesthesia, has local anesthetic effects and N-methyl-D-aspartate (NMDA) receptor antagonist action. Because recent studies emphasized the role of peripherally distributed NMDA receptors in processing the nociceptive information, we investigated whether peripheral application of the ointment containing KET is able to attenuate the symptoms of local neuropathic pain. CASE REPORTS: We applied ointment containing KET (0.25%-1.5%) to the affected area on limbs in 5 patients with complex regional pain syndrome type I (CRPS I) and in 2 patients with type II (CRPS II). One to 2 weeks later, we observed improvement of the report of pain intensity, measured by the visual analog scale, in 4 patients with acute early dystrophic stage of CRPS I. Swelling of the affected limbs subsided as well. No apparent changes were noticed in 1 patient with chronic atrophic stage of CRPS I and in both patients with CRPS II. CONCLUSION: Topical application of KET appears to be beneficial for the patients with acute early dystrophic stage of CRPS I because of either its local anesthetic effect or NMDA receptor antagonist action. Patients with chronic atrophic stage of CRPS I and CRPS II patients do not appear to respond to this treatment. PMID- 12373706 TI - Changes in regional cerebral blood flow in the thalamus after electroconvulsive therapy for patients with complex regional pain syndrome type 1 (preliminary case series). AB - BACKGROUND AND OBJECTIVE: The aim of the present case series was to examine whether changes in regional cerebral blood flow (rCBF) induced by electroconvulsive therapy (ECT) in the thalamus are related to the efficacy of ECT. Four chronic pain patients with complex regional pain syndrome (CRPS) type-1 (age, 33 to 58 years) who had failed to respond to standard pain treatments received a course of ECT. To investigate the possible mechanisms of the analgesic effect of ECT on chronic CRPS type-1, we measured significant changes in the rCBF of the thalamus using technetium-99m ethyl cysteinate dimer single photon emission computed tomography (99mTc ECD SPECT), before and after ECT and compared these values between responders and nonresponders. RESULTS: Two of 4 (50.0%) patients responded to ECT treatment (response defined as a reduction of at least 60% on the visual analog scale [VAS]). 99mTc ECD SPECT showed that the mean contralateral thalamus-to-cerebellum ratio increased 11.5% after ECT compared with the ratio before ECT in the 2 responders, but remained unchanged in nonresponders. CONCLUSIONS: The results from the SPECT suggest that normalization of the balance of rCBF in the thalamus may be related to the analgesic efficacy of the ECT on CRPS Type-1. PMID- 12373707 TI - High failure rate of small doses of ropivacaine when used alone for spinal anesthesia for cesarean delivery. PMID- 12373708 TI - A response to "Walking spinal anesthesia for cesarean delivery-have we walked too far?". PMID- 12373711 TI - Multimodal analgesia and intravenous nutrition after surgery. PMID- 12373712 TI - Postoperative multimodal analgesia and intravenous nutrition. PMID- 12373713 TI - Don't forget the anterior subdural space! PMID- 12373715 TI - "Santayana's prophecy fulfilled" requires a critique. PMID- 12373716 TI - Ondansetron is effective in treatment of pruritus after intrathecal fentanyl. PMID- 12373717 TI - Combined spinal-epidural technique for total hysterectomy in a patient with advanced, progressive multiple sclerosis. PMID- 12373718 TI - Pathological examination of sentinel lymph node in breast cancer: potential problems and possible solutions. AB - Sentinel lymph node (SLN) biopsy has emerged during the last few years as a viable option for staging the axilla in the treatment of breast carcinoma. This procedure can potentially identify patients who would be helped by full axillary lymph node dissection (the SLN-positive cases), and those who would not (the SLN negative cases). Review of the literature confirms the promise of SLN; however, the possible problems in the pathological handling of SLN, including the microscopic misinterpretation of benign structures and "spurious" immunohistochemical staining, need wider recognition. PMID- 12373719 TI - Histologic classification of ductal carcinoma in situ. AB - Prior to the current mammographic era, ductal carcinoma in situ (DCIS) usually presented as a large mass, was classified morphologically by architecture, and treated by mastectomy. The introduction of screening mammography led to an increase in the incidence of DCIS, a decrease in the average size of DCIS, and an increased emphasis on its heterogeneous nature. Thus, a reproducible and prognostically relevant classification system for DCIS is necessary. The ultimate goal of this classification is proper selection of patients for whom lumpectomy would suffice rather than mastectomy. Features to evaluate include: extent and size of disease, adequacy of resection margins, and histology. While none of the proposed histological classification systems were endorsed at the recent Consensus Conference on the Classification of DCIS, nuclear grade was the most important feature common to most of them. Architecture was given secondary importance. By definition, DCIS is a non-invasive clonal proliferation of epithelial cells originating in the terminal duct lobular unit, which would be expected to be monomorphic; however, it is the degree of nuclear pleomorphism that is primarily used to separate DCIS into low, intermediate, and high grades. Architecturally, DCIS has been divided into the following types: comedo, solid, cribriform, micropapillary, and papillary. Different architectural patterns and grades may be present in a given particular case; however, some combinations of patterns occur more frequently than others. Interobserver studies have shown nuclear grading to be interpreted with greater consistency than architecture, and nuclear grading methods have correlated with biological and molecular marker studies. PMID- 12373720 TI - Prognostic and predictive value of HER2/neu oncogene in breast cancer. AB - Assessment of HER2/neu oncogene has been used as both a prognostic and predictive marker for breast cancer. However, the choice of the best method to assess the status of HER2/neu oncogene in breast cancer tissue remains controversial. A variety of techniques are available to detect HER2/neu gene amplification and overexpression. Tissue-based detection methods by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) offers a clear advantage over other approaches. FISH is a costly and relatively difficult assay and yet appears to be a better predictor of response to Herceptin (Trastuzumab) therapy and patient outcome. IHC is less expensive and is easier to perform; however, it suffers from a high rate of false negativity and positivity as well as inter observer variability among pathologists. Suggestions have been made to use IHC as a screening procedure followed by confirmation by FISH in selected cases. Considering the importance of an accurate assessment of HER2/neu oncogene in selecting therapy, a better alternative may be to use FISH as the primary testing for HER2/neu oncogene. Herceptin therapy is associated with several side effects and is expensive. Thus, in the long term, it may be more cost-effective to use the FISH procedure and reduce the possibility of under-treatment or over treatment of breast cancer patients. In addition, assessment of HER2/neu oncogene on every newly diagnosed early breast carcinoma may not be necessary. Metastatic lesions, when they occur, can be sampled by fine needle aspiration biopsy or core needle biopsy for assessment of HER2/Neu status. PMID- 12373721 TI - Image analysis and morphometry in the diagnosis of breast cancer. AB - Image Analysis, a complicated field still in the early stages of application to Pathology, has the capability of rendering major contributions to the diagnosis, prognosis, and management of malignancies of the breast. The present review summarizes the main problems and the general approach to the use of this technique for quantitating immunohistochemical stain results, obtaining DNA histograms, and making de novo diagnoses in routine materials of the Pathology service. In the case of diagnosis, the main steps are sampling, segmentation, and measures of chromatin texture. Currently, the limiting factor for all routine applications of image analysis is probably the absence of a reliable automatic nuclear segmentation. PMID- 12373722 TI - Applications of quantitative digital image analysis to breast cancer research. AB - Our studies of radiogenic carcinogenesis in mouse and human models of breast cancer are based on the view that cell phenotype, microenvironment composition, communication between cells and within the microenvironment are important factors in the development of breast cancer. This is complicated in the mammary gland by its postnatal development, cyclic evolution via pregnancy and involution, and dynamic remodeling of epithelial-stromal interactions, all of which contribute to breast cancer susceptibility. Microscopy is the tool of choice to examine cells in context. Specific features can be defined using probes, antibodies, immunofluorescence, and image analysis to measure protein distribution, cell composition, and genomic instability in human and mouse models of breast cancer. We discuss the integration of image acquisition, analysis, and annotation to efficiently analyze large amounts of image data. In the future, cell and tissue image-based studies will be facilitated by a bioinformatics strategy that generates multidimensional databases of quantitative information derived from molecular, immunological, and morphological probes at multiple resolutions. This approach will facilitate the construction of an in vivo phenotype database necessary for understanding when, where, and how normal cells become cancer. PMID- 12373723 TI - A simple and rapid scanning electron microscope preparative technique for delicate "gymnodinioid" dinoflagellates. AB - Light microscopy (LM) is routinely used to investigate delicate (unarmoured and lightly armoured) "gymnodinioid" dinoflagellate species but at this level of resolution, morphological features such as apical grooves, apical pores, thin thecal plates, and scales are often difficult to observe, thereby necessitating the use of scanning electron microscopy (SEM). Good results were obtained when harvested cells were fixed with osmium tetroxide (OsO(4)) as the primary fixative, adhered with poly-L-lysine to round glass coverslips, dehydrated in an ethanol series, and dried with hexamethyldisilazane (HMDS). Poly-L-lysine has in the past effectively been used to adhere biological material such as human red blood cells, mouse leukemic cells, and marine dinoflagellates to glass coverslips. HMDS has been used to substitute critical point drying (CPD) to dry soft insect tissues, rat hepatic endothelial cells, and the cilia of rat trachea. By combining and fine-tuning these two protocols in SEM studies of delicate "gymnodinioid" dinoflagellates, it is possible to overcome cell distortion such as shrinking and collapsing that result from centrifuging, filtering, and CPD. The combination of poly-L-lysine and HMDS not only produces good results but also requires limited expertise and equipment, is inexpensive, and is less time consuming. PMID- 12373724 TI - Effects of loud noise exposure on mouse myocardium: a comparison with the rat. AB - Loud noise is an environmental stressor of everyday life, which affects different organs and apparati, in particular the cardiovascular system. We have already reported that noise exposure produces significant alterations in the rat myocardium, consisting of mitochondrial damage, which is evident as lysis of the cristae and dilution of the matrix. Since there are high similarities between mouse and human species, the aim of our study was to investigate the effects of acute noise exposure on the mouse heart. We found that noise exposure affects mouse myocardium at similar subcellular sites to those already described in the rat; nonetheless, quantitative analysis of the percentage of altered mitochondria in both species disclosed a clear difference between mouse and rat myocardium, which strongly suggests a different sensitivity to noise stimulus. We hypothesize that the species differences on the extent of myocardial alterations here observed might be due to the zonal pattern of cardiac noradrenergic receptors, which should be the final effectors for noise-induced myocardial changes. PMID- 12373725 TI - Morphological alterations induced by loud noise in the myocardium: the role of benzodiazepine receptors. AB - Noise represents an environmental stress factor affecting several organs and apparati, including the cardiovascular system. In experimental animals undergoing noise exposure, subcellular myocardial changes have been reported, especially at mitochondrial level; in particular, after 6 hours of exposure only the atrium exhibited significant mitochondrial alterations, whereas after 12 hours as well as subchronic exposure both atrium and ventricle were damaged. The first part of the present article overviews the experimental evidence on effects of noise on the myocardium. In the second part, the review analyzes the role of benzodiazepine receptors and the potential efficacy of benzodiazepine ligands in preventing the mitochondrial damage induced by noise exposure. Drugs acting at both central and peripheral benzodiazepine receptors significantly prevent this damage. Differences in the amount and the duration of the protective effect might depend on variability in the potency and pharmacokinetics of the specific drug. The effects of the combined treatment with selective and non-selective peripheral benzodiazepine ligands on noise stimulation are discussed at biochemical level reviewing studies on the effects of noise exposure on mitochondrial fractions. PMID- 12373727 TI - Aortic arch variation analyzed by using plastination. AB - Different ramification patterns can be observed during the development of the aortic arch. In this study a common trunk (CT), which subsequently branched into the brachiocephalic trunk (BT) and left common carotid artery (LCCA), arose from the aortic arch. The LCCA arose from the CT 10.27 mm above the aortic arch. After crossing the ventral aspect of the trachea and esophagus, the LCCA became situated on the left side of the esophagus. The caliber and length of the main branches of the aortic arch were determined and compared to reports in the literature. This variation was discovered in the context of producing transverse body slices using an E12 plastination process. PMID- 12373728 TI - Anterior coracoscapular ligament and suprascapular nerve entrapment. AB - A reduction in the height of the suprascapular foramen may predispose to entrapment of the suprascapular nerve. In this study, 16 of 27 cadavers (60%) demonstrated a heretofore unreported ligament located on the anterior aspect of the suprascapular foramen. In 11 of the 27 cadavers (41%), the ligament was observed bilaterally. The ligament decreased the foraminal height from the normative value of 5.6 +/- 0.4 to 2.3 +/- 0.4 mm (mean +/- SEM). Because this ligament, for which we propose the term anterior coracoscapular ligament (ACSL), substantially narrows the suprascapular foramen, it should be considered as a possible etiologic factor in suprascapular nerve entrapment. PMID- 12373729 TI - Location of the ventral margin of the paracaval portion of the caudate lobe of the human liver with special reference to the configuration of hepatic portal vein branches. AB - The topographic anatomy of the ventral margin of the paracaval portion of the caudate lobe of the human liver has not been clearly described to date. To this end we hypothesize the existence of a precaudate plane, a flat or slightly curved plane defined by the ventral margins of the ligamentum venosum and the hilar plate. Using 76 cadaveric livers, we investigated whether the paracaval portion of the caudate lobe extended ventral to this plane and whether the paracaval caudate branch of the portal vein (PC) ran through this plane to its ventral side. In 28 of the specimens (36.8%), the PC extended over the plane to a variable depth: less than 10 mm in 10 specimens, 10-20 mm in 10, and more than 20 mm in eight specimens. This ventral extension of the PC consistently included its penetration into the dome-like area under the terminals of the three major hepatic veins; therefore, the ventrally extended PC often interdigitated with these veins and their tributaries (in practice, the ventral margin of the paracaval portion of the caudate lobe could generally be considered to run alongside the middle hepatic vein). Moreover, the ventral extension of the PC often reached the upper, diaphragmatic surface or the dorsal surface of the liver immediately to the right of the inferior vena cava. Several branches (termed border branches) in the ventral extension were difficult to identify as belonging to the PC. We discuss both the marginal configuration of the paracaval portion of the caudate lobe and how to identify and operate on the ventrally extended PC and related border branches during liver surgery. PMID- 12373730 TI - Anatomy online: presentation of a detailed WWW atlas of human gross anatomy- reference for medical education. AB - We present an online anatomy atlas based on the Visible Human Project (VHP) of the US National Library of Medicine. The objective is to provide original unlabeled as well as labeled sections of the human body of high quality and resolution on the Internet, for use in basic and continuing medical education. For a representative overview of the body, 370 axial sections were selected from the male and female data base of the VHP with special regard to regions of clinical interest. Each section is accompanied by its corresponding computer tomography (CT) image and, if available, magnetic resonance images (MRI) for quick and easy comparison of morphologic and radiologic structures. The sections can be studied unlabeled or labeled according to the current Terminologia Anatomica. A linked vocabulary with more than 850 terms explains the labeling. Animations of the sections as well as of CT and MR images allow for further visualization of the topographic relationships of anatomical structures. The responses to the project indicate that students and physicians regard the Internet Atlas of Human Gross Anatomy as a most useful aid for learning and reviewing anatomical details. The atlas is accessible on: http://www.uni mainz.de/FB/Medizin/Anatomie/workshop/vishuman/Eready.html. PMID- 12373731 TI - Analysis of medical students' use of web-based resources for a gross anatomy and embryology course. AB - An extensive Web site supporting our gross anatomy and embryology course, which includes various course management pages as well as online lectures, has been in use for the past 2 years. To determine how this Web site is being used by students, we examined server log files to track access to each of the Web pages on the site. Using this data, along with student responses on a course evaluation, we have been able to quantitatively characterize Web site use and gain some insight into students' perception of the site. This analysis showed that all of the resources available online, including course management information, exam reviews, online lectures, and dissection guides were heavily used and deemed useful by students. Despite universal computer ownership and Internet access from home, most use of the Web site was from on-campus computer labs, especially for lectures with audio streams. This was probably due to the limited bandwidth of off-campus connections. Data on the day of the week and time of the day of access showed peak activity at expected times, but also significant activity at all hours, as students took full advantage of 'access on demand.' This on-demand nature of the Web was also evident in students' viewing of lectures in short sessions rather than in one sitting. Online lectures were used regularly by a majority of students both before and after corresponding class sessions, however, this was not the preferred venue for all students. Although the flexibility of Web-based resources accommodates students' varying study habits, the alternative of traditional print material and live lectures should not be abandoned lightly. PMID- 12373732 TI - Re-inventing anatomy: the impact of plastination on how we see the human body. AB - Over the past 20 years the development of plastination has opened up new vistas for gross anatomy. In particular, it has led to a major expansion in the range of human anatomic specimens available for teaching and its potential value in research is increasingly being appreciated. More recently, it has burst into the public arena through what has become known as 'Anatomy Art,' as depicted in the von Hagens exhibition, Korperwelten (Bodyworlds). In this exhibition, the lifeless cadavers of the dissecting room have been transformed into standing, sitting, and jumping lifelike plastinated 'models' that demonstrate spinal cords, tumorous lungs, cirrhotic livers, joint prostheses, and sagittally sectioned whole bodies. Not surprisingly, the exhibition has raised considerable ethical debate about treating human cadavers in this way, an issue of particular relevance to anatomists. This article is an attempt to further this debate by considering the nature of plastinated human specimens, and the context within which they should be examined. The only rationale for displaying (plastinated) human material in the public domain is an educational one, with a basis in a museum ethos. The boundaries of this educational rationale are discussed, as are the opportunities and challenges presented by plastination to the anatomical community. PMID- 12373733 TI - Re-inventing anatomy: the impact of plastination on how we see the human body. PMID- 12373734 TI - Re-inventing anatomy: the impact of plastination on how we see the human body. PMID- 12373735 TI - Preservation and plastination. PMID- 12373736 TI - Muscarinic receptors involved in the subthreshold cholinergic actions of neostriatal spiny neurons. AB - Administration of the peptide MT-1 (48 nM), a selective agonist of muscarinic M(1)-type receptors, mimicked the subthreshold actions of muscarine (1 microM) on neostriatal neurons, i.e., it produced a reduction in subthreshold inward rectification leading to an enhancement in input resistance (R(N)) and evoked discharge. In all recorded cells, MT-1 effects remained in the presence of the specific peptidergic antagonist of the M(4)-type receptor, MT-3 (10 nM), but were blocked by the specific M(1)-type receptor antagonist MT-7 (5 nM). These results suggest that most muscarinic facilitatory actions in the subthreshold voltage range occur through M(1)-type receptors. However, in a fraction of cells (40%) muscarine produced an excitability enhancement not blocked by MT-7. This additional facilitatory action, not present when using MT-1, was blocked by MT-3, suggesting it was mediated by M(4)-type receptor activation. This facilitation could not be blocked by Cs(+), TTX, or Cd(2+), but only by a reduction in extracellular sodium. This result is the first evidence that M(4)-type receptor activation enhances a cationic inward current in a fraction of neostriatal projection neurons. PMID- 12373737 TI - Systemic administration of dizocilpine maleate (MK-801) or L-dopa reverses the increases in GAD65 and GAD67 mRNA expression in the globus pallidus in a rat hemiparkinsonian model. AB - This study examined the consequences of systemic treatment with either L-dopa or MK-801 on the levels of mRNAs encoding the 65 and 67 kDa isoforms of glutamate decarboxylase (GAD65 and GAD67) in the striatum and globus pallidus (GP) of rats rendered hemiparkinsonian by intranigral 6-hydroxydopamine injection. GADs mRNA levels were assessed by means of in situ hybridization histochemistry. In the striatum, dopamine denervation resulted in increased GAD67 mRNA levels at the rostral and caudal levels, whereas GAD65 showed selective increase at the caudal level. L-dopa and MK-801 treatments showed differential effects on the two GAD isoform levels in rats with 6-hydroxydopamine lesion. The lesion-induced increases in GAD67 transcripts were potentiated by L-dopa but unaffected by MK 801, whereas the increases in GAD65 were suppressed by MK-801 but unaffected by L dopa. These data suggest a heterogeneity of glutamate-dopamine interaction in the anteroposterior extent of the striatum and show that NMDA-mediated mechanisms are involved in the 6-hydroxydopamine lesion-induced transcriptional changes in striatal GAD65 but not GAD67. In GP, the 6-OHDA lesion elicited increases in both GAD65 and GAD67 mRNA levels. L-dopa or MK-801 treatment suppressed the lesion induced augmentations in the two GADs mRNA levels. These results indicate that dopamine denervation-induced changes in the functional activity of GP neurons involve both dopamine and glutamate NMDA receptor-mediated mechanisms. Comparison between the effects of L-dopa and MK-801 treatments on markers of the activity of striatal and pallidal GABA neurons further suggest that the impact of these treatments at the GP level do not depend solely on the striatopallidal input. PMID- 12373738 TI - Amphetamine-sensitized animals show a marked increase in dopamine D2 high receptors occupied by endogenous dopamine, even in the absence of acute challenges. AB - While a range of dopamine D(2)-related behaviors are exaggerated in amphetamine sensitized animals, studies of the dopamine D(2) receptor have reported either no change or a decrease in dopamine D(2) receptor density--especially when measured using radioraclopride. We hypothesized that a decrease in D(2) receptors may actually be "apparent" and that these receptors may still be present, but are noncompetitively "occupied" by endogenous dopamine. Animals sensitized to amphetamine (and their saline controls) were examined 4 weeks after their last injection. We first measured the [(3)H]raclopride binding in vivo, and observed that sensitized animals showed a 29% lower level of raclopride binding in vivo, suggesting an apparently lower level of dopamine D(2) receptors. To assess the reason for this we examined the density of receptors (using Scatchard analysis in vitro) measured by [(3)H]raclopride in the presence and absence of guanilylimidodiphosphate. This guanine nucleotide converts the dopamine-bound high-affinity state of D(2) receptors into low-affinity states, thereby making measurable the absolute density of the sites. As previously reported, the amphetamine-sensitized animals showed a 31% lower number of D(2) receptors in conventional binding (B(max) 15.6 vs. 22.7 pmol/g). However, with the addition of guanilylimidodiphosphate there was an equalization of both groups (B(max) 25.9 vs. 25.6 pmol/g), revealing an additional 10.3 pmol/g in the sensitized animals, but only 2.9 pmol/g in saline controls. There were no changes in the dissociation constant of [(3)H]raclopride for the receptors. The nearly four-fold increase of dopamine D(2) receptors in the high-affinity state occupied by dopamine may explain why amphetamine-sensitized animals show almost an order of magnitude greater response to dopamine-releasing challenges or dopamine agonists, even though the absolute receptor number is unchanged and the "apparent" receptor number is decreased. PMID- 12373739 TI - Gamma-vinyl GABA, an irreversible inhibitor of GABA transaminase, alters the acquisition and expression of cocaine-induced sensitization in male rats. AB - We examined the effect of (+/-)-gamma-vinyl GABA (GVG, Vigabatrin), an irreversible inhibitor of the enzyme GABA transaminase, on the acquisition and expression of cocaine-induced sensitization in albino male Sprague-Dawley rats. Animals received a single injection of 1 ml/kg i.p. of 0.9% saline or 15 mg/kg i.p. of (-)-cocaine and locomotor activity was assessed using automated locomotor cages and stereotyped behaviors were scored using a 4-point rating scale (Day 1). Subsequently, animals were given 15 mg/kg i.p. of cocaine every 48 h in their home cage for 1 week (Days 3, 5, and 7) and then given no treatment for 1 week. A challenge injection of 15 mg/kg i.p. of cocaine, but not vehicle, produced a significant increase in locomotor activity and stereotyped behaviors on Day 15 compared to animals that received cocaine on Day 1. Administration of 75 mg/kg i.p. of GVG 2.5 h before the cocaine injections did not significantly alter the acquisition of cocaine-induced locomotor sensitization. However, 150 mg/kg i.p. of GVG significantly attenuated the acquisition of cocaine-induced locomotor sensitization. Administration of 150 mg/kg i.p. of GVG 2.5 h before the cocaine challenge injection on Day 15 significantly attenuated the expression of cocaine induced locomotor sensitization. Acquisition and expression of cocaine-induced sensitization of stereotypy was also significantly attenuated by 150 mg/kg i.p. of GVG. Since sensitization may be one of the factors involved in relapse to drug use, the present results, in combination with previous findings that GVG blocks the rewarding and incentive motivating effects of cocaine, suggest that GVG might prove useful in the treatment of cocaine addiction. PMID- 12373740 TI - Adenosine A2A antagonism reverses levodopa-induced motor alterations in hemiparkinsonian rats. AB - To evaluate the possible involvement of adenosine A(2A) receptor-mediated mechanisms in levodopa-induced motor fluctuations, we investigated the effects of CSC (8-(3-chlorostryryl) caffeine), a selective adenosine A(2A) receptor antagonist, on levodopa-induced motor alterations in rats with unilateral 6-OHDA lesion. Acute and chronic administration of CSC was studied to evaluate the possible reversion or prevention of these levodopa effects. In a first set of experiments, rats were treated with levodopa (25 mg/kg with benserazide, twice daily, i.p.) for 22 days and on day 23 CSC (5 mg/kg, i.p.) was administered immediately before levodopa. In a second set of experiments, rats were treated daily for 22 days with levodopa and CSC (5 mg/kg/day, i.p.). The duration of the rotational behavior induced by chronic levodopa decreased after 22 days (P < 0.05). Acute administration of CSC on day 23 reversed levodopa-induced shortening in motor response duration (P < 0.01). Chronic CSC administration did not prevent the shortening in response duration induced by levodopa. Our results demonstrate that the adenosine A(2A) receptor antagonist CSC reverses but does not prevent levodopa-induced motor alterations in parkinsonian rats. These results suggest a role for adenosine A(2A) receptor-mediated mechanisms in the pathophysiology of levodopa-induced motor response complications. These findings suggest that the antagonism of adenosine A(2A) receptors might confer clinical benefit to parkinsonian patients under levodopa therapy suffering from motor complication syndrome. PMID- 12373741 TI - Alpha-2A-adrenergic receptors are present on neurons in the central nucleus of the amygdala that project to the dorsal vagal complex in the rat. AB - The descending pathway between the central nucleus of the amygdala (CeA) and the dorsal vagal complex (DVC) is an important substrate for autonomic functions associated with emotion. Activity in this circuit is crucially modulated by catecholamines and agonists of the alpha-2A-adrenergic receptor (alpha(2A)-AR), which relieve cardiovascular and gastrointestinal symptoms associated with experience of aversive stimuli. The subcellular distribution of alpha(2A)-AR within the CeA, however, has not been characterized. It is also not known if any alpha(2A)-AR-expressing neurons in the CeA project to the dorsal vagal complex. In order to address these questions, we examined the immunocytochemical labeling of alpha(2A)-AR in the CeA of rats receiving microinjection of the retrograde tracer fluorogold (FG) into the dorsal vagal complex at the level of the area postrema, an area involved in cardiorespiratory and gastrointestinal functions. Of all alpha(2A)-AR-labeled profiles in the CeA, the majority were either dendrites (42%) or somata (24%). alpha(2A)-AR labeling was often present on the plasmalemma in dendrites and was mainly found in endosome-like organelles in somata. Of all alpha(2A)-AR immunoreactive somata, 62% also contained immunolabeling for FG and 23% of all dendrites also showed labeling for the retrograde tracer. The intracellular distribution of alpha(2A)-AR did not differ in somata or dendrites with or without detectable FG. The remaining singly labeled alpha(2A)-AR profiles consisted of axons (11%), axon terminals (12%), and glial processes (13%). In numerous instances, alpha(2A)-AR-labeled glia or axon terminals were apposed to DVC projecting neurons. Together, this evidence suggests that the principal site for alpha(2A)-AR activation is at extrasynaptic sites on dendrites of CeA neurons, many of which project to the DVC and also show endosomal receptor labeling. In addition, these results indicate that activation of alpha(2A)-AR in the CeA may influence the activity of DVC projecting neurons through indirect mechanisms, including changes in presynaptic transmitter release or glial function. These results suggest that alpha(2A)-AR agonists in the CeA may modulate numerous processes including stress-evoked autonomic reactions and feeding behavior. PMID- 12373742 TI - Role of brain alpha 1B-adrenoceptors in modafinil-induced behavioral activity. AB - These studies show that either central pharmacological blockade or genetic ablation of alpha(1B)-adrenoceptors markedly attenuates the behavioral activation caused by modafinil, implicating these receptors in the drug's action. PMID- 12373743 TI - Widespread but regionally specific effects of experimenter- versus self administered morphine on dendritic spines in the nucleus accumbens, hippocampus, and neocortex of adult rats. AB - We studied the effects of self-administered (SA) vs. experimenter-administered (EA) morphine on dendritic spines in the hippocampal formation (CA1 and dentate), nucleus accumbens shell (NAcc-s), sensory cortex (Par1 and Oc1), medial frontal cortex (Cg3), and orbital frontal cortex (AID) of rats. Animals in the SA group self-administered morphine in 2-h sessions (0.5 mg/kg/infusion, i.v.) for an average of 22 sessions and animals in the EA group were given daily i.v. injections of doses that approximated the total session dose for matched rats in Group SA (average cumulative dose/session of 7.7 mg/kg). Control rats were given daily i.v. infusions of saline. One month after the last treatment the brains were processed for Golgi-Cox staining. In most brain regions (Cg3, Oc1, NAcc-s) morphine decreased the density of dendritic spines, regardless of mode of administration (although to a significantly greater extent in Group SA). However, only SA morphine decreased spine density in the hippocampal formation and only EA morphine decreased spine density in Par1. Interestingly, in the orbital frontal cortex morphine significantly increased spine density in both Groups SA and EA, although to a much greater extent in Group SA. We conclude: 1) Morphine has persistent (at least 1 month) effects on the density of dendritic spines in many brain regions, and on many different types of cells (medium spiny neurons, pyramidal cells, and granule cells); 2) The effect of morphine on spine density (and presumably synaptic organization) varies as a function of both brain region and mode of drug administration; and 3) The ability of morphine to remodel synaptic inputs in a regionally specific manner may account for the many different long-term sequelae associated with opioid use. PMID- 12373745 TI - Applications of pulsed ultrafiltration-mass spectrometry. AB - Pulsed ultrafiltration-mass spectrometry (PUF-MS) is a method with a variety of uses for the discovery and development of biologically active small molecules, including the screening of combinatorial libraries and natural product extracts for biologically active compounds, investigation of thermodynamic and kinetic ligand-receptor binding parameters, high-throughput metabolic screening, and the screening of combinatorial libraries and botanical extracts for electrophilic metabolites. Solution-phase ligand-screening assays that use pulsed ultrafiltration-mass spectrometry are useful for "reverse pharmacology" studies in which a macromolecular receptor of interest has been isolated, but ligands for the receptor are needed. Protein-binding studies that involve pulsed ultrafiltration can be used to rapidly determine classical binding parameters for interactions between a macromolecular receptor and a compound of interest. Metabolic screening assays can identify substrates for cytochromes p450, and should be capable of characterizing phase I metabolites with a throughput of at least 60 compounds/hr. Pulsed ultrafiltration can also be used in conjunction with LC-MS-MS to screen mixtures for compounds that might be activated metabolically to electrophilic quinoid and epoxide metabolites by cytochrome p450; that screening can provide early warning of compounds likely to be toxic when administered in large doses. The combination of pulsed-ultrafiltration extraction and mass spectrometric detection provides the sensitivity and selectivity necessary to characterize compounds present at low concentrations in complex chemical mixtures, and is applicable to the analysis of biologically active compounds from combinatorial libraries and botanical extracts. PMID- 12373746 TI - Collision-induced fragmentations of the (M-H)- parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages. AB - This article describes the fundamental cleavage reactions of (M-H)(-) anions of underivatized peptides that contain up to 25 amino acid residues. The experimental observations of these cleavages have been backed up by molecular modeling, generally at the AM1 level of theory. The basic cleavages are the ubiquitous alpha- and beta-backbone cleavage reactions, which provide information similar to that of the B and Y + 2 cleavages of MH(+) ions of peptides. The residues Asp and Asn also effect cleavages of the backbone (called delta- and gamma-cleavages), by reactions initiated from side chain enolate anions, causing elimination reactions that cleave the backbone between the Asp (Asn) N bond;C backbone bond. Glu and Gln also direct analogous delta- and gamma-cleavages of the backbone, but in this case the processes are initiated by attack of the side chain CO(2) (-) (CONH(-)) to form a lactone (lactam). Ser and Thr residues undergo characteristic fragmentations of the side chain. These processes, losses of CH(2)O (Ser) and MeCHO (Thr), convert these residues into Gly. In larger peptides, Ser and Thr can effect two backbone cleavage reactions, called gamma- and epsilon -processes. The C-terminal CO(2) (-) (or CONH(-)) forms a hydrogen bond with the side chain OH (of Ser or Thr), placing the C-terminal residue in a position where it may affect S(N) (2) attack at the electrophilic backbone CH of Ser, with concomitant cleavage of the backbone. All of the above negative ion cleavages require the peptide backbone to be conformationally flexible. However, there is a backbone cleavage that requires the peptide to have an alpha-helical conformation in order for the two reacting centers to approach. This cleavage is illustrated for the Glu 23-initiated backbone cleavage at Ile 21 for the (M-H)(-) anion of the antimicrobial peptide caerin 1.1. PMID- 12373747 TI - Mass spectra of copolymers. AB - Recent and older literature (covering the last 12-13 years) in the field of mass spectra of random and block copolymers is reviewed. A detailed description is given of the information on copolymer properties that can be recovered from the analysis of the low-mass region of the spectrum (the region below 500 Da) and the high-mass region. The features of mass spectra of copolymers obtained by different synthetic routes are discussed, such as free radical, condensation, ring-chain equilibration, microbial synthesis, ring-opening, simple anionic, cationic, Ziegler-Natta, and/or metallocene catalysis, along with some random and block copolymers that occur in Nature. The emphasis is on copolymer composition and average molar mass determination, and on the benefits of coupling mass spectrometry (MS) with separation techniques such as size-exclusion chromatography (SEC) and high performance liquid chromatography (HPLC). PMID- 12373750 TI - Finite element simulation of Off-Gel trade mark buffering. AB - The protonation of an aqueous solution of two ampholytes AH and BH next to a gel buffered by immobilized acid moieties IH has been studied by finite element simulation in an iterative scheme. A ten species model has been formulated, taking into account transient diffusion and equilibrium kinetics of the two amphoteric species AH and BH, of water and of the immobilized species IH. This model has been developed to illustrate the pH evolution between an ampholyte solution and an Immobiline gel, and to study the influence of the Immobiline concentration on protons and ampholyte distributions. It has been demonstrated that a minimum initial Immobiline concentration of 10(-2) M is necessary to maintain the pH in the gel in contact with a closed chamber, when the difference between the isoelectric points of AH and BH is 4 and when the initial concentration of the ampholytes in solution is in the micromolar range. This approach provides a first theoretical framework for the recently developed Off Gel trade mark electrophoresis. PMID- 12373751 TI - Generation of multicolored, prestained molecular weight markers for gel electrophoresis. AB - Molecular weight marker proteins are routinely used in sodium dodecyl sulfate polyacrylamide gel electrophoresis to estimate the relative molecular mass of specific proteins within a sample. This report describes a simple procedure for the generation of multicolored molecular weight proteins using a variety of Remazol-reactive textile dyes. These multicolored proteins provide a set of unambiguous markers for gel electrophoresis. Furthermore, the colored markers can be used in conjunction with Western blotting techniques to provide a visual display of marker proteins on the transfer membrane. PMID- 12373752 TI - Fully reversible procedure for silver staining improves densitometry of complex mixtures of biopolymers resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AB - Due to its high sensitivity, silver staining is a widely popular method for the revelation of biopolymers separated by both native and denaturing electrophoresis. A step-by-step method for the destaining and restaining of overdeveloped/overloaded silver-stained bands is described that is applicable to both proteins and nucleic acids. The procedure significantly improves densitometric analysis of gels that have been silver stained with either commercial kits or solutions made in-house. The method permits reproducible densitometry of silver-stained gels and allows quantification of both main and minor components in complex mixture of molecules resolved on the same gel slab. All steps may be interrupted and are readily reversible, allowing for facile densitometric analyses and photographic recording under optimized conditions. Furthermore, common artifacts such as differential staining of the two gel surfaces, localized uneven yellow-ochre background, and the presence of fold marks and fingerprints can be easily removed. PMID- 12373753 TI - Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation. AB - We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization. PMID- 12373754 TI - Electrophoretically unique amylases in rat livers: phylogenic and ontogenic study on the mammalian liver. AB - Liver amylase activity in rodents was assayed with Blue Starch as substrate, and found to be higher than in humans or pigs. Based on the result of concanavalin A affinity chromatography, we found that the sugar moieties of amylase molecules increased in parallel with amylase activity in the tested mammals. However, the amounts of amylase proteins determined by Western bloting with anti-human salivary-type antibody as the probe, were similar to the levels in mammalian livers. Moreover, a similar expression of amylase mRNA was also detected in the mammalian livers by a reverse transcriptional-polymerase chain reaction using primers specific for the human salivary and/or pancreatic amylase complementary DNA (cDNA) sequences. The amylase was detected at the catalytic activity, protein molecule and mRNA levels in rat liver at all ages from fetus to adult. Salivary type liver amylase activity increased up to one week after birth, and was maintained at the adult level thereafter. However, based on the results of the electrophoretic mobility test, livers with accelerated amylase activity, e.g., at 2-4 weeks after birth or during liver regeneration after partial hepatectomy, were also found to express an amylase electrophoretical identical to pancreatic type amylase in addition to salivary-type activity. These results suggest that the liver may express an etopic amylase in a certain condition. PMID- 12373755 TI - Vanadium speciation in serum by means of blue native gel electrophoresis. AB - Slab-gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the (48)V radiotracer and the phosphor-screen technology. The method was first developed using transferrin, incubated with (48)V, as a model. After it was proved that the vanadium-transferrin complex was stable during separation, the method was validated by separating serum incubated with (48)V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations. PMID- 12373756 TI - Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing. AB - Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated. PMID- 12373757 TI - Effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability. AB - Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability. PMID- 12373758 TI - Denaturing gradient gel electrophoretic multilocus sequence typing of Staphylococcus aureus isolates. AB - To obviate the need for multilocus sequencing, a method using denaturing gradient gel electrophoresis (DGGE) was developed for the multilocus sequence typing (MLST) of Staphylococcus aureus isolates. Sequence types (STs) were obtained on the basis of sequences of polymerase chain reaction (PCR) products from seven housekeeping genes and compared to the reference MLST database. The melt curves, sequences and DGGE profiles were compared for 100 STs (i) to determine PCR conditions with 40-mer GC-clamps attached to the forward and reverse primers; (ii) to choose single restriction enzyme sites for digestion of PCR products into two fragments each with a GC-clamp attached and (iii) to optimize DGGE conditions. When the DGGE types (DT) were analyzed, the majority of DTs (76/100) were accurately classified into one ST (95% of nucleotide changes were detected), 10 DTs were classified into one of two STs corresponding to a single nucleotide ambiguity and 14 DTs were classified into 3 or 4 STs corresponding to 4 or 5 nucleotide ambiguities. A combination of STs and DTs were used to obtain septuplet sets of STs (7-ST) for 25 S. aureus isolates. When compared to the reference MLST database, one methicillin-resistant S. aureus (MRSA) isolate had the same genotype as the first MRSA clone. The DGGE-MLST method can be used as a rapid, accurate and 20-fold less expensive method than DNA sequencing for the detection of all sequence types. This combined laboratory and in silico approach could have wide applicability not only to MLST methods for other bacteria but to the screening of multilocus nucleotide differences deposited in other mutation databases. PMID- 12373759 TI - Pulsed-field gel electrophoretic analysis of the genome of Lactobacillus gasseri ATCC33323, and construction of a physical map. AB - Some species of Lactobacillus are of major industrial and health significance as fermenting agents in the manufacturing of food products, as food preservatives, as "probiotic" bacteria or as vaccine delivery vehicles. In spite of their importance, there is a paucity of published information on their genome organization and structure. In this study, a combination of pulsed field gel electrophoresis (PFGE) and hybridization approaches was used to investigate the genome of L. gasseri neotype strain ATCC33323. PFGE analysis of chromosomal DNA (after digestion with the rare-cutting restriction enzymes I-CeuI, CspI, SmaI, ApaI, and SgrAI) allowed the chromosome size of L. gasseri to be estimated at 1.96 Mbp, and also revealed the presence of a linear plasmid of 48.5 kbp. A physical map of the L. gasseri chromosome, containing 6 sites for the enzymes I CeuI and 12 for CspI, was constructed. Placed on the map were the genes dnaA and gyrB (usually located close to the origin of replication on the bacterial chromosome) and 18 ribosomal RNA (rrn) genes. Mapping analysis also revealed that the chromosome contained six rrn operons, and that one of them was inverted in orientation with respect to the others. Each rrn operon contained a single copy of each of the three rrn genes, 23S rRNA (rrl), 16S rRNA (rrs) and 55 rRNA (rrf) gene. The constructed physical map should be a useful foundation for genomic and genetic studies of the lactobacilli and provides a platform for applied research, such as the engineering of Lactobacillus strains with improved characteristics for industrial and probiotic applications. PMID- 12373760 TI - A powerful, novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations. AB - A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field. PMID- 12373761 TI - Electrophoretic identification of new genomic profiles with a modified selective amplification of microsatellite polymorphic loci technique based on AT/AAT polymorphic repeats. AB - The present paper introduces improvements of the conventional selective amplification of microsatellite polymorphic loci (SAMPL) technique, that exploit AT-rich microsatellite primers. Generally, AT/AAT microsatellites are frequent components of eukaryotic genomes, but their ubiquity and polymorphic information content (PIC) could not be exploited yet, because standard SAMPL conditions did not allow amplifications. Here we report (i) on the design of new versatile AT rich microsatellite primers, that are combined with (ii) a modified SAMPL adapter primer (called EcoRI-Short), and (iii) special polymerase chain reaction (PCR) amplification regimes. The novel SAMPL procedure expands the range of useful microsatellite primers to AT-rich sequences and produces a high number of bands and a clear banding pattern, and detects polymorphisms in otherwise nonpolymorphic genomes of plants (Dioscorea alata, D. rotundata) and a fungus (Mycosphaerella fijiensis). PMID- 12373762 TI - Phylogenetics of worldwide human populations as determined by polymorphic Alu insertions. AB - Alu elements, the largest family of interspersed repeats, mobilize throughout the genomes of primates by retroposition. Alu are present in humans in an excess of 500 000 copies per haploid genome. Since some of the insertion alleles have not reached fixation, they remain polymorphic and can be used as biallelic DNA marker systems in investigations of human evolution. In this study, six polymorphic Alu insertional (PAI) loci were used as genetic markers. These markers are thought to be selectively neutral. The presence of these six PAIs was determined by a polymerase chain reaction (PCR)-based assay in 1646 individuals from 47 populations from around the world. Examination of the populations by plotting the first and second principal components, shows the expected segregation of populations according to geographical vicinity and established ethnic affinities. Centroid analysis demonstrated that sub-Sahara populations have experienced higher than average gene flow and/or represent larger populations as compared to groups in other parts of the globe and especially to known inbreed populations. This is consistent with greater heterogeneity and diversity expected of source groups. In addition, maximum likelihood (ML) analyses were performed with these 47 populations and a hypothetical ancestral group lacking the insertion in all six loci. Analysis of our data supports the Out of Africa hypothesis. African populations and admixed groups of African descent formed a single monophyletic group with a basal placement on the tree, which grouped closest to the hypothetical ancestor. PMID- 12373763 TI - Mutation scanning analysis of mitochondrial cytochrome c oxidase subunit 1 reveals limited gene flow among bovine lungworm subpopulations in Sweden. AB - A mutation scanning approach was employed to investigate the population genetic structure of the bovine lungworm, Dictyocaulus viviparus (Nematoda: Trichostrongyloidea), in southern Sweden. A total of 252 individual nematodes were collected from cattle representing 17 farms. A portion of the mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) was amplified from genomic DNA isolated from individual lungworms by the polymerase chain reaction (PCR), and then subjected to single-strand conformation polymorphism (SSCP). Samples with distinct SSCP profiles were then sequenced. In total, 12 distinct pcox1 haplotypes (393 bp) were defined for the 252 individuals, and pairwise sequence differences among the haplotypes ranged from 0.3-2.3%. Average haplotype diversity and nucleotide diversity values were 0.16 and 0.002, respectively. There was no particular correlation between pcox1 haplotypes and their geographical origin. The "overall fixation" indices F(ST) and N(ST) were calculated to be 0.77 and 0.65, respectively. The results of this study revealed that both the mitochondrial DNA sequence diversity within populations and the gene flow among populations of D. viviparus were low. This is similar to findings for some parasitic nematodes of plants and insects, but distinctly different from gastrointestinal trichostrongyloid nematodes of domesticated ruminants considered to have relatively high levels of genetic diversity and gene flow. Such differences were interpreted to relate mainly to differences in host movement as well as parasite biology, population sizes and transmission patterns, and should therefore be of epidemiological relevance.* PMID- 12373764 TI - Fast choice of separation conditions for analyses by capillary zone electrophoresis using an information system Xemic. AB - An information system Xemic applicable in analytical chemistry is described and its use in capillary electrophoresis for searching suitable separation conditions is demonstrated. This system is capable to provide suitable separation conditions even for analytes for which no electrophoretic experiments have been published so far. The system works with a database of components of anionic character the analyses of which have been performed, published in reviewed scientific journals, and included into a database created by an expert. Moreover, the system learned to search also in abstracts or complete scientific articles to find articles dealing with the determination of a substance in a given sample matrix. When no experiments have been published so far for a defined substance in a specific matrix, Xemic shows the separation conditions for determination of the substance regardless of the matrix. When no response can be found for the analyte of interest at all, the system Xemic works like an expert in the field and searches chemically similar substances and offers a series of substances the physicochemical properties of which are close to the followed analyte with respect to the behavior in the electric field, and shows working conditions for their analysis. Thus, the analyst puts only the order in the form of a given analyte in a given matrix and obtains a recommendation of a separation system that should enable to perform a successful separation. The system is not rigid and enables the operator to change the importance of individual attributes used in similarity search so as to obtain a broader or narrower group of similar components. With a certain probability the analyte of interest can be successfully analyzed under separation conditions that suited for the analysis of the most similar substances in the given matrix. PMID- 12373765 TI - Selective genotyping of individual cells by capillary polymerase chain reaction. AB - On-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection was successfully demonstrated for individual human cells. A single 50 num inner diameter (ID) fused-silica capillary served both as the reaction vessel and for isolating single cells. SYBR Green I dye was added into the reaction mixture for dynamic fluorescence labeling. Because of the small ID of the capillary, PCR-amplified DNA fragments from single cells were localized in the capillary, providing discrete product zones with concentrations at readily detectable levels. With selective primer design, only cells containing the DNA of interest were amplified. By counting the number of peaks in the capillary via electromigration past a detection window, the number of targeted cell templates could be determined. Identification of the 295 bp fragment beta-actin gene from individual human lymphoblast cell was demonstrated. Independent on-column cell counting provided positive correlation between the starting cell templates and the final PCR products. This opens up the possibility of highly selective and sensitive disease diagnosis at an early stage, when only a few cells in the population are defective. PMID- 12373766 TI - Method for immobilization of liposomes in capillary electrophoresis by electrostatic interaction with derivatized agarose. AB - A novel procedure for immobilization of liposomes inside fused-silica capillaries is demonstrated. First, the inner wall of the capillaries was coated with a positively charged polymer, composed of derivatized agarose. Subsequently, negatively charged liposomes were immobilized by electrostatic interaction on the polymer coating. The developed liposome coated capillaries were used as a nanoseparation tool for studying interactions between small drug compounds and liposomes. Part of this work was presented at the 15th International Symposium on Microscale Separations and Analysis, HPCE 2002, Stockholm, Sweden, April 2002. PMID- 12373767 TI - Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers. AB - Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated. PMID- 12373768 TI - Field-amplified sample stacking in capillary electrophoresis for on-column concentration of alkaloids in Sophora flavescens Ait. AB - A simple and rapid capillary zone electrophoresis method was developed for the separation of the main alkaloids from Sophora flavescens Ait. with the optimum buffer solution containing 110 mM NaH(2)PO(4) and 15% 2-propanol (pH 3.0). The field-amplified sample stacking (FASS) technique was applied to the on-line concentration of the alkaloids. The data presented in this work demonstrate that the use of a short water plug at the column inlet is essential for improving the reproducibility of FASS with electro-injection, and that the water plug injection time affected the sensitivity significantly. The sample concentration was further increased by about 2-3-fold by the introduction of a relatively longer water plug. With this stacking measure, the concentration sensitivity was about 3-4 orders of magnitude higher than in hydrodynamic injection. PMID- 12373769 TI - Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection. AB - Enzymatic farnesylation of oncogenic forms of Ras proteins is the initial step in a series of posttranslational modifications essential for Ras activity. The modification is catalyzed by the enzyme, protein farnesyltransferase (PFTase), which transfers a farnesyl moiety from farnesyl diphosphate to the protein. We employed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to develop a rapid and sensitive method for the determination of PFTase activity in vitro. The limited substrate specificity of PFTase allowed us to use a fluorescently labeled pentapeptide instead of a Ras protein as a substrate for the enzyme; the product of the enzymatic reaction was the farnesylated pentapeptide. The product was separated from the substrate by CE and quantified with LIF detection. Under optimal conditions, the separation was achieved within 10 min with a resolution of 86. The mass and concentration limits of detection for the farnesylated product were 10(-19) mol and 0.28 nM, respectively. By measuring the rate of accumulation of the farnesylated product, we were able to determine the kinetic parameters of the enzymatic reaction. For yeast PFTase as an enzyme and difluorocarboxyfluorescein-labeled GCVIA peptide as a substrate, the values of k(cat) and K(M) were found to be (3.1 +/- 0.3)x10(-3) s(-1) and (12.0 +/- 1.2) nuM, respectively. Our results suggest that CE-LIF can be efficiently used for the determination of enzymatic activity of PFTase in vitro. After minor modifications, the developed method can be also applied to other reactions of enzymatic prenylation of proteins. PMID- 12373770 TI - Simultaneous determination of dopa and carbidopa enantiomers by capillary zone electrophoresis. AB - Dopa and carbidopa, components of the dual therapy for Parkinson's disease treatment, are both provided as single enantiomers, since their D-forms are inactive. To ensure the efficiency and safety of the therapy, these D enantiomers, therefore, should be considered as impurities. In this paper, the enantioseparation power of different types of cyclodextrins, both neutral and charged ones, on dopa and carbidopa enantiomers was tested. Three methods of simultaneous separation of dopa and carbidopa enantiomers were developed, using highly sulfated beta-cyclodextrin and sulfated beta-cyclodextrin as chiral selector, in normal and reversed polarity mode. Two methods among these three were found sensitive enough for the quantitation of 0.1% D-enantiomers in L-forms (impurity level). After the optimization study, the best method was selected, using 16 mM sulfated beta-cyclodextrin in 15 mM sodium phosphate buffer pH 2.45, an uncoated fused-silica capillary (50 num inner diameter, 30 cm total length), and an applied voltage of -12 kV. This method is robust and efficient, with very high resolution for all peaks within a short analysis time of 10 min. Quantitatively, the method offers a limit of detection (LOD) of 0.2 nug/mL and a limit of quantitation (LOQ) of 0.5 nug/mL for both D-dopa and D-carbidopa, which is equivalent to 0.02% and 0.05% against the respective L-enantiomers. A linear relationship was found between the concentration of the analyte and the corresponding peak area in a range of 0.5-2.0 nug/mL. PMID- 12373771 TI - Fast capillary electrochromatographic analysis of parabens and 4-hydroxybenzoic acid in drugs and cosmetics. AB - A fast capillary electrochromatographic method was developed for the analysis of paraben preservatives in drugs and cosmetics in the presence of their main metabolite and/or impurity, 4-hydroxybenzoic acid. The separation was optimized in a 75 num ID capillary, fully packed with 5 num C18 stationary phase, studying the effects of mobile phase pH and composition (buffer type and organic solvent content). The mobile phase 5 mM ammonium formate, pH 3.0, containing 65% acetonitrile allowed us to obtain the baseline separation of methyl-, ethyl-, propyl-, butyl-, and benzylparabens from a mixture in less than 2.5 min with repeatability and linearity using the short-end injection method (8 cm separation capillary effective length). Under the optimum experimental conditions, the method provided high separation efficiency for parabens, in the range of 129 312 140 325 number of theoretical plates per meter, and analyte quantitation limits (LOQs) in the range of 1.25-2.50 nug/mL. The method was successfully applied to the quantitative analysis of paraben preservatives in pharmaceutical and cosmetic industrial samples with direct injection or after reduced sample pretreatment. PMID- 12373772 TI - Strategies for proteomics with incompletely characterized genomes: the proteome of Bos taurus serum. AB - A reference map for Bos taurus serum was obtained using proteomic tools: 21 proteins, plus several serum albumin fragments, have been identified in 47 spots. One of the major acute-phase reactants, haptoglobin, was also detected in a pathological serum. A number of technical problems had to be solved. (i) Spot resolution in two-dimensional electrophoresis (2-DE) is not easily optimized, as several proteins have similar molecular mass; different polyacrylamide concentration gradients were used for the analysis of various size ranges. (ii) Identification of proteins through mass spectrometry (MS) procedures is also difficult as the genome of Bos taurus is incompletely characterized. The program FASTS proved particularly useful, since it allows simultaneous searching of several unordered sequence fragments, which may be individually too short to provide a statistically valid match using BLAST. PMID- 12373773 TI - Proteomic analysis of protein oxidation in Alzheimer's disease brain. AB - There is a growing body of evidence that oxidative stress plays a major role in Alzheimer's disease (AD) pathogenesis. Identification of oxidatively altered proteins in AD is important for understanding the relationship between protein oxidation, protein aggregation and neurodegeneration. In this communication, we report a method that can be applied to study oxidative changes of individual proteins in brain. In order to analyze protein oxidation by detection of protein bound carbonyls, cytosolic protein extracts were derivatized with 2,4 dinitrophenylhydrazine (DNPH) and then separated by two-dimensional (2-D) gel electrophoresis. After electrotransfer to polyvinylidene difluoride (PVDF) membranes, proteins were first stained with Sypro Ruby protein stain, and then the oxidized proteins were detected with anti-dinitrophenyl (DNP) antibody. About 150 proteins and more than 100 oxidized proteins were detected and quantified in both AD and control cases by 2-D image analysis. The amount of protein-bound carbonyls was decreased for six and increased for one protein in AD. The amount of protein was increased for three proteins in AD. Furthermore, the degree of oxidation was calculated as the ratio of protein-bound carbonyls to the total amount of an individual protein. Two proteins showed a significant decrease in the degree of oxidation in AD. Our results suggest that the balance of protein oxidation and degradation is altered in AD. PMID- 12373774 TI - Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study. AB - We used two-dimensional (2-D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty-eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests. Comparison of the BALF protein maps of the two groups of patients showed 32 spots with statistically significant disease-related variations in relative abundance. In sarcoidosis we found an increase in the amount of several plasma proteins, while in idiopathic pulmonary fibrosis we observed a statistically significant increase in low molecular-weight proteins, many of which are involved in inflammatory processes (such as MIF and calgranulin) or antioxidant response (such as antioxidant peroxysomal enzyme and thioredoxin peroxidase 2). 2-D electrophoresis allowed us to identify new BALF proteins and to characterize protein composition in patients with sarcoidosis and idiophatic pulmonary fibrosis. Comparison of the gels of the two diseases showed that they differ in BALF protein profiles as they do in type of immune response. PMID- 12373775 TI - Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. AB - Differential proteome analysis is used to study body fluids from patients suffering from rheumatoid arthritis (RA), reactive arthritis (reaA) or osteoarthritis (OA). Mass spectrometric structure characterization of gel separated proteins provided a detailed view of the protein-processing events that lead to distinct protein species present in the respective body fluids. (i) Fibrin(ogen) beta-chain degradation products, presumably plasmin-derived, appeared solely in synovial fluids (SF) from both patient collectives, (ii) calgranulin B (MRP14) was exclusively identified in SF samples derived from 5 out of 6 patients suffering from RA. Calgranulin B was not observed in synovial fluids from OA patients, nor in plasmas from either patient group. In all cases where calgranulin B was detected, calgranulin C was identified as well. (iii) Serum amyloid A protein spots were determined in plasmas and synovial fluids from patients with RA, but not in patients with OA. In addition to disease-relevant differences, interindividual differences in haptoglobin patterns of the patients under investigation were observed. Hence, in-depth proteome analysis of body fluids has proven effective for identification of multiple molecular markers and determination of associated protein structure modifications, that are thought to play a role for specifically determining a defined pathological state of diseased joints. PMID- 12373778 TI - Amygdalo-cortical sprouting continues into early adulthood: implications for the development of normal and abnormal function during adolescence. AB - Adolescence is a critical stage for the development of emotional maturity and diverse forms of psychopathology. The posterior basolateral nucleus of the amygdala is known to mediate fear and anxiety and is important in assigning emotional valence to cognitive processes. The medial prefrontal cortex, a homologue of the human anterior cingulate cortex, mediates emotional, attentional, and motivational behaviors at the cortical level. We postulate that the development of connectivity between these two corticolimbic regions contributes to an enhanced integration of emotion and cognition during the postnatal period. In order to characterize the development of this relay, injections of the anterograde tracer biocytin were stereotaxically placed within the posterior basolateral nucleus of the amygdala of rats at successive postnatal time points (postnatal days 6-120). Labeled fibers in the medial prefrontal cortex were evaluated using a combination of brightfield, confocal, and electron microscopy. We found that the density of labeled fibers originating from the posterior basolateral nucleus shows a sharp curvilinear increase within layers II and V of the anterior cingulate cortex and the infralimbic subdivisions of medial prefrontal cortex during the late postweanling period. This increase was paralleled by a linear rise in the number of axospinous and axodendritic synapses present in the neuropil. Based on these results, we propose that late maturation of amygdalo-cortical connectivity may provide an anatomical basis for the development and integration of normal and possibly abnormal emotional behavior during adolescence and early adulthood. PMID- 12373779 TI - Neurogenesis and gliogenesis in the spinal cord of turtles. AB - A 5-bromo-3'-deoxyuridine (BrdU) pulse administered to juvenile turtles resulted in cell labeling throughout the gray matter (GM) and white matter (WM) of the spinal cord. One and twenty-four hours postinjection, larger densities of BrdU labeled nuclei (LN) occurred within the GM, with a density peak localized in the central region (CR). Seven days later, density differences between GM and WM disappeared, accompanying a more uniform distribution of LN in the GM (absence of the central peak). Multiple injection experiments also showed similar evolution in the distribution of LN. Morphometric studies revealed that the size of LN had undergone time-related increments: Larger nuclei appeared at protracted fixation time points. Double-labeling experiments indicated that BrdU-labeled cells expressed neuroactive substances, such as gamma-aminobutyric acid (GABA), neuron specific nuclear protein (NeuN), and the cytoplasmic early postmitotic neuronal marker (TUC-4). Other BrdU-labeled cells expressed the glial-specific protein (GFAP). GABA-BrdU, TUC-4-BrdU, and GFAP-BrdU double-labeled cells were recognized 6 days after the first BrdU injection. NeuN-BrdU double-labeled cells were found at 50 days postinjection. Three-dimensional transmission electron microscopy revealed the presence of synapses and typical kinocilia in putative immature nerve cells. Kinocilia were also found in putative immature glial cells. In consideration of the scattered distribution pattern of BrdU-labeled cells, in animals fixed 1 hour postinjection, the existence of a single proliferating center was discarded. The CR, including the ependymal epithelium, showed the highest density of LN. PMID- 12373780 TI - PSA-NCAM immunocytochemistry in the cerebral cortex and other telencephalic areas of the lizard Podarcis hispanica: differential expression during medial cortex neuronal regeneration. AB - The lizard medial cortex, a region homologous to the mammalian dentate gyrus, shows postnatal neurogenesis and the surprising ability to replace its neurons after being lesioned specifically with the neurotoxin 3-acetylpyridine. As the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is expressed during neuronal migration and differentiation, we have studied its distribution in adult lizards and also during the lesion-regeneration process. In the medial cortex of control animals, many labeled fusiform somata, presumably corresponding to migratory neuroblasts, appeared in the inner plexiform layer. There were also scattered immunoreactive granule neurons in the cell layer. Double immunocytochemistry with 5'-bromodeoxyuridine revealed that some of the PSA-NCAM expressing cells in the inner plexiform and cell layers were generated recently. PSA-NCAM immunoreactivity was also present in the dorsomedial, dorsal, and lateral cortices, as well as in the dorsal ventricular ridge, the nucleus accumbens, and the nucleus sphericus. Twelve hours after the injection of 3 acetylpyridine, some medial cortex granule neurons appeared degenerated, although some of them still expressed PSA-NCAM. One to 2 days after the injection, most granule neurons appeared degenerated and no PSA-NCAM immunoreactivity was detected in the medial cortex cell layer. Four to 7 days after treatment, abundant labeled fusiform cells populated the inner plexiform layer and some immunoreactive somata were seen in the cell layer. Fifteen to 30 days after the neurotoxin injection, the number of PSA-NCAM expressing granule neurons augmented considerably and the level was still above control levels in lizards that survived 42 days. Our results show for the first time the expression of PSA-NCAM in a reptile brain, where it appears to participate in the migration and differentiation of granule neurons during adult neurogenesis and regeneration. PMID- 12373781 TI - Immunoreactivity against choline acetyltransferase, gamma-aminobutyric acid, histamine, octopamine, and serotonin in the larval chemosensory system of Dosophila melanogaster. AB - We have studied the distribution of choline acetyltransferase (ChAT), gamma aminobutyric acid (GABA), histamine, octopamine and serotonin in the larval chemosensory system of Drosophila melanogaster. Colocalization at the confocal level with green fluorescent protein (GFP) or Tau-GFP reporters, expressed in selected P[GAL4] enhancer trap lines, was used to identify the cells making up these neurotransmitters. As in the adult fly, larval olfactory afferents project into the (larval) antennal lobe (LAL), where they synapse onto local interneurons and projection neurons, whereas gustatory afferents terminate essentially in the tritocerebral-subesophageal (TR-SOG) region. We demonstrate that the neuropils of the LAL and the TR-SOG are immunoreactive to ChAT and GABA. In addition, serotonin- and octopamine-immunoreactive fibers are present in the LAL. ChAT immunostaining is localized in subsets of olfactory and gustatory afferents and in many of the projection neurons. In contrast, GABA is expressed in most, and perhaps all, of the local interneurons. Serotonin immunoreactivity in the LAL derives from a single neuron that is situated close to the LAL and projects to additional neuropil regions. Taken together, these findings resemble the situation in the adult fly. Hence, given the highly reduced numbers of odorant receptor neurons in the larva, as shown in a previous study (Python and Stocker [2002] J. Comp. Neurol. 445:374-387), the larval system may become an attractive model system for studying the roles of neurotransmitters in olfactory processing. PMID- 12373782 TI - A new look at calretinin-immunoreactive amacrine cell types in the monkey retina. AB - We have examined amacrine cells that are calretinin-immunoreactive (-IR) in the macaque monkey retina with the aim of classifying them into morphological and functional subtypes. There are calretinin-IR cells in the fovea and throughout the retina. Their highest density is reached at 1.0 mm from the foveal pit (10500 cells/mm(2)) and falls to 2600/mm(2) by 10 mm of eccentricity. Nearest-neighbor statistics for the calretinin-IR cell body distribution indicate a nonregular pattern, with a regularity index of 1.4-1.6. There is an increase or "bump" of cell density 3.5-4.0 mm from the foveal pit, corresponding to the rod photoreceptor density peak. Based on morphological differences, there appear to be three types of amacrine cell that are calretinin-IR. To determine the types, we doubly immunolabeled retinas, from fovea to periphery, for calretinin-IR in combination with other calcium binding proteins and inhibitory amino acid neurotransmitters. Labeling with parvalbumin and calretinin antibodies indicated that 70% of the amacrine cells were solely calretinin-IR, and 30% contained parvalbumin-IR as well. In the same way, 70% of the calretinin-IR amacrine cells colocalized calbindin, but 30% were only calretinin-IR. Among the calretinin/calbindin-colocalized cells, there were small-field and wide-field types. Double labeling with antibodies to calretinin and gamma-aminobutyric acid (GABA) and to calretinin and glycine revealed the majority to be glycine-IR, but some were GABA-IR. The glycine-IR population consists mainly of AII amacrine cell types, but clearly another non-AII type is involved. The non-AII glycine-IR population resembles a small- to medium-field diffuse type. The calretinin-IR wide-field type is GABAergic and corresponds to an A19 type. The central, rod free, fovea contains the calretinin-IR, non-AII glycine-IR type and the calretinin-IR, GABAergic type only. To learn more concerning the circuitry of the calretinin/glycine-IR, non-AII amacrine cell type in isolation from AII amacrine cells, we concentrated on the rod-free fovea, where AII amacrine cells are absent. We performed a serial section electron microscopy (EM) study on four calretinin-IR cells. They were involved with cone pathway circuitry. They got input from ON and OFF midget bipolar cells, reciprocated synapses to these bipolar cells, and provided synapses to ON-center ganglion cells. Thus we have obtained new information on a cone pathway amacrine cell of the central monkey fovea that is involved in the midget system. PMID- 12373783 TI - Distribution of urocortin-like immunoreactivity in the central nervous system of the frog Rana esculenta. AB - Corticotropin-releasing factor (CRF), sauvagine, and urotensin I are all members of the so-called CRF neuropeptide family. Urocortin (Ucn), a 40-amino-acid neuropeptide recently isolated from the rat brain, is the newest member of this family. Until now, the distribution of Ucn in the central nervous system (CNS) has been studied only in placental mammals. We used a polyclonal antiserum against rat Ucn to determine the distribution of Ucn-like immunoreactivity in the CNS of the green frog, Rana esculenta. The great majority of Ucn-immunoreactive perikarya was seen in the anterior preoptic area, ventromedial thalamic nucleus, posterior tuberculum, nucleus of the medial longitudinal fasciculus, and Edinger Westphal nucleus. Urocortin-immunoreactive nerve cells were also observed in the motor nuclei of the trigeminal and facial nerves and in the hypoglossal nucleus. Immunoreactive fibers were found in the medial and lateral septal nuclei, bed nucleus of the stria terminalis, many of the thalamic and hypothalamic nuclei, mesencephalic tectum, tegmental nuclei, torus semicircularis, and dorsal horn and central field of the spinal cord. Only scattered Ucn-immunoreactive axon terminals were observed in the external zone of the medial eminence. The densest accumulations of Ucn-immunoreactive nerve terminals were seen in the granular layer of the cerebellum and cochlear nuclei. Our results suggest that an ortholog of mammalian Ucn occurs in the CNS of the green frog. The distribution of Ucn like immunoreactivity in Rana esculenta showed many similarities to the distribution in placental mammals. The distribution of Ucn-like immunoreactivity in the anuran CNS was different from that of CRF and sauvagine, so our results suggest that at least three different lineages of the CRF neuropeptide family occur in the anuran CNS. PMID- 12373784 TI - Early development of the hypothalamus of a wallaby (Macropus eugenii). AB - We have studied the development of the hypothalamus of an Australian marsupial, the tammar wallaby (Macropus eugenii), to provide an initial anatomic framework for future research on the developing hypothalamus of diprotodontid metatheria. Cytoarchitectural (hematoxylin and eosin), immunohistochemical (CD 15 and growth associated protein, GAP-43), tritiated thymidine autoradiography, and carbocyanine dye tracing techniques were applied. Until 12 days after birth (P12), the developing hypothalamus consisted of mainly a ventricular germinal zone with a thin marginal layer, but by P25, most hypothalamic nuclei were well differentiated, indicating that the bulk of hypothalamic cytoarchitectural development occurs between P12 and P25. Strong CD 15 immunoreactivity was found in radial glial fibers in the rostral hypothalamus during early developmental ages, separating individual hypothalamic compartments. Immunoreactivity for GAP 43 was used to reveal developing fiber bundles. The medial forebrain bundle was apparent by P0, and the fornix appeared at P12. Tritiated thymidine autoradiography revealed lateral-to-medial and dorsal-to-ventral neurogenetic gradients similar to those seen in rodents. Dye tracing showed that projections to the posterior pituitary arose from the supraoptic nucleus at P5 and from the paraventricular nucleus at P10. Projections to the medulla were first found from the lateral hypothalamic area at P0 and paraventricular nucleus at P10. In conclusion, the pattern of development of the wallaby hypothalamus is broadly similar to that found in eutheria, with comparable neurogenetic compartments to those identified in rodents. Because most hypothalamic maturation takes place after birth, wallabies provide a useful model for experimentally manipulating the developing mammalian hypothalamus. PMID- 12373785 TI - Appreciative inquiry: a radically different approach to change. AB - Appreciative Inquiry, or Al, seeks to identify what went right and duplicate the experience. Adjustment in thinking may be difficult for defensive-minded health care professionals. Likelihood of success appears greater when smaller groups are involved. PMID- 12373786 TI - Benchmark results spur action in hospital ED. AB - Benchmarking stresses the importance of rate, not absolute numbers. Facility goes from dead last to first in just a few quarters. Sustaining change is much more difficult than achieving change. PMID- 12373787 TI - Want to innovate? Look outside of health care. AB - Innovation center will teach employees to be more creative, differentiate facility. Other companies are more than willing to meet and share their experiences. Innovation seems to be a red-hot topic in every industry except health care. PMID- 12373788 TI - MedMARx report may aid in error prevention. AB - Sentinel events represent only the tip of the iceberg in uncovering errors. Use targeted reporting in areas where errors are likely to occur. A high number of reported errors does not necessarily mean low quality. PMID- 12373789 TI - In e-communications, walk before you run. AB - Since cultures vary across clinical groups, customization is a must. Addressing concerns of physicians and patients makes e-mail more palatable. Web site offers greater privacy, improves documentation process. PMID- 12373790 TI - Patient safety alert. Poor supervision can contribute to a higher rate of errors. PMID- 12373791 TI - Patient safety alert. Injury prevention model broadens safety scope. PMID- 12373792 TI - Join the battle to help milk win out over soda. Milk vs soda. PMID- 12373793 TI - Pill splitting cuts rx costs but can pose medical risks. PMID- 12373794 TI - Stat! Pennsylvania's health care at risk. PMID- 12373795 TI - Enjoy summer sun safely. PMID- 12373796 TI - Petting zoo rules: have fun but wash your hands! PMID- 12373798 TI - Sing a song of summer salads. PMID- 12373797 TI - That old summer time glow. Even northerners need protection from the sun. PMID- 12373799 TI - [Diagnostic imaging of Alzheimer's disease]. PMID- 12373800 TI - [Prospects for diagnostic imaging of schizophrenia utilizing magnetic resonance techniques]. PMID- 12373801 TI - [Pathophysiology of mood disorders and the therapy]. PMID- 12373802 TI - [Imaging of brain lesions in mutism]. PMID- 12373803 TI - [Genetic study on schizophrenia and its recurrence utilizing pharmacological models]. PMID- 12373804 TI - [Characteristic gene expression in autopsy brain tissues of patients with schizophrenia--analysis by DNA chip]. PMID- 12373805 TI - [Study of 5-HT related genes as possible etiological agents in schizophrenia]. PMID- 12373806 TI - [Mitochondrial genes in bipolar disorder]. PMID- 12373807 TI - [Behavioral treatment for chronic insomnia]. AB - The efficacy of non-pharmacological intervention for chronic insomnia has been proven by several meta-analytic reviews, an NIH report, an American Academy of Sleep Medicine review, and numerous clinical trials. Behavior therapy for chronic insomnia consists of relaxation, stimulus control, sleep restriction, cognitive restructuring and sleep hygiene education, which has produced reliable and durable changes in total sleep time, sleep onset latency, number and duration of awakening. These studies also showed that the post-treatment effect of behavior therapy is equal to that of hypnotic therapy, and that these effects were maintained for 6 months on follow-up assessment. Elderly insomniac patients would gain considerable benefit from behavioral treatments because there are no adverse physical effects as there are from pharmacological therapy. The authors present the basic theory, techniques of behavior therapy for insomnia, and the results of two important key meta-analytic reviews. Any behavioral approach such as convenient education, self-care enhancement by bibliotherapy, and individual face to-face counseling, seem to be fruitful not only for American but also Japanese insomnia patients. Nonetheless, there are no currently actual intervention studies using behavior therapy in Japan. We have discussed the methodology of intervention study and published a behavioral self-help manual for people with sleep problems. Development of a behavioral approach to chronic insomnia seemed to be very beneficial and a useful contribution to mental health services. PMID- 12373808 TI - [Posttraumatic stress disorder in victims of sexual assault--related to depression or physical symptoms]. AB - To clarify the clinical characteristics of mental disorders in sexual assault victims, we investigated the victims focusing on PTSD, depression, physical symptoms, and their relationships. SUBJECTS: Participants were 46 treatment seeking female victims of sexual assault who consulted four hospitals, one clinic and one psychological services center, between February 2000 and April 2001. The mean +/- SD age of the participants was 28.0 +/- 8.9 years, the mean +/- SD period from the traumatic event was 94.5 +/- 88.0 months. PTSD was diagnosed and evaluated using a structured interview (Clinician-Administered PTSD Scale for DSM IV: CAPS). Depressive symptoms were assessed using Self-rating Depression Scales (SDS). Physical symptoms were assessed using the Physical symptom scale developed by the authors. RESULTS: Thirty-two participants (69.6%) met the criteria for PTSD in their current diagnosis, and 41 (89.1%) had the disorder at some point during their lives. SDS score and Physical symptom scale score of the PTSD group were significantly higher than those scores of the non-PTSD group. The SDS score correlated with the Avoidant-numbing score. The Physical symptoms scale score correlated with the Intrusion score and Hyperarousal score. We think that the PTSD group had the co-existing depression secondary to PTSD. Although previous studies have discussed the relationship between physical symptoms and Hyperarousal symptoms, this study suggested that physical symptoms were related to Intrusion symptoms as much as Hyperarousal symptoms. We found 2 patterns when PTSD patients reported physical symptoms related to Intrusion symptoms. The patterns were caused (1) by physiological reactivity on exposure to internal or external cues that symbolize an aspect of the traumatic event, and caused (2) by somatic reenactment symptoms. CONCLUSION: We discuss the importance for clinicians to distinguish Intrusion symptoms from physical symptoms as well as Avoidant-numbing symptoms from depressive symptoms on PTSD diagnosis. Because sexual assault victims have difficulty in talking about the traumatic experience, clinicians should pay attention to these findings in developing therapeutic plans for the victims. PMID- 12373809 TI - [Molecular diagnosis for diagnosis of leukemia]. AB - Molecular methods are emerging as important tools for diagnosis and therapy of patients with leukemia. First, the development of conventional Southern blot analysis has facilitated the detection of rearrangements in immunoglobulin and T cell receptor genes. Moreover, many chimeric genes involved in balanced translocation have identified significant classifications of leukemia patients by cytogenetic techniques such fluorescent in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). These techniques provide an advantage in monitoring minimal residual disease (MRD). Furthermore, recent gene expression analysis with quantitative PCR assays such as real-time PCR have developed the early diagnosis and monitoring of MRD. These molecular-based diagnoses have contributed to the clinical decision-making process in the diagnosis of leukemia. PMID- 12373810 TI - [Cell surface and intracellular marker analysis as a laboratory test for the diagnosis of hematological disorders]. AB - Immunophenotyping of hematopoietic malignancies is representative application of cell (surface) marker analysis by flow cytometry and monoclonal antibodies in the clinical laboratory. The multitude of available monoclonal antibodies demands a standardization of the selection and combination of antibodies. Therefore, some international committee or working group proposed the panels or guidelines for the selection of antibodies. Intracellular antigens are of major importance for immunophenotyping of hematological malignancies, and flow cytometric detection of intracellular antigens was improved by the development of new permeabilization/fixation solutions. Recently new gating method was recommended for better isolating the target cells in the flow cytometric analysis. Finally CD55 and CD59 assay for the diagnosis of PNH was mentioned. PMID- 12373811 TI - [Reticulated platelet and its clinical significance]. AB - Reticulated platelets (RP) retain some residual mRNA in their cytoplasm and are thought to be the most recently produced platelets in circulation. They can be visualized on a blood film with new methylene blue staining. RP is a flow cytometric assay utilizing a fluorescent dye, either thiazole orange or auramine O. There is a difference in reference values for RP between thiazole orange and auramine O. From the analytic results of RP in patients with thrombocytopenic disorders, RP measurement is considered useful for estimating thrombopoiesis in bone marrow and for differential diagnosis and elucidating the pathophysiology of thrombocytopenic disorders. In the future, test for RP might have a possibility of routine examination because RP can be rapidly and simply measured using whole blood stained with auramine O using an automated reticulocyte counter modified to determine RP. Furthermore, it is anticipated that standardization and morphological definition for RP are established and accuracy for RP is improved. PMID- 12373812 TI - [Hemostatic abnormalities in DIC]. AB - There are global coagulation tests and hemostatic molecular markers in the diagnosis of disseminated intravascular coagulation (DIC). In the global coagulation tests, the sensitivity of prothrombin time ratio and fibrinogen for the diagnosis of DIC is low, but their specificity is high. In platelet count and FDP, the sensitivity for the diagnosis of DIC is good, but the specificity is low. Fibrinogen may be unsuitable for the diagnosis of DIC, because it increases of the inflammatory reaction. It is possible to theoretically diagnose DIC by increased tissue factor production. It is currently considered that hemostatic molecular marker should be utilized to diagnose DIC. Thrombin-antithrombin complex and soluble fibrin are reflected to hypercoagulable state, thrombomodulin to vascular endothelial cell injuries, and plasminogen activator inhibitor-I to hypofibrinolytic state. In leukemia with DIC, hyperfibrinolysis and marked bleeding symptoms are often observed. In septicemia with DIC, hypofibrinolysis and severe organ failure often occur. Early diagnosis and treatment of DIC are important to improve the prognosis, and hemostatic molecular markers should be useful for that purpose. PMID- 12373813 TI - [Detection method for platelet activation markers]. AB - The assessment of platelet activation levels may be useful for identifying patients who would benefit from antiplatelet therapy and prediction of ischemic events. Laboratory markers of platelet activation include activation-dependent changes in glycoprotein (GP) IIb/IIIa complex, exposure of granule membrane proteins, binding of secreted platelet proteins, and development of procoagulant surfaces. Whole blood flow cytometry is a popular and useful method for the detection of these markers of platelet activation. Monoclonal antibodies against platelet surface glycoproteins are used to identify activated platelets. PMID- 12373814 TI - [Prothrombin time and its standardization]. AB - This review regarding prothrombin time and its standardization is described around some recent topics as the followings. 1. History of standardization for prothrombin time and revised WHO guideline for thromboplastin; A short history of standardization is summarized to understand a scheme of International Normalized Ratio (INR) based on International Sensitivity Index (ISI) that is calibrated by International Reference Preparation (IRP) for thromboplastin, and some key points in revised WHO guideline for thromboplastin and plasma used to control oral anticoagulant therapy are interpreted for research and practical use. 2. Point-of care prothrombin time monitoring; A portable device to measure prothrombin time with whole blood sample, such as CoagChek (Roche), contributes to self-management by patients required long-term oral anticoagulation. Some investigators reported clinical agreement to use this monitoring system and improvement of patient's QOL and cost-effectiveness in overseas. 3. New types of thromboplastins; Two new types of thromboplastins have been available since the last year in Japan. One is a human plain thromboplastin, Simplastin HTF (Biomereux) from extract of cultured human lung cancer cell, and another is IL test PT-Fibrinogen Recombinant (Iatron) from recombinant rabbit tissue factor relipidated in a synthetic phospholipid blend. For control of oral anticoagulation, good performance are expected in either thromboplastins because of their sufficient low ISI values. 4. INR methodology for other diseases; INR/ISI system is designed as a standardized methodology for control of oral anticoagulation. Prothrombin time, however is utilized as a global coagulation test for diagnosis or criteria of other disorders, such as congenital coagulation factor deficient, severe liver dysfunction and disseminated intravascular coagulation. Previous our study indicated that discrepancy of sensitivities to plasma absorbed multiple coagulation factors and plasma from patients under oral anticoagulation was revealed in rabbit brain thromboplastins, but not in human origins. Discrepancy of sensitivities observed in rabbit thromboplastins was emphasized in convert to INR values. These results suggested that the use of human thromboplastin of which ISI is close to 1.0 leads possibility for introducing INR methodology to evaluate PT of other disorders. PMID- 12373815 TI - [Hematologic and hemostatic tests availability in view of remuneration for medical services]. AB - Hematologic and hemostatic tests are important and indispensable for diagnosing any kind of hematologic disease. Recent advances in hematologic tests are seen in automation of the blood cell counts and morphological analyses that are required for diagnosing hematologic diseases, and in the accumulation and availability of genetic and chromosomal examinations related to their diagnosis. Although hemostatic tests used to be mainly aimed at checking bleeding tendency, they are now being directed toward diagnosing thrombotic tendency. Medical expenses have hardly been increased in past revisions of reimbursement schedules for medical services, which has driven hospital management into an even tighter corner. In the field of laboratory examination, test fees were heavily slashed and calculated on an all-inclusive basis, forcing budgetary restrictions on the management of test laboratories. I discuss the medical reimbursement for hematologic and hemostatic tests, focusing on the fiscal 2002 revision of the medical service fee schedule implemented in April. PMID- 12373816 TI - [Risk factors for atherosclerotic vascular disease in patients on maintenance hemodialysis--with especial respect to reverse cholesterol transport system and hyperhomocysteinemia]. AB - Hemodialysis (HD) patients have a high mortality rate due to vascular disease (VD). Therefore, we investigated the effect of uremic dyslipidemia on VD in HD patients, with special consideration of the reverse cholesterol transport (RCT) system including high-density-lipoprotein cholesterol (HDL-C), cholesteryl ester transfer protein (CETP) and its genetic (D442G) mutation. In 414 HD patients, a sub-median HDL-C level (< 48 mg/dl) was an independent risk factor for VD. In the lower HDL-C status, the CETP mutation leading to CETP levels was independently associated with VD. In 210 selected patients, the CETP level was an independent protective factor against VD among those with higher HDL-C levels (> 45 mg/dl). We also measured serum homocysteine (Hcy) levels and examined its association with VD considering that hyperhomocysteinemia is a newly identified risk factor for atheroma. HD Patients (n = 545) had about 3 times the Hcy levels of the general population. A common C677T mutation in the gene of methylenetetrahydrofolate reductase (MTHFR) involved in Hcy metabolism was independently and directly related to serum Hcy levels with TT genotype patients having the highest levels. Patients with the TT genotype were younger and had a shorter duration of dialysis than those with the CT or CC genotype after adjustment for age at the initiation of dialysis, although there was no difference in VD prevalence among the genotypes and no association between Hcy levels and VD prevalence. In conclusion, lower HDL-C and CETP status was a risk factor for VD in HD patients, suggesting the importance of RCT. Serum Hcy levels were markedly increased in HD patients and the TT genotype may be associated with higher mortality. However, a large-scale prospective study is required to clarify whether hyperhomocysteinemia or the TT genotype is a VD risk factor among HD patients. PMID- 12373817 TI - [Pathophysiology and diagnosis for arteriosclerosis obliterans]. AB - Patients with arteriosclerosis obliterans, or peripheral arterial disease have been conventionally diagnosed and treated from only the viewpoint of peripheral arterial circulation. These concepts may have improved the quality of life for patients, but could not contribute the prognosis of life, because peripheral arterial disease is associated with an increased risk of the coronary disease and cerebrovascular disease. Intermittent claudication, the most common symptom of peripheral arterial disease, results from flow-reducing lesions in the arteries of the lower extremity that cause exercise-induced muscle ischemia. In order to evaluate intermittent claudication, many kinds of noninvasive diagnostic studies, including ABPI (ankle brachial pressure index) and the measurement of claudication distance, et al have been proposed. We have used the recovery time of the ischemic reaction at foot sole, plethysmography, thermography, laser doppler flowmetry, or NIRS (near-infrared spectroscopy) after walking test, rather than ABPI. These examinations will be superior to ABPI to evaluate effects after ergotherapy or pharmacotherapy for patients with intermittent claudication. Carotid artery sclerosis may be a good marker of systemic atherosclerosis. By our assessment of risk factors, the progression of atherosclerotic change in carotid artery was strongly correlated with two risk factors, such as smoking and systolic blood pressure. In the cholesterol analysis, Lp (a) was only high risk factor for atherosclerotic change of carotid artery. Recent technical advances, adequate evaluation of systemic atherosclerosis, and reduction of risk factors should improve the prognosis of patients with peripheral arterial disease. PMID- 12373818 TI - [Homocysteine as a risk factor for ischemic heart disease]. AB - Hyperhomocysteinemia is currently regarded as a risk factor for the development of ischemic heart disease (IHD). We examined the relation between plasma total homocysteine (tHCY) concentrations and the severity and morphology of coronary stenosis in 238 Japanese patients. Coronary stenosis score (CS) in the first quartile of plasma tHCY levels was 4.9 and the second, third and fourth quartiles were 6.5, 7.5, and 8.9, respectively and plasma tHCY levels were 8.5, 10.9, 13.5, and 22.4 mumol/l, respectively. CS was higher in the fourth quartile than in the first, suggesting that plasma tHCY levels are related to the coronary stenosis severity. Among 238 patients, 123 did not have any significant coronary stenosis (group 1), 98 had focal coronary stenosis (group 2) and 17 had diffuse coronary stenosis (group 3). Plasma tHCY level was higher in group 3 than in group 1 or 2, suggesting that plasma tHCY levels are related to the diffuse coronary stenosis. We also examined whether hyperhomocysteinemia is concerned in coronary spasm. Plasma tHCY levels did not differ between 43 patients with vasospastic angina and 43 patients without ischemic heart disease, suggesting that plasma tHCY levels are not concerned in coronary spasm. Therefore, in Japanese patients, plasma tHCY contributes to the development of IHD through the influence on chronic coronary atherosclerotic background, but not through the influence on coronary spasm, i.e., one of the acute ischemic stimuli. Whether or not tHCY influences coronary thrombosis should be clarified in Japanese patients. PMID- 12373820 TI - [Validation of total ejection isovolume index measurement by continuous-wave Doppler echocardiography]. AB - We evaluated the validity of total ejection isovolume (TEI) index measurement by continuous wave Doppler method in 82 patients with various heart diseases. Validity was evaluated by correlation and regression analyses of the values measured by the continuous wave Doppler method and those recorded by the standard pulsed Doppler method. In addition, we also studied the effect on left ventricular dilatation, which may account for a difference between the values obtained by the pulsed Doppler and continuous wave Doppler methods. Our results showed a good correlation between the TEI indices measured by the continuous wave Doppler method and those recorded by the pulsed Doppler method. However, a significant constant systematic error was observed, and the value obtained by the continuous wave Doppler method was larger. Therefore, while it is possible to measure TEI index using the continuous wave Doppler method, evaluation criteria different from those adopted in the pulsed Doppler method may have to be used. Left ventricular dilatation has little influence on the difference between the two methods. PMID- 12373819 TI - [Study on CH50 levels in factor D-depleted serum]. AB - It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2 methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay. PMID- 12373821 TI - [Studies on the gonadal function in patients with anorexia nervosa using a highly sensitive radioimmunoassay kit for estradiol]. AB - We measured serum estradiol (E2) using a highly sensitive radioimmunoassay kit in patients with anorexia nervosa (AN). It is possible to determine the ovarian function with hypogonadism in patients with AN whose levels of E2 were below 10 pg/ml. In patients with extremely low levels of BMI (less than 15 kg/m2), basal levels of E2, LH, FSH and IGF-I increased significantly with gain of body weight. Recovery of the hypothalamic-pituitary-ovarian function in AN patients were correlated with weight gain and nutritional status. The levels of IGF-I and E2 raised in advance of gonadotropins. Using highly sensitive assay of E2, we recognized the clinical usefulness for evaluation of the ovarian function in patients with AN in the course of treatment. PMID- 12373822 TI - [Investigation of practical application of fluorescence in situ hybridization (FISH) analysis using microwave irradiation in formalin-fixed, paraffin-embedded tissue sections]. AB - We investigated fluorescence in situ hybridization (FISH) analysis using microwave irradiation in formalin-fixed, paraffin-embedded tissue sections of breast fibroadenoma. Higher percentage of cells with 2 signal copies of chromosome 3 centromere could be obtained in the condition of 5 microns thick sections, when we counted cells of more than 4 microns of nuclei in thickness. This method showed about the same results as FISH using cells separated from the same tissues. Percentage of cells with 2 signal copies of chromosome 17 centromere in 14 cases was 80.6 +/- 4.0% (Mean +/- S.D.). This method is expected in the application of the prognosis estimation of the breast cancer. PMID- 12373823 TI - [Progress in the field of neurology in Japan]. PMID- 12373824 TI - [Dawn of neurology in Japan]. PMID- 12373825 TI - [History of neurology in Japan and France]. PMID- 12373826 TI - [History of neurology in Japan and United States]. PMID- 12373827 TI - [Progress in the field of neurology in the last 100 years: Outbreak of beriberi and neurology]. PMID- 12373828 TI - [Progress in the field of neurology in the last 100 years--from the discovery of serum CPK to the discovery of localization of dystrophin]. PMID- 12373829 TI - [Progress in the field of neurology in the last 100 years: Triplet repeat diseases]. PMID- 12373830 TI - [Progress in the field of neurology in the last 100 years: Diagnostic imagings of brain]. PMID- 12373831 TI - [Progress in the field of neurology in the last 100 years: Electrophysiological diagnosis]. PMID- 12373832 TI - [History of neuropsychology in Japan]. PMID- 12373833 TI - [History of neuropathology in Japan]. PMID- 12373834 TI - [Progress in the field of neurology in the last 100 years: Therapeutics for neurological diseases]. PMID- 12373835 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Spontaneous occlusion of the circle of Willis(Moyamoya disease)]. PMID- 12373836 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Dentatorubral-pallidoluysian atrophy]. PMID- 12373837 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: SMON]. PMID- 12373838 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Minamata disease]. PMID- 12373839 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: HTLV-I-associated myelopathy (HAM)]. PMID- 12373840 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Hirayama's disease]. PMID- 12373841 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Crow-Fukase (POEMS)]. PMID- 12373842 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Congenital progressive muscular dystrophy]. PMID- 12373843 TI - [Contribution of Japanese researchers to progress in the field of neurology in the last 100 years: Satoyoshi syndrome]. PMID- 12373844 TI - [Progress in the field of neurology in the last 100 years: History of research in stroke]. PMID- 12373845 TI - [Progress in the field of neurology in the last 100 years: History of research in multiple sclerosis]. PMID- 12373846 TI - [Progress in diagnosis and therapy for Parkinson's disease]. PMID- 12373847 TI - [Progress in the field of neurology in the last 100 years: History of research in amyotrophic lateral sclerosis]. PMID- 12373849 TI - [Progress in the field of neurology in the last 100 years: History of research in Guillain-Barre syndrome]. PMID- 12373848 TI - [Progress in the field of neurology in the last 100 years: Myasthenia gravis]. PMID- 12373850 TI - [Progress in the field of neurology in the last 100 years: History of research in Alzheimer's disease]. PMID- 12373851 TI - [Progress in the field of neurology in the last 100 years: Migraine and tension type headache]. PMID- 12373852 TI - [Retrospective view of neurology in the 20th century and prospects for further progress in the 21st century(discussion)]. PMID- 12373854 TI - [Clinical guideline review: Headache]. PMID- 12373853 TI - [Clinical guideline review: Parkinson's disease]. PMID- 12373855 TI - [Clinical guideline review: Stroke]. PMID- 12373856 TI - [Clinical guideline review: Guillain-barre syndrome and chronic inflammatory demyelinating polyneuropathy]. PMID- 12373858 TI - [Behcet's disease with huge supratentorial white matter lesions in brain MRI]. PMID- 12373857 TI - [Myeloid/natural killer cell precursor acute leukemia initiated with pleural effusion]. PMID- 12373859 TI - [A fulminant case of autoimmune hepatitis, who received living-related liver transplantation]. PMID- 12373860 TI - [IgA nephropathy with malignant hypertension]. PMID- 12373861 TI - [Adult T-cell leukemia with pleural effusion and infiltration in bilateral anterior chambers]. PMID- 12373862 TI - [Paradoxical embolism in a case with Graves' ophthalmopathy during steroid therapy]. PMID- 12373863 TI - [The role of lipid metabolism in Alzheimer's disease]. AB - Lipid metabolism in the central nervous system has been focused as an important factor of Alzheimer's disease, since the apolipoprotein E gene was discovered as a genetic risk for the disease. Lipid metabolism in the brain, showing relatively closed environment, necessitates lipid reutilization. Cerebrospinal fluid contains only high-density lipoproteins composed of apoE and apoJ secreted from astrocytes and of apoA-I and apoA-II transported via the blood brain barrier. These apolipoproteins can bind to beta amyloid and possibly relate to its clearance. The aggregation of phosphorylated tau, found in neurofibrillary tangles in Alzheimer's brain, is also found in the brain with Niemann-Pick disease, suggesting that the impairment of lipid transport in neuronal cells participates in Alzheimer's disease. Mitochondrial function, lipid production, and acetylcholine production are closely related, and these alterations could be involved in cholinergic dysfunction in Alzheimer's disease. The regulation of lipid metabolism in and outside the brain could be a therapeutic and preventive target for Alzheimer's disease. PMID- 12373864 TI - [The neurobiological approaches to obsessive-compulsive disorder]. AB - The lifetime prevalence rate of obsessive-compulsive disorder (OCD) is more than 2 percent of the population. Its contemporary pathophysiological models have been explored. As serotonin reuptake inhibitors and cognitive behavior therapy are both considered first-line treatments of OCD, the treatment interventions provide us with clues. In this review, the authors summarized that genetics, neuropathology in the cortico-striatal-thalamic-cortical (CTSC) circuits, the association between OCD and Tourette's syndrome, the possibility of autoimmune mediated pathophysiology containing PANDAS, serologic surveys of patients, and animal models including transgenic mice. Further research, genetic, neuroimmunological, and neuroimaging works may ultimately be useful in developing new treatments of OCD. PMID- 12373865 TI - [Modifications of several pharmacological actions by diabetes: effects on the opioid receptor agonist and benzodiazepines]. AB - Diabetic neuropathy is a most-convoluted complication. Diabetic gastropathy, ulcers, diarrhea, and bladder dysfunction are the major peripheral neuropathies. Peripheral neuropathies have been the primary neuroscience focus of diabetes research. In contrast to the periphery, the brain is not usually thought to be a target of chronic diabetic complications. However, the impact of diabetes mellitus on the central nervous system has recently gained attention. It is well known that diabetes or hyperglycemia influences the sensitivity of laboratory animals to various pharmacological agents. An increased sensitivity of hyperglycemic or diabetic animals to barbiturates and a decreased sensitivity of D-amphetamine, p-chloroamphetamine, and carbon tetrachloride have been demonstrated. Furthermore, it was reported that mice and rats with streptozotocin induced diabetes and spontaneously diabetic mice are significantly less sensitive than non-diabetic mice to the antinociceptive effect of morphine. However, little information is available regarding the mechanism responsible for these changes. It is well established that anxiety and depression are common in patients with diabetes. Moreover, diabetic animals showed significantly more anxiogenic activity than non-diabetic animals did. However, the mechanisms through which diabetes may contribute to the development of, or be a risk factor for, psychiatric disorders are not clear. We provide an overview of our current understanding of the effects of streptozotocin-induced diabetes on the opioid receptor and the benzodiazepine receptor. PMID- 12373866 TI - [Human striatal D-neurons and their significance]. AB - It has recently been reported that the human striatum, especially its ventral part, the nucleus accumbens, contains numerous neurons immunoreactive for aromatic L-amino acid decarboxylase (AADC; the second-step monoamine synthesizing enzyme), but not for tyrosine hydroxylase (TH; the first-step catecholamine synthesizing enzyme) or tryptophan hydroxylase (TPH; the first-step serotonin synthesizing enzyme). These AADC (+)/TH(-)/TPH(-) neurons are named D-neurons. AADC is also the rate-limiting synthesizing enzyme of phenylethylamine (PEA). Although the functions of striatal D-neurons are yet unclear, their functions were discussed in the present review based on recent findings in the literature. D-neurons may participate in the manifestation of efficacy of pharmacotherapy for Parkinson's disease by uptaking monoamine precursors, including L-dopa or droxidopa (L-threo-DOPS), and by converting them to dopamine (DA) or noradrenaline (NA), respectively. Because the nucleus accumbens is one of the brain regions involved in the pathogenesis of schizophrenia and drug dependence, D-neurons might be related to the etiology of these mental disorders. It has also been suggested that striatal D-neurons are the pluripotential cells that have compensating functions against aging or degeneration. Further studies should be conducted to elucidate the functions of this unique cell group in the human striatum. PMID- 12373867 TI - [Future progress in the study of allergy and collagen disease]. PMID- 12373868 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Steroidal anti-inflammatory agents]. PMID- 12373869 TI - [Progress in the study of allergy and collagen disease in the last 100 years: History of antinuclear antibody]. PMID- 12373870 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Rheumatoid factor]. PMID- 12373871 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Concept of allergy]. PMID- 12373872 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Concept of anaphylaxis]. PMID- 12373873 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Concept of collagen disease]. PMID- 12373874 TI - [Progress in the study of allergy and collagen disease in the last 100 years: The LE cell phenomenon]. PMID- 12373875 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Immunoglobulin E]. PMID- 12373876 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Complement]. PMID- 12373877 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Major histocompatibility complex]. PMID- 12373878 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: Fas and autoimmune diseases]. PMID- 12373879 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: IL-6 and autoimmune diseases]. PMID- 12373880 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: Kimura disease]. PMID- 12373881 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: IgE and Allergy]. PMID- 12373882 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: Takayasu's arteritis]. PMID- 12373883 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: Cytokines and cytokine receptors]. PMID- 12373884 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: Pollinosis]. PMID- 12373885 TI - [Contribution of Japanese researchers to progress in the study of allergy and collagen disease in the last 100 years: Antiphospholipid syndrome]. PMID- 12373887 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Sjogren syndrome]. PMID- 12373886 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Behcet disease]. PMID- 12373888 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Systemic lupus erythematosus]. PMID- 12373889 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Vasculitis syndrome--special reference to periarteritis nodosa]. PMID- 12373890 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Gout]. PMID- 12373891 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Rheumatoid arthritis]. PMID- 12373892 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Rheumatic fever]. PMID- 12373893 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Mixed connective tissue disease]. PMID- 12373894 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Occupational allergy]. PMID- 12373895 TI - [Progress in the study of allergy and collagen disease in the last 100 years: Drug allergy]. PMID- 12373896 TI - [Retrospective thoughts on studies of allergy and collagen disease in the 20th Century and prospects for the 21st Century (discussion)]. PMID- 12373897 TI - [Clinical guideline review: Bronchial asthma]. PMID- 12373898 TI - [Clinical guideline review: Rheumatoid arthritis (ACR core set)]. PMID- 12373899 TI - [Clinical guideline review: Perennial rhinitis and pollinosis]. PMID- 12373900 TI - [Chronic idiopathic ataxic neuropathy improved by high-dose intravenous immunoglobulin therapy]. PMID- 12373901 TI - [Acute neuropathy associated with vitamin B12, B6, and folate deficiency after total gastrectomy]. PMID- 12373902 TI - [Case of familial hypoalphalipoproteinemia, type 2 diabetes mellitus and markedly advanced atherosclerosis with ABCAlexon 4 minus transcript in macrophages]. PMID- 12373903 TI - [Case of pulmonary Langerhans-cell histiocytosis]. PMID- 12373904 TI - [Case of Takayasu's arteritis (type V) together with Sjogrens's syndrome and Basedow's disease]. PMID- 12373906 TI - Knowledge is power, and peace of mind. PMID- 12373905 TI - [Chronic myelogenous leukemia and myelofibrosis occurring in the same family at about the same time]. PMID- 12373907 TI - Discipline decisions. PMID- 12373908 TI - Nursing in Africa. PMID- 12373909 TI - Specialty certification exam preparation. PMID- 12373910 TI - Benefits of telehealth. PMID- 12373911 TI - Update on nurse practitioners. PMID- 12373912 TI - Nebraska nurses victorious! NNA staves off proposed cuts to nursing programs! PMID- 12373913 TI - "I want to be a nurse": how the nursing shortage affects Nebraska nurses. PMID- 12373916 TI - The challenge of correctional nursing. PMID- 12373917 TI - Dementia: common types, interventions and advocacy. PMID- 12373918 TI - [What can we expect of raloxifene in the treatment of postmenopausal osteoporosis -views of a gynecologist]. AB - OBJECTIVE: Evaluation of positive properties and side effects of raloxifene treatment with respect to its potential use as agent to improve women's health and quality of life in postmenopausal years. DESIGN: A review article. SETTING: Obstetrics and Gynaecology Department, Charles University 2nd Medical Faculty and Teaching Hospital Motol, Prague. SUBJECT: Estrogen use may protect against osteoporosis and cardiovascular disease, but may increase the risk of breast cancer in long-term treated women and also may increase the risk of irregular uterine bleeding (in combination with gestagen in non-hysterectomized women) in perimenopause and postmenopause. Drugs with tissue-specific estrogenic effects are termed selective estrogen receptor modulators (SERM). Tamoxifen is the first SERM successfully used in the prevention and treatment of breast cancer. Another SERM raloxifene is widely used in the prevention and treatment of postmenopausal osteoporosis, especially in women without climacteric complaints. Therapy with raloxifene increases bone mineral density, lowers serum concentrations of total and low-density lipoprotein cholesterol, and does not stimulate endometrium and breast. Evaluation of another potential positive effects (reducing size of uterine leiomyomas, etc.) warrants further investigation. CONCLUSION: Raloxifene can be used in postmenopausal women free of climacteric symptoms for the prevention and treatment of postmenopausal osteoporosis with no increased risk of thrombosis and with the advantage of positive side effects during the treatment. PMID- 12373919 TI - [Advanced oxidation protein products in pregnancy]. AB - OBJECTIVE: Pregnancy and mainly its complications are associated with increased oxidative stress. Advanced oxidation protein products (AOPP) can serve as one of its markers. SETTING: First Institute of Medical Chemistry and Biochemistry and Institute for Clinical Biochemistry, First Medical Faculty, Charles University; Institute for Care of Mother and Child, Prague. METHODS: Together with parameters of prenatal screening, AOPP were measured in the serum of 23 pregnant women in the 2nd trimester of pregnancy. A group of healthy blood donors--women and men was used for comparison. AOPP were determined spectrophotometrically according to Witko-Sarsat (absorbance at 340 nm) and are expressed in chloramin units (mumol/l). RESULTS: Serum AOPP concentrations in pregnant women are significantly higher in comparison with blood donors--women (85.90 +/- 18.70 mumol/l vs 57.34 +/- 16.31 mumol/l, P < 0.0001) but there is no statistically significant difference between pregnant women and blood donors--men (85.90 +/- 18.70 mumol/l vs 78.60 +/- 44.01 mumol/l). AOPP level does not correlate either with the age of pregnant women or with the parameters of prenatal screening (human chorionic gonadotrophin--HCG, alpha-1-fetoprotein--AFP and trophoblast-specific--beta-1 glycoproteion--SP1). CONCLUSION: AOPP as a marker of oxidative stress is increased in the serum of pregnant women in comparison with women--blood donors but is similar as in men--blood donors which supports the hypothesis of hormonal influence. Nevertheless, AOPP do not correlate with the parameters of prenatal screening (HCG, AFP and SP1). PMID- 12373920 TI - [Analysis of the zona pellucida in human oocytes and embryos in an assisted reproduction program]. AB - OBJECTIVE: Our aim was to design simple method for quantitative evaluation of oocyte and embryo morphology in in vitro fertilization (IVF) programme efforting of embryo implantation prediction to be able to set the optimal number of embryos for transfer for given specific case. This study deals with zona pellucida (ZP)- the one interesting and for oocyte unique structure. The work is the part of comprehensive research of oocyte, zygote and embryo morphology. DESIGN: Prospective study. SETTING: Center of Assisted Reproduction, Department of Gynaecology and Obstetrics, Medical Faculty of Charles University, Pilsen. METHODS: The study includes analysis of 66 microphotography pictures of oocytes, zygotes and embryos of patients who were treated by IVF on our department between September and December 2000. The microphotographs were taken by digital microphotographic system. Analysis of zona pellucida parameters was conducted by image analysis software called AnalySIS, Soft Imaging System GmbH. RESULTS: It was found out the average width of the zona pellucida 18.4 microns +/- 2.1 microns, the minimum width was 12.3 microns a maximum 23.1 microns. CONCLUSION: There was found significant narrower zona pellucida in women who conceived in this IVF cycle then in the women who did not. PMID- 12373921 TI - [Incidence of congenital defects in selected areas and countries 1998-1998]. AB - OBJECTIVE: Presentation of the incidence of selected types of birth defects in registers of birth defects abroad and in comparison with data from the Czech Republic in 1988-1998. DESIGN: Retrospective epidemiological analysis of the incidence of birth defects. SETTING: Institute for the Care of Mother and Child, Prague. METHODS: Analysis of the incidence of 15 types of selected birth defects in liveborn and stillborn infants in 1988-1998 according to published data and publications of the International Clearing-house for Birth Defects Monitoring Systems (ICBDMS) and European Registration of Congenital Anomalies (EUROCAT). Comparison of the incidence of 15 selected types of congenital defects from 24 registers worldwide incl. the Czech Republic. RESULTS: In the resultant incidences of selected types of congenital defects during the presented period participate geographical and ethnic differences as well as a series of other factors, in particular primary and secondary prevention of birth defects. The influence of other biosocial factors is assumed. CONCLUSION: Long-term well functioning registration of birth defects is an essential prerequisite not only for national analyses but also for possible more detailed collaboration and analyses on an international scale. PMID- 12373922 TI - [Occurrence and characteristics of staphylococcal enterotoxin antibodies in gynecologic patients]. AB - OBJECTIVE: To control the occurrence of the antibodies against Staphylococcal type A and type B enterotoxin in gynaecological patients and in selected patients to determine the thermodynamic parameters of antibodies against Staphylococcal enterotoxins in their blood samples. DESIGN: Retrospective clinical study. SETTING: 2nd Department of Obstetrics and Gynaecology, P. J. Safarik University Kosice, Slovak Republic; Research Institute of Veterinary Medicine, Kosice, Slovak Republic; Department of Microbiology and Immunology, University of Veterinary Medicine, Kosice, Slovak Republic; Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, Madison, USA. METHODS: The occurrence of antibodies against Staphylococcal type A and type B enterotoxin was determined in 68 patients hospitalized in Department of Obstetrics and Gynecology in Kosice. RESULTS: The occurrence of antibodies against Staphylococcal enterotoxins was determined by radioimmunoassay (RIA) in blood samples of 45 (66%) patients. The antibodies against Staphylococcal type A enterotoxin were determined in 36 (53%) patients and the antibodies against Staphylococcal type B enterotoxin were determined in 9 (13%) patients. The antibodies against both type A and type B enterotoxins were determined simultaneously in blood samples of 10% of all patients. The thermodynamic parameters of the antibodies were determined in 5 patients with positive serum findings. CONCLUSION: With regard to the existence of heterogeneous clinical findings in large amount of patients with antibodies against Staphylococcal enterotoxins, the next study of Staphylococcal enterotoxins role in pathogenesis of wide spectrum of diseases is necessary. PMID- 12373923 TI - [Anthropologic study of women with hyperphenylalaninemia]. AB - OBJECTIVE: Anthropometric investigation in women with hyperphenylalaninemia. DESIGN: Retrospective clinical study. SETTING: Department of Obstetrics and Gynecology and Institute of Inherited Metabolic Disorders of General Faculty Hospital and 1st Medical Faculty of Charles University in Prague, Department of clinical Biochemistry, Hematology and Immunology of Hospital Na Homolce in Prague. METHODS: 44 anthropometric and 12 anthroposcopic parameters were studied in a group of 16 women with reproduction normality. RESULTS: Smaller body heights, pathological body posture and dysplastic findings were found. CONCLUSION: Registered differences and dysplastic findings are motivating for studies in a larger group of women with hyperphenylalaninemia. PMID- 12373924 TI - [Recommendations for care of the breast]. PMID- 12373925 TI - [The Web of Science--publications of Czech authors in periodicals with impact factors in gynecology and obstetrics in 2001]. PMID- 12373926 TI - [Impaired development of uteroplacental circulation]. AB - OBJECTIVE: To sum up the knowledge of uteroplacental circulation, their dysfunction and etiology and pathogenesis of preeclampsia. TYPE OF STUDY: Review. SETTING: Department of Obstetrics and Gynaecology, 1st Faculty of Medicine, Charles University, Prague, Institut of Pathological Physiology, 1st Faculty of Medicine, Charles University, Prague. SUBJECT OF STUDY: A summary of what is known about development of uteroplacental circulation predispose women to the development of preeclampsia and IUGR but the etiology of preeclampsia is still unknown. PMID- 12373927 TI - [Transplantation of the human uterus--a new era?]. PMID- 12373928 TI - DM effort produces dramatic gains in HIV/AIDS care. PMID- 12373929 TI - Alternative approaches to caring for complex patients show promise. AB - Providers have long complained that traditional managed care systems fail to the meet the needs of highly complex cases; the typical time constraints, reimbursement rates, and utilization management patterns are simply inadequate, in many cases, to keep such persons stabilized and out of the hospital. But now there is proof that there may, indeed, be a better way to manage these cases so that patients will be more satisfied, costs will be reduced, and outcomes improved. Sound too good to be true? Check out the "plan within a plan" concept implemented at Massachusetts-based Neighborhood Health Plan. PMID- 12373930 TI - Health plans jump on the generics bandwagon as more drugs become available. AB - More and more generics are becoming available in the blockbuster classes commonly prescribed within many DM programs, and some health plans are already saving themselves--and their members--millions by aggressively encouraging the use of generics as opposed to the much more expensive branded alternatives. Keep in mind, however, that while the approach sounds simple enough, getting providers and members to rely more often on generics is a tall order that will require action on several fronts. PMID- 12373931 TI - New smoking cessation effort offers multiple delivery models. AB - Providers know that doing everything they can to help their patients quit smoking is the right thing to do. But health care organizations often have a tough time justifying cessation programs from the standpoint of ROI. However, one way to make the financial equation fit is by offering the program to chronically ill enrollees in DM programs. In this population, ROI from such an effort will come quickly, according to experts at National Jewish Medical and Research Center. PMID- 12373932 TI - Facility profile. Modern showplace evolves from two old hospitals. River Region Medical Center, Vicksburg, Miss. PMID- 12373933 TI - Weighing the options. Small details can add up to big savings when managing construction costs. PMID- 12373934 TI - Getting personal. Biometric security devices gain access to health care facilities. PMID- 12373935 TI - It's pharma--bot! Robotics manufacturers aim to revolutionize hospital pharmacies. PMID- 12373936 TI - First of its kind. Certification for health care architects becomes a reality. PMID- 12373937 TI - Haste makes (infectious) waste. Saving money by segregating hospital refuse. PMID- 12373938 TI - Branches of evolution. PMID- 12373939 TI - Hepatitis C: a new pandemic? PMID- 12373940 TI - Early intraoral splinting and loading of one-stage dental implants in the edentulous mandible: literature review and case report. AB - Recent studies of immediately loaded screw-retained dentures, bar overdentures, and single-tooth replacements have demonstrated success rates comparable to similar restorations using a two-stage procedure. An international study of immediately loaded bar overdentures on a novel single-stage implant is currently being conducted in the United States and Europe. This article presents a case report that illustrates the intraoral construction of an overdenture bar pattern at the time of implant placement, followed by soldering and immediate loading. The elimination of transfer procedures may significantly reduce treatment time and enhance the passive fit of the bar. PMID- 12373941 TI - Clinical procedure for producing aesthetic stratified composite resin restorations. PMID- 12373942 TI - 2002 dental delights: the aesthetic legends. PMID- 12373943 TI - Aesthetic design preservation in multidisciplinary therapy: philosophy and clinical execution. AB - Complex perio-prosthetic cases that require multidisciplinary therapy often result in compromised aesthetics. Traditional treatment planning philosophies, as well as existing interdisciplinary relational patterns, do not promote the achievement of predictable aesthetic results. Implementation of a restorative driven approach requires the development of an aesthetic blueprint that will serve as a guide through treatment. This article illustrates the clinical techniques and sequence for an outcome-based protocol that enhances therapeutic cohesiveness and ensures the sequential transfer of design objectives for the preservation of aesthetics in multidisciplinary therapy. PMID- 12373944 TI - Applied techniques for predictable suture placement: Part 3. PMID- 12373945 TI - Dental lasers: Part 5. The significance of education. PMID- 12373946 TI - The proximal precinct in direct posterior composite restorations: interproximal integrity. AB - Resourceful efforts of researchers and clinicians have made for an ever-expanding restorative repertoire. The search for improved operative techniques must continue as the objectives of an ideal direct restoration reach beyond natural aesthetics and include pulpal health considerations, occlusal stability, anatomical restitution, marginal perfection, and interproximal integrity. This article focuses on the proximal precinct and provides solutions to the challenge of restoring proximal physiological form to contiguous surfaces when utilizing the direct composite resin technique. PMID- 12373947 TI - The dental management pyramid: Part 3--Case presentation. PMID- 12373948 TI - Financial and clinical professionals: a clash of values. PMID- 12373949 TI - Face sheet data exchanges. PMID- 12373950 TI - Putting greater "value" in your organization's PFS operations. PMID- 12373951 TI - Data trends. Ultrasound transducers: replace or repair? PMID- 12373952 TI - It's the economy, stupid--not health care! PMID- 12373953 TI - Charting the course to economic vitality. PMID- 12373954 TI - Sarbanes-Oxley raises red flag for not-for-profits. AB - In the near future, not-for-profits likely will feel the effects of these Sarbanes-Oxley directives: Establish audit committees with independent membership (precluding senior managers from being members) and make corresponding changes to corporate bylaws; Meet a higher standard for financial reporting, including increased disclosure and system certification representations as part of annual audits; Adopt a code of ethics for senior financial officers; Avoid senior executive compensation packages involving personal loans from the corporation; Ensure that board members are appropriately qualified and free of conflicts of interest.l PMID- 12373955 TI - Who is responsible for business failures? AB - Two high-profile business failures should be a wake-up call for hospital leaders. Management and boards cannot rely on unaudited financial statements as the principal indicator of financial health. Boards must ensure diligent analysis of key financial measures. Board members need better education about healthcare finance. Hospitals are not immune to financial collapse. PMID- 12373956 TI - Identifying and validating managed care data. AB - In a managed care organization, data can be the key to facilitating high-quality care and to managing patient care delivery systems effectively, in addition to monitoring costs. Reviewing electronic data requests before contacting data producers, asking the right questions about data, and knowing how to identify good data can help financial managers use data effectively to provide information. In the managed care environment, information is only as good as the steps taken to obtain and validate the data. PMID- 12373957 TI - Strategic planning: looking beyond the next move. AB - Effective strategic planning can help healthcare organizations better plan for the future and understand when change is needed. Strategic planning is a dynamic process. Unified communication throughout the organization is necessary. Progress should be tracked with quantifiable goals. PMID- 12373958 TI - Are you ready to invest in business development? AB - A hospital's strategy to increase market share should focus on physicians and the hospital's community. Organization managers should actively seek to enhance relations with physicians. The hospital should seek opportunities to collaborate with physicians. Promotional budgets should focus on informing consumers about service areas that interest them most, such as emergency and obstetrics services. A sales strategy should focus on acquainting community physicians with the hospital's specialists. PMID- 12373959 TI - The return of the heart hospital. A hospital that specializes in providing cardiovascular services can meet community needs but will compete with existing community hospitals for market share. AB - A hospital that provides cardiovascular services and embraces a heart-hospital brand and strategy can achieve competitive advantage. Providers that want to compete aggressively for cardiovascular services are developing a specialty-based carve-out strategy. A heart-hospital initiative can cannibalize revenues from a hospital's other programs and services. A successful heart-hospital strategy requires physician buy-in. A heart hospital needs a brand that customers will value. PMID- 12373960 TI - Journey to the frozen zone: a health plan's recovery after 9/11. PMID- 12373961 TI - Optimizing revenue by reducing medical necessity claims denials. AB - A proactive approach will help avoid Medicare claims denials related to medical necessity requirements. Providers should familiarize themselves with applicable local medical review policies. A self-audit should target areas where the most money is lost to medical necessity denials. Educational tools and a letter that highlights the provider's approach to medical necessity can encourage physicians to comply with Medicare requirements when providing a diagnosis. PMID- 12373962 TI - Profit opportunities still exist....in the operating room. AB - This piece is the first in a new series focused on improving profitability in the OR. Subsequent articles will present case studies, tools and best practices to help healthcare organizations improve resource management and increase profitability. The healthcare industry is at a financial impasse. While costs have increased, reimbursement has decreased. And just when the aging baby boomers are at the cusp of needing more care, the current nursing population is retiring, with too few new nurses to fill their shoes. Furthermore, healthcare organizations are experiencing the phenomenon of "profitless growth," a situation in which beds are full and resources appear to be fully utilized, yet profits are stagnant. Where can organizations focus to help close these gaps? The OR remains the single biggest opportunity. This project is a collaboration between McKesson Information Solutions and HFMA. The series will uncover hidden profit opportunities in the OR by focusing on three key areas: streamlining workflow, improving resource standardization and utilization, and integrating information and decision-making. PMID- 12373963 TI - The next little thing. AB - Nanomedicine is a blockbuster, with huge implications for health care (not to mention human life). Have you noticed how far we've come? PMID- 12373964 TI - Staying ahead of the future. AB - Most health care leaders fail to comprehend the newest technology and how it may change their systems. But a few leaders are ahead of the game. PMID- 12373965 TI - 2002 AHA environmental assessment. PMID- 12373966 TI - Take the time. PMID- 12373967 TI - The short life of a medical device. AB - One hospital studied the life cycle of emerging cardiovascular technologies so it could form better investment and strategy decisions. PMID- 12373968 TI - Most wired innovator awards. AB - Three winners and three finalists have been applying creativity and online technology to solve some of health care's thorniest business problems. PMID- 12373969 TI - Flying lessons. PMID- 12373970 TI - C.A.M. Complementary & Alternative Medicine. More CAM available to patients. PMID- 12373971 TI - Wary of choices. PMID- 12373972 TI - Separate and unequal. PMID- 12373974 TI - It's a waiting game. PMID- 12373973 TI - Promoting continence. PMID- 12373976 TI - First of many. PMID- 12373975 TI - Sharing the burden. PMID- 12373977 TI - Keep the faith. PMID- 12373978 TI - Modern, but not matrons. PMID- 12373979 TI - A team effort. PMID- 12373980 TI - It may take time, but there are ways to include people with dementia in research studies. PMID- 12373981 TI - Safety in numbers? PMID- 12373982 TI - 'The journey from cradle to grave can be pitifully short and desperate'. PMID- 12373983 TI - Life without the cover. PMID- 12373984 TI - Where did people's trust go? PMID- 12373985 TI - Nurse practitioner training in breast examination. AB - BACKGROUND: To date, there have been no published guidelines, either locally or nationally, to advise nurse practitioners on training and assessment in breast and axillary examination. This study is a prospective audit of the clinical competence of a nurse practitioner in breast and axillary clinical examination, following an 18-month period of clinical training and supervision by two consultant breast surgeons. CONCLUSION: The results of the audit show that the nurse achieved a high level of concordance with the findings of consultant breast surgeons. This training and audit process could be incorporated into the training and assessment of future nurse practitioners in this specialist area. PMID- 12373986 TI - Nursing knowledge: defining new boundaries. AB - Nursing knowledge covers those aspects of knowledge that are relevant to nursing. The types of knowledge in nursing are many and varied, the generation of knowledge therefore becomes complex. Nurses need to go beyond traditional ways of understanding nursing to redefine nursing knowledge. This could determine the nature of the profession in the 21st century. Nursing must explore new ways of thinking about and explaining the profession--there will always be new directions to take and new avenues to explore. PMID- 12373987 TI - Nutrition support in palliative care. AB - Nutrition is an important aspect of caring for patients with life-threatening illness. Good nutrition support is essential, not only for meeting the body's physical requirements but also because of associated social, cultural and psychological benefits for patients. The author uses examples from cancer and motor neurone disease to illustrate the nutrition needs, legal and ethical issues and specific symptoms that affect dietary intake in patients receiving palliative care. PMID- 12373988 TI - Who has the most contact with patients? PMID- 12373989 TI - What to do when an adolescent self harms. PMID- 12373990 TI - Paracetamol overdose. PMID- 12373991 TI - Self harm on increase. PMID- 12373992 TI - Using an evidence-based approach to thrombolysis. PMID- 12373993 TI - Diagnosing radiological abnormalities. PMID- 12373994 TI - Managing emergency pressures. PMID- 12373995 TI - ED patient's suicide is wake-up call: are you putting psychiatric patients at risk? AB - Psychiatric patients require expedited care, quiet areas, and other measures to reduce risks. Restraint use can be reduced by having staff attend a training course. Remember that a psychiatric patient's triage status may change, so monitor for worsening agitation or anxiety. Avoid placing psychiatric patients in a remote area, since they need continuous observation. PMID- 12373996 TI - Florida ED revamps its decontamination plan. AB - Your decontamination plans should address both small-scale events and treatment of hundreds or more patients. To protect team members, use rescue signals and medical screenings before staff enter the decontamination unit. Use covered walkways, overhangs, or covered parking garages to decontaminate large numbers of patients. Choose a site at an appropriate distance from the hospital to prevent contaminated patients or staff from entering. PMID- 12373997 TI - Use score card to boost quality. AB - Keeping a score card can identify problem areas and track improvements. When specific goals are reached, staff are given rewards such as thank-you letters, tokens, or pizza parties. Staff are kept informed about the results of the score card through bulletin board postings, staff meetings, and the hospital Intranet. Data are collected with manual entry by nursing staff, chart review by performance improvement, and a computerized program. PMID- 12374001 TI - How many words are enough? PMID- 12374000 TI - Postscript: follow-up after manuscript submission. AB - Tracking your manuscript is one way to help the review process go smoothly and it keeps you actively involved. Verifying the arrival of your manuscript and politely asking for feedback helps reduce needless frustrations. If a journal encourages you to revise and resubmit, the chances of acceptance and publication improve. PMID- 12374002 TI - Duplicate publication, Part 2: A case analysis. AB - Most authors, reviewers, editors, and scholars agree that it is the specific situation that has to be analyzed to determine if a particular manuscript duplicates a previously published one. Duplicate Publication, Part 1, which was published in the Summer 2002 issue of Nurse Author & Editor, described criteria to identify duplication. This article, Part 2, presents a case analysis on the degree of duplication of articles in a case situation. PMID- 12374003 TI - Past failures prompt drive for innovation to tackle child abuse. PMID- 12374004 TI - Stay, just a little bit longer. PMID- 12374005 TI - Does the NHS make use of too much jargon? PMID- 12374006 TI - The best days of your life. PMID- 12374007 TI - For those who are about to begin.... PMID- 12374008 TI - Fighting fever in Uganda. PMID- 12374009 TI - Helping students to learn in the clinical environment. AB - If students are to achieve a positive learning experience, it is vital that they receive adequate supervision and mentoring. However, while student numbers are increasing, staff numbers have been depleted in many hospitals, as a result of which the students' learning experiences can suffer. The author recognised that both parties need support, and so developed a student learning model, to ensure that learning activities were structured during clinical placements. The model focuses on five key areas: clinical work with a mentor or other nurse; observing practice; researching a relevant subject; the use of a learning pack related to the clinical area of the placement; and visits to departments outside the base placement. The article describes how to use the model and gives feedback on the project. PMID- 12374010 TI - Respecting a patient's care needs after death. AB - The delivery of care to patients who have died is a nursing intervention that most nurses will have to perform at some time. This article aims not only to provide the rationale behind the care given to patients during last offices, but also to explore the professional, legal and ethical considerations that must be addressed within the provision of holistic care. PMID- 12374011 TI - Providing nursing care in a children's hospice. AB - Children who are admitted to hospices need specialist treatment that enables them to enjoy their childhood as much as possible while they receive the care they require. Their parents also have particular needs. During Children's Hospice Week, which started on September 21, the Association of Children's Hospices aims to raise awareness of the work done by children's hospices and the services they provide. PMID- 12374012 TI - The nurse's role in managing psychosis. AB - This article provides a nursing perspective on psychosis. It looks at how psychotic behaviour is often no more than an exaggerated version of normal behaviour and explains that many people can have a psychotic experience, albeit temporarily, in unusual or exceptional circumstances. This article looks at how nurses can help people to make some sense of the phenomenon and begin the process of integrating it into their life experience. The importance of building a therapeutic relationship and maintaining a consistent clinical approach is discussed. Particular attention is paid to the stress-vulnerability model as a means of understanding the experience while looking at potential triggers and the prevention of relapse. PMID- 12374013 TI - Special focus. Pain. Assessment and diagnosis. PMID- 12374014 TI - Day in the life. PMID- 12374016 TI - Protein characterization by two-dimensional gel electrophoresis. PMID- 12374015 TI - The pathogenesis of disease due to nontypeable Haemophilus influenzae. AB - To summarize, the pathogenesis of disease due to nontypeable H. influenzae involves multiple steps and the interplay of a number of bacterial and host factors, as shown in Fig. 1. Following entry into the upper respiratory tract, bacteria encounter the mucociliary escalator. The P2 and P5 outer-membrane proteins and probably other factors promote bacterial binding to mucus, and elaboration of LOS causes damage to ciliated cells and impairs mucociliary function. Subsequently, several adhesins, including HMW1 and HMW2, pili, Hia, Hap, and others, mediate direct adherence to nonciliated epithelial cells. Cleavage of IgA1, invasion into cells and the subepithelial space, and phase and antigenic variation facilitate evasion of local immune mechanisms. Binding and uptake of iron and heme allow organisms to persist on the respiratory mucosa despite the relative scarcity of these nutrients. In the setting of a viral infection, allergic disease, or exposure to cigarette smoke, bacteria spread from the nasopharynx to other sites within the respiratory tract and produce symptomatic disease. PMID- 12374017 TI - Monitoring gene expression using DNA arrays. PMID- 12374018 TI - Gene expression technology. PMID- 12374019 TI - The genome sequence of Haemophilus influenzae. PMID- 12374020 TI - Structural profiling of short-chain lipopolysaccharides from Haemophilus influenzae. PMID- 12374021 TI - Mutagenesis of H. influenzae. PMID- 12374022 TI - Transposon Tn10. PMID- 12374023 TI - In vivo expression of bacterial genes during human infections. PMID- 12374024 TI - ELISA. PMID- 12374025 TI - Opsonophagocytosis assay using flow-cytometry. PMID- 12374026 TI - In vitro models of infection I--human respiratory tissue organ culture. PMID- 12374027 TI - The pathogenesis of disease due to type b Haemophilus influenzae. PMID- 12374028 TI - In vitro models of infection II--human umbilical vein endothelial cells (HUVECs) system. PMID- 12374029 TI - Animal models. PMID- 12374030 TI - General methods for culturing Haemophilus influenzae. PMID- 12374031 TI - Transformation of Haemophilus influenzae. PMID- 12374032 TI - Diagnosis of infection. PMID- 12374033 TI - Characterization of plasmids. PMID- 12374034 TI - JCO has a 35th birthday. PMID- 12374035 TI - Modified removable transpalatal bar for rapid uprighting of impacted second molars. PMID- 12374036 TI - Accurate band positioning in impressions. PMID- 12374037 TI - The psychology of influence in orthodontics. PMID- 12374038 TI - Relief of soft-tissue irritation from orthodontic appliances. PMID- 12374039 TI - Extraction decision-making wigglegram. PMID- 12374040 TI - Superelastic nickel titanium spring clips for the SPEED appliance. PMID- 12374041 TI - A new wraparound retainer design. PMID- 12374042 TI - [Allergens and risk factors in pediatric patients with allergic seasonal conjunctivitis]. AB - BACKGROUND: The seasonal allergic conjunctivitis is an inflammatory disease of the ocular surface that affects mainly children, with predominance of male sex. It is an immunological disease with a typical reaction of hypersensitivity type 1 (IgE), resulted from several biological reactions (antigen-antibody) and it's associated to several risk factors. OBJECTIVE: To determine the more frequently identified allergens and the associated risk factors to the seasonal allergic conjunctivitis in children living in Mexico City. MATERIAL AND METHODS: Ophthalmologic clinical study done to 50 patients with diagnosis of seasonal allergic conjunctivitis, during the months of March to October, 2001, at the Ophthalmology Department of the National Pediatric Institute. A control group was integrated by 50 patients of the consultation of ophthalmology with non-allergic ocular pathology, and percutaneous skin tests were made. A direct interrogation was applied to both groups to investigate associated risk factors of atopy. RESULTS: The associated risk factors to the development of allergic conjunctivitis are: family atopic background, negative antecedent of breast feeding, asthma o rhinitis (statistically significant). The most frequently identified allergens were Dermatophagoides pteronissinus, Dermatophagoides farinae, Lolium perenne and Atriplex bacteosa, of the group of dust mites and pollen, respectively. CONCLUSION: It is important to see our patients in an integral way. In the case of patients with seasonal allergic conjunctivitis, it should not be forgotten to make an interrogation of the factors associated to atopy and, if it is possible, to inform to the patient and their relatives about these, in order to prevent them. PMID- 12374043 TI - [Knowledge of flowmetry among asthmatic children and adolescents]. AB - BACKGROUND: The correct control of asthmatic children and teenagers substantially improves their quality of life. The use of the peak flowmeter permits to monitor the pulmonary function and to control this disease. OBJECTIVE: To gather information about the use of the peak flowmeter in asthmatic children and teenagers. MATERIAL AND METHODS: It was a descriptive and observational study done in 81 asthmatic children and teenagers who attended a summer camp. They answered a questionnaire in order to know their knowledge about the peak flowmeter's use. RESULTS: The age of the studied group ranked from five to 18 years (54.3% between five to 10 years). Out of the 81 children and teenagers, 64 knew about the peak flow. In spite of the knowledge, only 38 (46.9%) had used it in at least one occasion and 20 (24.7%) in an ambulatory manner (16 used it when they feel bad, and four, every day). Only nine of these 20 children knew the correct way to use it, to interpret the results and what the normal peak flow was. All of these children and teenagers were under medical control. Allergists were attending 66.7%. Independently of the specialty of their doctors, the average of the children that didn't use the peak flowmeter in their control of asthma was always over than 50%. CONCLUSIONS: In spite that all these children and teenagers were on medical care, the knowledge of the peak flowmeter usage was not enough to take advantage. PMID- 12374045 TI - [Apoptosis]. AB - The apoptosis phenomenon was identified approximately 40 years ago. In 1972, Kerr coined the term of apoptosis (programmed cellular death) to indicate that this way of cellular death was related to organic damage; but in contrast to other ways of death characterized by active cellular necrosis, in this, there is very little tissular reaction surrounding apoptotic cells. Apoptosis refers to the morphologic findings characterized by cellular shrink, nuclear condensation, fading of the membrane and fragmentation of this in apoptotic bodies with changes that possibly guide to the phagocytosis of the affected cell. Although there is great advance on phagocytosis mechanisms, signaling roads and pathology findings; a little is known about the molecular ways of apoptosis, intervening a great quantity of genes, surface receptors of T and B cells; ligand/receptor of death systems (particularly CD95), receptor of the tumoural necrosis factor (TNF-R), several interleukins with antiapoptotic activity, (specially IL-2), costimulatory receptors such as CD28 and proteins of the family Bcl-2 also with antiapoptotic activity. However, at this moment, the attention is focused in the apoptosis signaling pathways mediated by caspases, from which, 14 are known. Apoptosis has an important biological role in the development and homeostasis of cellular populations and in the pathogenesis and expression of diseases' processes. An excessive or insufficient apoptosis contributes to the pathogenesis of a wide variety of ischemic, neurodegenerative, and autoimmune diseases and viral infections, besides of participating in the growth and regression of tumoural processes. This article presents a general outline of the different apoptosis signaling pathways, the integration of multiple involved genes and receptors and the participation in several diseases of this programmed cellular death, either in excess or insufficient. PMID- 12374044 TI - [Use of cyclosporin A in patients with severe, active, treatment-resistant rheumatoid arthritis ]. AB - BACKGROUND: Rheumatoid arthritis (RA) is a multisystemic and chronic disease whose etiology still remains to know. RA and their complications implicate a huge cost, calculated in 1% of total national internal product in US. OBJECTIVE: To evaluate the use of cyclosporine A in RA patients refractory to conventional treatment. MATERIAL AND METHODS: Several improvement variables were used, such as Ritchie index, modified Sharp radiological scale, functional status, HAQ index, analog scale of pain, and PCR. 13 female were included with a mean age of 44 years (range 30-50), with a mean of RA evolution of 6 years (range 0.5-22); each patient received an initial dosage of 2.5 mg/day by oral microemulsion of cyclosporine A, with increasing dosage of 0.5 mg/kg/day, adjusting by seric values of creatinine. RESULTS: RA patients were evaluated, initially and after three and six months. Data were analyzed using Mann-Whitney U and Kruskal Wallis tests. In three months evaluation there was an improvement in functional status (p = 0.009). In six months evaluation there was an improvement in painful joints (p = 0.006), functional status (p = 0.007) and general comfort sensation (p = 0.014). CONCLUSION: The cyclosporine use is considered an effective therapy, like disease modifier in RA refractory patients to conventional treatment in at least six months of the treatment. PMID- 12374046 TI - [Cyclosporin A in atopic dermatitis]. AB - Atopic dermatitis is a chronic inflammatory skin disease, with inherited predisposition. It has typical morphology and distribution. Patients generally can be controlled with the use of moisturizers and topical steroids. In severe cases, it is recommended the use of alternative management. Cyclosporine is an immunosuppressor drug which inhibit the expression of T activated cells. Many open and placebo-controlled trials have been made evaluating its use, efficacy and security, in adults and children. The results suggest an initial dose of 5-6 mg/kg per day and reducing the amount according to response (load dose and maintenance dose) at long term in order to reach complete remission after withdrawal of treatment and limit adverse effects, like renal toxicity and hypertension. The immunological changes in AD patients treated with cyclosporine include eosinophil count reduction, besides lower levels of E-selectin, and soluble CD30 (known as disease markers), but overall, it corrects the imbalance between Th1 and Th2 response present in these kinds of diseases. PMID- 12374047 TI - The role of DDE, PCB, coplanar PCB and eggshell parameters for reproduction in the white-tailed sea eagle (Haliaeetus albicilla) in Sweden. AB - The reproduction of white-tailed sea eagles was monitored in 1964-1999 in 3 differently contaminated sub-populations: Baltic Sea coast (Bp), inland central Sweden (Ip) and Lapland (Lp). 249 dead eggs from 205 clutches were obtained for analyses of DDE and PCBs and for eggshell measurements. A desiccation index (Di) value was calculated for each egg as a measure of water loss through the shell. In the highly contaminated Bp, p,p'-DDE concentrations in the eggs decreased continuously and 5-fold during the study period and PCB concentrations decreased 3-fold from the mid 1980s. The PCB pattern changed slightly over time towards more high-chlorinated congeners but the relative toxicity of the PCB mixture, expressed as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ), remained constant and TEQ can be assumed to have decreased in a similar way as PCB over time. Productivity (P), shell thickness (St), shell index (Si) and Di increased over time in the Bp but no change in Di or productivity occurred in the Lp, where residue concentrations were 5-8 times lower. P of the Bp was not correlated to St or Si but was negatively correlated to Di, DDE and PCB. An S-shaped dose-response relationship was indicated between P and DDE. After 1988, when the PCB/DDE ratio was considerably higher than previously, PCB but not DDE concentrations were significantly higher in eggs with dead embryos as compared to undeveloped eggs, implying lethal concentrations of PCB, and a LOEL of 320 pg g-1 TEQ is suggested for embryo mortality. In a subset of 21 eggs, representing productive and unproductive females, analyzed for a selection of coplanar PCB congeners, tris(4 chlorophenyl) methanol and bis(4-chlorophenyl) sulphone, there was no evidence for a correlation between P and any of these compounds. A reduction in residue concentrations in old females did not lead to increased P or improved Di-values, indicating a remaining effect from a previous, higher exposure to contaminants. The inability to reproduce included a high rate of undeveloped eggs, indicating effects at a prezygotic stage. P showed the strongest correlation with Di, and Di was most strongly correlated to DDE. Thus, the remaining effect of previous exposure resulted in a stronger correlation to the symptom (Di) rather than to the suggested causative agent (DDE). LOEL values for depressed P were estimated at 120 micrograms g-1 DDE and 500 micrograms g-1 PCB (lipid basis). It is concluded that the major reason for depressed P during the study period was DDE, but that effects also from PCB were largely concealed by the effects from DDE. PMID- 12374048 TI - Possibilities for reducing nitrate leaching from agricultural land. AB - Agricultural soil is a contributor of nitrate to natural waters. High nitrate levels in water leached from soils are related to high nitrate concentrations in drinking water, and excess levels change the ecological balance of rivers and lakes. In this paper, sound solutions to the major environmental issue of limiting nitrate leaching by modifying agricultural practices are discussed. The causes of nitrate leaching from agricultural land are briefly explained and existing measures for the reduction of nitrate losses are described, analyzed and evaluated. Reduction of nutrient leaching is not a question of organic or conventional farming, but rather of the introduction and use of appropriate countermeasures. We propose the following guiding principles to minimize leaching from agricultural soils. To some extent these principles require a new way of thinking: i) environmental indexing of fields and consideration of spatial variability within fields in relation to their contribution to leaching losses within a catchment; ii) reduction of nitrogen inputs to soil to levels slightly below those expected to give the optimum yield by applying less nitrogen fertilizer and by a further reduction in animal density; and iii) use of a range of counter-measures (catch crops, minimum tillage, control of biological processes, etc.) depending on how sensitive the farming system, soil and climate are to the risk of nitrate leaching. PMID- 12374049 TI - A wasting syndrome in Swedish moose (Alces alces): background and current hypotheses. AB - In the 1980s, people in Sweden frequently responded to moose (Alces alces) found dead or in poor physical condition. The number of moose submitted for routine investigations to the National Veterinary Institute (SVA) increased tenfold and the hunters in Alvsborg County were especially concerned. Later, a complex wasting syndrome was described and reports of moose suffering from the syndrome have been collected since 1991. Today, there is no definitive answer as to the underlying causel(s) of the syndrome, but there are several plausible hypotheses that can be divided into two groups: food-related and host-parasite related. The food-related hypotheses are postulated to have any of the following ultimate causes: acidification/liming, browser density/food production or pollution. Our view is that few of the hypotheses have been critically tested. Most of the hypotheses are supported by some observations, which is to be expected because these are post-hoc attempts to explain these very observations. PMID- 12374050 TI - The problem of predicting global food production. AB - This paper examines the problem of the development of models capable of predicting the capacity of the global food production system. In particular, it identifies the various factors influencing the food production, and estimates their relative influence and predictability. The paper discusses also the problems connected with coupling of models representing the "driving" forces, the Earth system consisting of the atmosphere, the ocean and land surface, and food production. The overall conclusions drawn are: i) The time is not yet ripe for designing a comprehensive coupled model for predicting the global food production that takes into account all the factors having a significant influence; ii) the main difficulties are the modelling of the driving forces, e.g. socioeconomic and political factors, and iii) despite these problems, it is judged that results obtained with existing models are capable of providing concrete information for implementation of adaptation and mitigation measures. PMID- 12374051 TI - The past impact of livestock husbandry on dispersal of plant seeds in the landscape of Denmark. AB - The recent decline in species richness in (semi)-natural habitats in northern Europe has largely been attributable to habitat destruction, and to subsequent limitation in seed dispersal among fragments. However, some habitat types were probably split up already in the historical landscape, but the segregated parts were probably not isolated to the present degree. This paper seeks evidence for livestock as vectors for propagules at 3 spatial scales in the past cultural landscape. Three main scales at which livestock acted as seed dispersers are important: free movement in the landscape (1-10 km), driving animals to mast feeding or to manors (10-50 km), and the export of living animals (hundreds of km). The emerging picture is for most plant species a dramatically decreased chance of dispersal in the modern landscape. The consequence is probably decreasing species richness in (semi)-natural plant communities, such as pasture, meadow, and heathland. PMID- 12374052 TI - On purpose in science, conservation and government. The functional integrity of the earth is at issue not biodiversity. AB - The objectives of conservation have been focused ever more intensively for two decades on the preservation of "biodiversity." Emphasis has been on the losses of species through extinction. The cure has been the establishment of parks and reserves to protect "hot spots," especially in the tropics, where the diversity of species is high. The efforts in preservation have often extended to the development of connecting links among reserves to allow movements among them. The approach has been codified in law in the form of the Endangered Species Act in the United States and the Biodiversity Treaty, both of which address the issue species by species and each of which has obvious weaknesses. Such efforts may be appropriate but they are totally inadequate as the sum of activities in conservation in a world of 6 billion people with exploding technologies for exploiting virtually all of the earth for immediate human benefit. The biosphere is decaying rapidly as a habitat for all life, including people, not because of the extinction of species, but because of the progressive impoverishment of natural communities through human-induced chronic disruption that is now global and ubiquitous. The improverishment leads to progressive environmental dysfunction that is cumulative, but only in its later stages leads to extinction of species. Long before extinction becomes important, genetically distinct, local ecotypes are lost and the natural communities in which they were developed become improverished and dysfunctional. The most conspicuous disruption is that of climate, a global change in the environment of every ecosystem. The most elaborate and carefully interlinked array of natural reserves will succumb as climate is moved out from under them... and biodiversity will suffer the very extinctions the parks were established to avoid. But long before that, the human environment will suffer conspicuous and progressive impoverishment. The objective of conservation is the preservation of the integrity of function of landscapes (and waterbodies). Emphasis falls on forests in the normally naturally forested parts of the earth because forests are so large in area globally and have such a large influence on virtually every aspect of environment. Functional integrity requires structural integrity over 85% or more of the naturally forested zone in most areas. It also requires objective measurement and definition by the scientific community. Suddenly, conservation has become, not the preservation of biodiversity, honorable as that may be, but the preservation of the functional integrity of the human environment. That purpose is the central purpose that we assign to the governments that we establish in democracies to define and defend the public interest. It is past time for the scientific and conservation communities to recognize the urgency of this transition, join in defining competent new objectives for conservation, and to convey to the public the urgency of the need for governmental responsibility in protecting the public interest in a habitable biosphere. PMID- 12374053 TI - Resilience and sustainable development: building adaptive capacity in a world of transformations. AB - Emerging recognition of two fundamental errors underpinning past polices for natural resource issues heralds awareness of the need for a worldwide fundamental change in thinking and in practice of environmental management. The first error has been an implicit assumption that ecosystem responses to human use are linear, predictable and controllable. The second has been an assumption that human and natural systems can be treated independently. However, evidence that has been accumulating in diverse regions all over the world suggests that natural and social systems behave in nonlinear ways, exhibit marked thresholds in their dynamics, and that social-ecological systems act as strongly coupled, complex and evolving integrated systems. This article is a summary of a report prepared on behalf of the Environmental Advisory Council to the Swedish Government, as input to the process of the World Summit on Sustainable Development (WSSD) in Johannesburg, South Africa in 26 August 4 September 2002. We use the concept of resilience--the capacity to buffer change, learn and develop--as a framework for understanding how to sustain and enhance adaptive capacity in a complex world of rapid transformations. Two useful tools for resilience-building in social ecological systems are structured scenarios and active adaptive management. These tools require and facilitate a social context with flexible and open institutions and multi-level governance systems that allow for learning and increase adaptive capacity without foreclosing future development options. PMID- 12374054 TI - The circumboreal tundra-taiga interface: late Pleistocene and Holocene changes. AB - Creating a global perspective on past treeline changes is problematic due to the varying methods and definitions used. A general lack of a detailed description of the modern treeline position and vegetation complicates any comparative analysis of the magnitude of the most important changes. However, one seemingly common factor in most regions was an extremely rapid dispersal of trees when climate warmed drastically from full glacial conditions. Most Arctic treelines reached their northernmost positions in the early Holocene and receded to present positions starting at about 5.8 ka. The early occupation of the northernmost sites in ice-free and early deglaciated areas was possible because of the close proximity of invading trees in nearby glacial refugia, particularly in Fennoscandia and northern Russia. In Canada, the Northwest Territories and Quebec Labrador were out of phase with this general trend due to their late deglaciation. However, even here colonization was rapid, indicating that the tree species were present adjacent to the glaciers. Following this trend and based on the present evidence, we propose a scenario of a continuous but modest occupation of eastern Beringia by spruce during the late-Pleistocene instead of an exceptionally rapid spread of conifers from the glacial refugium south of the Laurentide ice sheet (2000 to 3000 km in about 200 years), which typically has been assumed. Macrofossil evidence of scattered occurrences of "exotic species" (for instance Siberian larch in central Sweden) far from their natural range limits in the early Holocene highlight the disparity between pollen and macrofossil analyses. It questions the validity of assigned pollen percentages to indicate the presence of a species within a region as these species were not observed in the pollen record. Thus, it is likely that trees were present at any given site well before the rise in pollen abundance. There is still a large potential to improve our knowledge about the environmental history of the circumboreal treeline areas. In particular, future research should concentrate not only on patterns of species displacement, but on finding the factors, apart from climate, which cause treeline shifts. PMID- 12374055 TI - Natural causes of the tundra-taiga boundary. AB - The tundra-taiga interface is characterized by a change in tree cover or density, tree size and shape, tree growth, and reproduction. Generally, trees get denser, taller, and less damaged as one moves from the tundra into the taiga proper. The environmental covariates and possible mechanisms resulting in these patterns are addressed in the paper. Low seed rain density, lack of safe sites caused by microclimatic variation, low surface substrate moisture, and low soil nutrient availability may limit the density of the tree species. Tree growth may be limited by a short growing season and further diminished, by shoot and root damage reducing carbon and nutrient stores as well as by reducing carbon and nutrient uptake capacities. Positive and negative feedbacks of tree density on tree growth exist at treeline. Increased tree density leads to increased air temperature and decreased wind damage, but also to lower soil temperature, reduced nutrient availability, and greater nutrient competition. PMID- 12374056 TI - The dynamics of the tundra-taiga boundary: an overview and suggested coordinated and integrated approach to research. AB - The tundra-taiga boundary stretches for more than 13,400 km around the Northern Hemisphere and is probably the Earth's greatest vegetation transition. The trees that define the boundary have been sensitive to climate changes in the past and models of future vegetation distribution suggest a rapid and dramatic invasion of the tundra by the taiga. Such changes would generate both positive and negative feedbacks to the climate system and the balance could result in a net warming effect. However, the boundary is becoming increasingly affected by human activities that remove trees and degrade forest-tundra into tundra-like areas. Because of the vastness and remoteness of the tundra-taiga boundary, and of methodological problems such as problematic definitions and lack of standardized methods to record the location and characteristics of the ecotone, a project group has been established under the auspices of the International Arctic Science Committee (IASC). This paper summarizes the initial output of the group and focuses on our uncertainties in understanding the current processes at the tundra taiga boundary and the conflicts between model predictions of changes in the location of the boundary and contrasting recently observed changes due to human activities. Finally, we present recommendations for a coordinated international approach to the problem and invite the international community to join us in reducing the uncertainties about the dynamics of the ecotone and their consequences. PMID- 12374057 TI - Human impacts on the tundra-taiga zone dynamics: the case of the Russian lesotundra. AB - The tundra-taiga zone is considered not only as a natural ecotone, but as a unique fringe zone with socioeconomic peculiarities. This holistic approach enables us to analyze several significant types of human impacts (industrial impacts and those associated with renewable resources development, including traditional reindeer herding and human settlements) and their role in the displacement of the lesotundra zone. In Russia, there is much evidence of deforestation and ecosystem degradation in different regions of the lesotundra zone and the northern taiga which borders the lesotundra zone. One indicator of this is that in the Archangelsk region and the Komi Republic, the observed current southern border of the lesotundra zone lies 40-100 km to the south of the southern boundary of the Protection Belt of Pretundra Forests, established in 1959. Human impacts also displace the northern boundary of the lesotundra zone (the boundary with the tundra zone) to the south. As a result, according to published estimations, the total area of human-made tundra and lesotundra stretching from the Kola Peninsula to Chukotka, is c. 470-500,000 km2. The increases in man-made tundra lead to negative consequences for the sociocultural sustainability of the lesotundra zone, a decrease in the quality of life (notably for permanent residents and native people and increases in mortality and depopulation. It cannot be predicted with any certainty that climate warming in the tundra-taiga zone will lead to a northward movement of the boreal forest treeline. We need also to consider the human impacts discussed in this article, which may actually lead to a southward movement of the lesotundra zone. PMID- 12374058 TI - How will the tundra-taiga interface respond to climate change? AB - The intuitive and logical answer to the question of how the tundra-taiga interface will react to global warming is that it should move north and this is mirrored by many models of potential treeline migration. Northward movement may be the eventual outcome if climatic warming persists over centuries or millennia. However, closer examination of the tundra-taiga interface across its circumpolar extent reveals a more complex situation. The regional climatic history of the tundra-taiga interface is highly varied, and consequently it is to be expected that the forest tundra boundary zone will respond differently to climate change depending on local variations in climate, evolutionary history, soil development, and hydrology. Investigations reveal considerable stability at present in the position of the treeline and while there may be a long-term advance northwards there are oceanic regions where climatic warming may result in a retreat southwards due to increased bog development. Reinforcing this trend is an increasing human impact, particularly in the forest tundra of Russia, which forces the limit of the forested areas southwards. Local variations will therefore require continued observation and research, as they will be of considerable importance economically as well as for ecology and conservation. PMID- 12374059 TI - Climate feedbacks at the tundra-taiga interface. AB - Feedbacks, or internal interactions, play a crucial role in the climate system. Negative feedback will reduce the impact of an external perturbation, a positive feedback will amplify the effect and could lead to an unstable system. Many of the feedbacks found in the climate system are positive; thus, for example, increasing CO2 levels will increase temperature, reduce the snow cover, increase the absorption of radiation and hence increase temperature further. The most obvious feedbacks, such as the snow example quoted above, are already included within our models of the climate and earth system. Others, such as the impact of increasing forest cover due to global warming, are only just being included. Others, such as, the impact of global warming on the northern peatlands and the impact of freshwater flows on the Arctic Ocean are not yet considered. The contrast in surface characteristics between low tundra vegetation to high taiga forest is considerable. The contrast is greatest in the winter, when the tundra is snow covered but the trees of the taiga protrude through the snow pack, and is probably the greatest contrast found on the land surface anywhere. This variation causes massive changes in the energy fluxes at the surface and hence the temperature conditions on the ground and within the atmosphere. There will be large resultant changes in the vegetation development, the carbon fluxes, the permafrost and the hydrology. The Arctic is already experiencing change and it is essential for us to understand the basic processes, and how these interact, to be confident of our predictions of environmental change in the future. PMID- 12374060 TI - How can the dynamics of the tundra-taiga boundary be remotely monitored? AB - This paper discusses some of the difficulties in establishing the location of the Arctic treeline and forest line on a circumpolar basis, and the contribution that remote sensing, particularly from spaceborne platforms, can make in resolving them. Spaceborne techniques can provide spatial resolutions as fine as a few meters, although the requirements for regional or global coverage are likely to limit the resolution to 30 to 100 m. Since this will preclude the identification of individual trees, the definition of the treeline will be based on statistical parameters estimated from satellite images. The optimum criteria for these parameters remain to be determined. Most remote-sensing observations that are suited to the measurement of the distribution of vegetation, and identification of its type, are based on the visible and near-infrared (VIR) parts of the electromagnetic spectrum, although there is increasing interest in the use of active microwave (radar) techniques. We discuss the basis of both types of approach and the techniques that follow from them, and present 3 case studies from the Russian Arctic. PMID- 12374061 TI - The tundra-taiga interface and its dynamics: concepts and applications. AB - The tundra-taiga interface is a dominant vegetation boundary that is related to climate and has an importance at a global level for its contribution to land atmosphere interactions, biodiversity and land use. However, our understanding of the precise location, dynamics and characteristics of the boundary, and its environmental and biotic drivers at a circumpolar level is poor. Our understanding has been constrained for various reasons, perhaps including a quest by researchers to denote 2- or even 3-dimensional tree distribution limits to a single line on a map. Current rapid sociological and environmental changes in the north necessitate better definitions to be made of characteristics associated with the tundra-taiga interface so that changes can be monitored and identified, and implications of these changes can be assessed. This concept paper introduces some of the complexities of adequately defining the boundary and suggests characteristics and processes that could focus future research at a collaborative, circumpolar level to create baseline data and to monitor and predict changes in the boundary zone. PMID- 12374062 TI - Experience with disaster yields lessons in preparedness. PMID- 12374063 TI - Medicare to partly cover extra cost of sepsis therapy, drug-eluting stents. PMID- 12374064 TI - Guidelines address prevention of catheter-related bloodstream infections. PMID- 12374065 TI - Partnering with nurses to handle personnel shortages. PMID- 12374066 TI - Counseling hospitalized pediatric patients with asthma. PMID- 12374067 TI - Full disclosure. PMID- 12374068 TI - Cost-efficacy analysis of ondansetron regimens for control of emesis induced by noncisplatin, moderately emetogenic chemotherapy. AB - The cost efficacy of various ondansetron regimens for the control of emesis induced by noncisplatin, moderately emetogenic chemotherapy was examined from a hospital perspective. Previous cost-efficacy analyses of ondansetron provide limited insight into the economic impact of ondansetron when used with moderately emetogenic chemotherapies. Clinical efficacy results of trials that used ondansetron to control emesis were retrieved from the biomedical literature published between January 1966 and December 2000. Only direct antiemetic treatment costs were considered. A total of 55 trials were analyzed, with a total of 22 regimens identified. Costs of antiemetic treatments were calculated by multiplying the milligrams of each product by the cost per milligram. For each injectable dose, the average cost of an administration device was Can$1.28. Pharmacy time required for preparing an i.v. dose was 2.5 minutes, at an average hourly rate of Can$25. Nursing time to administer each i.v. dose was estimated at one minute at an average rate of Can$23 per hour. The efficacy rate of the 22 regimens varied between 24.2% and 90.4%, while the cost varied from Can$20 to Can$413. A cost-efficacy analysis of ondansetron regimens for control of emesis caused by noncisplatin, moderately emetic antineoplastic treatment revealed regimens that should be avoided as well as regimens that are, in comparison, at least as efficacious and less expensive. The analysis supported the concomitant use of a corticosteroid, twice-daily administration of ondansetron, and limitation of ondansetron administration to a period not exceeding four days. PMID- 12374069 TI - Building a better online formulary. AB - The history of and improvements made to the University of Michigan Health System (UMHS) inpatient online formulary are described. The current formulary at UMHS is the third version of the Web-based formulary. The original effort in 1997 consisted of converting word-processing documents to HTML format and exporting this information to the university's intranet. There was no mechanism to search for formulary items, no therapeutic class cross-referencing, and no cost information. Documents and their conversion had to be manually maintained. The second version incorporated a series of automatic daily computer downloads from the inpatient pharmacy computer system. Web pages were built to dynamically display the formulary information from the database based on users' requests. The formulary enabled searching by brand or generic names, provided therapeutic category cross-references, listed the location of products within automated dispensing cabinets, provided accurate cost information, and was always up-to date. Maintenance efforts drastically decreased. The current version has incorporated additional logic to meet users' needs. If no matches are found, the system expands its search by automatically linking to UMHS's inpatient pharmacy system repository of all drugs, finding matches to what the user entered, and then returning the names of therapeutically similar formulary agents to the user. A cross-index feature allows the system to return all the drugs that fall under the searched therapeutic category. Dramatic improvements have been made to UMHS's inpatient online formulary in the past two years. The current formulary provides a very low-cost, easily maintainable, and effective means to access the formulary and clinically relevant and timely information specific to each medication. PMID- 12374070 TI - Stability of flucytosine 50 mg/mL in extemporaneous oral liquid formulations. PMID- 12374071 TI - Pharmacokinetics of amikacin and effect of ascites in Korean patients. PMID- 12374072 TI - Sterility of intravenous fat emulsion in plastic syringes. PMID- 12374073 TI - Use of botulinum toxin type B for migraine and tension headaches. PMID- 12374074 TI - Voluntary undertaking rule and duty to warn. PMID- 12374075 TI - Speaking for hire. PMID- 12374076 TI - Relationship between the pharmaceutical industry and pharmacy practitioners: undue influence? PMID- 12374077 TI - Corporate compliance: an emerging issue for pharmacists. PMID- 12374078 TI - Combination antithrombotic therapy in acute myocardial infarction. PMID- 12374079 TI - Possible adverse reactions to preservatives in high-dose pyridoxine hydrochloride i.v. injection. PMID- 12374080 TI - Establishing clinical pharmacy services in a Pakistani intensive care unit. PMID- 12374081 TI - Retaining staff by moving from quality control to quality assurance. PMID- 12374082 TI - Accuracy of medication histories. PMID- 12374083 TI - Oral gatifloxacin-induced ataxia. PMID- 12374084 TI - New drugs and lower costs. PMID- 12374085 TI - New drugs and lower costs. PMID- 12374086 TI - New drugs and lower costs. PMID- 12374087 TI - GPO reform. PMID- 12374088 TI - Musculoskeletal systems. PMID- 12374089 TI - The Mulligan concept: its application in the management of spinal conditions. AB - The Mulligan concept encompasses a number of mobilising treatment techniques that can be applied to the spine, these include 'NAGs' (natural apophyseal glides), 'SNAGs' (sustained natural apophyseal glides), and 'SMWLMs' (spinal mobilisations with limb movements). These techniques are described and the general principles of examination and treatment are outlined. Clinical examples are used to illustrate the concept's application to the spine, how it has evolved and been integrated into constantly changing physiotherapy practice. New applications are considered which can assist in the correction of dysfunctional movement. The paper reflects on the possible role that this concept has to play within evidence based practice. A future research direction is proposed in the light of presently available preliminary research results. PMID- 12374090 TI - Whither the mouse genome? PMID- 12374091 TI - Syntheses, electronic absorption, emission, and ion-binding studies of platinum(III) C empty set N empty set C and terpyridyl complexes containing crown ether pendants. AB - A series of platinum (II) C empty set N empty set C complexes, [Pt(C empty set N empty set C) (L)] (HC empty set N empty set CH=2,6-diphenylpyridine (dppy); L=Ph(2)PB15C5 (1, B15C5=benzo[15]crown-5), Ph(2)PDMP (2, DMP=3,4 dimethoxyphenyl), pyCOA15C5 (3, A15C5=aza[15]crown-5), pyCON(CH(2)CH(2)OCH(3))(2) (4), pyC[triple bond]CB15C5 (5), pyC[triple bond]CDMP (6)) and terpyridyl complexes, [Pt(trpy)(L)](X)(2) (trpy=2,2':6',2''-terpyridine; L=Ph(2)PB15C5, X=OTf (7 a), PF(6) (7 b); X=PF(6), L=Ph(2)PDMP (8), pyC[triple bond]CB15C5 (9), and pyC[triple bond]CDMP (10)) have been successfully synthesized and characterized. The structures of 1, 3, and 7 a have been determined by X-ray crystallography. Excitation of complexes 1-6 in EtOH/MeOH (4:1 v/v) glass gave high-energy structured emission bands, assigned as derived from states of metal perturbed intraligand (IL) origin. At higher concentrations, complexes 3-6 each displayed an additional, structureless emission band at 600-615 nm, with complexes 5 and 6 showing an obvious increase in the intensity of this emission band when the concentration was increased further. In dichloromethane at room temperature, complexes 3-6 showed, in addition to the high-energy emission at 490 505 nm, an extra, broad emission band at 620-625 nm when the concentration was increased. The emission origins of the low-energy band in glass and in fluid solutions are suggested to be derived from the ground-state oligomerization or aggregation process of the complexes. In the solid state at room temperature, complexes 1-6 each showed a broad, unstructured emission band at 560-600 nm, which was shifted to lower energy upon cooling to 77 K. On the other hand, the terpyridyl analogues 7-10 displayed intense vibronic-structured intraligand (IL) emissions at 460-472 nm in butyronitrile glass at 77 K. Solid-state samples of 9 and 10 displayed strong phosphorescence upon photoexcitation at 298 K and 77 K, tentatively assigned as derived from states of Pt(d pi)-->pi*(trpy) (3)MLCT origin(MLCT=metal-to-ligand charge transfer). The ion-binding properties of complexes 5 and 9 for Na(+), Ba(2+), and K(+) ions have been studied by UV/Vis spectrophotometric methods, and confirmed by ESI mass spectrometric studies. The ion-binding properties for Na(+) ions have also been probed by (1)H NMR experiments. For the same crown ether-containing ligand and the same metal ions, the neutral cyclometalated complexes gave larger binding constants than the positively charged terpyridyl analogues. PMID- 12374092 TI - In the skies over Cleveland. Interview by Georgia L. Narsavage. PMID- 12374093 TI - Nursing the families of critically ill patients. PMID- 12374094 TI - Caring for patients after an ICU admission. PMID- 12374095 TI - The role of dUTPase and uracil-DNA repair in cancer chemotherapy. AB - Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the molecular mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion observed following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metabolism as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metabolism. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clinical studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer. PMID- 12374096 TI - The herpesvirus encoded dUTPase as a potential chemotherapeutic target. AB - The human herpesviruses are a well characterized group of viruses that are responsible for a wide spectrum of human diseases. Included in this group of pathogens are the alphaherpesviruses (herpes simplex types 1 and 2 and varicella zoster virus), the betaherpesviruses (cytomegalovirus, human herpesvirus types 6 and 7) and the gammaherpesviruses (Epstein-Barr virus and human herpesvirus 8). An important feature of these viruses is that they cause latent infections that can be reactivated to cause disease. The herpesviruses encode for a large number of structural and non-structural proteins, and several of the non-structural proteins, such as thymidine kinase, DNA polymerase, and ribonucleotide reductase, have been utilized as targets for the development of anti-herpesvirus agents. Another herpesvirus encoded enzyme that has received little attention as a potential target for the development of specific anti-herpesvirus agents is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). Furthermore, little is known concerning the role of the herpesviruses' encoded dUTPases in virus replication and in modulating the chemotherapeutic efficiency of other anti herpes agents. Because of recent advances in molecular virology and biochemistry, it is now possible to rationally develop "designer" drugs based upon the structural/functional interaction of the drug with a specific viral protein. The purpose of this review is to describe previous studies demonstrating the potential use of the herpesvirus encoded dUTPase as a drug target, to describe problems associated with using the dUTPase as a target and to discuss new approaches that can be used. PMID- 12374097 TI - The role of retroviral dUTPases in replication and virulence. AB - Several retroviruses, including equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV) and feline immunodeficiency virus (FIV) encode dUTPase. The role of this enzyme in the replication of these viruses has been scrutinized, with particular emphasis on potential roles for dUTPase in virulence and viral mutation rate. Overall, the results of these studies have indicated a central role for dUTPase in facilitating productive viral replication in non-dividing cells. The requirement for dUTPase in EIAV, which replicates exclusively in macrophages, may be the most stringent. Studies of dUTPase mutants of virulent EIAV clones suggest that the enzyme is a major determinant of virulence. In contrast, FIV readily replicates in dividing cell populations such as CD4+ and CD8+ T cells, and B cells as well as in non-dividing macrophages. Thus, the virus burden and disease sequelae are lowered in cats infected with a dUTPase-minus FIV relative to cats infected with wild type FIV, but not totally abrogated. Growth in macrophages is attenuated with the DU-minus FIV with evidence of a 5 to 8-fold increase in G-->A transition mutations in viral integrants present in macrophages. These findings are consistent with an increase in uracil misincorporation in the absence of dUTPase, resulting in transition mutations that cripple the virus. Effects on virus replication and disease production have also been noted for dUTPase-deleted CEAV and visna virus. While HIV and SIV do not encode dUTPase some reports suggest that other viral and host cell factors may substitute for its activity. Betaretroviruses also encode dUTPase and while several of these cause significant disease, the role of dUTPase in their replication and pathogenesis is currently unknown. PMID- 12374099 TI - Bone growth protein expected to replace spinal fusion surgery. PMID- 12374100 TI - Artificial pancreas on the horizon? PMID- 12374098 TI - Trypanosomal dUTPases as potential targets for drug design. AB - Parasites of the Trypanosomatidae family are responsible for diseases that afflict several million people worldwide. Currently there is an urgent need for new drugs against these diseases and an approach to drug discovery is the study of biochemical and structural properties of a potential target and the subsequent design of specific compounds. Trypanosomatid genes coding for enzymes which distinctively hydrolyze dUTP have been isolated by genetic complementation in Escherichia coli mutants defective in dUTPase activity. An analysis of these sequences from Leishmania major and Trypanosoma cruzi showed that no significant similarity could be established with the family of known dUTPases and that the five consensus motifs were absent. However, limited similarity was identified for three motifs present in an enzyme related in function the dCTPase-dUTPase from T phages and 35 percent identity with a putative dUTPase identified in the eubacteria Campylobacter jejuni. T. cruzi and L. major dUTPases were highly similar and catalyzed in a specific fashion the hydrolysis of dUTP. A detailed kinetic study of both enzymes revealed that dUDP is also an efficient substrate of the enzyme while other nucleotides are poorly hydrolyzed. The enzyme is essential for viability in Leishmania and is up-regulated by inhibitors of dTMP synthesis. Thus, a new family of dUTPases might exist in certain organisms that bear no sequence or structure similarity with eukaryotic enzymes accomplishing the same function and that may constitute potential drug targets for the development of specific inhibitors. PMID- 12374101 TI - Business Week notes critical nursing shortage. PMID- 12374102 TI - [Treatments "substitute" to Hormonal Replacement Therapy of menopause]. PMID- 12374103 TI - Recurrence in the axilla after sentinel lymph node biopsy for breast cancer - EJSO 2002; 28: 199. PMID- 12374105 TI - US patent office, interfering again. PMID- 12374104 TI - Intraoperative hyperthermic intrathoracic perfusion chemotherapy for pleural metastases of thymic neoplasms. PMID- 12374106 TI - Therapeutic applications of sugar-mimicking glycosidase inhibitors. AB - Sugar-mimicking alkaloids inhibit the glycosidases involved in a wide range of important biological processes, principally owing to their structural resemblance to the sugar moiety of the natural substrate. The possibility of modifying and blocking these processes by using such inhibitors for therapeutic applications has attracted a lot of attention. PMID- 12374107 TI - [The history of modern andrology]. AB - The beginning of the history of modern Andrology as a clinical science can be located at the mid-century of 1900. The term itself of "Andrology" was first proposed in 1951. At that time, basic scientists coming from different cultural backgrounds - the main roots were urology, endocrinology and in some cases dermatology - "discovered" their common interest in the field of the male reproductive system, whose pathology and even physiology have been for a long time ignored or disregarded, often considered a shame more than a clinical entity. Very soon, many other specialists joined the team; among these biologists, genetics, psychologists etc., under, for the first time in history, the common definition of "Andrologists". They organized themselves in National and International Societies whose members, at the moment, are more than 8,000; holding national and international Meeting Workshops and Conventions all around the world and editing Textbooks and Journals. On the educational ground, pre - and post graduated courses are held in several Countries, both for medical students and doctors. Although a melting-pot of different cultural basis, today Andrology can be defined an unitary medical discipline dealing with the pathophysiology of the male reproductive system during all the life course of the male subject, from development to maturity and senescence. Shortly, and according to the WHO definition, it deals with male reproductive health. Quite similar to what is Gynecology for the female subject. The late arrival of the andrological discipline on the stage of modern medicine gave it the advantage of utilizing all the modern achievements of basic and clinical science, from molecular biology to ultrastructure, to genetic etc., reaching levels of high quality. Today, modern andrology, according to the principles of Evidence-Based Medicine, and although much is left to do, can effectively manage all the main pathologies of the male organism, form infertility to erectile dysfunction, but taking care of problems of puberty and senescence ("andropause") as well; giving to the male subject the "quality of life" that must be granted to every human being. PMID- 12374108 TI - Paleoandrology and prostatic hyperplasia in Italian mummies (XV-XIX century). AB - Prostatic hyperplasia, a very common condition today, was well known in the past as cause for bladder distension. The difficulty to identify, at autopsy of natural or artificial mummies, even a normal-sized prostate is probably the result of putrefaction processes and its usually dramatic size reduction as well. We report two ancient cases of prostatic hyperplasia recently observed in natural mummies from Italy. The first case regards Pandolfo III Malatesta (1370-1427), a leading figure of the Italian Renaissance. He was a valiant soldier and horseman with a very active life style. The tomb, containing his naturally mummified body, has recently been discovered in Fano (Marche, Central Italy). After careful X-ray and videographic examination, the autopsy showed good preservation of the skeletal muscles, cartilage, internal and external organs, included prostate gland and penis. Macroscopic examination revealed a staghorn calculus (calcium urate) of the left kidney and a severe enlargement of the prostate, with calcifications detectable by X-ray and large nodules protruding in the lumen of an ectatic urethra. Histology shows fibrous brands of connective and muscular tissue surrounding circular and oblong lacunae, with no preservation of epithelial structures. The macroscopic and histological picture allowed us to diagnose prostatic nodular hyperplasia. The second case (XIX century) concerns the natural mummy of an anonymous 50-60 years old man, found in ancient friary near L'Aquila (central Italy), which underwent computed tomography and a complete autopsy. Pelvic CT scans showed distended urinary bladder and a ring of dense tissue at the site of the prostate. At autopsy the bladder measured 7 x 6 x 5 cm and the prostate was 4 x 5 x 3 cm; prostatic urethra had a diameter of x 2 cm. Histology revealed dense fibrous tissue containing muscular fibers and roundish cavities of variable size, filled with eosinophil, PAS-positive material. Concretions were also present in some of these spaces. Strong immunohistochemical reactivy for PSA was observed in this material. The existence of glandular structures containing eosinophil, PAS-positive material, immunoreactive for PSA, confirmed the prostatic nature of the specimen, already suspected after CT scan and gross examination. The presence itself of the prostate, its histological picture, the preserved and distended urinary bladder, in addition to the age of the subject, supported the diagnosis of prostatic hyperplasia. PMID- 12374109 TI - The ancient name of rose. AB - The article is a survey of plants foods and drugs that Greeks and Romans thought to be aphrodisiac and to have a specific effect on the male libido. The article is a useful support to study the sexual therapy in ancient world. PMID- 12374110 TI - [Andrology in Celsus]. AB - The article presents the principal topoi of Celsus' De medicina in which andrological problems are discussed. Comparing them with other testimonies of ancient medicine, the author clarifies their peculiarity and explains them in the summary of the cultural and socio-economical conditions of ancient Rome. PMID- 12374111 TI - [Galen's pathological andrology]. AB - Galen, who was certainly not a great amateur of women, nevertheless was interested in some disfunctions of sexual activity in men. Although he develops a theory about the problem, he is matter of fact as far as daily life is concerned, and, even if he quotes him, quite different from what Epicurus used to be. PMID- 12374112 TI - [Anatomophysiology of the male sexual apparatus in Galen]. AB - Galen builds his andrology on animals dissections which he enlarges upon human anatomy by analogy, so it's very difficult to recognize the organs he describes, especially adenoeideis parastatai. His physiology of male sexual apparatus is based on the theory that semen comes from blood perfectly cooked in gonadics vessels and testicles. As usual, he tries to demonstrate Aristotle's principle on perfection of Nature's acts by experimentation, logic, imagination and sometimes insincerity. PMID- 12374113 TI - [About names and functions (testes, semen): from andrology to gynaecology]. AB - Philological study about how the making of a Gynecology in Ancient, Medieval and Renaissance world took as starting and reference point an Andrological understanding of Medicine (as 'Medicine of the human being'). The same terms (testes, semen, vasa, seminaria) were always used to name the sexual organs and functions of male and female bodies. PMID- 12374114 TI - [Andrology and anthropology in the Talmud]. AB - The article analyses andrological aspects of Hebrew medicine and legislation. A detailed analysis of the sources provides interesting testimonies about the practice of abstinence, sexual hygiene, evaluation of male pleasure, religious prohibitions, dangers of committing sexual excesses and forbidden practices as homosexuality. PMID- 12374115 TI - Sperm disorders according to the Byzantine medical writers (4th - 14th centuries). AB - Research in the works of the Byzantine medical authors brought to light significant information concerning disorders of the sperm as causes of infertility. The eminent Byzantine physicians give detailed accounts about the anatomy of the genitals, the creation of the sperm and its disorders as regards its quantity, quality, appearance, consistency, colour, ejaculation etc. According to those authors, the disorders of the sperm are due to, dietetic reasons (a list of foods and drugs influencing the production of sperm is referred to by many Byzantine physicians); constitution and age of the patient; obesity; diseases, such as "gonorrhea" (involuntary loss of sperm), oneirogmus, stenosis of the spermiducts, hypospadias and atrophy of the genitals; iatrogenic reasons (traumatic cutting off of the spermiducts during a lithotomy); castration. These concepts were based on the works of the ancient Greek physicians of the Hippocratic, Hellenistic and Roman eras. However, such ideas, enriched by the personal experience of the Byzantine doctors, were transmitted to and influenced Islamic and European medicine and thus the rest of the world. PMID- 12374116 TI - Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows. AB - High protein diets, which lead to excess production of nonprotein nitrogen such as ammonia and urea, have been associated with reduced fertility in dairy cows. In this study we test the hypothesis that diets containing high levels of quickly degradable urea nitrogen (QDN) compromise embryo development. Lactating dairy cows were fed mixed silage and concentrates twice daily. At 60 days postpartum, a synchronized estrus was induced and the cows were subsequently superovulated and inseminated using a standard protocol. On Day 7 after insemination, the uteri were flushed and embryos retrieved. At the start of treatment, cows were randomly allocated into three nutritional groups: control (CONT, n = 8), long (L-) QDN (n = 8) and short (S-) QDN (n = 9). The L-QDN cows were fed a supplement of urea from 10 days before insemination, and the S-QDN cows were fed the supplement from insemination until embryo collection. Both L- and S-QDN diets produced significant increases in plasma ammonia and urea 3 h post-feeding. The S-QDN but not the L-QDN diet was associated with a significant reduction in embryo yield. Embryo quality was also significantly reduced in the S-QDN cows. This study indicates that there is no deleterious effect on the yield and quality of embryos recovered 7 days after breeding when QDN feeding is initiated during the previous midluteal phase. However, introduction of a similar diet 10 days later, at the time of insemination, was deleterious. We suggest that QDN is toxic to embryos but cows can adjust within 10 days. PMID- 12374117 TI - Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment. AB - Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll centrifuged or accessory sperm cells. PMID- 12374118 TI - Effects of calcium salts of fatty acids and calcium salt of methionine hydroxy analogue on plasma prostaglandin F2alpha metabolite and milk fatty acid profiles in late lactation Holstein-Friesian cows. AB - Effects of a dietary lipid supplement containing calcium salts of fatty acids and methionine hydroxy analogue on plasma prostaglandin F2alpha (PGF2alpha) metabolite (PGFM) and milk fatty acid profiles were examined in 40 late lactation, nonpregnant, Holstein-Friesian cows for a period of 70 days. Effects on milk production, milk composition, and blood metabolites were also examined. Cows were paired on the basis of lactation number (first lactation, n = 8; second lactation, n = 32) and randomly assigned from within pairs to one of two dietary treatments: unsupplemented control (C) or 400 g per cow per day of the lipid supplement (S). Cows receiving the supplement had higher (P < 0.05) total milk production, total fat production (kg), and total lactose production (kg). Plasma cholesterol was significantly higher (P < 0.01) after 30 days of treatment in cows receiving the supplement. Cows receiving the supplement had lower (P < 0.01) concentrations of short chain milk fatty acids (C4:0 to C14:1) and higher concentrations of long chain fatty acids (C18:1 and C18:2; P < 0.01) than control animals. Oxytocin-induced prostaglandin release on Day 16 postovulation was increased (P < 0.01) in cows receiving the supplement. In conclusion, supplementation with calcium salts of fatty acids and methionine hydroxy analogue significantly increased milk yield and plasma PGFM. PMID- 12374119 TI - Factors affecting the uptake of DMSO by the eggs and embryos of medaka, Oryzias latipes. AB - High performance liquid chromatography (HPLC) was used to assess the uptake dynamics of the cryoprotectant DMSO by intact unfertilized eggs (stage 0), 8-cell (stage 5) and eyed embryos (stage 30) of medaka, Oryzias latipes, the relation of the internal concentration (Cin) of DMSO with fertilization and survival rates, and the effects of several factors on these processes. The factors examined were: cryoprotectant concentration (0.6, 1.2, 1.9 and 2.5 M), impregnation time (1, 3, 5, 10, 15 and 20 min), temperature (0, 5 and 20 degrees C), hydrostatic pressure (0 and 50 atm), and the osmotic conditions of the materials (normal or partially dehydrated). Cryoprotectant permeation, estimated from the initial rates of DMSO uptake, was higher in embryos than in eggs and increased with embryonic development; however, the DMSO Cin in eyed embryos reached a plateau at 1-5 min and could not be increased by prolonging impregnation. The highest fertilization and survival rates for any given DMSO Cin were obtained with high concentrations and short times of impregnation rather than low concentrations and long impregnation times. Application of hydrostatic pressure (50 atm) and exposure for 3 min to a 1 M trehalose solution prior to impregnation induced a substantial increase in the DMSO Cin of 8-cell embryos in comparison to untreated controls with no significant effect on survival. Hydrostatic pressure also promoted DMSO uptake in unfertilized eggs, but with rapid loss of viability, and was ineffective in eyed embryos. The uptake of DMSO and its toxicity to 8-cell embryos were directly proportional to the temperature of impregnation. The results of this study reveal important interactions between cryoprotectant concentration, impregnation time and the developmental stage (or type) of the materials and provide evidence that hydrostatic pressure, temperature of impregnation and the osmotic conditions of the materials can be manipulated to increase the uptake of cryoprotectant by fish eggs and embryos. PMID- 12374120 TI - Impaired semen quality of AI bulls fed with moldy hay: a case report. AB - The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T 2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay. PMID- 12374121 TI - Reproductive endocrinology and postweaning performance in the multiparous sow. Part 1. Influence of metabolic status during lactation. AB - The metabolic status of the sow during lactation might influence reproductive endocrinology and the postweaning reproductive performance. With regard to the multiparous sow, previous studies addressing this topic are scarce and the results inconsistent. Blood samples were collected from 18 multiparous sows during lactation and after weaning for analysis of nonesterified fatty acids (NEFA), triglycerides, creatinine, urea progesterone, LH, and estradiol-17beta. Based on the average preweaning NEFA levels the sows were divided into a "high" and a "low" catabolism group. The NEFA values were higher in the "high" group during each of the last 3 weeks of lactation. The levels of urea, creatinine and progesterone were similar (P > 0.05) in the two groups throughout the study. Reproductive functions seemed equally inhibited during lactation in the two groups and there were no differences in postweaning reproductive performance. The results suggest that metabolic rate during lactation varies considerably between equally nourished multiparous sows but this has no influence on postweaning reproductive performance. PMID- 12374122 TI - Reproductive endocrinology and postweaning performance in the multiparous sow. Part 2. Influence of nursing behavior. AB - The reason for variation in postweaning reproductive performance among multiparous sows is to a large extent unknown. In the present study, the influence of nursing behavior was explored. Blood samples were collected during lactation and after weaning from 18 multiparous sows for cortisol, LH, estradiol 17beta (E2), and progesterone analysis. Sow and piglet behavior was videotaped. The sows were fed according to litter size and slaughtered after the second postweaning estrus. The sows were divided into two groups based on average values for the different behavioral parameters. Sows with a long average nursing duration (long group) had lower average and basal LH levels on Day 14 and 21 of lactation as compared to the sows having a short average nursing duration (short group). In the long group, concentrations of E2 were lower the day after weaning, but on Day 15 and 21 of lactation no differences were noted between the two groups. Postweaning performance seemed impaired in the long group, though, differences were not significant. The sows in the long group were heavier and tended to lose less weight during lactation. To conclude, nursing duration seems to influence the extent to which reproductive functions are inhibited during lactation. PMID- 12374123 TI - Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. AB - We previously reported that germline chimeras could be produced by transfer of chicken gonadal primordial germ cells (gPGCs) cultured for a short term (5 days). This study was subsequently undertaken to examine whether gPGCs maintained in vitro for an extended period could retain their specific characteristics to induce germline transmission. Chicken (White Leghorn, WL) gPGCs were retrieved from embryos at stage 28 (5.5 days of incubation) and continuously cultured for 2 months in modified Dulbecco's minimal essential medium without subpassage and changing of the feeder cell layer. After the identification of gPGC characteristics using Periodic acid-Shiff's (PAS) reaction and anti stage specific embryonic antigen-1 (SSEA-1) antibody staining at the end of the culture, cultured gPGCs were injected into the dorsal aorta of Korean Ogol Chicken (KOC) recipient embryos at stage 17 (2.5 days of incubation). Nineteen chickens (13 males and 6 females) were hatched, grown to sexual maturity, and subsequently subjected to testcross analysis employing artificial insemination with adult KOC. Of these, four (three males and one female) hatched chickens with white coat color. The percentage of germline chimerism was 21% (4/19). The results of this study demonstrated that gPGCs could maintain their specific characteristics for up to 2 months in vitro, resulting in the birth of germline chimeras following transfer to recipient embryos. PMID- 12374124 TI - Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure. AB - The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos. PMID- 12374125 TI - Reproductive seasonality in female Iberian red deer (Cervus elaphus hispanicus). AB - This study characterized the seasonal pattern of luteal cyclicity in Iberian red deer (n=16), by measuring plasma progesterone concentrations in hinds (female red deer) twice per week from calving (May and June) 1996 until May 1997. In eight of these hinds we also examined plasma prolactin profiles to assess seasonal responses to photoperiod. Plasma progesterone concentration in the 16 hinds studied indicated that the reproductive pattern is seasonal, and lasts for 5.73 +/- 0.27 months. After calving, progesterone levels remained basal (no luteal activity) for several months, except in a hind that lost her calf just after calving, and thus did not have to suckle it. This hind showed two consecutive estrus cycles in the month following calving, which suggests that suckling has an inhibiting effect on the resumption of ovarian activity. These results also showed that as long as the hinds do not become pregnant, they show between 5 and 10 estrus cycles per reproductive season (8.06 +/- 0.35), ranging between 105 and 249 days from onset of the first cycle to end of the last one. Uninterrupted cycling lasted for 3.5-6.4 months (mean, 4.6 +/- 0.24). Cyclic luteal activity was found from October to February in all hinds, with a smaller, but notable proportion in September (56.25%) and March (68.8%), whereas it was negligible in the remaining period. Our results show a reproductive season similar to or longer than that recorded by other authors. Prolactin plasma concentrations showed a yearly trend following that of photoperiod, with peak concentrations from April to July, a decrease in August, minimal concentrations from September to February and a sharp increase in March. PMID- 12374126 TI - Semen cryopreservation of small abalone (Haliotis diversicolor supertexa). AB - Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa. PMID- 12374127 TI - Prediction of X and Y chromosome content in bovine sperm by using DNA pools through capillary electrophoresis. AB - Livestock resource management through gender offspring preselection is an efficient tool in terms of genetic improvement and farm management and additionally provides the opportunity to adjust offspring to market demands. In this study bull ejaculates were tested using PCR amplification of a segment of the X-Y homologous amelogenin gene in order to estimate the X and Y chromosome frequencies by capillary electrophoresis. Results were quantified against a regression function constructed with pools prepared with DNA from bulls and cows with known X and Y ratios. An average of 50.02 +/- 2.79% X chromosome content was found with normal distribution ranging from 38.7 to 58.2%. Bull effect was significant in the analysis of variance representing 8.5% of the total variance. This simple analysis provides a low-cost and quick method of evaluating an X-Y ratio in a high number of ejaculates, particularly when external factors can be manipulated to alter it. PMID- 12374128 TI - Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C. AB - Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log linear relationship with log(Y) = -0.912 + 0.007X; R2 = 0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions. PMID- 12374129 TI - Improvement of pregnancy rate in Japanese Black cows by administration of hCG to recipients of transferred frozen-thawed embryos. AB - Japanese Black primiparous and multiparous beef cows (n = 120) were selected as recipients and randomly divided into three groups (A, B, and C) of 40 recipients each. Group A received an intramuscular (i.m.) treatment of 1500 IU human chorionic gonadotropin (hCG) on day 1 (day 0 = onset of estrus), while Group B received an i.m. treatment of hCG on day 6. Group C received an i.m. treatment of 5 ml saline on day 6 as a control. On day 7, frozen-thawed embryo transfer was conducted in all groups, and pregnancy was diagnosed by palpated per rectum 40-50 days after the transfer. Twelve recipients were randomly selected from each group. Plasma progesterone (P) and estradiol-17beta (E2) concentrations were determined in these recipients on days 6, 7 and 14, and at the time of pregnancy diagnosis, and their ovaries were examined for a corpus luteum and follicles by palpated per rectum. The pregnancy rate in Group B was higher (67.5%. P < 0.05) than the rate in Group C (45.0%) and in Group A (42.5%). The plasma P concentration on day 14 tended to be higher although not significantly in Group B than in Groups C and A. At the time of pregnancy diagnosis, the blood P concentration of pregnant recipients in Group B was higher (P < 0.05) than that of those in Groups C and A. The plasma E2 concentrations on days 7 and 14 were lower (P < 0.05) in Group B than in Groups C and A. These results showed that administration of hCG 6 days after estrus improved the pregnancy rate for non surgical frozen embryo transfer 7 days after estrus by enhancing luteal function and depressing E2 secretion. PMID- 12374130 TI - Effect of follicular status on superovulatory response in ewes is influenced by presence of corpus luteum at first FSH dose. AB - The present study was developed to assess possible effects on ovulatory response and embryo yields arising from the presence of a corpus luteum (CL) at the time of initiation of the progestagen treatment used in superovulatory protocols in sheep. In breeding season, estrus was synchronized in 25 Manchega ewes using 40 mg FGA sponges for 14 days, together with a single dose of 125 microg of cloprostenol on Day 12, with Day 0 as day of progestagen insertion. Superovulatory treatment consisted of eight decreasing doses (1.5 x 3 ml, 1.25 x 2 ml, and 1 x 3 ml) of Ovagen twice daily from 60 h before to 24 h after sponge removal. The presence or absence of corpora lutea was assessed by transrectal ultrasonography at progestagen insertion and at first FSH dose. Number and size of all follicles > or = 2 mm were also evaluated at first FSH dose. The number of corpora lutea and the number and viability of recovered embryos in response to the treatment were evaluated 7 days after sponge removal. No significant effect on ovarian response of the presence of a CL at sponge insertion in 21 of the 25 ewes (84%) was detected. However, ewes with a CL at first FSH dose (16 ewes, 64%) yielded a higher number of transferable embryos (7.2 +/- 1.4 versus 2.7 +/- 0.7, P < 0.05), since the embryo degeneration rate was increased in sheep without a CL (42.5% versus 12.7%, P < 0.01). Analysis of possible effects derived from the presence of a large presumptively dominant follicle (> or = 6 mm) at first FSH dose showed that both recovery and viability rates were lowest (P < 0.05) in ewes bearing a large follicle in the absence of a CL (40.5 and 50.6%, respectively), and highest in ewes that did not show a large follicle but in which a CL was present (73.9 and 85.2%). The final number of transferable embryos was very different between groups (10.2 versus 1.8, P < 0.01). These results indicate that the number and quality of embryos obtained from superovulated ewes is affected by the presence of a CL prior to the first FSH dose (i.e. by the stage of the estrous cycle at progestagen insertion) and also by an interaction with suppressive effects from large dominant follicles. This finding suggests the existence of some effects on follicular population prior to the FSH treatment that may compromise follicle and oocyte developmental competence. It seems reasonable to hypothesize that superovulatory yields would be increased by beginning the treatment during the early-luteal phase of the estrous cycle, allowing for the presence of a CL along with the progestagen treatment. PMID- 12374131 TI - In vitro developmental competence of domestic cat embryos after somatic cloning: a preliminary report. AB - This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast's age and type of somatic cells used as donor nuclei. Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus-oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei. Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell-cytoplast complexes (SC-CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 micros. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 degrees C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24-35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level. PMID- 12374132 TI - Risk factors for postpartum ovarian cysts and their spontaneous recovery or persistence in lactating dairy cows. AB - Cystic ovarian disease is a major cause of reproductive failure and economic loss for the dairy industry. Many cysts that develop during the early postpartum period regress spontaneously. However, it is difficult to decide at what point it would be more cost effective to treat ovarian cysts than to wait for spontaneous recovery. The objective of this study was to analyze risk factors for the development of the ovarian cystic condition during early and late postpartum, and for its persistence or recovery during the pre-service period in lactating dairy cows. Using multiple logistic regression, we analyzed data derived from 873 lactating dairy cows from a single herd. An ovarian cyst was diagnosed if it was possible to observe a single follicular structure with an antrum diameter > or = 25 mm in the absence of a corpus luteum in three sonograms performed at 7-day intervals. The cystic condition was denoted as early if the cyst was diagnosed 43 49 days postpartum, and late if detected 57-63-day postpartum. Spontaneous cyst regression before 60-day postpartum was regarded as early cystic recovery. For the early cystic group, there were no significant effects of lactation number, body condition score on prepartum Day 60, at parturition or on postpartum Day 30, or of body condition loss from parturition to 30-day postpartum. Cows calving in summer were 2.6 times more likely to develop ovarian cysts than those giving birth in winter. The risk of having a cyst was 1.9 times higher in cows with an abnormal puerperium. A 1-kg increase in milk yield raised the risk of cysts by a factor of 1.05. A 1-unit increase in body condition score (scale from 1 to 5) from prepartum Day 60 to parturition increased the risk of cyst development 8.4 times. Milk production and lactation number were negatively correlated with spontaneous early cyst recovery. A 1-kg decrease in milk production increased the probability of cyst recovery by a factor of 1.06, and a 1-unit drop in lactation number was associated with a 1.4-fold increased probability of cyst recovery. For the late cystic group, there were no significant effects of abnormal puerperium and body score data, except for a prepartum change in body score. Calving season (Odds ratio: 2.3), lactation number (Odds ratio: 1.36), increased milk production (Odds ratio: 1.05) and increased body condition score during the prepartum period (Odds ratio: 4.3) were all related to an increased risk of ovarian cysts. The probability of having a late cyst was 36.6 times greater in cows with early cysts. These findings suggest that it would be profitable to treat multiparous cows having cysts very early in the postpartum period, while treatment of primiparous cows should be delayed, at least until the end of the pre-service period, to provide the opportunity for spontaneous recovery. PMID- 12374133 TI - Clinical Trials Council assesses nuclear medicine participation. PMID- 12374134 TI - Dose calculation pitfall. PMID- 12374135 TI - Corn oil emulsion as a cholecystagogue. PMID- 12374136 TI - A twist of fate. PMID- 12374137 TI - An assessment of guidelines for prevention of ischemic stroke. AB - OBJECTIVE: To compare methods and key management recommendations from recent stroke prevention guidelines. METHODS: Systematic review of guidelines for prevention of ischemic stroke published in English between 1996 and 2001 was conducted, and recommendations were independently abstracted and compared. RESULTS: Among 22 stroke prevention guidelines, information was provided about panel selection in 24%, funding source in 36%, consensus methods in 33%, and quantitative risk/benefit estimates in 38%. Eleven recommended anticoagulation for patients with atrial fibrillation at high risk for stroke, but eight different sets of criteria to identify high-risk patients were proposed. Recommendations regarding carotid endarterectomy for asymptomatic stenosis varied from general endorsement in a setting of low perioperative risk to routinely withholding surgery. All nine relevant guidelines endorsed aspirin in dosages between 50 and 325 mg/day for initial antiplatelet therapy following cerebral ischemia; six also suggested other antiplatelet agents as options for initial therapy. CONCLUSIONS: Current stroke prevention guidelines do not provide adequate methodologic information to permit assessment of their quality, potential bias, and clinical applicability. Management recommendations are relatively consistent but differ in several important areas. PMID- 12374138 TI - Familial infantile bilateral striatal necrosis: clinical features and response to biotin treatment. AB - BACKGROUND: Infantile bilateral striatal necrosis (IBSN) encompasses several syndromes of bilateral symmetric, spongy degeneration of the caudate nucleus, putamen, and globus pallidus. The familial form of IBSN is rare, and inheritance is either autosomal recessive or maternal. METHOD: The authors describe an Israeli Bedouin kindred in which 15 children born to consanguineous parents were affected with familial IBSN. They evaluated the clinical and radiologic evolution of the disease in 11 patients and the cerebral pathologic findings in one patient. Three of the children were treated with oral biotin 100 mg/day. RESULTS: Inheritance was apparently autosomal recessive. The untreated children had a similar clinical picture including developmental arrest beginning at the age of 7 to 15 months, choreoathetosis, and dysphagia. Pendular nystagmus appeared at a late stage. MRI, performed at various stages of the disease, showed severe basal ganglia atrophy. Postmortem study in one patient showed severe atrophy of the lenticular nuclei with gliosis and loss of neurons. Biotin, 100 mg/day, administered to the proband over a period of 15 months, may have slowed progression. In two other children treatment was initiated earlier and appeared to arrest or improve disease. CONCLUSIONS: Familial infantile bilateral striatal necrosis was inherited as an autosomal recessive trait. Clinical features included developmental arrest, dysphagia, and choreoathetosis. Imaging and pathology showed atrophy and degeneration of the basal ganglia. Oral biotin may have benefited three children. PMID- 12374139 TI - Calcium phosphate crystallization under terrestrial and microgravity conditions. AB - Calcium phosphate crystalline powders grown under terrestrial and space (EURECA 1992-1993 flight) conditions in the Solution Growth Facility are analyzed and compared by optical and electron microscopy (scanning and transmission), electron and X-ray microdiffraction and microanalyses. On earth, only small, micrometer size scale, spherolites of hydroxyapatite (HAP) grow. In space, the HAP spherolites reach hundreds of micrometer. Also, octacalcium phosphate (OCP) spherolites up to 3 mm have been obtained. Computer modelling of diffusion in a real chamber has been performed. It suggests high spatial supersaturation gradients at zero gravity which may provide much higher local supersaturations on earth, where convection takes place. The analyses suggest that the dramatic difference between the terrestrial and space samples should come from much lower supersaturation in space. PMID- 12374140 TI - Terrestrial and space-grown HAP and OCP crystals: effect of growth conditions on perfection and morphology. AB - This paper reports comparative characterizations of calcium phosphate crystals grown on earth and in space. At the CaCl2 and KH2PO4 + K2HPO4 solution concentrations and the pH used, only hydroxyapatite (HAP) crystals grow under terrestrial condition while both HAP and octacalcium phosphate (OCP) crystals grew during the space experiment. The space-grown OCP crystals reach 3 mm in size, the space-grown HAP crystals reach sizes up to 100 times larger than the earth-grown crystallites. It was found also that the space-grown crystallites are more perfect than the terrestrial ones, being more stable under electron beam during HRTEM examination. Spherolites of hydroxyapatite consist of small and thin HAP crystals with different orientations. Space-grown OCP crystals containing almost pure OCP phase show strong striations along the c direction due to thickness variations. Terrestrial OCP crystals grown at lowest supersaturation on earth may be almost as large as the space-grown ones, possess a regular habit and are homogeneous in thickness. However, they always contain substantial regions of HAP structure. Also, in these crystals electron irradiation induces phase transformation from crystalline to amorphous (disordered) state during transmission electron microscopy observations. In the space-grown crystals, such transformation needs longer radiation time. We believe that the differences described above come from much lower supersaturation and different pH for crystals nucleating and growing in space compared to those formed on earth. PMID- 12374141 TI - Depression program found more effective but more costly than usual care. PMID- 12374142 TI - Landmark collaboration yields diabetes guidelines in California. PMID- 12374143 TI - Lung surgery patients' hospital stays reduced. PMID- 12374144 TI - Surgeon General calls for eliminating mental health disparities. PMID- 12374145 TI - Reconciling patients' rights and God's wisdom: medical decision making in Pakistan. PMID- 12374146 TI - Habermas's proceduralism tries to tackle bioethics. PMID- 12374147 TI - Responding to the nightmare of bioterrorism. PMID- 12374148 TI - Public health in the age of bioterrorism. PMID- 12374149 TI - Embryonic stem cell research in Jewish law. PMID- 12374150 TI - Perceptions of intentional wrongdoing and peer reporting behavior among registered nurses. AB - How a person perceives a wrongdoing being committed by a coworker will affect whether the incident is reported within the organization. A significant factor that may influence the decision to report a wrongdoing is the perceived intentionality of the wrongdoer. This study sought to examine if differences in perceptions of a wrongdoing could affect the disclosure of unethical behavior. Three hundred seventy-two registered nurses (N=372) responded to a survey consisting of both intentional and unintentional wrongdoings that could occur by a nurse. Results of a paired t-test were as predicted. More wide ranging revelations found that respondents were more likely to discuss the unintentional wrongdoings with the wrongdoer in lieu of officially reporting to an immediate supervisor, or a member of upper management. Discussion, limitations, and suggestions for future research are provided. PMID- 12374151 TI - Restraining the paternalism of attorneys and families in end-of-life decision making while recognizing that patients want more than just autonomy. PMID- 12374152 TI - Cyberadvice: the ethical implications of giving professional advice over the internet. PMID- 12374154 TI - Competing clinical trials in the same institution: ethical issues in subject selection and informed consent. PMID- 12374155 TI - When IRBs disagree: waiving parental consent for sexual health research on adolescents. PMID- 12374156 TI - How shall we die? PMID- 12374157 TI - Where the president's ethics lecture went wrong. PMID- 12374158 TI - Challenging Singer. PMID- 12374159 TI - Reproductive and therapeutic cloning. PMID- 12374160 TI - "We've decided to have a clone". PMID- 12374161 TI - Take care when using prisoners as research subjects. PMID- 12374162 TI - Curiouser and curiouser: teshuvah on genetic engineering. PMID- 12374163 TI - The synagogue as health proxy: a proposal. PMID- 12374164 TI - Artificial hydration and nutrition: revisiting the Dorff and Reisner teshuvot. PMID- 12374165 TI - Responses to Leonard Sharzer. PMID- 12374166 TI - Theological reflections on the end of life: a theologian's address to physicians. PMID- 12374167 TI - It isn't yours: campaigners call for a ban on all genetic patents. PMID- 12374168 TI - Tough choices: would a separate measles vaccination be a disaster for Britain? PMID- 12374169 TI - Come clean: Britain's stance on MMR won't wash, and people know it. PMID- 12374170 TI - Don't mention the 'c' word. PMID- 12374171 TI - We can copy cats: but if you think cloning will bring back a pet you'll be disappointed. PMID- 12374172 TI - Catch-22: this gene therapy could work so well it'll have to be banned. PMID- 12374173 TI - Yes, Prime Minister: be open about the science, but don't expect an easy ride. PMID- 12374174 TI - Institutional review boards and financial conflicts of interest. PMID- 12374175 TI - Research subjects to be told about conflicts of interest. PMID- 12374176 TI - University later admits that regulations were not followed. PMID- 12374177 TI - Lawsuit claims that informed consent procedures did not follow regulations. PMID- 12374178 TI - Agency proposes to combat "IRB shopping" with a new regulation. PMID- 12374179 TI - IRBs, data sharing, and privacy of human subjects. PMID- 12374180 TI - IRBs and financial conflicts of interest. PMID- 12374181 TI - Research administrator should not serve on institutional review board. PMID- 12374182 TI - Multiple sources for financial damages after research subject dies in experiment. PMID- 12374183 TI - Ethnicity is issue in new law affecting research with children as subjects. PMID- 12374184 TI - What do patients, their relatives and medical staff know about genetic therapy? AB - OBJECTIVES: To investigate the knowledge of, and attitudes towards gene therapy of cancer patients, their relatives, and the staff treating the patients. DESIGN: Cross sectional questionnaire study. SUBJECTS: a) One hundred patients with ovarian cancer either 1) in current treatment with chemotherapy, or 2) in follow up after chemotherapy. b) The relatives accompanying these patients to clinic. c) Fifty doctors and fifty nurses at the hospital in question. MAIN OUTCOME MEASURES: Knowledge about genes and gene therapy, attitudes toward gene therapy and its introduction in the NHS, willingness to accept additional side-effects for a given-therapeutic gain. RESULTS: Patients and relatives have only limited knowledge about genes and gene therapy, but knowledge is also limited about how chemotherapy works. The attitude towards gene therapy is positive, and the worry about gene therapy being like 'playing God' is accepted by less than 10%. There is a strong belief that if gene therapy is effective it should be paid by the NHS (over 85% agree with this). PMID- 12374185 TI - Moral sensibility and ethics. PMID- 12374186 TI - Good practice in consent. PMID- 12374187 TI - Towards the abolition of man: the voice of disabled persons cannot be ignored. PMID- 12374188 TI - Mimicking gangliosides by design: mimics of GM1 headgroup. AB - Detailed knowledge of the three-dimensional structure of ganglioside headgroups has allowed the successful design of structural and functional mimics of ganglioside GMI oligosaccharide. Our recent work in this area is reviewed in this paper. PMID- 12374189 TI - Biochemical and structural information transduction at the mesoscopic level in biointerfaces containing sphingolipids. AB - In this work we describe two aspects of molecular and supramolecular information transduction. The first is the biochemical and structural information content and transduction associated with sphingomyelinase activity. The results disclose a lipid-mediated cross-communication between the sphingomyelinase and phospholipase A2 pathways. In addition, the two-dimensional degradation of sphingomyelin by sphingomyelinase affects the surface topography and the latter modulates the enzyme activity. The second is the information contained in the compositionally driven lateral organization of whole glial and neuronal membrane interfaces. The myelin monolayer exhibits microheterogeneous topographical structuring and nonhomogeneous lateral thickness of phase separated regions, depending dynamically on the lateral surface pressure. On the other hand, the differential response of functional living cells depends on information contained in the molecular organization of the contacting membrane interface. PMID- 12374190 TI - Cooperative behavior of ganglioside molecules in model systems. AB - A concise discussion of the role of different geometrical conformational states in the process of self-assembling of gangliosides is given. The report focuses on the effects of the geometrical variations occurring in the head group region of gangliosides as reflected on the geometrical properties of the whole assembly. Collective phenomena happening at the water interfacial region are found to be coupled to the phase transition of the lipid moiety, that is, to the well-known order-disorder conformational transition involving the hydrophobic tails. The possible biological relevance of the head group bistability is envisaged. PMID- 12374191 TI - The role of sphingolipids in neuronal development: lessons from models of sphingolipid storage diseases. AB - The study of sphingolipids has undergone a renaissance over the past decade due to the realization that these lipids are involved in a variety a biological processes, such as differentiation, apoptosis, cell growth, and cell migration. In the nervous system, sphingolipids, particularly gangliosides, have attracted particular attention as they occur at high levels and their levels change in a developmentally regulated program. Despite the fact that a large body of data has accumulated on the expression and metabolism of individual gangliosides within specific brain regions, the role of individual gangliosides in neuronal development is still poorly understood, and their specific functions are only now beginning to be elucidated. In the present article, we discuss various aspects of our current knowledge concerning the involvement of sphingolipids and gangliosides in neuronal development, and then discuss some recent findings that shed light on the role of sphingolipids and gangliosides obtained with animal models of sphingolipid and other lysosomal storage diseases. PMID- 12374192 TI - Cationic glycosphingolipids in neuronal tissues and their possible biological significance. AB - During the course of studies on natural occurrence of sphingosine base in brain, cationic glycosphingolipids bound to carboxymethyl-Sephadex and eluted with triethylamine in organic solvents were isolated and characterized. Four classes of compounds were identified: (i) plasmalopsychosine-A and -B; (ii) glyceroplasmalopsychosine; (iii) glycosphingolipids having de-N-acetyl hexosamine, e.g., de-N-acetyl-Lc3Cer; (iv) glycosylsphingosine, i.e., lysoglycosphingolipid. Only two kinds, galactosylsphingosine (psychosine) and lactosylsphingosine, were found to occur naturally in brain. All these compounds were isolated from extract of brain white matter. Their occurrence, quantity, and distribution pattern differ from one species to another. Their quantity is much lower than that of regular acidic and neutral glycosphingolipids. They may interact with regular glycosphingolipids in glycosphingolipid-enriched microdomains to elicit signal transduction, to modify cellular phenotype, although studies along this line are highly limited at this time. PMID- 12374193 TI - Gangliosides with O-acetylated sialic acids in tumors of neuroectodermal origin. AB - Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues. PMID- 12374194 TI - Carbohydrate structural units in glycosphingolipids as receptors for Gal and GalNAc reactive lectins. AB - Glycosphingolipids (GSLs) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. Many of them are in the nervous system and function as important receptors in various life processes. During the past two decades, 11 mammalian structural units have been used to express the binding domain of applied lectins. They are: F, GalNAc alpha1 --> 3GalNAc; A, GalNAc alpha1 --> 3Gal; T, Gal beta1 --> 3GalNAc; I, Gal beta1 --> 3GlcNAc; II, Gal beta1 --> 4GlcNAc; B, Gal alpha1 --> 3Gal; E, Gal alpha1 --> 4Gal; L, Gal beta1 - > 4Glc; P, GalNAc beta1 --> 3Gal; S, GalNAc beta1 --> 4Gal, and Tn, GalNAc alpha1 --> Ser(Thr). Although 10 of them occur in GSLs, only 3 (Lbeta, Sbeta, and Tbeta) are found in human brain, and 2 (Lbeta and IIbeta) are present in the inner structures of human blood group active GSLs. In the families of gangliosides, Lbeta and IIbeta represent 55% of the total structural units, while the other three units (Tbeta, Palpha, and Sbeta) constitute the rest. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding specificities of Gal/GalNAc reactive lectins have been classified according to their highest affinity for the structural units and their binding properties expressed by decreasing order of reactivity. Hence, the binding relation between GSLs and Gal/GalNAc specific lectins can be established. PMID- 12374196 TI - Ceramide regulation of apoptosis versus differentiation: a walk on a fine line. Lessons from neurobiology. AB - One of the characteristics of ceramide-mediated biology is the variety of biological outcomes observed in response to its intracellular accumulation. The molecular mechanisms that govern the cell "decision-making" in response to ceramide remain largely unclear. In this perspective, the study of neural models has begun to provide important insight into the understanding of these mechanisms that regulate differentiation and cell death. Indeed, differentiation and cell death are among the most common effects elicited by ceramide in most cell types and in neural cells, too. Therefore, the lessons we may learn from the study of ceramide regulation of neurobiology would also shed light on the regulation of ceramide-mediated biology in other cellular models. Since increasing evidence links aberrant metabolism of ceramide to different pathologies, the understanding of the mechanisms underlying these events may represent the key to the design of novel therapeutic approaches. PMID- 12374195 TI - Ceramide in apoptosis: a revisited role. AB - The sphingolipid ceramide has recently emerged as a new transducer or modulator of apoptotic cell death. This function, however, has recently been challenged. Here, in the light of recent observations, the role of ceramide in apoptosis signaling is discussed. PMID- 12374197 TI - Lysophospholipid receptors in the nervous system. AB - The lysophospholipid mediators, lysophosphatidic acid (LPA) and sphingosine-1 phosphate (S1P), are responsible for cell signaling in diverse pathways including survival, proliferation, motility, and differentiation. Most of this signaling occurs through an eight-member family of G-protein coupled receptors once known as the endothelial differentiation gene (EDG) family. More recently, the EDG receptors have been divided into two subfamilies: the lysophosphatidic acid subfamily, which includes LPA1, (EDG-2/VZG-1), LPA2 (EDG-4), and LPA3 (EDG-7), and the sphingosine-1-phosphate receptor subfamily, which includes S1P1 (EDG-1), S1P2 (EDG-5/H218/AGR16), S1P3 (EDG-3), S1P4 (EDG-6), and S1P5 (EDG-8/NRG-1). The ubiquitous expression of these receptors across species, coupled with their diverse cellular functions, has made lysophospholipid receptors an important focus of signal transduction research. Neuroscientists have recently begun to explore the role of lysophospholipid receptors in a number of cell types; this research has implicated these receptors in the survival, migration, and differentiation of cells in the mammalian nervous system. PMID- 12374198 TI - Understanding the stepwise synthesis of glycolipids. AB - Glycolipid expression is highly regulated during development and differeniation. The control relies mainly on transcriptional modulation of key glycosyltransferases acting at the branching points of the pathway of biosynthesis. Transferases are Golgi residents that depend on N-glycosylation and oligosaccharide processing for proper folding in the endoplasmic reticulum. The N terminal domain bears information for their transport to the Golgi, retention in the organelle and differential concentration in sub-Golgi compartments. In the Golgi, some transferases associate forming functional multienzyme complexes. It is envisaged that the machinery for synthesis in the Golgi complex, and its dynamics, constitute a potential target for fine tuning of the control of glycolipid expression according to cell demands. PMID- 12374199 TI - Ganglioside function in calcium homeostasis and signaling. AB - Ganglioside function in eukaryotic cells encompasses a variety of modulatory interactions related to both development and mature cellular behavior. In relation to the nervous system this includes induction of neurite outgrowth and trophic/neuroprotective phenomena; more generally this applies to ganglioside effects on receptor function, adhesion reactions, and signal transduction mechanisms in neural and extraneural systems. Underlying many of these trophic effects are ganglioside-induced changes in cellular calcium, accomplished through modulation of Ca2+ influx channels, Ca2+ exchange proteins, and various Ca2+ dependent enzymes that are altered through association with gangliosides. A clear distinction needs to be drawn between intrinsic functions of gangliosides as naturally expressed by the cell and activities created by application of exogenous ganglioside(s) that may or may not reflect natural function. This review attempts to summarize findings in this area and point to possible future directions of research. PMID- 12374200 TI - Recent development in mammalian sialidase molecular biology. AB - This review summarizes the recent research development on mammalian sialidase molecular cloning. Sialic acid-containing compounds are involved in several physiological processes, and sialidases, as glycohydrolytic enzymes that remove sialic acid residues, play a pivotal role as well. Sialidases hydrolyze the nonreducing, terminal sialic acid linkage in various natural substrates, such as glycoproteins, glycolipids, gangliosides, and polysaccharides. Mammalian sialidases are present in several tissues/organs and cells with a typical subcellular distribution: they are the lysosomal, the cytosolic, and the plasma membrane-associated sialidases. Starting in 1993, 12 different mammalian sialidases have been cloned and sequenced. A comparison of their amino acid sequences revealed the presence of highly conserved regions. These conserved regions are shared with viral and microbial sialidases that have been characterized at three-dimensional structural level, allowing us to perform the molecular modeling of the mammalian proteins and suggesting a monophyletic origin of the sialidase enzymes. Overall, the availability of the cDNA species encoding mammalian sialidases is an important step leading toward a comprehensive picture of the relationships between the structure and biological function of these enzymes. PMID- 12374201 TI - Sphingolipids in neuroblastoma: their role in drug resistance mechanisms. AB - Disseminated neuroblastoma usually calls for chemotherapy as the primary approach for treatment. Treatment failure is often attributable to drug resistance. This involves a variety of cellular mechanisms, including increased drug efflux through expression of ATP-binding cassette transporters (e.g., P-glycoprotein) and the inability of tumor cells to activate or propagate the apoptotic response. In recent years it has become apparent that sphingolipid metabolism and the generation of sphingolipid species, such as ceramide, also play a role in drug resistance. This may involve an autonomous mechanism, related to direct effects of sphingolipids on the apoptotic response, but also a subtle interplay between sphingolipids and ATP-binding cassette transporters. Here, we present an overview of the current understanding of the multiple levels at which sphingolipids function in drug resistance, with an emphasis on sphingolipid function in neuroblastoma and how modulation of sphingolipid metabolism may be used as a novel treatment paradigm. PMID- 12374202 TI - A new aspect in glycolipid biology: glycosphingolipids as antigens recognized by T lymphocytes. AB - T cells may recognize a large variety of ligands with different chemical structures. Recently, glycosphingolipids have also been shown to stimulate human T lymphocytes. Recognition of glycosphingolipids is restricted by the nonpolymorphic CD1 molecules, expressed by professional antigen-presenting cells and by macrophages infiltrating inflammatory sites. CD1 molecules have a structure resembling that of classical MHC class I molecules, with the terminal extracellular domains characterized by two antiparallel alpha helices placed on two hydrophobic pockets. The glycosphingolipids bound to CD1 insert the lipid tails in the two pockets and position the hydrophilic head on the external part of CD1. The TCR interacts with aminoacids present in the two alpha helices and with residues provided by the carbohydrate moiety of glycosphingolipids and discriminates their structural variations. T cells recognizing self glycosphingolipids release proinflammatory cytokines and may have a pathogenetic role in autoimmune diseases such as multiple sclerosis. PMID- 12374203 TI - The origin of anti-GM1 antibodies in neuropathies: the "binding site drift" hypothesis. AB - Elevated titers of serum antibodies against GM1-ganglioside are associated with a variety of autoimmune neuropathies. The origin of these autoantibodies is still unknown, although there is evidence that they are produced by CD5+ B-lymphocytes and that antigen mimicry is involved. Anti-GM, IgM-antibodies in the normal human immunological repertoire are low affinity antibodies that cross-react with other glycoconjugates carrying Gal beta1-3GalNAc and probably do not have GM1-mediated biological activity. Other anti-GM1 IgM-antibodies with higher affinity and/or different fine specificity are present in patients with motor syndromes. Based on our studies of structural requirement for binding, we hypothesize that disease associated anti-GM1 antibodies originate at random by mutations affecting the binding site of naturally-occurring ones. The hypothesis is conceptually similar to the established phenomenon of "genetic drift" in species evolutionary biology and is therefore termed "binding site drift". PMID- 12374205 TI - Metabolic formation of ceramide-1-phosphate in cerebellar granule cells: evidence for the phosphorylation of ceramide by different metabolic pathways. AB - Aiming to investigate the possible production of ceramide-1-phosphate from complex sphingolipid metabolism in neurons, we administered radiolabeled sphingolipids to cerebellar granule cells and inspected the formation of labeled ceramide-1-phosphate in different experimental conditions. We report that differentiated granule cells are capable to form Cer-1-P via ceramide derived from SM degradation at the plasma membrane level. Moreover we observed that ceramide-1-phosphate can be also produced from a metabolic pathway not involving SM degradation. In particular, we obtained evidence that ceramide, synthesized via the recycling of sphingosine produced from ganglioside catabolism, can also be the precursor of ceramide-1-phosphate. We also found that undifferentiated and differentiated granule cells display different capacities to phosphorylate Cer produced by the two different metabolic pathways. The results here obtained demonstrate that cerebellar neurons are able to metabolically produce ceramide-1 phosphate and support that this molecule may serve a potential role in sphingoid mediated signaling in the nervous system. PMID- 12374204 TI - Complex gangliosides as autoantibody targets at the neuromuscular junction in Miller Fisher syndrome: a current perspective. AB - Glycosphingolipid biology has increasingly interfaced with the field of human autoimmune neuropathy over the last two decades. There are currently over 20 distinct glycolipids that have been identified as autoantibody targets in a wide range of clinical neuropathy syndromes. This review sets out the clinical and experimental background to one interesting example of anti-glycolipid antibody associated neuropathy termed Miller Fisher syndrome. This syndrome, comprising the triad of ataxia, areflexia, and ophthalmoplegia, correlates highly with the presence of serum anti-GQ1b antibodies, arising through molecular mimicry with microbial oligosaccharides. Anti-GQ1b antibodies mediate neural injury through binding to GQ1b-enriched sites in the peripheral nervous system, including extraocular nerves. Animal experimental evidence, along with a hypothetical background, indicates the motor nerve terminal may be a key site for anti-GQ1b antibody binding with consequent defects in synaptic transmission, as occurs in botulism and other toxinopathies. Our work in recent years on this hypothesis is summarized. PMID- 12374206 TI - Identification of ceramide binding proteins in neuronal cells: a critical point of view. AB - Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl diazirinyl)phenyl-ethyl-2-[35S]-2-thioacetyl-D-erythro-C18-sphingosine ([35S]-TDS ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain. PMID- 12374207 TI - Palmitic is the main fatty acid carried by lipids of detergent-resistant membrane fractions from neural and non-neural cells. AB - Lipids extracted from detergent-resistant membrane fractions, thought to derive from membrane domains, were analyzed for fatty acid composition. The proportion of palmitic acid in fractions isolated from neurons (cerebellar granule cells) and from neural-like cell lines (neuroblastomaglioma NG108-15) nearly doubled (reaching about 54% of total fatty acids) with respect to cell WCL, indicating their enrichment in palmitic acid-carrying lipids. The proportion of palmitic acid in detergent-resistant fractions obtained from caveolin-transfected NG108-15 cells was comparable with that obtained from caveolin-negative cells, ruling out a specific role of this protein in recruiting palmitoylated lipid species. The enrichment in palmitic acid was remarked also in membrane fractions isolated from non-neuronal cell lines (A431) using either detergents or detergent-free techniques. Lipid fractionation and mass spectrometry experiments show that palmitic acid-rich phosphatidylcholine species are responsible of the peculiar fatty acid composition of these fractions. All together these results suggest that the enrichment in palmitic acid-rich phosphatidylcholine species is a common feature of neural and non-neural cell lines and may play a major role in the biogenesis of membrane domains. PMID- 12374208 TI - CrmA protects against apoptosis and ceramide formation in PC12 cells. AB - TNF-alpha activated caspase 8 and caspase 3 in PC12 cells, leading to cell death by apoptosis (DNA fragmentation). TNF-alpha caspase activation and cell killing were blocked by transfection and overexpression of the viral protein CrmA, which specifically inhibits caspase 8. CrmA was also able to block the TNF-alpha induced increase in ceramide formation in PC12 cells. Conversely, if caspase 8 was activated by light-activated Rose Bengal, there was an increase in both ceramide and caspase 3-mediated apoptosis, which was blocked by CrmA overexpression. This suggested that caspase 8 increases ceramide either by increasing its synthesis or by activating sphingomyelinase. Since fumonisin B1 did not block and sphingomyelin decreased when ceramide increased, we concluded that activation of sphingomyelinase is the most likely mechanism. The Rose Bengal activation of caspase 8 and increased ceramide formation was blocked with IETD CHO, to show that reactive oxygen species (also generated by Rose Bengal) were not responsible for the observed increase in ceramide. Thus in PC12 pheochromocytoma cells, ceramide appears to amplify the death signal and there appears to be a sequence of events: TNF; TRADD, pro-caspase 8, caspase 8, sphingomyelinase, ceramide, caspase 3, apoptosis. PMID- 12374209 TI - Association of cellular prion protein with gangliosides in plasma membrane microdomains of neural and lymphocytic cells. AB - In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrPc presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrPc and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrPc-enriched microdomains. In lymphoblastoid T cells PrPc molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrPc was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrPc-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrPc immunoprecipitates. The physical association of PrPc with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrPc-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions. PMID- 12374210 TI - Different metabolic effects of ganglioside GM1 in brain synaptosomes and phagocytic cells. AB - The metabolic effects of ganglioside GM1 were found to be quite different in brain synaptosomes and phagocytic cells. Incubation of rat brain cortex synaptosomes with GM1 was shown to decrease the production of reactive oxygen species induced by Fe2+-H2O2 system and measured by chemiluminometric method in the presence of luminol. Gangliosides GM1, GD1a, and GT1b significantly diminished the induced accumulation of lipid peroxidation product in brain synaptosomes, but protein kinase inhibitor (polymyxin B) abolished this effect. Incubation with antioxidants or GM1 significantly diminished the increase of 45Ca2+ influx and oxidative inactivation of Na+,K+-ATPase in brain synaptosomes exposed to glutamate, the effect of GM1 was concentration-dependent in the range 10(-11)-10(-8) M. But the incubation of human neutrophils and mouse peritoneal macrophages with 10(-11)-10(-10) M GM1, on the contrary, increased several times the luminol-dependent chemiluminescence response of these cells to activation by low concentrations of 12-myristate-13-acetate phorbol ester. The opposite effects of GM1 in the nerve endings and phagocytic cells seem to be protective in both cases as the inhibition of reactive oxygen species production in the nerve cells may enhance their viability in damaged brain, while the intensification of their production in phagocytic cells may promote the resistance of organism to infection. PMID- 12374211 TI - Influence of gangliosides on the IL-2- and IL-4-dependent cell proliferation. AB - Ganglioside-induced apoptosis in the cells of IL-2-dependent cytotoxic murine cell line CTLL-2 was shown to be caspase dependent: GM1-, GM2-, and GD3-induced suppression of cell proliferation was cancelled by a general caspase inhibitor Z VAD-FMK. Ganglioside-induced apoptosis pathways are different for different individual glycolipids; the differences exist both at the initiation and effector stages of the caspase cascade. Only for GM1-induced process, molecular mechanisms of signal transduction coincide with the ones for CD95 and TNFalpha: the participation of both the main initiation caspases 8, 1, and 4, and caspases 3 and 9 as well, has been shown. Caspase 3 participates in the pathway induced by GM3, GD1a, GD1b, and GT1b, but not by GM2. As morphological features show, tumor associated ganglioside GM2 is also a stimulus of programmed cell death (PCD) for CTLL-2 cell line: addition of GM2 into cell culture has resulted in appearance of annexin V-positive cells and in accumulation of DNA breaks (shown by the TUNEL direct dyeing of the open ends). But a caspase 3 inhibitor Z-DEVD-FMK did not restore the cell proliferation suppressed by GM2, and addition of a fluorescent substrate of caspase 3 Ac-DEVD-AFC did not result in the fluorescence development. So caspase 3 does not participate in downstream pathways of GM2 induced cell apoptosis, and a PCD-effector system other than the apoptosome mediated one is involved here. PMID- 12374212 TI - Effects of ganglioside GM3 on phospholipid turnover of human leukemic J6-2 cells. AB - Ganglioside GM3 was reported to induce the differentiation of HL-60 cells to differentiate along the macrophage-monocytic route. We used human monocytoid leukemia J6-2 cells and successfully induced differentiation by GM3. Because differentiation is accompanied by retarded growth rate and cell cycle is intimately related to phospholipid metabolism, so we explored how GM3 was related to phospholipid metabolism. By using [32P]Pi, [3H-CH3]choline, [3H-CH3]SAM, and [3H]inositol as radioactive tracers, we studied the turnover changes of phospholipids and their metabolites induced by GM3. For the morphological changes of differentiation to occur, the cells had to be treated with GM3 at a concentration of 50 microM for 5-6 days, but the phospholipid changes occurred at a very early stage of GM3 treatment (only 1 h). Our results indicate that GM3 stimulated PE methylation pathway inhibited both CDP-choline pathway and PI cycle. The phospholipid changes may constitute the early events in differentiation induced by GM3. PMID- 12374213 TI - Molecular cloning and characterization of galactosylceramide expression factor-1 (GEF-1). AB - A rat brain cDNA clone has been isolated using a eukaryotic cell transient expression system with anti-galactosylceramide (GalCer) monoclonal antibody (MAb), that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), but no homology to UDP-galactose: ceramide galactosyltransferase. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes and cell growth suppression. Overexpression of GEF 1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of CGT increased significantly in MDCK/GEF-1 cells compared with control cells. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer and sulfatide expression in the epithelial cells as well as in the brain. PMID- 12374214 TI - Study of interaction of GM2 activator protein with GM2 using circular dichroism and fluorescence spectroscopy. AB - GM2 activator protein (GM2AP) is a cofactor for stimulating the enzymatic hydrolysis of the glycolipid GM2 by beta-hexosaminidase A to produce GM3. We have examined the conformation of GM2AP before and after its interaction with GM2, GM3, and GA2 using circular dichroism and fluorescence spectroscopy techniques. In the presence of GM2, a blue shift of the fluorescence emission maximum and a strong decrease of molar ellipticity values in circular dichroism spectra were observed only at pH 4.5 and at GM2/GM2AP molar ratio higher than 10:1 (up to 50:1). These results suggest that GM2AP assumed a more organized alpha-helical conformation with the tryptophan residues moving from the polar medium toward the hydrophobic environment of the protein. The conformation of GM2AP in the presence of the downstream reaction product, GM3, or a less favorable substrate, GA2, clearly differed from that in the presence of GM2. The relationships between spectroscopic changes and enzymatic activity, herein discussed, strongly suggest that the specific conformation exhibited by GM2AP in the presence of GM2 is functional to serve as an activator for the enzymatic hydrolysis of GM2. PMID- 12374215 TI - Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model. AB - The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype. PMID- 12374216 TI - Stable transfection of GM1 synthase gene into GM1-deficient NG108-15 cells, CR-72 cells, rescues the responsiveness of Trk-neurotrophin receptor to its ligand, NGF. AB - Previous studies from this laboratory and others have suggested the evidences that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. The present study was aimed to examine whether Trk expressed in cells that are deficient in endogenous GM1 due to the mutation of GM1 synthase gene (NG-CR72 cells) is responsive to its ligand nerve growth factor and how genetic restoration of GM1 synthase gene by a stable transfection of the gene affects the function of the Trk protein. The data clearly showed that (1) confocal lazor microscopic studies disclosed NG-CR72 cells are really deficient in GM1, (2) stable transfection of GM1 synthase cDNA into these cells (NG-CR72G cells) restores the expression of GM1 in the cells, and (3) Trk protein is expressed in NG-CR72 cells but its location seemed not to be on the plasma membrane, whereas we clearly observed that the Trk protein is expressed on the plasma membrane in NG-CR72G cells. (4) NGF did not elicit the autophosphorylation of the Trk protein in GM1 deficient NG-CR72 cells but did elicit the activation of the Trk protein in NG-CR72G cells with an activation of mitogen activated protein kinase. These studies strongly suggested that GM1 is necessary for the normal expression of the Trk protein function and for normal targeting of the Trk protein to the plasma membrane. PMID- 12374217 TI - Effect of globotriaosyl ceramide fatty acid alpha-hydroxylation on the binding by verotoxin 1 and verotoxin 2. AB - Variation in the lipid moiety of the verotoxin (VT) receptor glycosphingolipid, globotriaosyl ceramide (Gb3) can modulate toxin binding. The binding of VT1 and VT2 to C18 and C22 alpha hydroxy and nonhydroxy fatty acid isoforms of Gb3 were compared using a receptor ELISA and a 125I-labeled toxin/glycolipid microtitre plate direct binding assay. Increased binding to the hydroxylated species, particularly C220H, was observed for both toxins. Increased RELISA binding at low glycolipid concentrations only, suggested the binding affinity is increased following Gb3 fatty acid hydroxylation. Nonlinear regression analysis of direct binding assay to these Gb3 isoforms confirmed the increased affinity of both toxins for the C22 hydroxylated Gb3. The capacity was also significantly increased. The increased binding of VTs for hydroxylated fatty acid Gb3 isoforms may be a factor in the selective renal pathology which can follow systemic verotoxemia, particularly in the mouse model. The more pronounced effect at lower glycolipid concentrations prompted investigation of VT1 binding affinity at different Gb3 concentrations. Unexpectedly, the VT1 Kd for Gb3 was found to decrease as an inverse function of the Gb3 concentration. This shows that glycolipids have "nonclassical" receptor properties. PMID- 12374218 TI - Glycosyltransferases in different brain regions during chick embryo development. AB - Glycosphingolipids, in particular gangliosides, play a crucial role in neuronal development and are known to change dramatically in total content and distribution in different brain areas during embryogenesis. In the present work we analyzed the activity of enzymes involved in the metabolism of gangliosides, at different periods of functional maturation in different regions of chick embryo brain. Our data demonstrate differences in the enzymatic activities in the examined areas; these differences might be correlated with the functional lateralization occurring in the brain during development. Significative differences were found in glycosphingolipid composition between controlateral cerebral hemispheres and optic lobes; these results together with previous data we found, contribute to reinforce our hypothesis on the occurrence of biochemical lateralization during early brain development. PMID- 12374219 TI - Association of GPI-anchored protein TAG-1 with src-family kinase Lyn in lipid rafts of cerebellar granule cells. AB - We have demonstrated that antibody-mediated crosslinking of GPI-anchored TAG-1 induced activation of src-family kinase Lyn and rapid tyrosine phosphorylation of an 80-kDa protein (p80), a putative substrate for Lyn, in the lipid raft fraction prepared from primary cerebellar cultures, suggesting the functional association of TAG-1 with Lyn in lipid rafts of the rat cerebellum. In this study, the association was confirmed using a cDNA expression system. TAG-1-expressing CHO transfectants exhibited enhanced self-aggregation and promoted neurite outgrowth of primary cerebellar cultures as a culture substrate. The anti-TAG-1 antibody co immunoprecipitated Lyn with TAG-1 and induced co-patching of TAG-1 with Lyn in both TAG-1 and Lyn-expressing CHO transfectants. Density gradient analysis revealed that TAG-1 is present in the lipid raft fraction of the CHO transfectants. Furthermore, pretreatment with a sphingolipid biosynthesis inhibitor ISP-1 reduced the extent of tyrosine phosphorylation of p80 by the antibody-mediated crosslinking of TAG-1. Immunocytochemical study showed that both TAG-1 and Lyn are present in cerebellar granule cells. These observations suggest that TAG-1 associates with Lyn in lipid rafts of rat cerebellar granule cells. PMID- 12374220 TI - Sphingolipid metabolism and caveolin expression in gonadotropin-releasing hormone expressing GN11 and gonadotropin-releasing hormone-secreting GT1-7 neuronal cells. AB - In this paper, we show that caveolin-1 is abundantly present in a cell line of immortalized gonadotropin-releasing hormone-expressing neurons (GN11). In contrast to GN11, caveolin is undetectable in a cognate cell line of immortalized gonadotropin-releasing hormone-secreting neurons (GT1-7). These two cell lines are characterized by a radically different sphingolipid metabolism. After incubation in the presence of tracer amount of [1-(3)H]sphingosine, GN11 and GT1 7 neurons incorporated similar amounts of radioactivity. In GT1-7 neurons, [1 (3)H]sphingosine metabolism was markedly oriented toward the biosynthesis of complex sphingolipids. In fact, almost all the radioactivity in the lipid extracts from GT1-7 cells was associated with biosynthetic products (ceramide, sphingomyelin, and glycosphingolipids). In particular glycosphingolipids represented more than 65% of total lipid radioactivity in these cells, and the main glycosphingolipid was GM3 ganglioside (about 47% of total lipid radioactivity). In the case of GN11 neurons, a high portion of [1 (3)H]sphingosine underwent complete degradation, as indicated by the formation of high levels of radioactive phosphatidylethanolamine (about 23% of lipid radioactivity). Moreover, the main complex sphingolipid in GN11 neurons was not a glycolipid, but sphingomyelin (its level in these cells, about 54% of lipid radioactivity, was two-fold higher than in GT1-7). Glycolipids, gangliosides in particular, were present in low amount (9.5% of lipid radioactivity) if compared with the cognate GT1-7 cell line, and GM3 was almost absent in GN11 neurons. Despite the radical differences in ganglioside and caveolin content, from both cell types a membrane fraction similarly enriched in sphingolipids was prepared. In the case of GN11 cells, this fraction was also enriched in caveolin. The presence of caveolin or GM3 may correlate with different functional properties linked to the stage of neuronal maturation, since GN11 and GT1-7 are representative, respectively, of immature, migrating, and differentiated, postmigratory gonadotropin-releasing hormone-positive neurons. PMID- 12374221 TI - Modulation of neuritogenesis by ganglioside-specific sialidase (Neu 3) in human neuroblastoma NB-1 cells. AB - Plasma membrane-associated sialidase (Neu 3), which specifically hydrolyzes gangliosides, is relatively abundantly present in the nervous system. To understand the role of Neu 3 in neuronal differentiation, we studied the relationship between neurite outgrowth and Neu 3 expression in human neuroblastoma NB-1 cells. The expression of Neu 3 in NB-1 cells increased when neurite outgrowth in these cells was induced by dibutyryl cAMP. While treatment with dibutyryl cAMP alone enhanced the outgrowth of dendrite-like processes, transfection of the Neu 3 gave rise to a more prominent outgrowth of neurites with axon-like characteristics, even in the absence of dibutyryl cAMP. Neu 3 induction by dibutyryl cAMP is probably attributable, in part, to transactivation of the Neu 3 gene through cAMP responsive elements in the 5'-upstream region, as revealed by the promotor activity assay using Neu 3 promotor expression plasmid. These results indicate that Neu 3 regulates neurite formation in NB-1 cells, and suggest that this effect may be enhanced by dibutyryl cAMP via a cAMP-dependent pathway. PMID- 12374223 TI - Influence of short-chain fatty acids on iron absorption by proximal colon. AB - BACKGROUND: Short-chain fatty acids produced by bacterial fermentation in the colon enhance the local absorption of cations, such as calcium, that could be used to improve the bioavailability of iron if a significant colonic absorption of iron were to occur. METHODS: Iron (iron gluconate, 100 microM) absorption by the caecum of the rat was compared with that in proximal sites of the small bowel using the Ussing chamber model; the influence of probiotic bacteria (Propionibacterium freudenreichii) on iron absorption was assessed and compared with that of two of their fermentation products (acetic and propionic acids) using the Ussing chamber and the ligated colon with gamma emitting iron as experimental models. RESULTS: The caecum absorbed less iron than the duodenum, but significantly more than the jejunum and ileum. This occurred mainly through an enhanced mucosal transfer of iron uptake. Propionibacteria enhanced iron absorption from the proximal colon; the same effect was observed in the presence of viable bacteria, or the culture medium free of viable bacteria, or acetate and propionate or propionate alone. CONCLUSIONS: The proximal colon could be a significant site available for iron absorption; this absorption can be enhanced by local production of short-chain fatty acids such as propionate. PMID- 12374222 TI - Anti-ganglioside antibodies bind with enhanced affinity to gangliosides containing very long chain fatty acids. AB - Gangliosides function in both physiological and pathological molecular recognition. Although much research has focused on the role of ganglioside glycans in recognition, fewer studies have addressed the role of the ceramide moiety. Ceramides of major brain gangliosides are composed predominantly of monounsaturated 18-carbon and 20-carbon long chain bases with a saturated 18 carbon fatty acid amide. In contrast, gangliosides of X-linked adrenoleukodystrophy patients are characterized by abnormal very long chain fatty acids that are proposed to be associated with autoimmune inflammation. In the current study we synthesized and characterized derivatives of the major brain ganglioside GD1a bearing defined very long chain fatty acid amides (C24:0, C24:1, and C26:0). When tested in a solid phase binding assay in the presence of auxiliary membrane lipids, GD1a species with long chain fatty acids were up to 8 fold more potent than normal brain GD1a in binding four different anti-GD1a monoclonal antibodies. These data support the hypothesis that gangliosides bearing very long chain fatty acids are differentially displayed on membranes, which may lead to altered antigenicity. PMID- 12374224 TI - Autoimmune enteropathy in an adult with autoimmune multisystemic involvement. AB - We describe the case of a 58-year-old woman with autoimmune enteropathy associated with thyroiditis, gastritis, transitory neutropenia, sicca syndrome and severe axonal polyneuropathy of autoimmune origin. Enterocyte autoantibodies were not detected. However, predisposition to autoimmune disease was indicated by the presence of high titres of anti-gastric parietal cell, anti-thyroglobulin, anti-thyroid peroxidase and anti-neutrophil antibodies. CD4+ and CD8+ lymphocytes were equally distributed in the lamina propria of the small intestine, but CD8+ cells were highly represented among intraepithelial lymphocytes. PMID- 12374225 TI - Immunolocalization of cholecystokinin-2 receptors in rat gastric mucosa. AB - BACKGROUND: Gastrin exerts trophic effects on the gastric mucosa by mechanisms not yet completely elucidated. Our aim was to localize the cholecystokinin-2 (CCK2) receptor in epithelial cells of foetal and adult rat stomachs in order to determine the cell types that are directly affected by gastrin. METHODS: Gastric tissue was subjected to indirect double immunofluorescence staining with antiserum against the C-terminal decapeptide of the CCK2 receptor and antibodies against 5' bromo-2-deoxyuridine, which had been injected into the rats I h before they were killed, the acid pump H,K-ATPase, the membrane-cytoskeletal linker ezrin, pepsin/pepsinogen or histidine decarboxylase. RESULTS: Undifferentiated foetal gastric epithelial cells expressed CCK2 receptors, whereas stem cells of adult gastric glands did not exhibit immunoreactivity. However, other epithelial cells in the progenitor zone of adult gastric glands did express CCK2 receptors. Some of these cells were faintly stained for H,K-ATPase; pepsin/pepsinogen was also detected in this region. Parietal cells in the isthmus/pit region of the glands contained ezrin, and some showed weak immunoreactivity for the CCK2 receptor. As expected, enterochromaffin-like cells also expressed CCK2 receptors. CONCLUSION: Our findings are consistent with the hypothesis that a CCK2 receptor mediates direct effects of gastrin on gastric epithelial cells during both stomach organogenesis and adult life. PMID- 12374226 TI - Surgery for late-onset ulcerative colitis: predictors of short-term outcome. AB - BACKGROUND: Onset of ulcerative colitis and Crohn disease after the age of 65 (late-onset disease) is not common, and is usually associated with a worse prognosis. We review our experience with late-onset ulcerative colitis and define the predictors of short-term outcome. METHODS: A retrospective analysis of our surgical experience with 33 patients suffering from late-onset ulcerative colitis. The medical records of 17 women and 16 men who had surgery between 1984 and 1999 were reviewed for age at surgery, sex, duration of disease, extent of disease, indications for surgery, surgical procedures and outcome. Additionally, we identified predictors of outcome. RESULTS: The median age at surgery was 74 years (range 65-83). The most common indication for surgery was refractoriness to medical treatment. There were 4 deaths for a mortality rate of 12%, and 7 major complications. There was no mortality for elective procedures. On univariate analysis, albumin levels of 2.8 g/dl or less and urgent surgery were predictors of poor outcome. Disease of short duration (3 years or less from onset of disease to surgery) was also associated with a poor outcome, but this did not reach statistical significance. CONCLUSIONS: We conclude that in the elderly population suffering from late-onset ulcerative colitis and requiring an operation, urgent surgery and hypoalbuminemia are predictors of adverse outcome. Age at surgery, sex and the extent of colonic involvement did not influence outcome. Low complication and death rates should be expected for elective procedures in the elderly. PMID- 12374227 TI - Comparison of faecal and intestinal concentrations of granulocyte marker protein and localization of gastrointestinal tumours in rats. AB - BACKGROUND: Increased faecal concentrations of the granulocyte marker protein (GMP) have been found in rats with azoxymethane (AOM) induced carcinoma of the colon, but the origin of this GMP is unknown. The aims were to investigate the concentrations of GMP in different parts of the gastrointestinal (GI) tract in rats with or without AOM-induced carcinoma and to correlate the GMP concentrations to localization of the carcinomas. METHODS: Nineteen rats were given intramuscular injections of AOM, 15 mg/kg, once weekly for 6 weeks and were killed after 22 weeks. Five rats that were not given AOM injections served as controls. RESULTS: All rats given AOM developed tumours; 18 developed a total of 33 adenocarcinomas in the GI tract and one developed an adenoma in the colon. Nine animals had carcinoma in the small bowel, seven of which also had carcinoma of the colon, and nine animals had carcinomas in the large bowel only. No other tumours were found. All except one of the animals that had carcinoma of the colon had elevated faecal GMP concentrations, and from week 11 there was a significant difference in the GMP values between the control group and the group that developed colon carcinoma. In all rats that developed carcinoma in the small bowel, the tumour was localized in the proximal part. In the rats that had been given AOM, the luminal GMP concentrations were significantly higher in the proximal part of the small bowel than in the distal part, but there were no significant differences in the GMP concentrations between animals with and without carcinoma in the small bowel. Sixteen rats developed a total of 24 carcinomas in the colon, and one rat developed an adenoma. Luminal GMP concentration in the distal part of the colon was elevated in all animals with carcinomas in the colon, and the GMP concentrations were significantly higher in the distal part than in the proximal part. Rats with one carcinoma in the colon had significantly lower GMP values in the distal part, compared to rats that had two carcinomas in the colon. CONCLUSIONS: The animal model described is suitable for further studies on many aspects of tumour development in the colon. Furthermore, it is likely that increased faecal GMP concentration in rats with colon carcinoma is a result of an inflammatory process in or around tumours. PMID- 12374228 TI - Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls. AB - BACKGROUND: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. METHODS: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. RESULTS: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls (P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria, the Enterobacteriaceae, the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. CONCLUSION: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon. PMID- 12374229 TI - Galacto-oligosaccharides stimulate the growth of bifidobacteria but fail to attenuate inflammation in experimental colitis in rats. AB - BACKGROUND: Galacto-oligosaccharides potentially attenuate colonic inflammation by two mechanisms: through beneficial effects on intestinal microflora and by increasing the colonic short-chain fatty acid concentration. The purpose of this study was to investigate the effects of galacto-oligosaccharides on the development of inflammation and on the growth of bifidobacteria in trinitrobenzene sulphonic acid (TNBS)-induced colitis, a model that has been shown to benefit from short-chain fatty acid administration and to be associated with alterations in the colonic microflora. METHODS: Rats were given daily either whey-derived or lactose-derived galacto-oligosaccharides (4 g kg(-1) day(-1), p.o.); starting 10 days before colitis induction, or dexamethasone (2 mg kg(-1) day(-1), s.c., a positive control), starting at colitis induction. Colon wet weight, macroscopic damage and myeloperoxidase activity were assessed 72 h after the induction of colitis. Faecal bifidobacteria were counted at the beginning of the study, and immediately before and 72 h after colitis induction. RESULTS: Galacto-oligosaccharides increased the colonic levels of bifidobacteria but also the levels of other bacterial species. Neither whey-derived nor lactose-derived galacto-oligosaccharides reduced the severity of inflammation. CONCLUSIONS: Galacto-oligosaccharides are able to modify gut microflora in severe TNBS-induced colitis, but unable to attenuate the inflammation. PMID- 12374230 TI - Mutations of the APC gene in human sporadic colorectal cancers. AB - BACKGROUND: Mutations of the APC gene are reported to occur frequently in sporadic colorectal adenomas and adenocarcinomas. We studied APC gene mutations in cases of human sporadic colorectal cancer in order to evaluate their correlation with pathologic characteristics and clinical prognosis. METHODS: Most of the mutations of the APC gene (95%) are nonsense or frame shift mutations, encoding for truncated APC proteins. For this reason, mutation detection of the APC gene was performed using the in vitro synthesized protein (IVSP) assay, analysing the region between nucleotide 2058 and nucleotide 5079 of the gene, containing the mutation cluster region. RESULTS: Out of 58 cases of colorectal cancer, 29 presented a mutated form of APC (mutation frequency 50%). We did not find a statistically significant correlation between APC gene mutation and age, sex, localization of the primary tumour, grading, Crohn-like lymphoid reaction or presence of residual adenoma. Tumours with low invasivity (Dukes' stages A and B) were less frequently mutated (12/27, 44.5%) than tumours of Dukes' stage C (15 out of 21, 71.4%), which developed macroscopically secondary metastasis with variable latency after surgery. Highly invasive tumours with synchronous metastases (Dukes' stage D) had, instead, a low frequency of APC mutations (20%, 2/10) (P = 0.02, compared with Dukes' stages A, B and C). CONCLUSIONS: These data suggest that more aggressive Dukes' stage D tumours develop metastasis by means of an unknown mechanism, independent of APC mutation. PMID- 12374231 TI - Colon neoplasia co-existing with coeliac disease in older patients: coincidental, probably; important, certainly. AB - BACKGROUND: Coeliac disease and colorectal neoplasia are both common, present most often in patients over 40 and cause similar symptoms. Greater awareness and early use of serological tests have improved the diagnosis of coeliac disease, but raise the concern that co-existing colorectal neoplasia may be missed. This study assessed the prevalence of colorectal neoplasia among patients with coeliac disease diagnosed after the age of 40 who presented with altered bowel habit or iron deficiency. METHODS: All patients meeting the above criteria underwent colonoscopy unless this or barium enema had been performed shortly before. RESULTS: Of 69 patients with coeliac disease undergoing colonoscopy, 7 (10%) had colon neoplasia: 5 had tubulovillous polyps, and 2 had carcinoma. The prevalence figures for coeliac patients undergoing colonoscopy with iron deficiency and altered bowel habit alone were 11% (5 of 47) and 10% (2 of 22), respectively None of a further 13 who had undergone previous colon investigation (all by barium enema) had neoplasia, although these were probably a selected population. The seven patients with colorectal neoplasia had not reported rectal bleeding. The prevalence of colorectal neoplasia was not significantly higher than in two series of non-coeliac patients undergoing colonoscopy for investigation of iron deficiency (12%) or altered bowel habit (8%). CONCLUSIONS: There is a high prevalence of colorectal neoplasia among older patients with coeliac disease who present with iron deficiency or altered bowel habit, though this is no higher than for non-coeliac patients with these presentations. The possibility of dual pathology should be considered and excluded by colon investigation. PMID- 12374233 TI - Elevated arterial compliance in patients with cirrhosis is not related to arterial endothelin-1. AB - BACKGROUND: Patients with cirrhosis and portal hypertension have a hyperkinetic systemic circulation. A number of circulating vasoactive peptides, including endothelin-1 (ET-1), are elevated and, recently, increased arterial compliance has been described in these patients. The aim of the present study was to investigate a potential relation between altered arterial compliance and arterial ET-1 in patients with cirrhosis. As ET-1 may be manipulated by somastostatin, the study includes infusion of octreotide in a subset of patients. METHODS: A total of 67 patients with cirrhosis and 27 controls were studied during a haemodynamic investigation. Arterial ET-1 was determined by two different radioimmunoassays and arterial compliance was determined as the stroke volume/pulse pressure index. RESULTS: Arterial compliance was elevated by 32%-40% in the cirrhotic patients as compared to the controls (P < 0.005). Arterial ET-1 was elevated by 26%-170% in the cirrhotic patients (P<0.001). No significant relationships could be established between arterial compliance and arterial ET-1 (r = -0.15 to 0.23, ns). Intravenous bolus injection and infusion of octreotide (100 pg + 100 microg/h, n = 9) did not significantly change either arterial compliance or arterial ET-1. CONCLUSION: Both arterial compliance and arterial ET- I are substantially elevated in patients with cirrhosis, but there is no significant relation between arterial compliance and arterial ET- I in these patients. PMID- 12374232 TI - Hepatic preservation, liposomally entrapped adenosine triphosphate and nitric oxide production: a study of energy state and protein metabolism in the cold stored rat liver. AB - BACKGROUND: Liposomally entrapped adenosine triphosphate (ATP) has been demonstrated to improve energy state and function of the cold-stored liver. The increased nitrite release associated with liposome administration led us to investigate the interactions between liposome supply and nitric oxide (NO) production through the use of L-NAME, a non-selective inhibitor of NO synthesis. METHODS: Twenty-four livers from fasted rats were stored for 18 h at +4 degrees C in University of Wisconsin solution directly (control group) or after infusion with ATP-containing liposomes (Lip-ATP), L-NAME (L-NAME) or both (Lip-ATP-L NAME). Metabolic fluxes, cell volume and energy state were studied during reperfusion. RESULTS: After storage, nitrite release was increased by 61% in the Lip-ATP group, markedly decreased in the Lip-ATP-L-NAME group and almost abolished in the L-NAME group. The ATP content was increased by 20% in the Lip ATP group (P < 0.05 versus control) and on reperfusion this was associated with an increase in cell volume (17%; P < 0.05) and a decrease in branched-chain amino acid release (21%; P < 0.01). The simultaneous addition of L-NAME did not affect these results, but induced a large (6-fold) increase in glucose production, possibly related to the metabolism of glycerol supplied by the liposomes. In the L-NAME group, global amino acid release was 50% lower and was associated with a dramatic decrease in urea production while the energy state deteriorated rapidly. CONCLUSIONS: The improvement in energy state and anabolic cell swelling induced by ATP-containing liposomes seems to be independent of NO synthesis. On the other hand, inhibition of NO synthesis appears to exert a detrimental effect on the liver, presumably through the decrease in hepatic energy content. PMID- 12374234 TI - Hemodynamic effects of propranolol and nitrates in cirrhotics with transjugular intrahepatic portosystemic stent-shunt. AB - BACKGROUND: The combination of tailored TIPS with vasoactive drugs might allow reduction of the rate of subsequent shunt-related sequelae. METHODS: We studied cirrhotic patients 8 weeks (median) after TIPS insertion (8-10 mm) for variceal bleeding. Nitrate (0.1 mg/kg) and propranolol (0.15 mg/kg) alone or combined (same dosages) were infused (I h) sequentially at 1-h intervals (n = 17). Similarly, propranolol was randomly compared to placebo (NaCl, n = 14). We measured mean arterial pressure (MAP, mmHg), heart rate (HR) and portal pressure gradient (PPG: portal minus central venous pressure) prior to and after drugs. RESULTS: Propranolol reduced PPG (mean +/- s, mmHg) significantly (14.8 +/- 3.7 versus 12.1 +/- 3.7; -21% +/- 10%; P < 0.001), while nitrates alone (14.3 +/- 3.4 versus 13.7 +/- 3.4; -11% +/- 3%; P=0.06) or nitrates plus propranolol (12.9 +/- 4 versus 12.4 +/- 4; -7% +/- 8%; P=0.2) induced only minor additive effects on portal pressure. However, nitrate reduced MAP (P < 0.001) and increased HR (P < 0.01), whereas propranolol reduced only HR (P < 0.001) with unchanged MAP, and the combination decreased MAP (P < 0.001). Compared to placebo (no effect), propranolol decreased PPG (14.4 +/- 5.6 versus 11.1 +/- 5.5; -23% +/- 11%; P < 0.001) and HR (P < 0.001). Overall, most patients (92%) responded to propranolol and 54% showed a marked PPG decrease (>20%). CONCLUSIONS: Propranolol significantly reduced portal pressure in cirrhotic patients after TIPS, whereas nitrates induced only minor benefit. TIPS-treated patients might therefore profit from additive propranolol therapy allowing limited shunts to be applied initially and/or to reduce the need for TIPS revisions in the case of shunt-dysfunction during follow-up. PMID- 12374235 TI - Ontogenetic development and spatial distribution of the ileal apical sodium dependent bile acid transporter and the ileal lipid-binding protein in apoE knockout and C57BL/6 mice. AB - BACKGROUND: Although apoE-/- mice are characterized by hypercholesterolemia, the bile acid enterohepatic circulation, which plays a crucial role in cholesterol homeostasis, has not been examined in these mice. The differences between apoE-/- and C57BL/6 mice in expression of the ileal ASBT and ILBP and in intestinal bile acid absorption were studied. METHODS: The intestinal tissues of the fetal, neonatal and post-weaning mice were processed for immunohistochemistry. Body retention and fecal excretion of 75SeHCAT were measured. The bile acid pool size and its composition were analysed by HPLC. RESULTS: In apoE-/- and C57BL/6 mice, the bile acid pool size was 75 +/- 13 and 78 +/- 13 micromol/ 100 g body weight, respectively, while the ratio of cholic acid/beta-muricholic acid was 1.8 +/- 0.3 and 1.4 +/- 0.3 (P < 0.05), respectively. The daily body retention of 75SeHCAT was 48% = 1.8% in C57 black mice and 58.4% +/- 2.7% in apoE-/- mice (P < 0.05). In both mouse strains, ASBT expression in the small intestine was found in the near-term fetal and post-weaning mice, while ILBP expression was found in all postnatal mice. In the post-weaning mice, ILBP expression was limited to the distal 25%-30% of the small intestine, while ASBT expression was limited to the distal 18%. CONCLUSIONS: The bile acid enterohepatic circulation in apoE-/- mice probably does not differ greatly from that in C57BL/6 mice. PMID- 12374236 TI - Serum profiles of interleukin-18 in different severity forms of human acute pancreatitis. AB - BACKGROUND: Interleukin 18 (IL-18) is a new mediator and modulator of the immune response; its role in acute pancreatitis (AP), however, has not yet been fully explained. The aim of our study was to evaluate the profile IL-18 serum concentrations in the course of acute pancreatitis. METHODS: The prospective study involves 30 patients with AP (n = 15 with mild AP and n = 15 with severe AP) as well as 10 healthy subjects. AP severity was defined according to Ranson's and Balthazar's criteria, supplemented by serum CRP concentration measurements. In the course of hospitalization, 2 patients with severe AP died. Serum IL-18 and plasma polymorphonuclear leukocyte elastase (PMN-E) concentrations were measured at admission (day 1) and on days 2, 3, 5 and 10. RESULTS: In both the mild and the severe forms of AP, serum IL-18 concentration was significantly higher than in the healthy controls. In severe AP, serum IL-18 reached the highest levels in all observed periods compared to that in patients with mild AP. Significant correlations, calculated for day 1, were found between serum IL-18 and plasma PMN E (Rs = 0.514. P < 0.001) and between IL-18 and CRP (Rs = 0.463, P < 0.001) levels. CONCLUSIONS: Serum profile IL-18 during AP indicates that this cytokine was released early after AP onset and may play the key role in inflammatory and immune response. Positive correlation between serum IL-18 and commonly known early prognostic markers of AP severity suggest that serum IL-18 concentrations may represent another early marker indicating severe course of AP. PMID- 12374237 TI - Carbon dioxide insufflation reduces discomfort due to flexible sigmoidoscopy in colorectal cancer screening. AB - BACKGROUND: Flexible sigmoidoscopy is currently recommended as a screening modality for colorectal cancer. However, a substantial number of patients experience discomfort because of the procedure. possibly limiting compliance and thus screening success. During endoscopy, air is commonly used to insufflate the bowel. Carbon dioxide rather than air insufflation has been shown to reduce procedure-related pain and discomfort in colonoscopy. The aim of the present study was to evaluate whether carbon dioxide insufflation reduces discomfort during and after flexible sigmoidoscopy for colorectal cancer screening. METHODS: In a randomized, double-blinded design, 230 consecutive participants in a population-based flexible sigmoidoscopy colorectal cancer screening trial were assigned to have their examination performed with either carbon dioxide or air insufflation. Patients were asked to grade discomfort experienced both during and in the hours after the procedure on a visual analogue scale. RESULTS: Carbon dioxide insufflation significantly reduced the amount of discomfort at 1, 3 and 6 h after the sigmoidoscopy. One hour after the examination. 84% of patients in the CO2 group reported no discomfort, compared to 64% in the air group (P = 0.006). No differences between the groups were observed during the examination. CONCLUSIONS: Carbon dioxide insufflation significantly reduced post-examination discomfort. The use of carbon dioxide rather than air insufflation may contribute to better public acceptance for flexible sigmoidoscopy screening. PMID- 12374238 TI - Pyoderma gangrenosum associated with crohn disease: effect of TNF-alpha blockade with infliximab. AB - Eight patients with pyoderma gangrenosum associated with Crohn disease were treated with infliximab. All had active mucosal inflammation indicated by endoscopic examination. Within 1-4 months, infliximab treatment resulted in complete healing of the pyoderma gangrenosum in 3 cases (1 parastomal, 2 lower limb), partial healing in 3 (2 parastomal, 1 lower limb) and temporary improvement in 2. Adverse effects such as skin rash, pneumonia and diarrhoea were seen in three patients. Our results imply that infliximab has a therapeutic potential on skin manifestations associated with inflammatory bowel disease, even though successful treatment may require repeat courses of infliximab infusions. PMID- 12374239 TI - Steroidal management and serum cytokine profile of a case of alcoholic hepatitis with leukemoid reaction. AB - Leukemoid reactions (LRs) are rare in alcoholic hepatitis (AH), but they are a sign of poor prognosis. The treatment of AH with corticosteroids is controversial, though several reports suggest that these should be used in severe cases of AH. We report a case of AH-associated LRs that presented with an increase of the serum concentrations of the proinflammatory cytokines interleukin (IL)-18 (an initiator of inflammation) and IL-1beta (likely responsible for the neutrophilia of the LRs). These findings provided a pathogenic indication for the use of corticosteroids (that block the transcription of IL-1beta), and this approach achieved a clinical and analytical recovery in our patient. This pathogenic mechanism might also underlie other cases of LRs and other complications of AH, thus providing a rationale for the benefits of corticotherapy in these rare but severe conditions. PMID- 12374240 TI - Mycobacteria and inflammatory bowel diseases: a cumulative association due to immunosuppressive therapy? PMID- 12374241 TI - Possible association of Helicobacter pylori infection with lymphoma development in the gastric stump. PMID- 12374242 TI - Further report of familial occurrence of collagenous colitis. PMID- 12374243 TI - Laparoscopic treatment of uncomplicated common bile duct stones: what is the evidence? PMID- 12374244 TI - 'Alarm symptoms' in patients with dyspepsia: a three-year prospective study from general practice. AB - BACKGROUND: Ten percent of patients consulting a general practitioner (GP) because of dyspepsia report one or more alarm symptom(s): anemia, black stools, blood in stools, dysphagia, jaundice, weight loss. We observed the consequence of such symptoms prospectively over 3 years. METHODS: Postal questionnaires were sent to GPs to obtain recorded information from patients who had consulted the GP because of dyspepsia. Mortality and gastrointestinal morbidity per 1000 person years were studied in two cohorts of patients, one presenting with, the other without, alarm symptoms and compared to expected rates from the general population. The incidence of ulcers was compared between the two cohorts. The predictive value of alarm symptoms for the development of cancer and ulcer was calculated. RESULTS: Compared to the general population, dyspeptic patients without alarm symptoms had an insignificant increase in mortality (OR = 1.5 (0.9 2.4)) and a significant increase in gastrointestinal (GI) cancers (OR = 2.4 (1.1 7.1)), whereas in patients with alarm symptoms both mortality (OR = 2.3 (1.7 3.2)) and GI cancers (OR = 6.3 (3.6-11.0)) were significantly raised. In dyspeptic patients, the presence of alarm symptoms increased the risk of developing peptic ulcers significantly (OR = 5.3 (3.1-9.1)) and gastrointestinal cancer insignificantly (OR = 1.9 (0.9-4.1)). Positive predictive values for development of cancer and ulcer were 4% and 14%, respectively. During 3 years of observation, patients with alarm symptoms were diagnosed with a malignancy in 4%, ulcers in 11% and minor gastrointestinal diseases in 25% of cases. CONCLUSION: Although the presence of alarm symptoms predicts a bad prognosis, the positive predictive values were low and negative predicted values high, reflecting low incidences of the diseases in the population at risk. The majority of patients who developed cancer or ulcer did not present with alarm symptom(s) at the initial consultation. PMID- 12374245 TI - Jane Delano--organizer and recruiter of nurses. PMID- 12374246 TI - Bisphosphonates and the upper gastrointestinal tract: skeletal gain without visceral pain? PMID- 12374247 TI - Oral bisphosphonates and upper gastrointestinal tract problems: what is the evidence? AB - OBJECTIVE: To review and evaluate the evidence regarding possible associations of bisphosphonate use with upper gastrointestinal (GI) tract adverse events (AEs). METHODS: We reviewed and summarized published information and abstracts regarding upper GI tract safety and tolerability of bisphosphonates, including laboratory and animal studies, epidemiological (observational) studies, endoscopy studies, and randomized controlled trials (RCTs). The evidence was summarized by using the principles of evidence-based medicine, giving the greatest credence to high quality RCTs. RESULTS: Clinical reports of esophagitis associated with bisphosphonate use appear to have declined in frequency once the importance of proper administration was explained to physicians after early reports of complications. Conflicting results have been reported in endoscopy studies; some reported no significant increase in upper GI tract lesions, whereas others reported a higher incidence of gastric (but not esophageal) lesions among patients taking oral bisphosphonates. Endoscopy studies that reported differences were of short duration (2 weeks) and were not of double-blind design. Results from large RCTs involving thousands of participants detected no increase in upper GI tract AEs among individuals treated with bisphosphonates. Other studies of patients who discontinued taking bisphosphonates and were randomized to blinded re-treatment with either a bisphosphonate or placebo show that most patients (>85%) were able to continue treatment, with no difference in AEs between the bisphosphonate and placebo groups. CONCLUSIONS: The highest level of evidence, RCTs, suggests little or no increase in risk of upper GI tract problems if bisphosphonates are administered properly. Upper GI tract symptoms are common among patients with osteoporosis. The evidence suggests that many upper GI tract AEs reported during therapy with bisphosphonates may reflect a high background incidence of upper GI tract complaints and an increased sensitivity to detection rather than a causal relationship to therapy. PMID- 12374248 TI - Tolerability of once-weekly alendronate in patients with osteoporosis: a randomized, double-blind, placebo-controlled study. AB - OBJECTIVE: To compare the upper gastrointestinal (GI) tract tolerability of once weekly oral alendronate, 70 mg, and placebo. PATIENTS AND METHODS: This was a 12 week multicenter, randomized, double-blind, placebo-controlled study. The first patient initiated treatment on June 5, 2000, and the last patient completed treatment on March 1, 2001. The study enrolled 450 postmenopausal women and men with osteoporosis (224 took alendronate, 226 took placebo) who were ambulatory and community dwelling at 48 outpatient study centers in the United States. By design, approximately half of the patients were naive to bisphosphonates. The primary end point was upper GI tract tolerability based on the incidence of any upper GI tract adverse events. Secondary end points included the number of discontinuations due to drug-related upper GI tract adverse events and the change from baseline in bone resorption, assessed by the urinary N-telopeptide creatinine ratio at 12 weeks. A subgroup analysis of the primary and secondary end points was performed on the patients stratified by prior bisphosphonate use. The safety and tolerability of the weekly alendronate and placebo regimens were captured as clinical and laboratory adverse events. RESULTS: A total of 11% of the alendronate patients and 13% of the placebo patients reported an upper GI tract adverse event. Discontinuations due to drug-related upper GI tract adverse events occurred in 3% of alendronate patients and 1% of placebo patients. The differences between the treatment groups for the primary and secondary end points were not significant. For the primary end point, the upper limit of the 95% confidence interval of the difference was well within the prespecified 14% comparability bound (-2.2%; 95% confidence interval, -8.3% to 3.9%). The overall incidence of upper GI tract adverse events was lower in the subgroup of patients with prior bisphosphonate exposure (8%) than in those who were bisphosphonate naive (16%). However, regardless of prior bisphosphonate exposure, the incidence of upper GI tract adverse events was similar between the alendronate and placebo patients. The urinary N-telopeptide-creatinine ratio showed a significant decrease in the alendronate patients (72% of baseline, P<.001) compared with a slight increase in the placebo patients (106% of baseline) at week 12. CONCLUSION: In this 3-month study, the incidence of upper GI tract adverse events in patients treated with once-weekly alendronate, 70 mg, was comparable to that with placebo. PMID- 12374249 TI - Comparison of processes and outcomes of pneumonia care between hospitalists and community-based primary care physicians. AB - OBJECTIVE: To compare medical care provided by hospitalists and primary care physicians to patients with community-acquired pneumonia in order to identify specific practices that might explain the improved efficiency of care provided by hospitalists. PATIENTS AND METHODS: We retrospectively reviewed the medical charts of 455 patients hospitalized with pneumonia at a community-based tertiary care center between January 1, 1998, and January 1, 1999. Exclusion criteria included human immunodeficiency virus infection, lung cancer, active tuberculosis, hospitalization within 7 days, length of stay (LOS) more than 14 days, and requirement of mechanical ventilation. All patients were cared for by either a full-time hospitalist or a primary care physician. Data collected included patient insurance status, variables to calculate each patient's Pneumonia Severity Index score, initial antibiotic selection, door-to-needle time, time to patient stability for switch to oral antibiotics, time to actual switch, unstable variables at discharge, and subspecialty consultation rate. Each patient's initial chest x-ray film was reviewed and classified as diagnostic of pneumonia, indeterminate, or clear. Outcomes measured via administrative database were mortality, LOS, costs, and readmission rate. RESULTS: Primary care physicians cared for 270 patients, and hospitalists cared for 185. Primary care physician patients were older, and this group had a higher proportion of the highest-risk patients. The mean time to stability was 3.2 days for hospitalists and 3.3 days for primary care physicians, and the mean time from stability to actual switch from intravenous to oral antibiotics was 1.6 days and 23 days, respectively (P=.003). The mean adjusted LOS was 5.6 days for hospitalists and 6.5 days for primary care physicians. Similarly adjusted costs were $594 less per patient treated by hospitalists. A difference in door-to-needle time of 0.9 hour favoring primary care physicians did not contribute to LOS. No significant differences were noted in adjusted inpatient mortality or the appropriateness of initial antibiotics used. Primary care physicians were more likely to prescribe clindamycin and ceftazidime, and they requested infectious disease consultations more often. At discharge, 14% of hospitalist patients and 7% of primary care physician patients had at least 1 unstable variable. Differences in hospital readmission rates at 15 and 30 days were not statistically significant in combined or risk-stratified analyses. CONCLUSIONS: Inpatients with community acquired pneumonia cared for by hospitalists had a shorter adjusted LOS than those seen by primary care physicians primarily because of earlier recognition of stability and more rapid conversion from intravenous to oral antibiotics. Adjusted costs were likewise reduced. However, patients seen by hospitalists were discharged with an unstable clinical variable more often. Other than earlier switch to oral antibiotics, less use of clindamycin and ceftazidime, and fewer infectious disease consultations, hospitalists' processes of care were similar to those of primary care physicians. PMID- 12374250 TI - Efficacy of brief couples support groups developed to manage the stress of in vitro fertilization treatment. AB - OBJECTIVE: To assess the efficacy of brief couples support groups offered concurrently with in vitro fertilization (IVF) treatment. PATIENTS AND METHODS: Couples in IVF treatment were given the option of participating in a biweekly support group at the IVF clinic at Wilford Hall Medical Center, San Antonio, Tex. At least 1 member of 26 couples participated in the brief couples support groups, and at least 1 member of 19 other couples completed the questionnaires but did not attend the support group sessions and so comprised the control group. Facilitators used cognitive behavioral techniques to help participants process their feelings and cognitions about their infertility. Emotional and cognitive factors were assessed both before and after group attendance by using the Beck Depression Inventory; the Beck Anxiety Inventory; the Life Orientation Test, which assesses optimism and pessimism; the Survey of Personal Views, which measures irrational beliefs; and the Social Provisions Scale, which measures social support. RESULTS: Women who attended group sessions were significantly less anxious after the IVF treatment than they were before the cycle (P < .001). Men who attended the group sessions were more optimistic than nongroup men or the women at the completion of the IVF cycle (P < .001) but endorsed greater numbers of irrational beliefs (P < .001). CONCLUSIONS: Despite the fact that the service was relatively inexpensive compared with IVF in the civilian community, the complexity of IVF treatment and the logistic and psychological stress experienced by couples made it hard to form and maintain such groups. Nevertheless, both men and women derived psychological benefit from the group: women reported less anxiety and men greater optimism on completion of the group sessions. PMID- 12374251 TI - Genetics and etiopathophysiology of schizophrenia. AB - Schizophrenia is one of the most common, devastating, and least understood neuropsychiatric illnesses present in the human population. Despite decades of research involving neurochemical, neuroanatomical, neuropathologic, neurodevelopmental, neuropsychological, and genetic approaches, no clear etiopathophysiology has been elucidated. Among the most robust findings, however, is the contribution of genetics to disease development. Statistical models suggest that susceptibility to the disorder is governed by the effects of multiple genes, coupled with environmental and stochastic factors. This review briefly summarizes recent etiopathologic findings and hypotheses, with special attention to genetics. PMID- 12374252 TI - Statin lipid-lowering therapy for acute myocardial infarction and unstable angina: efficacy and mechanism of benefit. AB - The use of statin agents in patients with acute coronary syndromes (ACSs) remains an area of intense clinical interest. Statin therapy has an established secondary preventive benefit in patients with coronary artery disease, and its extension to ACS seems logical. A number of observational studies have shown an association between initiation of statin therapy early in ACS and improved clinical outcome. Additionally, 4 randomized controlled trials have examined the use of statin therapy for ACS: the Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study, the Pravastatin Turkish Trial, the Fluvastatin on Risk Diminishing After Acute Myocardial Infarction (FLORIDA) study, and the Lipid Coronary Artery Disease (L-CAD) study. Three of these trials showed a benefit with early initiation of statin therapy, whereas 1 trial demonstrated neither benefit nor harm. All the available trials lacked the power and design to sufficiently evaluate whether early initiation of statin therapy reduces mortality and reinfarction in patients with ACS. Four ongoing trials have been designed and sufficiently powered to determine whether statin therapy reduces the risk of death and reinfarction when initiated early in ACS treatment. A body of evidence suggests that the pleiotropic actions of statin agents might modulate benefit in ACS. This article summarizes the available data and provides a rationale for early initiation of statin therapy for patients with ACS. PMID- 12374253 TI - Presenting syndromes of human immunodeficiency virus. AB - Over the past 2 decades, numerous changes have occurred in the demographics and clinical course of the human immunodeficiency virus (HIV) infection. Since the initial reports of mucosal candidiasis, severe weight loss, and Pneumocystis carinii pneumonia as presenting manifestations of the late stages of immunodeficiency, clinicians have recognized a wide spectrum of manifestations associated with HIV disease. The original reports of severe immunodeficiency were simply the "tip of the iceberg." Advances in antiretroviral therapy and prevention of opportunistic infections have made early diagnosis important. Recognition of cases in earlier stages facilitates opportunities to prevent transmission throughout the population, especially in high-risk groups and pregnant women. Recent evidence suggests that antiviral therapy for primary HIV infection may beneficially alter the course and long-term outcome in persons infected with HIV. This article reviews common presenting syndromes of HIV to aid clinicians in establishing an earlier diagnosis. PMID- 12374254 TI - 23-year-old woman with increasing frequency of migraine headaches. PMID- 12374255 TI - Clinical indications and diagnostic yield of video-electroencephalographic monitoring in patients with seizures and spells. AB - Video-electroencephalographic (EEG) monitoring is an important neurodiagnostic technique that may be used for selected patients who present with recurrent and unprovoked spells. For most patients who have epilepsy, the "routine" EEG is sufficient for physicians to classify seizure types and initiate medical therapy; however, routine EEG has substantial limitations for approximately 20% of patients who do not have epilepsy but are referred to comprehensive epilepsy programs because of medically refractory "seizures." These patients may have physiological or psychological disorders that may cause diagnostic confusion with epilepsy and result in the patients being treated unnecessarily with antiepileptic drugs. Video-EEG monitoring, ie, ictal EEG monitoring, performed either on an outpatient basis or in an epilepsy monitoring unit, can help physicians identify ictal EEG patterns that may be necessary for classifying seizure types and determining surgical localization. The sensitivity and specificity of EEG recordings during clinical episodes are superior to those of the routine interictal EEG. Video-EEG monitoring may prove to be an essential procedure for helping physicians confirm diagnoses of seizure disorders, classify seizure types, and select surgical candidates who have intractable epilepsy. PMID- 12374256 TI - Pseudomonas aeruginosa causing a right carotid artery mycotic aneurysm after a dental extraction procedure. AB - Mycotic aneurysms of the carotid arteries are rare. We describe a right carotid artery mycotic aneurysm in a 70-year-old man. His symptoms began immediately after a complicated molar extraction and persisted until the diagnosis was made and surgical resection and repair were undertaken. Pseudomonas aeruginosa was isolated from multiple blood cultures and excised tissues. We review another 73 cases uncovered by an extensive literature search. Bacteremia, recent surgery, head and neck infections, dental infections, and endocarditis are the most common predisposing conditions. Computed tomography and magnetic resonance imaging are techniques for accurately confirming the suspicion of any aneurysm, but angiography is the gold standard. Primary resection of the aneurysm with native vein interposition, in conjunction with prolonged antibiotic therapy, is the preferred strategy. A total of 6 cases thus far, including ours, have been clearly associated with dental surgical procedures. These cases are characterized by rapidly enlarging neck masses in the presence of fever. Microorganisms, particularly gram-negative rods, in contrast to normal oral flora, eg, streptococci and anaerobes, are often isolated. With prompt diagnosis and treatment, outcome is often satisfactory. PMID- 12374258 TI - Use of low-molecular-weight heparin in pregnant women with mechanical valves. PMID- 12374257 TI - Enterocolitis as initial presentation of acute myelogenous leukemia exacerbated by induction chemotherapy with idarubicin-cytosine arabinoside. PMID- 12374259 TI - Why practice culturally sensitive care? Integrating ethics and behavioral science. PMID- 12374260 TI - Longitudinal analysis of student performance in a dental hygiene distance education program. AB - The purpose of the study was to determine if learners who receive face-to-face instruction in an educational program performed statistically better on established benchmark assessments (GPA, course averages, and NBDHE) than learners at a distance from the didactic course instructor. A comparative, quasi experimental, ex-post facto study was conducted. The treatment variable was program type: face-to-face vs. distance. The performance of five consecutive classes was analyzed, from 1997 to 2001. These five classes consisted of 221 learners, 105 of them at the host site and 115 using distance learning. The experimental groups were divided based upon location--host or cooperating college (distance) site learners. Study results identified no significant difference between host and distance learner performance for the entire educational program. The use of interactive television (ITV) for delivery of an educational program using distance education technology provided acceptable results in learner didactic performance. Learners at both the host and cooperating college (distance) sites performed equally well. The results were used to document program outcomes. PMID- 12374261 TI - Sources of stress and psychological disturbance among dental students in the West Indies. AB - The aim of this study was to investigate sources of stress and psychological disturbance in dental students across the five years of undergraduate study at a dental school in Trinidad. Eighty-three percent of students completed a modified version of the Dental Environment Stress questionnaire (DES) and the Brief Symptom Inventory (BSI). On a scale ranging from 0 (not stressful) to 5 (highly stressful), overall mean DES scores for each of the five years of study were 1.58, 1.83, 2.65, 2.39, and 2.61 respectively, suggesting that levels of stress increase over the five years with a noticeable spike at the transition between the preclinical and clinical phases. Significant differences were found between specific stressors across the five years of study. Seven specific stressors and the stressor domains of Academic work and Clinical factors were more stressful for female students (t-test p < 0.05). The Global Severity Index of the BSI indicated that 54.8 percent of males and 44.2 percent of females were in the clinical range indicating significant psychological disturbance. Psychological disturbance was significantly associated with stress levels for male students (Spearmans rank correlation r = 0.56; p < 0.001), but not generally for female students. Further development is needed of dental educational programs that enhance students' psychosocial well-being. PMID- 12374262 TI - A measure of agreement between clinicians and a computer-based decision support system for planning dental treatment. AB - This study was conducted to estimate agreement and explain differences between treatment decisions and associated fees recommended by dentists and by a computer based decision-support program (TxDENT 2.0). The treatment fees associated with forty-eight clinical records of patients attending a dental school clinic provided a measure of correlation and agreement between treatments recommended by TxDENT and by clinical instructors with students. The average difference between the two methods of forecasting fees was $466, and a regression line (y=0.43x+407) with an r-value of 0.54 indicated the strength of the relationship. The differences between methods increased as the cost of treatment increased, due largely to disagreements about the need to restore or replace weak or missing teeth. There is reasonable agreement between TxDENT and the collaborative treatment plans of clinical instructors with their students, which suggests that this computer-based decision-support system for screening patients in a standardized way could be a helpful predictor of treatment provided in a dental school clinic. PMID- 12374263 TI - Faculty appointment policies and tracks in U.S. dental schools with clinical or research emphases. AB - The 1995 Institute of Medicine study of the future of dental education, Dental Education at the Crossroads: Challenges and Change, recommended that dental schools increase the use of nontenure-track positions in their employment of faculty. As part of a larger investigation of faculty appointment processes in U.S. dental schools, dental deans were queried about institutional policies governing faculty appointments and the use of tenure and nontenure faculty tracks. Response from the fifty-four U.S. schools exceeded 90 percent for each of two mailed questionnaires. Dental schools were classified according to one of two emphases: clinical or research. Deans classified faculty into one of seven appointment tracks. Nontenure-track appointments were less common in clinical emphasis schools. Research-emphasis schools had a greater mean proportion of their faculties in both the nontenure research track (7.0 percent vs. 2.7 percent) and the nontenure clinical track (20.8 percent vs. 17.4 percent). Compared to faculty appointment data reported in 1990, there were more nontenure track faculty in 81 percent of research-oriented schools and 55 percent of clinical-oriented schools. The most frequently cited reasons for more nontenure faculty in these thirty-three schools were greater administrative flexibility, better fulfillment of mission, and increased difficulty achieving tenure. This study showed the number of faculty holding nontenure-track appointments had increased since 1990, especially among research-emphasis dental schools. PMID- 12374264 TI - Assessment of evidence-based dental prophylaxis education in postdoctoral pediatric dentistry programs. AB - The objective of the study was to investigate various aspects of evidence-based dental prophylaxis education in postdoctoral pediatric dentistry training programs in the United States. An anonymous nationwide postal survey of fifty-two postdoctoral pediatric dentistry program directors was conducted in September 2001. The survey had a response rate of 75 percent with all geographic regions of the nation represented and with a preponderance of university-based programs (62 percent). Most of the training programs (74 percent) routinely recommended dental prophylaxis for all recall patients. The proportion of programs that recommended dental prophylaxis for the following indications were: plaque, stain and/or calculus removal--97 percent; caries prevention--59 percent; prior to topical fluoride application--67 percent; prior to sealant application--62 percent; and for behavioral modification--77 percent. Most training programs (77 percent) defined dental prophylaxis as both rubber cup pumice prophylaxis and toothbrush prophylaxis. However, only one-half of the training programs (51 percent) had modified their teaching to substitute toothbrush prophylaxis in lieu of rubber cup pumice prophylaxis. In conclusion, only one half of postdoctoral pediatric dentistry training programs in the United States teach evidence-based practice of dental prophylaxis for recall patients. PMID- 12374265 TI - Tobacco use, prevention, and cessation: introduction to the special section. IADR Symposium on Tobacco Use, Prevention, and Cessation. March 9, 2002. PMID- 12374266 TI - Reducing tobacco use: what works in the population? AB - One-half of those who have ever smoked cigarettes are currently former smokers. This cessation coincided with a forty-year effort to educate and inform smokers about the risks of smoking. This paper examines the effects of several tobacco control interventions for smokers in the general population using population based survey data. States with large media-led tobacco control programs have higher rates of smoking cessation, suggesting that these comprehensive approaches can alter smoking behavior. This paper also presents evidence supporting effects on smoking cessation for restrictions on where people can smoke, increases in the cost of cigarettes, provision of physician advice to quit coupled with cessation assistance, pharmacological assistance, and telephone hotlines. It also provides evidence that many of these interventions are being implemented in the general population in ways that are less effective than expected based on clinical trials. Increasing the effectiveness of these interventions and linking multiple interventions to provide synergy offer great opportunities to improve rates of population-based cessation. PMID- 12374267 TI - Managing nicotine addiction. AB - Nicotine addiction has been identified as the primary contributor to continued widespread tobacco use worldwide. Although the health benefits of smoking cessation are well publicized, few smokers successfully quit on a long-term basis. A number of pharmacological agents have been shown to approximately double long-term smoking cessation rates and have, therefore, been recommended as first line therapy for the treatment of nicotine dependence in the clinical practice guidelines recently released by the Agency for Healthcare Research and Quality (AHRQ). These include the currently available dosage forms of nicotine replacement therapy (gum, patch, nasal spray, and inhaler) and bupropion. Other agents that have exhibited some efficacy in increasing smoking cessation rates are nortriptyline and clonidine. All pharmacological treatments are most effective in conjunction with behavioral therapy. Other approaches to treating tobacco use disorder now being investigated include additional ways to administer nicotine, a vaccine to prevent nicotine from crossing the blood-brain barrier, and agents that alter the metabolism of nicotine. This review summarizes the characteristics of nicotine addiction, reviews the pharmacological agents currently used to treat tobacco use disorder, and describes possible approaches to treat nicotine dependence in the future. PMID- 12374268 TI - Impact of a dental/dental hygiene tobacco-use cessation curriculum on practice. AB - Tobacco use is the chief avoidable cause of morbidity and mortality in North America and is associated with increased risk for oral cancer and increased prevalence and severity of periodontitis and other oral conditions. By delivering two- to three-minute tobacco-use cessation counseling (TUCC), oral health professionals can achieve quit rates substantially higher than the spontaneous quit rate. However, many clinicians report lack of training and knowledge in TUCC as barriers to providing cessation counseling. The purpose of this study was to evaluate whether implementation of a comprehensive, dental school-based, tobacco use cessation program would increase the extent to which tobacco-using patients received TUCC. The school's program was based on the critical administrative, cultural, structural, and policy components of effective TUCC interventions outlined by Fiore et al. A pre- and post-program telephone interview of tobacco using patients assessed TUCC intervention by students. A significantly greater proportion of patients received TUCC post-program compared to pre-program in terms of consequences associated with tobacco use as well as advice to quit. A comprehensive TUCC program resulted in an improvement of 11.7 percent for consequences and 23 percent for advice to quit. PMID- 12374269 TI - Effectiveness of tobacco counseling in the dental office. AB - This article describes the results of studies among dental care providers regarding tobacco cessation in the past two decades. In the early period, surveys described what dentists were doing in their own practices. The results suggested that they were not adequately communicating to their patients the importance of quitting. There is good evidence that brief interventions from health professionals can increase rates of smoking cessation. The outcome from a number of trials that examined the feasibility of conducting smoking cessation in dental practices is reviewed here. The pivotal role of a team approach is highlighted in many studies. Dentists who implement an effective smoking cessation program can expect to achieve quit rates up to 10-15 percent each year among their patients who smoke or use smokeless tobacco. The challenge is implementing effective treatment in one's practice or institution while using available primary care resources to provide additional benefit. PMID- 12374270 TI - Oral screening and brief spit tobacco cessation counseling: a review and findings. AB - This paper reviews five randomized controlled trials of brief spit (smokeless) tobacco (ST) cessation treatment by dental professionals consisting of oral cancer screening, cessation advice, self-help materials, and brief cessation counseling by a dental hygienist. In addition, original two-year findings from a randomized controlled trial to determine the effect of a dental-directed, peer assisted ST intervention among high school baseball athletes in rural California (n=1084) are reported. In the latter study, results show sustained quitting at two years of 23 percent (32/141) in the intervention group and 13 percent (21/166) in the control group (OR=2.0, 95% CI 1.1-3.9) with subjects lost-to follow-up considered non-quitters. The evidence presented supports the efficacy of oral screening and brief cessation counseling by dental professionals to promote ST cessation in the dental office or in athletic facilities. In addition, recommendations for policy and future research are presented. PMID- 12374271 TI - Where to from here? PMID- 12374272 TI - Meeting the demand for future dental school faculty: trends, challenges, and responses. AB - This report presents data from ADEA's 2001-02 survey of vacant budgeted faculty positions and examines challenges likely to exacerbate faculty shortages in the immediate future. The fifty-four dental schools responding to the survey reported 344 vacant budgeted positions, a decrease of 4 percent from 2000 to 2001. Seventy nine percent of these vacancies are for full-time positions. Approximately one out of four dental schools has ten or more vacancies. Of just over 1,000 faculty separations during 2001-02, 53 percent were reported to be individuals leaving to enter private practice. There is no indication of a near-term reversal of the decade-long trend toward increasing budgeted vacancies, and the current economic environment along with other factors delineated in this report makes the challenge to recruit and retain dental faculty more difficult. ADEA and other stakeholders are currently pursuing a number of strategies to meet the demand for future dental school faculty. PMID- 12374273 TI - The issue of using patients for dental licensing examinations in the United States. PMID- 12374274 TI - Students have numerous obstacles to overcome during their first four years of dental education. PMID- 12374275 TI - Family violence content in dental hygiene curricula: a national survey. AB - Dental personnel are in an excellent position to recognize suspected abuse of dental patients because 65-75 percent of abuse occurs in the head and neck area. While most dental and dental hygiene curricula include the topic of child abuse, it has previously been unknown if other types of abuse, such as intimate partner abuse, elder abuse, and abuse of disabled persons, are addressed. This study was conducted to determine the extent to which dental hygiene programs have incorporated these family violence topics into the curriculum. Specific data on content, teaching methods, faculty, and resources were collected. Reasons for not including family violence in the curricula, attitudes on mandatory continuing education, and support services available for abuse victims were also examined. A fifteen-item survey was sent to all 229 U.S. accredited dental hygiene programs. Surveys were returned from 173 programs for a response rate of 77.5 percent. Child abuse was taught in most programs (N=122, 70.5 percent), while elder abuse (N=95, 54.9 percent), intimate partner abuse (N=81, 46.8 percent), and abuse of individuals with disabilities (N=80, 46.2 percent) were taught in fewer programs. Reasons for not including family violence in the curricula (N=31, 18 percent) varied. The need is critical for increased curriculum attention in U.S. dental hygiene programs to help stem the epidemic of family violence. Raising dental hygienists' awareness of the problem and potentially increasing the number of reports of and referrals for suspected violence may help more victims. PMID- 12374276 TI - Coordinate regulation of translation by the PI 3-kinase and mTOR pathways. AB - Control of translation initiation is an important means by which cells tightly regulate the critical processes of growth and proliferation. Multiple effector proteins contribute to translation initiation of specially modified mRNAs that modulate these processes. Coordinated regulation of these translational effectors by multiple signaling pathways allows the integration of information regarding mitogenic signals, energy levels, and nutrient sufficiency. The mTOR protein, in particular, serves as a sensor of all of these signals and is thought to thus serve as a crucial checkpoint control protein. Signals from the mTOR pathway converge with mitogenic inputs from the phosphoinositide (PI) 3-kinase pathway on translational effector proteins to coordinately control cellular growth, size, and cell proliferation. The translational effectors regulated by the PI 3-kinase and mTOR pathways and their roles in regulation of cellular growth will be the primary focus of this review. PMID- 12374277 TI - The cell-mediated immune response to human papillomavirus-induced cervical cancer: implications for immunotherapy. PMID- 12374278 TI - The T-cell response in patients with cancer. PMID- 12374279 TI - The life and death of a B cell. AB - Regulation of apoptosis in the B cell lineage has implications for homeostasis, quality control of the antibody response, and tolerance. In this chapter we examine the different checkpoints that control life and death decisions of B cells during the antigen-independent and antigen-dependent phases of their development. We discuss the cell death mechanism involved in elimination of unwanted B cells at different stages of their development as well as the signals that trigger or repress the apoptotic process. At the steady state, before or after development of an immune response, B cell apoptosis ensures that the antigen receptor (BCR) on newly produced B cells is functional and does not recognize self-antigens with high avidity. It also ensures that the size of the peripheral B cell compartment remains constant in spite of the continuous input of B cells from the bone marrow. All these processes are controlled by the mitochondrial death pathway and are thus perturbed by overexpression of the antiapoptotic members of the bcl-2 gene family. By contrast, the death receptor pathway plays a prominent role during the antigen-dependent phase of B cell development. Three sets of membrane molecules stand as crucial regulators of B cell survival. First, the BCR which plays a central but ambiguous role. On the one hand, it triggers death of B cells that recognize self-antigens or have been exposed to repeated antigenic stimulations. On the other hand, it promotes survival of the peripheral mature B cell pool and protects activated B cells from CD95-induced killing. Second, the death receptor Fas/CD95 which is instrumental in censoring B cells activated in a bystander fashion at the initiation of the response to T-dependent antigens. It also drives elimination of low-affinity and self-reactive B cell clones that arise through the process of somatic mutations during the germinal center reaction. As such, it contributes to the affinity maturation of the antibody response. Finally, three membrane receptors (TACI, BCMA, and BAFF-R) which bind a newly discovered member of the tumor necrosis factor family named BAFF. BAFF acts specifically on peripheral B cells but its cellular targets seem to be restricted to two splenic B cell populations: (i) transitional immature B cells and (ii) marginal zone B cells, known to be responsible for the response to thymus-independent type 2 antigens. This suggests its possible implication in positive selection of peripheral B cells and in the antibacterial B cell responses. PMID- 12374280 TI - Histone acetyltransferases and deacetylases in the control of cell proliferation and differentiation. AB - Histone acetylation and deacetylation are chromatin-modifying processes that have fundamental importance for transcriptional regulation. Transcriptionally active chromatin regions show a high degree of histone acetylation, whereas deacetylation events are generally linked to transcriptional silencing. Many of the acetylating and deacetylating enzymes were originally identified as transcriptional coactivators or repressors. Their histone-modifying enzymatic activity was discovered more recently, opening up a whole new area of research. Histone acetyltransferases such as CREB-binding protein (CBP) and PCAF are involved in processes as diverse as promoting cell cycle progression and regulating differentiation. A controlled balance between histone acetylation and deacetylation seems to be essential for normal cell growth. Both histone acetyltransferases and deacetylases are involved in the development of diseases, including neurodegenerative disorders and cancer. Treatments that target these enzymes are already under clinical investigation. PMID- 12374281 TI - Molecular pathogenesis of human hepatocellular carcinoma. PMID- 12374282 TI - The von Hippel-Lindau tumor suppressor complex and regulation of hypoxia inducible transcription. PMID- 12374283 TI - Cellular immunity to the Her-2/neu protooncogene. AB - Her-2/neu (HER-2) is a 185-kDa receptor-like glycoprotein that is overexpressed by a variety of tumors such as breast, ovarian, gastric, and colorectal carcinomas. Overexpression of this oncogene is directly associated with malignant transformation of epithelial cells. The frequency of HER-2 overexpression varies among the different types of cancers, but universally represents a marker of poor prognosis. The critical role of HER-2 in epithelial oncogenesis as well as its selective overexpression on malignant tissues makes it an ideal target for immunotherapy. Antibodies and T cells reactive to HER-2 are known to naturally occur in patients with HER-2 positive tumors, confirming the immunogenicity of the molecule. Both antibodies as well as T cells reactive to HER-2 have been utilized for immunotherapy of HER-2 positive tumors. The "humanized" monoclonal antibody Herceptin has been tested in several clinical trials and found to be an effective adjuvant therapy for HER-2 positive breast and ovarian cancer patients. However, the frequency of patients responding to Herceptin is limited and a majority of patients initially responding to Herceptin develop resistance within a year of treatment. The use of vaccination strategies that generate T cell responses with or without accompanying antibody responses may serve to mitigate the problem. Various strategies for generating T cell-mediated responses against HER-2 are currently being examined in animal models or in clinical trials. The potential advantages of the various approaches to immunotherapy, their pitfalls, and the mechanisms by which HER-2 positive tumors can evade immune responses are discussed in this review. PMID- 12374284 TI - Retinoblastoma tumor suppressor and genome stability. AB - Retinoblastoma gene (Rb) is the prototype of tumor suppressors. Germline mutation in the retinoblastoma gene is susceptible to cancer and reintroduction of wild type Rb is able to suppress neoplastic phenotypes. The fundamental cellular functions of Rb in the control of cell growth and differentiation are important for its tumor suppression. In general, cancer susceptibility caused by inactivation of a tumor suppressor gene results from genome instability. Accordingly, Rb may function in the maintenance of chromosome stability by influencing mitotic progression, faithful chromosome segregation, and structural remodeling of mitotic chromosomes. Rb is also implicated in the regulation of replication machinery and in the control of cell cycle checkpoints in response to DNA damage, further supporting such a role for Rb. Moreover, the mechanistic basis for Rb-mediated transcriptional repression has revealed its connection to global chromatin remodeling. It is likely that Rb suppresses tumor formation by virtue of its multiple biological activities, and a theme throughout its multiple cellular functions is its central role in controlling activities that involve chromatin remodeling. A model in which Rb controls global genome fluidity is thus proposed. Finally, a recent study provides direct evidence indicating that loss of Rb function leads to genome instability. Therefore, tumor suppressors have a common role in the maintenance of genome stability, and such a role may be pivotal for their functions in tumor suppression. PMID- 12374285 TI - A new challenge for successful immunotherapy by tumors that are resistant to apoptosis: two complementary signals to overcome cross-resistance. AB - Tumor resistance to conventional therapies is a major problem in cancer treatment. While tumors initially respond to radiation or chemotherapies, subsequent treatments with these conventional modalities are ineffective against relapsed tumors. The problem of tumor resistance to chemotherapy and radiation has led to the development of immunotherapy and gene-based therapies. These alternative therapeutic approaches are intensely explored because they are supposed to be more tumor specific and better tolerated than the conventional therapies. Recent advances in apoptosis have revealed that resistance to apoptosis is one of the major mechanisms of tumor resistance to conventional therapies. Resistance to apoptosis is a naturally acquired characteristic during oncogenesis and is selected for after successive rounds of conventional therapies. Resistance to apoptosis involves dysregulation and/or mutation of apoptotic signaling molecules that render tumor cells unresponsive to apoptotic stimuli. Since both immunotherapy and chemotherapy kill tumors by apoptosis and the killings are signaled through a central core apoptotic program, dysregulation of this central program and development of resistance to apoptosis in chemoresistant cells could render them cross-resistant to immunotherapy. Therefore, in order to establish an effective antitumor response and to complement immunotherapy and gene-based therapies, cross-resistance due to resistance to apoptosis must be overcome. In this review, based on prior findings and recent evidence, we put forth a model, verified experimentally, in which chemoresistant tumor cells can be sensitized to immune-mediated killing by subtoxic concentrations of chemotherapeutic drugs/factors. The model involves two complementary signals. The first signal is a sensitizing signal that regulates pro/antiapoptotic targets, thus facilitating the apoptotic signal. The second apoptotic signal initiates a partial activation of the apoptotic signaling pathway, and activation is completed by complementation with signal one. Thus, effective killing of immunoresistant cells is achieved by both signals. The two signal approach provides a new strategy to overcome cancer cross-resistance to immunotherapy and opens new avenues for the development of more effective and selective immunosensitizing agents. PMID- 12374286 TI - Cell volume and ion changes during apoptotic cell death. PMID- 12374287 TI - Mitochondria and apoptosis: new therapeutic targets. PMID- 12374288 TI - The Abl family kinases: mechanisms of regulation and signaling. PMID- 12374289 TI - Federal antitrust policy and physician discontent: defining moments in the struggle for congressional relief. AB - Organized medicine has battled the Federal Trade Commission (FTC) since the 1970s over enforcement of the antitrust laws. Physicians' discontent stems from the belief that federal policy allows managed care organizations to achieve dominance in health care markets just as it discourages physicians from taking collective action to protect their interests. This article examines two important efforts by organized medicine over a twenty-year interval to alter federal antitrust policy. In 1982, physicians and other professionals sought a special exemption from FTC jurisdiction; beginning in 1998, physicians promoted legislation that would exempt independent practitioners from the antitrust laws for collective bargaining purposes. Both initiatives passed in the House of Representatives but failed in the Senate. This article uses an advocacy coalition framework to reinterpret the events and to assess the reasons for legislative failure. The evidence suggests that in both instances, although twenty years apart, consumer groups and federal bureaucrats determined the outcome in favor of corporate medicine. PMID- 12374290 TI - Will physician unions improve health system performance? AB - In the public debate over the extension of collective bargaining rights to independent physicians, union proponents' primary argument has been that patients would benefit from allowing physicians to bargain collectively with health plans. This article examines the likely effects of physician unions on the U.S. health care system. Specifically considered are likely effects on economic efficiency, quality, access, and cost. Under none of these criteria are physician unions likely to improve health system performance, particularly when compared with available alternative strategies for dealing with problems identified by union proponents. PMID- 12374291 TI - Foul weather friends: big business and health care reform in the 1990s in historical perspective. AB - Existing accounts of the Clinton health reform efforts of the early 1990s neglect to examine how the change in big business reform interests during the short period between the late 1980s and 1994 might have altered the trajectory of compulsory health insurance legislation in Congress. This article explores evidence that big employers lost their early interest in reform because they believed their private remedies for bringing down health cost inflation were finally beginning to work. This had a discouraging effect on reform efforts. Historical analysis shows how hard times during the Great Depression also aligned big business interests with those of reformers seeking compulsory social insurance. Unlike the present case, however, the economic climate did not quickly improve, and the social insurance reform of the New Deal succeeded. The article speculates, therefore, that had employer health expenditures not flattened out, continuing and even growing big business support might have neutralized small business and other opposition that contributed heavily to the failure of reform. Thus in light of the Clinton administration's demonstrated willingness to compromise with business on details of its plan, some kind of major reform might have succeeded. PMID- 12374292 TI - The politics of discretionary Medicaid spending, 1980-1993. AB - Why do some states choose to spend more than four times as much as others to provide health care to the disadvantaged? Political scientists who have traditionally explored this question by analyzing trends in overall Medicaid expenditures lumped states' discretionary spending in with other money that states are mandated to spend. Analyses of total expenditures found that socioeconomic factors drove spending but that party control of state legislatures made no difference in health policy making. By isolating discretionary state Medicaid expenditures from total spending figures, I reexamine the influences of political as well as economic and demographic factors. The often-doubted importance of party control becomes clear. This study investigates spending patterns in the discretionary portions of state Medicaid programs in forty-six states from 1980 to 1993 and analyzes both incremental program changes and absolute differences in state spending. To discover how greatly the researcher's choice of dependent variables can affect results, optional spending is separated from total spending levels and the variation is modeled in both. Focusing not on the spending that the federal government requires of state officials but on the policies that state officials actually choose allows a balanced exploration of both political and economic effects on welfare expenditures. This research also provides new insights about which forces will shape policy decisions if more and more control of the public health care system is devolved to the states. PMID- 12374293 TI - Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects. AB - IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis. PMID- 12374294 TI - Subcellular localization of MURA and MURB proteins encoded by the maize MuDR transposon. AB - MuDR controls transposition of the Mu transposable element family in Zea mays L. It produces two major transcripts: mudrA and mudrB, mudrA encodes the MURA transposase, but no specific function has been ascribed to mudrB, which lacks strong homology to known genes. Using transient expression assays in onion epidermal cells, we defined three monopartite nuclear localization signals (NLSs) of MURA; each was functionally sufficient for nuclear targeting of MURA:GUS fusion proteins. Interestingly, one NLS (NLS-A3) is produced by the splicing of the third intron. In contrast, there were no clear NLS in MURB, and the major form of MURB aggregated in the cytoplasm. Self-interaction of MURA and of MURB was also shown in a yeast two-hybrid assay. To test whether interactions of MURA and MURB can occur at the level of protein translocation into the nucleus, a cytoplasmically localized MURB:GFP was co-expressed with MURA or with the GUS fusion proteins. Co-expression did not change the localization pattern of either MURA or MURB; MURA and MURB do not detectably interact in a yeast two-hybrid assay. These results suggest that MURA and MURB do not mutually affect their localization, at least in the forms examined here. PMID- 12374296 TI - Molecular cloning and functional expression in bacteria of the potassium transporters CnHAK1 and CnHAK2 of the seagrass Cymodocea nodosa. AB - The cDNAs CnHAK1 and CnHAK2, encoding K+ transporters, were amplified from the leaves of the seagrass Cymodocea nodosa. None of these transporters suppressed the K+ deficiency of a Saccharomyces cerevisiae mutant, but both suppressed the equivalent defect of an Escherichia coli mutant. Overexpression of the transporter CnHAKI, but not CnHAK2, mediated very rapid K+ or Rb+ influxes in the E. coli mutant. The concentration dependence of these influxes demonstrated that CnHAK1 is a low-affinity K+ transporter, which does not discriminate between K+ and Rb+. CnHAK1 expressed in E. coli worked in reverse when the external K+ concentrations were low, and we established the condition of a simple functional test of K+ loss for transporters of the Kup-HAK family. In comparison with its homologue barley transporter HvHAK2, CnHAKI was insensitive to Na+. PMID- 12374297 TI - Maize C4 and non-C4 NADP-dependent malic enzymes are encoded by distinct genes derived from a plastid-localized ancestor. AB - NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO2 intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene. PMID- 12374298 TI - Characterization of gene expression during potato tuber development in individuals and populations using the luciferase reporter system. AB - Analysis of gene expression and enzyme activity in pooled tuber samples has previously indicated different developmental events occurring in a fixed sequential order during tuber development, starting with the up-regulation of starch synthesis then induction of protein storage followed by cell division and cell enlargement. In this report we analysed in vivo promoter activity of genes related to cell division and storage of reserves during tuber development in individual in vitro tubers, using the non invasive firefly luciferase reporter system. The average activity of the storage related promoters (AGPaseS and lambdaPat21) was up-regulated prior to visible swelling, while the average activity of both cell cycle genes (cycB1;1 and CDC2a) showed an up-regulation after the onset of swelling. However, this novel system allowed expression analysis in individual tubers, which showed a variable up-regulation of both storage genes in relation to the moment of swelling, from 4 days before to 10 days after the onset of swelling. We conclude that during the first stages of tuber development, the moment of storage gene induction is independent from swelling. These results indicate that the developmental program of potato tubers does not consist of a fixed sequential order of events, but consists of independent developmental programs (storage and swelling), together resulting in the formation of a potato tuber. It is concluded that analysis of developmental programs by studying individuals may result in new insights, possibly obscured when using pooled samples. PMID- 12374299 TI - The proline-rich, extensin-like receptor kinase-1 (PERK1) gene is rapidly induced by wounding. AB - We report the isolation and characterization of PERKI (Proline Extensin-like Receptor Kinase 1), a novel plant RLK from Brassica napus that is predicted to consist of a proline-rich extracellular domain with sequence similarity to extensins, a transmembrane region, and a catalytic domain possessing serine/threonine kinase activity. Database searches with the predicted PERK1 amino acid sequence also led to the identification of a predicted family of related genes in the Arabidopsis genome. Using biolistic bombardment of onion epidermal cells, we have shown that a PERK1-GFP fusion is localized to the plasma membrane as predicted for a receptor kinase. Given the similarity of PERK1's extracellular domain to extensins, a possible role in plant defense responses was investigated by treating B. napus tissue with mechanical stresses and infection with the fungal pathogen, Sclerotinia sclerotiorum. Various wounding stimuli resulted in a dramatic and rapid accumulation of PERK1 mRNA. Levels of PERK1 mRNA also increased moderately in response to infection by the fungal pathogen S. sclerotiorum. Given the kinetics of PERK1 mRNA accumulation in response to these treatments, PERK1 may be involved early on in the general perception and response to a wound and/or pathogen stimulus. PMID- 12374300 TI - Characterization of the expression of a wheat cystatin gene during caryopsis development. AB - A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation. PMID- 12374301 TI - Powerful effect of an atypical bifactorial endosperm box from wheat HMWG-Dx5 promoter in maize endosperm. AB - The proximal region of the high-molecular-weight glutenin promoter of the Dx5 gene (PrHMWG-Dx5) carries an atypical bifactorial endosperm box containing two cis-acting elements, namely a G-box like motif followed by a prolamin-box motif (Pb1). Transient expression assays in maize endosperm indicate that a promoter fragment containing at least the G-box like element is necessary and sufficient for maximal expression of the HMWG-Dx5 promoter. In transformed maize, we have shown that a 89 bp sequence bearing the bifactorial endosperm box behaves like a functional cis-acting unit. Its repetition in tandem confers a strong specific additive effect specifically in endosperm tissue. In contrast, the fusion of the activation sequences 1 (as-1) and 2 (as-2) of the cauliflower mosaic virus (CaMV) 35S promoter with HMWG-Dx5 derived promoter sequences deregulates its activity in transformed maize. By gel mobility shift assays we have demonstrated that the G box like motif may alternatively bind two protein groups which have the same DNA binding affinities as the transcription factors of either the Opaque2 (O2) family and/or the ASF-1 family. PMID- 12374295 TI - Leaf Ests from Stevia rebaudiana: a resource for gene discovery in diterpene synthesis. AB - Expressed sequence tags (ESTs) are providing a new approach to gene discovery in plant secondary metabolism. Stevia rebaudiana Bert. leaves produce high concentrations of diterpene steviol glycosides and should be a rich source of transcripts involved in diterpene synthesis. In order to create a resource for gene discovery and increase our understanding of steviol glycoside biosynthesis, we sequenced 5,548 ESTs from a S. rebaudiana leaf cDNA library. The EST collection was fully annotated based on database search results. ESTs involved in diterpene synthesis were identified using published sequences as electronic probes, by keyword searches of search results, and by differential representation. A significant portion of the ESTs were specific for standard leaf metabolic pathways; energy and primary metabolism represented 17.6% and 13.1% of total transcripts respectively. Diterpene metabolism in S. rebaudiana represented 1.1% of total transcripts. This study identified candidate genes for 70% of the known steps in the steviol glycoside pathway. One candidate, kaurene oxidase, was the 8th most abundant EST in the collection. Identification of many candidate genes specific to the I -deoxyxylulose 5-phosphate pathway suggests that the primary source of isopentenyl diphosphate, a precursor of geranylgeranyl diphosphate, is via the non-mevalonic acid pathway. The use of ESTs has greatly facilitated the identification of candidate genes and increased our understanding of diterpene metabolism. PMID- 12374302 TI - Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells. AB - RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber viroid, hop latent viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNA(Tyr) gene or near the 3' end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub cellular compartments. PMID- 12374303 TI - The four subunits of mitochondrial respiratory complex II are encoded by multiple nuclear genes and targeted to mitochondria in Arabidopsis thaliana. AB - Mitochondrial respiratory complex II contains four subunits: a flavoprotein (SDHI), an iron-sulphur subunit (SDH2) and two membrane anchor subunits (SDH3 and SDH4). We have found that in Arabidopsis thaliana SDH I and SDH3 are encoded by two, and SDH4 by one nuclear genes, respectively. All these encoded polypeptides are found to be imported into isolated plant mitochondria. While both SDHI proteins are highly conserved when compared to their counterparts in other organisms, SDH3 and SDH4 share little similarity with non-plant homologues. Expression of SDH1-1, SDH3 and SDH4 genes was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the second SDH1 gene (SDH1-2) is expressed at a low level. PMID- 12374304 TI - Regulation of alternative oxidase gene expression in soybean. AB - Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 degrees C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways. PMID- 12374305 TI - Post-phloem protein trafficking in the maize caryopsis: zmTRXh1, a thioredoxin specifically expressed in the pedicel parenchyma of Zea mays L., is found predominantly in the placentochalaza. AB - The pedicel of the maize (Zea mays L.) caryopsis is a complex structure consisting of layers of specialized cell-types involved with solute transfer into the developing kernel. A molecular marker for one of these cell layers, the phloem parenchyma, has been obtained by differential screening of a cDNA library from 7 days after pollination (DAP) kernels. The clone encodes a novel processed type of thioredoxin, ZmTRXh1, with a variant active site sequence. The transcript is exclusively present in phloem parenchyma cells of the pedicel. The protein accumulates predominantly in the adjacent placentochalazal layer up to 21 DAP, declining thereafter until, at 31 DAP, only traces remain. ZmTRXh1, which is catalytically active, is present in the cytosol, and is restricted to a fraction of the placentochalazal cells at 12 DAP, where it accumulates to high levels. ZmTRXh1 represents the first example of post-phloem protein trafficking in the pedicel. The reasons for this transfer and possible functions for the protein in the placentochalaza are discussed. PMID- 12374306 TI - Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems. AB - The binding protein BiP is an endoplasmic reticulum (ER)-resident member of the HSP70 stress-related protein family, which is essential for the constitutive function of the ER. In addition to responding to a variety of environmental stimuli, plant BiP exhibits a tissue-specific regulation. We have isolated two soybean BiP genomic clones, designated gsBiP6 and gsBiP9, and different extensions of their 5' flanking sequences were fused to beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum by Agrobacterium tumefaciens mediated transformation. Transgenic plants displayed prominent GUS activity in the vascular bundles of roots and shoots as well as in regions of intense cell division, such as procambial region and apical meristems. Promoter deletion analyses identified two cis-regulatory functional domains that are important for the spatially-regulated activation of BiP expression under normal plant development. While an AT-rich enhancer-like sequence, designated cis-acting regulatory domain 1, CRD1 (-358 to -211, on gsBiP6), activated expression of the BiP minimal promoter in all organs analyzed, BiP promoter activity in meristematic tissues and phloem cells required the presence of a second activating domain, CRD2 (-211 to -80). Apparently, the CRD2 sequence also harbors negative cis-acting elements, because removal of this region caused activation of gsBiP6 promoter in parenchymatic xylem rays. These results suggest that the tissue-specific control of BiP gene expression requires a complex integration of multiple cis-acting regulatory elements on the promoter. PMID- 12374307 TI - Expression analysis of the two ferrochelatase genes in Arabidopsis in different tissues and under stress conditions reveals their different roles in haem biosynthesis. AB - The Arabidopsis thaliana genome has two genes (AtFC-I and AtFC-II), encoding ferrochelatase, the terminal enzyme of haem biosynthesis. The roles of the two enzymes in the synthesis of haem for different haemoproteins was investigated using reporter gene analysis. A 1.41 kb fragment from the 5' upstream region of the AtFC-II gene was fused to the luciferase gene, and then introduced into tobacco plants, followed by luciferase activity measurements. AtFC-II-LUCwas expressed in all aerial parts of the plant, and was highest in flowers, but it was not expressed in roots. It was unaffected by viral infection, and considerably reduced by wounding or oxidative stress. Similarly, a 1.76 kb region of the AtFC-I promoter was fused to the uidA gene encoding beta-glucuronidase. AtFC-I-GUS was expressed in all tissues of the plant, but was higher in roots and flowers than in leaves or stems. It was induced by sucrose, wounding and oxidative stress and, most markedly, by plants undergoing the hypersensitive response to TMV infection. Levels of endogenous ferrochelatase activity were increased in pea chloroplasts isolated from wounded leaves, indicating that the induction in promoter activity is likely to result in increased haem biosynthetic potential. Salicylic acid, but not methyl-jasmonate was able to replace the stress treatment in induction of AtFC-I expression, suggesting that the requirement for haem synthesis is part of the defence response. The implications of the results for the different roles of the two ferrochelatases in haem biosynthesis are discussed. PMID- 12374308 TI - Expansins in the bryophyte Physcomitrella patens. AB - Expansins are cell wall proteins which play a key function in basic processes of plant growth and differentiation. It has been proposed that expansins are likely to be present in all land plants and, to date, they have been reported in angiosperms, gymnosperms and pteridophytes. In this paper, we provide the first report and analysis of genes encoding expansin-like proteins in the bryophyte, Physcomitrella patens. Our analysis indicates that both alpha- and beta-expansins are present as gene families in this plant and expression analysis indicates that these genes are subject to a complex regulation by both hormonal and environmental factors. In particular, the expression of many expansin genes in P. patens is upregulated by stress conditions, suggesting that they play a role in the specific cellular differentiation displayed by P. patens in response to such stress. Finally, we provide the first report on the generation and analysis of a series of knockout mutants for individual expansin genes. PMID- 12374309 TI - AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome. AB - The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is approximately 4 x deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones. PMID- 12374310 TI - Medical image computing at the Institute of Mathematics and Computer Science in Medicine, University Hospital Hamburg-Eppendorf. PMID- 12374311 TI - Quantitative analysis of reconstructed 3-D coronary arterial tree and intracoronary devices. AB - Traditional quantitative coronary angiography is performed on two-dimensional (2 D) projection views. These views are chosen by the angiographer to minimize vessel overlap and foreshortening. With 2-D projection views that are acquired in this nonstandardized fashion, however, there is no way to know or estimate how much error occurs in the QCA process. Furthermore, coronary arteries possess a curvilinear shape and undergo a cyclical deformation due to their attachment to the myocardium. Therefore, it is necessary to obtain three-dimensional (3-D) information to best describe and quantify the dynamic curvilinear nature of the human coronary artery. Using a patient-specific 3-D coronary reconstruction algorithm and routine angiographic images, a new technique is proposed to describe: 1) the curvilinear nature of 3-D coronary arteries and intracoronary devices; 2) the magnitude of the arterial deformation caused by intracoronary devices and due to heart motion; and 3) optimal view(s) with respect to the desired "pathway" for delivering intracoronary devices. PMID- 12374312 TI - The use of visual search for knowledge gathering in image decision support. AB - This paper presents a new method of knowledge gathering for decision support in image understanding based on information extracted from the dynamics of saccadic eye movements. The framework involves the construction of a generic image feature extraction library, from which the feature extractors that are most relevant to the visual assessment by domain experts are determined automatically through factor analysis. The dynamics of the visual search are analyzed by using the Markov model for providing training information to novices on how and where to look for image features. The validity of the framework has been evaluated in a clinical scenario whereby the pulmonary vascular distribution on Computed Tomography images was assessed by experienced radiologists as a potential indicator of heart failure. The performance of the system has been demonstrated by training four novices to follow the visual assessment behavior of two experienced observers. In all cases, the accuracy of the students improved from near random decision making (33%) to accuracies ranging from 50% to 68%. PMID- 12374313 TI - Statistical analysis of nonlinearly reconstructed near-infrared tomographic images: Part I--Theory and simulations. AB - Near-infrared (NIR) diffuse tomography is an emerging method for imaging the interior of tissues to quantify concentrations of hemoglobin and exogenous chromophores non-invasively in vivo. It often exploits an optical diffusion model based image reconstruction algorithm to estimate spatial property values from measurements of the light flux at the surface of the tissue. In this study, mean squared error (MSE) over the image is used to evaluate methods for regularizing the ill-posed inverse image reconstruction problem in NIR tomography. Estimates of image bias and image standard deviation were calculated based upon 100 repeated reconstructions of a test image with randomly distributed noise added to the light flux measurements. It was observed that the bias error dominates at high regularization parameter values while variance dominates as the algorithm is allowed to approach the optimal solution. This optimum does not necessarily correspond to the minimum projection error solution, but typically requires further iteration with a decreasing regularization parameter to reach the lowest image error. Increasing measurement noise causes a need to constrain the minimum regularization parameter to higher values in order to achieve a minimum in the overall image MSE. PMID- 12374314 TI - Statistical analysis of nonlinearly reconstructed near-infrared tomographic images: Part II--Experimental interpretation. AB - Image error analysis of a diffuse near-infrared tomography (NIR) system has been carried out on simulated data using a statistical approach described in Part I of this paper (Pogue et al., 2002). The methodology is used here with experimental data acquired on phantoms with a prototype imaging system intended for characterizing breast tissue. Results show that imaging performance is not limited by random measurement error, but rather by calibration issues. The image error over the entire field of view is generally not minimized when an accurate homogeneous estimate of the phantom properties is available; however, local image error over a target region of interest (ROI) is reduced. The image reconstruction process which includes a Levenberg-Marquardt style regularization provides good minimization of the objective function, yet its reduction is not always correlated with an overall image error decrease. Minimization of the bias in an ROI which contains localized changes in the optical properties can be achieved through five to nine iterations of the algorithm. Precalibration of the algorithm through statistical evaluation of phantom studies may provide a better measure of the image accuracy than that implied by minimization of the standard objective function. PMID- 12374315 TI - Reconstruction of time-varying 3-D left-ventricular shape from multiview X-ray cineangiocardiograms. AB - This paper reports on the clinical application of a system for recovering the time-varying three-dimensional (3-D) left-ventricular (LV) shape from multiview X ray cineangiocardiograms. Considering that X-ray cineangiocardiography is still commonly employed in clinical cardiology and computational costs for 3-D recovery and visualization are rapidly decreasing, it is meaningful to develop a clinically applicable system for 3-D LV shape recovery from X-ray cineangiocardiograms. The system is based on a previously reported closed-surface method of shape recovery from two-dimensional occluding contours with multiple views. To apply the method to "real" LV cineangiocardiograms, user-interactive systems were implemented for preprocessing, including detection of LV contours, calibration of the imaging geometry, and setting of the LV model coordinate system. The results for three real LV angiographic image sequences are presented, two with fixed multiple views (using supplementary angiography) and one with rotating views. 3-D reconstructions utilizing different numbers of views were compared and evaluated in terms of contours manually traced by an experienced radiologist. The performance of the preprocesses was also evaluated, and the effects of variations in user-specified parameters on the final 3-D reconstruction results were shown to be sufficiently small. These experimental results demonstrate the potential usefulness of combining multiple views for 3-D recovery from "real" LV cineangiocardiograms. PMID- 12374316 TI - Estimation of 3-D left ventricular deformation from medical images using biomechanical models. AB - The quantitative estimation of regional cardiac deformation from three dimensional (3-D) image sequences has important clinical implications for the assessment of viability in the heart wall. We present here a generic methodology for estimating soft tissue deformation which integrates image-derived information with biomechanical models, and apply it to the problem of cardiac deformation estimation. The method is image modality independent. The images are segmented interactively and then initial correspondence is established using a shape tracking approach. A dense motion field is then estimated using a transversely isotropic, linear-elastic model, which accounts for the muscle fiber directions in the left ventricle. The dense motion field is in turn used to calculate the deformation of the heart wall in terms of strain in cardiac specific directions. The strains obtained using this approach in open-chest dogs before and after coronary occlusion, exhibit a high correlation with strains produced in the same animals using implanted markers. Further, they show good agreement with previously published results in the literature. This proposed method provides quantitative regional 3-D estimates of heart deformation. PMID- 12374317 TI - Computation of unmeasured third-generation VCT views from measured views. AB - We compute unmeasured cone-beam projections from projections measured by a third generation helical volumetric computed tomography system by solving a characteristic problem for an ultrahyperbolic differential equation [John (1938)]. By working in the Fourier domain, we convert the second-order PDE into a family of first-order ordinary differential equations. A simple first-order integration is used to solve the ODEs. PMID- 12374318 TI - Time-domain reconstruction for thermoacoustic tomography in a spherical geometry. AB - Reconstruction-based microwave-induced thermoacoustic tomography in a spherical configuration is presented. Thermoacoustic waves from biological tissue samples excited by microwave pulses are measured by a wide-band unfocused ultrasonic transducer, which is set on a spherical surface enclosing the sample. Sufficient data are acquired from different directions to reconstruct the microwave absorption distribution. An exact reconstruction solution is derived and approximated to a modified backprojection algorithm. Experiments demonstrate that the reconstructed images agree well with the original samples. The spatial resolution of the system reaches 0.5 mm. PMID- 12374319 TI - Exact frequency-domain reconstruction for thermoacoustic tomography--I: Planar geometry. AB - We report an exact and fast Fourier-domain reconstruction algorithm for thermoacoustic tomography in a planar configuration assuming thermal confinement and constant acoustic speed. The effects of the finite size of the detector and the finite length of the excitation pulse are explicitly included in the reconstruction algorithm. The algorithm is numerically and experimentally verified. We also demonstrate that the blurring caused by the finite size of the detector surface is the primary limiting factor on the resolution and that it can be compensated for by deconvolution. PMID- 12374321 TI - Comments on "Frequency decomposition and computing of ultrasound medical images with wavelet packets". AB - In this paper, errors and discrepancies in the subject paper [Cincotti et al., (2002)] are highlighted. A comment, concerning the axial resolution associated to the adopted processing procedure is also reported. PMID- 12374320 TI - Exact frequency-domain reconstruction for thermoacoustic tomography--II: Cylindrical geometry. AB - Microwave-induced thermoacoustic tomography (TAT) in a cylindrical configuration is developed to image biological tissue. Thermoacoustic signals are acquired by scanning a flat ultrasonic transducer. Using a new expansion of a spherical wave in cylindrical coordinates, we apply the Fourier and Hankel transforms to TAT and obtain an exact frequency-domain reconstruction method. The effect of discrete spatial sampling on image quality is analyzed. An aliasing-proof reconstruction method is proposed. Numerical and experimental results are included. PMID- 12374322 TI - The relational self: an interpersonal social-cognitive theory. AB - The authors propose an interpersonal social-cognitive theory of the self and personality, the relational self, in which knowledge about the self is linked with knowledge about significant others, and each linkage embodies a self-other relationship. Mental representations of significant others are activated and used in interpersonal encounters in the social-cognitive phenomenon of transference (S. M. Andersen & N. S. Glassman, 1996), and this evokes the relational self. Variability in relational selves depends on interpersonal contextual cues, whereas stability derives from the chronic accessibility of significant-other representations. Relational selves function in if-then terms (W. Mischel & Y. Shoda, 1995), in which ifs are situations triggering transference, and thens are relational selves. An individual's repertoire of relational selves is a source of interpersonal patterns involving affect, motivation, self-evaluation, and self regulation. PMID- 12374323 TI - Conditionals: a theory of meaning, pragmatics, and inference. AB - The authors outline a theory of conditionals of the form If A then C and If A then possibly C. The 2 sorts of conditional have separate core meanings that refer to sets of possibilities. Knowledge, pragmatics, and semantics can modulate these meanings. Modulation can add information about temporal and other relations between antecedent and consequent. It can also prevent the construction of possibilities to yield 10 distinct sets of possibilities to which conditionals can refer. The mental representation of a conditional normally makes explicit only the possibilities in which its antecedent is true, yielding other possibilities implicitly. Reasoners tend to focus on the explicit possibilities. The theory predicts the major phenomena of understanding and reasoning with conditionals. PMID- 12374324 TI - The neural basis of human error processing: reinforcement learning, dopamine, and the error-related negativity. AB - The authors present a unified account of 2 neural systems concerned with the development and expression of adaptive behaviors: a mesencephalic dopamine system for reinforcement learning and a "generic" error-processing system associated with the anterior cingulate cortex. The existence of the error-processing system has been inferred from the error-related negativity (ERN), a component of the event-related brain potential elicited when human participants commit errors in reaction-time tasks. The authors propose that the ERN is generated when a negative reinforcement learning signal is conveyed to the anterior cingulate cortex via the mesencephalic dopamine system and that this signal is used by the anterior cingulate cortex to modify performance on the task at hand. They provide support for this proposal using both computational modeling and psychophysiological experimentation. PMID- 12374325 TI - Signal detection theory with finite mixture distributions: theoretical developments with applications to recognition memory. AB - An extension of signal detection theory (SDT) that incorporates mixtures of the underlying distributions is presented. The mixtures can be motivated by the idea that a presentation of a signal shifts the location of an underlying distribution only if the observer is attending to the signal; otherwise, the distribution is not shifted or is only partially shifted. Thus, trials with a signal presentation consist of a mixture of 2 (or more) latent classes of trials. Mixture SDT provides a general theoretical framework that offers a new perspective on a number of findings. For example, mixture SDT offers an alternative to the unequal variance signal detection model; it can also account for nonlinear normal receiver operating characteristic curves, as found in recent research. PMID- 12374326 TI - Ecological and evolutionary validity: comments on Johnson-Laird, Legrenzi, Girotto, Legrenzi, and Caverni's (1999) mental-model theory of extensional reasoning. AB - The mental-model account of naive probabilistic reasoning by P. N. Johnson-Laird, P. Legrenzi, V. Girotto, M. S. Legrenzi, and J.-P. Caverni (1999) provides an opportunity to clarify several similarities and differences between it and ecological rationality (frequentist) accounts. First, ambiguities in the meaning of Bayesian reasoning can lead to disagreements and inappropriate arguments. Second, 2 conflated effects of using natural frequencies are noticed but not actually tested separately because of an artificial dissociation of frequency representations and natural sampling. Third, similarities are noted between the subset principle and the principle of natural sampling. Finally, some potentially misleading portrayals of the role of evolutionary factors in psychology are corrected. Mental-model theory, rather than better explaining probabilistic reasoning, may be able to use frequency representations as a key element in clarifying its own ambiguous constructs. PMID- 12374327 TI - Does a prosocial-selfish distinction help explain the biological affects? Comment on Buck (1999). AB - R. Buck (1999) argued that a conceptual distinction between prosocial and selfish motivation is necessary to understand the biological affects (consciously experienced feelings and desires having an innate neurochemical basis). However, at a biological level of analysis, a prosocial-selfish distinction is doubtful empirically and conceptually. For this reason, Buck's proposed typology of biological affects is unclear. Moreover, a prosocial-selfish distinction is not necessary to explain hemispheric differences in brain activity associated with affect. In contrast, an approach-withdrawal distinction explains some data uniquely well, although numerous exceptions imply that simple models are inadequate. To extend hemispheric models of experienced emotion, a prosocial selfish distinction is unlikely to be explanatory, whereas an alternative account based on a distinction between verbal and nonverbal working memory may be useful. PMID- 12374328 TI - Sex differences in behavioral and hormonal response to social threat: commentary on Taylor et al. (2000). AB - Taylor and colleagues proposed that women uniquely respond to stressors by tending to children and befriending other women rather than by fighting or fleeing (S. E. Taylor et al., 2000). In this article, the authors expand Taylor et al.'s evolutionary frame and incorporate several unique aspects of human social dynamics. First, humans are characterized by extensive paternal investment, and thus men's tending is predicted and observed in some stressful contexts. Second, the dynamics of women's befriending suggest an evolutionary elaboration of the mechanisms that support reciprocal altruism. Third, coalitional male-male competition indicates that men's befriending is a predicted component of their fight-or-flight response. Finally, men's tending should result in the evolution of female-female competition over this form of parental investment. PMID- 12374329 TI - The IQ paradox: resolved? Still an open question. AB - A generalized Dickens-Flynn (2001) model is presented and various simulations undertaken with it to give readers a better sense of the properties of such models. In particular, the inclusion of moderate degrees of persistence of intelligence and intelligence-relevant environment did not have much impact on the overall behavior of the models, although more extreme degrees of persistence did. Even moderate degrees, however, affected the internal relationships in the models. The importance of specifying the time scale and of addressing developmental aspects of the models is emphasized. It is noted that the translation of individual changes to population changes is not a simple matter in resolving the "IQ paradox" of large population gains over time in intelligence test scores. PMID- 12374330 TI - Expanding variance and the case of historical changes in IQ means: a critique of Dickens and Flynn (2001). AB - The Flynn effect is the rise in mean IQ scores during the 20th century, amounting to about 0.33 IQ points per year. Many theoretical explanations have been proposed, though none are universally accepted. W. Dickens and J. R. Flynn's (2001) new approach explains the large IQ changes by means of recursive models of IQ growth. A salient feature of their models is that IQ phenotypes and their supportive environments are correlated; in addition, environmental effects can rebound on phenotypic IQ to increase or lower IQ. In this critique, the authors examine an empirical challenge to their models, which typically imply large changes in IQ variance. However, the historical rise in IQ mean level has not been accompanied by substantial variance changes, a finding inconsistent with the properties of the proposed model. PMID- 12374331 TI - Model intestinal microflora in computer simulation: a simulation and modeling package for host-microflora interactions. AB - The ecology of the human intestinal microflora and its interaction with the host are poorly understood. Though more and more data are being acquired, in part using modern molecular methods, development of a quantitative theory has not kept pace with this increase in observing power. This is in part due to the complexity of the system and to the lack of simulation environments in which to test what the ecological effect of a hypothetical mechanism of interaction would be, before resorting to laboratory experiments. The MIMICS project attempts to address this through the development of a cellular automaton for simulation of the intestinal microflora. In this paper, the design and evaluation of this simulator is discussed. PMID- 12374332 TI - Use of topological charge to determine filament location and dynamics in a numerical model of scroll wave activity. AB - The unique time course of an excitable element in cardiac tissue can be represented as the phase of its trajectory in state space. A phase singularity is defined as a spatial point where the surrounding phase values changes by a total of 2 pi, thereby forming the organizing center for a reentrant excitatory wave, a phenomenon which occurs in cardiac fibrillation. In this paper, we describe a methodology to detect the singular filament in numeric simulations of three dimensional (3-D) scroll waves by using the concept of topological charge. Here, we use simple two-variable models of cardiac activity to construct the state space, generate the phase field, and calculate the topological charge as a summation of 3-D convolution operations. We illustrate the usage of the algorithm on the basic dynamics of vortex ring filament behavior as well as the more complex spatiotemporal behavior observed in fibrillation. We also compare the motion of filament wavetips as determined by the phase field produced by two variable state space and single-variable, time-delay embedded state space. Finally, we examine the state spaces produced by a more complex three-variable model. We conclude that the use of state-space analysis, along with the unique properties of topological charge, allows for a novel means of filament localization. PMID- 12374333 TI - The use of the SPSA method in ECG analysis. AB - The classification, monitoring, and compression of electrocardiogram (ECG) signals recorded of a single patient over a relatively long period of time is considered. The particular application we have in mind is high-resolution ECG analysis, such as late potential analysis, morphology changes in QRS during arrythmias, T-wave alternants, or the study of drug effects on ventricular activation. We propose to apply a modification of a classical method of cluster analysis or vector quantization. The novelty of our approach is that we use a new distortion measure to quantify the distance of two ECG cycles, and the class distortion measure is defined using a min-max criterion. The new class-distortion measure is much more sensitive to outliers than the usual distortion measures using average-distance. The price of this practical advantage is that computational complexity is significantly increased. The resulting nonsmooth optimization problem is solved by an adapted version of the simultaneous perturbation stochastic approximation (SPSA) method of. The main idea is to generate a smooth approximation by a randomization procedure. The viability of the method is demonstrated on both simulated and real data. An experimental comparison with the widely used correlation method is given on real data. PMID- 12374334 TI - Optimal excitation wavelengths for discrimination of cervical neoplasia. AB - Fluorescence spectroscopy has shown promise for the in vivo, real-time detection of cervical neoplasia. However, selection of excitation wavelength has in the past been based on in vitro studies and the availability of light sources. The goal of this study was to determine optimal excitation wavelengths for in vivo detection of cervical neoplasia. Fluorescence excitation-emission matrices (EEMs) were measured in vivo from 351 sites in 146 patients. Data were analyzed in pairs of diagnostic classes to determine which combination of excitation wavelengths yields classification algorithms with the greatest sensitivity and specificity. We find that 330-340-, 350-380-, and 400-450-nm excitation yield the best performance. The sensitivity and specificity for discrimination of squamous normal tissue and high-grade squamous intraepithelial lesion (HGSIL) were 71% and 77% on cross validation using three excitation wavelengths. These results are comparable with those found in earlier in vivo studies; however, in this study we find that the proportion of samples which are HGSIL influences performance. Furthermore stratification of samples within low-grade squamous intraepithelial lesion and HGSIL also appears to influence diagnostic performance. Future diagnostic studies should be carried out at these excitation wavelengths in larger groups so that data can be stratified by diagnostic subcategory, age and menopausal status. Similarly, large studies should be done in screening populations. PMID- 12374335 TI - Time-varying properties of renal autoregulatory mechanisms. AB - In order to assess the possible time-varying properties of renal autoregulation, time-frequency and time-scaling methods were applied to renal blood flow under broad-band forced arterial blood pressure fluctuations and single-nephron renal blood flow with spontaneous oscillations obtained from normotensive (Sprague Dawley, Wistar, and Long-Evans) rats, and spontaneously hypertensive rats. Time frequency analyses of normotensive and hypertensive blood flow data obtained from either the whole kidney or the single-nephron show that indeed both the myogenic and tubuloglomerular feedback (TGF) mechanisms have time-varying characteristics. Furthermore, we utilized the Renyi entropy to measure the complexity of blood flow dynamics in the time-frequency plane in an effort to discern differences between normotensive and hypertensive recordings. We found a clear difference in Renyi entropy between normotensive and hypertensive blood flow recordings at the whole kidney level for both forced (p < 0.037) and spontaneous arterial pressure fluctuations (p < 0.033), and at the single-nephron level (p < 0.008). Especially at the single-nephron level, the mean Renyi entropy is significantly larger for hypertensive than normotensive rats, suggesting more complex dynamics in the hypertensive condition. To further evaluate whether or not the separation of dynamics between normotensive and hypertensive rats is found in the prescribed frequency ranges of the myogenic and TGF mechanisms, we employed multiresolution wavelet transform. Our analysis revealed that exclusively over scale ranges corresponding to the frequency intervals of the myogenic and TGF mechanisms, the widths of the blood flow wavelet coefficients fall into disjoint sets for normotensive and hypertensive rats. The separation of the scales at the myogenic and TGF frequency ranges is distinct and obtained with 100% accuracy. However, this observation remains valid only for the whole kidney blood pressure/flow data. The results suggest that understanding of the time-varying properties of the two mechanisms is required for a complete description of renal autoregulation. PMID- 12374336 TI - Model selection in electromagnetic source analysis with an application to VEFs. AB - In electromagnetic source analysis, it is necessary to determine how many sources are required to describe the electroencephalogram or magnetoencephalogram adequately. Model selection procedures (MSPs) or goodness of fit procedures give an estimate of the required number of sources. Existing and new MSPs are evaluated in different source and noise settings: two sources which are close or distant and noise which is uncorrelated or correlated. The commonly used MSP residual variance is seen to be ineffective, that is it often selects too many sources. Alternatives like the adjusted Hotelling's test, Bayes information criterion and the Wald test on source amplitudes are seen to be effective. The adjusted Hotelling's test is recommended if a conservative approach is taken and MSPs such as Bayes information criterion or the Wald test on source amplitudes are recommended if a more liberal approach is desirable. The MSPs are applied to empirical data (visual evoked fields). PMID- 12374337 TI - Automated breath detection on long-duration signals using feedforward backpropagation artificial neural networks. AB - A new breath-detection algorithm is presented, intended to automate the analysis of respiratory data acquired during sleep. The algorithm is based on two independent artificial neural networks (ANN(insp) and ANN(expi)) that recognize, in the original signal, windows of interest where the onset of inspiration and expiration occurs. Postprocessing consists in finding inside each of these windows of interest minimum and maximum corresponding to each inspiration and expiration. The ANN(insp) and ANN(expi) correctly determine respectively 98.0% and 98.7% of the desired windows, when compared with 29,820 inspirations and 29,819 expirations detected by a human expert, obtained from three entire-night recordings. Postprocessing allowed determination of inspiration and expiration onsets with a mean difference with respect to the same human expert of (mean +/- SD) 34 +/- 71 ms for inspiration and 5 +/- 46 ms for expiration. The method proved to be effective in detecting the onset of inspiration and expiration in full night continuous recordings. A comparison of five human experts performing the same classification task yielded that the automated algorithm was undifferentiable from these human experts, falling within the distribution of human expert results. Besides being applicable to adult respiratory volume data, the presented algorithm was also successfully applied to infant sleep data, consisting of uncalibrated rib cage and abdominal movement recordings. A comparison with two previously published algorithms for breath detection in respiratory volume signal shows that the presented algorithm has a higher specificity, while presenting similar or higher positive predictive values. PMID- 12374338 TI - Bayesian nonstationary autoregressive models for biomedical signal analysis. AB - We describe a variational Bayesian algorithm for the estimation of a multivariate autoregressive model with time-varying coefficients that adapt according to a linear dynamical system. The algorithm allows for time and frequency domain characterization of nonstationary multivariate signals and is especially suited to the analysis of event-related data. Results are presented on synthetic data and real electroencephalogram data recorded in event-related desynchronization and photic synchronization scenarios. PMID- 12374339 TI - Noninvasive myocardial activation time imaging: a novel inverse algorithm applied to clinical ECG mapping data. AB - Linear approaches like the minimum-norm least-square algorithm show insufficient performance when it comes to estimating the activation time map on the surface of the heart from electrocardiographic (ECG) mapping data. Additional regularization has to be considered leading to a nonlinear problem formulation. The Gauss-Newton approach is one of the standard mathematical tools capable of solving this kind of problem. To our experience, this algorithm has specific drawbacks which are caused by the applied regularization procedure. In particular, under clinical conditions the amount of regularization cannot be determined clearly. For this reason, we have developed an iterative algorithm solving this nonlinear problem by a sequence of regularized linear problems. At each step of iteration, an individual L-curve is computed. Subsequent iteration steps are performed with the individual optimal regularization parameter. This novel approach is compared with the standard Gauss-Newton approach. Both methods are applied to simulated ECG mapping data as well as to single beat sinus rhythm data from two patients recorded in the catheter laboratory. The proposed approach shows excellent numerical and computational performance, even under clinical conditions at which the Gauss-Newton approach begins to break down. PMID- 12374340 TI - Interdigitated humidity sensors for a portable clinical microsystem. AB - This paper presents and compares two capacitive humidity sensors with interdigitated electrodes for a portable clinical application. A polyimide sensitive layer covers the first structures and the optimized ones include a benzocyclobutene-sensitive layer and a heating resistor. Humidity measurements results are presented, in particular sensors response time in absorption, which are very small (inferior to 500 ms). Next, mechanisms of absorption and adsorption in a polymer layer are described and the two structures are compared. Suitability of optimized sensors for our application is discussed: new structures allow us to increase sensitivity and decrease response time. When the structure is maintained at 40 degrees C by its heater, the response time is 200 ms and the total desorption time is 11 s. PMID- 12374341 TI - Fiber-optic confocal reflectance microscope with miniature objective for in vivo imaging of human tissues. AB - We have built a fiber-optic confocal reflectance microscope capable of imaging human tissues in near real time. Miniaturization of the objective lens and the mechanical components for positioning and axially scanning the objective enables the device to be used in inner organs of the human body. The lateral resolution is 2 micrometers and axial resolution is 10 micrometers. Confocal images of fixed tissue biopsies and the human lip in vivo have been obtained at 15 frames/s without any fluorescent stains. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. PMID- 12374342 TI - Binaural sonar electronic travel aid provides vibrotactile cues for landmark, reflector motion and surface texture classification. AB - Electronic travel aids (ETAs) for the blind commonly employ conventional time-of flight sonars to provide range measurements, but their wide beams prevent accurate determination of object bearing. We describe a binaural sonar that detects objects over a wider bearing interval compared with a single transducer and also determines if the object lies to the left or right of the sonar axis in a robust manner. The sonar employs a pair of Polaroid 6500 ranging modules connected to Polaroid 7000 transducers operating simultaneously in a binaural array configuration. The sonar determines which transducer detects the echo first. An outward vergence angle between the transducers improves the first-echo detection reliability by increasing the delay between the two detected echoes, a consequence of threshold detection. We exploit this left/right detection capability in an ETA that provides vibrotactile feedback. Pager motors mount on both sides of the sonar, possibly worn on the user's wrists. The motor on the same side as the reflecting object vibrates with speed inversely related to range. As the sonar or object moves, vibration patterns provide landmark, motion and texture cues. Orienting the sonar at 45 degrees relative to the travel direction and passing a right-angle corner produces a characteristic vibrational pattern. When pointing the sonar at a moving object, such as a fluttering flag, the motors alternate in a manner to give the user a perception of the object motion. When the sonar translates or rotates to scan a foliage surface, the vibrational patterns are related to the surface scatterer distribution, allowing the user to identify the foliage. PMID- 12374343 TI - Design and implementation of a brain-computer interface with high transfer rates. AB - This paper presents a brain-computer interface (BCI) that can help users to input phone numbers. The system is based on the steady-state visual evoked potential (SSVEP). Twelve buttons illuminated at different rates were displayed on a computer monitor. The buttons constituted a virtual telephone keypad, representing the ten digits 0-9, BACKSPACE, and ENTER. Users could input phone number by gazing at these buttons. The frequency-coded SSVEP was used to judge which button the user desired. Eight of the thirteen subjects succeeded in ringing the mobile phone using the system. The average transfer rate over all subjects was 27.15 bits/min. The attractive features of the system are noninvasive signal recording, little training required for use, and high information transfer rate. Approaches to improve the performance of the system are discussed. PMID- 12374344 TI - Intrabody three-dimensional position sensor for an ultrasound endoscope. AB - To avoid or reduce the X-ray exposure in endoscopic examinations and therapy, as an alternative to the conventional two-dimensional X-ray fluoroscopy we are developing an intrabody navigation system that can directly measure and visualize the three-dimensional (3-D) position of the tip and the trace of an ultrasound endoscope. The proposed system can identify the 3-D location and direction of the endoscope probe inserted into the body to furnish endoscopic images. A marker transducer(s) placed on the surface of the body transmits ultrasound pulses, which are visualized as a marker synchronized to the scanning of the endoscope. The position (direction and distance of the marker transducer(s) outside the body relative to the scanning probe inside the body) of the marker is detected and measured in the scanned image of the ultrasound endoscope. Further, an optical localizer locates the marker transducer(s) with six degrees of freedom. Thus, the proposed method performs inside-body 3-D localization by utilizing the inherent image reconstruction function of the ultrasound endoscope, and is able to be used with currently available commercial ultrasound image scanners. The system may be envisaged as a kind of global positioning system for intrabody navigation. PMID- 12374346 TI - A versatile microwave plethysmograph for the monitoring of physiological parameters. AB - A simple microwave technique for in vivo monitoring of human pulmonary and cardiac activity is here presented. The technique is based on detecting the changes in the modulation envelope of amplitude modulated waves passing through the human body. A simplified human chest model was developed, proving an unambiguous correlation between heart blood filling and microwave transmission through the chest. A prototype system for transmittance measurement was realized at the 868.5-MHz operating frequency, demonstrating the feasibility of a small, lowcost microwave plethysmograph. In vivo measurements showed a good agreement with numerical simulations. PMID- 12374345 TI - Investigating membrane breakdown of neuronal cells exposed to nonuniform electric fields by finite-element modeling and experiments. AB - High electric field strengths may induce high cell membrane potentials. At a certain breakdown level the membrane potential becomes constant due to the transition from an insulating state into a high conductivity and high permeability state. Pores are thought to be created through which molecules may be transported into and out of the cell interior. Membrane rupture may follow due to the expansion of pores or the creation of many small pores across a certain part of the membrane surface. In nonuniform electric fields, it is difficult to predict the electroporated membrane area. Therefore, in this study the induced membrane potential and the membrane area where this potential exceeds the breakdown level is investigated by finite-element modeling. Results from experiments in which the collapse of neuronal cells was detected were combined with the computed field strengths in order to investigate membrane breakdown and membrane rupture. It was found that in nonuniform fields membrane rupture is position dependent, especially at higher breakdown levels. This indicates that the size of the membrane site that is affected by electroporation determines rupture. PMID- 12374347 TI - Simplified calibration of single-plunge bipolar electrode array for field measurement during defibrillation. AB - In an earlier study, the authors presented a calibration technique for a triaxial bipolar electrode array (EA) that used 72 data points collected during a global sweep of the electric field vector relative to the EA axes. Although necessary for the initial characterization of the EAs, this data requirement has to be significantly reduced for the technique to become a practical tool. Therefore, in the present study, an analysis is performed to determine the relation between the number of data points used in the calibration and the mean root-mean-square error. The analysis shows that 18 data points can produce results nearly identical to those obtained with the 72-point calibration, thus reducing the required amount of data fourfold. PMID- 12374348 TI - Matched-filter template generation via spatial filtering: application to fetal biomagnetic recordings. AB - We have developed a two-step procedure for signal processing of fetal biomagnetic recordings that removes cardiac interference and noise. First, a modified matched filter (MF) is applied to remove maternal cardiac interference; then, a simple signal space projection (SSP) is applied to remove noise. The key difference between our MF and a conventional one is that the interference template and the template scaling are derived from a signal that has been spatially filtered to isolate the interference, rather than from the raw signal. Unlike conventional MFs, ours is able to separate maternal and fetal cardiac complexes, even when they have similar morphology and overlap strongly. When followed by a SSP that preserves only the signal subspace, the noise is reduced to a low level. PMID- 12374349 TI - Anode-break excitation during end-diastolic stimulation is explained by half-cell double layer discharge. AB - The phenomenon of anodal-break excitation during end-diastolic stimulation of the heart was discovered many years ago by B. Hoffman. Yet, the existence and mechanistic explanation of this effect remain controversial. We sought to confirm its existence and to determine a possible role of half-cell potential. We used isolated Langendorff-perfused rabbit hearts (n = 6) which were stained with di-4 ANEPPS and perfused with 15-mM butanedione monoxime (BDM). Transmembrane potentials were optically recorded at the left ventricular epicardium with a high spatial and temporal resolution (200 microm/343 micros) near the tip of a 120 microm platinum-iridium Teflon-coated unipolar pacing electrode to detect virtual electrode polarization and to reconstruct an activation pattern. Hearts were paced at a cycle length of 300 ms by anodal square pulses with an amplitude of 0.1-10 mA and a duration of 5-60 ms. Data revealed that the anodal-break excitation does exists and is accompanied by an overshoot in the recordings of the pacing current. Addition of a diode in the stimulation circuit eliminated both the overshoot and the break excitation. The findings suggest that a half cell surface potential at the pacing electrode metal-saline interface may influence the pacing currents during unipolar anodal cardiac stimulation providing "break"-like activation. We also confirmed that the threshold of "break"-like excitation is lower than make-excitation. We suggest that further exploration of this effect is needed in order to design improved multiphasic pacing waveforms. PMID- 12374350 TI - Elevating pain thresholds in humans using depolarizing prepulses. AB - Electrocutaneous stimulation is a potentially useful communication tool for applications in virtual reality, sensory substitution, and sensory augmentation. Many of these applications require the use of arrays of small electrodes. Stimulation through small electrodes is often painful, however, limiting the practicality of such arrays. The purpose of this study was to test a method for elevating the pain threshold to electrocutaneous stimulation through small (1-mm diameter) electrodes on the fingertip. We hypothesized that long, subthreshold, depolarizing prepulses (PP) would elevate the pain threshold so that a subsequent stimulus pulse (SP) would be less likely to be painful. We used psychophysical methods to measure the probability that an SP would be perceived as painful both by itself and when preceded by a PP that was 2, 4, 6, 8, or 10 dB lower in amplitude than the SP. We found that the PPs significantly increased the pain threshold, reducing the likelihood that the SP was painful (p < .0001). The dose effect of PP amplitude was also highly significant (p < .0001), with larger PPs elevating pain thresholds more. To our knowledge, this is the first report of PPs being used to elevate electrical stimulation thresholds in humans. PPs may be useful for selective inactivation of neural subpopulations in many human neuroprosthetic applications. PMID- 12374351 TI - Exploring the pathogenesis of necrotizing fasciitis due to Streptococcus pneumoniae. AB - Monobacterial necrotizing fasciitis is a rare form of soft tissue infection usually caused by the group A beta-hemolytic Streptococcus. Soft tissue infection is an uncommon clinical manifestation of invasive disease due to Streptococcus pneumoniae. We describe 3 cases of pneumococcal necrotizing fasciitis and explore potential pathogen-specific mechanisms of pathogenesis. The clinical characteristics of necrotizing fasciitis due to S. pneumoniae and group A beta hemolytic Streptococcus appear to overlap. The similarities include predominant occurrence in elderly adults with underlying chronic illness, predilection for lower extremity infection, progression to toxic shock-like syndrome and a high case fatality rate. No DNA fragments corresponding to speA, speB or speC were amplified by PCR from the 3 pneumococcal isolates. Western immunoblot revealed no evidence of SpeA, SpeB or SpeC protein expression. Evaluation for protease production and cytotoxicity was unrevealing. The similar clinical presentation of pneumococcal necrotizing fasciitis to the disease caused by the group A beta hemolytic Streptococcus has important therapeutic implications. The molecular mechanisms underlying the pathogenesis are unclear. Prospective population-based studies are required to define the epidemiology of this infection. PMID- 12374352 TI - Staphylococcus aureus in a single positive blood culture: causes and outcome. AB - Single positive culture was encountered in 61/235 patients (26%) with Staphylococcus aureus in blood culture over a 2-y period. It represented either true bacteremia (n = 52 cases; 85.2%) or contamination (n = 9; 14.8%). In comparison to cases with < or = 2 positive cultures, these patients did not have less severe disease or a lower incidence of complications. PMID- 12374353 TI - Incidence and clinical significance of non-tuberculous mycobacteria isolated from clinical specimens during a 2-y nationwide survey. AB - A 2-y nationwide survey of patients in Denmark with non-tuberculous mycobacteria (NTM) cultures was undertaken. Patients were identified by means of records held at the International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Denmark. The objectives were to identify isolated NTM to species level, to describe the incidence of the various species and to evaluate the clinical significance of pulmonary NTM isolates other than M. avium complex (MAC) and M. gordonae. Identification was performed by means of hybridization or sequencing of 16S rDNA. The clinical significance of pulmonary NTM isolates was evaluated by means of questionnaires concerning patients (was sent to the clinicians!) patients who had NTM isolated for the first time using bacteriologic, radiographic and clinical criteria. A total of 1110 specimens (2.1%) from 525 patients grew NTM. After MAC (n = 198) and M. gordonae (n = 168), most patients had M. abscessus (n = 21), M. malmoense (n = 20) and M. xenopi (n = 17) isolated. Of the pulmonary patients, 50.6% met bacteriologic criteria, 75.3% radiographic criteria and 53.4% clinical criteria for significant infection. Almost half of the pulmonary patients met all the criteria for significant NTM infection that could be evaluated. Clinically significant infection was associated with underlying disease in most patients. PMID- 12374354 TI - Prevalence and annual risk of tuberculosis infection in Edirne, Turkey. AB - Tuberculosis is still an important problem in developing countries. A total of 3,774 students from primary schools in Edirne, Turkey were included in this study in order to determine the annual infection risk for the year 1994. Five tuberculin units of purified protein derivative were applied using a Mantoux test and evaluated. The mean induration diameter was 14.6 mm. A total of 51.6% of the students were found to be sensitive to tuberculin. The annual infection risk of tuberculosis was found to be 1.51%, lower than that found in 1987. Other studies from Turkey have reported lower values than ours. We did not diagnose any cases of tuberculosis. We revealed that the annual infection risk among primary schoolchildren in Edirne is close to the average for Turkey and thus tuberculosis is still a serious risk for these children. Tuberculosis remains an important public health problem in Turkey and is partially attributable to socioeconomic difficulties. PMID- 12374355 TI - Educational intervention for parents and healthcare providers leads to reduced antibiotic use in acute otitis media. AB - We used a controlled before-and-after design with the aims of reducing both the total consumption of antibiotics and the use of broad-spectrum antibiotics against acute otitis media (AOM), and to study to what extent prescriptions for antibiotics against AOM were dispensed. Information on evidence-based treatment of uncomplicated AOM was provided to doctors and nurses, and written guidelines were implemented. Pamphlets and oral information concerning symptomatic treatment and the limited effect of antibiotic use in AOM were given to parents. Eligible patients were 819 children aged 1-15 y. The proportion of patients receiving a prescription for antibiotics was reduced from 90% at baseline to 74% during the study period. The proportion of prescriptions for penicillin V increased from 72% at baseline to 85% during the study period. There were no significant changes at the control site. The proportion of dispensed prescriptions was 70% both at baseline and during the study period. Educational efforts reduced the total consumption of antibiotics and the use of broad-spectrum antibiotics for AOM in children aged 1-15 y at an emergency call service. Data on antibiotic use in AOM based only on prescribing overestimates the use of antibiotics. PMID- 12374356 TI - Failure to detect Chlamydia pneumoniae in aortic valves and peripheral blood mononuclear cells from patients undergoing aortic valve replacement in Norway. AB - The association of Chlamydia pneumoniae with atherosclerosis is still controversial. Reports from different laboratories have varied widely and "gold standards" for the detection of C. pneumoniae are lacking. In the present study, aortic valves and peripheral blood mononuclear cells from 48 patients undergoing aortic valve replacement were examined for the presence of C. pneumoniae using a nested PCR. C. pneumoniae-specific DNA was not detected in any of the clinical samples. No PCR inhibition was observed by spiking the samples with target C. pneumoniae. A total of 31/46 patients (67%) were seropositive for C. pneumoniae IgG. These results do not support the association of C. pneumoniae with aortic valves and peripheral blood mononuclear cells in patients with atherosclerotic aortic heart valve disease. PMID- 12374357 TI - Self-reported herpes labialis in a Swedish population. AB - In a cross-sectional study, the occurrence of a self-reported history of labial herpes was evaluated. The study population comprised a stratified random sample of 5,000 individuals, aged 0-60 y, from south-west Sweden. A questionnaire, together with written and photographic descriptions of labial herpes lesions, was sent to the participants. Of 5,000 questionnaires sent out, 3597 (72%) were returned. In answer to the question "Have you ever had herpes?" the point estimate was 26.6% (95% confidence interval [CI] 25.1%-28.1%) and for the question "Have you had herpes in the last 2 y?" it was 19.4 (95% CI 18.0-20.8). The proportion of individuals who had had herpes was higher for the older age groups. About 5% of children aged < or = 5 y had experienced labial herpes. Herpes was more frequently reported by females than males: odds ratio 1.29 (95% CI 1.10-1.51). Compared to previous studies in Sweden, our data do not indicate that the occurrence of labial herpes lesions has decreased. PMID- 12374358 TI - Thymic involvement in immune recovery during antiretroviral treatment of HIV infection in adults; comparison of CT and sonographic findings. AB - In adult HIV-infected patients, thymic size evaluated from CT scans seems to be important to the degree of immune reconstitution obtainable during treatment with highly active antiretroviral therapy (HAART). To examine whether ultrasound is as reliable as CT for estimating thymic size and predicting immune recovery, CT and ultrasound scans were performed in 25 adult HIV-infected patients and 10 controls. CD4 counts and naive CD4 counts were measured in order to determine immune reconstitution. Furthermore, the CD4+ T-cell receptor excision circle (TREC) frequency and T-cell receptor (TCR) repertoire were determined. The study demonstrated no correlation between the 2 scanning methods (r = 0.201, p = 0.358 in patients and r = 0.457, p = 0.184 in controls). Among the patients, no association was found between the sonographically estimated thymic size and immunological parameters such as CD4 count (r = 0.083, p = 0.706), naive CD4 count (r = 0.067, p = 0.762), CD4 + TREC frequency (r = 0.028, p = 0.900) and CD4 + TCR repertoire (r = -0.057, p = 0.828). These findings show that CT remains superior for assessing thymic size in adults and is preferable to ultrasound when evaluating the importance of a large thymus to immune recovery during HAART. PMID- 12374359 TI - Percutaneous drainage of pyogenic lung abscess. AB - Although lung abscesses are successfully treated with antibiotics in 80-90% of cases, this conservative approach may occasionally fail. In cases of failure, pulmonary resection is usually advised. Although it remains controversial, an alternative therapy in such situations is percutaneous transthoracic tube drainage (PTTD). Herein we review the medical literature on PTTD from the last 25 y, focusing on its efficacy, indications, technique, complications and mortality. We conclude that PTTD is a safe, simple and efficacious tool for the management of refractory lung abscess. Complications relating to the procedure occurred in 9.7% of cases and included catheter occlusion, chest pain, pneumothorax and hemothorax. The overall mortality rate secondary to lung abscess was acceptable (4.8%). PMID- 12374360 TI - Prognostic value of quantitative blood cultures for the outcome of central venous catheters in children. AB - Quantitative blood cultures have been used in order to define catheter-related bloodstream infection (CRBI) in pediatric patients with malignancy and central venous catheters (CVCs). We prospectively followed 32 patients with a total of 38 CVCs for a period of 4 y (14,068 catheter-days). Of a total of 35 cases of bacteremia, 9 were considered to be CRBI (25%). The incidence of bacteremia in our study was 2.48 episodes/1,000 catheter-days and 20/38 CVCs (52%) were affected by bacteremia. The incidence of CRBI was 0.63 episodes/1,000 catheter days and it was detected in 9/38 CVCs (23%). The catheter salvage rate in cases of bacteremia, irrespective of etiology, was 30/35 (85%). The catheter salvage rate in cases of CRBI was only 4/9 (44%), whereas all the catheters (26/26) in non-catheter-related cases of bacteremia were salvaged. We suggest that the use of quantitative blood cultures is a useful tool for the evaluation of bacteremia in patients with CVCs and is of prognostic value. PMID- 12374361 TI - Streptococcus suis infection as a cause of severe illness: 2 cases from Croatia. AB - Streptococcus suis is a zoonotic agent that can be spread to humans, e.g. butchers, abattoir workers and farmers, by contact with pigs. Human infection is most frequently manifested as purulent meningitis, in combination with deafness and ataxia, but there have been rare reports of septic shock leading to multiorgan failure and death. We report 2 patients with S. suis type 1 infection. One patient suffered an abrupt and severe illness, with septic shock leading to multiorgan failure and death, whereas the other presented with purulent meningitis and deafness. Both patients were immunocompromised. They were most likely infected as a result of handling pork at home. In both cases, the infection was due to S. suis type 1, in contrast to previous reports indicating an association between human infection and S. suis type 2. Epidemiologic surveys of human infection may be of interest, especially among individuals exposed to pigs and pork. PMID- 12374362 TI - Surgical wound infection associated with Staphylococcus sciuri. AB - We describe a case of surgical wound infection due to Staphylococcus sciuri. The isolated strain was susceptible to trimethoprim-sulfamethoxazole, erythromycin, chloramphenicol, ciprofloxacin and vancomycin and resistant to gentamicin, clindamycin, rifampicin, methicillin, ampicillin and ceftriaxone. The multiresistance of the strain had a serious impact on the prolonged course of the infection. Although this bacterium is principally found in animals, our strain was probably of nosocomial origin. PMID- 12374363 TI - Plesiomonas shigelloides sepsis in a thalassemia intermedia patient. AB - Bacteremia due to Plesiomonas shigelloides was associated with rapidly fulminant septicemia, disseminated intravascular coagulation and massive adrenal hemorrhage in a splenectomized patient suffering from thalassemia intermedia who was treated with hydroxyurea. P. shigelloides was isolated in blood cultures; despite a vigorous combination of antibiotics the patient died after 24 h in the ICU. Lethal sepsis due to P. shigelloides has not previously been reported in Greece. PMID- 12374364 TI - Hemophagocytic syndrome as an initial presentation of miliary tuberculosis without pulmonary findings. AB - A 9-y-old girl was admitted with fever, weakness and weight loss. She had pancytopenia in peripheral blood, hypocellularity and hemophagocytosis in bone marrow. Disseminated tuberculosis was diagnosed after a long delay, with involvement of the lungs, bone marrow, liver, spleen and central nervous system. Tuberculosis can be a cause of hemophagocytosis and should be taken into account in the differential diagnosis of fever of unknown origin associated with pancytopenia and hemophagocytosis. PMID- 12374365 TI - Septic shock associated with Shigella flexneri dysentery. AB - Septic shock is a very unusual presentation of Shigella infection. We describe a 3-y-old child who developed severe septic shock and severe encephalopathy during an episode of dysentery caused by Shigella flexneri. PMID- 12374366 TI - Central venous catheter-related infection due to Agrobacterium radiobacter: a report of 2 cases. AB - We report 2 cases of Agrobacterium radiobacter bacteremia in immunocompromised patients. Removal of the central venous catheter and administration of antimicrobial therapy led to favorable outcomes in both patients. Infections due to A. radiobacter are rare and usually occur in patients with predisposing factors. PMID- 12374367 TI - Venous sinus thrombosis after Proteus vulgaris meningitis and concomitant Clostridium abscess formation. AB - A 19-y-old woman presented with Proteus vulgaris meningitis as a complication of chronic otitis media. Despite treatment with ceftazidime and amikacin no clinical improvement was observed. Cranial MRI revealed right-sided mastoiditis/otitis media and venous sinus thrombosis. After mastoidectomy, repeat cranial MRI demonstrated abscess formation in the venous sinuses. The abscess was drained. Clostridium spp. was isolated from the abscess culture. PMID- 12374368 TI - A case of Strongyloides stercoralis and mesenteric tuberculous infection with acute abdominal pain in an HIV-positive patient. AB - We describe an HIV-positive female patient who had acute abdominal pain as the initial presentation of Strongyloides stercoralis infection. The diagnosis was established by identifying rhabditiform larvae in stool. She also had intra abdominal tuberculosis without intestinal perforation. To our knowledge, this is the first reported case of such a presentation. PMID- 12374369 TI - HCV infection in a child without obvious risk factors for blood-borne infections. AB - The mother of an HCV-infected child had been subjected to neonatal exchange transfusion. It is likely that she contracted a chronic HCV infection which was later transmitted to her daughter. A similar route of infection is suggested in other HCV-infected patients lacking obvious risk factors. PMID- 12374370 TI - Corynebacterium macginleyi isolated from urine in a patient with a permanent bladder catheter. AB - An 82-y-old male patient with a neurogenic bladder and vesical stones presented with a urinary tract infection caused by Corynebacterium macginleyi. This is the first case of isolation of C. macginleyi from a non-conjunctival specimen. The patient recovered fully with antimicrobial treatment. PMID- 12374371 TI - Successful treatment of Paecilomyces lilacinus endophthalmitis with voriconazole. AB - Paecilomyces lilacinus is a rare cause of endophthalmitis and there are few reports of it in the literature. Herein we report a patient with P. lilacinus endophthalmitis who was treated with the new triazole, voriconazole, for 4 months, with a good clinical evolution. This treatment appears to be a valuable new therapeutic option but requires further clinical confirmation. PMID- 12374372 TI - Disseminated infection with encephalitozoon intestinalis in AIDS patients: report of 2 cases. AB - Microsporidiosis must be regarded as a late opportunistic infection when HIV is advanced. In this article we describe 2 cases of disseminated infection with Encephalitozoon intestinalis. The first case had a local intestinal infection for > 1 y before it disseminated and microsporidia were found intracellularly in sputum. In the second case, spores were initially found in conjunctival cells, sinus lavage, sputum and urine. This patient had clinical symptoms and radiological findings from the central nervous system. Signs of cerebral lymphoma developed after treatment of the opportunistic microsporidial infection. PMID- 12374373 TI - Reversible posterior leukoencephalopathy in an HIV-infected patient with thrombotic thrombocytopenic purpura. AB - We describe a 37-y-old male with advanced HIV disease who was diagnosed with thrombotic thrombocytopenic purpura after MRI of the brain revealed reversible posterior leukoencephalopathy. We discuss reversible posterior leukoencephalopathy as a diagnostic clue in HIV-infected patients with multi organ system disease. PMID- 12374374 TI - Listeria monocytogenes demonstrated in cerebrospinal fluid in the absence of inflammatory cells: a case of fever and seizures in a 13-y-old boy. PMID- 12374375 TI - High prevalence of hepatitis B virus (HBV) among male blood donors in a developing country: urgent need for systematic screening. PMID- 12374376 TI - An automated phantom-film QA procedure for intensity-modulated radiation therapy. AB - To verify that the calculated dose distribution is delivered accurately during intensity-modulated radiation therapy (IMRT), we have implemented an automated plan/film validation protocol. The cubic polystyrene film phantom provided with the Peacock IMRT system and the Radiation Imaging Technology (RIT) film dosimetry system were used to compare planned and delivered dose distributions. The calculated dose matrix from CORVUS was transferred to RIT and analyzed. The analysis included dose-difference histograms, dose comparison in low-gradient areas, distance to agreement in high-gradient areas, dose profiles, and isodose comparisons. Dose differences of up to 5% were commonly observed in the high-dose and low-gradient areas between verification films and treatment plans for prostate patients. The most prominent discrepancies were detected in the high gradient areas of dose distributions. The automated protocol is an efficient technique that provides information about spatial differences between calculated and delivered doses. PMID- 12374377 TI - A method of scaling the 3D electron pencil-beam dose calculation to obtain accurate monitor units for irregularly-shaped electron beams. AB - A method for determining monitor units (MU) for electron beams using a 3D pencil beam dose algorithm is described. A set of correction factors (OF(c)) to the pencil beam dose is generated that transforms the output into dose per MU. The OF(c)s depend on cone selection, field size, and source-to-surface distance (SSD) for a given electron energy. The dose per MU is determined by scaling the dose by the OF(c). The OF(c) value is determined using a measured set of relative output factors (OF(rel)) for various field sizes and SSD. The pencil-beam algorithm is used to compute the "raw" value (OF(p)) to each of the OF(rel) measurement points. The OF(c) is the ratio of the measured OF(rel) to the calculated OF(p). The OF(c) value for irregularly-shaped electron fields or for electron fields at extended SSD may be interpolated from the OF(c) table. The interpolation over SSD is performed linearly using the central axis SSD. The interpolation over field size is more complicated and uses the minimum area-circumscribing rectangle around the field shape. Due to the relative flatness of the OF(c), the interpolation is less error prone than the more common direct interpolation of the output factors. Computations were performed in the ADAC Pinnacle3 Planning System. Measurements were obtained using a Varian 2300c for 6 and 15 MeV. The results show that this method can predict the dose per MU within 1% to 2% for clinical fields and within 3% to 4% for extreme field shapes. PMID- 12374378 TI - Compensators for intensity-modulated beams. AB - This study describes the importance of attenuator scatter in the construction of compensators. The attenuator used in this study was Lipowitz metal, commonly referred to as cerrobend. Linear attenuation coefficients of cerrobend were measured in air for different thickness of cerrobend sheets and different field sizes for a 6-MV photon beam. The magnitude of the dose contribution from photons scattered by the attenuator was measured. The variations of beam hardening and the scatter to primary ratio as a function of the thickness of cerrobend and varying field size were investigated. The compensators in this study were produced using a simple exponential attenuation model and the measured linear attenuation coefficients. It was found that the beam hardening effect was significant, and can lead to an error of 6.2% in the transmission, for 6 cm of cerrobend in the beam. The maximum scatter contribution to the measured fluence was 19.8% of the transmitted primary dose for a 20 x 20-cm2 field size, and 6 cm of cerrobend in the beam. For a simple wedge-step compensator; there was a maximum deviation of 6% between the measured and our predicted fluence profile. For simple compensators, this deviation can be attributed to scatter. PMID- 12374379 TI - The effects of edema on urethral dose following palladium-103 prostate brachytherapy. AB - The effects of edema on urethral dose after interstitial prostate brachytherapy with palladium-103 (103Pd) were studied. Fifty patients underwent a 90-Gy 103Pd implant followed by dosimetric computed tomography (CT). Twenty-one days later, a Foley catheter was reinserted and a dosimetric CT was repeated. The mean reduction in prostate volume between day 0 and day 21 was 16%. Median prostate D90 on day 0 was 89.7 Gy (range 59.5 to 127) and 99.5 Gy (range 62.5 to 130) on day 21. Median prostate V100 was 90% (range 63 to 98%) on day 0 and 96% (range 66 to 99%) on day 21. Median V150 was 61% (range 31 to 85%) on day 0 and 75% (range 39 to 93%) on day 21. Median urethral D50 was 107 Gy (range 57 to 201) on day 0 and 126 Gy (range 64 to 193) on day 21. Regression analysis demonstrated a significant correlation between the decrease in the prostate volume and the increased urethral D50 (r 0.58, p < 0.05). Acute urinary toxicity was 32% grade 0, 38% grade 1, and 30% grade 2. The median urethral D50 increased by a mean of 18% with a correlation coefficient of 0.58 (p < 0.05). Catheterization of the urethra was well tolerated and was of value in better characterizing urethral dose after 103Pd brachytherapy. PMID- 12374380 TI - TMC-256A1 and C1, new inhibitors of IL-4 signal transduction produced by Aspergillus niger var niger TC 1629. AB - New inhibitors of IL-4 signal transduction, designated as TMC-256A1 and C1, were discovered together with TMC-256B1, a previously known dihydronaphthopyrone, from the fermentation broth of Aspergillus niger var niger TC 1629 by using an IL-4 driven reporter gene assay. Based on spectroscopic analyses, TMC-256A1 and C1 were found to be new members of the naphthopyrone antibiotics. TMC-256A1, B1 and C1 inhibited the IL-4 driven luciferase activity with IC50 values of 25 microM, 30 microM and 1.7 microM, respectively in this assay system. Furthermore, these compounds inhibited the expression of germline C epsilon mRNA with IC50 values of 6.6 microM , 34 microM and 0.31 microM, respectively. PMID- 12374381 TI - Aspergillin PZ, a novel isoindole-alkaloid from Aspergillus awamori. AB - Aspergillin PZ was obtained from the fermentation of Aspergillus awamori (Nakazawa) by activity-guided fractionation and purification. Its structure was elucidated on the basis of spectral data, especially by 2D NMR, and finally confirmed by an X-ray analysis. It could induce conidia of P. oryzae to deform moderately. PMID- 12374382 TI - New dithiolopyrrolone antibiotics from Saccharothrix sp. SA 233. I. Taxonomy, fermentation, isolation and biological activities. AB - Three new natural antibacterial and antifungal dithiolopyrrolone antibiotics were isolated along with the known iso-butyropyrrothine and thiolutine from the fermentation broth of an actinomycete strain which was isolated from a saharian palm grove soil collected at Adrar, south Algeria. The strain was identified as Saccharothrix sp. The three new antibiotics exhibited broad antimicrobial activity against gram-positive bacteria, yeasts and fungi in vitro. PMID- 12374383 TI - New dithiolopyrrolone antibiotics from Saccharothrix sp. SA 233. II. Physicochemical properties and structure elucidation. AB - Three new natural dithiopyrrolone antibiotics, 3-methyl-2-butenoylpyrrothine (1), tigloylpyrrothine (2), and n-butyropyrrothine (3) were isolated along with the known isobutyropyrrothine (4) and thiolutin (5) from the fermentation broth of Saccharothrix sp. SA 233. The structures of the novel compounds were established on the basis on their spectral data. PMID- 12374384 TI - Identification of L-glutamine: 2-deoxy-scyllo-inosose aminotransferase required for the biosynthesis of butirosin in Bacillus circulans. AB - Using inverse PCR, two new genes (btrN and btrS) were identified upstream of the putative glycosyltransferase gene btrM in the butirosin-biosynthetic btr gene cluster of Bacillus circulans. The upstream gene btrS showed significant homology with stsC of Streptomyces griseus, which encodes L-glutamine:scyllo-inosose aminotransferase in the biosynthesis of streptomycin. The function of BtrS was further confirmed by heterologous expression in Escherichia coli and chemical identification of the conversion of 2-deoxy-scyllo-inosose into 2-deoxy-scyllo inosamine. The identification of BtrS as L-glutamine:2-deoxy-scyllo-inosose aminotransferase is the first report of the aminotransferase gene responsible for 2-deoxystreptamine biosynthesis. PMID- 12374385 TI - Argyrins, immunosuppressive cyclic peptides from myxobacteria. II. Structure elucidation and stereochemistry. AB - The structures of argyrins A-H were elucidated by NMR spectroscopy, chemical degradation and X-ray analysis as cyclic octapeptides. Argyrins A and B, in addition to the common amino acids tryptophan, glycine, dehydroalanine and alanine or alpha-aminobutyric acid, sarcosine, contain 2-(1-aminoethyl)thiazol-4 caboxylic acid and the novel amino acid 4'-methoxytryptophan. In argyrins C and D the latter is replaced by 4'-methoxy 2'-methyltryptophan. According to NMR analysis the solution and crystal conformations of argyrins A and B are identical in CDCl3 and slightly different in acetone-d6. Argyrins A and B are identical with the antibiotics A21459 A and B, whose structures are revised with respect to 4'-methoxytryptophan. PMID- 12374386 TI - New anti-MRSA and anti-VRE carbapenems; synthesis and structure-activity relationships of 1beta-methyl-2-(thiazol-2-ylthio)carbapenems. AB - Discovery of novel antimicrobial agents effective against infections caused by drug-resistant pathogens is an important objective. In order to find a new parenteral carbapenem antibiotic, which has potent antibacterial activity especially against methicillin-resistant staphylococci, vancomycin-resistant enterococci and penicillin-resistant Streptococcus pneumoniae, a series of 1beta methylcarbapenems with thiazol-2-ylthio groups at the C-2 position have been synthesized. Structure-activity relationships were investigated which led to SM 197436 (27), SM-232721 (44) and SM-232724 (41), being selected for further evaluation. PMID- 12374387 TI - F-10748 A1, A2, B1, B2, C1, C2, D1 and D2, novel papulacandins. PMID- 12374388 TI - Pteridic acids A and B, novel plant growth promoters with auxin-like activity from Streptomyces hygroscopicus TP-A0451. PMID- 12374389 TI - Synthesis, and cytotoxic activity of N(ind)-alkoxy derivatives of antibiotic arcyriarubin and dechloro-rebeccamycin aglycon. PMID- 12374390 TI - Application of a risk assessment based approach to designing ambient air quality monitoring networks for evaluating non-cancer health impacts. AB - An ambient air quality monitoring network has been established using risk assessment techniques to evaluate adverse health effects from exposures to airborne contaminants. The risk assessment method was compared to traditional methods of establishing air quality monitoring networks: identifying m aximum concentration impacts or maximum total population. Results suggest that the health risk method best predicted the location of adverse, non-carcinogenic respiratory illnesses during the evaluation period. Spearman Rank Correlation Coefficient, r(s), values obtained using the risk assessment method were statistically greater than the values obtained using the concentration and population methods. The concentration method was the least accurate predictor of adverse effects. PMID- 12374391 TI - Indicators of wetland condition for the prairie pothole region of the United States. AB - We describe a study designed to evaluate the performance of wetland condition indicators of the Prairie Pothole Region (PPR) of the north central United States. Basin and landscape scale indicators were tested in 1992 and 1993 to determine their ability to discriminate between the influences of grassland dominated and cropland dominated landscapes in the PPR. Paired plots were selected from each of the major regions of the PPR. Among the landscape scale indicators tested, those most capable of distinguishing between the two landscapes were: 1) frequency of drained wetland basins. 2) total length of drainage ditch per plot, 3) amount of exposed soil in the upland subject to erosion, 4) indices of change in area of wetland covered by water, and 5) number of breeding duck pairs. Basin scale indicators including soil phosphorus concentrations and invertebrate taxa richness showed some promise: however, plant species richness was the only statistically significant basin scale indicator distinguishing grassland dominated from cropland dominated landscapes. Although our study found a number of promising candidate indicators, one of our conclusions is that basin scale indicators present a number of implementation problems. including: skill level requirements, site access denials, and recession of site access by landowners. Alternatively, we suggest that the use of landscape indicators based on remote sensing can be an effective means of assessing wetland integrity. PMID- 12374392 TI - Towards a long-term integrated monitoring programme in Europe: network design in theory and practice. AB - Long-term integrated monitoring is an important approach for investigating, detecting and predicing the effects of environmental changes. Currently. European freshwaters, glaciers, forests and other natural and semi-natural ecosystems and habitats are monitored by a number of networks established by different organisations. However, many monitoring programmes have a narrow focus (e.g. targeting individual ecosystems) and most have different measurement protocols and sampling design. This has resulted in poor integration of ecosystem monitoring at a European level, leading to some overlapping of efforts and a lack of harmonised data to inform policy decisions. The need for a consistent pan European long-term integrated monitoring of terrestrial systems programme is recognised in the scientific community. However, the design of such a system can be problematic, not least because of the constraints imposed by the need to make maximum use of existing sites and networks. Based on the outcomes of the NoLIMITS project (Networking of Long-term Integrated Monitoring in Terrestrial Systems). this article reviews issues that should be addressed in designing a programme based on existing monitoring sites and networks. Four major design issues are considered: (i) users' requirements, (ii) the need to address multiple objectives, (iii) role of existing sites and (iv) operational aspects. PMID- 12374393 TI - A methodology to estimate carbon storage and flux in forestland using existing forest and soils databases. AB - Sequestration of carbon through expansion and management of forestland can assist in reducing greenhouse gas concentrations in the atmosphere. Quantification of the amount of carbon presents an ongoing challenge that calls for new approaches. These new approaches must seek to simplify the science-based accounting of carbon storage and flux, while adhering to general principles of greenhous gas accounting. Quantifying change in carbon storage and carbon flux consists of two steps: developing a baseline of carbon storage, and measuring resulting storage and flux following a change of conditions. A methodology is proposed that accomplishes both steps, applicable to an aggregate-level analysis using the state of Iowa (U.S.A.) as a case study. The method combines existing databases from the U.S. Forest Service (USFS) and U.S. Department of Agriculture (USDA), and merges these with the methods of Birdsey (USDA. 1992. 1995: IPCC, 1997: EIIP, 1999) for partitioning carbon stocks into storage pools. Forested ecosystems in the study area contain approximately 137.3 metric tons organic carbon per hectare, or 114 million metric tons of carbon in aggregate. Of this total, 44.7 million tons are stored in biomass tissue, and 69.2 million tons of carbon are contained in soils. Carbon flux due to forests in the state of Iowa is estimated to be a net annual sequestration (removal from the atmosphere) of 4.3 million metric tons of CO2-equivalent, approximately 5% of the net annual CO2-equivalent emissions from the state (Ney et al.. 1996). PMID- 12374394 TI - Distribution and partitioning of trace metals in sediments of the lower reaches of the New Calabar River, Port Harcourt, Nigeria. AB - The distribution of trace metals in sediments of the lower reaches of the New Calabar River, Nigeria was evaluated together with the partitioning of their chemical species between five geochemical phases. Samplings were made in five zones at the lower reaches of the New Calaber River. All the trace metals were determined by AAS after selective chemical extractions and concentrations given in microg gm(-1) (dry weight basis). The average total concentrations found for trace metals in the sediment were ( mean +/- rsd.) Pb: 41.6 +/- 0.29, Zn: 31.60 +/- 0.42, Cd: 12.80 +/- 0.92, Co: 92 +/- 0.25, Cu: 25.5 +/- 0.65 and Ni: 3.2 +/- 0.25. Maxima and minima concentrations are inconsistent with previous studies in other rivers of this region. Spatial distribution revealed that the sources of trace metals into the river appeared to be of non-point. Five contamination indices were applied in studying the partitioning of the trace metals in the sediment. These indices provided bases for ascertaining the potential environmental risk of trace metals in the river system. The results denote high partition levels in the more mobile and more dangerous phases. PMID- 12374395 TI - Spectrophotometric determination of three anti-ulcer drugs through charge transfer complexation. AB - A simple charge-transfer complexation method is described for the spectrophotometric assay of nizatidine, ranitidine, and famotidine. This method is based on interaction of these drugs, as n-electron donors, with 7,7,8,8 tetracyanoquinodimethane, as the pi-acceptor, in acetonitrile to give highly colored green radical anions that are measured at 840 nm. Calibration graphs for the 3 compounds are linear over the concentration ranges of 1-6 microg/mL for nizatidine and ranitidine and 1-7 microg/mL for famotidine, with correlation coefficients (n = 6) of >0.999. The conditioned stability constants and the free energy changes were measured; the values obtained were generally high and negative, respectively, suggesting highly stable complexes. The proposed method was successfully applied to the determination of the drugs in pharmaceutical preparations. The assay results were in accordance with those obtained by using reference methods. PMID- 12374396 TI - Confirmation of phenylbutazone residues in bovine kidney by liquid chromatography/mass spectrometry. AB - A confirmatory method is described for phenylbutazone (PB) residues in bovine kidney tissue. Ground kidney tissue is diluted with water, and the mixture is made basic with 25% ammonium hydroxide in water; the lipids are extracted with ethyl and petroleum ethers. The ether layer is discarded, and the tissue is acidified with 6N HCl. PB residues are extracted with tetrahydrofuranhexane (1 + 4). The extract is passed through a silica solid-phase extraction column, and the eluate is evaporated to dryness. The residue is dissolved in acidified acetonitrile-water-acetic acid (50 + 49.4 + 0.6). A single quadrupole mass spectrometer coupled to a liquid chromatograph with an electrospray interface is used to confirm the identity of the PB residues in the kidney extract. Negative ion detection with selected-ion monitoring of 4 ions is used. Sets of control and fortified-control kidney tissues (at 50, 100, and 200 ppb PB) and several kidney tissue field samples were analyzed for method validation. The method was tested further during the course of a survey to determine the incidence of PB residues in bovine kidney samples obtained from slaughterhouses across the country. In addition, the method was tested for use with an ion-trap mass spectrometer coupled to a liquid chromatograph, which allowed confirmation of PB at lower levels (5-10 ppb) in kidney tissue. PMID- 12374397 TI - Determination of cimetidine, famotidine, and ranitidine hydrochloride in the presence of their sulfoxide derivatives in pure and dosage forms by high performance thin-layer chromatography and scanning densitometry. AB - A selective, precise, and accurate method was developed for the determination of cimetidine (C), famotidine (F), and ranitidine hydrochloride (R x HCl) in the presence of their sulfoxide derivatives. The method involves quantitative densitometric evaluation of mixtures of the drugs and their derivatives after separation by high-performance thin-layer chromatography on silica gel plates (10 x 20 cm) with ethyl acetate-isopropanol-20% ammonia (9 + 5 + 4, v/v) as the mobile phase for both C and F and ethyl acetate-methanol-20% ammonia (10 + 2 + 2, v/v) as the mobile phase for R x HCl; Rf values for C, F, and R x HCl and their corresponding derivatives were 0.85 and 0.59, 0.73 and 0.41, and 0.56 and 0.33, respectively. Developing time was approximately 20 min. For densitometric evaluation, peak areas were recorded at 218, 265, and 313 nm for C, F, and R x HCl, respectively. The relationship between concentration and the corresponding peak area was plotted for the ranges of 5-50 microg/spot for C and 2-20 microg/spot for F and R x HCl. Mean recoveries were 100.39 +/- 1.33, 99.77 +/- 1.30, and 100.09 +/- 0.69% for C, F, and R x HCl, respectively. The proposed method was used successfully for stability testing of the pure drugs in the presence of up to 90% of their degradates, in bulk powder and dosage forms. The results obtained were analyzed statistically and compared with those obtained by the official methods. PMID- 12374398 TI - Sensitive and rapid spectrophotometric methods for determination of anticancer drugs. AB - Sensitive, rapid, and simple spectrophotometric methods were developed for determination of the anticancer drugs vinblastine sulfate (VBS) and vincristine sulfate (VCS), which belong to the class of vinca alkaloids. The first method is based on the reaction of VBS and VCS with diazotized dapsone, forming yellow azo products with absorption maxima at 430 nm. The colored species obey Beer's law in the concentration range of 0.5-24 microg/mL for VBS and 0.5-12 microg/mL for VCS. The second method describes the reaction of VBS and VCS with iron(III) and subsequent reaction with ferricyanide in hydrochloric acid medium to yield blue products with absorption maxima at 750 nm. The Beer's law range for this method is 0.1-4 microg/mL for VBS and 0.5-10 microg/mL for VCS. With both methods, colored species were stable for 1 h. The methods are simple and reproducible and are applied for determination of VBS and VCS in pharmaceutical formulations. Commonly encountered pharmaceuticals added as excipients do not interfere in the analysis and the results obtained in the analysis of dosage forms agree well with the labeled contents. PMID- 12374399 TI - Specific detection of Clostridium botulinum types A, B, E, and F using the polymerase chain reaction. AB - Clostridium botulinum organisms generally produce 1 of 4 neurotoxin types (A, B, E, and F) associated with human illness. Neurotoxin type determination is important in identification of the bacterium. A polymerase chain reaction (PCR) method was developed to identify 24 h botulinal cultures as potential types A, B, E, and F neurotoxin producers as well as other clostridial species which also produce neurotoxins. Components of the PCR and amplification conditions were adjusted for optimal amplification of toxin gene target regions to enable simultaneous testing for types A, B, E, and F in separate tubes using a single thermal cycler. Each primer set was specific for its corresponding toxin type. A DNA extraction procedure was also included to remove inhibitory substances that may affect amplification. This procedure is rapid, sensitive, and specific for identification of toxigenic C. botulinum. PMID- 12374400 TI - Visual immunoprecipitate assay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.09): collaborative study. AB - AOAC Official Method 996.09, Visual Immunoprecipitate Assay (VIP) for Escherichia coli O157:H7, was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by VIP assay and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment procedure and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 15 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level, where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 396 test portions and controls by both methods, for a total of 792 test portions. Of the 396 paired test portions, 75 were positive and 230 were negative by both the VIP and culture methods. Eleven test portions were presumptively positive by VIP and could not be confirmed culturally; 32 were negative by VIP, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the VIP method. There was no statistical difference between results obtained with the VIP for EHEC 8 h method and the culture method except for cooked beef, where the VIP had significantly higher recovery for one inoculation level. PMID- 12374401 TI - Assurance enzyme immunoassay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.10): collaborative study. AB - AOAC Official Method 996.10, Assurance Enzyme Immunoassay (EIA) for Escherichia coli O157:H7 (EHEC), was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by EIA and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment conditions and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 14 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 378 test portions and controls by both the 8 h EIA and the USDA/FSIS enrichment methods, for a total of 756 test portions. Of the 378 paired test portions, 75 were positive and 212 were negative by both methods. Thirteen test portions were presumptively positive by EIA and could not be confirmed culturally; 30 were negative by EIA, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the EIA method. There was no statistical difference between results obtained with the Assurance EIA for EHEC 8 h method and the culture method for raw ground beef. The Assurance EIA had a significantly higher recovery for cooked beef. PMID- 12374402 TI - Detection of enteroviruses in shellfish by fluorogenic polymerase chain reaction integrated with 96-well microplate scanning. AB - A one-step procedure was developed to confirm viral targets by using a fluorometric 96-well microplate scanner following polymerase chain reaction (PCR). The fluorogenic PCR, integrated with fluorometric scanning, measured the end point fluorescence of viral PCR amplicon/probe hybrids and permitted the use of nonfluorogenic PCR conditions with addition of a Cy3 fluorophore-labeled linear probe for viruses. This linear probe generated higher ratios of viral signal-to-noise than a comparative beacon probe. Detection efficiency with a Cy3/quencher linear probe was comparable with Southern analysis at the level > or = 0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing < 0.27 PFU, the fluorometric measurements of the first-round PCR viral amplicon were not as sensitive as Southern analysis; however, equivalent sensitivities were achieved with fluorogenic nested PCR. Concentrates of 11 oyster samples exposed to municipal sewage were tested for enteroviruses; the fluorogenic detection correlated 100% with Southern analysis. This method using fluorometric scanning of viral amplicon is simple; it requires neither continuously monitoring equipment nor redesigning PCR primers; and it accurately detects enteroviruses in oyster sample concentrates in less time than classic spectrophotometry or Southern analysis. PMID- 12374403 TI - N-nitroso compounds and mutagens in Chinese fermented (sour) corn pancakes. AB - Stomach cancer rates in rural Linqu County, Shandong Province, China, are exceptionally high. A previous case-control study revealed that the risk of stomach cancer was 30% higher among those who consumed sour (fermented) corn pancakes at least daily. A previous study of the sour pancakes reported volatile nitrosamines in most specimens, and almost half reportedly showed mutagenic activity. Few households currently consume sour pancakes, and the duration of fermentation has been shortened. We tested specimens of pancake batter and sour pancakes from Linqu County for mutagenic activity using the Ames test; for N nitroso compounds (NOC) we used the Nitrolite-thermal energy analysis (TEA) method. Results of the Ames test were inconclusive: only 1 out of 15 cooked pancakes showed a positive mutagenic response, and all 15 batter specimens were negative; however, several batter specimens showed a weakly positive trend of mutagenicity with extract concentration. Our assay for total nitroso compounds was weakly positive in only 1 out of 15 specimens of sour pancake batter. That specimen was also tested by gas chromatography-TEA for nitrosaminoacids and volatile nitrosamines, but none were detected. It seems unlikely that the Chinese sour pancakes are significantly contaminated by NOC or other mutagens. PMID- 12374404 TI - Determination of brevetoxins in shellfish by the neuroblastoma assay. AB - A neuroblastoma assay for determination of brevetoxins in shellfish was developed together with a method for sample cleanup that allows separation of brevetoxins from most of the components that cause matrix interference in the assay. This improved assay method was applied to a range of shellfish samples with different characteristics. Extracts of naturally contaminated and nontoxic shellfish together with extracts spiked with known amounts of toxin were tested. The results demonstrated that brevetoxins could be reliably detected in shellfish extracts at concentrations below the regulatory limit. Brevetoxin activity was detected in 15 of 23 samples from 5 separate toxicity incidents in which shellfish tested positive in the neurotoxic shellfish poisoning (NSP) mouse bioassay. Twelve of these positive NSP results came from 2 toxicity incidents. Yessotoxin was the major contributor to toxicity in 2 other incidents, although some samples contained both yessotoxin and brevetoxin. The sample from the remaining incident contained an unidentified toxin bioactivity, together with gymnodimine. In contrast to earlier versions of the neuroblastoma assay, gymnodimine was not detected by this modified method. PMID- 12374405 TI - Determination of fat content and fatty acid composition in meat and meat products after supercritical fluid extraction. AB - Two different relatively simple, commercially available supercritical fluid extractors (SFE), Leco and Foss-Tecator, were tested for the determination of total fat content in meat and meat products. The fatty acid composition in meat and meat products was also determined after the Foss-Tecator extraction in an aliquot of the extract. Total fat was determined by weighing after the different extraction procedures and the fatty acid composition by gas chromatography after hydrolysis and methylation of the extract. The results for total fat content agreed well with results from a standard method of Schmid, Bondzynski, and Ratzlaff, which uses conventional solvent extraction. Fatty acid composition was compared with the Bligh and Dyer extraction, and showed good agreement. The average relative difference between SFE and Bligh and Dyer of all fatty acids in the sample was <3% for acids exceeding 0.5% of total fatty acid amount. The advantages of SFE over traditional methods are a much lower consumption of hazardous organic solvents and shorter extraction times. To obtain quantitative recoveries by SFE, ethanol was added to the extraction cells before extraction. PMID- 12374406 TI - Determination of Cry9C protein in processed foods made with StarLink corn. AB - StarLink (Aventis CropScience US) hybrid corn has been genetically modified to contain a pesticidal protein, Cry9C, which makes it more resistant than traditional varieties to certain types of corn insect pests. Unlike other varieties of genetically engineered corn, the U.S. Environmental Protection Agency authorized the use of StarLink corn for animal feed and industrial use only, not for human consumption. However, some Cry9C-containing corn was mistakenly or inadvertently comingled with yellow corn intended for human food use. Because corn containing the Cry9C construct was not approved for human use, the U.S. Food and Drug Administration considers food containing it to be adulterated. Consequently, this regulatory violation resulted in hundreds of recalls of corn-based products, such as taco shells, containing cry9C DNA. Detecting the novel protein in StarLink corn is an emerging issue; therefore, there is no standardized or established analytical method for detecting Cry9C protein in processed foods. We developed a procedure for quantitation of Cry9C protein, with validation data, in processed food matrixes with a limit of quantitation at 1.7 ng/g (ppb), using a commercial polyclonal antibody-based Cry9C kit that was intended for corn grain samples. Intra- and interassay coefficients of variation were 2.8 and 11.8%, respectively. Mean recoveries were 73 and 85% at 2 and 5 ng/g Cry9C fortifications, respectively, for 19 control non StarLink corn-based matrixes. Our data demonstrate only 0-0.5% of Cry9C protein survived the processing of tortilla chips and soft tortillas made from 100% StarLink corn, resulting in levels from below the detection limit to 45 ppb. PMID- 12374407 TI - Novel reference molecules for quantitation of genetically modified maize and soybean. AB - New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy. PMID- 12374408 TI - Determination of vegetal proteins in milk powder by sodium dodecyl sulfate capillary gel electrophoresis: interlaboratory study. AB - An interlaboratory study, with the participation of 8 laboratories, was conducted to evaluate a sodium dodecyl sulfate-capillary gel electrophoresis method for determination of adulteration of milk powder with soy and pea proteins. Calibration standards (0-8%, w/w, soy and pea protein in total protein) and adulterated skim milk powders (0-5%, w/w, soy and pea proteins in total protein) were produced. Vegetal proteins were determined after removal of milk proteins by pretreatment of the samples with tetraborate-EDTA buffer, pH 8.3. Repeatability standard deviations ranged from 9 to 15% and reproducibility standard deviations ranged from 25 to 30% in the samples containing 5% vegetal protein in total protein. PMID- 12374409 TI - Measurement of alpha-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the Ceralpha assay: collaborative study. AB - This study was conducted to evaluate the method performance of a rapid procedure for the measurement of alpha-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing alpha-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable alpha-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, alpha-glucosidase, and beta-amylase. When the substrate is cleaved by endo-acting alpha-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of alpha-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 micromol p-nitrophenyl (in the presence of excess quantities of alpha-glucosidase) in 1 min at 40 degrees C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal alpha-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%. PMID- 12374410 TI - Measurement of resistant starch by enzymatic digestion in starch and selected plant materials: collaborative study. AB - Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high. PMID- 12374411 TI - Temperature-sensitive resolution of cis- and trans-fatty acid isomers of partially hydrogenated vegetable oils on SP-2560 and CP-Sil 88 capillary columns. AB - This study examines the effect of the column operating temperature of 100 m SP 2560 and CP-Sil 88 capillary gas chromatographic (GC) columns on the separation of cis- and trans-octadecenoic (18:1) isomers in partially hydrogenated vegetable oils. The overlapping GC peaks were measured at column isothermal temperatures of 170, 175, 180, 185, and 190 degrees C. With both columns, isothermal operation at 180 degrees C produced the fewest overlapping peaks of the cis and trans isomers. At this temperature, all trans-18:1 isomers, except 13t-18:1 (t = trans), 14t 18:1, and 15t-18:1 isomers were resolved from the cis-18:1 isomers. The peaks of the 13t-18:1 and 14t-18:1 isomer pair, which always elute together, overlapped peaks of the 6c-18:1 (c = cis), 7c-18:1, and 8c-18:1 isomers; the peak of the 15t 18:1 isomer overlapped the major cis-18:1 peak, which was mainly due to 9c-18:1. Isothermal operations above or below 180 degrees C produced some additional overlapping problems. At 185 and 190 degrees C, the peaks of the 16t-18:1 and 13c 18:1 isomers overlapped. At 175 and 170 degrees C, the 16t-18:1 peak overlapped the 14c-18:1 peak, and the peaks of the 13t + 14t-18:1 isomer pair partially overlapped the major cis-18:1 peak. The separation of 11c-20:1 and alpha linolenic acid and its geometric isomers was also affected by the column operating temperature. Isothermal operation of the SP-2560 column at 180 degrees C produced a baseline separation of 11c-20:1 and alpha-linolenic acid and its geometric isomers, whereas with the CP-Sil 88 column the best resolution was obtained at 170 degrees C. The results of this study show that the SP-2560 capillary column has a slight advantage over the CP-Sil 88 column for the simultaneous resolution of all the fatty acids generally found in partially hydrogenated vegetable oils. PMID- 12374412 TI - Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules. AB - Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops. PMID- 12374413 TI - Simultaneous determination of vitamin A and beta-carotene in dietary supplements by liquid chromatography. AB - Several liquid chromatography (LC) methods for analysis of vitamin A in foods and feeds have been previously reported but only a few have been applied in non-food matrixes. A validated LC method is needed for determination of vitamin A and beta carotene in the various matrixes presented by dietary supplements. The performance of a reversed-phase method with methanol-isopropanol gradient elution was evaluated with standard retinyl derivatives and beta-carotene. The reversed phase method is capable of separating retinol from other derivatives such as retinyl acetate, retinyl palmitate, and beta-carotene. Two types of extraction were used to extract the analytes from the dietary supplements: a hexane methylene chloride extraction for soft-gel capsules containing beta-carotene, and a direct solvent extraction for dietary supplements in tablet form. The direct solvent extraction consisted of treatment with ethanol and methylene chloride following addition of hot water (55 degrees C). Results with the reversed-phase method for vitamin A and beta-carotene in the products examined (n = 8) indicated excellent method performance. The main form of vitamin A or beta-carotene in dietary supplements was the all-trans isomer. The reversed-phase method avoids saponification and is rapid, accurate, precise, and suitable for simultaneous determination of retinyl derivatives and beta-carotene in dietary supplements. PMID- 12374414 TI - Routine, high-sensitivity, cold vapor atomic absorption spectrometric determination of total mercury in foods after low-temperature digestion. AB - A cold vapor atomic absorption spectrometric method was developed for the subnanogram-per-gram determination of total Hg in a wide variety of foods. Foods were weighed into 50 mL polypropylene centrifuge tubes and dried without charring at 55 degrees C in a circulating oven. Samples were then digested at 58 degrees C with HNO3, HCl, and H2O2. After matrix modification with solutions of 2% Mg(NO3)2, 0.01% Triton X-100, and Cu(II) at 10 microg/mL, samples were analyzed by using a CeTAC Technologies M-6000A dedicated Hg analyzer. Based on a 2 g sample weight, the detection limit of the method over 12 batches averaged 0.30 ng/g wet weight and ranged from 0.03 to 0.6 ng/g. Recoveries of Hg added to 17 different foods, analyzed in a routine manner, averaged 97%, and individual recoveries ranged from 77 to 107%. Accuracy was confirmed by analysis of 7 biological reference materials from the National Research Council of Canada and the National Institute of Standards and Technology. Stabilization of low concentrations of Hg in solutions containing no sample was required to prevent loss of Hg from blanks. In a comparison of NaCl, potassium dichromate, and Au(II), chloride was much more effective for stabilization than the other two, and HCl was used for subsequent stabilization. PMID- 12374415 TI - Spectrofluorimetric determination of formaldehyde in maple syrup. AB - A quick and simple method was developed for determination of formaldehyde in maple syrup. In this method, formaldehyde reacts with Fluoral P to form a complex which is chemically extracted by isobutanol and determined by spectrofluorimetry. Performance, as gauged by the limits of detection (0.16 mg/kg) and quantitation (0.21 mg/kg), recovery (>79%), and variability (1.9-16.1%, depending on fortification level and class of syrup) were superior to the current official AOAC standard method. PMID- 12374416 TI - Determination of pesticide residues in nonfatty foods by supercritical fluid extraction and gas chromatography/mass spectrometry: collaborative study. AB - A collaborative study was conducted to determine multiple pesticide residues in apple, green bean, and carrot by using supercritical fluid extraction (SFE) and gas chromatography/mass spectrometry (GC/MS). Seventeen laboratories from 7 countries participated in the final study, and a variety of different instruments was used by collaborators. The procedure simply entails 3 steps: (1) mix 1.1 g drying agent (Hydromatrix) per 1 g frozen precomminuted sample, and load 4-5.5 g of this mixture into a 7-10 mL extraction vessel; (2) perform SFE for 20-30 min with a 1-2 mL/min flow rate of carbon dioxide at 0.85 g/mL density (320 atm, 60 degrees C); and (3) inject the extract, which was collected on a solid-phase or in a liquid trap, into the gas chromatograph/mass spectrometer, using either an ion-trap instrument in full-scan mode or a quadrupole-type instrument in selected ion monitoring mode. The ability of GC/MS to simultaneously quantitate and confirm the identity of the semivolatile analytes at trace concentrations is a strong feature of the approach. The selectivity of SFE and GC/MS avoids the need for post-extraction cleanup steps, and the conversion of the CO2 solvent to a gas after SFE eliminates the solvent evaporation step common in traditional methods. The approach has several advantages, but its main drawback is the lower recoveries for the most polar analytes, such as methamidophos and acephate, and the most nonpolar analytes, such as pyrethroids. Recoveries for most pesticides are >75%, and recoveries of nonpolar analytes are still >50%. The (within laboratory) repeatability relative standard deviation (RSDr) values of the recoveries are generally <15%. More specifically, the average results from the 9 14 laboratories in the final analysis of 6 blind duplicates at 3 concentrations for each pesticide are as follows: carbofuran in apple (75-500 ng/g), 89% recovery, 7% RSDr, 9% reproducibility relative standard deviation (RSDR); diazinon in apple (60-400 ng/g), 83% recovery, 13% RSDr, 17% RSDR; vinclozolin in apple (6-400 ng/g), 97% recovery, 13% RSDr, 18% RSDR; chlorpyrifos in apple (50 300 ng/g), 105% recovery, 11% RSDr, 13% RSDR; endosulfan sulfate in apple (150 1000 ng/g), 95% recovery, 15% RSDr, 17% RSDR; trifluralin in green bean (30-200 ng/g), 58% recovery, 11% RSDr, 27% RSDR; dacthal in green bean (60-400 ng/g), 88% recovery, 11% RSDr, 17% RSDR; quintozene in green bean (60-400 ng/g), 79% recovery, 13% RSDr, 18% RSDR; chlorpyrifos in green bean (50-300 ng/g), 84% recovery, 11% RSDr, 17% RSDR; p,p'-DDE in green bean (45-300 ng/g), 64% recovery, 14% RSDr, 27% RSDR; atrazine in carrot (75-500 ng/g), 90% recovery, 11% RSDr, 15% RSDR; metalaxyl in carrot (75-500 ng/g), 89% recovery, 8% RSDr, 12% RSDR; parathion-methyl in carrot (75-500 ng/g), 84% recovery, 14% RSDr, 15% RSDR; chlorpyrifos in carrot (50-300 ng/g), 77% recovery, 13% RSDr, 19% RSDR; and bifenthrin in carrot (90-600 ng/g), 63% recovery, 12% RSDr, and 25% RSDR. All analytes except for the nonpolar compounds trifluralin, p,p'-DDE, and bifenthrin gave average Horwitz ratios of <1.0 when AOAC criteria were used. These 3 analytes had high RSDr values but lower RSDR values, which indicated that certain SFE instruments gave consistently lower recoveries for nonpolar compounds. The collaborative study results demonstrate that the method meets the purpose of many monitoring programs for pesticide residue analysis, and the Study Director recommends that it be adopted Official First Action. PMID- 12374417 TI - Comparison of five extraction methods for determination of incurred and added pesticides in dietary composites. AB - The National Exposure Research Laboratory of the U.S. Environmental Protection Agency conducts research to measure exposure of individuals to chemical pollutants through the diet. In support of this research, methods are being evaluated for the determination of pesticides in dietary composite samples. In the present study, Soxhlet, blender, microwave-assisted, pressurized fluid, and supercritical fluid extraction methods were compared for the determination of incurred and added pesticides in 4 dietary composites, which varied in fat and water content. Incurred pesticides were chlorothalonil, chlorpyrifos, DDE, dicloran, dieldrin, endosulfan I, malathion, cis- and trans-permethrin, and trifluralin. Added pesticides were alpha- and gamma-chlordane, hexachlorobenzene, and fonofos. Concentrations of the individual pesticides were between 0.2 and 20 ng/g composite. All 5 methods tested could extract pesticides from dietary composites. Most incurred pesticides were recovered from the dietary composites within the range of 59-140% of expected values. Recoveries of added pesticides were between 60 and 130%. Microwave-assisted extraction led to significantly higher concentrations of 7 pesticides. Blender extraction yielded significantly higher concentrations of chlorothalonil and fonofos. Water content was a significant factor in the recovery of chlorothalonil, and fat content was a significant factor in the recovery of fonofos. In designing an exposure study, the selection of the extraction method would be determined by number of samples to be extracted, analyte stability, and cost. PMID- 12374418 TI - Comparison of magnesium sulfate and sodium sulfate for removal of water from pesticide extracts of foods. AB - Water-miscible solvents, such as acetone and acetonitrile, effectively extract both polar and nonpolar pesticide residues from nonfatty foods. The addition of sodium chloride to the resulting acetonitrile-water or acetone-water extract (salting out) results in the separation of the water from the organic solvent. However, the organic solvent layer (pesticide extract) still contains some residual water, which can adversely affect separation procedures that follow, such as solid-phase extraction and/or gas chromatography. Drying agents, such as sodium sulfate or magnesium sulfate, are used to remove the water from the organic extracts. In the present study, we used nuclear magnetic resonance spectroscopy to study the composition of the phases resulting from salting out and to compare the effectiveness of sodium sulfate and magnesium sulfate as drying agents. The study showed that considerable amounts of water remained in the organic phase after phase separation. Sodium sulfate was a relatively ineffective drying agent, removing little or no residual water from the organic solvent. Magnesium sulfate proved to be a much more effective drying agent. PMID- 12374420 TI - AOAC International methods committee guidelines for validation of qualitative and quantitative food microbiological official methods of analysis. AB - Responding to a need for a guide for conducting Official Method validation studies of microbiological methods, AOAC utilized the experience of three microbiologists who have been active in the field of method validation. In collaboration, a document was prepared which covered the following areas: terms and their definitions associated with the Official Methods program (e.g., reference methods, alternative methods, and ruggedness testing), protocols and validation requirements for qualitative methods versus those for quantitative methods, the concept of the precollaborative study, ruggedness testing, tests for significant differences, performance indicators, and the approval process. After its preparation, this document was reviewed by the members of the Methods Committee on Microbiology and Extraneous Materials and by members of the Official Methods Board. Herein is presented the approved version of that document. PMID- 12374419 TI - Rapid method for trace determination of organochlorine pesticides and polychlorinated biphenyls in yogurt. AB - A new multiresidue method was developed for the analysis of 19 organochlorine pesticides and 6 polychlorinated biphenyls in yogurt. The sample was extracted twice with acetone by homogenization with an Ultra-Turrax dispersing unit, and the combined extracts were filtered. The extract was then purified by reversed phase C18 columns and subjected to further cleanup with neutral alumina columns. The residues were determined by gas chromatography with electron capture detection. After the method was optimized, it was validated by determination of recovery percentages, precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) with yogurt samples fortified at 10 and 1 microg/kg concentration levels. The recovery of 23 organochlorine residues ranged from 77 to 95% at a level of 10 microg/kg, from 74 to 102% at a level of 1 microg/kg, and between 54 and 61% for dieldrin and alpha-endosulfan. The method is repeatable and reproducible, with relative standard deviation values <19% for all residues except dieldrin. Detection and quantitation limits were between 0.02 and 0.62 microg/kg. The analytical method proposed was quick, accurate, repeatable, and reproducible for the determination of organochlorine residues in yogurt samples. PMID- 12374421 TI - Comparison of visual immunoassay and chromogenic culture medium for the presence of Listeria spp. in foods. AB - Two rapid screening methods [the TECRA Listeria Visual Immunoassay (LIS-VIS) kit, an AOAC-approved 48 h visual test, which detects Listeria through colorimetry, and BCM Listeria isolation and differentiation plating agar] were used to screen U.S. Food and Drug Administration-regulated commodities for the presence of Listeria spp. Seventy-four different food samples were screened for the presence of Listeria spp. by using both protocols. Test results for the TECRA LIS-VIA showed 66 negative samples and 1 false positive, with 4 confirmed as L. monocytogenes and 3 as L. innocua. With the BCM agar, 67 samples were negative, 4 were confirmed as L. monocytogenes, and 3 were confirmed as L. innocua. Both methods showed similar results and were effective screening tools for Listeria spp. in foods. The BCM agar method proved to be a rapid, sensitive, and excellent tool for early screening and differentiation of Listeria spp. present in foods. PMID- 12374423 TI - Mistaken memories: remembering events that never happened. AB - Our memories can be accurate, but they are not always accurate. Eyewitness testimony, for example, is notoriously unreliable. Insights into both veridical and false remembering have come from recent investigations of memory distortion. Behavioral measures have been used to demonstrate false memory phenomena in the laboratory, and neuroimaging measures have been used to provide clues about the relevant events in the brain that support remembering versus misremembering. A central category of misremembering results from confusion between memories for perceived and imagined events, which may result from overlap between particular features of the stored information comprising memories for perceived and imagined events. PMID- 12374422 TI - Alzheimer's disease: what multiphoton microscopy teaches us. AB - A definitive diagnosis of Alzheimer's disease depends on postmortem analysis of brain tissue bearing the pathological hallmarks of the disease: plaques and tangles. Imaging techniques that allow visualization and characterization of these lesions in living animals permit a better understanding of the pathogenesis of the disease as well as paradigms for preventing or reversing the deposits. Multiphoton microscopy uses near infrared light that is benign to living tissue and penetrates more deeply than visible or UV light, permitting high-resolution imaging of these microscopic structures deep within the cortex of living transgenic mice over time. This in vivo imaging approach allows direct examination of the natural history of plaques and evaluation of antiplaque therapeutics in mouse models of the disease. PMID- 12374425 TI - Multiple sclerosis as a by-product of the failure to sustain protective autoimmunity: a paradigm shift. AB - Autoimmune diseases are traditionally viewed as an outcome of a chaotic situation in which an individual's immune system reacts against the body's own proteins. In multiple sclerosis, a disease of the white matter of the central nervous system (CNS), the immune attack is directed against myelin proteins. In this article, the authors propose a paradigm shift in the perception of autoimmune disease. They suggest that an autoimmune disease may be viewed as a by-product of the malfunctioning of a physiological autoimmune response whose purpose is protective. The proposed view is based on observations by their group suggesting that an autoimmune response is the body's own mechanism for coping with CNS damage. According to this view, all individuals are endowed with the potential ability to evoke an autoimmune response to CNS injuries. However, the inherent ability to control this response so that its beneficial effect will be expressed is limited and is correlated with the individual's inherent ability to resist autoimmune disease induction. The same autoimmune T cells are responsible for neuroprotection and for disease development. In patients with CNS trauma or neurodegenerative disorders, it might be possible to gain maximal autoimmune protection and avoid autoimmune disease induction by boosting the immune response, using myelin-associated peptides that are nonpathogenic or antigens that simulate the activities of such peptides. In patients with multiple sclerosis and other neurodegenerative diseases, where the aim is to block the autoimmune disorder while deriving the potential benefit of the autoimmune response, the effect of treatment should be immunomodulatory rather than immunosuppressive. In this article, the authors present a novel concept of protective autoimmunity and propose that autoimmune disease is a by-product of failure to sustain it. They summarize the basic findings that led them to formulate the new concept and offer an explanation for the commonly observed presence of cells and antibodies directed against self-components in healthy individuals. The therapeutic implications of the new concept and their experimental findings are discussed. PMID- 12374424 TI - Brains rule! fun = learning = neuroscience literacy. AB - Brains Rule! Neuroscience Expositions is a project designed to improve neuroscience literacy among children and the general public by applying a model where neuroscience professionals transfer knowledge and enthusiasm about neuroscience through fun, engaging hands-on activities. This educational model draws strength from many national and local partnerships of neuroscience professionals to coordinate expositions across the country in a variety of local communities. Brains Rule! Neuroscience Expositions uses a flexible science fair like format to engage children in the process of science and teach about neuroscience concepts, facts, and professions. Neuroscience literacy is important to everyday life and helps individuals better understand themselves, make informed decisions about health and drug use, participate knowledgeably in governmental and social issues, and better understand scientific advancements. In this study, children's ratings of Brains Rule! Neuroscience Expositions activities were analyzed both quantitatively and qualitatively. Analysis of the responses revealed that overall the children perceived the learning activities as fun and interesting and believed that they learned something about the brain and nervous system after engaging in the activities. The Brains Rule! Neuroscience Expositions education model can be an effective tool in improving neuroscience literacy for both children and adults. PMID- 12374426 TI - Activity-dependent synaptic plasticity: insights from neuromuscular junctions. AB - Experience-dependent editing shapes synaptic connections throughout the developing nervous system, but the underlying cellular mechanisms remain poorly understood. A useful model synapse for addressing these mechanisms is the neuromuscular junction, the connection between spinal motor neurons and skeletal muscle fibers. Here the authors review current ideas about the role of activity in editing neuromuscular synaptic connections. A variety of new tools are being used to address some unanswered questions in vivo and in vitro. Understanding activity-dependent plasticity at developing neuromuscular synapses may reveal how neural circuits in the central nervous system are altered by experience throughout life. PMID- 12374427 TI - Imaging databases and neuroscience. AB - Brain atlases are equivalent to neuroimage databases provided an appropriate coordinate system to enable multisubject comparisons, along with comprehensive descriptions of the data, are included. Warping tools, visualization, and statistical analyses that accommodate the various neuroimaging modalities can be used to integrate diverse data and form comprehensive maps describing a particular subpopulation's brain structure and function. By linking task performance and genetic information to brain morphology, complex interrelations between genotype, phenotype, and behavior can be established. Several examples of these multimodal, multisubject atlases, including those that are dynamic, are presented. PMID- 12374428 TI - Modular organization of spinal motor systems. AB - The vertebrate nervous system produces a wide range of movement flexibly and efficiently, even though the simplest of these movements is potentially highly complex. The strategies by which the nervous system overcomes these complexities have therefore been of interest to motor physiologists for decades. In this review, the authors present a number of recent experiments that propose one strategy by which the nervous system might simplify the production of movement. These experiments suggest that spinal motor systems are organized in terms of a small number of distinct motor responses, or "modules." These distinct modules can be combined together simply to produce a wide range of different movements. Such a modular organization of spinal motor systems can potentially allow the nervous system to produce a wide range of natural behaviors in a simple and flexible manner. PMID- 12374429 TI - Neural connections and receptive field properties in the primary visual cortex. AB - A cubic millimeter of primary visual cortex contains about 100,000 neurons that are heavily interconnected by intrinsic and extrinsic afferents. The effort of many neuroanatomists over the past has revealed the general outline of these connections; however, their function remains a mystery. Recently, combined physiological and anatomical approaches are beginning to reveal the role of these connections in the generation of cortical receptive fields. A common theme emerges from all these studies: cortical connections are remarkably specific and this specificity is determined in great extent by the type of connection and the neuronal response properties. Feedforward connections follow relatively rigid rules of wiring selectively targeting neurons with receptive fields matched in position and contrast polarity (thalamus --> cortical layer 4) or position and orientation selectivity (layer 4 --> layers 2 + 3). In contrast, horizontal connections follow more flexible rules connecting distant cells that are not retinotopically aligned and neighboring cells with different orientation preferences. These differences in connectivity may give a hint on how visual stimuli are processed in the primary visual cortex. An attractive hypothesis is that local stimuli use the highly selective feedforward inputs to reliably drive cortical neurons while background stimuli modulate their activity through more flexible horizontal (and feedback) connections. PMID- 12374430 TI - Cellular replacement therapy for Parkinson's disease--where we are today? AB - The concept of replacing lost dopamine neurons in Parkinson's disease using mesencephalic brain cells from fetal cadavers has been supported by over 20 years of research in animals and over a decade of clinical studies. The ambitious goal of these studies was no less than a molecular and cellular "cure" for Parkinson's disease, other neurodegenerative diseases, and spinal cord injury. Much research has been done in rodents, and a few studies have been done in nonhuman primate models. Early uncontrolled clinical reports were enthusiastic, but the outcome of the first randomized, double blind, controlled study challenged the idea that dopamine replacement cells can cure Parkinson's disease, although there were some significant positive findings. Were the earlier animal studies and clinical reports wrong? Should we give up on the goal? Some aspects of the trial design and implantation methods may have led to lack of effects and to some side effects such as dyskinesias. But a detailed review of clinical neural transplants published to date still suggests that neural transplantation variably reverses some aspects of Parkinson's disease, although differing methods make exact comparisons difficult. While the randomized clinical studies have been in progress, new methods have shown promise for increasing transplant survival and distribution, reconstructing the circuits to provide dopamine to the appropriate targets and with normal regulation. Selected promising new strategies are reviewed that block apoptosis induced by tissue dissection, promote vascularization of grafts, reduce oxidant stress, provide key growth factors, and counteract adverse effects of increased age. New sources of replacement cells and stem cells may provide additional advantages for the future. Full recovery from parkinsonism appears not only to be possible, but a reliable cell replacement treatment may finally be near. PMID- 12374431 TI - Is Alzheimer's disease a mitochondrial disorder? AB - Cell bodies of neurons at risk of death in Alzheimer's disease (AD) have increased lipid peroxidation, nitration, free carbonyls, and nucleic acid oxidation. These oxidative changes occur in all vulnerable neurons and are reduced in neurons that contain neurofibrillary pathology. In this review, the authors provide a summary of recent work that demonstrates key abnormalities that may play a part in initiating and promoting neuronal oxidative damage. Mitochondrial abnormalities are clearly involved as a source of reactive oxygen species that culminates in perikaryal oxidative damage. However, because mitochondria in AD do not exhibit striking evidence of oxidative damage, as would be expected if they produced free radicals directly, the authors suspected that abnormal mitochondria are responsible for supplying a key reactant, that once in the cytoplasm, releases radicals. Because abnormal mitochondria, H2O2 and redox active iron are juxtaposed in the same AD neuron, it has all the markings of a "radical factory." The proximal causes of mitochondrial abnormalities likely involve reentry into the cell cycle, where organellokinesis and proliferation results in an increase of mitochondria and intermediately differentiated cells, with a consequent increase in turnover. Supporting this, the authors have considerable in vivo and in vitro evidence for mitotic disturbances in AD. PMID- 12374432 TI - The Wnt signaling pathway in bipolar disorder. AB - The Wnt signaling pathway is a highly conserved pathway critical for proper embryonic development. However, recent evidence suggests that this pathway and one of its key enzymes, glycogen synthase kinase 3beta, may play important roles in regulating synaptic plasticity, cell survival, and circadian rhythms in the mature CNS-all of which have been implicated in the pathophysiology and treatment of bipolar disorder. Furthermore, two structurally highly dissimilar medications used to treat bipolar disorder, lithium and valproic acid, exert effects on components of the Wnt signaling pathway. Together, these data suggest that the Wnt signaling pathway may play an important role in the treatment of bipolar disorder. Here, the authors review the modulation of the Wnt/GSK-3beta signaling pathway by mood-stabilizing agents, focusing on two therapeutically relevant aspects: neuroprotection and modulation of circadian rhythms. The future development of selective GSK-3beta inhibitors may have considerable utility not only for the treatment of bipolar disorder but also for a variety of classical neurodegenerative disorders. PMID- 12374433 TI - Genealogy of the "grandmother cell". AB - A "grandmother cell" is a hypothetical neuron that responds only to a highly complex, specific, and meaningful stimulus, such as the image of one's grandmother. The term originated in a parable Jerry Lettvin told in 1967. A similar concept had been systematically developed a few years earlier by Jerzy Konorski who called such cells "gnostic" units. This essay discusses the origin, influence, and current status of these terms and of the alternative view that complex stimuli are represented by the pattern of firing across ensembles of neurons. PMID- 12374434 TI - Implicit self-theories of shyness. AB - Three studies examined implicit self-theories in relation to shy people's goals, responses, and consequences within social situations. Shy incremental theorists were more likely than shy entity theorists to view social situations as a learning opportunity and to approach social settings (Study 1). Shy incremental theorists were less likely to use strategies aimed at avoiding social interaction (Studies 2 and 3) and suffered fewer negative consequences of their shyness (Study 3). These findings generalized across both hypothetical and actual social situations as well as both self-reports and observer reports and could not be attributed to individual differences in level of shyness. Together, these studies indicate that implicit self-theories of shyness are important for understanding individual differences among shy people and suggest new avenues for implicit self theories research. PMID- 12374435 TI - Inferences about the morality of an aggressor: the role of perceived motive. AB - The research investigated perceivers' inferences about the morality of target persons who engaged in aggressive behavior. Across several experiments, inferences about the morality of an aggressor were based more on the perceived motives of the target than on the presence of facilitating situational forces. For example, when a target's aggression was facilitated by personal rewards for aggression (instrumental aggression), perceivers inferred more negative motives and attributed lower morality to the target than when the target's aggression was facilitated by situational provocation (reactive aggression). The results suggest that perceived motives play an important role in dispositional inference and pose a problem for models that focus primarily on perceived causality, assumptions about base rates (consensus), or diagnosticity. PMID- 12374436 TI - Emotion concepts and emotional states in social judgment and categorization. AB - An objection to conclusions of research investigating effects of emotions on cognitive processes is that the effects are due to the activation of semantic concepts rather than to emotional feelings. A sentence unscrambling task was developed to prime concepts of happiness, sadness, or neutral ideas. Pilot studies demonstrated that unscrambling emotional sentences did not affect emotional state but did prime semantically related words. Experiment 1 showed that the induction of emotional state but not the sentence unscrambling task produced emotion-congruent judgments. Results of Experiment 2 showed that individuals in emotional states categorized according to emotional equivalence more often than participants in a neutral state. Sentence unscrambling had no effect on emotional response categorization. The influences of emotions and emotion knowledge in cognition and emotion are discussed. PMID- 12374437 TI - Effects of social exclusion on cognitive processes: anticipated aloneness reduces intelligent thought. AB - Three studies examined the effects of randomly assigned messages of social exclusion. In all 3 studies, significant and large decrements in intelligent thought (including IQ and Graduate Record Examination test performance) were found among people told they were likely to end up alone in life. The decline in cognitive performance was found in complex cognitive tasks such as effortful logic and reasoning; simple information processing remained intact despite the social exclusion. The effects were specific to social exclusion, as participants who received predictions of future nonsocial misfortunes (accidents and injuries) performed well on the cognitive tests. The cognitive impairments appeared to involve reductions in both speed (effort) and accuracy. The effect was not mediated by mood. PMID- 12374438 TI - We talk, therefore we think? A cultural analysis of the effect of talking on thinking. AB - The Western assumption that talking is connected to thinking is not shared in the East. The research examines how the actual psychology of individuals reflects these different cultural assumptions. In Study 1, Asian Americans and European Americans thought aloud while solving reasoning problems. Talking impaired Asian Americans' performance but not that of European Americans. Study 2 showed that participants' beliefs about talking and thinking are correlated with how talking affects performance, and suggested that cultural difference in modes of thinking can explain the difference in the effect of talking. Study 3 showed that talking impaired Asian Americans' performance because they tend to use internal speech less than European Americans. Results illuminate the importance of cultural understanding of psychology for a multicultural society. PMID- 12374439 TI - Crowded minds: the implicit bystander effect. AB - Five studies merged the priming methodology with the bystander apathy literature and demonstrate how merely priming a social context at Time 1 leads to less helping behavior on a subsequent, completely unrelated task at Time 2. In Study 1, participants who imagined being with a group at Time 1 pledged significantly fewer dollars on a charity-giving measure at Time 2 than did those who imagined being alone with one other person. Studies 2-5 build converging evidence with hypothetical and real helping behavior measures and demonstrate that participants who imagine the presence of others show facilitation to words associated with unaccountable on a lexical decision task. Implications for social group research and the priming methodology are discussed. PMID- 12374440 TI - Motivation by positive or negative role models: regulatory focus determines who will best inspire us. AB - In 3 studies, the authors demonstrated that individuals are motivated by role models who encourage strategies that fit their regulatory concerns: Promotion focused individuals, who favor a strategy of pursuing desirable outcomes, are most inspired by positive role models, who highlight strategies for achieving success; prevention-focused individuals, who favor a strategy of avoiding undesirable outcomes, are most motivated by negative role models, who highlight strategies for avoiding failure. In Studies 1 and 2, the authors primed promotion and prevention goals and then examined the impact of role models on motivation. Participants' academic motivation was increased by goal-congruent role models but decreased by goal-incongruent role models. In Study 3, participants were more likely to generate real-life role models that matched their chronic goals. PMID- 12374441 TI - Comparison-level preferences after performance: is downward comparison theory still useful? AB - Although often credited with prompting a paradigm shift in social comparison theory, T. A. Wills's (1981) downward comparison (DC) theory has received some criticism recently. In particular, several recent studies have failed to find support for T. A. Wills's (1981) contention that threat and accompanying negative affect lead to a desire for DC. These apparent failures have led some investigators to question basic principles of the theory. To resolve this controversy, 5 studies were conducted examining preferred comparison level (PCL) after performance; 4 of the studies also assessed change in this preference. Results supported DC theory, but with modifications. Specifically, individuals who performed poorly lowered their PCLs. Under some circumstances, this "downward shift" included an increased interest in "true" DC--comparing with worse-off others. A reconciliation of these results with those of previous studies is offered. PMID- 12374442 TI - Activation of the attachment system in adulthood: threat-related primes increase the accessibility of mental representations of attachment figures. AB - Three studies explored the effects of subliminal threat on the activation of representations of attachment figures. This accessibility was measured in a lexical decision task and a Stroop task following threat- or neutral-word primes, and was compared with the accessibility of representations of other close persons, known but not close persons, and unknown persons. Participants also reported on their attachment style. Threat primes led to increased accessibility of representations of attachment figures. This effect was specific to attachment figures and was replicated across tasks and experiments. Attachment anxiety heightened accessibility of representations of attachment figures even in neutral contexts, whereas attachment avoidance inhibited this activation when the threat prime was the word separation. These effects were not, explained by trait anxiety. The discussion focuses on the dynamics of attachment-system activation in adulthood. PMID- 12374443 TI - Sensitivity to status-based rejection: implications for African American students' college experience. AB - The authors proposed a process model whereby experiences of rejection based on membership in a devalued group can lead people to anxiously expect, readily perceive, and intensely react to status-based rejection. To test the model, the authors focused on race-based rejection sensitivity (RS-race) among African Americans. Following the development and validation of the RS-Race Questionnaire (Studies 1 and 2), the authors tested the utility of the model for understanding African American students' experiences at a predominantly White university (Study 3). Students high in RS-race experienced greater discomfort during the college transition, less trust in the university, and relative declines in grades over a 2- to 3-year period. Positive race-related experiences, however, increased feelings of belonging at the institution among students high in RS-race. PMID- 12374444 TI - Protestant relational ideology and (in)attention to relational cues in work settings. AB - M. Weber (1947) proposed that exposure to Calvinist Protestantism is associated with limited attention to relational concerns in work settings. Two experiments provide support for this proposition. Study 1 showed that Protestant European Americans raised in traditions of Calvinism were less attentive to affect in spoken words when primed with a work context relative to a nonwork context, and to participants raised as Catholics in either context. Study 2 used an unconscious mimicry paradigm to measure relational focus and showed that within a work setting, male Protestants mimicked a confederate's foot shaking less than male non-Protestants and women in either group. Within a nonwork setting, male Protestants mimicked more and did not differ from male non-Protestants. Women showed greater mimicry than men. PMID- 12374445 TI - Coping with sexual harassment: reconceptualizing women's resistance. AB - This study explored the underlying structure of women's coping with sexual harassment from a rational-empirical approach. On the basis of multidimensional scaling, clustering, and confirmatory factor analysis across 8 data sets, 4 clusters of coping behaviors emerged, with little variance across the data sets. These clusters bear resemblance to Moos and colleagues' (C. J. Holahan, R. H. Moos, & J. A. Schaefer, 1996; R. Moos, 1992; R. H. Moos & J. A. Schaefer, 1993) distinction between coping strategies that differ in both method and foci. The four clusters that emerged are behavioral engagement, behavioral disengagement, cognitive engagement, and cognitive disengagement. This framework provides insight into the complex forms that women's coping with sexual harassment takes and has important legal implications. PMID- 12374446 TI - On being sad and mistaken: mood effects on the accuracy of thin-slice judgments. AB - A series of studies explored how sadness impacts the accuracy of social judgments. In Study 1, induced sadness led to reduced accuracy in judgments of teacher effectiveness from brief samples of nonverbal behavior (thin slices). In Study 2, sad participants showed reduced accuracy in judging relationship type from thin slices as well as diminished judgmental efficiency. Study 3 revealed that higher Beck Depression Inventory scores were associated with diminished accuracy on the Profile of Nonverbal Sensitivity. Finally, Study 4 tested the possibility that sadness impairs accuracy by promoting a more deliberative information-processing style. As expected, accuracy was higher among participants in a sad mood condition who completed the judgment task while simultaneously performing a distracting cognitive load task. PMID- 12374447 TI - The search for dimensional structure differences between normality and abnormality: a statistical review of published data on personality and psychopathology. AB - A statistical review of published data for 37 personality and psychopathology inventories was conducted to determine whether there are dimensional structure differences between clinical and nonclinical respondents. Correlation and factor loading matrices from multiscale inventories and from specialized measures were tested for structural invariance across populations. There was relatively consistent evidence for high levels of similarity between normal and abnormal populations both in the number of factors that exist in the data matrices and in the factor patterns. The dimensional universes of normality and abnormality are apparently the same, at least according to data derived from contemporary assessment instruments. Categorical-taxonic differences between clinical and nonclinical populations, which were not examined, may nevertheless exist within contexts of dimensional structure similarity. PMID- 12374448 TI - The substantive nature of psycholexical personality factors: a comparison across languages. AB - The psycholexical approach to personality structure in American English has led to the Big Five factors. The present study considers whether this result is similar or different in other languages. Instead of placing the usual emphasis on quantitative indices, this study examines the substantive nature of the factors. Six studies in European languages were used to develop a taxonomy of content categories. The English translations of the relevant terms were then classified under this taxonomy. The results support the generality of Big Five Factor III (Conscientiousness). Factors IV (Emotional Stability) and V (Intellect) generally did not cohere. Factors I (Extraversion) and II (Agreeableness) tended to split when this was necessary to produce 5 factors. The analysis was extended to several additional studies. PMID- 12374449 TI - Intelligence and information processing during an auditory discrimination task with backward masking: an event-related potential analysis. AB - The relation between mental ability and auditory discrimination ability was examined by recording event-related potentials from 60 women during an auditory oddball task with backward masking. Across conditions that varied in intensity and in the interval between the target and masking stimuli, the higher ability (HA) group exhibited greater response accuracy, shorter response times, larger P3 amplitude, and shorter P3 latency to target stimuli than the lower ability (LA) group. When instructed to ignore the stimuli, the HA group exhibited shorter mismatch negativity latency to deviant tones than the LA group. The greater speed and accuracy of auditory discrimination for the HA group, observed here with multiple measures, is not a consequence of response strategy, test-taking ability, or attention deployment. PMID- 12374450 TI - Parenteral busulfan: is therapeutic monitoring still warranted? PMID- 12374451 TI - Once-daily intravenous busulfan given with fludarabine as conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early clinical outcomes. AB - The availability of an i.v. form of busulfan (Bu) has prompted investigation of administration schedules other than the 4-times-daily dosage commonly used with oral Bu. We have studied an allogeneic stem cell transplantation (SCT) preparative regimen comprising fludarabine (FLU) 50 mg/m2 on days -6 to -2 plus i.v. Bu 3.2 mg/kg daily in a 3-hour infusion on days -5 to -2. The regimen was given to 70 patients aged 15 to 64 years (median, 41 years) with hematologic malignancy. Thirty-six patients (51%) had high-risk malignancy, 28 (40%) had unrelated or genotypically mismatched related donors (alternate donors [AD]) and 29 (41%) received bone marrow rather than blood as stem cell source. Acute GVHD prevention comprised antithymocyte globulin 4.5 mg/kg over 3 days pretransplantation, cyclosporin A, and short-course methotrexate with folinic acid. Hepatic toxicity was transient and there was no clinically diagnosed veno occlusive disease. Grade II stomatitis occurred in 49 patients (70%) and hemorrhagic cystitis in 9 patients (13%). One patient with subtherapeutic phenytoin levels had a convulsion 8 hours after the third i.v. Bu dose, but no other neurotoxicity was apparent. Incidence of acute GVHD grades II to IV was 8% and incidence of grade III-IV was 3%, with no deaths from this cause. Actuarial incidence of chronic GVHD at 2 years is 38%. There were 2 cases of graft failure in unrelated donor BMT recipients, 1 of which was reversed by asecond transplantation. With a median follow-up of 16 months (range, 6-27 months), transplantation-related mortality at 100 days and 2 years was 2% and 5% for matched related donor (MRD) SCT and 8% and 19% for AD SCT, respectively (P = not significant). Relapse rates were 21% for 34 patients with acute myeloid leukemia (AML) in complete remission or chronic myeloid leukemia in chronic phase (low risk), 66% for 19 patients with high-risk AML, and 18% for 17 patients with other active malignancy. Projected disease-free and overall survival rates at 2 years were 74% and 88% for low-risk disease, 26% and 37% for advanced AML, and 65% and 71% for other high-risk disease, respectively. Pharmacokinetic studies were done using 11 samples with the first and fourth doses of Bu. Kinetics were linear, and for the first and fourth doses, the half-lives were 2.60 +/- 0.44 and 2.57 +/- 0.36 hours, respectively. Clearances were 106.77 +/- 16.68 and 106.86 +/- 21.57 mL/min per m2, peak concentrations (Cmax) were 3.92 +/- 0.31 and 3.96 +/- 0.28 mcg/mL, and Bu areas under the plasma concentration versus time curve (AUC) were 4866.51 +/- 771.42 and 4980 +/- 882.80 microM x min, respectively. Bu was completely cleared within 24 hours and the day 4 pharmacokinetic values were very similar to those on day 1 for every patient. The cumulative AUC was comparable to the target range established for p.o. Bu. This regimen incorporating once-daily i.v. Bu is convenient to give, is relatively well tolerated, gives predictable blood levels, and deserves further study in circumstances in which cytoreduction as well as immune suppression is needed. PMID- 12374452 TI - Busulfan systemic exposure relative to regimen-related toxicity and acute graft versus-host disease: defining a therapeutic window for i.v. BuCy2 in chronic myelogenous leukemia. AB - Complete bioavailability of i.v. busulfan (Bu) provides dose assurance by reducing the interdose and interpatient variability in Bu systemic exposure (Bu SE) associated with the oral formulation. We hypothesized that Bu-SE, represented by the area under the plasma concentration versus time curve (AUC), would correlate with treatment outcome after allogeneic hematopoietic stem cell transplantation (HSCT) for chronic myelogenous leukemia (CML). Therefore, we analyzed the risk of death, incidence of regimen-related toxicity, and incidence of acute GVHD (aGVHD) as functions of the per dose i.v. Bu AUC in 36 CML patients who received a HSCT from an HLA-matched family donor after the i.v. BuCy2 regimen. Per-dose Bu AUCs were calculated for each subject using data obtained for doses 1, 5, 9, and 13. Toxicity was evaluated using the modified National Cancer Institute criteria. Because no patient developed veno-occlusive disease, increased serum bilirubin was used to characterize hepatotoxicity. We found that the probabilities of developing gastrointestinal toxicity (P = .01), hepatotoxicity (P < .01), mucositis (P = .09), and aGVHD (P < .01) all increased with increasing AUC. Further, the risk of death was significantly lower for patients having a per-dose AUC between approximately 950 and 1520 microMol-min, whereas the risk increased sharply with either lower or higher AUC values. These data suggest that an optimal Bu therapeutic window, based on per-dose AUC, exists. Given the ability of i.v. Bu to provide a more consistent per-dose AUC, these results should be useful in designing future i.v.V Bu-based treatment protocols for stem cell transplantation. PMID- 12374453 TI - Evaluation of safety and pharmacokinetics of administering intravenous busulfan in a twice-daily or daily schedule to patients with advanced hematologic malignant disease undergoing stem cell transplantation. AB - Intravenous busulfan (i.v. BU) has demonstrated safety when administered at 0.8 mg/kg per dose i.v. every 6 hours x 16 doses. We evaluated the safety and pharmacokinetics (PK) of giving the same total daily i.v. BU dose (3.2 mg/kg) either divided as a twice-daily infusion or as a single infusion to patients undergoing hematopoietic stem cell transplantation (HSCT). Twelve patients with hematologic malignant disease were treated; 7 patients had non-Hodgkin's lymphoma, 4 patients had acute myeloid leukemia, and 1 patient had chronic myelogenous leukemia. The first cohort (group A) received, on the basis of actual body weight, i.v. BU at 1.6 mg/kg per dose over 4 hours every 12 hours for 4 days (day -7 to day -4). The second cohort (group B) received 3.2 mg/kg per dose of i.v. BU (same total dose as group A) as a single infusion over 4 hours daily for 4 days. In both groups the i.v. BU was followed by cyclophosphamide 60 mg/kg daily for 2 days (day -3 and day -2). Blood specimens were collected on the first, fifth, and seventh doses for group A and on the first and fourth doses for group B to determine the disposition of i.v. BU. Peripheral blood stem cells (autologous in 7 cases and HLA-matched allogeneic in 5 cases) were given 2 days after completion of cyclophosphamide administration (day 0), and granulocyte colony-stimulating factor 5 microg/kg was started on the same day. GVHD prophylaxis consisted of tacrolimus plus methotrexate for recipients of allogeneic stem cells. One patient developed presumed fungal pneumonia and died of multisystem organ dysfunction on day +21 before hematologic reconstitution could be evaluated. Another was reported to have sudden death of undetermined cause at home on day 40. The remaining patients had engraftment (absolute neutrophil count >500/microL) at a median of 11 days and sustained platelet counts >20,000/microL at a median of 14 days. Significant regimen-related toxicity (grade III-IV) was limited to hepatic toxicity (2 cases) catheter infection (2 cases), epistaxis (3 cases), diarrhea (1 case), anorexia (1 case), mucositis (1 case), hyperglycemia (1 case), pneumonia (1 case), and sepsis (1). In group B there was 1 case of mild venoocclusive disease, which resolved without sequelae. No central nervous system or pulmonary toxicity was noted. Pharmacokinetic parameters, including clearance, half-life, maximum concentration, and area under the curve, demonstrated that the first dose profile was highly predictive of later dose PK profiles. No accumulation of the drug was noted. The change in dosing schedule did not increase toxicity or end-organ damage despite higher plasma concentration-times. Although further study for long term efficacy is warranted, i.v. BU can be given safely with reproducible results on a twice-daily divided or single-daily dosing schedule to patients undergoing HSCT. PMID- 12374454 TI - Intravenous versus oral busulfan as part of a busulfan/cyclophosphamide preparative regimen for allogeneic hematopoietic stem cell transplantation: decreased incidence of hepatic venoocclusive disease (HVOD), HVOD-related mortality, and overall 100-day mortality. AB - Hepatic venoocclusive disease (HVOD) is a complication of allogeneic hematopoietic stem cell transplantation (HSCT) and is a well-recognized dose limiting toxicity of oral busulfan (Bu)-based preparative regimens. The unpredictable absorption of oral Bu from the gastrointestinal (GI) tract and hepatic first-pass effects have led to the development of an intravenous Bu preparation (i.v. Bu). The purpose of this retrospective comparison was to evaluate the incidence rate of HVOD and the 100-day mortality rate in patients treated with a busulfan/cyclophosphamide (BuCy2) regimen in which either oral Bu or i.v. Bu was administered. Data from 2 similar groups of patients treated between March 1995 and December 1997 were analyzed. Thirty patients were treated with oral Bu (1 mg/kg x 16 doses) at City of Hope and 61 patients were treated with i.v. Bu (0.8 mg/kg x 16 doses) in a multicenter trial involving 7 sites. Bu was followed by Cy (60 mg/kg x 2 days) and a histocompatible-sibling-donor HSCT. In the i.v. Bu treatment group, 48% of the patients were classified as heavily pretreated (> or = 3 prior chemotherapy regimens, prior radiation, or prior HSCT) with 13% having had a prior HSCT and 75% having active disease at the time of transplantation. According to the same classification criteria, 33% of the patients in the oral-Bu treatment group were considered heavily pretreated, with 23% having had a prior HSCT and 80% having active disease at the time of transplantation. The incidence rates of clinically diagnosed HVOD were 5/61 (8%) and 10/30 (33%) after i.v. and oral Bu, respectively. HVOD-related mortality occurred in 2 (3.3%) of 61 i.v. and 6 (20%) of 30 oral Bu patients. The (standardized) Jones criteria for HVOD were met by 4.9% of i.v. and 20% of oral Bu patients. Univariate logistic regression analysis identified oral versus i.v. Bu (P = .001) and a diagnosis of myelodysplastic syndrome (P = .04) as statistically significant factors in the development of HVOD, with prior extensive treatment identified as marginally significant (P = .25). No other demographic parameter was found to be significant. After adjustment for prior treatment, multivariate analyses showed that the use of oral versus i.v. Bu was the strongest predictor for development of HVOD (odds ratio, 7.5; 95% confidence interval, 2.1-27.2; P = .002). This study showed that the incidence rate of HVOD is significantly lower (P = .002) and the 100-day survival rate significantly higher (P = .002) in patients treated with i.v. Bu than in patients treated with oral Bu when Bu is used as part of a BuCy2 preparative regimen for allogeneic HSCT. PMID- 12374455 TI - Cytomegalovirus-specific CD4+ and CD8+ T-cells follow a similar reconstitution pattern after allogeneic stem cell transplantation. AB - Cytomegalovirus (CMV) is a common herpes virus that can cause significant morbidity and mortality in immunocompromised individuals, particularly those undergoing allogeneic stem cell transplantation (SCT) for hematological malignancies. Recent studies have examined the kinetics of CMV-specific CD8+ T cell reconstitution after SCT transplantation and have found virus-specific cytotoxic T-lymphocyte regeneration to be dependent on CMV serologic status and CMV reactivation events. However, the reconstitution kinetics of CMV-specific CD4+ T-cells under these same circumstances were not addressed. In this study, we used HLA class I peptide tetramer for CMV pp65 and cytokine flow cytometry to follow the reconstitution of both CD4+ and CD8+ CMV-specific T-cells after allogeneic SCT. We found that following SCT in which both donors and recipients are CMV seropositive, virus-specific CD4+ T-helper cells show the same reconstitution kinetics as CD8+ cytotoxic T-cells. Following CMV reactivation, a synchronous but temporary increase in both CD4+ and CD8+ CMV-specific lymphocytes occurs. The pattern repeats itself after subsequent episodes of CMV reactivation. These data imply that both CD4+ and CD8+ lymphocytes are necessary for an efficient immune response to CMV and suggest that CD4+ and CD8+ CMV-specific T cells are required for the complete restoration of CMV immunity. These findings may have important implications in the development of CMV-specific adoptive immunotherapy strategies. PMID- 12374456 TI - Incidence and outcome of bacterial and fungal infections following nonmyeloablative compared with myeloablative allogeneic hematopoietic stem cell transplantation: a matched control study. AB - Infections contribute significantly to morbidity and mortality after myeloablative allogeneic hematopoietic stem cell transplantation (HSCT). Whether recipients of nonmyeloablative HSCT have different posttransplantation infection risk was unknown. We therefore analyzed the incidence and risk of bacteremia during the first 100 days and of fungal infection during the first 365 days posttransplantation for 56 consecutive patients with hematological malignant disease who received nonmyeloablative HSCT (case patients). We compared the results with those among 112 control patients who received conventional myeloablative HSCT during the same years (January 1997-April 2000). Control patients were matched (2:1) for cytomegalovirus (CMV) risk group, HSC source, donor type, age, and underlying disease. Most donors (93%) were HLA-matched and related. Case patients had shorter periods of neutropenia (absolute neutrophil count, <100/mm3) than did control patients (median, 0 days; range, 0-11 versus 9 days; range, 4-25; P < .0001). This finding was associated with fewer episodes of bacteremia during the first 30 days (9% versus 27%; P = .01) and a trend to fewer episodes of bacteremia during the first 100 days posttransplantation (27% versus 41%, P = .07). Overall survival was significantly improved in case patients compared with control patients (day 100, 93% versus 81%; P = .04). During the first year posttransplantation, invasive aspergillosis occurred at a similar rate (case patients, 15%; control patients, 9%; P value not significant). Multivariate risk factor analyses identified neutropenia and CMV disease as the major factors associated with bacteremia and aspergillosis, respectively. We conclude that shorter periods of severe neutropenia in nonmyeloablative HSCT are associated with decreased risk of early bacteremia, although risk of fungal infection late after HSCT persists. This risk is an important consideration for the future development of preventive strategies. PMID- 12374458 TI - A novel, complex heterozygous mutation within Gsalpha gene in patient with McCune Albright syndrome. AB - McCune-Albright syndrome (MAS) is caused by embryonic somatic mutations leading to the substitution of His or Cys for Arg at amino acid 201 of the alpha-subunit of the signal transduction protein Gs (Gsalpha). The mutations have been found in many affected tissues of patients with MAS. Recently, a new missense mutation was detected in a patient with MAS, leading to the substitution of glycine for arginine at amino acid 201 of the Gsalpha gene, whereas no mutations have been reported at other sites in this gene. In the present study, we identified the activating mutations in the gene encoding Gsalpha protein in the osseous lesions of fibrous dysplasia and peripheral blood leukocyte in a 17-yr-old male patient with MAS. In addition, a heterozygous mutation encoding substitution of Arg201 of Gsalpha with His was found. Interestingly, we also found the other two types of mutations within the Gsalpha gene in the patient's affected osseous tissue. One is a combination mutation in the same allele at codons 209 and 210 of the Gsalpha gene, and the other the missense mutation at codon 235. PMID- 12374459 TI - Thyroxine vs thyroxine plus triiodothyronine in treatment of hypothyroidism after thyroidectomy for Graves' disease. AB - It was recently demonstrated that treatment with levorotatory thyroxine (T4) plus triiodothyronine (T3) compared with treatment with T4 alone improves psychologic functioning in hypothyroid patients with thyroid cancer or autoimmune thyroiditis. In the present double-blind crossover study, we again compared the effects of combined thyroid replacement vs monotherapy on psychologic function, endocrine function, cardiovascular function, and body composition. The patients were women who were hypothyroid after thyroidectomy for Graves' disease. The substitution of 10 microg of T3 for 50 microg of T4 caused a statistically significant decrease in free T4 concentration but no significant change in T3 or thyroid-stimulating hormone concentration. Symptoms of hypothyroidism and of hyperthyroidism tended to decrease on a standard symptom scale after combined treatment. With combined hormone replacement, mental state tended to improve on some mood scales but not on cognitive tests. We found alterations in left ventricular diastolic function but no change in body composition after the combined treatment regimen. These preliminary findings in a small group of patients with Graves' disease are consistent with earlier findings that thyroid replacement with T4-T3 combination improves mental functioning. PMID- 12374460 TI - Low hemoglobin levels in children with in idiopathic growth hormone deficiency. AB - Multiple lines of evidence have implicated the growth hormone (GH) axis in the regulation of erythropoiesis. To test the hypothesis that GH deficiency is associated with hematologic abnormalities, we analyzed pretreatment hemoglobin levels in 100 children with GH deficiency. Hemoglobin levels were decreased in children with GH deficiency compared with age-corrected norms. PMID- 12374461 TI - Gender-related differences in basal and hypoxia-induced activation of signal transduction pathways controlling cell cycle progression and apoptosis, in cardiac fibroblasts. AB - Previously we showed that cardiac fibroblasts are cellular targets of estrogen and that there are significant differences in proliferative response of male and female cardiac fibroblasts under hypoxia, a condition of myocardial ischemia. Here, we tested the hypothesis that signaling pathways that control cell cycle progression and apoptosis in cardiac fibroblasts may be activated in a gender specific manner. Cardiac fibroblasts from adult, age-matched male and female rat heart were exposed to hypoxia (2% O2) and normoxia. Western analysis of cell lysate was used to compare the level of basal and hypoxia-induced expression of signal transduction proteins, known to control cell cycle progression and cell death. Hypoxia led to significant activation of MAP (mitogen-activated protein) kinase and Jun kinase pathways, as shown by phosphorylated extracellular signal regulated kinase (ERK1/2) and Jun kinase isotypes in male cells but this effect was modest in female cells. Male cells expressed higher levels of basal expression for transcription factors c-jun and NF-kB as well as the inhibitor of NF-kB (lk-B). Although hypoxia did not induce changes in the level of c-Jun in either cell type, it moderately increased the level of NF-kB in male cells but led to its decrease in female cells. Basal and hypoxia-induced expression of cyclin D1, c-fos, and PCNA seemed to be comparable in both male and female cells. However, hypoxia-induced activation of cyclin B1, which occurred in both cells, was stronger in female cells. Basal expression of apoptosis-associated transcription factor, p53, was comparable in both cells. However, under hypoxia, there was an increase in the p53 level only in female cells. Although female cells showed higher basal expression for survival-associated protein, Bcl-2, the level of this protein remained unchanged under hypoxia in both cells. Together, these data demonstrate differences in basal and hypoxia-induced expression of proteins with an established role in cell cycle progression and apoptosis in male and female cardiac fibroblasts. These differences may further point to gender related differences in signal transduction pathways that control the proliferative response of those cells under hypoxia. PMID- 12374457 TI - The endocrine system in diabetes mellitus. AB - The pathophysiology of diabetes mellitus is complex and not fully understood. However, it emerges as an abnormal metabolic condition associated with a systemic damage to the vascular bed. Cumulative evidence also reveals that the endocrine system is not intact in patients with diabetes mellitus. It is not clear whether the changes observed in the endocrine system represent a primary defect or reflect the effects of the impaired insulin action and abnormal carbohydrate and lipid metabolism on the hormonal milieu. Review of the literature reveals that the function of the entire endocrine system including the functions of hormones from the hypothalamus, pituitary, adrenal, thyroid, parathyroid, the vitamin D system, the gonads, and the endocrine function of the adipose tissue, is impaired. Good metabolic control and insulin treatment may reverse some of these abnormalities. It remains unanswered as to what extent these changes in the endocrine system contribute to the vascular pathologies observed in individuals affected by diabetes mellitus and whether part of the abnormalities observed in the endocrine system reflect a basic cellular defect in the diabetic syndrome. PMID- 12374462 TI - The angiogenic factor Cyr61 is induced by the progestin R5020 and is necessary for mammary adenocarcinoma cell growth. AB - Cyr61 is a secreted pro-angiogenic factor that belongs to an emerging family of growth regulators classified as CCN (CTGF/Cyr61/NOV). Work in our laboratory has focused on sex steroid regulation of Cyr61 and its role in hormonal carcinogenesis. In this study, both Cyr61 mRNA and protein were induced by the progestin, R5020, in T47D mammary adenocarcinoma cells in a dose- and time dependent fashion. Cyr61 gene induction by R5020 was transcriptionally regulated by progesterone receptor (PR) as the antiprogestin, RU486, and actinomycin D blocked induction completely. Moreover, Cyr61 was upregulated by epidermal growth factor (EGF) but not by R5020 in the PR-MDA-MB-431 mammary adenocarcinoma cell line, underscoring the necessity of PR. The functional significance of progestin induction of Cyr61 in breast cancer cell growth was demonstrated by anti-Cyr61 neutralizing antibodies, which diminished R5020 and EGF-dependent DNA synthesis by 30%. Moreover, anti-Cyr61 neutralizing antibodies reduced the synergistic effects of R5020 and EGF on T47D cell growth by 30%. Accordingly, protein lysates generated from stage II invasive ductal carcinomas (n = 20) were analyzed in order to determine the relevance of Cyr61 expression in the context of breast tumorigenesis. Remarkably, increased Cyr61 protein expression was observed in greater than 50% of primary breast tumor lysates that were progesterone receptor (PR)+ but estrogen receptor negative. Taken together, our data suggest that in addition to its proangiogenic activity, Cyr61 may be a novel mediator of progesterone activity in enhancing growth-factor-driven tumor growth in breast cancer. PMID- 12374463 TI - Reproductive endocrinology in Hatano high- and low-avoidance rats during the estrous cycle. AB - The high- and low-avoidance animals (HAA and LAA rats) were originally selected from Sprague-Dawley rats for their shuttle-box task. Reproductive endocrinology during the estrous cycle was compared between HAA and LAA rats. All HAA rats showed a regular 4-d estrous cycle, whereas most LAA rats (70.8%) showed a regular 5-d estrous cycle. The peak level of preovulatory luteinizing hormone (LH) surge level was significantly lower in LAA rats than in HAA rats on the day of proestrus. In contrast, the peak level of prolactin surge on the day of proestrus was significantly higher in LAA rats than in HAA rats. Plasma concentrations of follicle-stimulating hormone (FSH) and estradiol-17beta were significantly lower in LAA rats as compared with HAA rats at 12 h on the day of estrus and from 24 h on the day of diestrus to 18 h on the day of proestrus. On the other hand, plasma concentrations of progesterone were significantly higher in LAA rats compared with HAA rats on the day of diestrus. The number of antral follicles (300-600 microm in diameter) at 12 h on the day of proestrus was significantly fewer in LAA rats than in HAA rats. The size and number of corpus luteum at 12 h on the day of estrus were significantly greater in LAA rats than in HAA rats. These results clearly demonstrated that apparent differences are observed in reproductive endocrinology between two Hatano strains. These strain differences probably originated from neural regulation of pituitary hormones. PMID- 12374465 TI - Expression and localization of RAP250 mRNA in rat ovary: possible implications in follicular development and ovulation. AB - The expression levels of nuclear receptor coregulators in specific tissue compartments and cells are thought to influence the expression of hormone responsive genes involved in metabolism, development, and reproduction. RAP250 is a novel nuclear receptor coactivator highly expressed in brain and reproductive organs. To investigate the possible involvement of RAP250 in tissue-specific regulation of ovarian function, untreated immature, pregnant mare's serum gonadotropin luteinizing hormone (PMSG-LH)-primed, cycling, and pregnant rat models were used to study the localization and expression of RAP250 mRNA in rat ovary by in situ hybridization (ISH) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that RAP250 mRNA was primarily localized to granulosa cells of healthy follicles in immature, cycling, and pregnant rats and increased during PMSG-induced follicular development. In the preovulatory and ovulatory follicles from the LH-primed rats of 48-h post-PMSG administration, the signals for RAP250 mRNA increased further and remained high until early luteal formation. Only a subset of corpora lutea during diestrus 1, diestrus 2, and initiation of pregnancy was weakly positive, and atretic follicles were largely negative. The RT-PCR results confirmed the presence of RAP250 mRNA in the rat ovary and strengthen the data from ISH. These findings suggest that RAP250 may play potential roles in follicular development and ovulation. PMID- 12374464 TI - Characterization of oxytocin receptors and serotonin transporters in mast cells. AB - Oxytocin (OT) inhibits the uptake of serotonin (5HT) into uterine mast cells. This may modulate 5HT bioavailability in the myometrium. Because 5HT isan important endogenous uterotonic compound, it has been postulated that this effect of OT may contribute to its potency as a labor inducer. This also predicts the presence of oxytocin receptors (OTRs) and transducing signals that will interact with 5HT transporters (SERT) in mast cells. In this study, OTR and SERT were characterized in murine peritoneal mast cells by radioligand-binding studies. Saturation assays for OTR showed no changes in Kd along the estrous cycle (6.95 +/- 2.76 nM in estrus and 4.07 +/- 1.73 nM in diestrus) but an increase in Bmax in estrus (0.71 +/- 0.08 pmol/10(6) cells and 0.37 +/- 0.05 pmol/10(6) cells in estrus and diestrus, respectively). Bmax and Kd for SERT were not affected along the estrous cycle. The signaling between the OTR and the SERT was analyzed by measuring the extent of inhibition of OT and PMA (activator of protein kinase C on 5HT uptake and the capability of Ro318220 (specific inhibitor of PKC) to increase 5HT uptake and block the effect of the above compounds in mast cells. The results showed that in murine peritoneal mast cells in vitro (1) ovarian hormones modulate OTR but not SERT expression, (2) the magnitude of OT action on 5HT uptake depends on the number of OTRs expressed in mast cells, and (3) the signaling between OTR and the SERT is mediated through the activation of protein kinase C. It is concluded that the ovarian hormones have a modulatory action on 5HT uptake which involves OT-mediated mechanism. PMID- 12374466 TI - Direct in vitro effects of androgens and estrogens on ob gene expression and leptin secretion in human adipose tissue. AB - In the present study, we have explored, in vitro, the possibility that short exposure to androgens and estrogens for 24 h may directly influence leptin expression (ARNm and secretion) in sc adipose tissue from men and women. In men, only dihydrotestosterone at high concentration (100 nM) induced a reduction in leptin secretion and ob mRNA level. In women, 17beta-estradiol (10-100 nM) increased ob mRNA expression (+180 to +500%) and leptin release (+75%). Moreover, in adipose tissue of women, the estrogen precursors testosterone (100 nM) and dehydroepiandrosterone (1 microM) also induced an increase in leptin secretion (+84 and +96%, respectively), an effect that was prevented by the aromatase inhibitor letrozole. Finally, the stimulatory effect of 17beta-estradiol observed in women was antagonized by the antiestrogen ICI182780. Altogether, these results suggest that the sexual dimorphism of leptinemia in humans is mainly owing to the estrogen receptor-dependent stimulation of leptin expression in adipose tissue by estrogens and estrogen precursors in women. PMID- 12374467 TI - Role of type IV collagen in prolactin release from anterior pituitaries of male rats. AB - We previously demonstrated that laminin, a component of basement membranes, modulates pituitary hormone secretion. In the present study, we evaluated the effect of type IV collagen, another component of this membrane, on the release of prolactin (PRL) by anterior pituitary gland from adult male rats. Hemipituitaries were incubated for 3 h with type IV collagen or antibodies against it and PRL release was studied. Rabbit IgG to type IV collagen at concentrations of 10(-7) - 10(-5) M had a significant stimulatory effect on PRL release, in comparison to normal rabbit serum IgG or medium alone used as controls. Type IV collagen induced a significant inhibitory effect on basal release of PRL at a concentration of 30 microg/mL. A slight decrease in PRL release was detected in thyrotropin-releasing hormone-stimulated hemipituitaries incubated with type IV collagen at all concentrations used. These results suggest that type IV collagen, similar to laminin-1, modulates PRL released from hemipituitaries, in vitro. PMID- 12374469 TI - United Kingdom legislation on pack sizes of analgesics: background, rationale, and effects on suicide and deliberate self-harm. AB - Following increasing concern in the UK about the mortality and morbidity associated with self-poisoning with analgesics, especially paracetamol (Tylenol, acetaminophen), legislation was introduced in 1998 to modify packs sold over-the counter. The most important change was a reduction in the maximum size of packs. In this paper the background to the legislation, the rationale behind it, and its early impact are reviewed. The changes have had significant positive initial benefits on the mortality and morbidity associated with self-poisoning with analgesics. PMID- 12374468 TI - Impact of gender on insulin signaling pathway in lacrimal and salivary glands of rats. AB - The structure and function of lacrimal and salivary glands present gender differences. Previous works have indicated a synergic action between insulin and androgens over lacrimal gland, and insulin-signaling pathways were recently described in lacrimal gland and salivary gland. Our present study investigates whether gender modulates the early steps of the insulin-signaling system in vivo. Eight-week-old male and female Wistar rats (n = 8/group) were compared to evaluate insulin serum levels and insulin tolerance tests by radioimmunoassay and glucose oxidase method, respectively. To assess insulin receptor (IR), Shc, STAT 1, ERK, and Akt phosphorylation in response to insulin in lacrimal gland and salivary gland, tissues from female and male rats (n = 5-8/group) were submitted to immunoprecipitation and immunoblotting or Western blotting protocol, and phosphorylation level was determined by densitometry. No difference was found in insulin serum levels or insulin tolerance tests comparing both groups. Nevertheless, lacrimal gland and salivary gland of female rats had a significantly lower insulin-induced IR phosphorylation compared with males. IR phosphorylation was not affected by the estrous cycle stage in either tissue. In addition, in females an apparent but not significant lower STAT and Akt phosphorylation in response to insulin was observed in the lacrimal gland, compared with males. Our findings suggest that alterations in insulin signal transduction may play a role in lacrimal gland and salivary gland gender differences. PMID- 12374470 TI - Can the Edinburgh Risk of Repetition Scale predict repetition of deliberate self poisoning in an Australian clinical setting? AB - This study tested the ability of the Edinburgh Risk of Repetition Scale (ERRS) to identify patients at high risk for repeat deliberate self-poisoning (DSP). Consecutive DSP patients (N= 1,317) over a 3-year period were followed-up for 12 months. A statistically significant relationship between ERRS scores and repetition was observed; however, sensitivity and specificity were low. Logistic regression analysis revealed only "previous parasuicide" contributed significantly to repetition. The ERRS had limited value in identifying patients at high risk of repeat DSP in this clinical population. PMID- 12374471 TI - Alcohol and suicide death among American Indians of New Mexico: 1980-1998. AB - The relationship between alcohol use prior to suicide was explored among American Indian decedents in New Mexico for the years 1980 through 1998. The suicide data were collected from New Mexico Vital Statistics and toxicology reports from the New Mexico Office of the Medical Investigator and matched on a case-by-case basis. Detailed analyses were undertaken for all cases of resident New Mexico Indians from the Navajo, Pueblo, and Apache cultures. Alcohol was detected in 69% of all suicides of American Indians with some variance by major tribal cultural groups (range = 62.1% to 84.4%). This is higher than in suicides among the overall New Mexico population (44.3%). The mean blood alcohol concentration (BAC) of the drinking Indian decedents at suicide was 0.198 (+/- SD of .088). Mean BACs were high for both males (0.199) and females (0.180) who had been drinking. Over 90% of the Indian decedents who had been drinking had BACs greater than the legal intoxication level of 0.08. The Navajo had the lowest percentage of cases that were alcohol involved, and their mean BAC was lower than the other two cultural groups. Alcohol use for completed suicides also varied somewhat by age, sex, method of suicide, and place of occurrence, but very little by whether the decedent was an on or off reservation resident. Analyses indicated that alcohol use prior to suicide was significantly more associated with male suicides than for females, and it was negatively correlated for those who died by overdose and also those using other drugs at suicide. Otherwise, alcohol use did not significantly differentiate American Indian suicides by age, use of firearms, hanging, use of other methods, or residence, for the presence of alcohol was a factor very commonly associated with all of these variables. Heavy alcohol consumption is, therefore, an important factor in over two thirds of all completed suicides among the Indians of New Mexico. PMID- 12374472 TI - Suicidal behavior among urban, African American young adults. AB - The objectives of the present study were four-fold. First, to determine the lifetime, last year, and 6-month prevalence and demographic correlates of suicidal behavior in a defined population of urban, African American young adults. Second, to determine the degree of mental health service utilization among attempters. Third, to study the comorbidity between mental disorders and suicidal behavior, along with the variation in the numbers and types of psychiatric disorders associated with attempts versus ideation only. Fourth, to examine gender differences in the psychiatric diagnoses associated with attempts and ideation. Data relevant to each of these objectives were gathered through structured interviews of 1,157 economically disadvantaged, African American young adults. Lifetime, last year, and 6-month prevalence rates for attempts were 5.3%, 1.2%, and 0.4%, respectively, whereas the lifetime and 6-month prevalence of ideation were 14% and 1.9%, respectively. Approximately two thirds of those who reported lifetime ideation, and a similar proportion of those who reported lifetime attempts, had a history of at least one lifetime psychiatric disorder. There were no gender differences in terms of the degree of risk for suicidal behavior (ideation or attempts) associated with any of the comorbid psychiatric diagnoses assessed. Despite the severity of most attempts, few attempters received mental health services in their lifetime or at the time of their most recent attempt. PMID- 12374473 TI - Lesbian, gay, and bisexual suicidal behavior: testing a constructivist model. AB - The present investigation surveyed 162 self-identified lesbian, gay, and bisexual individuals recruited from LGB-related social organizations or contacted through networking procedures with regard to suicidal behaviors, suicide risk factors, and reasons for living. Approximately 41% of the respondents indicated a serious consideration of suicide including the identification of a specific suicide plan (23%) or a past suicide attempt (36%) with significant intent to die (13%). Forty six percent of the sample indicated at least some degree of chance of attempting suicide in the future. Grounded in the existential-constructivist theory of suicide (Rogers, 2001), empirically and theoretically identified suicide risk factors were found as a group to predict suicidal ideation (R2 = .16) and attempts (R2 = .17), with abuse-related items independently predicting both suicidal ideation (R2 = .03) and attempts (R2 = .08). Items related to self identity issues and social acceptance were predictive of suicidal ideation (R2 = .04), while substance abuse was predictive of suicidal ideation (R2 = .05) and attempts (R2 = .13) for males only. The established factor structure of the Reasons for Living Inventory (Linehan et al., 1983) was not supported in the current data, suggesting that it may not be an appropriate measure of reasons for living with LGB individuals. PMID- 12374474 TI - Mental health professionals' determinations of adolescent suicide attempts. AB - The degree of ambiguity in the term suicide attempt was examined among 14 expert suicidologists, and 59 general mental health clinicians who either did or did not receive a standard definition of the term. The participants judged whether each of ten vignettes of actual adolescent self-harm behaviors was a suicide attempt. Low levels of agreement were found within each group, although agreement was better for the most and least serious cases. Possible explanations were examined, including how professionals weight suicidal intent and medical lethality in their suicide attempt decisions, and the use of a "fuzzy," natural language conceptualization of suicide attempts was proposed. PMID- 12374475 TI - Psychiatric and substance use disorders as risk factors for attempted suicide among adolescents: a case control study. AB - The objective of this research was to test substance-related and non-substance related psychiatric disorders as predictors of attempted suicide among adolescents. Ninety-six psychiatrically disordered suicide attempters were matched one-to-one to 96 psychiatrically disordered non-attempters on age, race, gender, and the presence/absence of major depression. Conditional logistic regression was used to test psychiatric risk factors for their power to predict attempted suicide among adolescents. Bipolar disorder, cocaine use disorders, and conduct disorder were found to be predictive of attempted suicide in univariate testing. Bipolar disorder, inhalant use disorders, cocaine use disorders, and hallucinogen use disorders were found to be predictive of attempted suicide, after adjusting for all other covariates. Loglinear analyses revealed high odds ratios associated with the comorbidities of alcohol use disorder with conduct disorder and drug use disorders with conduct disorder in both groups. Higher rates of cocaine use disorder/conduct disorder, hallucinogen use disorder/conduct disorder, and alcohol use disorder/ conduct disorder were found among suicide attempters. Evaluation of these particular comorbid conditions should be part of the adolescent suicide risk assessment. PMID- 12374476 TI - Self-destructive behavior in patients with dissociative disorders. AB - Highrates of self-injury have been reported in patients with dissociative disorders, yet no prior study has directly compared these patients with other psychiatric patients. The present study assesses self-destructive behavior in a group of inpatients who have dissociative disorders compared to those who report few dissociative symptoms. These patients more frequently engage in self destructive behaviors, use more methods of self-injury, and begin to injure themselves at an earlier age then patients who do not dissociate. Results have important implications for understanding the relationship between dissociation, childhood trauma, and self-injury and for assessment and treatment of patients with dissociative disorders. PMID- 12374477 TI - Exposure to suicide: incidence and association with suicidal ideation and behavior: United States, 1994. AB - Exposure to the suicide of another is common, but the magnitude and effects of such exposure are not well quantified. From a national random telephone survey of U.S. adults, we estimated the 12-month incidence of exposure to suicide and its association with suicidal ideation, planning, and behavior. Of 5,238 respondents, 342 (a weighted 7.0% representing 13.2 million persons) reported knowing a suicide decedent from the previous year. Univariate analysis showed persons reporting such exposure were significantly more likely to describe suicidal ideation and behavior than those unexposed; multivariate analysis showed no association. Though the risk related to suicide exposure may be small, given the magnitude of exposure, it may warrant intervention efforts because of its potential societal impact. PMID- 12374478 TI - Association between serotonin transporter gene polymorphism and family history of attempted and completed suicide. AB - The purpose of this study was to examine the association of the serotonin transporter gene to family history of suicidality. Forty-seven volunteers responded to questionnaires about family history of suicide, and provided buccal swabs for analysis of the polymorphism. Allelic homozygocity (the short variant) was associated with family history of suicidality. These data, to be interpreted with the study's limitations in mind, suggest a link between the serotonin transporter gene polymorphism and suicide-related variables, which should be the focus of future research. PMID- 12374479 TI - Serum cholesterol levels and suicide: a meta-analysis. AB - A meta-analysis was performed on studies exploring the link between low levels of serum cholesterol and increased risk of suicide. Follow-up studies found that those with lower cholesterol levels do have a tiny but statistically significant increased risk of completing suicide. Individuals who have attempted suicide in the past have lower cholesterol levels, especially if they used violent methods for suicide. Cholesterol lowering studies, however, did not lead to a significant increase in completed suicide. PMID- 12374480 TI - Low-molecular-weight herapin. PMID- 12374481 TI - New techniques in wound management: vacuum-assisted wound closure. AB - Vacuum-assisted wound closure (VAC) is a wound management technique that exposes the wound bed to negative pressure by way of a closed system. Edema fluid is removed from the extravascular space, thus eliminating an extrinsic cause of microcirculatory embarrassment and improving blood supply during this phase of inflammation. In addition, the mechanical tension from the vacuum may directly stimulate cellular proliferation of reparative granulation tissue. Orthopaedic indications for VAC include traumatic wounds after debridement, infection after debridement, and fasciotomy wounds for compartment syndrome. VAC also can be used as a dressing for anchoring an applied split-thickness skin graft. The technique is contraindicated in patients with thin, easily bruised or abraded skin; those with neoplasm as part of the wound floor; and those with allergic reactions to any of the components that contact the skin. Clinical experience with the technique has resulted in a low incidence of minor, reversible irritation to surrounding skin and no major complications. Further experience is required, as well as clinical and basic research, to define optimal indications and benefits compared with traditional methods of wound management. PMID- 12374482 TI - Musculoskeletal manifestations of human immunodeficiency virus infection. AB - Musculoskeletal manifestations of the human immunodeficiency virus (HIV) are common and are sometimes the initial presentation of the disease. Knowledge of the conditions affecting muscle, bone, and joints in HIV-infected patients is essential for successful management. Myopathies may be caused by pyogenic infection (eg, pyomyositis), idiopathic inflammation (eg, polymyositis), or drug effect (eg, AZT myopathy). Characteristic skeletal infections, such as tuberculosis and bacillary angiomatosis, require a high index of suspicion for accurate diagnosis. Neoplastic processes, such as non-Hodgkin's lymphoma and Kaposi's sarcoma, occur more frequently as the immune system deteriorates. Inflammatory and reactive arthropathies are more prevalent in HIV-positive than HIV-negative individuals and include Reiter's syndrome, psoriatic arthritis, HIV associated arthritis, painful articular syndrome, acute symmetric polyarthritis, and hypertrophic osteoarthropathy. Patients with atypical musculoskeletal complaints and a suspected history of exposure should be tested for HIV. PMID- 12374483 TI - Surgical treatment of developmental dysplasia of the hip in adults: I. Nonarthroplasty options. AB - Hip dysplasia is a developmental disorder that results in anatomic abnormalities leading to increased contact pressure in the joint and, eventually, coxarthrosis. However, many patients with hip dysplasia become symptomatic before the development of severe degenerative changes because of abnormal hip biomechanics, mild hip instability, impingement, or associated labral pathology. Several nonarthroplasty treatment options are available. Because the primary deformity is mostly acetabular, for many patients, a reconstructive osteotomy that restores more nearly normal pelvic anatomy is preferable. The Bernese periacetabular osteotomy is presently favored because it provides good correction while creating little secondary pelvic deformity or destabilizing the pelvis. Proximal femoral osteotomy is occasionally needed as a complement to pelvic osteotomy and may also be indicated as an isolated procedure when most deformity is located on the femoral side (coxa valga subluxans). Arthroscopy can be beneficial when symptoms seem to be related only to labral tears or loose bodies in the absence of severe structural abnormalities about the hip. Fusion and resection arthroplasty are rarely indicated and are reserved for occasional patients who are not candidates for total hip replacement or other procedures but who complain of refractory hip pain. PMID- 12374484 TI - Surgical treatment of developmental dysplasia of the hip in adults: II. Arthroplasty options. AB - Total hip arthroplasty is the procedure of choice for most patients with symptomatic end-stage coxarthrosis secondary to hip dysplasia. The anatomic abnormalities associated with the dysplastic hip increase the complexity of hip arthroplasty. When pelvic bone stock allows, it is desirable to reconstruct the socket at or near the normal anatomic acetabular location. To obtain sufficient bony coverage of the acetabular component, the socket can be medialized or elevated, or a lateral bone graft can be applied. Uncemented acetabular components allow biologic fixation with potentially improved results compared with cemented cups, especially in young patients. The location of the acetabular reconstruction and the desired leg length influence the type of femoral reconstruction. Cemented and uncemented implants can be used in femoral reconstruction, depending on the clinical situation. Femoral shortening is required in some cases and can be performed by metaphyseal resection with a greater trochanteric osteotomy and advancement or by a shortening subtrochanteric osteotomy. The results of total hip arthroplasty demonstrate a high rate of pain relief and functional improvement. The long-term durability of cemented total hip arthroplasty reconstruction in these patients is inferior to that in the general population. The results of uncemented implants are promising, but only limited early and midterm data are available. PMID- 12374485 TI - Fractures around the knee in children. AB - Traumatic forces applied to the immature knee result in fracture patterns different from those in adults. The relative abundance of cartilage in the knee of the growing child may make the diagnosis of certain injuries more challenging. If plain radiographs fail to reveal a fracture, a stress radiograph, computed tomography scan, or magnetic resonance imaging study may help to establish the diagnosis. Certain fractures, such as hyperextension injuries to the distal femoral or proximal tibial epiphysis, or displaced tibial tuberosity fractures, may be especially susceptible to neurovascular problems. Although the use of appropriate treatment techniques may minimize the occurrence of late complications such as malunion and physeal bridging, not all problems are preventable. A careful discussion of the injury with both patient and parents should stress the importance of follow-up so that any problems that do occur can be promptly addressed. PMID- 12374486 TI - Arthroscopic management of osteoarthritis of the knee. AB - Recent advances in instrumentation and a growing understanding of the pathophysiology of osteoarthritis have led to increased use of arthroscopy for the management of degenerative arthritis of the knee. Techniques include lavage and debridement, abrasion arthroplasty, subchondral penetration procedures (drilling and microfracture), and laser/thermal chondroplasty. In most patients, short-term symptomatic relief can be expected with arthroscopic lavage and debridement. Greater symptomatic relief and more persistent pain relief can be achieved in patients who have acute onset of pain, mechanical disturbances from cartilage or meniscal fragments, normal lower extremity alignment, and minimal radiographic evidence of degenerative disease. Arthroscopic chondroplasty techniques provide unpredictable results. Concerns include the durability of the fibrocartilage repair tissue in subchondral penetration procedures and thermal damage to subchondral bone and adjacent normal articular cartilage in laser/thermal chondroplasty. Although recent prospective, randomized, double blinded studies have demonstrated that outcomes after arthroscopic lavage or debridement were no better than placebo procedure for knee osteoarthritis, controversy still exists. With proper selection, patients with early degenerative arthritis and mechanical symptoms of locking or catching can benefit from arthroscopic surgery. PMID- 12374487 TI - Classifications of thoracic and lumbar fractures: rationale and supporting data. AB - Classification systems are generalizations that attempt to identify common attributes within a group to predict behavior or outcome without sacrificing too much detail. Because of the inherent variability of fractures, classifying them can be difficult. To properly apply any of the commonly cited classification schemes for thoracic and lumbar fractures, one must not only know the injury categories described in the original studies but also be familiar with the rationale for developing the classification. Many original reports describing common thoracic and lumbar injury classifications lack a rigorous scientific foundation. They were based largely on the insights of experienced surgeons and researchers. Although the ideal classification for thoracic and lumbar fractures does not exist, it would incorporate neurologic as well as structural factors. Standardization of terminology as related to treatment decisions and prognosis is key to an improved understanding of the clinical behavior of these injuries and identification of optimal treatment options. PMID- 12374488 TI - Mitochondrial therapy for Parkinson disease. PMID- 12374489 TI - Glial cells under physiologic and pathologic conditions. AB - Glial cells have long been considered to play roles in the nervous system that are unexciting compared with those of neurons. They provide neurons with nutrients, guide migrating neurons and their precursors during development, and dispose of the brain's "waste." Recent evidence, however, suggests that glial cells play more sophisticated, neuronlike roles. They integrate neuronal input, modulate synaptic activity, and process signals related to learning and memory. These findings have significant implications for humans with neurodegenerative diseases. In addition to activation on nervous system injury and during neuronal degeneration, glial cells also degenerate in several neurodegenerative diseases. Therefore, glial cell loss may contribute to the impairment of learning and memory. Therapeutic approaches to combat human neurodegenerative diseases thus need to restore the function of both neurons and glial cells. PMID- 12374490 TI - Indications and usefulness of nerve biopsy. PMID- 12374491 TI - Effects of coenzyme Q10 in early Parkinson disease: evidence of slowing of the functional decline. AB - BACKGROUND: Parkinson disease (PD) is a degenerative neurological disorder for which no treatment has been shown to slow the progression. OBJECTIVE: To determine whether a range of dosages of coenzyme Q10 is safe and well tolerated and could slow the functional decline in PD. DESIGN: Multicenter, randomized, parallel-group, placebo-controlled, double-blind, dosage-ranging trial. SETTING: Academic movement disorders clinics. PATIENTS: Eighty subjects with early PD who did not require treatment for their disability. INTERVENTIONS: Random assignment to placebo or coenzyme Q10 at dosages of 300, 600, or 1200 mg/d. MAIN OUTCOME MEASURE: The subjects underwent evaluation with the Unified Parkinson Disease Rating Scale (UPDRS) at the screening, baseline, and 1-, 4-, 8-, 12-, and 16 month visits. They were followed up for 16 months or until disability requiring treatment with levodopa had developed. The primary response variable was the change in the total score on the UPDRS from baseline to the last visit. RESULTS: The adjusted mean total UPDRS changes were +11.99 for the placebo group, +8.81 for the 300-mg/d group, +10.82 for the 600-mg/d group, and +6.69 for the 1200 mg/d group. The P value for the primary analysis, a test for a linear trend between the dosage and the mean change in the total UPDRS score, was.09, which met our prespecified criteria for a positive trend for the trial. A prespecified, secondary analysis was the comparison of each treatment group with the placebo group, and the difference between the 1200-mg/d and placebo groups was significant (P =.04). CONCLUSIONS: Coenzyme Q10 was safe and well tolerated at dosages of up to 1200 mg/d. Less disability developed in subjects assigned to coenzyme Q10 than in those assigned to placebo, and the benefit was greatest in subjects receiving the highest dosage. Coenzyme Q10 appears to slow the progressive deterioration of function in PD, but these results need to be confirmed in a larger study. PMID- 12374492 TI - The role of cerebrospinal fluid hypocretin measurement in the diagnosis of narcolepsy and other hypersomnias. AB - CONTEXT: Narcolepsy, a neurological disorder affecting 1 in 2000 individuals, is associated with HLA-DQB1*0602 and low cerebrospinal fluid (CSF) hypocretin (orexin) levels. OBJECTIVES: To delineate the spectrum of the hypocretin deficiency syndrome and to establish CSF hypocretin-1 measurements as a diagnostic tool for narcolepsy. DESIGN: Diagnosis, HLA-DQ, clinical data, the multiple sleep latency test (MSLT), and CSF hypocretin-1 were studied in a case series of patients with sleep disorders from 1999 to 2002. Signal detection analysis was used to determine the CSF hypocretin-1 levels best predictive for International Classification of Sleep Disorders (ICSD)-defined narcolepsy (blinded criterion standard). Clinical and demographic features were compared in narcoleptic subjects with and without low CSF hypocretin-1 levels. SETTING: Sleep disorder and neurology clinics in the United States and Europe, with biological testing performed at Stanford University, Stanford, Calif. PARTICIPANTS: There were 274 patients with narcolepsy; hypersomnia; obstructive sleep apnea; restless legs syndrome; insomnia; and atypical hypersomnia cases such as familial cases, narcolepsy without cataplexy or without HLA-DQB1*0602, recurrent hypersomnias, and symptomatic cases (eg, Parkinson disease, depression, Prader-Willi syndrome, Niemann-Pick disease type C). The subject group also included 296 controls (healthy and with neurological disorders). INTERVENTION: Venopuncture for HLA typing, lumbar puncture for CSF analysis, primary diagnosis using the International Classification of Sleep Disorders, Stanford Sleep Inventory for evaluation of narcolepsy, and sleep recording studies. MAIN OUTCOME MEASURES: Diagnostic threshold for CSF hypocretin-1, HLA-DQB1*0602 positivity, and clinical and polysomnographic features. RESULTS: HLA-DQB1*0602 frequency was increased in narcolepsy with typical cataplexy (93% vs 17% in controls), narcolepsy without cataplexy (56%), and in essential hypersomnia (52%). Hypocretin-1 levels below 110 pg/mL were diagnostic for narcolepsy. Values above 200 pg/mL were considered normal. Most subjects with low levels were HLA-DQB1*0602-positive narcolepsy cataplexy patients. These patients did not always have abnormal MSLT. Rare subjects without cataplexy, DQB1*0602, and/or with secondary narcolepsy had low levels. Ten subjects with hypersomnia had intermediate levels, 7 with narcolepsy (often HLA negative, of secondary nature, and/or with atypical cataplexy or no cataplexy), and 1 with periodic hypersomnia. Healthy controls and subjects with other sleep disorders all had normal levels. Neurological subjects had generally normal levels (n = 194). Intermediate (n = 30) and low (n = 3) levels were observed in various acute neuropathologic conditions. CONCLUSIONS: Narcolepsy cataplexy with hypocretin deficiency is a genuine disease entity. Measuring CSF hypocretin-1 is a definitive diagnostic test, provided that it is interpreted within the clinical context. It may be most useful in cases with cataplexy and when the MSLT is difficult to interpret (ie, in subjects already treated with psychoactive drugs or with other concurrent sleep disorders). PMID- 12374493 TI - Diffuse axonal and tissue injury in patients with multiple sclerosis with low cerebral lesion load and no disability. AB - BACKGROUND: Although in situ pathological studies and in vivo magnetic resonance (MR) investigations have shown that axonal injury can be significant in the early stages of multiple sclerosis (MS), diffuse axonal injury is generally considered a secondary event. Cerebral axonal damage can be specifically assessed in vivo by measuring levels of brain N-acetylaspartate (NAA, a specific index of axonal integrity detected by MR spectroscopy). Other new MR measurements such as magnetization transfer ratio (MTr) or computed estimation of brain volume can provide less specific indexes of tissue damage. OBJECTIVE: To determine whether diffuse axonal and tissue injury is present in patients with definite MS who do not show clinically significant disability. METHODS: We measured brain NAA levels (normalized to creatine [Cr]), MTr values, and cerebral volumes in patients with definite MS who had low T2-weighted MR imaging lesion volumes and no clinical disability, and also in age-matched healthy control subjects. RESULTS: Values of central brain NAA/Cr and MTr in normal-appearing white matter were significantly lower in the MS patients than in controls (P<.001). In contrast, total brain volumes were not significantly different between these groups. Similar results were found for MS patients with early disease (duration, <3 years) and with a particularly low cerebral T2-weighted MR imaging lesion load (< or = 2 cm(3)). CONCLUSIONS: Cerebral NAA/Cr and MTr values are diffusely decreased in MS patients with early disease, low demyelinating lesion load, and no significant disability. This suggests that axonal and/or tissue injury begins very early in the course of MS and might be at least partially independent of cerebral demyelination. PMID- 12374494 TI - Longitudinal brain volume measurement in multiple sclerosis: rate of brain atrophy is independent of the disease subtype. AB - BACKGROUND: In multiple sclerosis (MS), brain atrophy depicted by magnetic resonance imaging reflects overall tissue loss, including axonal loss. OBJECTIVE: To determine the course of atrophy by studying the rate of development of brain atrophy in patients who have different subtypes of MS. METHODS: Eighty-three patients with MS (42 with relapsing-remitting, 21 with secondary progressive, and 20 with primary progressive) were studied longitudinally, with an interval of 2 to 4 years. Magnetic resonance imaging included T1- and T2-weighted images to obtain 2 brain volume measurements: (1) the parenchymal fraction as a marker of global brain atrophy and (2) the ventricular fraction as a marker of central atrophy. The annualized rate of global and central brain atrophy was compared between those with different subtypes of MS and related to clinical characteristics, including sex, age, disease duration, and disability. RESULTS: There was a significant decrease of the parenchymal fraction (-0.7% per year; SEM, 0.11% per year) and a significant increase of ventricular fraction (3.7% per year; SEM, 0.54% per year) in the total group. Significant tissue loss was also seen in all 3 subtypes of MS; the decrease in parenchymal fraction was not different between subtypes, whereas the increase in ventricular fraction tended to be larger in patients with secondary progressive MS compared with patients with primary progressive MS. Marginal associations were found between clinical determinants and the rate of brain atrophy. Annualized increase in the ventricular fraction was correlated with age (r = -0.26) and duration of symptoms (r = -0.22): younger patients (mainly patients with relapsing-remitting MS who have a limited disability) displayed a larger increase in ventricular fraction compared with older patients. CONCLUSIONS: The rate of development of brain atrophy is largely independent of the course of the disease and other clinical characteristics. The relentless loss of tissue occurring in MS is not restricted to later (progressive) phases of the disease, thereby stressing the need for early neuroprotective treatment in MS. PMID- 12374495 TI - Lesion patterns and mechanism of ischemia in internal carotid artery disease: a diffusion-weighted imaging study. AB - CONTEXT: Although embolism and low-flow phenomenon are the 2 main mechanisms of stroke in internal carotid artery (ICA) occlusive disease, the mechanism of border-zone infarction remains controversial. Diffusion-weighted imaging (DWI) can more easily detect small or multiple ischemic lesions than conventional imaging. OBJECTIVES: To investigate the ischemic lesion patterns on DWI and to discuss the mechanisms of stroke in ICA disease. DESIGN: Case series. SETTING: A tertiary referral center. PATIENTS: We enrolled 35 consecutive patients who had an acute ischemic stroke and (> or = 70%) stenosis or an occlusion of the extracranial ICA confirmed by cerebral angiography and an acute relevant stroke lesion on DWI within 1 week of onset, but without cardiac sources of embolism and tandem intracranial arterial disease. MAIN OUTCOME MEASURES: The lesion pattern on DWI was categorized as territorial or border zone. Multiple ischemic lesions were defined as noncontiguous lesions on DWI in more than 1 vascular territory. RESULTS: There were 3 distinctive stroke lesion patterns. (1) A territorial lesion without a border-zone lesion was found in 21 patients: superficial and superficial territorial in 9, superficial and deep territorial in 7, and single in 5. (2) A border-zone lesion with or without a territorial lesion was found in 10 patients: border zone and territorial in 9 and border zone alone in 1. (3) Bilateral hemispheric lesions were found in 4 patients. Multiple ischemic lesions were found in 29 (82.9%) of the 35 patients. No patient had episodes of hemodynamic compromise. CONCLUSIONS: An acute ischemic lesion in ICA occlusive disease is mainly multiple. Border-zone infarction was mostly associated with territorial infarction. These results support the fact that embolism is the predominant stroke mechanism in ICA occlusive disease. PMID- 12374496 TI - Etiology, duration, and prognosis of transient ischemic attacks: an analysis from the German Stroke Data Bank. AB - CONTEXT: A transient ischemic attack (TIA) has been arbitrarily defined as a focal cerebral ischemic deficit lasting less than 24 hours. OBJECTIVE: To determine if TIAs of short duration (<1 hour) and long duration (1 hour to <24 hours) differ from each other and from ischemic stroke (IS). DESIGN, SETTING, AND PATIENTS: Inception cohorts of 1429 patients with acute TIAs and 5206 patients with IS were prospectively documented in 15 German medical centers with neurology departments and acute stroke units. Outcome after 3 months was assessed in 72.8% of the patients with TIAs. MAIN OUTCOME MEASURES: Risk factor distribution, etiology, and prognosis of TIAs and IS. RESULTS: Patients with TIAs, especially those with symptoms lasting less than 1 hour, were significantly more likely to have a history of TIAs and less likely to have diabetes mellitus, arterial hypertension, or atrial fibrillation at admission compared with those with IS. Cardioembolic etiologies were less frequent and unknown etiologies more frequent among patients with TIAs than those with IS. Functional outcome and mortality did not differ significantly in patients with TIAs of different durations. CONCLUSION: This study demonstrates differences in comorbidity and etiology among patients with TIAs of different durations and IS. PMID- 12374497 TI - Dementia and Alzheimer disease incidence rates do not vary by sex in Rochester, Minn. AB - BACKGROUND: Incidence rates of Alzheimer disease (AD) were higher in women than in men in several recent European and Asian studies. Cohort studies in the United States, on the other hand, have consistently reported no difference in incidence across sex. OBJECTIVE: To measure age- and sex-specific incidence rates of dementia and AD for persons aged 50 years and older residing in Rochester, Minn, during 1985 to 1989. SUBJECTS AND METHODS: Cases were ascertained through the medical records linkage system of the Rochester Epidemiology Project, which encompasses the records of all medical care providers (including outpatient clinics, hospitals, general practitioners, and nursing homes) in Rochester. Computer indices of clinical diagnoses, histologic diagnoses, and medical procedures were screened for indications of dementia. All medical records of potential cases were reviewed and abstracted by a trained nurse abstractor. A neurologist (E.K.) confirmed the presence of dementia and established a differential diagnosis of AD using the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, and estimated the year of onset. RESULTS: A total of 482 incident cases of dementia were identified; 356 of them (73.9%) had AD. For both dementia and AD, incidence rates increased steeply with age, and there were no consistent differences between men and women. The sex pattern for AD did not change after removing cases with silent bilateral infarcts on imaging. CONCLUSIONS: Contrary to observations from European and Asian populations, women were not at increased risk of incident AD in Rochester. Our findings, based on a medical records linkage system, corroborate findings from several other US studies that involved the direct contact of cohort members. The consistency of findings across study designs suggests that sex or sex-related exposures do not consistently play a major role in AD causation in American populations. PMID- 12374498 TI - A clinicopathological study of vascular progressive supranuclear palsy: a multi infarct disorder presenting as progressive supranuclear palsy. AB - BACKGROUND: Clinical features suggesting a diagnosis of progressive supranuclear palsy (PSP) include early falls, axial rigidity, vertical supranuclear ophthalmoplegia, and levodopa unresponsiveness. When these clinical features are present, the diagnosis is almost always PSP, yet vascular disease sometimes has a similar presentation, referred to as vascular PSP. OBJECTIVE: To evaluate clinical and pathologic features of cases of vascular PSP submitted to a PSP brain bank. DESIGN: Review of gross and microscopic neuropathological features, determination of tau haplotype, and medical record review of 4 patients with an antemortem diagnosis of PSP who did not meet the pathologic criteria for PSP and instead had vascular pathologic abnormalities. RESULTS: All patients had vertical supranuclear ophthalmoplegia, a history of falls, and a gradually progressive disease course. Falls began 1 year after symptom onset, and all patients had asymmetric findings on a neurological examination. A magnetic resonance imaging scan revealed lacunar basal ganglia infarcts in one patient and an increased T2 weighted signal in the corona radiata and centrum semiovale in another. Gross and microscopic neuropathological studies demonstrated infarcts in the cerebral cortex (n = 4), thalamus (n = 4), basal ganglia (n = 3), and cerebellum (n = 4). The brainstem was affected in one patient, but no infarcts were detected in the subthalamic nucleus or substantia nigra. Of the 4 patients, 3 carried an H2 tau haplotype, a rare occurrence in the general population. CONCLUSIONS: Asymmetric signs, falls after 1 year of symptom onset, vascular lesions on a magnetic resonance imaging scan, and an H2 tau haplotype may help differentiate vascular PSP from PSP. Thalamic and basal ganglia infarcts are common in patients with vascular PSP and, when present, may contribute to misdiagnosis. PMID- 12374499 TI - Incidence of vascular dementia in Rochester, Minn, 1985-1989. AB - OBJECTIVE: To examine the contribution of cerebrovascular disease to dementia. METHODS: We used the records-linkage system of the Rochester Epidemiology Project to ascertain incident cases of dementia in Rochester, Minn, for 1985 through 1989. We defined dementia using the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. To define dementia types, we reviewed neuroimaging reports, which were available for two thirds of dementia cases, in addition to medical histories and neurologic examination results. Vascular dementia (VaD) was defined by 1 of the following criteria: dementia onset or worsening within 3 months of a clinical stroke or bilateral gray matter infarctlike lesions shown by imaging that fulfilled specified location criteria (critical imaging lesions). RESULTS: We found 482 incident cases of dementia. Overall, 10% of patients had onset or worsening of their dementia within 3 months of a stroke. Eleven percent of the incident dementia cases had bilateral gray matter lesions on imaging that were considered critical. Eighteen percent of patients had one or the other of these features (VaD by our criteria), but only 4% of patients had both. The incidence rate of VaD increased steeply with advancing age and was similar in men and women. Our incidence rates were similar to those from a recent European meta-analysis. CONCLUSION: The presence of either a stroke temporally related to dementia onset or worsening or of critical imaging lesions was common among dementia patients, whereas the occurrence of both features together was rare. PMID- 12374500 TI - Cognitive and physiologic correlates of subclinical structural brain disease in elderly healthy control subjects. AB - CONTEXT: Healthy elderly persons commonly show 4 types of change in brain structure-cortical atrophy, central atrophy, deep white-matter hyperintensities, and periventricular hyperintensities-as forms of subclinical structural brain disease (SSBD). OBJECTIVES: To characterize the volumes of SSBD present with aging and to determine the associations of SSBD, physiology, and cognitive function. DESIGN: Cross-sectional study. SETTING: University of California, Los Angeles, Neuropsychiatric Institute. SUBJECTS: Forty-three community-dwelling healthy control subjects, aged 60 through 93 years. MAIN OUTCOME MEASURES: Volumetric magnetic resonance imaging, neuropsychological testing, and quantitative electroencephalographic coherence (functional connectivity) between brain regions. RESULTS: Regression models demonstrated significant relationships between SSBD volumes, age, cognitive performance, and connectivity. Cortical and central atrophy and periventricular hyperintensities had significant associations with age while deep white-matter hyperintensities did not. Posterior atrophy showed stronger associations with age than did anterior atrophy. Only a subset of subjects at older ages showed large SSBD volumes; older subjects primarily showed increasing variance of SSBD. Although all subjects scored within the normal range on cognitive testing, SSBD volume was inversely related to performance, most notably on the Trail-Making Test part B and the Shipley-Hartford Abstract Reasoning test. Coherence had significant associations with SSBD. Path analysis supported mediation of the effects of deep white-matter hyperintensities and periventricular hyperintensities on cognition by altered connectivity. For several measures, cognitive performance was best explained by coherence, and only secondarily by SSBD. CONCLUSIONS: Modest volumes of SSBD were associated with decrements in cognitive performance within the normal range in healthy subjects. Lower coherence was associated with greater volumes of SSBD and increasing age. Path analysis models suggest that brain functional connectivity mediates some effects of SSBD on cognition. PMID- 12374501 TI - Familial dementia with lewy bodies: a clinical and neuropathological study of 2 families. AB - BACKGROUND: Dementia with Lewy bodies (DLB) is characterized by early dementia and associated visual hallucinations, parkinsonism, and fluctuations in cognition. Few families with DLB have been described with detailed clinical, pathological, and genetic assessments. OBJECTIVE: To investigate the clinical, neuropathological, and genetic characteristics of families with 2 or more autopsy proven cases of DLB. DESIGN: Consecutive cases with the neuropathological diagnosis of DLB were reviewed as part of a case series. Families included in this study have 2 or more autopsy-proven cases of DLB available and a positive family history of dementia. We obtained clinical and neuropathological data on all first-degree relatives. Neuropathological evaluations included alpha synuclein immunostaining for Lewy body detection. We conducted apolipoprotein E genotyping and sequenced the alpha-, beta-, gamma-synuclein, and parkin genes. SETTING: Subjects were selected from the neuropathology core of the University of Washington's Alzheimer's Disease Research Center. PATIENTS: The study investigated 2 families. Clinical information was obtained from 10 individuals in family 1 and 7 individuals in family 2. Neuropathological examinations were conducted in 3 individuals in family 1 and 2 individuals in family 2. MAIN OUTCOME MEASURES: Each subject was examined for the presence of clinical symptoms and neuropathological findings consistent with DLB. RESULTS: While all affected individuals presented with dementia in both families, only individuals in family 1 developed visual hallucinations and delusions. Parkinsonism, if present, occurred later in the course of illness. Neuropathological examination revealed Lewy bodies in all patients, while 1 patient from each family also met the neuropathological criteria for Alzheimer disease. All affected individuals carried at least 1 APOE (apolipoprotein E) epsilon 4 allele, while there were no nucleotide alterations in the synuclein or parkin genes. CONCLUSIONS: Familial DLB exists, although there is substantial clinical and neuropathological heterogeneity within and between families. Additional clinicopathologic and genetic studies are necessary to further our understanding of DLB. PMID- 12374502 TI - Bilateral neuroretinitis associated with mumps. AB - BACKGROUND: Involvement of the optic nerve is a rare complication of mumps infection. OBJECTIVES: To report a case of bilateral neuroretinitis complicating a mumps infection and to review 5 previously reported cases. DESIGN: Case report and literature review. SETTING: Tertiary hospital. PATIENT: A 7-year-old girl had sudden-onset blindness due to bilateral neuroretinitis. Approximately 3 weeks prior to the initial examination, she developed a self-limited febrile illness with parotid swelling and subsequent meningoencephalitis. RESULTS: Mumps was determined to be the underlying cause of the meningoencephalitis and bilateral optic neuritis because of the exposure history in this nonvaccinated child, the typical clinical signs and symptoms, and the positive serologic test results. Recovery of visual function was gradual but nearly complete. CONCLUSIONS: Physicians should be aware that optic nerve involvement may be a manifestation of mumps infection. The delayed onset of optic neuritis, the bilateral involvement, and the near complete recovery suggest an immune-mediated pathogenesis. PMID- 12374503 TI - The "spray can" sign: validation of a clinical observation in chronic inflammatory demyelinating polyneuropathy. AB - BACKGROUND: The presentation of chronic inflammatory neuropathies is variable. The decision regarding when to intervene with treatment is ideally determined by identifying early markers of loss of function. OBJECTIVE: To test the hypothesis that an observation of functional impairment, defined by a patient with demyelinating neuropathy, can be used as a reproducible and reliable measure of improvement with intravenous immune globulin. DESIGN: A 28-year-old woman presented with a chronic inflammatory demyelinating polyneuropathy. Her first complaint was the inability to use her deodorant spray because of hand weakness. A calibrated pincer gauge fixed on top of her usual spray can was used to objectively test finger flexion. Tip grip and lateral pinch were also measured. A calibrated dynamometer was used to measure grip strength. RESULTS: Power and precision grip force were reproducible in normal control subjects by means of the spray can test. This test proved to be a reliable indicator of reduced muscle strength in the patient and improved after treatment with intravenous immune globulin. CONCLUSIONS: The spray can test objectively quantified the daily function, nominated by the patient, of operating an aerosol can. This measurement, drawn from a functional loss observed by the patient, proved to be a portable and reliable indicator of decline and recovery in chronic inflammatory demyelinating polyneuropathy. PMID- 12374504 TI - Brain calcifications in systemic sclerosis. PMID- 12374505 TI - The pioneering work of Josef Breuer on the vestibular system. PMID- 12374506 TI - Gamma knife thalamotomy for disabling tremor. PMID- 12374507 TI - Gamma knife radiosurgery as an alternative form of therapy for movement disorders. PMID- 12374509 TI - Full access for government-funded clinical trials. PMID- 12374510 TI - The new National Cholesterol Education Program guidelines: clinical challenges for more widespread therapy of lipids to treat and prevent coronary heart disease. PMID- 12374511 TI - Discussing treatment options and risks with medical patients who have psychiatric problems. AB - Discussing medical treatment options and risks becomes a more complicated task when patients have psychiatric problems. Such patients may perceive risk and judge options differently from usual, they raise special issues about informed consent and competency, and they may present special needs and stresses in the physician-patient relationship. This article addresses how to approach such treatment discussions within the framework of 3 content areas of the medical interview (medical decision making, informed consent, and the physician-patient relationship) and 2 formal techniques of the interview (exploration and assertion). Clinical research regarding how psychiatric problems may affect each of these areas of concern is reviewed. Ultimately, the goal of understanding such variations--and of possessing methods to address them in discussing treatment options and risks--is to help the patient be as free as possible from the burden of biases or distortions in making his or her decisions and to promote the best fit between the patient's wishes and the physician's medical judgment. PMID- 12374512 TI - The prevention of dementia with antihypertensive treatment: new evidence from the Systolic Hypertension in Europe (Syst-Eur) study. AB - BACKGROUND: After the double-blind, placebo-controlled Systolic Hypertension in Europe (Syst-Eur) trial ended in February 1997, randomized patients were offered active study medication for a further period of observation. OBJECTIVE: To refine the estimates of the long-term effects of antihypertensive therapy on the incidence of dementia. METHODS: Eligible patients had no dementia and were at least 60 years old. Their systolic blood pressure at entry was 160 to 219 mm Hg, with diastolic blood pressure below 95 mm Hg. Antihypertensive therapy was started immediately after randomization in the active treatment group, but only after termination of the double-blind trial in the control patients. Treatment consisted of nitrendipine (10-40 mg/d), with the possible addition of enalapril maleate (5-20 mg/d), hydrochlorothiazide (12.5-25 mg/d), or both add-on drugs. RESULTS: Median follow-up increased from 2.0 years in the double-blind trial to 3.9 years overall. The incidence of dementia doubled from 32 to 64 cases, 41 of whom had Alzheimer disease. Throughout follow-up, systolic/diastolic blood pressure was 7.0/3.2 mm Hg higher in the 1417 control patients than in the 1485 subjects randomized to active treatment. At the last examination, the blood pressure difference was still 4.2/2.9 mm Hg; 48.1%, 26.4%, and 11.4% of the control patients were taking nitrendipine, enalapril, and/or hydrochlorothiazide, whereas in the active treatment group these proportions were 70.2%, 35.4%, and 18.4%, respectively. Compared with the controls, long-term antihypertensive therapy reduced the risk of dementia by 55%, from 7.4 to 3.3 cases per 1000 patient-years (43 vs 21 cases, P<.001). After adjustment for sex, age, education, and entry blood pressure, the relative hazard rate associated with the use of nitrendipine was 0.38 (95% confidence interval, 0.23-0.64; P<.001). Treatment of 1000 patients for 5 years can prevent 20 cases of dementia (95% confidence interval, 7-33). CONCLUSION: The extended follow-up of Syst-Eur patients reinforces the evidence that blood pressure-lowering therapy initiated with a long-acting dihydropyridine protects against dementia in older patients with systolic hypertension. PMID- 12374513 TI - Medical complications and outcomes after hip fracture repair. AB - BACKGROUND: Most evidence guiding perioperative medical risk management of patients undergoing hip fracture repair focuses on cardiac and thromboembolic risk. Little is known of the relative clinical importance of other complications. OBJECTIVE: To systematically map incidence and outcomes of a broad spectrum of medical complications after hip fracture repair. METHODS: Retrospective cohort study of patients 60 years or older in 20 academic, community, and Veterans Affairs hospitals. Data on complications and mortality were abstracted from medical records by trained abstractors using standardized, pretested forms or the National Death Index. RESULTS: Of 8930 patients, 1737 (19%) had postoperative medical complications. Cardiac and pulmonary complications were most frequent (8% and 4% of patients, respectively). Similar numbers of patients had serious cardiac or pulmonary complications (2% and 3%, respectively). Other complications were gastrointestinal tract bleeding (2%), combined cardiopulmonary complications (1%), venous thromboembolism (1%), and transient ischemic attack or stroke (1%). Renal failure and septic shock were rare. After the index complication, 416 patients had 587 additional complications. Mortality was similar for serious cardiac or pulmonary complications (30 day: 22% and 17%, respectively; 1 year: 36% and 44%, respectively) and highest for patients with multiple complications (30 day: 29%-38%; 1 year: 43%-62%). Complications and death occurred significantly earlier for serious cardiac than for serious pulmonary complications (1 vs 4 days, 2 vs 8 days, P<.001); length of stay for patients surviving these complications was similar. CONCLUSIONS: Most patients had no medical complications after hip fracture repair. Serious cardiac and pulmonary complications were equally important in frequency, mortality, and survivors' length of stay. Patients with multiple complications had especially poor prognosis. PMID- 12374514 TI - Pharmaceutical costs in obese individuals: comparison with a randomly selected population sample and long-term changes after conventional and surgical treatment: the SOS intervention study. AB - BACKGROUND: Obesity is associated with increased morbidity rates and pharmaceutical costs. To what extent various medication costs are affected by intentional weight loss is unknown. METHODS: A cross-sectional comparison of the use of prescribed pharmaceuticals was conducted in 1286 obese individuals in the Swedish Obese Subjects (SOS) intervention study and 958 randomly selected reference individuals. Medication changes for 6 years after bariatric surgery were evaluated in 510 surgically and 455 conventionally treated SOS patients. RESULTS: Compared with the reference group, obese individuals were more often taking diabetes mellitus, cardiovascular disease, nonsteroidal anti-inflammatory and pain, and asthma medications (risk ratios ranging from 2.3-9.2). Average annual costs for all medications were 1400 Swedish kronor (SEK) (US $140) in obese individuals and 800 SEK (US $80) in the reference population (P<.001). Average yearly medication costs during follow-up were 1849 (US $185) in surgically treated patients (weight change -16%) and 1905 SEK (US $190) in weight stable conventionally treated patients (P =.87). The surgical group had lower costs for diabetes mellitus (difference: -94 SEK/y (-US $9]) and cardiovascular disease medications (difference: -186 SEK/y (-US $19]) but higher costs for gastrointestinal tract disorder (difference: +135 SEK/y [US $13]) and anemia and vitamin deficiency medications (difference: +50 SEK/y [US $5]). CONCLUSIONS: Use and cost of medications are markedly increased in obese vs reference populations. Surgical obesity treatment lowers diabetes mellitus and cardiovascular disease medication costs but increases other medication costs, resulting in similar total costs for surgically and conventionally treated obese individuals for 6 years. PMID- 12374515 TI - Body mass index, waist circumference, and health risk: evidence in support of current National Institutes of Health guidelines. AB - BACKGROUND: No evidence supports the waist circumference (WC) cutoff points recommended by the National Institutes of Health to identify subjects at increased health risk within the various body mass index (BMI; calculated as weight in kilograms divided by the square of height in meters) categories. OBJECTIVE: To examine whether the prevalence of hypertension, type 2 diabetes mellitus, dyslipidemia, and the metabolic syndrome is greater in individuals with high compared with normal WC values within the same BMI category. METHODS: The subjects consisted of 14 924 adult participants of the Third National Health and Nutrition Examination Survey, which is a nationally representative cross sectional survey. Subjects were grouped by BMI and WC in accordance with the National Institutes of Health cutoff points. Within the normal-weight (18.5 24.9), overweight (25.0-29.9), and class I obese (30.0-34.9) BMI categories, we computed odds ratios for hypertension, diabetes, dyslipidemia, and the metabolic syndrome and compared subjects in the high-risk (men, >102 cm; women, >88 cm) and normal-risk (men, $35 000) in persons with a low risk of clinical UGI event with conventional NSAIDs (eg, 2.5% per year). If the baseline risk of clinical UGI events is moderately high (eg, 6.5%), using a COX-2-selective NSAID becomes the most effective and least costly (dominant) treatment strategy, followed closely by cotherapy with a daily proton-pump inhibitor. Because small changes in costs or assumed efficacy of these drugs could change the conclusions, the incremental cost-effectiveness ratios between any 2 strategies were presented in a nomogram that allows the flexible use of a wide range of values for costs and rates of clinical UGI events. CONCLUSIONS: The risk of clinical UGI events in NSAID users depends on their baseline risk, the added risk associated with the individual NSAID, and the protection conferred by cotherapy. A nomogram can be used to incorporate these factors and derive estimates regarding cost-effectiveness of competing strategies aimed at reducing the risk of clinical UGI events. PMID- 12374520 TI - Glucosamine sulfate use and delay of progression of knee osteoarthritis: a 3 year, randomized, placebo-controlled, double-blind study. AB - BACKGROUND: Conventional symptomatic treatments for osteoarthritis do not favorably affect disease progression. The aim of this randomized, placebo controlled trial was to determine whether long-term (3-year) treatment with glucosamine sulfate can modify the progression of joint structure and symptom changes in knee osteoarthritis, as previously suggested. METHODS: Two hundred two patients with knee osteoarthritis (using American College of Rheumatology criteria) were randomized to receive oral glucosamine sulfate, 1500 mg once a day, or placebo. Changes in radiographic minimum joint space width were measured in the medial compartment of the tibiofemoral joint, and symptoms were assessed using the algo-functional indexes of Lequesne and WOMAC (Western Ontario and McMaster Universities). RESULTS: Osteoarthritis was of mild to moderate severity at enrollment, with average joint space widths of slightly less than 4 mm and a Lequesne index score of less than 9 points. Progressive joint space narrowing with placebo use was -0.19 mm (95% confidence interval, -0.29 to -0.09 mm) after 3 years. Conversely, there was no average change with glucosamine sulfate use (0.04 mm; 95% confidence interval, -0.06 to 0.14 mm), with a significant difference between groups (P =.001). Fewer patients treated with glucosamine sulfate experienced predefined severe narrowings (>0.5 mm): 5% vs 14% (P =.05). Symptoms improved modestly with placebo use but as much as 20% to 25% with glucosamine sulfate use, with significant final differences on the Lequesne index and the WOMAC total index and pain, function, and stiffness subscales. Safety was good and without differences between groups. CONCLUSION: Long-term treatment with glucosamine sulfate retarded the progression of knee osteoarthritis, possibly determining disease modification. PMID- 12374521 TI - Coinfection with hepatitis viruses and outcome of initial antiretroviral regimens in previously naive HIV-infected subjects. AB - BACKGROUND: The effect of chronic coinfection with hepatitis viruses on the response to therapy against human immunodeficiency virus 1 (HIV-1) remains debated. METHODS: In a prospective cohort study, the effect of hepatitis B virus (HBV) and hepatitis C virus (HCV) serostatus on the outcome of potent HIV-1 therapy was analyzed in HIV-1-infected patients previously naive to antiretroviral therapy. Changes from baseline CD4+ cell counts and HIV RNA levels over time were analyzed by linear regression models. Time to clinical progression and time to reach virologic and immunologic response were analyzed by multivariate Cox proportional hazards regression models. RESULTS: We studied 1320 patients, among whom 600 were HCV antibody-positive and 90 were HBV surface antigen-positive. During a median follow-up of 37 months (range, 1-48 months), clinical progression was observed in 99 patients (56 new acquired immunodeficiency syndrome-defining events and 43 deaths). In multivariate models, HCV-positive HBV-negative patients showed a shorter time to clinical progression (hazard ratio, 1.55; 95% confidence interval, 1.00-2.41). Patients who were HCV positive also showed mean CD4+ recoveries over time that were at least 30 cells/ micro L fewer than those of seronegative patients. Hepatitis virus serostatus did not affect the virologic response to HIV-1 therapy. CONCLUSIONS: Clinical progression of HIV-1 disease after starting potent antiretroviral therapy is accelerated by concomitant infection with HCV. Compared with patients without coinfection, coinfected patients showed impaired CD4+ cell recovery, despite similar virologic response to HIV-1 therapy. These findings may have important implications for the treatment of HCV and for the timing of initiation of HIV-1 therapy in coinfected individuals. PMID- 12374522 TI - Office practice-based confirmation of onychomycosis: a US nationwide prospective survey. AB - BACKGROUND: Onychomycosis is sufficiently prevalent to be seen and treated by primary care physicians. The diagnosis of onychomycosis is most often confirmed from nail specimens by microscopy and fungal culture done at a central laboratory; these are relatively expensive tests with a turnaround time of a month or more. This study was conducted (1) to evaluate the use of in-office dermatophyte test medium (DTM) culture, and (2) to determine the epidemiology of onychomycosis in a large, nationwide sample of patients who were not participants in a clinical trial. METHODS: A nationwide sample of primary care physicians and podiatrists enrolled 670 patients with clinical signs of toenail onychomycosis. Dermatophyte test medium cultures were performed in the office and the results were compared with fungal cultures performed by a central laboratory. RESULTS: Central laboratory fungal cultures were positive in 44% (n = 297) of patients and DTM cultures in 51% (n = 345). Dermatophytes accounted for 93% of the confirmed infections and nondermatophyte molds the rest. In the 617 patients with paired dermatophyte test medium and laboratory fungal culture results, the 2 tests were in agreement (both positive or both negative) in 68% of cases (kappa, 0.37; asymptotic SE, 0.04; 95% confidence interval, 0.299-0.441). CONCLUSIONS: A DTM culture is a relatively rapid, easy, and inexpensive method to confirm dermatophyte infections in patients with signs of onychomycosis in the primary care setting. Because the available drugs for treating onychomycosis are effective against all dermatophyte species, the confirmation of dermatophyte infection, without further identification of genus and species, is sufficient evidence to begin treatment. PMID- 12374523 TI - Thrombolytics are not contraindicated in the very old. PMID- 12374525 TI - Chlamydia pneumoniae infection: which role in atherosclerosis? PMID- 12374527 TI - Blood-borne viruses and health care workers. PMID- 12374528 TI - Hereditary angioedema and hormones. PMID- 12374530 TI - Practical utility of case-management telephone intervention in heart failure? PMID- 12374532 TI - Use of an 800-nm pulsed-diode laser in the treatment of recalcitrant dissecting cellulitis of the scalp. PMID- 12374533 TI - Dermatologic manifestations and management of vascular steal syndrome in hemodialysis patients with arteriovenous fistulas. PMID- 12374534 TI - Problem-based learning: an approach to dermatology resident education. PMID- 12374535 TI - Viral disease transmitted by laser-generated plume (aerosol). AB - OBJECTIVE: To evaluate the possibility of disease transmission through liberated plume from virally infected tissue that is exposed to the carbon dioxide laser. DESIGN: Bovine papillomavirus-induced cutaneous fibropapillomas were exposed to the carbon dioxide laser. Laser settings were within the range of clinically used settings. The laser plume (aerosol) was suctioned and collected and then reinoculated onto the skin of calves. SETTING: University laboratory research center. MAIN OUTCOME MEASURES: Laser plume viral content and postinoculation tumor growth were analyzed and documented. RESULTS: Collected laser plume contained papillomavirus DNA in all tested laser settings. The viral DNA was most likely encapsulated. Tumors developed at laser plume-inoculated sites for all laser parameter settings. Histological and biochemical analyses revealed that these tumors were infected with the same virus type as present in the laser plume. CONCLUSIONS: Laser plume has been shown, for the first time to our knowledge, to actually transmit disease. Strict care must be maintained by the laser practitioner to minimize potential health risks, especially when treating viral-induced lesions or patients with viral disease. PMID- 12374536 TI - Awareness of the risks of tanning lamps does not influence behavior among college students. AB - HYPOTHESIS: Awareness of the risks of artificial tanning influences tanning behavior among college students. OBJECTIVE: To correlate the prevalence of tanning lamp use, the perceived benefits and risks associated with UV exposure, and knowledge about skin cancer among university students. DESIGN: A survey was designed and administered to college students seeking "walk-in" care at a university student health center from September 7, 1999, through September 30, 1999. SETTING: A large midwestern public university student health center. PARTICIPANTS: Undergraduate and graduate students attending the student health center for any medical condition. INTERVENTION: None. MAIN OUTCOME MEASURE: Completion of the survey. RESULTS: Of the surveyed students, 47% had used a tanning lamp during the preceding 12 months. Female students were more common users than male students. Of the students surveyed, 39% reported never having used tanning lamps. More than 90% of users of tanning lamps were aware that premature aging and skin cancer were possible complications of tanning lamp use. CONCLUSIONS: Despite adequate knowledge of the adverse effects of UV exposure, university students freely and frequently use tanning lamps, primarily for desired cosmetic appearance. To alter this risky behavior will require a fundamental change in the societal belief that tans are attractive and healthy. PMID- 12374537 TI - Application patterns among participants randomized to daily sunscreen use in a skin cancer prevention trial. AB - BACKGROUND: Despite many investigations of sunscreen use, there have been few among adults in the community at large. Better understanding of sunscreen application patterns will lead to more strategic skin cancer prevention strategies among sun-exposed populations. OBJECTIVE: To explore patterns of sunscreen use, particularly the quantity of sunscreen used and the application frequency, among participants in a community-based sunscreen intervention. DESIGN: Follow-up of patterns of sunscreen use over 4.5 years. SETTING: Nambour, a subtropical town in Queensland, Australia. PARTICIPANTS: People drawn randomly from the electoral register who were later randomized as part of a skin cancer prevention trial. INTERVENTIONS: Daily application of a standard sun protection factor 15+ broad-spectrum retail sunscreen to the head and neck, arms, and hands. OUTCOME MEASURES: Frequency of application of sunscreen, weight of sunscreen applied, and quantity applied per unit area of skin. RESULTS: Fifty-six percent of participants reported applying sunscreen on at least 5 days per week, with 27% using sunscreen infrequently on 2 or fewer days per week. The median daily amount of sunscreen applied averaged over the duration of the trial was 1.5 g/d (range, 0-7.4 g/d). The median quantity of sunscreen applied was 0.79 mg/cm(2), which was less than half the amount needed to achieve the labeled sun protection factor. CONCLUSIONS: It is possible to implement the daily application of sunscreen in sun-exposed populations, although protection would be increased if the quantity of sunscreen applied were greater. PMID- 12374538 TI - Dermoscopic examination of nail pigmentation. AB - BACKGROUND: Diagnosis of longitudinal melanonychia is usually difficult, and neither a single clinical criterion nor a combination of symptoms currently can be used to clearly distinguish malignant from benign bandlike pigmented nail lesions. Biopsy is painful and often leaves definitive dystrophic scars. OBJECTIVES: To describe and evaluate dermoscopic patterns associated with longitudinal nail pigmentation. PATIENTS AND METHODS: A total of 148 unselected consecutive cases of longitudinal melanonychia were included over a period of 4 years (20 melanoma, 37 nevi, 16 drug-induced nail pigmentation, 45 nail apparatus lentigo of various types, 8 ethnic-type nail pigmentation, and 22 subungual hemorrhages). All patients were recruited from the dermatology unit outpatient clinic of the Hotel Dieu de Lyon. All cases were photographed in vivo under oil immersion (dermoscopy). Patterns were recorded prior to final pathologic diagnosis. An independent biostatistics unit performed statistical evaluation using 7 semiologic patterns. RESULTS: Melanoma cases were significantly associated with a brown coloration of the background and the presence of irregular longitudinal lines (P =.001). Blood spots were mostly observed in subungual hemorrhages (P =.001); however, their presence could not rule out melanoma. Micro-Hutchinson sign was observed only in melanoma, but its rare occurrence did not allow any statistical evaluation of its specificity. Nail apparatus nevi were significantly associated with a brown coloration of the background and the presence of regular lines (P =.001). Nail apparatus lentigo, ethnic-type pigmentation, and drug-induced pigmentation were significantly associated with homogeneous longitudinal thin gray lines and gray coloration of the background (P =.001). Microscopic longitudinal grooves were unspecific, occurred in several conditions, and were associated with any type of ungual discoloration. CONCLUSIONS: We believe that dermoscopic examination of the nail plate in cases of longitudinal melanonychia provides useful information that could help clinicians to more accurately decide if a nail apparatus biopsy should be performed; however, histopathologic diagnosis remains the gold standard in doubtful cases. PMID- 12374539 TI - Treatment of chronic erosive oral lichen planus with low concentrations of topical tacrolimus: an open prospective study. AB - BACKGROUND: Chronic erosive oral lichen planus (EOLP) is a severe form of lichen of the buccal mucosa that is often resistant to systemic or topical therapies. OBJECTIVE: To evaluate the efficacy and safety of topical tacrolimus, 0.1 mg per 100 mL of water, in treating EOLP. DESIGN: Open-label, prospective, noncomparative study, with 6 months of treatment and 6 months of follow-up. SETTING: Dermatology department at a university hospital in Nice, France. PATIENTS: Ten patients with histologically proved EOLP that was refractory to treatment. Two patients were withdrawn because of noncompliance; findings in 8 were available for evaluation. INTERVENTIONS: Mouthwashes with tacrolimus, 0.1 mg per 100 mL of distilled water, 4 times daily for 6 months. MAIN OUTCOME MEASURES: Efficacy was assessed using a calculated score that combined the intensity of spontaneous and meal-triggered pain and the surface area of erosions. Safety assessment included the monitoring of adverse effects, clinical laboratory values, and blood concentrations of tacrolimus. RESULTS: Among the 8 patients evaluated, 1 had no improvement and 7 were improved. The mean score decreased from 7.00 at baseline to 5.43 (a 22.43% decrease) at 1 month, 4.14 (a 40.86% decrease) at 2 months, 3.00 (a 57.14% decrease) at 3 months, 2.43 (a 65.29% decrease) at 4 months, 2.57 (a 63.29% decrease) at 5 months, and 3.43 (a 51.00% decrease) at 6 months. A decrease of symptoms was reported by the 7 responding patients as soon as the first month of treatment. No severe adverse effects were observed. All patients had whole-blood concentrations of tacrolimus below the detection limit of the assay (1.5 ng/mL) at all intervals. At 9 months, 6 patients had had a relapse within a mean of 38.6 days. At 12 months, all patients had had a relapse and required treatment with topical corticosteroids or systemic hydroxychloroquine sulfate. CONCLUSION: Results of our study suggest a rapid and important palliating effect of low concentration of topical tacrolimus in distilled water in patients with EOLP. PMID- 12374540 TI - Clinical and immunologic assessment of patients with psoriasis in a randomized, double-blind, placebo-controlled trial using recombinant human interleukin 10. AB - BACKGROUND: In several open-label studies, recombinant human interleukin 10 (rhIL 10), a type 2 anti-inflammatory cytokine, has been reported to improve psoriasis, a disease characterized by type 1 cytokine inflammation. OBJECTIVE: To evaluate the safety, efficacy, and immunologic parameters in individuals with psoriasis treated with rhIL-10. DESIGN AND INTERVENTION: Patients received rhIL-10 (20 micro g/kg) or placebo subcutaneously 3 times weekly for 12 weeks in a randomized, double-blind manner. SETTING AND PATIENTS: National Institutes of Health Clinical Center in Bethesda. Twenty-eight patients with moderate-to-severe psoriasis as defined by a Psoriasis Area Severity Index (PASI) score of 10 or higher. MAIN OUTCOME MEASURE: The primary clinical end point was the mean percentage change in the PASI score comparing baseline and week 12 scores. Intracellular cytokine production by peripheral blood mononuclear cells (PBMCs) was measured by flow cytometry. RESULTS: There was no significant difference in the mean percentage change in the PASI score from baseline to week 12 between the rhIL-10-treated group and control patients (17% vs 13% improvement, respectively; P =.69), although a modest trend toward improvement in patients receiving rhIL-10 was documented at both the 6- and 8-week points. Interestingly, proinflammatory and type 1 cytokine production by PBMCs progressively declined in the rhIL-10 treated patients during the entire 12-week study period. CONCLUSIONS: Treatment with rhIL-10 resulted in only temporary clinical improvement in psoriasis, despite sustained systemic decreases in proinflammatory and type 1 cytokine production. These data suggest that immunotherapy that decreases the ratio of systemic type 1 and type 2 cytokine production does not necessarily lead to improvement of type 1 cytokine-mediated disease. PMID- 12374541 TI - Circulating CD4+CD7- lymphocyte burden and rapidity of response: predictors of outcome in the treatment of Sezary syndrome and erythrodermic mycosis fungoides with extracorporeal photopheresis. AB - BACKGROUND: Extracorporeal photopheresis (ECP) is an effective treatment for cutaneous T-cell lymphoma. Controversy has arisen regarding its ability to improve survival rates in Sezary syndrome (SS). We describe our experience with ECP in the treatment of SS and erythrodermic mycosis fungoides, with particular emphasis on early predictors of long-term outcome. OBSERVATIONS: We included 17 patients (15 with SS and 2 with erythrodermic mycosis fungoides) who received ECP as initial treatment. Four of these patients were moribund on presentation (Eastern Cooperative Oncology Group Performance Status score, 4) and underwent only 1 to 2 cycles of ECP. The median survival was 56 months for the 11 patients with SS and an Eastern Cooperative Oncology Group Performance Status score of less than 4. If all 15 patients with SS are considered, median survival was 34 months. Response after 5 months of ECP correlated with long-term survival. A low number (<6.0 x10(3)/ micro L) of circulating CD4(+)CD7(-) lymphocytes correlated with response after 5 months of ECP. CONCLUSIONS: Extracorporeal photopheresis is a safe, effective, and well-tolerated treatment for erythrodermic mycosis fungoides and SS. Low numbers of CD4(+)CD7(-) cells in the circulation and a positive response after 5 months of therapy predicted long-term survival. Moribund patients are much less likely to benefit from ECP. PMID- 12374542 TI - Extrafacial and generalized granulomatous periorificial dermatitis. AB - BACKGROUND: Granulomatous periorificial dermatitis is a well-recognized entity presenting most commonly in prepubertal children as yellow-brown papules limited to the perioral, perinasal, and periocular regions. The condition is self limiting and is not associated with systemic involvement. OBSERVATIONS: We reviewed the medical charts of 5 healthy children presenting with extrafacial granulomatous papules in addition to the typical periorificial papules. These extrafacial lesions were clinically and histologically identical to the facial lesions, were self-limiting, and were not associated with systemic involvement. Resolution seemed to be hastened with the use of systemic antibiotic therapy in 4 of the 5 patients. CONCLUSIONS: Extrafacial lesions can occur in granulomatous periorificial dermatitis and do not appear to adversely affect the duration, response to therapy, or risk of extracutaneous manifestations. Overly aggressive evaluation and inappropriate systemic therapy should be avoided. PMID- 12374543 TI - Long-term remission after allogeneic hematopoietic stem cell transplantation for refractory cutaneous T-cell lymphoma. AB - BACKGROUND: Allogeneic hematopoietic stem cell transplantation has proved to be an effective therapeutic option in various hematologic neoplastic disorders. Because patients with advanced cutaneous T-cell lymphoma have a poor prognosis, with minimal possibilities of sustained remission, we studied the therapeutic potential of hematopoietic stem cell transplantation. OBSERVATIONS: Three young patients with refractory tumor stage mycosis fungoides underwent allogeneic HLA matched sibling transplantation with combined marrow and CD34-enriched peripheral blood stem cell transplantation after cytoreductive chemotherapy and total-body irradiation. Complete and sustained clinical and histologic remission was achieved in 2 patients, and both remain disease free 4(1/2) years and 15 months later. One patient was in complete remission for 9 months, followed by limited cutaneous recurrence. Mild graft-vs-host disease and graft-vs-tumor effect have contained the recurring disease as a low-grade process. CONCLUSIONS: Allogeneic hematopoietic stem cell transplantation has the potential for sustained remission and the possibility of cure for young patients with advanced and recalcitrant cutaneous T-cell lymphoma. Even in the absence of complete remission, an allogeneic graft-vs-tumor effect may provide an immune mechanism to control the malignant T-cell process and alter the natural history of disease. PMID- 12374544 TI - Nonablative dermal remodeling: does it really work? PMID- 12374545 TI - Dermoscopy allows better management of nail pigmentation. PMID- 12374546 TI - Indurated plaques on the arms. PMID- 12374547 TI - A chronic draining plaque on the foot. PMID- 12374548 TI - Unilateral eruption in a child. PMID- 12374549 TI - Pustular eruption. PMID- 12374550 TI - Dermoscopy in melanoma screening. PMID- 12374551 TI - Can nondermatologists really recognize potentially dangerous skin lesions as well as dermatologists? PMID- 12374552 TI - Candy's dandy but cantharidin's quicker. PMID- 12374553 TI - Follow-up of melanocytic skin lesions with digital dermoscopy: risks and benefits. PMID- 12374554 TI - Usefulness of dermoscopy in treating pigmented tumors. PMID- 12374555 TI - Telepathology as a substitute for traditional glass slides in a pathology consultation practice. PMID- 12374556 TI - Nonablative wrinkle reduction: treatment results with a 585-nm laser. PMID- 12374557 TI - Histopathologic misdiagnoses and their clinical consequences. PMID- 12374558 TI - A case of air bag dermatitis. PMID- 12374559 TI - Juvenile horseplay purpura. PMID- 12374560 TI - IgM-mediated epidermolysis bullosa acquisita. PMID- 12374566 TI - Myeloid zinc finger (MZF)-like, Kruppel-like and Ets families of transcription factors determine the cell-specific expression of mouse extracellular superoxide dismutase. AB - Extracellular superoxide dismutase (EC-SOD or SOD3) is an important protective enzyme against the toxicity of superoxide radicals that are produced under both physiological and pathophysiological conditions. We have isolated and characterized over 11 kb of the mouse EC-SOD gene and its 5'- and 3'-flanking regions. The gene consists of two exons, with the entire coding region located within exon 2. In order to study the mechanism of cell-specific gene regulation for mouse EC-SOD, we characterized 2500 bp of its 5'-flanking region using cultured cells derived from mouse lung fibroblasts (MLg), kidney medulla (mIMCD3) and hepatocytes (Hepa 1-6). Real-time PCR showed that basal expression of EC-SOD was considerably higher in MLg cells compared with the other cell types. Reporter gene assays revealed that the proximal promoter region was sufficient to support this high expression in MLg cells. Although no obvious TATA box was identified, our results show that a highly purine-rich region from -208 to +104 contains active binding sites for both the Kruppel-like and Ets families of transcription factors. Using electrophoretic mobility shift, DNase footprinting and reporter gene assays, we identified myeloid zinc finger 1 and gut-enriched Kruppel-like factor-like nuclear transcription factors as repressors of EC-SOD expression, whereas nuclear transcription factors from the Ets family, such as Elf-1 and GA binding protein alpha and beta, were potent activators of EC-SOD transcription. We propose a model that highlights competition between Ets activators and Kruppel like repressors within the proximal promoter region that determines the level of EC-SOD expression in a particular cell type. PMID- 12374567 TI - Endogenous phospholipase D2 localizes to the plasma membrane of RBL-2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid. AB - We have examined the specificity of oleate as an activator of phospholipase D2 (PLD2) and whether it can be used to study PLD2 localization and its involvement in cell function. Oleate stimulates PLD activity in intact RBL-2H3 mast cells. Comparing PLD1- with PLD2-overexpressing cells, oleate enhanced PLD activity only in PLD2-overexpressing cells. Membranes were also sensitive to oleate and when membranes prepared from PLD1- and PLD2-overexpressing cells were examined, oleate further increased PLD activity only in membranes from PLD2-overexpressing cells. Overexpressed green fluorescent protein (GFP)-PLD2 fusion protein was localized at the plasma membrane and GFP-PLD1 was found in an intracellular vesicular compartment. Oleate was used to examine whether overexpressed PLD2 co-localized with endogenous PLD2. RBL-2H3 mast cell homogenates were fractionated on a linear sucrose gradient and analysed for both oleate-stimulated activity and ADP ribosylation factor 1-stimulated PLD1 activity. The oleate-stimulated activity co localized with markers of the plasma membrane including the beta-subunit of the FcepsilonRI and linker for activation of T cells. Fractionation of homogenates from PLD2-overexpressing cells demonstrated that the overexpressed PLD2 fractionated in an identical location to the endogenous oleate-stimulated activity and this activity was greatly enhanced in comparison with control membranes. Examination of membranes prepared from COS-7, Jurkat and HL60 cells indicated a relationship between oleate-stimulated PLD2 activity and PLD2 immunoreactivity. We examined whether oleate could be used to activate secretion and membrane ruffling in adherent RBL-2H3 mast cells. Oleate did not stimulate secretion but did stimulate membrane ruffling, which was short-lived. We conclude that oleic acid is a selective activator of PLD2 and can be used for localization studies, but its use as an activator of PLD2 in intact cells to study function is limited due to toxicity. PMID- 12374568 TI - Protein kinase B/Akt is essential for the insulin- but not progesterone stimulated resumption of meiosis in Xenopus oocytes. AB - In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling. PMID- 12374569 TI - Effects of osmolarity, ions and compatible osmolytes on cell-free protein synthesis. AB - To mimic what might happen in cells exposed to hypertonicity, the effects of increased osmolarity and ionic strength on cell-free protein synthesis have been examined. Translation of globin mRNA by rabbit reticulocyte lysate decreased by 30-60% when osmolality was increased from 0.35 to 0.53 osmol/kg of water by the addition of NaCl, KCl, CH(3)CO(2)Na or CH(3)CO(2)K. In contrast, equivalent additions of the compatible osmolytes betaine or myo -inositol caused a 40-50% increase in the rate of translation, whereas amino acids (50-135 mM) that are transported via system A had no significant effect. Addition of 75 mM KCl caused a dramatic fall in the amount of the 43 S pre-initiation complex, whereas it was totally preserved when osmolarity was similarly increased by the addition of 150 mM betaine. The formation of a non-enzymic initiation complex between rabbit [(3)H]Phe-tRNA, poly(U) and the 80 S ribosomes was unaffected by the addition of 75 mM NaCl or KCl, but translation of the complex decreased by 70%. Density gradient centrifugation of reticulocyte extracts translating endogenous mRNA revealed that addition of 150 mM betaine had no effect, whereas addition of 75 mM KCl caused a marked decrease in the polysome peak, concomitant with an increase in the proportion of 80 S ribosomes and ribosomal subunits, even when elongation was inhibited with fragment A of diphtheria toxin. These results are consistent with the notion that both initiation and elongation are inhibited by unusually high concentrations of inorganic ions, but not by the compatible osmolytes betaine or myo -inositol. PMID- 12374572 TI - Modeling molecular networks: a systems biology approach to gene function. AB - A report on the European Science Foundation Workshop on Modeling of Molecular Networks, Granada, Spain, 11-14 June 2002. PMID- 12374570 TI - Insulin stimulation of pyruvate dehydrogenase in adipocytes involves two distinct signalling pathways. AB - In isolated rat adipocytes, the insulin stimulation of pyruvate dehydrogenase can be partially inhibited by inhibitors of PI3K (phosphoinositide 3-kinase) and MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase). In combination, U0126 and wortmannin completely block the insulin stimulation of pyruvate dehydrogenase. It is concluded that the effect of insulin on pyruvate dehydrogenase in rat adipocytes involves two distinct signalling pathways: one is sensitive to wortmannin and the other to U0126. The synthetic phosphoinositolglycan PIG41 can activate pyruvate dehydrogenase but the activation is only approx. 30% of the maximal effect of insulin. This modest activation can be completely blocked by wortmannin alone, suggesting that PIG41 acts through only one of the pathways leading to the activation of pyruvate dehydrogenase. PMID- 12374573 TI - Developmental biologists cast a net over sequenced genomes. AB - A report on the annual meeting of the Society of Developmental Biology, Madison, Wisconsin, USA, 21-24 July 2002. PMID- 12374571 TI - Regulatable liver expression of the rabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in mice lacking endogenous APOBEC-1 leads to aberrant hyperediting. AB - Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which results in translation of the apoB-48 protein instead of the genomically encoded apoB-100. ApoB-48-containing lipoproteins are cleared more rapidly from plasma and are less atherogenic than apoB-100-containing low-density lipoproteins (LDLs). In humans, the intestine predominantly produces apoB-48 whereas the liver secretes apoB-100 only. To evaluate a potential therapeutic use for liver-induced apoB mRNA editing in humans, we investigated the efficiency and safety of transgenic expression of apoB mRNA-editing enzyme catalytic polypeptide 1 (APOBEC 1) in the absence of endogenous editing in the mouse model. Here we show that regulatable tetO-mediated APOBEC-1 expression in the livers of gene-targeted mice lacking endogenous APOBEC-1 results in 30% apoB mRNA editing. In a time-course experiment, the expression of tetO-APOBEC-1 mRNA was suppressed within 2 days after mice were fed doxycycline and apoB mRNA editing and apoB-48 formation were suppressed within 4 days. However, tetO-APOBEC-1 expression resulted in regulatable aberrant hyperediting of several cytidines downstream of C(6666) in apoB mRNA and in novel APOBEC-1 target 1 (NAT1) mRNA. Several of the cytidines in apoB mRNA were hyperedited to a level similar to that of C(6666), although editing at C(6666) was lower than that in wild-type mice. These results demonstrate that even moderate APOBEC-1 expression can lead to hyperediting, limiting the single-gene approach for gene therapy with APOBEC-1. PMID- 12374574 TI - Viruses in and out. AB - A report on the twelfth Congress of Virology, part of 'The world of microbes', the joint meeting of the three divisions of the International Union of Microbiological Societies, Paris, France, 27 July to 1 August 2002 PMID- 12374575 TI - Genomics, proteomics and bioinformatics: all in the same boat. AB - A report on the Genomics, Proteomics and Bioinformatics for Medicine (GPBM) 2002 meeting, St. Petersburg to Moscow, Russia, 22-30 June 2002. PMID- 12374576 TI - Pantothenate kinase-associated neurodegeneration (Hallervorden-Spatz syndrome). AB - The arguments over the nomenclature of the syndrome are reviewed. Ethical considerations favour replacing the present eponyms with the title of panthothenate kinase-associated neurodegeneration (PKAN), now that more is known about the cause of the condition. The symptoms and signs of the syndrome are described, and these can present from infancy to adult life. Dystonia, involuntary movements and spasticity are prominent causes of disability. If the onset is delayed the presentation can be unusual. Tests that can help in diagnosis are reviewed, especially the "eye of the tiger" revealed by magnetic resonance imaging scanning. Death usually occurs about 10 years after the onset, but the course may be more prolonged. The findings on autopsy are also considered, with the typical findings of iron pigment deposits and axonal spheroids. Then the causes are discussed. Once the responsible gene PANK2 had been discovered on chromosome 20 it was found that this encoded for pantothenate kinase which is essential for the synthesis of coenzyme A from pantothenate; and this is integral to fatty acid synthesis and energy metabolism. Also this can lead to a concentration of cysteine in the basal ganglia, and then to an accumulation of iron in these areas. The cysteine-iron complex will result in tissue damage by promoting oxidative stress, as in some other neurodegenerative diseases. The syndrome of PKAN can therefore be identified as a disorder of pantothanate, vitamin B5, metabolism. Infantile neuroaxonal dystrophy is briefly described as there have been suggestions that it is a variety of PKAN, but the evidence is in favour of the two diseases being separate entities. There may as yet be no specific treatment for this syndrome, but much can be done to help these children. Drugs may be needed to control epilepsy, and when dystonia is severe it may be possible to alleviate this by medical or surgical means. Also there will be other problems needing expert management, such as the provision of alternative means of communication if dysarthria is marked. The hope for the future is that now the cause has been found it will be possible to use methods such as antioxidative therapy and gene induction procedures. PMID- 12374577 TI - Auditory event-related brain potentials in parents of children with specific language impairment. AB - Auditory event-related brain potentials evoked in response to tone stimuli and to speech stimuli were recorded in a group of parents of children with specific language-impairment and a group of parents of normally speaking children. The parents of the language-impaired children showed longer P3 latencies than the parental control group in the speech task requiring a phonological discrimination, but did not differ from the controls in the linguistically non demanding tone discrimination task. The longer P3 latency was associated with a positive parental history of language delay. There were no group differences concerning the N1 component in any of the tasks. The findings indicate that parents of children with specific language impairment show signs of deficient late-stage perceptual higher order linguistic processing, whereas the earlier central sensory detection stage of the phonological information is no different from the controls. Our observations are particularly interesting with regard to a study of the children of these two parental groups, where the language-impaired children showed longer P3 latencies than controls in both a tone task and a speech task, whereas there were no differences between the children concerning the N1 component. We propose that deficient late-stage auditory higher order perceptual processing as indexed by the longer P3 latency to speech stimuli observed both in children with specific language-impairment and in their parents may represent a constitutional trait, contributing to the language acquisition difficulties in these children. PMID- 12374578 TI - Occipital epilepsies in children. AB - Occipital lobe seizures, as defined by subjective symptoms and objective signs, can be recognized by clinical seizure characteristics in most cases. Visual symptoms such as hallucinations and amaurosis are the most common occipital lobe seizure symptoms. The patients must be classified in order to be able to define the prognosis. In this study, we classified patients with occipital epilepsy as childhood epilepsy with occipital paroxysms (19 patients), idiopathic photosensitive occipital epilepsy (10 patients) and symptomatic occipital epilepsy (25 patients). They were evaluated according to clinical, electrographic and neuroimaging characteristics. PMID- 12374579 TI - Benign familial infantile convulsions: a clinical study of seven Dutch families. AB - Benign familial infantile convulsions (BFIC) is a recently identified partial epilepsy syndrome with onset between 3 and 12 months of age. We describe the clinical characteristics and outcome of 43 patients with BFIC from six Dutch families and one Dutch-Canadian family and the encountered difficulties in classifying the syndrome. Four families had a pure BFIC phenotype; in two families BFIC was accompanied by paroxysmal kinesigenic dyskinesias; in one family BFIC was associated with later onset focal epilepsy in older generations. Onset of seizures was between 6 weeks and 10 months, and seizures remitted before the age of 3 years in all patients with BFIC. In all, 29 (67%) of the 43 patients had been treated with anti-epileptic drugs for a certain period of time. BFIC is often not recognized as (hereditary) epilepsy by the treating physician. Seizures often remit shortly after the start of anti-epileptic drugs but, because of the benign course of the syndrome and the spontaneous remission of seizures, patients with low seizure frequency do not necessarily have to be treated. If prescribed, anti-epileptic drugs can probably be withdrawn after 1 or 2 years of seizure freedom. PMID- 12374580 TI - Acute lymphoblastic leukaemia presenting with low back pain. AB - We report a 13-year-old boy with a 3-month history of low back pain following a mild trauma. Extensive osteoporosis and vertebral collapses were seen on conventional X-ray and computed tomography scan. Laboratory findings were non specific. Bone marrow infiltration was observed on magnetic resonance imaging, suggesting a myeloproliferative disorder. Although not diagnostic, marrow infiltration in a child with osteoporosis should raise the suspicion of leukaemia. PMID- 12374581 TI - Magnetic resonance demonstration in the newborn of generalized cerebral venous dilation with spontaneous resolution. AB - The authors present serial magnetic resonance (MR) images of an infant with cardiac failure and generalized cerebral venous dilation, which was initially misdiagnosed in the first week of life on cranial ultrasound as a vein of Galen malformation. At 3 months of age, repeat MR imaging demonstrated complete resolution of this marked cerebral venous distension with no evidence for cerebral injury. This case illustrates the value of MR in the identification of this disorder and its distinction from more serious conditions, such as vein of Galen malformation and venous sinus thrombosis. Complete resolution of the venous dilation and the lack of definable parenchymal injury suggest a good prognosis for this disorder. PMID- 12374585 TI - Collagen type VI and related disorders: Bethlem myopathy and Ullrich scleroatonic muscular dystrophy. PMID- 12374586 TI - Hyperandrogenism in girls with juvenile neuronal ceroid lipofuscinosis. AB - Acne and hirsutism are common findings in girls with juvenile neuronal ceroid lipofuscinosis (JNCL). A study on their hormonal status was conducted to investigate the mechanisms underlying these symptoms. Sixteen girls with JNCL entered the study. Ten of the girls had periodic menstruation, while three were given medroxyprogesterone acetate therapy to prevent menstrual bleeding, and three had earlier undergone an ovariectomy. Ten age- and weight-matched healthy girls served as controls. Age at menarche, menstrual cycle length, acne, and hirsutism were assessed. Extensive hormonal laboratory tests were made in the early follicular phase. In addition, 1.5 Tesla magnetic resonance imaging of the lower abdomen was performed to search for structural abnormalities of the ovaries. The mean age at menarche in these JNCL patients was 11.6 years. Of the patients with periodic menstruation, four of ten had irregular (prolonged) cycles, but, in patients with regular cycles, the mean ovarian cycle was short (26 days). Hyperandrogenism, characterized by acne, hirsutism and/or hyperandrogenaemia, was found significantly more often in the patients than in the controls (p<0.01). No significant differences were found in the laboratory parameters. Polycystic ovaries were found in two of seven of the patients who menstruated, but in none of the healthy controls. Hyperandrogenism is common in patients with JNCL. In addition, there is an early menarche and signs of anovulation. The factors underlying these hormonal changes seem complex, possibly including a neurodegenerative process, the obesity associated with JNCL, and the drugs used for symptomatic treatment of the patients. PMID- 12374587 TI - Visual pathway glioma in children treated with chemotherapy. AB - Visual pathway gliomas occur predominantly in young children. Chemotherapy has been increasingly used as a first-line treatment because of the complications caused by radiotherapy and surgery. Nine children between 6 months and 9 years (median age of 4.8 years) were treated with vincristine and carboplatin according to the SIOP (Societe Internationale d'Oncologie Pediatrique) low-grade glioma 1996 protocol. Five patients had evidence of neurofibromatosis type 1. Magnetic resonance imaging (MRI) and ophthalmological assessment were performed during and after treatment. There was a positive response in all children (100%). Three patients developed progressive disease between 8 and 12 months after ceasing treatment. One of them, being only 2.5 years old, was again treated by chemotherapy with partial response on MRI. Patients with neurofibromatosis type 1 never developed progressive disease. Our data suggest that chemotherapy is an effective treatment option in young children with visual pathway gliomas. MRI is an important means of monitoring the tumour response provided that a rigid imaging protocol is used to detect the early tumoral changes. PMID- 12374588 TI - Systematic review of the effect of therapeutic dietary supplements and drugs on cognitive function in subjects with Down syndrome. AB - The objective was to evaluate the effects of therapeutic dietary supplements and drugs on cognitive function in subjects with Down syndrome. The study design was a systematic review of randomized controlled trials of dietary supplements and/or drugs reporting any assessment of cognitive function in subjects with Down syndrome. Eleven trials were identified with 373 randomized participants. None of the trials reported cognitive enhancing effect in subjects with Down syndrome. Meta-analysis was not conducted due to the heterogeneous nature of the population, interventions and outcome measures used. Overall, the quality of the trials was poor with few subjects and generally inadequate allocation concealment of the treatments given. This comprehensive systematic review provides no positive evidence that any combination of drugs, vitamins and minerals enhance either cognitive function or psychomotor development in people with Down syndrome. However, because of the small number of subjects involved and the overall unsatisfactory quality of the trials, an effect cannot be excluded at this point. At present there is no justification for the use of such regimes outside the context of large well designed trials. Parents of children with Down syndrome should be actively discouraged from giving these 'miracle drugs' to their children. PMID- 12374589 TI - Frequent seizures with elevated interleukin-6 at the eruptive stage of exanthema subitum. AB - A 15-month-old girl developed frequent seizures at the eruptive stage of exanthema subitum. The eruption persisted for 2 weeks. Serum immunoglobulin G antibody to human herpes virus type 6 (HHV-6) increased markedly. Interleukin-6 was elevated whereas HHV-6 deoxyribonucleic acid was not detected in cerebrospinal fluid. These findings suggest that immune-mediated reactions after HHV-6 infection rather than direct action of active HHV-6 are responsible for frequent seizures in this case. PMID- 12374590 TI - Isolated hypoglossal nerve palsy in a 14-year-old girl. AB - Isolated hypoglossal nerve palsy is rare, but occasionally it appears as the initial or solitary sign of an intracranial or extracranial space-occupying lesion or a vascular abnormality of the internal carotid artery. We present a 14 year-old girl who, following an upper respiratory tract infection, presented with isolated right hypoglossal nerve palsy. Anti-streptolysin O titre was increased to >1280 suggesting a preceding streptococcal infection. Magnetic resonance imaging of the brain did not show any intracranial or extracranial abnormality. She had a partial improvement at 3 months. This case emphasizes the value of recognizing the existence of benign self-limiting, post-infectious causes of isolated hypoglossal nerve palsies in children. PMID- 12374591 TI - Bilateral symmetrical frontoparietal polymicrogyria. AB - A patient with bilateral symmetrical frontoparietal polymicrogyria is reported. Severe developmental delay, mental retardation, spastic tetraplegia, and seizures were the main clinical features. Magnetic resonance imaging revealed bilateral thick cortex with irregular gyri and festoon-like grey-white matter junction in the frontoparietal areas. Bilateral frontoparietal polymicrogyria might represent either a severe form of a spectrum of malformations involving the frontoparietal area or a further variety of the congenital bilateral symmetrical polymicrogyria syndromes in addition to bilateral frontal polymicrogyria, bilateral perisylvian syndrome, and bilateral parasagittal parieto-occipital polymicrogyria. PMID- 12374613 TI - Protein oxidation during aging of the nematode Caenorhabditis elegans. AB - The nematode Caenorhabditis elegans has proven a robust genetic model for studies of aging, including the roles of oxidative stress and protein damage. In this review, we focus on the genetics of select long-lived (e.g., age-1, daf-2, daf 16) and short-lived (e.g., mev-1) mutants that have proven useful in revealing the relationships that exist among oxidative stress, life span, and protein oxidation. The former are known to control an insulin/IGF-1-like pathway in C. elegans, while the latter affect mitochondrial function. PMID- 12374614 TI - Hypercholesterolemia promotes inflammation and microvascular dysfunction: role of nitric oxide and superoxide. AB - Relatively brief periods (days) of hypercholesterolemia can exert profound effects on endothelium-dependent functions of the microcirculation, including dilation of arterioles, fluid filtration across capillaries, and regulation of leukocyte recruitment in postcapillary venules. Hypercholesterolemia appears to convert the normal anti-inflammatory phenotype of the microcirculation to a proinflammatory phenotype. This phenotypic change appears to result from a decline in nitric oxide (NO) bioavailability that results from a reduction in NO biosynthesis, inactivation of NO by superoxide (O(2)(*)(-)), or both. A consequence of the hypercholesterolemia-induced microvascular responses is an enhanced vulnerability of the microcirculation to the deleterious effects of ischemia and other inflammatory conditions. Hence, therapeutic strategies that are directed towards preventing the early microcirculatory dysfunction and inflammation caused by hypercholesterolemia may prove effective in reducing the high mortality associated with ischemic tissue diseases. Agents that act to maintain the normal balance between NO and reactive oxygen species (ROS) in vascular endothelial cells may prove particularly useful in this regard. PMID- 12374615 TI - The labile iron pool: characterization, measurement, and participation in cellular processes(1). AB - The cellular labile iron pool (LIP) is a pool of chelatable and redox-active iron, which is transitory and serves as a crossroad of cell iron metabolism. Various attempts have been made to analyze the levels of LIP following cell disruption. The chemical identity of this pool has remained poorly characterized due to the multiplicity of iron ligands present in cells. However, the levels of LIP recently have been assessed with novel nondisruptive techniques that rely on the application of fluorescent metalosensors. Methodologically, a fluorescent chelator loaded into living cells binds to components of the LIP and undergoes stoichiometric fluorescence quenching. The latter is revealed and quantified in situ by addition of strong permeating iron chelators. Depending on the intracellular distribution of the sensing and chelating probes, LIP can be differentially traced in subcellular structures, allowing the dynamic assessment of its levels and roles in specific cell compartments. The labile nature of LIP was also revealed by its capacity to promote formation of reactive oxygen species (ROS), whether from endogenous or exogenous redox-active sources. LIP and ROS levels were shown to follow similar "rise and fall" patterns as a result of changes in iron import vs. iron chelation or ferritin (FT) degradation vs. ferritin synthesis. Those patterns conform with the accepted role of LIP as a self-regulatory pool that is sensed by cytosolic iron regulatory proteins (IRPs) and feedback regulated by IRP-dependent expression of iron import and storage machineries. However, LIP can also be modulated by biochemical mechanisms that override the IRP regulatory loops and, thereby, contribute to basic cellular functions. This review deals with novel methodologies for assessing cellular LIP and with recent studies in which changes in LIP and ROS levels played a determining role in cellular processes. PMID- 12374616 TI - Redox signaling in vascular angiogenesis. AB - Angiogenesis is thought to be regulated by several growth factors (EGF, TGF alpha, beta-FGF, VEGF). Induction of these angiogenic factors is triggered by various stresses. For instance, tissue hypoxia exerts its pro-angiogenic action through various angiogenic factors, the most notable being vascular endothelial growth factor, which has been mainly associated with initiating the process of angiogenesis through the recruitment and proliferation of endothelial cells. Recently, reactive oxygen species (ROS) have been found to stimulate angiogenic response in the ischemic reperfused hearts. Short exposure to hypoxia/reoxygenation, either directly or indirectly, produces ROS that induce oxidative stress which is associated with angiogenesis or neovascularization. ROS can cause tissue injury in one hand and promote tissue repair in another hand by promoting angiogenesis. It thus appears that after causing injury to the cells, ROS promptly initiate the tissue repair process by triggering angiogenic response. PMID- 12374617 TI - Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. AB - Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated. PMID- 12374618 TI - Ammonia aggravates stress-induced gastric mucosal oxidative injury through the cancellation of cytoprotective heat shock protein 70. AB - The relationship between Helicobacter pylori colonization and the formation of stress-induced gastric mucosal injury remains unknown. Since ammonia (NH(3)) is known as one of the injurious factors in H. pylori-colonized gastric mucosa, the present study is designed to investigate the level of stress-induced gastric mucosal oxidative injury with or without intragastric NH(3) overloading. To apply emotional stress, the communication box paradigm was used in the mouse model. Mice (C57BL/6, male) were pretreated with distilled water (responder-H(2)O) or 0.01% NH(3) (responder-NH(3)) through a gastric tube once a day for a week. Emotional stress was then applied to the responder mice for 3 h per day for 3 d by watching and hearing the behavior of the sender mice subjected to electric shocks to the feet (2 mA, 10 s, 50 s interval). After the communication box protocol, the tissue MPO activity, the contents of TBA-reactive substances (TBARS), and the level of gastric mucosal HSP70 were examined. Responder-NH(3) mice developed more severe gastric lesions than the responder-H(2)O subjects. MPO activity and TBARS contents were enhanced significantly in the responder-NH(3) group compared with the responder-H(2)O subjects. Although the contents of HSP70 in the gastric mucosa increased in the responder-H(2)O group compared with the control-H(2)O animals, they were significantly attenuated in the responder-NH(3) mice. Excess intragastric NH(3) was able to enhance the formation of emotional stress-induced gastric mucosal lesions. This injury may be associated with the enhanced production of oxygen free radicals from accumulated neutrophils under the NH(3)-mediated cancellation of gastric mucosal cytoprotective HSP70. PMID- 12374619 TI - Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced nephrosis. AB - The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity. PMID- 12374620 TI - Dermal wound healing properties of redox-active grape seed proanthocyanidins. AB - Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants, such as hydrogen peroxide, mostly contributed by neutrophils and macrophages. We proposed that oxidants in the wound microenvironment support the repair process. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Previously we have reported that a grape seed proanthycyanidin extract containing 5000 ppm resveratrol (GSPE) potently upregulates oxidant and tumor necrosis factor-alpha inducible VEGF expression in human keratinocytes (Free Radic. Biol. Med. 31:38 42, 2001). Our current objective was to follow up on that finding and test whether GSPE influences dermal wound healing in vivo. First, using a VEGF promoter-driven luciferase reporter construct we observed that the potentiating effect of GSPE on inducible VEGF expression is at the transcriptional level. The reporter assay showed that GSPE alone is able to drive VEGF transcription. Next, two dermal excisional wounds were inflicted on the back of mice and the wounds were left to heal by secondary intention. Topical application of GSPE accelerated wound contraction and closure. GSPE treatment was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. GSPE treatment also increased VEGF and tenascin expression in the wound edge tissue. Tissue glutathione oxidation and 4-hydroxynonenal immunostaining results supported that GSPE application enhanced the oxidizing environment at the wound site. Oxidants are known to promote both VEGF as well as tenascin expression. In summary, our current study provides firm evidence to support that topical application of GSPE represents a feasible and productive approach to support dermal wound healing. PMID- 12374621 TI - Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes. AB - We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3 gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints. PMID- 12374622 TI - Inhibition of tobacco smoke-induced lung inflammation by a catalytic antioxidant. AB - Cigarette smokers experience airway inflammation and epithelial damage, the mechanisms of which are unknown. One potential cause may be free radicals either in tobacco smoke or produced during persistent inflammation. Inflammation may also be a driving force to cause airway epithelium to undergo changes leading to squamous cell metaplasia. To test whether tobacco smoke-induced inflammation could be reduced by a catalytic antioxidant, manganese(III)meso-tetrakis(N,N' diethyl-1,3-imidazolium-2-yl) porphyrin (AEOL 10150) was given by intratracheal instillation to rats exposed to filtered air or tobacco smoke. Exposure to tobacco smoke for 2 d or 8 weeks (6 h/d, 3 d/week) significantly increased the number of cells recovered by bronchoalveolar lavage (BAL). AEOL 10150 significantly decreased BAL cell number in tobacco smoke-treated rats. Significant reductions in neutrophils were noted at 2 d and macrophages at 8 weeks. Lymphocytes were significantly reduced by AEOL 10150 at both time points. Squamous cell metaplasia following 8 weeks of tobacco smoke exposure was 12% of the total airway epithelial area in animals exposed to tobacco smoke without AEOL 10150, compared with 2% in animals exposed to tobacco smoke, but treated with AEOL 10150 (p <.05). We conclude that a synthetic catalytic antioxidant decreased the adverse effects of exposure to tobacco smoke. PMID- 12374623 TI - Apolipoprotein E deficiency promotes increased oxidative stress and compensatory increases in antioxidants in brain tissue. AB - The epsilon 4 allele of the apolipoprotein E gene (ApoE) is associated with Alzheimer's disease (AD). The extent of oxidative damage in AD brains correlates with the presence of the E4 allele of ApoE, suggesting an association between the ApoE4 genotype and oxygen-mediated damage in AD. We tested this hypothesis by subjecting normal and transgenic mice lacking ApoE to oxidative stress by folate deprivation and/or excess dietary iron. Brain tissue of ApoE-deficient mice displayed increased glutathione and antioxidant levels, consistent with attempts to compensate for the lack of ApoE. Folate deprivation and iron challenge individually increased glutathione and antioxidant levels in both normal and ApoE deficient brain tissue. However, combined treatment with folate deprivation and dietary iron depleted antioxidant capacity and induced oxidative damage in ApoE deficient brains despite increased glutathione, indicating an inability to compensate for the lack of ApoE under these conditions. These data support the hypothesis that ApoE deficiency is associated with oxidative damage, and demonstrate a combinatorial influence of genetic predisposition, dietary deficiency, and oxidative stress on oxidative damage relevant to AD. PMID- 12374624 TI - Inhibition of PTPs by H(2)O(2) regulates the activation of distinct MAPK pathways. AB - It has been shown that endogenous production of reactive oxygen species (ROS) during T cell activation regulates signaling events including MAPK activation. Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS which modify the catalytic cysteine residues of the enzymes. We have analyzed the interplay between the inhibition of PTPs and the activation of MAPK by H(2)O(2). Stimulation of Jurkat T cells with H(2)O(2) induces the phosphorylation of ERK, p38, and JNK members of MAPK family. H(2)O(2) stimulation of T cells was found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of cells with wtSHP-1 decreased H(2)O(2)-induced ERK and JNK phosphorylation without affecting p38 phosphorylation. Transfection with wtHePTP inhibited H(2)O(2)-induced ERK and p38 phosphorylation without inhibiting JNK phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H(2)O(2)-induced phosphorylation of ERK, p38, and JNK. The phospholipase C (PLC) inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked H(2)O(2)-induced ERK phosphorylation, whereas the same treatment did not inhibit p38 or JNK phosphorylation. Taken together, these results suggest that inhibition of PTPs by H(2)O(2) contributes to the induction of distinct MAPK activation profiles via differential signaling pathways. PMID- 12374625 TI - Dual effect of glucose on LDL oxidation: dependence on vitamin E. AB - The aim of the present study was to determine the direct effect of glucose on LDL oxidation, a key step in the development of atherosclerosis. Purified human LDL were incubated with glucose (500 mg/dl) and LDL oxidation was started by adding CuCl(2) to the media. Glucose delayed the vitamin E consumption, but accelerated the formation of conjugated dienes and increased both the formation of thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility. When LDL were incubated with increasing concentrations of glucose and submitted to oxidation, the formation of conjugated dienes, TBARS, and the electrophoretic mobility increased in a concentration-dependent manner. When LDL was enriched with vitamin E, it showed a delay in the formation of conjugated dienes, even in the presence of glucose. To determine whether glucose had any effect on LDL oxidation, once the process was started and vitamin E consumed, LDL were submitted to oxidation and, at different times thereafter, glucose was added into the media. Under these conditions glucose also accelerated the LDL oxidation. In summary, present results show that in LDL submitted to oxidation, glucose delays the early phases of the oxidation, slowing the vitamin E consumption, but it accelerates the rate of LDL oxidation once LDL vitamin E has been consumed; the effect being concentration-dependent. PMID- 12374626 TI - A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke. AB - Oxidative stress is a major source of injury from cerebral ischemia and reperfusion. We hypothesized that a catalytic antioxidant AEOL 10150 [manganese (III) meso-tetrakis (di-N-ethylimidazole) porphyrin] would attenuate changes in brain gene expression in a mouse model of transient middle cerebral artery occlusion (MCAO). C57BL/6J mice were subjected to either sham surgery or 60 min of right MCAO. AEOL 10150 or phosphate-buffered saline was given intravenously 5 min after onset of reperfusion (n = 6 per group). Six hours later, parenchyma within the MCA distribution was harvested. RNA from the six brains in each group was pooled and mRNA expression determined using an Affymetrix murine MG_U74A v. 2.0 expression microarray. Each experiment was performed three times. The largest changes in expression occurred in stress response and inflammatory genes such as heat shock protein, interleukin-6, and macrophage inflammatory protein-2. Treatment with AEOL 10150 attenuated only the increase in expression of inflammatory genes. This suggests that AEOL 10150 protects brain by attenuating the immune response to ischemia and reperfusion. PMID- 12374627 TI - A role for the myoglobin redox cycle in the induction of endothelial cell apoptosis. AB - This study investigates the potential role of the ferric/ferryl redox cycle of myoglobin (Mb) in the development of endothelial cell injury. Bovine aortic endothelial cells were incubated with ferric Mb (0.5-100 micro M) in the presence or absence of low steady states of H(2)O(2) (3-4 micro M) generated by glucose oxidase (GOX). The reaction of ferric Mb with H(2)O(2) generated ferryl Mb as monitored spectrophotometrically. Ferryl Mb formation correlated with the induction of apoptosis as indicated by morphological criteria, caspase 3 activation, phosphatidylserine (PS) externalization, and nuclear condensation by Hoechst 33342 staining. The addition of ascorbate or catalase inhibited the formation of ferryl Mb and the onset of apoptosis, whereas apoptosis was enhanced in cells depleted of intracellular glutathione by pretreatment with buthionine sulfoximine. Mb and Mb/GOX suppressed cell cycle progression, but only Mb/GOX produced significant cell loss revealed by the accumulation of sub G1 events. These results suggest a role for the Mb redox cycle in the induction of endothelial cell apoptosis, which may be relevant in the pathophysiology of diseases characterized by the release of Mb from damaged muscle. PMID- 12374628 TI - Different distributions of the 5-HT reuptake complex and the postsynaptic 5 HT(2A) receptors in Brodmann areas and brain hemispheres. AB - The aim of the present study was to determine the distribution of the presynaptic 5-HT reuptake complex and the 5-HT(2A) receptors through Brodmann areas from two control subjects, together with the possible existence of laterality between both brain hemispheres. A left laterality was observed in the postsynaptic 5-HT(2A) binding sites, with significantly higher B(max) values in the left frontal and cingulate cortex. In frontal cortex, [3H]imipramine and [3H]paroxetine binding showed the highest B(max) values in areas 25, 10 and 11. In cingulate cortex, the highest [3H]imipramine and [3H]paroxetine B(max) values were noted in Brodmann area 33 followed by area 24, while postsynaptic 5-HT(2A) receptors were mainly distributed through Brodmann areas 23 and 29. In temporal cortex, the highest [3H]imipramine and [3H]paroxetine B(max) was noted in Brodmann areas 28 and 34, followed by areas 35 and 38. All Brodmann areas from parietal cortex (1, 2, 3, 4, 5, 6, 7, 39, 40 and 43) showed similar presynaptic and postsynaptic binding values. In occipital cortex no differences were observed with regard to the brain hemisphere or to the Brodmann area (17, 18 and 19). These results suggest the need to carefully define the brain hemisphere and the Brodmann areas studied, as well to avoid comparisons between studies including different Brodmann areas or brain hemispheres. PMID- 12374630 TI - Visual processing and neuropsychological function in schizophrenia and schizoaffective disorder. AB - Persons with schizophrenia and schizoaffective disorder exhibit deficits in both visual processing and neuropsychological tasks. Little is known, however, about whether these deficits are related to one another. We administered psychophysical tests of visual discrimination and recognition, and neuropsychological tests of abstract flexibility, verbal learning, visual memory, working memory and attention to 42 outpatients with stable but chronic schizophrenia or schizoaffective disorder. Multiple regression analyses were performed to determine the relationship between these measures of neuropsychological function and visual psychophysical performance. Results indicated that motion perception was associated with working memory, and that the addition of a memory component to motion perception (motion recognition) was associated with both working memory and visual memory. Visual performance was not associated with symptom severity as measured by the PANSS. These results suggest that psychophysical tests of visual processing may contribute to deficits on neuropsychological tests of visual cognition, and may also reflect cross-modal disturbances of working memory function. PMID- 12374629 TI - Interaction of lithium with 5-HT(1B) receptors in depressed unipolar patients treated with clomipramine and lithium versus clomipramine and placebo: preliminary results. AB - Lithium is commonly used in combination with antidepressant drugs as a treatment for refractory depression; less often, it is used in non-resistant depression. The aim of this study was to examine the interaction of lithium with 5-HT(1B) receptors in 10 non-resistant unipolar depressed patients treated with clomipramine+lithium (C+L) vs. clomipramine+placebo (C+P). A mediation of the serotonergic system has been proposed in the literature to explain the clinical effect of lithium. Indeed, in a previous study of healthy human blood platelets, we demonstrated the interaction of lithium with adenylate cyclase activity coupled to 5-HT(1B) receptors. The functional activity of these receptors was measured by studying the inhibitory effect of L694,247, a 5-HT(1B) receptor agonist, on the adenylate cyclase activity determined by the production of cAMP. Using the same technique in the present study, we found that lithium significantly reduced the inhibition of adenylate cyclase activity induced by 5 HT(1B) receptor activation. This result confirms the specific interaction of lithium with 5-HT(1B) receptors. Moreover, a correlation between the percentage of 5-HT(1B) receptor-dependent adenylate cyclase inhibition and the clinical benefit of lithium was established, suggesting 5-HT(1B) receptors may be a target for the therapeutic effect of lithium. PMID- 12374631 TI - Written but not oral verbal production is preserved in young schizophrenic patients. AB - The aim of this study is to discover whether the language capabilities of young schizophrenic patients are more affected in speaking than in writing or whether the disorders are equivalent in the two modes. To do this, we compared spoken and written descriptions of pictures obtained from 10 schizophrenic patients with those produced by 10 control subjects. These productions were analysed on the basis of objective indices. The syntax and coherence of the productions were evaluated by judges. The comparison of the performances of the controls and schizophrenic patients supports the hypothesis that the latter suffer from a language disorder affecting the oral mode but impacting less frequently and less severely on the written mode. These results are discussed in the light of the cognitive mechanisms which may provide an explanation of these language disorders. PMID- 12374632 TI - Clinical characteristics and risk factors for Kraepelinian subtype of schizophrenia: replication of previous findings and relation to summer birth. AB - The aims of the study were: (1) to replicate findings that patients with Kraepelinian schizophrenia constitute a distinct subgroup and (2) to examine the relationship between season of birth and the Kraepelinian subtype. Thirty-one Kraepelinian patients, defined on the basis of a longitudinal criterion--at least 5 years of continuous and complete dependence on others to maintain the basic necessities of life, including food, clothing and shelter--were compared with 279 non-Kraepelinian schizophrenic patients. All patients met ICD-10 criteria for schizophrenia and were evaluated with the Positive and Negative Syndrome Scale. Kraepelinian schizophrenic patients had more negative symptoms and were more disorganized than non-Kraepelinian patients. Positive and anxious-depressive symptoms did not differ between the two groups. Among Kraepelinian patients, there was an excess number of births in the month of July. These findings are consistent with previous reports that Kraepelinian patients could have a disease with an etiopathophysiology separate from that of other schizophrenic patients. PMID- 12374633 TI - Expressed emotion is not associated with disorder severity in first-episode mental disorder. AB - A family atmosphere characterized by expressed emotion (EE) is a robust predictor of clinical outcome of patients with schizophrenia and mood disorders. However, there is ongoing discussion as to whether EE is more a cause of clinical outcome or a parental reaction to disorder severity. This cross-sectional study examines a sample of 42 consecutive first-episode patients from a defined geographical area with severe mental disorders (schizophrenia-related disorders, psychotic mood disorders, and non-psychotic mood disorders). Their 42 relatives were interviewed, and the relationships between EE variables derived with the five minute speech sample method (FMSS) and the patients' demographic, premorbid and clinical measures were analyzed. A high EE score was found in 40% of the relatives. High EE was associated with the interviewed relative's not being a spouse and the patient's being young and unmarried. It was not associated with premorbid characteristics, symptom dimensions or the diagnostic group of the patient. These results do not support the hypothesis that EE is a reaction to the clinical features of the patient. Instead, demographic factors may partly mediate the effect of EE on prognosis. PMID- 12374634 TI - N2 and P3 components of event-related potential in first-episode schizophrenic patients: scalp topography, medication, and latency effects. AB - Auditory N2 and P3 components of event-related potentials were assessed in first episode schizophrenic and normal control subjects (n=12/group). P3 amplitude was decreased in the patients most prominently over the frontal areas in contrast to a widespread P3 amplitude decrease reported in chronic schizophrenia. Moreover, frontal attenuation of P3 amplitude was greater in the non-medicated compared with medicated patients, a finding that suggests frontal areas are primarily affected at the onset of the first schizophrenic episode. Prolongation of N2 and P3 latencies was also observed in the patients, which indicates that stimulus classification and memory updating processes were slowed even in early stages of schizophrenia. These findings indicate that first-episode schizophrenic patients produce N2 and P3 abnormalities that are distinct from those in chronic patients, and that psychotropic medication can attenuate event-related potential effects in specific ways. PMID- 12374635 TI - Children with attention-deficit/hyperactivity disorder and comorbid oppositional defiant disorder: an EEG analysis. AB - This study investigated EEG differences between two groups of children with attention-deficit/hyperactivity disorder combined type (ADHD), with or without comorbid oppositional defiant disorder (ODD), and normal control subjects. Each group consisted of 20 males. All subjects were between the ages of 8 and 12 years, and groups were matched on age. EEG was recorded during an eyes-closed resting condition from 21 monopolar derivations, which were clustered into nine regions for analysis. The EEGs were Fourier transformed to provide absolute and relative power estimates for the delta, theta, alpha and beta bands. Values were also calculated for the theta/alpha and theta/beta ratios. The ADHD groups had more absolute and relative theta than the control group. Regionally, the ADHD groups had less relative alpha and more relative delta in posterior regions, and less relative beta in the frontal regions, than the control group. These differences were also apparent in both ratio measures. Only two significant topographic differences were found between the ADHD groups, with both of these being less deviant from normality in the ADHD+ODD group than the ADHD group. These results indicate that EEG correlates of ADHD are not clouded by the presence of comorbid ODD, which suggests possible applications in clinical practice. PMID- 12374636 TI - Inpatient diagnostic assessments: 3. Causes and effects of diagnostic imprecision. AB - Preceding studies found that clinicians using the Traditional Diagnostic Assessment (TDA, the standard of clinical practice) often made imprecise diagnoses, compared with gold standards. Those same studies found excellent diagnostic agreement (kappa>0.75) between Computer Assisted Diagnostic Interview (CADI) and gold standards, thus warranting CADI's use as the standard for data collection and diagnosis in this study. When TDA and CADI users independently examined 106 inpatient-subjects, TDA users agreed only 45.3% (48/106) with CADI's primary diagnosis and found only 50.5% as many total diagnoses. This study searched for the causes and effects of those differences. To test the hypothesis that insufficient data collection was the cause, the 106 TDA write-ups were analyzed word-by-word. Only 46.2% (49/106) of the TDA write-ups listed enough symptom criteria (e.g. hallucinations, depression) to meet DSM-IV requirements for diagnosis, a likely cause of TDA's inaccuracy. TDA write-ups evaluated only 52.9% of the 18 Key Criteria necessary to screen for 10 diagnostic groups, a likely cause of TDA's incompleteness. TDA's diagnostic imprecision had effects on (1) length of stay (LOS) for hospitalized patients and (2) associated costs. Patients evaluated with TDA had a mean LOS of 12.5 days versus 7.7 days for CADI patients, a reduction of 4.8 days (12.5-7.7). If CADI replaced TDA, then annual savings of 3,000,000 dollars system-wide could be projected for inpatient care. Remedies for TDA's diagnostic imprecision are proposed. PMID- 12374637 TI - Internal validity of an anxiety disorder screening instrument across five ethnic groups. AB - We tested the factor structure of the National Anxiety Disorder Screening Day instrument (n=14860) within five ethnic groups (White, Black, Hispanic, Asian, Native American). Conducted yearly across the US, the screening is meant to detect five common anxiety syndromes. Factor analyses often fail to confirm the validity of assessment tools' structures, and this is especially likely for minority ethnic groups. If symptoms cluster differently across ethnic groups, criteria for conventional DSM-IV disorders are less likely to be met, leaving significant distress unlabeled and under-detected in minority groups. Exploratory and confirmatory factor analyses established that the items clustered into the six expected factors (one for each disorder plus agoraphobia). This six-factor model fit the data very well for Whites and not significantly worse for each other group. However, small areas of the model did not appear to fit as well for some groups. After taking these areas into account, the data still clearly suggest more prevalent PTSD symptoms in the Black, Hispanic and Native American groups in our sample. Additional studies are warranted to examine the model's external validity, generalizability to more culturally distinct groups, and overlap with other culture-specific syndromes. PMID- 12374638 TI - Body shape preference and body satisfaction in Taiwanese college students. AB - This study examined differences in measures of perception of body shape and body satisfaction among 25 male and 165 female undergraduates. The students completed a body image questionnaire, comprised of four parts: (1) anthropometric data; (2) figure rating scale; (3) cognitive attitude toward body size; and (4) satisfaction with body characteristics. This study found that male ratings of the Ideal figure, the figure of how they currently thought they looked, and the figure of how they felt most of time were almost identical, as were the figure of what they thought most attractive to women and the figure of how they thought others saw them. On the contrary, female ratings of the Ideal figure were significantly thinner than their perception of their current figure, the figure as others saw them, and the figure they felt most of time. Furthermore, the female figure that women rated as most attractive to males was thinner than the figure that males actually preferred. On the other hand, male judgments of the male figure most attractive to females were heavier than female ratings of the same. Both males and females erred in estimating what the opposite sex would find attractive. Males overestimated and females underestimated the body size attractive to the other sex. Although over half of the male and female subjects reported their body size as 'In-between', more males wanted to gain weight and more females wanted to lose weight to present their Ideal body shape. Furthermore, males were biased toward being satisfied with their body and females were biased toward being dissatisfied with their body. Additionally, the number of females satisfied with hands and dissatisfied with upper thighs and buttocks was higher than that of males. PMID- 12374639 TI - Norepinephrine transporter gene polymorphism is not associated with susceptibility to alcohol dependence. AB - Abnormalities in monoamine neurotransmission have been implicated in the pathogenesis of alcoholism, mood disorders and schizophrenia. Murine norepinephrine transporter gene (NET) has been mapped to a region on chromosome 8 where a quantitative trait locus for ethanol sensitivity. Therefore we tested whether norepinephrine transporter (NET) gene variants confer susceptibility to either alcohol dependence or severe alcohol withdrawal symptoms. There is a highly polymorphic silent G1287A mutation in the NET gene. In our study 157 alcoholics and 185 healthy unrelated matched control subjects were analyzed for a silent G1287A mutation. No significant differences in allele and genotype distribution between control subjects f(A)=0.33 and alcoholics f(A)=0.29 were found. No significant results were found in more homogenous subgroups, i.e. alcoholics with severe alcohol withdrawal (seizures, delirium), early onset age<26 nor dependent patients with positive familial history of alcoholism. These results suggest that the NET gene polymorphism in exon 9 accession number: mRNA: NM_001043, genomic contig.: NT_019610, is unlikely to be involved in the susceptibility to alcoholism and severe alcohol withdrawal. PMID- 12374640 TI - A variable number of tandem repeats in the serotonin transporter gene does not affect the antidepressant response to fluvoxamine. AB - A variable number of tandem repeats (VNTR) in the second intron of the serotonin transporter gene (STin2) has been studied in association with the susceptibility to affective disorders. Recently, it was reported that selective serotonin reuptake inhibitors were more effective in patients with major depressive disorder having the homozygous allele pair (12-copy/12-copy) of VNTR in the STin2 than in ones having other allele combinations. As the study had methodological problems, further studies are needed to confirm the above finding. Therefore, the authors investigated whether the allelic variation of VNTR in the STin2 was associated with the antidepressant response to fluvoxamine in 66 patients with major depressive disorder. Fluvoxamine was prescribed up to 200 mg/day in the dosing protocol for 6 weeks. The present study showed no significant association between the polymorphism of VNTR in the STin2 and the treatment response to fluvoxamine. PMID- 12374641 TI - Factor-analytic study of the Anorectic Behavior Observation Scale in Japan: comparisons with the original Belgian study. AB - The Anorectic Behavior Observation Scale (ABOS) is a questionnaire developed to obtain information from relatives about behaviors and attitudes of patients with eating disorders. The original report of the ABOS revealed three factors. This is the first study to confirm the factor structure by use of confirmatory analyses. Relatives of 102 patients with eating disorders completed the ABOS, and exploratory factor analyses were performed. Confirmatory factor analyses were performed for this Japanese sample, and the fitness of the factor structure was compared between that in the original report proposed by Vandereycken and that used in the present study. A three-factor structure was revealed by the data of Japanese patients, including factor I (eating behavior meeting the criteria of anorexia; concern with weight, foods, and denial), factor II (bulimia; including bingeing, disposing, furtive eating and purging, and talk about thinness and dieting), and factor III (hyperactivity, eating slowly, and chopping food into very small pieces). Confirmatory analyses supported the improvement of fitness in this modified three-factor structure in comparison with the original three-factor model. This Japanese ABOS study revealed three dimensions, which differed from the original subscales in Belgium. Cross-cultural differences were observed, though the ABOS may be useful in Japan as an instrument for assessing eating disorders. PMID- 12374642 TI - Introversion and individual differences in middle ear acoustic reflex function. AB - A growing body of psychophysiological evidence points to the possibility that individual differences in early auditory processing may contribute to social withdrawal and introverted tendencies. The present study assessed the response characteristics of the acoustic reflex arc of introverted-withdrawn and extraverted-sociable individuals. Introverts displayed a greater incidence of abnormal middle ear acoustic reflexes and lower acoustic reflex amplitudes than extraverts. These findings were strongest for stimuli presented at a frequency of 2000 Hz. Results are discussed in light of the controversy concerning the anatomic loci (peripheral vs. central neuronal activity) of the individual differences between introverts and extraverts in early auditory processing. PMID- 12374643 TI - Event-related potential correlates of serial-position effects during an elaborative memory test. AB - Twenty undergraduate students participated in an elaborative learning test to evaluate the relationship between electrical brain activity and subsequently recalled and not-recalled words. Data collected from the midline (Fz, Cz, Pz) and lateral scalp sites (F3, F4, C3, C4, P3, P4) were analysed. The difference between event-related potentials (ERPs) elicited by subsequently recalled and not recalled words, the ERP memory effect, was evaluated for each portion (primacy, plateau and recency) of the serial-position curve (SPC). We compared peak amplitudes for the P1, N1, P2, N400, P3 and frontal positive slow wave (FPSW) components. The electrophysiological data support the hypothesis that different mechanisms underlie primacy and recency effects during free recall paradigms. There was no support for the hypothesis that an association arises between memory and the FPSW when subjects utilise elaborative learning strategies. The P2 component predicted subsequent recall at the primacy portion of the SPC, and P1 predicted recall at the primacy and plateau portions of the curve. The findings suggest that the early positive components of the ERP (i.e. P1 and P2) are useful indices of the differential stimulus processing during elaborative learning which predicts later recall. PMID- 12374644 TI - One-year test-retest reliability of auditory ERPs in young and old adults. AB - The reliability of ERP measures was investigated in a sample covering the adult life span (n = 59, age 21-92). This sample was divided into a young and an old group. ERPs to an auditory two-stimuli oddball task were recorded in the sample at two occasions separated by 12-14 months (T1 and T2). The recordings of T1 were split in half, to assess within session reliability. Correlations were calculated for N1, P2 and P3 peak latency and amplitude, for average amplitude during 50 ms epochs within the defined P3-window 250-550, and for average amplitude in successive 15 ms epochs from 1 to 705 ms. The results show that amplitude measures were more reliable than the latency measures at all electrodes. Time window/epoch amplitude measures yielded reliabilities in the same range as peak amplitude. Reliabilities peaked around the conventionally studied N1, P2 and P3, and this is seen as a validation of the components. In general, the old group exhibited weaker P3 peak latency reliabilities than the young group. However, many of these differences did not reach statistical significance. Implications of the findings are discussed. PMID- 12374645 TI - Propagation of repetitive alpha waves over the scalp in relation to subjective preferences for a flickering light. AB - Paired-comparison tests were performed to examine subjective preferences for a flickering light. Electroencephalograms were then recorded from seven electrodes (10-20 system) during presentations of the most and least preferred flickering light conditions. As a way of investigating the flow of alpha waves on the scalp over both the left and right hemispheres in relation to subjective preference, the alpha waves were analyzed by means of the cross-correlation function (CCF). The maximum value of the CCF, /phi(tau)/(max), between the alpha waves measured at different electrodes and its delay time, tau(m), were analyzed. Results show that the most preferred flickering light has a significant larger /phi(tau)/(max) than the least preferred flickering light, and that /phi(tau)/(max) decreases with increasing distance between comparison (O(1) or O(2)) and test electrodes. On the other hand, the delay time of the maximum value of the CCF, tau(m), increases with the distance between comparison and test electrodes. PMID- 12374646 TI - Variability of EEG synchronization during a working memory task in healthy subjects. AB - Working memory is associated with an increase in EEG theta synchronization and a decrease in lower alpha band synchronization. We investigated whether such changes in mean synchronization level are accompanied by changes in small scale fluctuations of synchronization. EEGs (19 channels; average reference; sample frequency 250 Hz) were recorded in 21 healthy subjects (12 males; mean age 62.5 years; S.D. 2.1) at rest and during a visual working memory condition. EEG synchronization was computed in six frequency bands (2-6; 6-10; 10-14; 14-18; 18 22; 22-50 Hz) using the synchronization likelihood. Variability of the synchronization was quantified with synchronization entropy. During the working memory condition synchronization increased in the 2-6 Hz band, and decreased in the 6-10, 14-18 and 18-22 Hz bands. Working memory was associated with increased variability in the 2-6 Hz band, and decreased variability in the 6-10 Hz band and, to a lesser extent, in the 14-18 and 18-22 Hz bands. Working memory is accompanied not only by characteristic changes in the mean level of interactions between neural networks, but also by changes in small scale fluctuations in such interactions. Strong, but rapidly fluctuating coupling between neural systems might provide a mechanism to optimize the balance between local differentiation and global integration of brain activity. PMID- 12374647 TI - Airway response of healthy individuals to affective picture series. AB - We studied the effects of viewing pictures in three series of homogeneous affects on respiratory resistance. Results from prior studies had been equivocal with respect to modulation of resistance by affective valence or arousal. Forty-two healthy participants viewed three series of affective slides preselected for global categories of positive, neutral and negative valence. Oscillatory resistance (R(os)), time, volume and flow parameters of respiration, cardiac interbeat-interval, respiratory sinus arrhythmia, and skin conductance were measured throughout the picture series, and individual pictures were rated in pleasure, arousal and interest. During the slide presentation, R(os) increased with decreasing picture valence. Other physiological parameters did not parallel these changes. R(os) changes were restricted to the expiratory portion of the breathing cycle, thus suggesting a contribution of upper airway sites to the response. It is concluded that R(os) is modulated by affective valence when pictures are selected in global categories of positive, neutral and negative affect. Observed airway responses are probably less informative with respect to affect-induced exacerbation in lower airway disease. PMID- 12374648 TI - Auditory novelty oddball allows reliable distinction of top-down and bottom-up processes of attention. AB - An auditory novelty-oddball task, which is known to evoke a P3 event-related potential (ERP) in a target condition and a novelty-P3 ERP in response to task irrelevant unique environmental sounds, was repeatedly applied to healthy participants (n = 14) on two separate recording sessions, 7 days apart. Both target-P3 and novelty-P3 were internally consistent and test-retest reliable. Interestingly, novelty-P3 amplitude declined from the first to the second half of each recording session, whereas no systematic alteration between both sessions occurred. The target-P3 showed the opposite pattern, i.e. a reduced amplitude from the first to the second session, but no systematic change within each session. These findings suggest that novelty-P3 amplitude changes reflect habituation, whereas target-P3 session effects may indicate the adjusted amount of processing resources invested into the task. In general, the results support the interpretation of the novelty-P3 as indicating automatic, bottom-up related aspects of attention, whereas the target-P3, in the present paradigm, seems to reflect voluntary, top-down related aspects of attention. PMID- 12374649 TI - Effect of negative air ions on computer operation, anxiety and salivary chromogranin A-like immunoreactivity. AB - The effects of negative air ions on computer operation were examined using a biochemical index of the activity of the sympathetic/adrenomedullary system (i.e. salivary chromogranin A-like immunoreactivity (CgA-like IR)) and a self-report questionnaire (State-Trait Anxiety Inventory, Anxiety State--STAI-S). Twelve female students carried out a word processing task for 40 min. The salivary CgA like IR increased more than three times on the task, but the salivary cortisol did not change. The increase in the CgA-like IR level was attenuated by the exposure to negative air ions during the task. The exposure to the ions during the recovery period following the task was effective for rapidly decreasing the CgA-like IR level that had increased after the task. These effects by negative air ions were also observed using STAI-S. Task performance was slightly but significantly improved by the presence of negative air ions. These results suggest that negative air ions are effective for the reduction of and the prompt recovery from stress caused by computer operation. PMID- 12374650 TI - Introduction of Dr. John Edward Niederhuber. PMID- 12374651 TI - The things that matter most. PMID- 12374652 TI - Breast cancer and leukemia: the forest for the trees? PMID- 12374653 TI - The prognosis of patients with thick primary melanomas: is regional lymph node status relevant, and does removing positive regional nodes influence outcome? PMID- 12374654 TI - Melanoma and human leukocyte antigen status: the missing link? PMID- 12374655 TI - Minimally invasive surgery in the diagnosis and treatment of upper gastrointestinal tract malignancy. PMID- 12374656 TI - Therapy-induced leukemias and myelodysplastic syndromes after breast cancer treatment: an underemphasized clinical problem. PMID- 12374657 TI - Comparison of side effects between sentinel lymph node and axillary lymph node dissection for breast cancer. AB - BACKGROUND: Axillary lymph node dissection (ALND) is often associated with permanent arm side effects. Side effects after sentinel lymph node dissection (SLND) should be less common, because the surgery is less extensive. METHODS: The study compared side effects and interference with daily life between 169 women who underwent an SLND and 78 who underwent an ALND for breast cancer. Patients rated symptom severity and interference with daily life caused by pain, numbness, limitation of arm range of motion (ROM), and arm swelling at 1, 6, and 12 months after surgery by using the Measure of Arm Symptom Survey. Repeated-measures and regression analyses for each time period were used to determine associations between symptoms and dissection type. RESULTS: At 1 month, SLND patients reported less pain, numbness, limitation in ROM, and seromas than ALND patients. At 6 months, SLND patients had less pain, numbness, and arm swelling, and at 12 months, SLND patients had less numbness, arm swelling, and limitation in ROM than ALND patients. At 1 month, pain, numbness, and limitation in ROM interfered significantly more with daily life for ALND patients. At 6 and 12 months, only numbness interfered more with daily life for ALND patients. CONCLUSIONS: SLND was associated with fewer side effects than ALND at all time points. PMID- 12374658 TI - Prognostic implications of thick (>or=4-mm) melanoma in the era of intraoperative lymphatic mapping and sentinel lymphadenectomy. AB - BACKGROUND: Lymphatic mapping/sentinel lymphadenectomy (LM/SL) has become a routine part of our treatment algorithm for primary melanoma, yet its role in the management of thick (>or=4-mm) lesions is unknown. METHODS: One hundred twenty one patients with thick primaries underwent LM/SL at our institute. Survival curves were constructed from Kaplan-Meier estimates and analyzed by Cox proportional hazards methods. RESULTS: Sixty-three percent of patients were men, median age 54 years. The primary tumor sites were trunk (46%), extremities (32%), and head and neck (21%). Primary thickness ranged from 4 to 15 mm (median, 6.0 mm). Forty-five percent of primary tumors were ulcerated. Thirty-five percent of patients had tumor-positive dissections. Median follow-up was 31 months. The overall 5-year survival was no different (P =.726) for ulcerated and nonulcerated lesions. There was no difference (P =.159) in overall survival after tumor negative (60% +/- 7%) and tumor-positive (50% +/- 10%) dissections. The 5-year disease-free survival was significantly (P =.012) lower in patients with tumor positive (34% +/- 9%) than tumor-negative (47% +/- 7%) dissections. CONCLUSIONS: Although LM/SL has become a popular technique for staging the regional lymph nodes in early-stage melanoma, our results suggest that sentinel node status is predictive of disease-free survival for thick primary tumors but is not yet reflective of overall survival. The role of LM/SL for patients with thick primary tumors is not clearly defined. PMID- 12374659 TI - The surgical management of metastatic melanoma. AB - When deciding whether or not to perform a resection for metastatic melanoma, one should follow general principles that apply to the patient with melanoma as well as to the patient with metastases from other types of primary tumors. When the resection is palliative, the success of surgical treatment will be governed by the presence of identifiable symptoms, the morbidity of the procedure, the course of the disease, and the ability to communicate treatment goals among surgeon, patient, and family. When the resection is performed with curative intent, long term survival depends on the ability of the surgeon to select patients with a pattern of recurrence suggestive of a less aggressive tumor biology. Regardless of the extent of the operative procedure, resection of metastases in patients whose disease recurs early after the treatment of the primary tumor, in those who present with multiple lesions, and in those who present with disease that cannot be completely resected will only rarely be associated with subsequent long-term survival. PMID- 12374660 TI - Prognostic effect of lymph node micrometastasis in patients with histologically node-negative gastric cancer. AB - BACKGROUND: There is no consensus as to the impact of lymph node micrometastasis on survival of patients with gastric cancer. The aim of this study was to clarify the prognostic significance of lymph node micrometastasis in patients with histologically node-negative gastric cancer. METHODS: Lymph nodes (n = 2039) from 64 patients with histologically node-negative gastric cancer (T2, T3) were evaluated for micrometastasis. Three serial 5- micro m sections of the resected lymph nodes were prepared for immunohistochemical staining with the anti cytokeratin antibody CAM 5.2. RESULTS: Micrometastasis was found in 73 of 2039 nodes (4%) and 20 of 64 patients (32%). The 5-year survival rate was significantly lower for patients with lymph node micrometastasis than for those without lymph node micrometastasis (66% vs. 95%, P <.01). The 5-year survival rate was significantly lower when there were four or more positive micrometastatic nodes (94% vs. 29%, P <.01) and when there were extragastric micrometastatic nodes (89% vs. 53%, P <.01). CONCLUSIONS: Lymph node micrometastasis was associated with poor outcome in patients with histologically node-negative gastric cancer. The number and the level of lymph node micrometastases are useful prognostic markers for deciding treatment strategies for additional therapy and follow-up. PMID- 12374661 TI - Outcome of ratio of lymph node metastasis in gastric carcinoma. AB - BACKGROUND: The purpose of this study was to clarify the outcome of the ratio of the metastatic lymph nodes (RML) in gastric cancer patients. METHODS: The postoperative survival of 650 patients with gastric cancer who underwent D2 curative gastrectomy was analyzed with regard to the RML. The location, number, and RML in the N1 station and in all (N1 and N2) stations were analyzed. These data were compared from the viewpoints of staging accuracy and patient survival. RESULTS: The RML was classified as follows: RML 0, no involvement; RML 1, 0 to.1; RML 2,.1 to.25; and RML 3, >or=.25. The 5-year survival rates stratified by RML were RML 0, 86%; RML 1, 68%; RML 2, 35%; and RML 3, 16%. Cox model identified all methods of classifying lymph node metastases as independent prognostic indicators in each calculation. However, a second Cox regression revealed that RML was the only independent prognostic factor among the three methods (P <.001). Stage migration was present in 35 cases (15%) when the number was considered. However, only 15 cases (7%) were underdiagnosed when RML was used. CONCLUSIONS: RML is a useful classification of patients with gastric cancer. It may prevent the phenomenon of stage migration. PMID- 12374662 TI - Malignancy after renal transplantation: incidence and role of type of immunosuppression. AB - BACKGROUND: Cancer, particularly skin cancer and lymphoma, is a complication of posttransplantation immunosuppression. We investigated the characteristics of cancers in our renal transplant population, the role of type of immunosuppression on cancer incidence, and whether newer, more potent immunosuppressive agents produce cancers sooner after transplantation. METHODS: The charts of patients who developed cancer after renal transplantation between 1958 and 2000 were reviewed. Statistical analyses were performed with the mid-P version of Fisher's exact test for 2 x 2 tables for incidence comparison of cancer and with Student's t-test for differences between mean times to cancer. RESULTS: Between 1958 and 2000, 924 transplantations in 760 patients were performed. We found a cancer incidence of 12.2%. The most frequent cancers were skin and genitourinary. The overall mortality was 54%. We found an increased incidence of cancer in the group of patients in the cyclosporine era and for patients >or=45 years at transplantation. Cancer did not develop sooner in the cyclosporine group. CONCLUSIONS: The distribution of types of cancer was similar to that reported in the literature. The mortality rate was high. The incidence of cancer was higher in the cyclosporine era in patients >or=45 years at transplantation. PMID- 12374663 TI - Recurrent differentiated thyroid carcinoma: biological implications of age, method of detection, and site and extent of recurrence. AB - BACKGROUND: We identified factors predictive of outcome for recurrent differentiated thyroid carcinoma (DTC). METHODS: Fifty-seven patients with local (LR), regional (RRec), and/or distant recurrence (DR) of 431 recurrent DTCs were studied. Disease-specific survival (DSS) rate was estimated with the Kaplan-Meier method. Univariate and multivariate comparisons were conducted by log-rank and Cox regression analysis. RESULTS: The median follow-up was 13 years. Distribution of the first relapse was LR only (35%), LR and RRec (23%), LR and DR (30%), and LR, RRec, and DR (12%). Factors predictive of resectability were a long (>or=5 year) disease-free interval (DFI) and subclinical and thyroid remnant recurrence. Only 26% of symptomatic and 45% of thyroid bed LR, and 43% with DFI <5 years, could be resected completely. No isolated thyroid remnant and 75% of thyroid bed LR resulted in tumor-related mortality. Age <45 years, subclinical recurrence, isolated LR, and the ability to render the patient disease free independently predicted DSS. Fifteen-year DSS for LR only; LR and RRec; LR and DR; and LR, RRec, and DR were 49%, 28%, 15%, and 0%, respectively. CONCLUSIONS: Isolated thyroid remnant recurrence defines a benign phenotype. Age, method of detection, site and extent of recurrence, and the ability to render the patient disease free predict outcome for recurrent DTC. Multimodality long-term follow-up is warranted to detect recurrence at a subclinical potentially curative stage. PMID- 12374664 TI - Clinical utility of positron emission tomography in the diagnosis and management of periampullary neoplasms. AB - BACKGROUND: This study examined the effect that 18-fluorodeoxyglucose positron emission tomography ((18)FDG-PET) imaging had on the clinical management of patients with suspected periampullary malignancy. METHODS: Fifty-four patients with suspected pancreatic neoplasms underwent both whole-body (18)FDG-PET and abdominal computed tomography (CT). Malignant or benign disease was confirmed pathologically in 47 patients. RESULTS: Of the 41 patients with malignancy, (18)FDG-PET failed to identify the primary tumor in 5 patients. (18)FDG-PET demonstrated increased uptake suggesting primary malignancy in 37 patients. Malignant pathology was confirmed in 36 cases. (18)FDG-PET identified malignant locoregional lymph node metastases in six of ten patients. All nodes identified before surgery by (18)FDG-PET were also seen on preoperative CT. Six patients who were thought to have resectable disease by CT were found to have distant metastasis at laparotomy. (18)FDG-PET did not detect metastasis in any of these cases. Before surgery, (18)FDG-PET identified distant metastases that were not detected by CT in one patient. CONCLUSIONS: Despite high sensitivity and specificity in diagnosing periampullary malignancy, (18)FDG-PET did not change clinical management in the vast majority of patients previously evaluated by CT. In addition, (18)FDG-PET missed >10% of periampullary malignancies and did not provide the anatomical detail necessary to define unresectabilty. PMID- 12374665 TI - Frequency of nodal metastases to the upper mediastinum in Barrett's cancer. AB - BACKGROUND: In Barrett's cancer, the frequency of lymph node metastases to the middle and upper mediastinum has rarely been analyzed because it requires a complete mediastinal lymphadenectomy. METHODS: Fifty-one patients with esophageal adenocarcinoma underwent transthoracic en-bloc esophagectomy with two-field lymph node dissection. A meticulous work-up of the resected specimen allowed a specific assignment of each single lymph node to defined groups of the abdominal and mediastinal compartment. Histopathology classified the lymph nodes as metastatic or nonmetastatic. RESULTS: A total of 1706 lymph nodes were resected, with a mean of 33.5 lymph nodes per patient (range, 13-74). Of 51 patients, 28 (54.9%) were classified as pN1; 7 (25%) of 28 pN1 patients had nodal metastases at the level of the tracheal bifurcation (3 of 28 patients) or in the upper mediastinum (5 of 28 patients). In all 28 pN1 patients, the abdominal compartment was involved. The distribution of nodal metastases demonstrated that the main lymphatic spread occurred close to the primary tumor, along the lesser curvature and the left gastric artery. CONCLUSIONS: Adenocarcinomas of the distal esophagus have a bidirectional lymphatic spread to the mediastinum and the abdomen. Two-field lymphadenectomy seems to be an adequate surgical approach for this tumor entity to achieve a complete nodal clearance. PMID- 12374666 TI - Improved antitumor response to isolated limb perfusion with tumor necrosis factor after upregulation of endothelial monocyte-activating polypeptide II in soft tissue sarcoma. AB - BACKGROUND: Experiments with tumor necrosis factor alpha (TNF) in rodents have shown that a high dose can lead to hemorrhagic necrosis in tumors. Endothelial monocyte-activating polypeptide II (EMAP-II) is a novel tumor-derived cytokine, and its expression increases the TNF-1 receptor on tumor endothelium, enhances the induction of tissue factor on tumor endothelial cells, and has an antiangiogenic effect. It has recently been shown that in vivo sensitivity of tumor vasculature to TNF is determined by tumor production of EMAP-II. METHODS: We measured the level of EMAP-II in a TNF-resistant soft tissue sarcoma. We subsequently stabile-transfected this cell line with a retroviral construct containing the EMAP gene. In an extremity perfusion model in tumor-bearing rats, we measured response rates to TNF therapy. RESULTS: Functional EMAP-II production was increased after this transfection. Immunostaining of paraffin-embedded tumor tissue sections in rats showed an overexpression of human EMAP-II. Results of the TNF perfusions in rats suggest that this tumor is more sensitive to TNF therapy. CONCLUSIONS: EMAP-II is produced in various levels. One can increase the sensitivity of tumor for TNF therapy in vivo by upregulating the EMAP-II production. This result leaves an opportunity for enhanced TNF response of tumors in future settings. PMID- 12374667 TI - Immediate breast reconstruction after skin-sparing mastectomy for the treatment of advanced breast cancer: radiation oncology considerations. PMID- 12374668 TI - Nuclear matrix proteins as proteomic markers of preneoplastic and cancer lesions : commentary re: G. Brunagel et al., nuclear matrix protein alterations associated with colon cancer metastasis to the liver. Clin. Cancer Res., 8: 3039 3045, 2002. PMID- 12374669 TI - Approval summary: imatinib mesylate in the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors. AB - PURPOSE: Imatinib mesylate (Gleevec; Novartis, East Hanover, NJ)is a receptor tyrosine kinase inhibitor approved previously in 2001 by the United States Food and Drug Administration for the treatment of chronic myelogenous leukemia in blast crisis, accelerated phase, or in chronic phase after failure of IFN-alpha therapy. We review herein the clinical profile of this drug and the regulatory review leading to the approval of a supplemental New Drug Application for the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors (GISTs). EXPERIMENTAL DESIGN: We discuss the efficacy and side effects of imatinib mesylate in a Phase II trial of 147 patients with metastatic and/or unresectable malignant GISTs, the basis for marketing approval, and postmarketing commitments by the drug's manufacturer. RESULTS: Imatinib was assessed in a single, open-label trial involving one European center and three centers in the United States. Seventy-three patients were randomly allocated to receive 400 mg of imatinib daily, and 74 patients received 600 mg daily. At the study report cutoff date, an objective response was confirmed in 56 patients; the overall response rate for the combined study arms was 38% (95% confidence interval, 30 46%). These responses were all partial responses. There was no statistically significant difference in response rates between the two dose groups. Adverse events included edema, fluid retention, nausea, vomiting, diarrhea, myalgias, skin rash, bone marrow suppression, bleeding, and elevations in aspartate aminotransferase, alanine aminotransferase, or bilirubin. Bleeding into the gastrointestinal tract or intratumoral sites occurred in 7 patients (5%) and was not correlated with thrombocytopenia or tumor bulk. The pharmacokinetics of imatinib in GIST patients were similar to those of chronic myelogenous leukemia patients. CONCLUSIONS: On February 1, 2001, imatinib mesylate was approved by the United States Food and Drug Administration for the treatment of malignant metastatic and/or unresectable GISTs. The recommended dose is 400 or 600 mg daily. PMID- 12374670 TI - Nuclear matrix protein alterations associated with colon cancer metastasis to the liver. AB - PURPOSE: The development of colon cancer markers that can detect liver metastases early and predict which patients are at risk to develop liver metastases would have a major impact on this disease. We have previously identified G. Brunagel, et al., Cancer Research, 62:2437-2442, 2002, nuclear matrix proteins (NMPs), which are associated with colon cancer. The objective of this study is to identify the existence of a specific NMP "fingerprint" for human liver metastasis from colon cancer. EXPERIMENTAL DESIGN: Using high-resolution two-dimensional gel electrophoresis, we analyzed the NMP expression of 12 matched liver metastases and adjacent normal samples and three normal donor liver samples. These were compared with colon cancer NMP patterns, along with several primary cell systems and lines. RESULTS: Analysis of multiple gels for each sample revealed three proteins present in all liver metastases, which are not present in normal liver tissue and normal hepatocytes. These three proteins were also present in colon cancer samples. CONCLUSION: Data provided here demonstrate that the NMP composition is able to differentiate liver metastases from normal liver tissue and normal hepatocytes and that these proteins are also expressed in colon cancer. These results further show that the adjacent normal liver tissue changes its NMP pattern of expression. Development of an assay to detect these specific NMPs in tissue and/or serum specimens is a promising modality for early detection of liver metastases from colon cancer or potentially as a prognostic tool. In addition the functional characterization of these proteins will significantly enhance our understanding of the development of liver metastases of this disease. PMID- 12374671 TI - Expression of eukaryotic initiation factor 4E in atypical adenomatous hyperplasia and adenocarcinoma of the human peripheral lung. AB - The overexpression of eukaryotic initiation factor 4E (eIF4E), a key regulator of protein synthesis, is involved in the malignant progression of various human cancers. We investigated eIF4E expression in atypical adenomatous hyperplasia (AAH) and adenocarcinomas of the human peripheral lung. On the basis of the WHO criteria with minor modifications, adenocarcinomas were classified as bronchioloalveolar carcinoma (BAC), mixed subtypes with a bronchioloalveolar pattern and minor invasion (early MX), and mixed subtypes with a papillary pattern and marked invasion (overt MX). eIF4E immunohistochemistry was performed in 143 tissue samples (31 AAH, 38 BAC, 43 early MX, and 31 overt MX). Both tumoral and stromal eIF4E levels were elevated from AAH, BAC, and early MX to overt MX and significantly associated with histological grade (P < 0.001 and P < 0.001, respectively). Tumoral and stromal eIF4E staining intensities were significantly correlated (P < 0.01). Immunoblot analysis of 51 tissue samples (2 AAH, 11 BAC, 18 early MX, and 20 overt MX) demonstrated that eIF4E expression in adenocarcinomas was 3.4-7.4-fold higher than in normal lung and that its expression progressively increased in the following order: AAH (lowest expression), BAC, early MX, and overt MX (highest expression). Multiple regression analysis revealed that both tumoral and stromal eIF4E expressions were significant independent factors for the histological subtype (P < 0.01 and P < 0.01, respectively). These results suggest that translational control is dysregulated during the development of peripheral lung adenocarcinoma and that progressive increases of tumoral and stromal eIF4E may be part of a positive feedback loop for malignant progression. PMID- 12374672 TI - ERBB receptor signaling promotes ependymoma cell proliferation and represents a potential novel therapeutic target for this disease. AB - PURPOSE: This study was designed to investigate the biological and therapeutic significance of ERBB1, ERBB2, ERBB3, and ERBB4 in childhood ependymoma. EXPERIMENTAL DESIGN: The expression frequency and clinical significance of ERBB1 4 was analyzed in a large cohort of pediatric ependymoma (n = 121) using immunohistochemistry, Western blotting, and reverse transcription-PCR analysis. Histological markers of anaplasia (necrosis, microvascular proliferation, and Ki 67 proliferative index) were also determined. Functional assessment of ERBB dependent cell signaling and proliferation, in addition to novel therapeutic inhibition of these processes, was conducted using short-term cultures of human ependymoma cells. RESULTS: Coexpression of ERBB2 and ERBB4 was identified in over 75% of tumors. High-level coexpression of these receptors was significantly related to tumor proliferative activity [P < 0.05; Ki-67 labeling index (LI)] and, in combined survival analysis of clinical (degree of surgical resection) and molecular (ERBB2/ERBB4 expression status and Ki-67 LI) factors, enabled a greater resolution of patient prognosis than any individual variable alone. Ligand dependent activation of ERBB receptor-signaling in cultured ependymoma cells resulted in AKT phosphorylation and cellular proliferation that was significantly blocked in a dose-dependent manner using WAY-177820, a novel inhibitor of ERBB2 tyrosine kinase activity. CONCLUSIONS: This study suggests that ERBB receptor signaling results in aggressive disease behavior in ependymoma by promoting tumor cell proliferation. An analysis of ERBB2 and ERBB4 expression, in association with Ki-67 LI and the degree of surgical resection, may provide an accurate tool for assessing disease risk in children with this disease. In addition, these receptors may serve as a target for novel therapeutic approaches in ependymoma. PMID- 12374673 TI - Prognostic significance of signal transducer and activator of transcription 1 activation in breast cancer. AB - PURPOSE: Signal transducers and activators of transcription (STATs)were shown to be activated in mammary carcinoma. Because different STAT factors are likely to have different functions in these tumors, an assessment of their individual role is mandatory. EXPERIMENTAL DESIGN: In this study we have separately determined activation of STAT1, STAT3, and STAT5 by measuring their DNA binding activity and tyrosine phosphorylation in breast cancer tissue samples. The predictive value of STAT activation on relapse-free and overall survival among women who received treatment for primary breast cancer was evaluated in a retrospective study. RESULTS: Survival analysis demonstrated that patients with high STAT1 activation have substantially longer overall and relapse-free survival, irrespective of whether STAT1 activation was determined by its DNA binding activity (P = 0.003 and 0.010, respectively) or by its tyrosine phosphorylation (P = 0.046 and 0.011, respectively). In accordance, Cox proportional hazard regression analysis revealed an enhanced hazard of death (hazard ratio, 3.77; P = 0.018) and relapse of disease (hazard ratio, 6.55; P = 0.013) for the group of women with low STAT1 activation. After adjusting for known prognostic variables (lymph node status, stage of disease, estrogen receptor status, and cathepsin D), STAT1 activation remained an independent prognostic value. Activation of STAT3 and STAT5 DNA binding did not significantly correlate with prognosis. CONCLUSION: Our study reveals a favorable and independent prognostic significance of STAT1 activation in mammary carcinoma, and is in accordance with the documented role of STAT1 in growth arrest, and in pro-apoptotic signaling pathways. PMID- 12374674 TI - A phase II pilot trial of concurrent biochemotherapy with cisplatin, vinblastine, temozolomide, interleukin 2, and IFN-alpha 2B in patients with metastatic melanoma. AB - PURPOSE: In an effort to reduce the frequency of central nervous system (CNS) progression in patients with metastatic melanoma with ongoing systemic response to biochemotherapy, we modified our standard concurrent biochemotherapy regimen by replacing dacarbazine (DTIC) with oral temozolomide. EXPERIMENTAL DESIGN: Patients received cisplatin, vinblastine, and temozolomide (20 mg/m(2) cisplatin and 1.2 mg/m(2) vinblastine i.v., days 1-4; 150 mg/m(2) p.o. temozolomide, days 1 4) concurrent with interleukin 2 (9 MIU/m(2)/day) by continuous i.v. infusion on days 1-4 and IFN-alpha (5 MU/m(2)/day) on days 1-5, 8, 10, and 12. Prophylactic antibiotics and a maximum of four cycles were administered. Routine granulocyte colony stimulating factor and aggressive antiemetics were also provided. Tumor staging included torso computed tomography scans and brain magnetic resonance imaging pretreatment, after cycle 4 and then every 3 months for 2 years. Torso computed tomography scans were also performed after cycle 2. RESULTS: A total of 147 treatment cycles were administered to 48 patients. No patients had received prior chemotherapy or interleukin 2; however, 19 (40%) had received prior adjuvant IFN-alpha. Significant toxicities included 2 deaths from cardiac events (pericarditis al tamponade and posttreatment myocardial infarction with associated ventricular arrhythmia) and 3 gastrointestinal serious adverse events (pancreatitis, appendicitis, and upper GI bleed). No other nonhematological grade 4 toxicities were observed. Tumor responses were seen in 22 of 47 evaluable patients (relative risk, 47%) with 7 complete responses (15%). Response durations ranged from 1 to 29+ months with 1 currently ongoing. Median survival was 7.5 months. The CNS was the initial site of progression in 2 responding patients. An additional 6 responding patients developed CNS progression within 3 months of systemic progression. Initial CNS progression was significantly less frequent what was seen with the prior DTIC-based biochemotherapy regimen (2 of 22 versus 12 of 19; P = 0.001). CONCLUSION: This regimen appears to be active and reasonably well tolerated in patients with metastatic melanoma. Although the substitution of temozolomide for DTIC reduced the incidence of initial CNS progression, this effect did not appear to result in an improved overall outcome. PMID- 12374675 TI - Phase I trial of an infrared pulsed laser device in patients with advanced neoplasias. AB - PURPOSE: The objective of this study was to evaluate the toxicity/radiant exposure/time relationship of an infrared pulsed laser device (IPLD) treatment in patients with advanced neoplasias. Karnofsky performance status (KPS), Spitzer quality of life index (QLI), and potential antitumor activity, if any, were also assessed. EXPERIMENTAL DESIGN: Seventeen patients (n = 17) received a daily IPLD radiant exposure of 4.5 x 10(5) J/m(2) (904 nm pulsed at 3 MHz) under a one-dose schedule and procedure design. Toxicity was evaluated under the parameters of the WHO; indirect toxic ocular effects were also monitored. KPS and QLI measurements were conducted before treatment and at six 3-months intervals. Scores for the seventh interval are the last available (range, 19-39 months). For statistical purposes, patients were classified into group 1, those alive at the end of the study, and group 2, those who had died. RESULTS: Dose-limiting toxicity was not observed. Five patients (n = 5) reported occasional headaches (grade 2), and four (n = 4) referred local pain (grade 2). In group 1 (n = 7), statistically significant increases in KPS and QLI were observed in all of the follow-up intervals compared with pretreatment values. One patient had a complete response, 1 a partial response, 4 stable diseases > or =12 months, and 1 progressive disease. In group 2 (n = 10), statistically significant increases in QLI were observed during the first two intervals. Eight patients had stable disease > or =6 months and 2 had uninterrupted progressive diseases. CONCLUSIONS: The IPLD treatment studied is safe for clinical use and may have potential effects on KPS, QLI, and antitumor activity in patients with advanced neoplasias. PMID- 12374676 TI - A phase I trial of the single-chain immunotoxin SGN-10 (BR96 sFv-PE40) in patients with advanced solid tumors. AB - PURPOSE: Our purpose in the study was to establish the maximum tolerated dose and toxicity profile of SGN-10 (or BR96 sFv-PE40), a single-chain immunotoxin. SGN-10 is composed of the fused gene products encoding the translocating and ADP ribosylating domains of Pseudomonas exotoxin (PE40) and the variable heavy (V(H)) and variable light (V(L)) regions of BR96 monoclonal antibody. This antibody is specific for a Lewis(Y) (Le(Y))-related carbohydrate antigen expressed on multiple carcinomas. EXPERIMENTAL DESIGN: A total of 46 patients with Le(Y) positive metastatic carcinoma were enrolled in a Phase I dose-escalation study in cohorts of three to six patients who received SGN-10 at doses ranging from 0.024 to 0.962 mg/m(2), administered on days 1, 4, 8, and 11, followed by 2 weeks of rest and a second cycle of therapy. Pharmacokinetics and human antibody response to SGN-10 were also determined. RESULTS: The maximum tolerated dose of SGN-10 was 0.641 mg/m(2) with gastrointestinal dose-limiting toxicity. Pharmacokinetic studies performed in eight patients at the 0.641-mg/m(2) dose revealed a t([1/2]) of 2.5 +/- 0.3 h and a C(max) of 389 +/- 112 ng/ml. Pharmacodynamic analyses demonstrated a rapid clearance of the drug by day 11 associated with an antitoxin human antitoxin antibody (HATA) response in most patients. Signs consistent with a modest vascular leak syndrome, specifically, transient hypoalbuminemia, were observed in patients treated with doses of > or =0.384 mg/m(2). No complete or partial tumor responses were observed at an 8-week evaluation, although 31% of patients had stable disease. CONCLUSIONS: The maximal tolerated dose of SGN-10 given twice weekly for 2 weeks is 0.641 mg/m(2) with gastrointestinal dose limiting toxicity. The immunogenicity of the toxin moiety limits the ability of SGN-10 to circulate by day 11 of therapy. Studies are ongoing to evaluate strategies to ameliorate toxicities and to inhibit the development of the anti SGN-10 immune response. PMID- 12374677 TI - Phase I and pharmacokinetic trial of the proapoptotic sulindac analog CP-461 in patients with advanced cancer. AB - CP-461 is a member of a class of novel proapoptotic drugs that specifically inhibit cyclic GMP phosphodiesterases but not cyclooxygenase-1 or -2. CP-461 inhibits the growth of a broad range of human tumor cell lines in vitro at micromolar concentrations and selectively induces apoptosis in cancer cell lines but not normal cells. Preclinical studies revealed good oral bioavailability and no toxicity in dogs and rats at single doses up to 500 mg/kg. In a Phase I trial, 21 patients with a range of solid tumors and good performance status received CP 461 p.o. twice daily for 28 consecutive days. Cycles were repeated without a treatment-free interval. CP-461 doses ranged from 100 to 800 mg/day. Therapy was well tolerated overall, and a maximum tolerated dose was not reached. Grade 3 asymptomatic aspartate aminotransferase/alanine aminotransferase elevation in 1 patient treated at 800 mg/day was the only dose-limiting toxicity. No hematologic toxicity was noted. Peak plasma concentrations occurred between 1 and 2 h after dosing, and doses above 200 mg/day exceeded the known in vitro EC(50) (1-2 micro M) for apoptosis in cancer cells. No drug was detectable after 24 h of administration, and the terminal half-life was 6.7 h. The area under the plasma concentration-time curve was dose-proportional from 200 to 800 mg/day. Four patients exhibited disease stability after two cycles of treatment. CP-461 is minimally toxic at doses up to 800 mg/day when administered p.o. on a twice-daily schedule. PMID- 12374678 TI - A phase II breast cancer chemoprevention trial of oral alpha difluoromethylornithine: breast tissue, imaging, and serum and urine biomarkers. AB - PURPOSE: A double-blind randomized Phase II chemoprevention trial of alpha difluoromethylornithine (DFMO) was conducted in a group of women at high risk for development of breast cancer. DFMO is an irreversible inhibitor of ornithine decarboxylase, the limiting enzyme of polyamine synthesis that is often up regulated in breast cancer. EXPERIMENTAL DESIGN: Study entrants were required to have random periareolar fine-needle aspiration cytology prior to entry that exhibited hyperplasia or hyperplasia with atypia, as well as a mammogram and clinical breast exam judged as not suspicious for breast cancer and no clinical hearing loss. Subjects were randomized to 6 months of oral DFMO (0.5 g/m(2)/day) or placebo, followed by repeat fine-needle aspiration and biomarker assessment. The main study end point was an improvement in cytologic pattern. RESULTS: Of 119 subjects entered, 96% completed the study and were evaluable for the main study end point. A modest reduction (28%) in average total urine polyamines was obtained in the DFMO group, but there was no reduction in the spermidine:spermine ratio. There was no difference in cytologic improvement between DFMO and placebo. Likewise, there was no difference between DFMO and placebo for the secondary end points of breast molecular marker changes (immunocytochemical expression of proliferating cell nuclear antigen, p53, and epidermal growth factor receptor), mammographic breast density, serum insulin-like growth factor I: insulin-like growth factor binding protein 3 ratio, adverse events, quality of life indices, or subsequent cancer development. CONCLUSIONS: DFMO at a dose level of 0.5 g/m(2)/day administered for 6 months does not modulate breast risk biomarkers tested in this study. PMID- 12374679 TI - Identification of gene expression profiles that segregate patients with childhood leukemia. AB - To identify genes whose expression correlated with biological features of childhood leukemia, we prospectively analyzed the expression profiles of 4608 genes using cDNA microarrays in 51 freshly processed bone marrow samples from children with acute leukemia, over a 24-month period, at a single institution. Two supervised methods of analysis were used to identify the 20 best discriminating genes between the following cohorts: acute myelogenous leukemia (AML) versus acute lymphoblastic leukemia (ALL); B-lineage versus T-lineage ALL; newly diagnosed B-lineage standard-risk versus high-risk ALL; and B-lineage leukemia harboring the TEL-AML 1 fusion versus patients without a molecularly characterized translocation. These methods identified overlapping sets of genes that segregated patients within described subgroups. Cross-validation demonstrated that the majority of patients could be correctly classified based on these genes alone, and hierarchical clustering grouped patients with similar clinical and biological disease features. The potential for select genes to discriminate patients was validated using real-time PCR in samples that were analyzed by microarray profiling and in other uniformly processed leukemic marrow samples. As expected, microarray technology can successfully segregate patients defined by traditional measures such as immunophenotype and cytogenetic alterations. However, among specific subgroups, this preliminary analysis also suggests that microarrays can identify unanticipated similarities and diversity in individual patients and thus may be useful in augmenting risk-group stratification in the future. PMID- 12374680 TI - Inducible nitric oxide synthase and survivin messenger RNA expression in hepatocellular carcinoma. AB - PURPOSE: Proliferative activity and suppression of apoptosis of cancer cells are important to tumor progression in hepatocellular carcinoma (HCC). Recently, the expressions of inducible nitric oxide synthase (iNOS) and survivin mRNA have been reported to correlate with suppression of apoptosis in some tumors. However, the clinical importance of expression of these genes in HCC progression remains unclear. In the present study, the correlation between the expression of iNOS and survivin mRNA and the occurrence of spontaneous apoptosis and proliferative activity of cancer cells and prognostic importance of expression of these genes in HCC were investigated. EXPERIMENTAL DESIGN: Tissues were obtained by surgical resection of livers from 61 patients with HCC and 8 without HCC. Expressions of iNOS and survivin mRNA were evaluated using the reverse transcription-PCR in 61 tumors, 61 adjacent histologically noncancerous livers, and 8 normal livers. Apoptotic cancer cells and the proliferative activity of cancer cells were detected by immunohistochemistry. RESULTS: iNOS mRNA expression was detected in 34 of 61 (55.7%) HCCs, 19 of 61 (31.1%) noncancerous liver tissues adjacent to carcinoma, and none of the 8 normal livers. In addition, survivin mRNA was detected in 19 of 61 (31.1%) HCCs, none of 61 noncancerous liver tissues, and none of the 8 normal livers. iNOS mRNA expression did not correlate with the proliferative activity of cancer cells or with the occurrence of apoptosis in HCCs. In contrast, survivin mRNA expression strongly correlated with a high proliferative activity of cancer cells and a low apoptotic index. Disease specific survivals did not differ between patients with iNOS-positive or negative HCCs. Although, the disease-specific survival of patients with survivin positive HCCs was significantly poorer than that of patients with survivin negative HCCs. CONCLUSIONS: These results indicate that iNOS may not correlate with cancer cell-proliferative activity or apoptosis; survivin, however, may not only suppress apoptosis but also accelerate cancer cell-proliferative activity and play an important role in tumor progression in HCC. PMID- 12374681 TI - Signaling abnormalities, apoptosis, and reduced proliferation of circulating and tumor-infiltrating lymphocytes in patients with oral carcinoma. AB - We have reported earlier that T cells found in the tumor microenvironment of head and neck cancer showed evidence of apoptosis as well as decreased expression of signaling molecules. In this prospective study, spontaneous apoptosis in tumor infiltrating lymphocytes (TILs) and in paired circulating peripheral blood lymphocytes (PBLs) was evaluated in 28 patients with oral carcinoma and correlated with zeta-chain expression and anti-CD3 antibody-induced proliferation of the PBL obtained from each patient. In addition, expression of CD3, CD4, and CD8 molecules on TIL and Fas ligand (FasL) on the tumor was studied by immunohistochemistry. Soluble FasL was measured in the patients' sera. PBL obtained from 20 age-matched normal donors was used as a control. Reduced zeta chain expression was observed in TIL-T of 9 of 28 patients and in PBL-T of 12 of 28 patients. Low zeta expression in autologous TIL-T and PBL-T was correlated (P < 0.0012), and it was associated with high levels of expression of FasL on the tumor (P = 0.0002 and P < 0.0013, respectively). Low zeta expression in PBL-T was also associated with the poor ability of these cells to proliferate in response to anti-CD3 antibodies (P = 0.0012). Increased proportions of apoptotic cells were detected in PBL of 6 of 28 (21%) patients versus 13 of 28 patients (46%) in TIL. Apoptosis in autologous PBL and TIL was found to correlate (P = 0.0322) and was significantly associated with reduced zeta-chain expression. Serum levels of soluble FasL were decreased in patients relative to normal controls but did not correlate with PBL apoptosis or FasL expression on the tumor. Decreased expression of TcR-associated zeta chain, depressed immune function, and apoptosis of T cells were observed to occur concomitantly in TIL and circulating PBL-T of a subset of patients with oral carcinoma. These alterations correlated with high levels of FasL expression on the tumor but not with the disease stage. The results suggest that tumor exerts systemic suppressive effects on immune cells, which may be, in part, mediated via the Fas/FasL pathway. PMID- 12374682 TI - Tissue content of hydroxyestrogens in relation to survival of breast cancer patients. AB - PURPOSE: The main goal of our study was to assess estrogen contents of breast tumor tissues, having different estrogen receptor status, in relation to long term follow-up of patients. EXPERIMENTAL DESIGN: Twenty-one breast cancer cases, all collected from January 1986 to January 1988 at the M. Ascoli Cancer Hospital Centre in Palermo, were included in the study and compared with 6 healthy women as a control group. Average follow-up time of patients was 144 +/- 10 months. The estrogen receptor status of tissues was determined by both ligand binding and immunohistochemical assays. A high performance liquid chromatography-based approach, jointly with gas chromatography/mass spectrometry, was used to identify and measure main estrogens, various hydroxyestrogens, and their methoxy derivatives in both normal and tumor tissues. RESULTS: Although variable concentrations of hydroxylated estrogens were detected, they consistently accounted for >80% of all of the estrogens. Significantly greater amounts of both 2- and 4-hydroxyestradiol, along with a marked increase of 16 alpha hydroxyestrone (OHE(1)), were observed in cancer with respect to normal breast tissues. A significant positive association was observed with elevated 16 alpha OHE(1) (P = 0.015) in patients alive, leading to significantly lower (P = 0.043) 2OHE(1):16 alpha OHE(1) ratio values. Conversely, ratio values of 4:2 hydroxy+methoxy estrogens was significantly lower (P = 0.006) in deceased patients. Using cutoff values of 1.2 for 4:2 hydroxy+methoxy ratio and 150 fmol/mg tissue for 16 alpha OHE(1) we achieved a clear-cut separation of patients, with over-cutoff patients having 147 months and under cutoff patients showing only 47 months median survival time (P = 0.00008). CONCLUSIONS: Our data imply that individual hydroxyestrogens may have a distinct role in the onset and the clinical progression of breast cancer, with greater 16 alpha OHE(1) levels being in turn associated to cancer with respect to normal tissues and to a prolonged survival of breast cancer patients. PMID- 12374683 TI - K-ras mutation, p53 overexpression, and microsatellite instability in biliary tract cancers: a population-based study in China. AB - PURPOSE: The genetic alterations in biliary tract cancer and clinicopathological associations have not been studied in large population-based studies. EXPERIMENTAL DESIGN: We evaluated genetic alterations such as K-ras mutation, p53 overexpression, microsatellite instability (MSI), and alterations of the polyadenine tract present in the transforming growth factor beta receptor type II (TGFbetaRII) gene in 126 biliary tract cancers: 75 gallbladder cancers, 33 bile duct cancers, and 18 ampullary cancers. These genetic alterations were compared with patient demographics and clinicopathological characteristics of the tumors. RESULTS: Mutation of the K-ras gene was present in 18 of 126 (14.3%) biliary tract cancers. K-ras mutation was present in 11 of 18 (61.1%) ampullary cancers, 5 of 33 (15.2%) bile duct cancers, and 2 of 75 (2.7%) gallbladder cancers (P = 0.000001). The mean survival of patients who had bile duct carcinomas with K-ras mutation was 3.0 +/- 2.2 months compared with 15.5 +/- 12.5 months for those without mutation (P = 0.03) but was not different for other tumor sites. p53 overexpression was present in 34 of 123 (27.6%) cancers. MSI-high (allelic shifts in 40% or more loci or alteration of the TGFbetaRII gene) was present in 4 of 126 (3.2%) biliary tract cancers without hereditary nonpolyposis colorectal cancer. MSI-high was more common in mucinous adenocarcinomas (P = 0.006) and in patients with early age of onset of cancer (P = 0.04). CONCLUSIONS: The genetic alterations in biliary tract cancers are dependent on the tumor subsite, histology, and age of onset and are associated with prognosis. PMID- 12374684 TI - Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma. AB - The purpose of this study was to examine the methylation profile of various oral squamous cell carcinomas and to correlate the methylation of particular chromosomal loci with the clinicopathological features of the tumors. A semiquantitative analysis of the methylation status of 12 loci in 96 primary tumors and 13 cell lines was carried out. Methylation frequency was calculated as the percentage of methylated alleles detected by bisulfate-PCR. Of the 12 loci examined, 9 (p16INK4A, p15INK4B, p14ARF, DCC, DAP kinase, MINT1, MINT2, MINT27, and MINT31) exhibited aberrant methylation at various frequencies, whereas 3 (hMLH1, HRK, and CACNA1G) showed no methylation. Dense methylation of the 5' CpG island of DAP kinase and MINT1 was well correlated with loss of gene expression. In addition, methylation of DCC was correlated with bone invasion by gingival tumors (P = 0.036), with aggressive invasiveness of tumors of the tongue (P = 0.046), and with reduced survival (P = 0.050). Methylation of MINT1 and MINT31 also correlated with poor prognoses (P = 0.058 and 0.041), whereas methylation of p14ARF correlated with a good prognosis (P = 0.021). Cox regression analysis showed methylation of MINT31 to be an independent predictor of outcome (hazard ratio, 3.79; 95% confidence interval, 1.58-9.10) and to be associated with the T4 disease group (hazard ratio, 5.71; 95% confidence interval, 1.25-26.07). Analysis of DNA methylation is a useful approach to evaluation of the biological characteristics of oral cancers and may be a useful diagnostic indicator of patient prognosis. PMID- 12374685 TI - Coexpression of parathyroid hormone related protein and its receptor in early breast cancer predicts poor patient survival. AB - PURPOSE: Parathyroid hormone-related protein (PTHRP) is in part responsible for the clinical syndrome of hypercalcaemia of malignancy and has been implicated as an important factor in the development of bone metastases. The aim of this study was to determine the coexpression of PTHRP and its receptor in early breast cancer (EBC) and bone metastases (BM), and correlate these findings to clinical outcome. EXPERIMENTAL DESIGN: Samples of surgically excised EBC (n = 176) and BM (n = 43) were collected and stored in liquid nitrogen. PTHRP protein was determined using immunohistochemistry and receptor mRNA using in situ hybridization (n = 107) or semiquantitative reverse transcription-PCR (n = 69). RESULTS: PTHRP protein was expressed in 115 of 170 (68%) EBC compared with 100% of BM (P < 0.001), whereas its receptor mRNA was expressed in 88 of 176 (50%) EBC compared with 35 of 43 (81%) BM (P < 0.001). Coexpression of both PTHRP and its receptor was present in 62 EBC samples (37%) and in 35 BM samples (81%; P < 0.001). The PTHRP receptor correlated well with increasing patient age, but not with tumor size, grade, estrogen receptor, progesterone receptor, or lymph node status. Individually PTHRP and PTHRP receptor both correlated well with a reduced disease-free survival (P < 0.004) and receptor alone with reduced overall survival (P < 0.003). Coexpression of both PTHRP and receptor predicted the worst clinical outcome at 5 years, with a mortality rate of 20 of 62 (32%) compared with the ligand and receptor-negative group with 2 of 32 (6%; P < 0.004). CONCLUSIONS: Overall these results show that the PTHRP receptor is expressed more frequently in BM than EBC, and is associated with poor clinical outcome and survival. PMID- 12374686 TI - Investigation in liver tissues and cell lines of the transcription of 13 genes mapping to the 16q24 region that are frequently deleted in hepatocellular carcinoma. AB - Many studies have associated chromosomal deletions in the 16q24 region with human cancers, including hepatocellular carcinoma. A more limited region around the microsatellite D16S402 has been shown implicated in the metastatic spread of hepatocellular carcinoma, prostate cancer, and Wilms' tumors. It is likely that one or more tumor suppressor genes are located in this 16q24 area. We used SYBR Green reagents to quantify, by real-time quantitative RT-PCR, the production of mRNA for 13 genes mapping to 16q24. The locations of these genes were determined from published human genome sequencing data. We studied mRNA levels in 10 liver tumor tissues, 10 nontumor liver tissues, five hepatoma cell lines, and in isolated hepatocytes. Results were compared with those for loss of heterozygosity observed in the D16S402 region and recurrence. No down-regulation was observed in tumor tissues. Two genes were consistently overexpressed: OKL38 and CDH13. CDH13, which functions in cell-cell adhesion, seems to be involved in liver carcinogenesis. However, no relationship was observed between the expression of this gene and changes in the D16S402 microsatellite or tumor recurrence. None of the other genes tested seemed to be a good candidate for a major tumor suppressor gene in liver carcinogenesis. Our results suggest that additional unknown genes involved in carcinogenesis are located in the 16q24 area. PMID- 12374687 TI - Human papillomavirus type 16 and squamous cell carcinoma of the head and neck. AB - PURPOSE: Human papillomavirus (HPV) has previously been reported to be associated with squamous cell carcinoma of the head and neck. Our objective was to investigate the presence and type of HPV infection in head and neck tumors and determine whether infection was associated with individual tumor characteristics, patients' pattern of tobacco and alcohol exposure, or with clinical outcome. EXPERIMENTAL DESIGN: Using a case series design, fresh tumor samples were obtained from a series of 89 head and neck squamous cell carcinoma patients, including 64 men and 25 women. The majority of tumors were located in the oral cavity, followed by the oropharynx. A PCR-based technique with restriction fragment length polymorphism analysis was used to detect and type HPV. RESULTS: Of the 89 patients, 18 (20%) had detectable HPV 16 in their tumor samples. HPV 16 was detected in 64% of tonsil tumors, 52% oropharyngeal tumors, and 5% oral cavity tumors. The mean age of subjects with HPV 16-positive tumors was younger than the patients with HPV-negative tumors. Also, this group consumed less alcohol on a weekly basis and had a better clinical outcome compared with the HPV negative group. Smoking, clinical stage, tumor grade, and tumor-node-metastasis status were not associated with HPV 16 presence. CONCLUSIONS: Our study supports the previous reports that suggest HPV 16 is associated with squamous cell cancers located in the oropharynx and oral cavity. The fact that HPV-positive tumors were observed in younger, lighter alcohol-consuming individuals with a better overall and disease-specific survival suggests a distinct disease process in these patients. PMID- 12374688 TI - Preoperative serum vascular endothelial growth factor levels: significance in ovarian cancer. AB - PURPOSE: To assess the clinical relevance of serum vascular endothelial growth factor (VEGF) levels in distinguishing patients with ovarian cancer from those with benign adnexal masses. EXPERIMENTAL DESIGN: Preoperative serum VEGF levels were assessed in 101 women with invasive epithelial ovarian cancer, 16 with low malignant potential (LMP) ovarian tumors, and 34 women with benign ovarian tumors. VEGF levels were determined using an ELISA (R&D Systems, Minneapolis, MN). RESULTS: Ovarian cancer patients had a mean preoperative VEGF level of 549 pg/ml (median 379 pg/ml), which was significantly higher than those with benign adnexal masses (mean 228 pg/ml, median 155 pg/ml; P < 0.001) and LMP tumors (mean 200 pg/ml, median 129 pg/ml; P < 0.001). There were no significant differences in VEGF levels between individuals with benign masses and LMP tumors. The ability of VEGF to differentiate malignancy from benign masses at a cutoff VEGF level of 246 pg/ml gave a sensitivity of 74%, a specificity of 71%, a positive predictive value of 88%, and a negative predictive value of 48%. VEGF levels were also significantly higher in patients with stage I ovarian cancer compared with those with benign disease or LMP tumors. Among patients with ovarian cancer, there were no significant differences in VEGF levels based on age, stage, grade, or level of cytoreduction. The presence of ascites was associated with a significantly higher VEGF level (mean 667 pg/ml, median 445 pg/ml versus mean 317 pg/ml, median 293 pg/ml; P < 0.001). Various preoperative VEGF levels were assessed as a predictor of survival, and a VEGF level >380 pg/ml was associated with a hazard ratio of 2.13 (P = 0.009) by univariate analysis. In multivariate analysis of age, stage, cytoreduction, preoperative CA-125, grade, ascites, and VEGF levels above 380 pg/ml, only VEGF levels >380 pg/ml (hazard ratio 2.33; P = 0.02) and advanced stage (hazard ratio 9.03; P = 0.004) were significant. CONCLUSIONS: Preoperative VEGF levels may be useful in differentiating benign adnexal masses from malignancy. Preoperative VEGF levels >380 pg/ml are an independent risk factor for death because of disease. PMID- 12374689 TI - Clonotypic myeloma cells able to xenograft myeloma to nonobese diabetic severe combined immunodeficient mice copurify with CD34 (+) hematopoietic progenitors. AB - The identity of the multiple myeloma (MM) precursor(s) is unknown. Our objective was to determine the myelomagenic capabilities of CD34-enriched autografts. Hematopoietic progenitor fractions from fresh or cryopreserved granulocyte-colony stimulating factor mobilized blood from myeloma patients were obtained by sorting or enrichment, followed by RT-PCR analysis of clonotypic transcripts and/or their ability to transfer myeloma to immunodeficient mice. CD34(+) enrichment using immunomagnetic methods comparable with those used clinically results in copurification of MM cells able to xenograft nonobese diabetic severe combined immunodeficient mice. Highly purified CD34(+) progenitors from granulocyte-colony stimulating factor mobilized blood of myeloma patients include, on average, 31% clonotypic MM cells. CD34(+) progenitors also include 31% DNA aneuploid cells. For six of six MM patients, enriched progenitors were myelomagenic as measured by engraftment of clonotypic cells and/or the development of lytic bone lesions. Intrasternal injection of enriched progenitor fractions led to clonotypic cells in the femoral bone marrow and bone lesions at distant skeletal locations, confirming dissemination of myelomagenic cells. MM precursors copurify with normal hematopoietic progenitors, emphasizing the need for tumor-free grafts. Autologous MM engraftment is likely to be considerably more efficient than in a xenogeneic host, strongly suggesting that MM autografts contribute to posttransplant relapse. The xenografting myelomagenic component(s) is unlikely to be plasma cells, given the lack of morphologically identified plasma cells among enriched progenitors. Xenografting MM precursors appear to be CD34(+)CD45(low), similar to normal progenitors. Precursor function within the MM clone seems to be complex and may involve multiple components of the MM hierarchy. PMID- 12374690 TI - Overexpression of the peripheral benzodiazepine receptor is a relevant prognostic factor in stage III colorectal cancer. AB - PURPOSE: The peripheral benzodiazepine receptor (PBR) has been implicated in the growth control of colorectal cancer, where PBR-specific ligand-binding is increased 3-4-fold. However, the prognostic relevance of PBR (over) expression has not yet been evaluated in colorectal cancer. EXPERIMENTAL DESIGN: A 5-year follow-up was performed in 116 consecutive patients undergoing surgery for colorectal cancer with regional or distant metastases [Union International Contre le Cancer (UICC) stage III, 59 patients; UICC stage IV, 57 patients]. The monoclonal anti-PBR antibody 8 D7 was used for immunohistochemical examination of paraffin-embedded sections. PBR-specific staining was compared in cancer tissues and normal mucosa. Kaplan-Meier survival curves were calculated. RESULTS: Twenty eight % of the colorectal cancers strongly overexpressed PBR. The mean survival of patients with stage III cancer was 56.2 +/- 9.2 months with and 86.8 +/- 6.6 months without high overexpression of PBR (P = 0.006). Univariate and multivariate analyses revealed that high PBR overexpression is an independent unfavorable prognostic factor in stage III colorectal cancer. In stage IV, however, the PBR status did not correlate with different survival times. CONCLUSIONS: Strong PBR overexpression is a new independent prognostic marker in stage III colorectal cancer. Evaluating PBR overexpression may be useful for stratifying risk and developing risk-adapted strategies of adjuvant therapy. PMID- 12374691 TI - Thioredoxin reductase plays a critical role in IFN retinoid-mediated tumor-growth control in vivo. AB - The IFN and retinoic acid (RA) combination suppresses cell growth by inducing apoptosis in the cultured tumor cells. Using a genetic technique, we have isolated several "genes associated with retinoid-IFN-induced mortality" (GRIM) that participate in this death pathway. One such gene, GRIM-12, encodes the redox enzyme thioredoxin reductase (TR). Antisense-mediated inhibition of TR abrogates cell death. To test the in vivo relevance of TR for growth suppression, we have conducted the following study. A wild-type TR or a catalytically defective mutant were expressed in MCF-7 breast carcinoma cells and transplanted into athymic nude mice. These mice were treated with IFN-beta and all-trans RA combination. Tumors expressing the vector or wild-type TR were readily suppressed by the IFN/RA combination. In contrast, the tumors bearing a mutant TR were resistant to regression. We further show that markers of apoptosis are stimulated in the regressing tumors. These studies show a prominent role for TR in tumor-growth suppression. PMID- 12374692 TI - Identification of an antigenic epitope for helper T lymphocytes from carcinoembryonic antigen. AB - PURPOSE: The product of the carcinoembryonic antigen (CEA) gene is an attractive candidate for T-cell-based immunotherapy because it is frequently expressed in epithelial solid carcinomas. Although many CEA peptide epitopes capable of stimulating CTLs have been identified, no MHC class II-restricted T helper epitope has yet been reported. EXPERIMENTAL DESIGN: The amino acid sequence of CEA was examined for the presence of potential T helper epitopes, and candidate peptides were used to stimulate in vitro T-cell responses. RESULTS: We describe here that using an algorithm to identify promiscuous helper T-cell epitopes, a peptide of CEA occupying residue positions 653 to 667 (CEA(653-667)), was effective in inducing in vitro T helper responses in the context of the HLA-DR4, HLA-DR7, and HLA-DR 9 alleles. Most significantly, some of the peptide-reactive helper T lymphocytes were also capable of recognizing naturally processed antigen in the form of recombinant CEA protein or cell lysates from tumors that express CEA. Interestingly, the newly identified helper T-cell epitope was found to overlap with a previously described HLA-A24-restricted CTL epitope, CEA(652-660), which could facilitate the development of a therapeutic vaccine capable of eliciting both CTL and T helper responses in patients suffering from epithelial carcinomas. CONCLUSION: These results indicate that T helper lymphocytes are capable of recognizing CEA as a tumor antigen and that epitope CEA(653-667) could be used for immunotherapy against tumors expressing CEA. PMID- 12374694 TI - Leukotriene B4 receptor antagonist LY293111 inhibits proliferation and induces apoptosis in human pancreatic cancer cells. AB - PURPOSE: The effects of leukotriene (LT) B4 and its receptor antagonist LY293111 on proliferation and apoptosis of human pancreatic cancer cells were investigated, both in vitro and in vivo. EXPERIMENTAL DESIGN: Six human pancreatic cancer cell lines (MiaPaCa-2, HPAC, Capan-1, Capan-2, PANC-1, and AsPC 1) were used. Expression of LTB4 receptors, BLT1 and BLT2, was measured by reverse transcription-PCR. Cell proliferation was measured by [methyl (3)H]thymidine incorporation and cell number counting. Extracellular signal regulated kinase (ERK) 1/2 activation was measured by Western blotting. Apoptosis was assessed by morphology, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and poly(ADP-ribose) polymerase cleavage. The effect of LY293111 on growth of AsPC-1 and HPAC cell xenografts was assessed in BALB/c nu/nu athymic mice. RESULTS: Both LTB4 receptor types were found to be expressed in human pancreatic cancer cells. The LTB4 receptor antagonist LY293111 caused both time- and concentration-dependent inhibition of proliferation of all six human pancreatic cancer cell lines studied. In contrast, LTB4 stimulated proliferation of these cell lines and induced ERK1/2 phosphorylation. The growth stimulatory effect and ERK1/2 phosphorylation induced by LTB4 were inhibited by LY293111. Coincident with growth inhibition, LY293111 induced apoptosis in these pancreatic cancer cell lines, as indicated by morphology, TUNEL assay, and poly(ADP-ribose) polymerase cleavage. In studies using AsPC-1 and HPAC cell xenografts in athymic mice, LY293111 treatment markedly inhibited tumor growth over a 24-day treatment period, as measured by both tumor volume and tumor weight. In situ tissue TUNEL assay showed massive apoptosis in LY293111-treated tumor tissues. CONCLUSIONS: LTB4 can directly regulate the growth of human pancreatic cancer cells and control their survival. Additional studies will clarify the underlying mechanisms of LTB4-regulated pancreatic cancer cell growth and apoptosis. LTB4 receptor blockade and inhibition of the downstream signal pathway are likely to be valuable for the treatment of human pancreatic cancer. PMID- 12374693 TI - Antibody to vascular endothelial growth factor slows growth of an androgen independent xenograft model of prostate cancer. AB - PURPOSE: Human tumors are dependent on angiogenesis for growth, and vascular endothelial growth factor (VEGF) is a major regulator of this process. We aimed to study clinical utility of a recombinant humanized monoclonal anti-VEGF antibody (rhu alpha VEGF) in the treatment of the CWR22R androgen-independent xenograft model of prostate cancer. EXPERIMENTAL DESIGN: rhu alpha VEGF has previously shown clinical activity in several xenograft cancer models. We administered 5 mg/kg rhu alpha VEGF i.p. twice weekly as a single agent and together with paclitaxel to established CWR22R xenografts. RESULTS: rhu alphaVEGF inhibited established tumor growth by 85% (P < 0.01 for trajectories of the average tumor volumes of the groups) at 3 weeks, but after cessation of rhu alpha VEGF treatment, tumor regrowth ensued. A paclitaxel dosage of 6.25 mg/kg s.c. five times/week slowed tumor growth (72% compared with controls at 3 weeks, P = 0.02). The combination of paclitaxel and rhu alpha VEGF resulted in greater inhibition of tumor growth than that observed with either agent alone (98% growth inhibition, P = 0.024 versus rhualpha VEGF alone and P = 0.02 versus paclitaxel alone). Paclitaxel alone had no antiangiogenic effects at the dosage studied, whereas rhu alpha VEGF had significant inhibition of angiogenesis, noted by microvessel density and CD34 staining. CONCLUSIONS: rhu alpha VEGF has cytostatic clinical activity in this androgen-independent prostate cancer xenograft model, and the addition of paclitaxel demonstrates increased clinical activity. PMID- 12374695 TI - Tumor suppression through angiogenesis inhibition by SUIT-2 pancreatic cancer cells genetically engineered to secrete NK4. AB - NK4, composed of the N-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts not only as a competitive antagonist of HGF but also as an inhibitor of angiogenesis. By studying the antitumor effect of NK4, we evaluated the potential of gene therapy with NK4 as a treatment for pancreatic cancer. Expression vector pcDNA3-NK4 containing NK4 cDNA was used to transfect human pancreatic cancer cell line SUIT-2. Although the established NK4 transfectant continuously expressed NK4 protein, the expression was shown by migration assay to be insufficient to antagonize HGF in vitro. Proliferation of the NK4 transfectant did not differ significantly from that of a mock transfectant. In vivo, we used models of orthotopic implantation and liver metastasis to transplant NK4-transfected clone or mock-transfected clone into nude mice. Cell proliferation in vivo, evaluated by immunohistochemical staining of proliferating cell nuclear antigen, did not differ between NK4 and mock transfectants, and this was also the finding in the in vitro assay. However, the NK4-transfected clone showed significant inhibition of tumor progression in both the orthotopic implantation and liver metastasis models. The number of vessels within tumors was significantly decreased, and the apoptotic tumor cells were increased in number. The results of these experiments show that genetic modification of tumor cells with NK4 cDNA yields a significant antitumor effect and that this effect is mainly obtained by NK4's function as an angiogenesis inhibitor rather than as an HGF antagonist. We conclude that the potent angiogenesis inhibitor NK4 may be a promising molecule for gene therapy of pancreatic cancer. PMID- 12374696 TI - Enhancement of antitumor activity of ionizing radiation by combined treatment with the selective epidermal growth factor receptor-tyrosine kinase inhibitor ZD1839 (Iressa). AB - PURPOSE: The epidermal growth factor receptor (EGFR) is expressed in the majority of human epithelial cancers and has been implicated in the development of cancer cell resistance to cyotoxic drugs and to ionizing radiation. EXPERIMENTAL DESIGN: We used ZD1839, a selective small molecule EGFR tyrosine kinase inhibitor currently in clinical development. We tested the antiproliferative and the proapoptotic activity of ZD1839 in combination with ionizing radiation in human colon (GEO), ovarian (OVCAR-3), non-small cell lung (A549 and Calu-6), and breast (MCF-7 ADR) cancer cell lines. The antitumor activity of this combination was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: With ionizing radiation or ZD1839, a dose-dependent growth inhibition was observed in all of the cancer cell lines growing in soft agar. A cooperative antiproliferative and proapoptotic effect was obtained when cancer cells were treated with ionizing radiation followed by ZD1839. This effect was accompanied by inhibition in the expression of the antiapoptotic proteins bcl-xL and bcl-2, and by a suppression of the activated (phosphorylated) form of akt protein. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT) resulted in a dose-dependent tumor growth inhibition that was reversible upon treatment cessation. Long term GEO tumor growth regressions were obtained after RT in combination with ZD1839. This resulted in a significant improvement in survival of these mice as compared with the control group (P < 0.001), the RT-treated group (P < 0.001), or the ZD1839-treated group (P < 0.001). The only mice alive 10 weeks after tumor cell injection were in the RT plus-ZD1839 group. Furthermore, 10% of mice in this group were alive and tumor free after 26 weeks. Similar results were obtained in mice bearing established human A549 lung adenocarcinoma xenografts. Finally, the combined treatment with RT plus ZD1839 was accompanied by a significant potentiation in the inhibition of transforming growth factor alpha, vascular epidermal growth factor, and basic fibroblast growth factor expression in cancer cells, which resulted in significant antiangiogenic effects as determined by immunohistochemical count of neovessels within the GEO tumors. CONCLUSION: This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective EGFR tyrosine kinase inhibitors such as ZD1839. PMID- 12374697 TI - Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer. AB - PURPOSE AND EXPERIMENTAL DESIGN: Insulin-like growth factors (IGFs) I and II and their principle receptor, IGF-I receptor (IGF-IR), are frequently expressed in human colon cancers and play a role in preventing apoptosis, enhancing cell proliferation, and inducing expression of vascular endothelial growth factor (VEGF). To elucidate the in vitro and in vivo effects of IGF-IR in human colon cancer growth and angiogenesis, HT29 cells were transfected with a truncated dominant-negative (DN) form of IGF-IR or vector alone. RESULTS: IGF-I increased VEGF expression in parental and vector-transfected cells, whereas IGF-I induction of VEGF mRNA and protein was abrogated in IGF-IR DN cells. The IGF-IR DN cells demonstrated inhibited growth in both monolayer culture and soft agar (P < 0.05). s.c. injections of IGF-IR DN cells in nude mice led to significantly decreased tumor growth (P < 0.05). Immunohistochemical analyses revealed that IGF-I DN tumors demonstrated decreased tumor cell proliferation, VEGF expression, and vessel count and increased tumor cell apoptosis (P < 0.05 for all parameters compared with controls). Furthermore, IGF-IR DN-transfected cells yielded significantly decreased tumorigenicity and growth in the liver. CONCLUSIONS: These studies demonstrate that the IGF ligand-receptor system plays an important role in multiple mechanisms that mediate human colon cancer growth including regulation of VEGF and angiogenesis. PMID- 12374698 TI - Prevention and treatment of experimental breast cancer with the combination of a new selective estrogen receptor modulator, arzoxifene, and a new rexinoid, LG 100268. AB - The selective estrogen receptor modulator arzoxifene and the rexinoid LG 100268 were active not only as single agents for prevention and treatment of breast cancer in the rat model that uses nitrosomethylurea as the carcinogen but also showed striking synergy, both preventively and therapeutically, in a series of six experiments with a total of 465 rats. Mechanistic studies in cell culture reported here suggest that enhancement of stromal-epithelial interactions may contribute to this synergy. The possible clinical use of the combination of arzoxifene and LG 100268 for prevention of breast cancer in women at high risk, for treatment of women in the adjuvant setting, or for treatment of end-stage disease should now be considered. PMID- 12374699 TI - Enhanced radiation sensitivity in prostate cancer by inhibition of the cell survival protein clusterin. AB - PURPOSE: The purpose of this study is to evaluate the role of the cell survival gene clusterin in radiation-induced cell death in human LNCaP and PC-3 prostate cancer models. EXPERIMENTAL DESIGN: Radiation sensitivities were compared in parental and clusterin-overexpressing LNCaP cells and in PC-3 cells and tumors treated with antisense or mismatch clusterin oligonucleotides. RESULTS: Clusterin overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell death rates (23% after 8 Gy) compared with parental LNCaP cells (50% after 8 Gy) 3 days after irradiation. Clusterin expression in PC-3 cells after radiation was found to be up-regulated in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by 84% up to 30 Gy. Inhibition of clusterin expression in PC-3 cells using antisense oligonucleotides (ASOs) occurred in a sequence- and dose-dependent manner and significantly enhanced radiation-induced apoptosis and decreased PC-3 cell growth rate and plating efficiency. Compared with mismatch control oligonucleotide treatment, clusterin ASO treatment enhanced radiation therapy and significantly reduced PC-3 tumor volume in vivo by 50% at 9 weeks. In addition, TUNEL staining revealed increased number of apoptotic cells in clusterin ASO-treated and irradiated PC-3 tumors, compared with treatment with mismatch control oligonucleotides plus radiation. CONCLUSIONS: These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis. In vivo results further suggest that inactivation of clusterin using ASO technology might offer a novel strategy to improve results of radiation therapy for prostate cancer patients. PMID- 12374700 TI - Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors. AB - Overexpression of the human epidermal growth factor receptor (HER) 2 has been linked to the development and maintenance of malignant phenotypes in breast tumors. In addition, the growth and dissemination of human cancers are regulated in part by the autocrine motility factor (AMF)/phosphoglucose isomerase shown to be up-regulated by heregulin (HRG) in breast cancer cells. This study was undertaken to explore the effect of anti-HER2 monoclonal antibody 4D5 [Herceptin (HCT)] on AMF expression and the potential of its augmentation by specific simple sugar AMF inhibitors. Here we show that HCT treatment of high HER2-expressing breast cancer SK-BR3, BT-474, and ZR-75R cells resulted in down-regulation of AMF mRNA and protein. HCT inhibited the ability of HRG to induce AMF expression in cells with a normal HER2 level, and HCT-mediated down-regulation could be reversed by HRG treatment in breast cancer cells with a high HER2 level. HCT also inhibited transcription from a chimeric pGL3-Luc vector-based reporter system containing the 1.8-kb promoter region of human AMF. Treatment of breast cancer cells with the combination of HCT and specific AMF inhibitors, erythrose 4 phosphate or D-mannose 6-phosphate, resulted in an additive inhibitory effect on both the growth rate and invasiveness of cells as compared with treatment with each agent alone. Results presented here suggest that HCT can effectively block both ligand-induced and constitutive expression of AMF associated with high HER2 overexpression, implying a role of the AMF pathway in the action of HCT. Accordingly, the combination of AMF inhibitor with HCT can potentiate the growth inhibitory and anti-invasive action of HCT in breast cancer cells. PMID- 12374701 TI - Proapoptotic and antitumor activities of adenovirus-mediated p202 gene transfer. AB - PURPOSE AND EXPERIMENTAL DESIGN: p202, a mouse IFN-inducible protein, is a member of the 200-amino acid repeat family. Enforced p202 expression in stable cancer cell lines resulted in growth inhibition in vitro and tumor suppression in vivo. However, to study the immediate effect of p202 and test the potential efficacy of p202 treatment, an efficient gene delivery system for p202 is required. For these purposes, an adenoviral vector expressing the p202 gene (Ad-p202) was generated. We examined the effects of Ad-p202 infection on human breast cancer cells. Furthermore, we tested the efficacy of Ad-p202 treatment on breast and pancreatic cancer xenograft models. RESULTS: We found that Ad-p202 infection induces growth inhibition and sensitizes the otherwise resistant cells to tumor necrosis factor alpha-induced apoptosis. In addition, we demonstrated for the first time that Ad p202 infection induces apoptosis and that activation of caspases is required for the full apoptotic effect. More importantly, we showed the efficacy of Ad-p202 treatment on breast cancer xenograft models, and this antitumor effect correlated well with enhanced apoptosis in Ad-p202-treated tumors. CONCLUSIONS: We conclude that Ad-p202 is a potent growth-inhibitory, proapoptotic, and tumor-suppressing agent. Ad-p202 may be further developed into an efficient therapeutic agent for human cancer gene therapy. PMID- 12374702 TI - Ad-IFN gamma induces antiproliferative and antitumoral responses in malignant mesothelioma. AB - PURPOSE: The aim of the work was to investigate the effects of adenovirus(Ad) mediated IFN gamma gene transfer on human mesothelioma (HM) cell proliferation in vitro and growth in nude mice. EXPERIMENTAL DESIGN: We constructed an E1E3 deleted replication-defective recombinant Ad carrying the human IFN gamma gene (Ad-IFN gamma) and tested its activity in vitro on HM cell lines established in our laboratory and in a nude mice model. RESULTS: In vitro, infection of HM cells with Ad-IFN gamma led to a prolonged production of an active cytokine in the 10 HM cell lines tested and also led to an antiproliferative effect on the HM cells previously demonstrated as responsive to exogenous recombinant human IFN gamma. In nude mice, s.c. inoculation of HM cells from one responsive HM cell line previously infected with Ad-IFN gamma resulted in a delay in tumor development, and injection of Ad-IFN gamma in preestablished tumors restrained tumor development. CONCLUSIONS: These results indicate for the first time that HM cells are efficiently transduced by Ad-IFN gamma and produce an active cytokine for several days. IFN gamma produced by gene transfer is shown to have both an antiproliferative effect in vitro and an antitumoral effect in vivo in a nude mice model. PMID- 12374703 TI - Airway management: the good, the bad, and the ugly. PMID- 12374704 TI - Changing anesthetic practice: walking to the OR. PMID- 12374705 TI - Myocardial protection by anesthetic agents against ischemia-reperfusion injury: an update for anesthesiologists. AB - PURPOSE: The aim of this review of the literature was to evaluate the effectiveness of anesthetics in protecting the heart against myocardial ischemia reperfusion injury. SOURCE: Articles were obtained from the Medline database (1980-, search terms included heart, myocardium, coronary, ischemia, reperfusion injury, infarction, stunning, halothane, enflurane, desflurane, isoflurane, sevoflurane, opioid, morphine, fentanyl, alfentanil sufentanil, pentazocine, buprenorphine, barbiturate, thiopental, ketamine, propofol, preconditioning, neutrophil adhesion, free radical, antioxidant and calcium). PRINCIPAL FINDINGS: Protection by volatile anesthetics, morphine and propofol is relatively well investigated. It is generally agreed that these agents reduce the myocardial damage caused by ischemia and reperfusion. Other anesthetics which are often used in clinical practice, such as fentanyl, ketamine, barbiturates and benzodiazepines have been much less studied, and their potential as cardioprotectors is currently unknown. There are some proposed mechanisms for protection by anesthetic agents: ischemic preconditioning-like effect, interference in the neutrophil/platelet-endothelium interaction, blockade of Ca2+ overload to the cytosolic space and antioxidant-like effect. Different anesthetics appear to have different mechanisms by which protection is exerted. Clinical applicability of anesthetic agent-induced protection has yet to be explored. CONCLUSION: There is increasing evidence of anesthetic agent-induced protection. At present, isoflurane, sevoflurane and morphine appear to be most promising as preconditioning-inducing agents. After the onset of ischemia, propofol could be selected to reduce ischemia-reperfusion injury. Future clinical application depends on the full elucidation of the underlying mechanisms and on clinical outcome trials. PMID- 12374706 TI - The Amsterdam preoperative anxiety and information scale provides a simple and reliable measure of preoperative anxiety. AB - PURPOSE: To compare three anxiety scales; the anxiety visual analogue scale (VAS), the anxiety component of the Amsterdam preoperative anxiety and information scale (APAIS), and the state portion of the Spielburger state-trait anxiety inventory (STAI), for assessment of preoperative anxiety levels in same day admission patients. METHODS: Patients completed the three anxiety assessment scales both before and after seeing the anesthesiologist preoperatively. The scales used were the STAI, the six-question APAIS, and the VAS. APAIS was further subdivided to assess anxiety about anesthesia (sum A), anxiety about surgery (sum S) and a combined anxiety total (i.e., sum C = sum A + sum S). These scales were compared to one another. Pearson's correlation (pair-wise deletion) was used for validity testing. Cronbach's alpha analysis was used to test internal validity of the various components of the APAIS scale. A correlation co-efficient (r) > or = 0.6 and P < 0.05 were considered significant. RESULTS: Four hundred and sixty three scale sets were completed by 197 patients. There was significant and positive correlation between VAS and STAI r = 0.64, P < 0.001), VAS and APAIS r = 0.6, P < 0.001), sum C and STAI r = 0.63, P < 0.001) and between VAS and sum C r = 0.61, P < 0.001). Sum C and STAI r value were consistent with repeated administration. Cronbach's alpha-levels for the anxiety components of the APAIS (sum C) and desire for information were 0.84 and 0.77 respectively. CONCLUSION: In addition to VAS, the anxiety component of APAIS (sum C) is a promising new practical tool to assess preoperative patient anxiety levels. PMID- 12374707 TI - Silica zeolite scavenging of exhaled isoflurane: a preliminary report. AB - PURPOSE: We evaluate the effectiveness of a silica zeolite (Deltazite) hydrophobic molecular sieve adsorbent, in removing exhaled isoflurane. METHODS: In three experiments, a simulated anesthesia mannequin was ventilated using 1% isoflurane in nitrous oxide and oxygen (1:1 ratio) at a gas flow of 3 L x min-1. Airway pressures, end-tidal carbon dioxide [ETCO2], inspired and end-tidal isoflurane were measured. The scavenging line was connected to a canister containing 750 g of the silica zeolite. Concentrations of isoflurane entering and exiting the canister were measured, as well as the pressure gradient across the canister and gas flow through the canister. In phase 1 (n = 3), the mannequin was ventilated for 6.5 hr, followed by phase 2 where a test lung replaced the simulator. The time (phase 1 plus phase 2) until isoflurane 'breakthrough' (> 0.02%) was noted. RESULTS: The average canister weight increase was 68 g, however 92 g of isoflurane were used. The isoflurane concentration exiting the canister remained undetectable throughout phase 1 in each experiment. The pressure gradient across the canister averaged 0.13 cm H2O and did not increase throughout phase 1. The time to 'breakthrough' (phase 1 plus phase 2) was 8.0 hr, 8.8 hr and 9.0 hr. CONCLUSIONS: Silica zeolite was effective at completely removing 1% isoflurane from exhaled gases for periods of eight hours. The technology shows promise in removing isoflurane emitted from anesthesia machine scavenging systems. PMID- 12374708 TI - QT interval and QT dispersion increase in the elderly during laparoscopic cholecystectomy: a preliminary study. AB - PURPOSE: To compare the influence of a longer duration of intraperitoneal CO2 insufflation with head-up tilt on electrocardiogram indices during laparoscopic cholecystectomy between elderly and younger patients. METHODS: Twelve elderly and 12 younger patients were studied. In all patients, intraperitoneal CO2 insufflation was performed for more than 150 min in the head-up position. RR interval, QT interval, the rate-corrected QT (QTc) interval, QT dispersion (QTD) and the rate-corrected QTD (QTcD) were measured. RESULTS: The QT interval and the QTc interval increased significantly from 120 to 150 min after CO2 insufflation in the elderly. The QTD and QTcD increased significantly during CO2 insufflation in both groups. Those were significantly greater in the elderly than in younger patients from 120 to 150 min after CO2 insufflation. CONCLUSION: Longer duration of CO2 insufflation with head-up tilt is associated with a prolongation of the QT interval and the QTD in elderly patients. The clinical significance of these findings remains to be determined. PMID- 12374709 TI - Total spinal anesthesia provides transient relief of intractable pain. AB - PURPOSE: Intentional total spinal anesthesia (TSA) has been used for intractable pain treatment. However, the long-term effect of pain-relief is controversial. We investigate the short- and long-term effects of pain-relief by TSA. METHODS: Twelve patients with intractable pain participated in a crossover study. All participants received two different treatments in random order at a 30-day interval: i.v. infusion with 300 mg of lidocaine (i.v.-Lido), and TSA with 20 mL of 1.5% lidocaine (TSA-Lido). Pain level at rest was scored with the visual analogue scale (VAS: 0-100), and blood pressure and heart rate were measured before and at two hours, 24 hr, seven days, and 30 days after treatment. Plasma lidocaine concentrations were measured at 0.5, one, and two hours. RESULTS: Heart rate and mean arterial pressure during or after TSA-Lido were similar to those before TSA-Lido. Plasma lidocaine concentrations were similar between the two treatments. No significant difference in any value occurred in the i.v.-Lido treatment. VAS were similar before both treatments (87 +/- 6 for TSA-Lido; 86 +/- 7 for i.v.-Lido). After TSA-Lido, VAS decreased significantly until day seven (two hours, 17 +/- 22, P < 0.01; 24 hr, 43 +/- 20, P < 0.01; seven days, 66 +/- 16, P < 0.01). However, VAS returned to the pre-block values 30 days after TSA Lido. CONCLUSION: Intractable pain was decreased significantly for several days after TSA, but pain-relief was not sustained. PMID- 12374710 TI - Audit of an early feeding program after Cesarean delivery: patient wellbeing is increased. AB - PURPOSE: Early feeding is well tolerated after Cesarean delivery. However, patient wellbeing and nurses' attitudes toward implementation of early feeding have rarely been investigated. METHODS: A quality-assurance program of 18 months duration was implemented because evaluation of traditional practice demonstrated significant deficiencies (phase I). Drinking was then allowed within one hour and feeding within six to eight hours after delivery. Gradual dietary expansion followed according to a detailed program. Three consecutive evaluations (phase II IV) were performed: 1) to measure implementation by the ward nurses; 2) to record the type of food and the volume of water effectively received; 3) to evaluate patients' gastrointestinal tolerance and patients' levels of hunger and thirst and patients' overall satisfaction. RESULTS: In phase I, 60% of patients received nothing by mouth and 28% received only water on the day of surgery (D0). Moderate or severe hunger and thirst were seen in a large portion of these patients (D0, hunger: 38%, thirst: 63%, D1, hunger: 40%, thirst: 28%). Introduction of the program significantly improved patient wellbeing as well as patient satisfaction. No side effects were encountered. CONCLUSION: Hunger and thirst are frequently encountered after Cesarean delivery when patients are allowed to eat only after return of the first flatus. By using a quality-assurance program, it was possible to reduce the incidence and the severity of these distressing symptoms and to improve patients' satisfaction while no side effects were encountered. These beneficial effects were maintained in phase IV suggesting a high acceptance rate from the nursing staff. PMID- 12374711 TI - Intracranial subdural hematoma following dural puncture in a parturient with HELLP syndrome. AB - PURPOSE: To present a case of postpartum bilateral intracranial subdural hematoma after dural puncture during attempted epidural analgesia for labour. CLINICAL FEATURES: This complication occurred following accidental dural puncture in a parturient with thrombocytopenia (99,000 x microL-1) who subsequently developed the syndrome of hemolysis, elevated liver enzymes and low platelets. On the first postoperative day, postdural puncture headache (PDPH) developed. An epidural blood patch (EBP) was deferred to the third postoperative day because of a platelet count of 21,000 x micro L-1. However, the headache intensified from a typical PDPH to one which was not posturally related. A second EBP was abandoned after the injection of 5 mL of blood because of increasing headache during the procedure. Magnetic resonance imaging revealed bilateral temporal subdural hematomas. The patient was managed conservatively and discharged home without any sequelae. CONCLUSION: It is conceivable that thrombocytopenia together with possible abnormal platelet function increased the risk of subdural hematoma. Alternative diagnoses to PDPH should be considered whenever headache is not posturally related. PMID- 12374712 TI - Transcutaneously measured near-infrared spectroscopic liver tissue oxygenation does not correlate with hepatic venous oxygenation in children. AB - PURPOSE: To compare transcutaneous near-infrared spectroscopic (NIRS) measured liver tissue oxygenation with hepatic vein oxygen saturation (SvhO2) in children undergoing cardiac catheterization. METHODS: A NIRS optode (containing an emitter and a receiver of near-infrared light) was placed directly below the right costal arch above the palpable liver in 40 children aged 0.02 to 7.28 yr (median: 1.8 yr). Spatially resolved spectroscopic measured tissue oxygenation index (TOI) was recorded using the NIRO-300. Paired blood samples from the hepatic vein were taken under radiological control for determination of SvhO2 in a co-oxymeter. TOI values were compared with hepatic vein oxygenation, with simultaneously obtained arterial oxygen saturation (SaO2), inferior vena cava SO2 and hemoglobin concentration using simple linear and multi-regression analysis. RESULTS: TOI values ranged from 35% to 73% (58.6 +/- 8.4%); SvhO2 from 32% to 80% (58.4 +/- 14.4%), and arterial SO2 from 54% to 100% (90.0 +/- 11.4%). TOI and hepatic vein oxygen saturation failed to correlate (r = 0.052/P = 0.752). A regression model containing arterial saturation (Delta R2 = 0.177) and the ratio of pulmonary to systemic resistance (Delta R2 = 0.095) explained 27.3% of the observed variance in TOI. In this model, hepatic vein oxygen saturation was no longer significant; explaining only 3.4% of the variance. No other variable retained a significant association. CONCLUSION: Transcutaneously measured NIRS tissue oxygenation with an optode placed over the palpable liver does not correlate with SvhO2. The value is dominated by non-hepatic variables such as arterial saturation and vascular resistances. PMID- 12374713 TI - Propofol decreases cerebral blood flow velocity in anesthetized children. AB - PURPOSE: Propofol, by virtue of its favourable pharmacokinetic profile, is suitable for maintenance of anesthesia by continuous infusion during neurosurgical procedures in adults. It is gaining popularity for use in pediatric patients. To determine the effects of propofol on cerebral blood flow in children, middle cerebral artery blood flow velocity (Vmca) was measured at different levels of propofol administration by transcranial Doppler (TCD) sonography. METHODS: Twelve ASA I or II children, aged one to six years undergoing elective urological surgery were randomized to receive one of two propofol dosing regimens. Half of the patients received propofol in an escalating fashion, initially targeting an estimated steady-state serum concentration of 3 microg x mL-1, which was then doubled. The other half received propofol designed initially to target the high concentration followed by the lower one. In each child anesthesia was induced and maintained with propofol according to the protocol, rocuronium was given to facilitate tracheal intubation, and a caudal epidural block was performed. A TCD probe was placed appropriately to measure Vmca. Cerebral blood flow velocity (CBFV), mean arterial pressure (MAP) and heart rate (HR) were recorded simultaneously at both levels of propofol administration. RESULTS: Twelve patients were studied. At the higher estimated target serum propofol concentration there were significant decreases in Vmca (17%, P < 0.001), MAP (6%, P < 0.002) and HR (8%, P < 0.05) when compared to the lower targeted concentration. CONCLUSION: This study shows that a higher rate of propofol infusion is associated with lower CBFV and MAP values in children. Propofol's cerebral vasoconstrictive properties may be responsible for this finding. PMID- 12374714 TI - Mechanisms of hemodynamic changes during off-pump coronary artery bypass surgery. AB - PURPOSE: To describe the mechanisms of hemodynamic changes during off-pump coronary artery bypass graft surgery (OP-CABG). SOURCE: Pertinent medical literature in the English and French languages was identified through a Medline computerized literature search and a manual search of selected articles, using off-pump coronary artery surgery, beating heart surgery, hemodynamic, and transesophageal echocardiography as key words. Human and animal studies were included. PRINCIPAL FINDING: Hemodynamic variations in OP-CABG may be due to mobilization and stabilization of the heart, or myocardial ischemia occurring during coronary occlusion. Suction type and compression type stabilizers produce hemodynamic effects through different mechanisms. Heart dislocation (90 degrees anterior displacement) and compression of the right ventricle to a greater extent than the left ventricle are responsible for hemodynamic alterations when using suction type stabilizers. Compression of the left ventricular outflow tract and abnormal diastolic expansion secondary to direct deformation of the left ventricular geometry are proposed mechanisms for hemodynamic derangements with compression type stabilizer. Coronary occlusion during the anastomosis can have additional effects on left ventricular function, depending on the status of collateral flow. The value and limitations of electrocardiographic (ECG), hemodynamic and echocardiographic monitoring modalities during OP-CABG are reviewed. CONCLUSIONS: In summary, hemodynamic changes which can either be secondary to the stabilization technique or to transient ischemia represent an important diagnostic challenge during off-bypass procedures. The mechanism can vary according to the stabilization system. Current monitoring such as ECG and hemodynamic monitoring are used but remain limited in establishing the cause of hemodynamic instability. Transesophageal echocardiography is used in selected patients to diagnose the etiology of hemodynamic instability and can direct therapy, particularly in those with severe myocardial systolic and diastolic dysfunction, mild to moderate mitral regurgitation, or for patients who are unstable during the procedure. PMID- 12374715 TI - Management choices for the difficult airway by anesthesiologists in Canada. AB - PURPOSE: This study assessed difficult airway management, training and equipment availability among Canadian anesthesiologists. METHODS: A postal survey of active members of the Canadian Anesthesiologists' Society was conducted in 2000. Respondents chose an induction condition and intubation technique for each of ten difficult airway scenarios. Availability of airway devices in their workplaces was assessed. Chi square analyses were used to compare groups. A P value of < 0.05 was considered statistically significant. RESULTS: Eight hundred and thirty three of 1702 (49%) surveys were returned. Staff comprised 88%, and residents 12%. Fifty-five percent had attended a difficult airway workshop within five years and 30% received mannequin airway training during residency. Direct laryngoscopy (48%) or fibreoptic bronchoscopy (34%) were the preferred techniques for intubation. For laryngeal, subglottic and unstable cervical spine scenarios, awake intubation with fibreoptic bronchoscope was most widely chosen. Asleep intubation with direct laryngoscopy was most commonly selected for trauma scenarios. Availability of difficult airway equipment varied between regions and types of hospital. Cricothyroidotomy equipment and difficult airway carts were not universally available. CONCLUSIONS: Our survey assessed current preferences, training and equipment availability for the difficult airway amongst Canadian anesthesiologists. Direct laryngoscopy and fibreoptic bronchoscopy were the preferred technique for intubation despite widespread availability of newer airway equipment. Lack of certain essential airway equipment and difficult airway training should be addressed. PMID- 12374716 TI - The LMA-ProSeal is an effective alternative to tracheal intubation for laparoscopic cholecystectomy. AB - PURPOSE: To compare LMA-ProSeal (LMA-PS) with endotracheal tube (ETT) with respect to pulmonary ventilation and gastric distension during laparoscopic cholecystectomy. METHODS: We randomized 109 ASA I-III adults to LMA-PS or ETT after stratifying them as non-obese or obese (body mass index > 30 kg x m-2). After preoxygenation, anesthesia was induced with propofol, fentanyl and rocuronium. An LMA-PS (women #4, men #5) or ETT (women 7 mm, men 8 mm) was inserted and the cuff inflated. A #14 gastric tube was passed into the stomach in every patient and connected to continuous suction. Anesthesia was maintained with nitrous oxide, oxygen and isoflurane. Ventilation was set at 10 mL x kg-1 and 10 breaths x min-1. The surgeon, blinded to the airway device, scored stomach size on an ordinal scale of 0-10 at insertion of the laparoscope and upon decompression of the pneumoperitoneum. RESULTS: There were no statistically significant differences in SpO2 or P(ET)CO2 between the two groups before or during peritoneal insufflation in either non-obese or obese patients. Median (range) airway pressure at which oropharyngeal leak occurred during a leak test with LMA-PS was 34 (18-45) cm water. Change in gastric distension during surgery was similar in both groups. Four of 16 obese LMA-PS patients crossed over to ETT because of respiratory obstruction or airway leak. CONCLUSIONS: A correctly seated LMA-PS or ETT provided equally effective pulmonary ventilation without clinically significant gastric distension in all non-obese patients. Further studies are required to determine the acceptability of the LMA-PS for laparoscopic cholecystectomy in obese patients. PMID- 12374717 TI - The reinforced laryngeal mask airway (RLMA) protects the airway in patients undergoing nasal surgery--an observational study of 200 patients. AB - PURPOSE: The laryngeal mask airway (LMA) is used in nasal surgery but there is some concern of tracheal or laryngeal contamination with blood and secretions. We have evaluated the ability of the LMA to prevent airway contamination until full emergence from anesthesia. METHODS: Two hundred adults, ASA I-III patients, undergoing nasal surgery under general anesthesia were included in a prospective observational study. A reinforced LMA, sizes 3-5, was used during surgery and removed with its cuff inflated, in recovery, when the patients awoke. The LMA was examined on its laryngeal aspect for contamination of blood and secretions and scored (0-3) independently by two observers according to soiling (score of 0 = no blood; score of 1 = staining on the cuff; score of 2 = staining on the inside of mask; score of 3 = blood in the tube). RESULTS: The contamination scores were [n (%)]: 0 =174 (87%); 1 = 22 (11%); 2 = 4 (2%); 3 = 0 (0%). CONCLUSION: Ninety eight percent of patients had no or minimal contamination of the LMA. The 2% incidence of grade 2 LMA soiling is low and probably acceptable, since it did not result in symptoms of airway contamination. We suggest that the use of the LMA for nasal surgery may be appropriate. PMID- 12374719 TI - Laryngeal mask cuff inflation at removal does not affect early postoperative laryngopharyngeal morbidity. AB - PURPOSE: We assessed the effect of cuff inflation of the laryngeal mask airway at removal on sore throat, pharyngeal morbidity and airway complications. METHODS: In a prospective randomized trial, we used a standardized technique of anesthesia and of laryngeal mask insertion in 126 consecutive day-case patients. Postoperatively, on eye opening, the masks were removed either inflated (Group A) or deflated (Group B) and examined for blood by a blinded observer. Episodes of coughing, gagging, laryngospasm, hiccups and retching, and symptoms of sore throat and hoarseness were recorded by the same observer. RESULTS: Demographics were similar. Bloodstaining occurred in 21% of patients in Group A (n = 63) vs 13% in Group B (n = 63; P = 0.23); the incidence of sore throat was identical (19%). Group A experienced more hoarseness (22% vs 9%; P = 0.05). Overall airway complications did not differ between groups (19% vs 11%; P = 0.21). CONCLUSION: We conclude that removal of the laryngeal mask airway inflated does not reduce the incidence of sore throat, pharyngeal morbidity or airway complications. PMID- 12374718 TI - The ProSeal laryngeal mask airway: fibreoptic visualization of the glottic opening is associated with ease of insertion of the gastric tube. AB - PURPOSE: To verify if correct ProSeal laryngeal mask airway (PLMA) placement may condition blind insertion of a gastric tube via the PLMA. METHODS: The PLMA was studied in 150 anesthetized patients using a size #4 in (females) and #5 in (males). Its position was determined by inserting a fibrescope in the airway tube. A lubricated gastric tube was inserted through the PLMA drainage tube, recording the number of attempts at insertion. The relationship between fibreoptic glottic visualization score and attempts at gastric tube insertion using the PLMA was tested statistically. RESULTS: Insertion success rate of the PLMA and of the gastric tube was 93.3% and 99.3%, respectively. Ventilation was satisfactory in all patients, irrespective of fibreoptic score value. A significant correlation (Spearman's rank correlation, P = 0.0186) was present between attempts at gastric tube insertion and fibreoptic score. CONCLUSION: Partial or total visualization of the vocal cords makes the success of gastric tube insertion more probable. Considering that fibreoptic visualization of the glottic aperture is associated with ease of insertion of a gastric tube (P < 0.02), the authors recommend adjusting or reinserting the PLMA if difficulty during the initial positioning of the gastric tube is experienced. PMID- 12374720 TI - Case report: pulmonary soiling after one-lung ventilation with a bronchial blocker. AB - PURPOSE: To report a case of pulmonary soiling of the dependent and of the non dependent remaining lung when a Univent tube was used to achieve one-lung ventilation (OLV). CLINICAL FEATURES: A 61-yr-old, 158-cm, 61-kg woman was scheduled for the resection of a lung cancer in the left lower lobe. An internal diameter 7.0-mm Univent tube was inserted under direct laryngoscopy and positioned via fibreoptic bronchoscopy. Prior to termination of OLV, there was no discharge through the blocker's lumen, aspirated just before deflating the cuff. As soon as the cuff was deflated, however, abundant blood-tinged secretions were aspirated. At the end of the operation, the chest radiograph showed haziness in the right upper lobe and in the remaining left upper lobe. The ineffective removal of secretions through the lumen of the blocker may be one of its main disadvantages. The bronchial blocker is always placed in the non-dependent bronchus for OLV, which may increase the probability of contaminating the dependent lung. Before deflating the blocker, we recommend the steep Trendelenburg position and the presence of a fibreoptic bronchoscope with a suction port at the tracheal carina to prevent overflow of secretions and soiling of the dependent lung. CONCLUSION: Whenever a bronchial blocker is used for OLV, we should be cautious about the possibility that secretions accumulated distal to the blocker may contaminate the dependent or the non-dependent remaining lung. PMID- 12374721 TI - High-dose colforsin daropate increases diaphragmatic contractility in dogs. AB - PURPOSE: To evaluate the effects of colforsin daropate, a water-soluble derivate known to improve contractility in fatigued canine diaphragm, at two different doses (low-dose and high-dose) on contractility of the non-fatigued diaphragm of dogs. METHODS: Twenty-four pentobarbitone-anesthetized dogs were divided into three groups of eight each: Group I received no study drug; Group II received low dose (0.2 microg x kg-1 x min-1) colforsin daropate; Group III received high-dose (0.5 microg x kg-1 x min-1) colforsin daropate. Diaphragmatic contractility was assessed by transdiaphragmatic pressure (Pdi). RESULTS: In Group III, with an infusion of high-dose colforsin daropate, Pdi at low-frequency (20 Hz) and high frequency (100 Hz) stimulation increased from baseline values (P < 0.05). Compared with Group I, Pdi at both stimuli increased during colforsin daropate administration in Group III (P < 0.05). In Group II, with an infusion of low- dose colforsin daropate, Pdi to each stimulus did not change. CONCLUSION: Colforsin daropate, only when administered at high-dose, increases contractility of non-fatigued diaphragm in dogs. PMID- 12374723 TI - Audio and video on the Web. PMID- 12374722 TI - Primary cutaneous mucormycosis complicating the use of adhesive tape to secure the endotracheal tube. AB - PURPOSE: To report a rare case of primary cutaneous mucormycosis (PCM), complicating securing of the endotracheal tube with adhesive tape. CLINICAL FEATURES: A 39-yr-old woman with systemic lupus erythematosus (SLE) developed four annular, punched out ulcers with a necrotic centre and elevated border in a linear distribution over the left cheek, under the tape securing the endotracheal tube. A tissue biopsy revealed broad, branching, nonseptate hyphae found in epidermis and dermis consistent with mucormycosis, best demonstrated with silver staining. Cultures were positive for Rhizopus species. Treatment with iv amphotericin B was successful. CONCLUSION: Because of the rarity of the disease and the difficulty of culturing the causative organism, diagnosis of mucormycosis is often elusive. Tissue biopsy and microscopic visualization of nonseptate hyphae with right-angled branching are the only methods for making the diagnosis. Skin biopsy of new ulcerative or plaque-like lesions should be obtained in immunocompromised patients. Early diagnosis and prompt treatment are critical for favourable outcomes in PCM. PMID- 12374724 TI - Relieving anxiety by entering the operating room on foot. PMID- 12374725 TI - Preventing contamination of propofol infusions. PMID- 12374726 TI - Short- and long-term efficacy of oral ketamine in eight chronic-pain patients. PMID- 12374727 TI - Hemodynamic effects of stellate ganglion block: analysis using a model of aortic input impedance. PMID- 12374728 TI - Decreased neck mobility and postoperative complications. PMID- 12374729 TI - Posterior-beveled vs lateral-beveled tracheal tube for fibreoptic intubation. PMID- 12374730 TI - [Effects of intra-hospital transport of severely head injured patients on the parameters of cerebral perfusion]. PMID- 12374731 TI - Use of a laryngeal mask in acute airway obstruction after carotid endarterectomy. PMID- 12374732 TI - Getting across--bacterial type III effector proteins on their way to the plant cell. AB - Pathogenicity of most Gram-negative bacterial plant pathogens depends on hrp (hypersensitive response and pathogenicity) genes, which control the ability to cause disease and to elicit specific defense responses in resistant plants. hrp genes encode a specialized type III secretion (TTS) system that mediates the vectorial delivery of bacterial effector proteins across both bacterial membranes as well as across the eukaryotic plasma membrane into the host cell cytosol. One well-studied effector protein is AvrBs3 from Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot in pepper and tomato. AvrBs3 induces hypertrophy symptoms in susceptible plants and triggers a resistance gene specific cell death reaction in resistant plants. Intriguingly, AvrBs3 has characteristic features of eukaryotic transcription factors, suggesting that it modulates the host's transcriptome. Here, we discuss the TTS system of X.campestris pv. vesicatoria in the light of current knowledge on type III dependent protein secretion in plant pathogenic bacteria. PMID- 12374733 TI - Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli. AB - Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli. Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress. Much less is known of the third protein, MscK (KefA). Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS. While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension. In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K(+), NH(4)(+), Rb(+) or Cs(+) to gate; no activity is observed with Na(+), Li(+) or N-methyl-D glucamine (NMDG). An MscK gain-of-function mutant gates spontaneously in the presence of K(+) or similar ions, and will gate in the presence of Na(+), Li(+) and NMDG, but only when stimulated by membrane tension. Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels. PMID- 12374734 TI - Functions of LIM proteins in cell polarity and chemotactic motility. AB - LimC and LimD are two novel LIM proteins of Dictyostelium, which are comprised of double and single LIM domains, respectively. Green fluorescent protein-fused LimC and LimD proteins preferentially accumulate at areas of the cell cortex where they co-localize with actin and associate transiently with cytoskeleton-dependent dynamic structures like phagosomes, macropinosomes and pseudopods. Furthermore, both LimC and LimD interact directly with F-actin in vitro. Mutant cells that lack either LimC or LimD, or both, exhibit normal growth. They are, however, significantly impaired in growth under stress conditions and are highly sensitive to osmotic shock, suggesting that LimC and LimD contribute towards the maintenance of cortical strength. Moreover, we noted an altered morphology and F actin distribution in LimD(-) and LimC(-)/D(-) mutants, and changes in chemotactic motility associated with an increased pseudopod formation. Our results reveal both unique and overlapping roles for LimC and LimD, and suggest that both act directly on the actin cytoskeleton and provide rigidity to the cortex. PMID- 12374735 TI - Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism. AB - F1 is a 33.5 kDa serine peptidase of the alpha/beta-hydrolase family from the archaeon Thermoplasma acidophilum. Subsequent to proteasomal protein degradation, tricorn generates small peptides, which are cleaved by F1 to yield single amino acids. We have solved the crystal structure of F1 with multiwavelength anomalous dispersion (MAD) phasing at 1.8 A resolution. In addition to the conserved catalytic domain, the structure reveals a chiefly alpha-helical domain capping the catalytic triad. Thus, the active site is accessible only through a narrow opening from the protein surface. Two structures with molecules bound to the active serine, including the inhibitor phenylalanyl chloromethylketone, elucidate the N-terminal recognition of substrates and the catalytic activation switch mechanism of F1. The cap domain mainly confers the specificity for hydrophobic side chains by a novel cavity system, which, analogously to the tricorn protease, guides substrates to the buried active site and products away from it. Finally, the structure of F1 suggests a possible functional complex with tricorn that allows efficient processive degradation to free amino acids for cellular recycling. PMID- 12374736 TI - Genes expressed in the Drosophila head reveal a role for fat cells in sex specific physiology. AB - The downstream effectors of the Drosophila sex determination cascade are mostly unknown and thought to mediate all aspects of sexual differentiation, physiology and behavior. Here, we employed serial analysis of gene expression (SAGE) to identify male and female effectors expressed in the head, and report 46 sex biased genes (>4-fold/P < 0.01). We characterized four novel, male- or female specific genes and found that all are expressed mainly in the fat cells in the head. Tsx (turn on sex-specificity), sxe1 and sxe2 (sex-specific enzyme 1/2) are expressed in males, but not females, and are dependent on the known sex determination pathway, specifically transformer (tra) and its downstream target doublesex (dsx). Female-specific expression of the fourth gene, fit (female specific independent of transformer), is not controlled by tra and dsx, suggesting an alternative pathway for the regulation of some effector genes. Our results indicate that fat cells in the head express sex-specific effectors, thereby generating distinct physiological conditions in the male and female head. We suggest that these differences have consequences on the male and female brain by modulating sex-specific neuronal processes. PMID- 12374737 TI - Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes. AB - Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases. PMID- 12374738 TI - CD40 regulates the processing of NF-kappaB2 p100 to p52. AB - The nf-kb2 gene encodes the cytoplasmic NF-kappaB inhibitory protein p100 from which the active p52 NF-kappaB subunit is derived by proteasome-mediated proteolysis. Ligands which stimulate p100 processing to p52 have not been defined. Here, ligation of CD40 on transfected 293 cells is shown to trigger p52 production by stimulating p100 ubiquitylation and subsequent proteasome-mediated proteolysis. CD40-mediated p52 accumulation is dependent on de novo protein synthesis and triggers p52 translocation into the nucleus to generate active NF kappaB dimers. Endogenous CD40 ligation on primary murine splenic B cells also stimulates p100 processing, which results in the delayed nuclear translocation of p52-RelB dimers. In both 293 cells and primary splenic B cells, the ability of CD40 to trigger p100 processing requires functional NF-kappaB-inducing kinase (NIK). In contrast, NIK activity is not required for CD40 to stimulate the degradation of IkappaBalpha in either cell type. The regulation of p100 processing by CD40 is likely to be important for the transcriptional regulation of CD40 target genes in adaptive immune responses. PMID- 12374739 TI - Tr-kit-induced resumption of the cell cycle in mouse eggs requires activation of a Src-like kinase. AB - Microinjection in mouse eggs of tr-kit, a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa, causes resumption of meiosis through activation of phospholipase Cgamma1 (PLCgamma1) and Ca(2+) mobilization from intracellular stores. We show that the Src-like kinase Fyn phosphorylates Tyr161 in tr-kit and that this residue is essential for tr-kit function. Fyn is localized in the cortex region underneath the plasma membrane in mouse oocytes. Using several approaches, we demonstrate that Fyn associates with tr-kit and that the interaction requires Tyr161. The interaction between tr-kit and Fyn triggers activation of the kinase as monitored by both autophosphorylation and phosphorylation of PLCgamma1. Co-injection of tr-kit with the SH2 domain of Fyn, or pre-treatment with a Fyn inhibitor, impairs oocyte activation, suggesting that activation of Fyn by tr-kit also occurs in vivo. Finally, microinjection of constitutively active Fyn triggers oocyte activation downstream of tr-kit but still requires PLC activity. We suggest that the mechanism by which tr-kit triggers resumption of meiosis of mouse eggs requires a functional interaction with Fyn and phosphorylation of PLCgamma1. PMID- 12374740 TI - A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation. AB - The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases. PMID- 12374741 TI - Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1. AB - Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP). However, topological differences in cleavage resulting in amyloid beta-peptide (Abeta) or the Notch-1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N-terminal Notch-1 Abeta-like fragment (Nbeta). Analysis of Nbeta by MALDI TOF MS revealed that Nbeta is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of betaAPP at position 40 and 42 of the Abeta domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer's disease-associated PS1 mutations similar to the pathological endoproteolysis of betaAPP. Considering these similarities between intramembranous processing of Notch and betaAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/gamma-secretase. PMID- 12374742 TI - SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. AB - Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin-dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch-IC. Notch-IC associates with the DNA-binding protein RBP-Jkappa/CBF 1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP-Jkappa/CBF-1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP-Jkappa/CBF-1-interacting corepressor in a yeast two-hybrid screen. In cotransfection experiments, SHARP-mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP Jkappa-interacting protein. SHARP repressed Hairy/Enhancer of split (HES)-1 promoter activity, inhibited Notch-1-mediated transactivation and rescued Notch-1 induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP-Jkappa to repress transcription of target genes in the absence of activated Notch. PMID- 12374743 TI - Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy. AB - In chronic diseases such as diabetes mellitus, continuous stress stimuli trigger a persistent, self-reinforcing reprogramming of cellular function and gene expression that culminates in the pathological state. Late-onset, stable changes in gene expression hold the key to understanding the molecular basis of chronic diseases. Renal failure is a common, but poorly understood complication of diabetes. Diabetic nephropathy begins with mesangial cell hypertrophy and hyperplasia, combined with excess matrix deposition. The vasoactive peptide endothelin promotes the mesangial cell hypertophy characteristic of diabetic nephropathy. In this study, we examined the signaling pathways and changes in gene expression required for endothelin-induced mesangial cell hypertrophy. Transcriptional profiling identified seven genes induced with slow kinetics by endothelin. Of these, p8, which encodes a small basic helix-loop-helix protein, was most strongly and stably induced. p8 is also induced in diabetic kidney. Mesangial cell hypertrophy and p8 induction both require activation of the ERK, JNK/SAPK and PI-3-K pathways. Small interfering RNA (siRNA)-mediated RNA interference indicates that p8 is required for endothelin-induced hypertrophy. Thus, p8 is a novel marker for diabetic renal hypertrophy. PMID- 12374744 TI - P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium. AB - Stimulation of p21-activated kinase-1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant-negative fragment or by short interference RNA markedly reduced the estrogen receptor-alpha (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the beta lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen-activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function-2 domain of the ER at the N-terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1-mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1-ER pathway in promoting hyperplasia in mammary epithelium. PMID- 12374745 TI - Gcn4 co-ordinates morphogenetic and metabolic responses to amino acid starvation in Candida albicans. AB - Candida albicans is a major fungal pathogen of humans. It regulates its morphology in response to various environmental signals, but many of these signals are poorly defined. We show that amino acid starvation induces filamentous growth in C.albicans. Also, starvation for a single amino acid (histidine) induces CaHIS4, CaHIS7, CaARO4, CaLYS1 and CaLYS2 gene expression in a manner reminiscent of the GCN response in Saccharomyces cerevisiae. These morphogenetic and GCN-like responses are both dependent upon CaGcn4, which is a functional homologue of S.cerevisiae Gcn4. Like ScGcn4, CaGcn4 activates the transcription of amino acid biosynthetic genes via the GCRE element, and CaGcn4 confers resistance to the histidine analogue, 3-aminotriazole. CaGcn4 interacts with the Ras-cAMP pathway to promote filamentous growth, but the GCN-like response is not dependent upon morphogenetic signalling. CaGcn4 acts as a global regulator in C.albicans, co-ordinating both metabolic and morphogenetic responses to amino acid starvation. PMID- 12374746 TI - Control of CBP co-activating activity by arginine methylation. AB - The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. Recently, the KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Here, we report that another domain of CBP is specifically methylated by CARM1 on conserved arginine residues in vitro and in vivo. We also provide functional evidence that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, our data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity. PMID- 12374747 TI - Phosphorylation of CIITA directs its oligomerization, accumulation and increased activity on MHCII promoters. AB - The class II transactivator (CIITA) is the master regulator of major histocompatibility complex class II (MHCII) transcription. Its activity is regulated at the post-transcriptional level by phosphorylation and oligomerization. This aggregation mapped to and depended on the phosphorylation of residues between positions 253 and 321 in CIITA, which resulted in a dramatic accumulation of the protein and increased expression of MHCII genes in human promonocytic U937 cells, which represent immature antigen-presenting cells. Thus, the post-transcriptional modification of CIITA plays an important role in the immune response. PMID- 12374748 TI - Two isoforms of Serpent containing either one or two GATA zinc fingers have different roles in Drosophila haematopoiesis. AB - serpent (srp) encodes a GATA transcription factor essential for haematopoiesis in Drosophila. Previously, Srp was shown to contain a single GATA zinc finger of C terminal type. Here we show that srp encodes different isoforms, generated by alternative splicing, that contain either only a C-finger (SrpC) or both a C- and an N-finger (SrpNC). The presence of the N-finger stabilizes the interaction of Srp with palindromic GATA sites and allows interaction with the Friend of GATA factor U-shaped (Ush). We have examined the respective functions of SrpC and SrpNC during embryonic haematopoiesis. Both isoforms individually rescue blood cell formation that is lacking in an srp null mutation. Interestingly, while SrpC and SrpNC activate some genes in a similar manner, they regulate others differently. Interaction between SrpNC and Ush is responsible for some but not all aspects of the distinct activities of SrpC and SrpNC. Our results suggest that the inclusion or exclusion of the N-finger in the naturally occurring isoforms of Srp can provide an effective means of extending the versatility of srp function during development. PMID- 12374749 TI - SSRP1 functions as a co-activator of the transcriptional activator p63. AB - The p53 homolog p63 is a transcriptional activator. Here, we describe the identification of an HMG1-like protein SSRP1 as a co-activator of p63. Over expression of wild-type, but not deletion mutant, SSRP1 remarkably enhanced p63gamma-dependent luciferase activity, G1 arrest, apoptosis and expression of endogenous PIG3, p21(Waf1/cip1) and MDM2 in human p53-deficient lung carcinoma H1299 cells and mouse embryonic fibroblasts. Also, SSRP1 interacted to p63gamma in vitro and in cells, and resided with p63gamma at the p53-responsive DNA element sites of the cellular endogenous MDM2 and p21(Waf1/cip1) promoters. Moreover, N-terminus-deleted p63 (DeltaN-p63) bound to neither SSRP1 nor its central domain in vitro. Accordingly, SSRP1 was unable to stimulate DeltaN-p63 mediated residual luciferase activity and apoptosis in cells. Finally, the ectopic expression of the central p63-binding domain of SSRP1 inhibited p63 dependent transcription in cells. Thus, these results suggest that SSRP1 stimulates p63 activity by associating with this activator at the promoter. PMID- 12374751 TI - Effects of DNA strand breaks on transcription by RNA polymerase III: insights into the role of TFIIIB and the polarity of promoter opening. AB - Certain deletion mutants of the Brf1 and Bdp1 subunits of transcription factor (TF) IIIB retain the ability to recruit RNA polymerase (pol) III to its promoters, but fail to support promoter opening: deletions within an internal Bdp1 segment interfere with initiation of DNA strand separation, and an N terminal Brf1 deletion blocks propagation of promoter opening past the transcriptional start site. The ability of DNA strand breaks to restore pol III transcription activity to these defective TFIIIB assemblies has been analyzed using U6 snRNA gene constructs. Breaks in a 21 bp segment spanning the transcriptional start rescue transcription in DNA strand-specific and subunit/mutation-specific patterns. A cluster of Bdp1 internal deletions also reverses the inactivation of transcription with wild-type TFIIIB generated by certain transcribed (template) strand breaks near the transcriptional start site. A structure-based model and topological considerations interpret these observations, explain how Bdp1 and Brf1 help to enforce the general upstream--> downstream polarity of promoter opening and specify requirements for polarity reversal. PMID- 12374750 TI - Hmo1, an HMG-box protein, belongs to the yeast ribosomal DNA transcription system. AB - Hmo1 is one of seven HMG-box proteins of Saccharo myces cerevisiae. Null mutants have a limited effect on growth. Hmo1 overexpression suppresses rpa49-Delta mutants lacking Rpa49, a non-essential but conserved subunit of RNA polymerase I corresponding to the animal RNA polymerase I factor PAF53. This overexpression strongly increases de novo rRNA synthesis. rpa49-Delta hmo1-Delta double mutants are lethal, and this lethality is bypassed when RNA polymerase II synthesizes rRNA. Hmo1 co-localizes with Fob1, a known rDNA-binding protein, defining a narrow territory adjacent to the nucleoplasm that could delineate the rDNA nucleolar domain. These data identify Hmo1 as a genuine RNA polymerase I factor acting synergistically with Rpa49. As an HMG-box protein, Hmo1 is remotely related to animal UBF factors. hmo1-Delta and rpa49-Delta are lethal with top3 Delta DNA topoisomerase (type I) mutants and are suppressed in mutants lacking the Sgs1 DNA helicase. They are not affected by top1-Delta defective in Top1, the other eukaryotic type I topoisomerase. Conversely, rpa34-Delta mutants lacking Rpa34, a non-essential subunit associated with Rpa49, are lethal in top1-Delta but not in top3-Delta. PMID- 12374752 TI - Pre-spliceosome formation in S.pombe requires a stable complex of SF1-U2AF(59) U2AF(23). AB - We have initiated a biochemical analysis of splicing complexes in extracts from the fission yeast Schizosaccharomyces pombe. Extracts of S.pombe contain high levels of the spliceosome-like U2/5/6 tri-snRNP, which dissociates into mono snRNPs in the presence of ATP, and supports binding of U2 snRNP to the 3' end of introns, yielding a weak ATP-independent E complex and the stable ATP-dependent complex A. The requirements for S.pombe complex A formation (pre-mRNA sequence elements, protein splicing factors, SF1/BBP and both subunits of U2AF) are analogous to those of mammalian complex A. The S.pombe SF1/BBP, U2AF(59) and U2AF(23) are tightly associated in a novel complex that is required for complex A formation. This pre-formed SF1- U2AF(59)-U2AF(23) complex may represent a streamlined mechanism for recognition of the branch site, pyrimidine tract and 3' splice site at the 3' end of introns. PMID- 12374753 TI - Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins. AB - During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis. PMID- 12374754 TI - 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. AB - 60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm. PMID- 12374755 TI - Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex. AB - The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed. PMID- 12374756 TI - RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. AB - Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway. PMID- 12374757 TI - Site-specific ORC binding, pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins. AB - Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences. In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa. PMID- 12374759 TI - Aggregate formation inhibits proteasomal degradation of polyglutamine proteins. AB - Insoluble protein aggregates are consistently found in neurodegenerative disorders caused by expanded polyglutamine [poly(Q)] repeats. The aggregates contain various components of the ubiquitin/proteasome system, suggesting an attempt of the cell to clear the aberrant substrate. To investigate the effect of expanded poly(Q) repeats on ubiquitin/proteasome-dependent proteolysis, we targeted these proteins for proteasomal degradation by the introduction of an N end rule degradation signal. While soluble poly(Q) proteins were degraded, they resisted proteasomal degradation once present in the aggregates. Stabilization was also observed for proteins that are co-aggregated via interaction with the expanded poly(Q) domain. Introduction of a degradation signal in ataxin-1/Q92 reduced the incidence of nuclear inclusions and the cellular toxicity, conceivably by accelerating the clearance of the soluble substrate. PMID- 12374758 TI - Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities. AB - Enzymatic activities that cleave Holliday junctions are required for the resolution of recombination intermediates and for the restart of stalled replication forks. Here we show that human cell-free extracts possess two distinct endonucleases that can cleave Holliday junctions. The first cleaves Holliday junctions in a structure- and sequence-specific manner, and associates with an ATP-dependent branch migration activity. Together, these activities promote branch migration/resolution reactions similar to those catalysed by the Escherichia coli RuvABC resolvasome. Like RuvC-mediated resolution, the products can be religated. The second, containing Mus81 protein, cuts Holliday junctions but the products are mostly non-ligatable. Each nuclease has a defined substrate specificity: the branch migration-associated resolvase is highly specific for Holliday junctions, whereas the Mus81-associated endonuclease is one order of magnitude more active upon replication fork and 3'-flap structures. Thus, both nucleases are capable of cutting Holliday junctions formed during recombination or through the regression of stalled replication forks. However, the Mus81 associated endonuclease may play a more direct role in replication fork collapse by catalysing the cleavage of stalled fork structures. PMID- 12374760 TI - Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy. AB - X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder with impaired very long-chain fatty acid (VLCFA) metabolism. The disease-associated ABCD1 (ALD) gene encodes a peroxisomal membrane protein, which belongs to the superfamily of ATP-binding cassette transporters. Several treatment regimes have been tried without satisfactory clinical benefit. Recently, the cholesterol lowering drug lovastatin was reported to normalize VLCFA levels in two out of three clinical studies. This investigation aimed to disclose the molecular mechanism of successful reduction of VLCFA accumulation in order to fill in the gap in the understanding how dietary cholesterol lowering affects the levels of VLCFA in patients with X-ALD and to allow more efficacious treatment. Overexpression of ABCD2 (ALDR), the closest relative of ABCD1, restores VLCFA accumulation in cultured ABCD1-deficient cells. Here we show by real-time PCR that the ABCD2 gene is induced in cultured human fibroblasts and monocytes upon sterol depletion via a mechanism requiring the activation of sterol regulatory element-binding proteins (SREBPs), a family of transcription factors that control the metabolism of cholesterol and fatty acids. This is unexpected and the first report that extends the mechanism of transcriptional regulation by SREBPs to a peroxisomal protein, thus providing a closer link between peroxisomes, cholesterol and fatty acid biosynthesis. Using reporter gene studies, site directed mutagenesis and gel shift assays, we identified a functional sterol regulatory element in the proximal promoter region of ABCD2. Finally, we demonstrated that ABCD2 induction by sterol depletion significantly reduced the accumulation of VLCFA in X-ALD fibroblasts. Thus, lowering cholesterol leads to SREBP maturation, increased ABCD2 expression and reduced VLCFA accumulation. PMID- 12374761 TI - Cln3(Deltaex7/8) knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth. AB - Juvenile-onset neuronal ceroid lipofuscinosis (JNCL; Batten disease) features hallmark membrane deposits and loss of central nervous system (CNS) neurons. Most cases of the disease are due to recessive inheritance of an approximately 1 kb deletion in the CLN3 gene, encoding battenin. To investigate the common JNCL mutation, we have introduced an identical genomic DNA deletion into the murine CLN3 homologue (Cln3) to create Cln3( Deltaex7/8) knock-in mice. The Cln3( Deltaex7/8) allele produced alternatively spliced mRNAs, including a variant predicting non-truncated protein, as well as mutant battenin that was detected in the cytoplasm of cells in the periphery and CNS. Moreover, Cln3( Deltaex7/8) homozygotes exhibited accrual of JNCL-like membrane deposits from before birth, in proportion to battenin levels, which were high in liver and select neuronal populations. However, liver enzymes and CNS development were normal. Instead, Cln3( Deltaex7/8) mice displayed recessively inherited degenerative changes in retina, cerebral cortex and cerebellum, as well as neurological deficits and premature death. Thus, the harmful impact of the common JNCL mutation on the CNS was not well correlated with membrane deposition per se, suggesting instead a specific battenin activity that is essential for the survival of CNS neurons. PMID- 12374763 TI - Domain-specific mutations in sequestosome 1 (SQSTM1) cause familial and sporadic Paget's disease. AB - Paget's disease of bone (PDB) is a common disorder characterized by focal abnormalities of increased and disorganized bone turnover. Genetic factors are important in the pathogenesis of PDB, and in previous studies, we and others identified a locus for familial PDB by genome-wide search on 5q35-qter (PDB3). The gene encoding sequestosome 1 (SQSTM1/p62) maps to within the PDB3 critical region, and recent studies have identified a proline-leucine amino acid change at codon 392 of SQSTM1 (P392L) in French-Canadian patients with PDB. We conducted mutation screening of positional candidate genes in the PDB3 locus in patients with PDB, and also identified mutations in the gene encoding SQSTM1 as a common cause of familial and sporadic PDB. Three different mutations were found, all affecting the highly conserved ubiquitin-binding domain. The most common mutation was the P392L change in exon 8, which was found in 13 of 68 families (19.1%). Another mutation-a T insertion that introduces a stop codon at position 396 in exon 8-was found in four (5.8%) families. A third mutation affecting the splice donor site in intron 7 was found in one (1.5%) family. The P392L mutation was also found in 15 of 168 (8.9%) of patients with sporadic PDB and 0 of 160 of age- and sex-matched controls (P<0.0001). These studies confirm that mutations affecting the ubiquitin-binding domain of SQSTM1 are a common cause of familial and sporadic Paget's disease of bone. PMID- 12374762 TI - The inherited blindness associated protein AIPL1 interacts with the cell cycle regulator protein NUB1. AB - Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene have been found in patients with Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. To determine the normal function of AIPL1 and to better understand how mutations in this gene cause disease, we performed a yeast two-hybrid screen to identify AIPL1-interacting proteins in the retina. One of the identified interacting proteins corresponds to NUB1 (NEDD8 Ultimate Buster 1), which is thought to control many biological events, especially cell cycle progression, by downregulating NEDD8 expression. The AIPL1 NUB1 interaction was verified by co-immunoprecipitation studies in Y79 retinoblastoma cells, demonstrating that this interaction occurs within cells that share a number of features with retinal progenitor cells. Furthermore, we examined the localization of the AIPL1 protein within developing and adult retinas, and found that AIPL1 is present in the developing photoreceptor layer of the human retina and within the photoreceptors of the adult retina. Similar to AIPL1, NUB1 is also expressed in the developing and adult retina. Therefore, it is possible that the early-onset form of retinal degeneration seen in LCA patients with AIPL1 mutations may be due to a defect in the regulation of cell cycle progression during photoreceptor maturation. These data raise the possibility that AIPL1 is important for appropriate photoreceptor formation during development and/or survival following differentiation. PMID- 12374764 TI - Pseudohypoparathyroidism type Ib with disturbed imprinting in the GNAS1 cluster and Gsalpha deficiency in platelets. AB - Pseudohypoparathyroidism Ib (PHPIb), characterized by parathyroid hormone resistant hypocalcemia and hyperphosphatemia, is caused by a deregulation in the imprinting status of the GNAS1 cluster, comprising exons XL, NESP55 and 1A and the coding exons of Gsalpha. Differences in methylation of exon 1A and sporadically also of exons XL and NESP55 were found and thought to result in long range effects on Gsalpha expression, limited to the proximal renal tubules. The exact imprinting defect is not precisely localized, and the expected differences in Gsalpha protein level and function are mainly hypothetical. We describe a PHPIb patient with lack of methylation of the exon XL and 1A promoters, and biallelic methylation of the NESP55 promoter. Platelets of this patient show a functional Gs defect, decreased cAMP formation upon Gs-receptor stimulation, normal Gsalpha sequence but reduced Gsalpha protein levels. Transcriptional deregulation between the now biallelically active promoters of both exon 1A and exon 1 of Gsalpha could explain the decreased Gsalpha expression in platelets and presumably in the proximal renal tubules. We found decreased NESP55 and increased XLalphas protein levels in platelets, in agreement with the methylation status of their corresponding first exons. In a megakaryocytic cell line MEG-01, exon 1A is methylated on both alleles, in contrast to the normally maternally methylated exon 1A in leukocytes. Experimental demethylation of exon 1A in MEG-01 cells led to reduced Gsalpha expression, in agreement with the observations in the patient. Platelet studies may therefore allow easy evaluation of disturbances of the GNAS1 cluster in PHPIb patients. PMID- 12374765 TI - Involvement of survival motor neuron (SMN) protein in cell death. AB - Infantile spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN)1 gene. We investigated the role of human (h) SMN protein on cell death in PC12 and Rat-1 cells. hSMN prolonged cell survival in PC12 cells deprived of trophic support and in Rat-1 cells induced to die by activation of the proto-oncogene c-Myc, to similar magnitude as Bcl-2 or IAP-2. While hSMN was ineffective in inhibiting apoptosis induced by ultraviolet light (UV) or etoposide treatment in proliferating PC12 or Rat-1 cells, a protective effect was observed in terminally NGF/dBcAMP-differentiated PC12 cells. hSMN inhibited the onset of apoptosis in NGF/dBcAMP-deprived or UV-treated co-differentiated PC12 cells by preventing cytochrome c release and caspase-3 activation, indicating that its effects are through suppression of the mitochondrial apoptotic pathway. Expressing hSMN deleted for exon 7 (Delta7) or for exons 6 and 7 (Delta6/7), or with the SMA point mutant Y272C, resulted in loss of survival function. Moreover, these mutants also exhibited pro-apoptotic effects in Rat-1 cells. The localization pattern of full-length hSMN in PC12 and Rat-1 cells was similar to that of endogenous SMN: granular labelling in the cytoplasm and discrete fluorescence spots in the nucleus, some of which co-localized with p80 coilin, the characteristic marker of Cajal bodies. However, cytoplasmic and nuclear aggregates were often seen with hSMNDelta7, whereas the hSMNDelta6/7 mutant showed homogenous nuclear labelling that excluded the nucleolus. Thus, our results show that the C-terminal region is critical in suppression of apoptosis by SMN. PMID- 12374766 TI - Molecular and pathological effects of a modifier gene on deficiency of the sodium channel Scn8a (Na(v)1.6). AB - Scn8a encodes an abundant, widely distributed voltage-gated sodium channel found throughout the central and peripheral nervous systems. Mice with different mutant alleles of Scn8a provide models of the movement disorders ataxia, dystonia, tremor and progressive paralysis. We previously reported that the phenotype of the hypomorphic allele of Scn8a, medJ, is dependent upon an unlinked modifier locus, Scnm1. Strain C57BL/6J carries a sensitive allele of the modifier locus that results in juvenile lethality. We now provide evidence that the modifier acts on the splicing efficiency of the mutant splice donor site. Mutant mice display either 90% or 95% reduction in the proportion of correctly spliced mRNA, depending on modifier genotype. The abundance of the channel protein, Na(v)1.6, is also reduced by an order of magnitude in medJ mice, resulting in delayed maturation of nodes of Ranvier, slowed nerve conduction velocity, reduced muscle mass and reduction of brain metabolic activity. medJ mice provide a model for the physiological effects of sodium channel deficiency and the molecular mechanism of bigenic disease. PMID- 12374767 TI - A novel transgenic line of mice exhibiting autosomal recessive male-specific lethality and non-alcoholic fatty liver disease. AB - We have isolated a Meis1a transgenic mouse line exhibiting recessive male specific lethality and non-alcoholic fatty liver disease (NAFLD), which coincides with pubescence and is androgen-dependent. The phenotype is due to disruption of an endogenous locus, since other Meis1a transgenic lines do not exhibit these phenotypes. Necropsy analysis revealed hepatic microvesicular steatosis in pubescent male homozygous mice, which is absent in transgenic females. The transgene insertion site was localized to chromosome 1 and further refined by cloning the flanking regions. Sequence analysis shows that the integration site disrupts a putative metallo-beta-lactamase gene with a 21.3 kb deletion encompassing exons 5-7. PMID- 12374768 TI - Functional association of the parkin gene promoter with idiopathic Parkinson's disease. AB - Loss-of-function mutations in the parkin gene were first identified in autosomal recessive juvenile parkinsonism (AR-JP). Subsequently, parkin mutations were found in many early-onset patients with Parkinson's disease (PD) (<45 years at onset). We hypothesized that parkin gene expression also may contribute to the age-associated risk of idiopathic PD (>50 years at onset). Two single-nucleotide polymorphisms within the parkin core promoter have been identified and assessed. We show one of the variants, -258 T/G, is located in a region of DNA that binds nuclear protein from human substantia nigra in vitro and functionally affects gene transcription. Furthermore, the -258 T/G polymorphism is genetically associated with idiopathic PD, as assessed in a large population-based series of cases and controls. Our results further implicate the parkin gene in the development of Parkinson's disease. PMID- 12374769 TI - Craniofacial expression of human and murine TBX22 correlates with the cleft palate and ankyloglossia phenotype observed in CPX patients. AB - Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a Mendelian, semidominant X-linked disorder and has been described in several large families from different ethnic origins. It is a useful genetic model for non-syndromic cleft palate, a common congenital disorder. Recently, the underlying genetic defect in CPX was identified, where unique mutations were found in the T-box containing transcription factor TBX22. Here we report two new familial cases with novel missense and insertion mutations, each occurring within the T-box domain and highlighting the functional significance of this DNA-binding motif. We describe TBX22 expression in early human development, where expression is found in the palatal shelves and is highest prior to elevation to a horizontal position above the tongue. mRNA is also detected in the base of the tongue in the region of the frenulum that corresponds to the ankyloglossia seen in CPX patients. Other sites of expression include the inferior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop, and the tooth buds themselves. We have also identified the orthologous mouse Tbx22 gene and performed expression analysis in E12.5-E17.5 mouse embryos. The location of mRNA expression closely correlates between mouse and human, while at later stages of development, we also detected expression in mouse lung and whisker follicles. We conclude that expression of TBX22 is entirely consistent with the CPX phenotype and that the mouse should provide a useful model for elucidating its role in craniofacial development. PMID- 12374770 TI - Does the coronary risk factor low density lipoprotein alter growth and signaling in vascular smooth muscle cells? AB - There is increasing evidence that hypertension promotes low density lipoprotein (LDL) transportation into the subendothelial space of the vascular wall. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development and progression of cardiovascular diseases. Recently, several studies have demonstrated that LDL acts as a classic growth factor promoting VSMC growth via mitogenic signals normally elicited by classic growth factors. The present work summarizes current nontraditional concepts regarding possible cellular mechanisms through which hypertension and LDL may promote the development of atherosclerosis. Especially addressed are the possible effects of an elevated blood pressure in combination with LDL on VSMC growth. The new research concept concerning LDL as a growth factor and carrier for biological active phospholipids such as sphingosine-1-phosphate and sphingosylphosphorylcholine may contribute to an understanding of the pathogenesis of atherosclerosis by elevated high blood pressure. PMID- 12374771 TI - Severely reduced neutrophil adhesion and impaired host defense against fecal and commensal bacteria in CD18-/-P-selectin-/- double null mice. AB - Leukocyte recruitment to sites of inflammation requires the functions of selectins and integrins. P-selectin null (CD62P-/-) mice show a mild and CD18 null (CD18-/-) mice a more severe neutrophil recruitment defect in some inflammatory models. To investigate the possible cooperative interactions between CD18 integrins and P-selectin in mediating neutrophil recruitment, we generated CD18-/-CD62P-/- double null mice. CD18-/-CD62P-/- mice were apparently normal at weaning and fertile but later failed to gain weight, showed increased susceptibility to infection by fecal and commensal bacteria, and survived only 5 6 months. Some CD18-/-CD62P-/- mice showed severe spontaneous skin lesions; most showed neutrophil infiltration in the lungs and liver, and positive bacterial cultures from internal organs. The number and velocity of rolling leukocytes in tumor necrosis factor alpha treated venules of CD18-/-CD62P-/- mice was similar to those in wild-type mice, but neutrophil adhesion was severely reduced. Only 25% of adhered leukocytes were neutrophils in CD18-/-CD62P-/- mice vs. >90% in wild-type, CD62P-/-, and CD18-/- single mutants. Our data show that removing both P-selectin and CD18 integrins from mice leads to severe neutrophil recruitment defects and spontaneous pathology. PMID- 12374772 TI - IL-1 beta potentiates heat-activated currents in rat sensory neurons: involvement of IL-1RI, tyrosine kinase, and protein kinase C. AB - Interleukin 1 beta (IL-1 beta) is a proinflammatory cytokine that maintains thermal hyperalgesia and facilitates the release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Brief applications of IL-1 beta to nociceptive neurons yielded a potentiation of heat-activated inward currents (Iheat) and a shift of activation threshold toward lower temperature without altering intracellular calcium levels. The IL-1 beta-induced heat sensitization was not dependent on G-protein-coupled receptors but was mediated by activation of protein kinases. The nonspecific protein kinase inhibitor staurosporine, the specific protein kinase C inhibitor bisindolylmaleimide BIM1, and the protein tyrosine kinase inhibitor genistein reduced the sensitizing effect of IL-1 beta whereas negative controls were ineffective. RT-PCR and in situ hybridization revealed IL-1RI but not RII expression in neurons rather than surrounding satellite cells in rat dorsal root ganglia. IL-1 beta acts on sensory neurons to increase their susceptibility for noxious heat via an IL-1RI/PTK/PKC-dependent mechanism. PMID- 12374773 TI - Negative regulatory role of overexpression of PLC gamma 1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts. AB - The early growth response 1 (Egr-1) gene product is a transcription factor that functions as an oikis factor. Loss of Egr-1 expression is closely associated with tumor formation. Phospholipase Cgamma1 (PLCgamma1) is overexpressed in some tumors, and its overexpression causes anchorage-independent growth. Here we report that overexpression of PLCgamma1 and SH2-SH3 domain of PLCgamma1 decreased induction of Egr-1 and the Egr-1-regulated genes TSP-1 and PAI-1. Results from the nuclear run-on assay and transfection experiment with the proximal 455 base pair region of the Egr-1 promoter (-454 to +1) showed that Egr-1 transcriptional activity was suppressed in PLCgamma1-3Y1 cells whereas decay of Egr-1 mRNA was similar in both cell lines. Serum response element- and ternary complex factor Elk-1-mediated transcriptional activation of the reporter gene in response to EGF were also inhibited in PLCgamma1-3Y1 cells. Pretreatment with the protein synthesis inhibitor cycloheximide (CHX) partially abrogated the serum-induced suppression of Egr-1 transcription in PLCgamma1-3Y1 cells, suggesting that a CHX sensitive factor(s) is involved in the suppression of Egr-1 transcription in PLCgamma1-3Y1 cells. Our results demonstrated that overexpression of PLCgamma1 functions as a negative modulator of the tumor suppressor Egr-1 gene expression, possibly through inhibition of Elk-1-dependent transcriptional activity. PMID- 12374774 TI - Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway. AB - Proteinase-activated receptor 1 (PAR-1) is activated by thrombin and induces chloride secretion by intestinal epithelial cells. To elucidate further the mechanisms whereby PAR-1 stimulates secretion, monolayers of SCBN intestinal epithelial cells were studied in modified Ussing chambers. Short circuit current responses were determined after basolateral application of thrombin and the PAR-1 activating peptide, Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-Citrulline-Tyr (Cit NH2) in the presence or absence of a variety of signal transduction and cyclo oxygenase (COX) pathway inhibitors. Increased kinase activity was monitored by immunoprecipitation and Western blot analysis of target phosphoproteins. The PAR 1-induced chloride secretory response was significantly attenuated by inhibitors of the EGF receptor tyrosine kinase, Src-kinase, MEK1/2, as well as by inhibitors of cytosolic phospholipase (cPL) A2, COX-1 and COX-2. PAR-1-induced activation of cPLA2, as shown by Western blot of phosphoserine residues, was blocked in cells treated with the MEK inhibitor U0126, indicating that the MEK-ERK1/2 MAP kinase pathway mediated PAR-1-induced cPLA2 phosphorylation. Our data show that PAR-1 induced chloride secretion in SCBN cells involves Src, EGF receptor trans activation, activation of a MAPK pathway, phosphorylation of cPLA2, COX activity, but not PGF2alpha or PGE2. These findings may be of clinical importance in inflammatory diseases of the intestine where secretory dysfunction is evident and thrombin levels are elevated. PMID- 12374775 TI - Plasma membrane cholesterol controls the cytotoxicity of Alzheimer's disease AbetaP (1-40) and (1-42) peptides. AB - Cell degeneration in Alzheimer's disease is mediated by a toxic mechanism that involves interaction of the AbetaP peptide with the plasma membrane of the target cell. We report here that PC12 cells become resistant to the cytotoxic action of AbetaP when incubated in a medium that enriches cholesterol levels of the surface membrane. On the other hand, making cholesterol-deficient membranes by either cholesterol extraction with cyclodextrin or by inhibiting de novo synthesis of cholesterol makes PC12 cells more vulnerable to the action of AbetaP. Increasing cholesterol content of PS liposomes also suppresses AbetaP-dependent liposome aggregation. We suggest that by modifying the fluidity of the neuronal membranes, cholesterol modulates the incorporation and pore formation of AbetaP into cell membranes. This idea is supported by our finding that the enhanced cytotoxicity generated by lowering the membrane cholesterol content can be reversed by AbetaP calcium channel blockers Zn2+ and tromethamine. PMID- 12374776 TI - Testosterone, cytochrome P450, and cardiac hypertrophy. AB - Cytochrome P450 mono-oxygenases (CYP) play an essential role in steroid metabolism, and there is speculation that sex hormones might influence cardiac mass and physiology. As CYP mono-oxygenases activity is frequently altered during disease, we tested our hypothesis that CYP mono-oxygenase expression and testosterone metabolism are altered in cardiac hypertrophy. We investigate major CYP mono-oxygenase isoforms and other steroid-metabolizing enzymes and the androgen receptor in normal, hypertrophic, and assist device-supported human hearts and in spontaneously hypertensive rats (SHR). We show increased and idiosyncratic metabolism of testosterone in hypertrophic heart and link these changes to altered CYP mono-oxygenase expression. We show significant induction of 5-alpha steroid reductase and P450 aromatase gene expression and enhanced production of dihydrotestosterone, which can be inhibited by the 5-alpha reductase inhibitor finasteride. We show increased gene expression of the androgen receptor and increased levels of lipid peroxidation in diseased hearts, the latter being markedly inhibited by CYP mono-oxygenase inactivation. We show alpha-MHC to be significantly repressed in cardiac hypertrophy and restored to normal on testosterone supplementation. We conclude that heart-specific steroid metabolism is of critical importance in cardiac hypertrophy PMID- 12374777 TI - Characterization of a novel metabolic strategy used by drug-resistant tumor cells. AB - Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug-resistant cells from drug-sensitive cells. This strategy may serve to protect drug-resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug-resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug-resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance. PMID- 12374778 TI - Stem cell differentiation requires a paracrine pathway in the heart. AB - Members of the transforming growth factor beta1 (TGF-beta) superfamily--namely, TGF-beta and BMP2--applied to undifferentiated murine embryonic stem cells up regulated mRNA of mesodermal (Brachyury) and cardiac specific transcription factors (Nkx2.5, MEF2C). Embryoid bodies generated from stem cells primed with these growth factors demonstrated an increased potential for cardiac differentiation with a significant increase in beating areas and enhanced myofibrillogenesis. In an environment of postmitotic cardiomyocytes, stem cells engineered to express a fluorescent protein under the control of a cardiac promoter differentiated into fluorescent ventricular myocytes beating in synchrony with host cells, a process significantly enhanced by TGF-beta or BMP2. In vitro, disruption of the TGF-beta/BMP signaling pathways by latency-associated peptide and/or noggin prevented differentiation of stem cells. In fact, only host cells that secrete a TGF-beta family member induced a cardiac phenotype in stem cells. In vivo, transplantation of stem cells into heart also resulted in cardiac differentiation provided that TGF-beta/BMP2 signaling was intact. In infarcted myocardium, grafted stem cells differentiated into functional cardiomyocytes integrated with surrounding tissue, improving contractile performance. Thus, embryonic stem cells are directed to differentiate into cardiomyocytes by signaling mediated through TGF-beta/BMP2, a cardiac paracrine pathway required for therapeutic benefit of stem cell transplantation in diseased heart. PMID- 12374779 TI - Protection of innate immunity by C5aR antagonist in septic mice. AB - Innate immune functions are known to be compromised during sepsis, often with lethal consequences. There is also evidence in rats that sepsis is associated with excessive complement activation and generation of the potent anaphylatoxin C5a. In the presence of a cyclic peptide antagonist (C5aRa) to the C5a receptor (C5aR), the binding of murine 125I-C5a to murine neutrophils was reduced, the in vitro chemotactic responses of mouse neutrophils to mouse C5a were markedly diminished, the acquired defect in hydrogen peroxide (H2O2) production of C5a exposed neutrophils was reversed, and the lung permeability index (extravascular leakage of albumin) in mice after intrapulmonary deposition of IgG immune complexes was markedly diminished. Mice that developed sepsis after cecal ligation/puncture (CLP) and were treated with C5aRa had greatly improved survival rates. These data suggest that C5aRa interferes with neutrophil responses to C5a, preventing C5a-induced compromise of innate immunity during sepsis, with greatly improved survival rates after CLP. PMID- 12374780 TI - Angiogenesis stimulated by PDGF-CC, a novel member in the PDGF family, involves activation of PDGFR-alphaalpha and -alphabeta receptors. AB - A newly discovered PDGF isoform, PDGF-CC, is expressed in actively angiogenic tissues such as placenta, some embryonic tissues, and tumors. We test the possibility that PDGF-CC promotes angiogenesis in vivo. The core domain (mature form) of human PDGF-CC is sufficiently potent to stimulate neovascularization in the mouse cornea. The corneal angiogenic response induced by PDGF-CC is robust although the area of neovascularization is smaller than those of FGF-2- and VEGF stimulated angiogenesis. Similarly, PDGF-BB and PDGF-AB induce angiogenic responses virtually indistinguishable from PDGF-CC-stimulated vessels. In contrast, PDGF-AA displays only a weak angiogenic response in the mouse cornea. Although there was no significant difference in incorporation of mural cells to the newly formed blood vessels induced by PDGF-BB and -CC, the percentage of mural cell positive vessels induced by PDGF-AA was greater than those induced by FGF-2, PDGF-BB, and PDGF-CC. In the developing chick embryo, PDGF-CC induced branch sprouts from established blood vessels. In PDGF receptor-transfected endothelial cells, PDGF-CC activated the PDGF receptor alpha subunit (PDGFR alpha). PDGF-CC, but not PDGF-AA, was able to activate PDGFR-beta receptor in endothelial cells that coexpress both alpha and beta forms of receptors. Thus, the PDGF-CC-mediated angiogenic response is most likely transduced by PDGF alphaalpha and -alphabeta receptors. These data demonstrate that the PDGF family is a complex and important group of proangiogenic factors. PMID- 12374782 TI - Genetic and pharmacological dissection of pathways involved in the angiotensin II mediated depression of baroreflex function. AB - Heart failure and hypertension are associated with increases in angiotensin II (ANG II) activity. One brain area where ANG II effects may be particularly important in these situations is the nucleus of the solitary tract (NTS). Located in the dorsomedial medulla, the NTS is the termination site of baroreceptor afferents and is essential for mediating the baroreflex. In hypertensive animals the baroreflex is impaired; this may be reversed by antagonizing ANG II AT1 receptors in the NTS. Recently, we showed that the baroreflex depressant action of ANG II in the NTS is mediated by activation of endothelial nitric oxide synthase (eNOS) and enhanced release of GABA. Using conventional pharmacological tools and a range of adenoviral-mediated expression of dominant negative proteins, we have determined the intracellular pathway(s) in the NTS by which ANG II activates eNOS. Our data indicate that ANG II acting in the NTS depresses the baroreflex via a Gq protein-mediated activation of phospholipase C, which through 1,4,5-inositol triphosphate causes release of calcium from the IP3-sensitive intracellular stores and calcium-calmodulin formation. In contrast, multiple site disruption of a pathway leading to eNOS activation via the serine/threonine kinase Akt was ineffective PMID- 12374781 TI - Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism. AB - Adenosine is released from the myocardium, endothelial cells, and skeletal muscle in ischemia and is an important regulator of coronary blood flow. We have already shown that acute (2 min) activation of A2a purinoceptors stimulates NO production in human fetal umbilical vein endothelial cells (1) and now report a key role for p42/p44 mitogen-activated protein kinases (p42/p44MAPK) in the regulation of the l-arginine-nitric oxide (NO) signaling pathway. Expression of mRNA for the A2a-, A2b-, and A3-adenosine receptor subtypes was abundant whereas A1-adenosine receptor mRNA levels were negligible. Activation of A2a purinoceptors by adenosine (10 microM) or the A2a receptor agonist CGS21680 (100 nM) resulted in an increase in l-arginine transport and NO release that was not mediated by changes in intracellular Ca2+, pH, or cAMP. Stimulation of endothelial cells with adenosine was associated with a membrane hyperpolarization and phosphorylation of p42/p44MAPK. l-NAME abolished the adenosine-induced hyperpolarization and stimulation of l-arginine transport whereas sodium nitroprusside activated an outward potassium current. Genistein (10 microM) and PD98059 (10 microM), an inhibitor of MAPK kinase 1/2 (MEK1/2), inhibited adenosine-stimulated l-arginine transport, NO production, and phosphorylation of p42/p44MAPK. We found no evidence for activation of eNOS via the serine/threonine kinase Akt/PKB (protein kinase B) in adenosine-stimulated cells. Our results provide the first evidence that adenosine stimulates the endothelial cell l-arginine-NO pathway in a Ca2+ insensitive manner involving p42/p44MAPK, with release of NO leading to a membrane hyperpolarization and activation of l-arginine transport. PMID- 12374783 TI - Ezrin turnover and cell shape changes catalyzed by proteasome in oxidatively stressed cells. AB - We find that ezrin, a cytoskeletal protein involved in anchoring actin to the cell membrane, is preferentially degraded and resynthesized after oxidative stress. Ezrin was identified using 2-dimensional gels and amino-terminal microsequencing as one of a select few [35S]methionine prelabeled proteins degraded in clone 9 rat liver cells exposed to hydrogen peroxide (H2O2). Metabolic labeling of cellular proteins with [35S]methionine after oxidative stress showed that resynthesis of ezrin rose dramatically but carboxyl terminus anti-ezrin monoclonal antibodies revealed constant intracellular ezrin levels; in other words, degradation and resynthesis were exactly matched. Ezrin degradation was blocked by selective inhibitors of the proteasome (lactacystin, NLVS, and epoxomycin) and by an antisense oligonucleotide directed against the proteasome C2 subunit. H2O2 also caused major changes in cell shape, including significant increases in cell diameter, which must require substantial cytoskeletal rearrangement. Peroxide-induced increases in cell diameter were, however, blocked by the selective proteasome inhibitor lactacystin. The degradation and resynthesis of ezrin may therefore be an underlying mechanism for overall cell shape changes observed during oxidative stress. Oxidative stress induces extensive protein oxidation and degradation and significant increases in cell blebbing, rounding-up, and overall size. Our results indicate that all these oxidant-induced changes may actually be catalyzed by the proteasome. PMID- 12374784 TI - Nitric oxide promotes intracellular calcium release from mitochondria in striatal neurons. AB - Overproduction of nitric oxide by NMDA receptor stimulation is implicated in calcium deregulation and neurodegeneration of striatal neurons. We investigated the involvement of nitric oxide (NO) in inducing intracellular calcium release and in modifying calcium transients evoked by NMDA. NO application (4-10 microM) reversibly and repeatedly increased the intracellular calcium concentration [Ca2+]i in Fura-2- or fluo-3-loaded cultured mouse striatal neurons. NO-induced [Ca2+]i responses persisted in the absence of extracellular calcium, indicating that Ca2+ was released from intracellular stores. The source of calcium was distinct from [Ca2+]i-activated (ruthenium red and ryanodine sensitive) or IP3 activated (thapsigargin-sensitive) Ca2+ stores and was not dependent on cGMP production because a cell permeant analog, 8-bromo-cGMP, did not increase basal [Ca2+]i. Glucose removal potentiated the NO-induced release of [Ca2+]i. In contrast, pretreatment with either the mitochondrial uncoupler carbonyl cyanide m chlorophenylhydrazone or cyclosporin A, a blocker of the mitochondrial permeability transition pore, prevented the [Ca2+]i increase after NO. The rise in [Ca2+]i during NO exposure was preceded by a decrease in mitochondrial membrane potential that was partly reversible during washout. Repeated applications of NMDA induced irreversible [Ca2+]i responses in a subpopulation of striatal cells that were greatly reduced by the NOS inhibitor N omega-nitro-l arginine. Calcium transients were prolonged by conjoint application of NMDA and NO. We conclude that NMDA-evoked [Ca2+]i transients are modulated by endogenous NO production, which leads to release of calcium from the mitochondrial pool. An NO-activated mitochondrial permeability transition pore may lead to cell death after overstimulation of NMDA receptors. PMID- 12374785 TI - Family ties of gated pores: evolution of the sensor module. AB - The six-transmembrane channels are thought to be composed of two modules: pore and sensor. Whereas the modular design of the pore has been established, the modularity of the sensor remains hypothetical. As a first step toward establishing the modularity of this region, we searched for genes where the sensor is found independent of the pore and have identified new members of the sensor superfamily. Analysis of these sensors reveals a motif shared among not only these newly discovered members and voltage-gated, transient receptor potential, and polycystin channel sensors, but also MscL, a bacterial mechanosensitive channel. Mutational analyses presented here and in previous studies demonstrate that highly conserved residues within this motif are required for normal channel activity; mutations of residues within this motif in different subfamilies lead to consistent channel phenotypes. Previous studies have demonstrated that peptides containing this motif and the adjacent conserved transmembrane domain elicit channel activities when reconstituted into lipid membranes. These data provide evidence for the modularity of the sensor, imply a model for its evolution, suggest a common origin for mechano- and voltage sensing, and may offer a glimpse of the properties of the first sensor/channel. PMID- 12374786 TI - Contribution of the Kir3.1 subunit to the muscarinic-gated atrial potassium channel IKACh. AB - The muscarinic-gated atrial potassium (I(KACh)) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I(KACh) is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I(KACh), the contribution of Kir3.1 to I(KACh) is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cell-attached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I(KACh). We conclude that Kir3.1 confers properties to I(KACh) that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current. PMID- 12374787 TI - Impaired proliferation and survival of activated B cells in transgenic mice that express a dominant-negative cAMP-response element-binding protein transcription factor in B cells. AB - The cAMP-response element-binding protein (CREB) is activated by phosphorylation on serine 133 and mediates the proliferative response to a number of different signals. A mutant CREB with a serine to alanine substitution at position 133 (CREBM1) functions as a dominant-negative inhibitor. Transgenic mice that express the dominant-negative CREB protein in B lymphocytes were developed as a means to study the effects of the inhibition of CREB function on B-cell proliferation and survival. We have shown previously that CREB up-regulates Bcl-2 expression in B cells in response to activation signals. B cells from CREBM1 transgenic mice expressed lower levels of Bcl-2 with and without stimulation. Proliferation of B cells from the transgenic mice was impaired in part by lack of induction of activator protein 1 (AP1) transcription factors. B cells from the transgenic mice were more susceptible to induction of apoptosis with several different agents, consistent with the decreased expression of Bcl-2. These studies demonstrate that B-cell activation requires phosphorylation of CREB for the proliferative response and to protect against activation-induced apoptosis. PMID- 12374788 TI - Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro. AB - Membrane-bound transferrin-like protein (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H10T1/2 cells. In ATDC5 cultures, bone morphogenetic protein-2 and transforming growth factor-beta as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf protein level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to 10 microg/ml insulin. MTf overexpression in C3H10T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype. These findings suggest that the expression of MTf on the cell surface facilitates the differentiation of prechondrogenic cells, although MTf overexpression alone seems to be insufficient to commit pluripotent mesenchymal cells to the chondrocyte lineage. PMID- 12374789 TI - Utilization of a novel recombinant myoglobin fusion protein expression system to characterize the tissue inhibitor of metalloproteinase (TIMP)-4 and TIMP-2 C terminal domain and tails by mutagenesis. The importance of acidic residues in binding the MMP-2 hemopexin C-domain. AB - Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A induced cellular activation of the enzyme. Measurement of k(on) rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by approximately 2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C TIMP-4 (V193E/Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by approximately 75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu(192) and Asp(193) in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2. PMID- 12374790 TI - Location of N-unsubstituted glucosamine residues in heparan sulfate. AB - Functional properties of heparan sulfate (HS) are generally ascribed to the sulfation pattern of the polysaccharide. However, recently reported functional implications of rare N-unsubstituted glucosamine (GlcNH(2)) residues in native HS prompted our structural characterization of sequences around such residues. HS preparations were cleaved with nitrous acid at either N-sulfated or N unsubstituted glucosamine units followed by reduction with NaB(3)H(4). The labeled products were characterized following complementary deamination steps. The proportion of GlcNH(2) units varied from 0.7-4% of total glucosamine in different HS preparations. The GlcNH(2) units occurred largely clustered at the polysaccharide-protein linkage region in intestinal HS, also more peripherally in aortic HS. They were preferentially located within N-acetylated domains, or in transition sequences between N-acetylated and N-sulfated domains, only 20-30% of the adjacent upstream and downstream disaccharide units being N-sulfated. The nearest downstream (toward the polysaccharide-protein linkage) hexuronic acid was invariably GlcUA, whereas the upstream neighbor could be either GlcUA or IdoUA. The highly sulfated but N-unsubstituted disaccharide unit, -IdoUA2S-GlcNH(2)6S-, was detected in human renal and porcine intestinal HS, but not in HS from human aorta. These results are interpreted in terms of a biosynthetic mechanism, whereby GlcNH(2) residues are formed through regulated, incomplete action of an N deacetylase/N-sulfotransferase enzyme. PMID- 12374791 TI - Osteoclast inhibitory lectin, a family of new osteoclast inhibitors. AB - We have identified two novel type II membrane-bound C-lectins, designated mOCILrP1 and mOCILrP2, of 218 and 217 amino acids, respectively, that share substantial identity with the murine osteoclast inhibitory lectin (OCIL). The extracellular domains of mOCILrP1 and mOCILrP2 share 83 and 75% identity, respectively, with the extracellular domain of mOCIL. When the extracellular domains were expressed as recombinant proteins, each inhibited osteoclast formation in murine bone marrow cultures treated with M-CSF and RANKL with similar potencies to mOCIL (IC(50) of 0.2 ng/ml). Distinct but highly related genes encoded the three OCIL family members, with mOCIL and mOCILrP2 controlled by an inverted TATA promoter, and mOCILrP1 by a TTAAAA promoter. However only mOCIL was robustly regulated by calciotropic agents, while mOCILrP1 was not expressed, and mOCILrP2 was constitutively expressed in osteoblasts. Immunohistochemistry using antipeptide antibodies to the intracellular domain of mOCILrP1/mOCILrP2 and to mOCIL demonstrated that mOCIL and mOCILrP1/mOCILrP2 were concordantly expressed in osteoblasts, chondrocytes, and in extraskeletal tissues. Further, their cellular distribution was identical to that of RANKL. The identification of three distinct genes that were functionally related implies redundancy for OCIL, and their concordant expression with that of RANKL suggests that the RANKL:OPG axis may be further influenced by OCIL family members. PMID- 12374792 TI - Usage of tautomycetin, a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo. AB - Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 microm TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS 7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo. PMID- 12374793 TI - Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells. AB - Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3 deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1 stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1. PMID- 12374794 TI - Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region. AB - The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3. PMID- 12374795 TI - Stoichiometry of active smad-transcription factor complexes on DNA. AB - Transforming growth factor (TGF)-beta signals through a heteromeric complex of serine/threonine kinase receptors. The type I receptor phosphorylates and activates the receptor-regulated Smads (R-Smads), Smad2 and Smad3, which form hetero-oligomeric complexes with the co-Smad, Smad4, and translocate to the nucleus. Smad3 and Smad4 can bind directly to consensus DNA-binding elements in the promoters of target genes, whereas Smad2/Smad4 complexes are targeted to DNA by interacting with sequence-specific DNA-binding transcription factors that contain a well-defined Smad interaction motif (SIM). The exact stoichiometry of Smad homo- and hetero-oligomers both before and after ligand stimulation is controversial. Here we determine the stoichiometry of TGF-beta-induced Smad transcription factor complexes on DNA. We show that complexes of Smad2/Smad4 with the transcription factors Fast-1 or Fast-3 contain one Fast, two Smad2s, and one Smad4. In contrast, Smad3/Smad4 complexes that bind the Smad-binding element from the c-jun promoter, are heterodimers. Furthermore, these Smad3/Smad4 complexes contain at least two additional components essential for complex formation, one of which contains a SIM. Our data suggest that the R-Smads can form heterodimers or heterotrimers with Smad4, and we propose that the exact stoichiometries of active Smad complexes on DNA may be determined by the transcription factors with which they associate. PMID- 12374796 TI - The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment. AB - The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine. PMID- 12374797 TI - Purification, characterization, cloning, and expression of a novel xyloglucan specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase. AB - A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed in Escherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained. PMID- 12374798 TI - Transcriptional activation of the MUC2 gene by p53. AB - MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types. PMID- 12374799 TI - Regulation of WNK1 by an autoinhibitory domain and autophosphorylation. AB - WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity. PMID- 12374800 TI - Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation. AB - The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2 dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions. PMID- 12374801 TI - Follicle-stimulating hormone interacts with exoloop 3 of the receptor. AB - The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of approximately 330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of (580)KVPLITVSKAK(590) was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH alpha but not FSH beta. This is in contrast to photoaffinity labeling of FSH beta by the peptide mimic of the N-terminal region of the receptor. Leu(583) and Ile(584) are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu(583) and Ile(584) side chains appear to project in opposite directions. Ile(584) appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu(583) is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu(583) improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile(584) and Lys(590) abolished the cAMP and IP induction. Our results open a logical question whether Leu(583), Ile(584), and Lys(590) interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation. PMID- 12374803 TI - The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism. AB - Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli. PMID- 12374802 TI - Acetylation of interferon regulatory factor-7 by p300/CREB-binding protein (CBP) associated factor (PCAF) impairs its DNA binding. AB - Interferon regulatory factor 7 (IRF7) is an interferon-inducible transcription factor required for induction of delayed early interferon alpha genes and the onset of a potent antiviral state. After induction of IRF7 by autocrine interferon, latent IRF7 is activated by virus-induced phosphorylation on serine residues within the C-terminal regulatory domain. Although it is likely that IRF7 is subjected to a cascade of events responsible for regulating its biological activity, to date no mechanism other than phosphorylation has been reported to modulate IRF7 activity. Here, we report that IRF7 is acetylated in vivo by the histone acetyltransferases p300/CBP-associated factor (PCAF) and GCN5. The single lysine residue target for acetylation, lysine 92, is located in the DNA-binding domain and is conserved throughout the entire IRF family. Mutation of lysine 92 resulted in complete abolition of DNA binding ability. However, a mutant that cannot be acetylated by PCAF due to a change in the surrounding amino acid context of lysine 92 showed increased DNA binding and activity compared with wild type IRF7. Conversely, we showed that acetylated IRF7 displayed impaired DNA binding capability and that over-expression of PCAF led to decreased IRF7 activity. Together, our results strongly suggest that acetylation of lysine 92 negatively modulates IRF7 DNA binding. PMID- 12374804 TI - Hypertonicity is involved in redirecting the aquaporin-2 water channel into the basolateral, instead of the apical, plasma membrane of renal epithelial cells. AB - In renal collecting ducts, vasopressin increases the expression of and redistributes aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane, leading to urine concentration. However, basolateral membrane expression of AQP2, in addition to AQP3 and AQP4, is often detected in inner medullary principal cells in vivo. Here, potential mechanisms that regulate apical versus basolateral targeting of AQP2 were examined. The lack of AQP2-4 association into heterotetramers and the complete apical expression of AQP2 when highly expressed in Madin-Darby canine kidney cells indicated that neither heterotetramerization of AQP2 with AQP3 and/or AQP4, nor high expression levels of AQP2 explained the basolateral AQP2 localization. However, long term hypertonicity, a feature of the inner medullary interstitium, resulted in an insertion of AQP2 into the basolateral membrane of Madin-Darby canine kidney cells after acute forskolin stimulation. Similarly, a marked insertion of AQP2 into the basolateral membrane of principal cells was observed in the distal inner medulla from normal rats and Brattleboro rats after acute vasopressin treatment of tissue slices that had been chronically treated with vasopressin to increase interstitial osmolality in the medulla, but not in tissues from vasopressin deficient Brattleboro rats. These data reveal for the first time that chronic hypertonicity can program cells in vitro and in vivo to change the insertion of a protein into the basolateral membrane instead of the apical membrane. PMID- 12374805 TI - UV stimulation of nucleophosmin/B23 expression is an immediate-early gene response induced by damaged DNA. AB - Nucleophosmin/B23 (NPM/B23), a nucleolar protein, was rapidly up-regulated after UV irradiation (at 254 nm; 30 J/m(2)) in NIH 3T3 cells and HeLa/S3 cells. Levels of NPM/B23 mRNA peaked 45-60 min after UV treatment and returned to baseline by 12 h. Transcription inhibitor actinomycin D (5 microg/ml) prevented the UV induced increase of NPM/B23 mRNA, suggesting that UV induction of NPM/B23 was mediated at the transcriptional level. Moreover, UV-induced NPM/B23 expression was super-induced by cycloheximide (20 microg/ml), which was characteristic of immediate-early gene response. The transcriptional activation of NPM/B23 by UV was also confirmed by NPM/B23 promoter activity assay. Thymine dinucleotide, mimicking the effects of UV-induced DNA damage, was able to trigger NPM/B23 expression in the absence of genomic DNA damage. UV-induced activation of NPM/B23 promoter could not be blocked by UV-inducible pathway inhibitors, such as those of growth factor tyrosine kinase, mitogen-activated protein kinase, AP-1, NF kappaB, and DNA-dependent kinase. Our results indicate that UV stimulation of NPM/B23 expression may be mediated through a novel UV-inducible pathway and is an immediate-early gene response induced by damaged DNA. Induction of immediate early gene is an initial step in the regulation of cellular and genomic responses to external stimuli. Our results thus provide important evidence for an involvement of NPM/B23 in the acute response of mammalian cells to environmental stress. PMID- 12374806 TI - Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1. AB - The Ipl protein consists of a single pleckstrin homology (PH) domain with short N and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs. PMID- 12374808 TI - Modulation of T cell cytokine production by interferon regulatory factor-4. AB - Production of cytokines is one of the major mechanisms employed by CD4(+) T cells to coordinate immune responses. Although the molecular mechanisms controlling T cell cytokine production have been extensively studied, the factors that endow T cells with their ability to produce unique sets of cytokines have not been fully characterized. Interferon regulatory factor (IRF)-4 is a lymphoid-restricted member of the interferon regulatory factor family of transcriptional regulators, whose deficiency leads to a profound impairment in the ability of mature CD4(+) T cells to produce cytokines. In these studies, we have investigated the mechanisms employed by IRF-4 to control cytokine synthesis. We demonstrate that stable expression of IRF-4 in Jurkat T cells not only leads to a strong enhancement in the synthesis of interleukin (IL)-2, but also enables these cells to start producing considerable amounts of IL-4, IL-10, and IL-13. Transient transfection assays indicate that IRF-4 can transactivate luciferase reporter constructs driven by either the human IL-2 or the human IL-4 promoter. A detailed analysis of the effects of IRF-4 on the IL-4 promoter reveals that IRF-4 binds to a site adjacent to a functionally important NFAT binding element and that IRF-4 cooperates with NFATc1. These studies thus support the notion that IRF-4 represents one of the lymphoid-specific components that control the ability of T lymphocytes to produce a distinctive array of cytokines. PMID- 12374807 TI - Identification of three NFAT binding motifs in the 5'-upstream region of the human CD3gamma gene that differentially bind NFATc1, NFATc2, and NF-kappa B p50. AB - Human immunodeficiency virus, type 1 (HIV-1) infection of CD4(+) T cells progressively abrogates T cell receptor (TCR).CD3 function and surface expression by specifically interfering with CD3gamma gene transcription. Our data show that the loss of CD3gamma transcripts begins very early after infection and accumulates to a >90% deficiency before a significant effect on surface receptor density is apparent. Blocking TCR.CD3-directed NFAT activation with cyclosporin A provokes a partial re-expression of CD3gamma gene transcripts and surface complexes in a time- and dose-dependent manner. We have identified three NFAT consensus sequences (5'-GGAAA-3') in the 5'-upstream region of the human CD3gamma gene at: -124 to -120 (NFAT(gamma1)), -384 to -380 (NFAT(gamma2)), and +450 to +454 (NFAT(gamma3)) from the first transcription initiation site. Using electrophoretic mobility shift and supershift assays, we show that NFATc2 alone binds to the NFAT(gamma2) motif; however, complexes containing either NFATc2 or NFATc1 plus NF-kappaB p50 bind to the NFAT(gamma1) and NFAT(gamma3) sites. We further demonstrate that NFATc1 and NF-kappaB p50 bind in the same protein.DNA complex and that a fourth Ala added to the core sequence (5'-GGAAAA-3') in NFAT(gamma1), and NFAT(gamma3) is critical for their binding. Finally, we have shown that an increase in the binding of nuclear NFATc2, NFATc1, and NF-kappaB p50 to these three motifs is correlated with a progressive loss of CD3gamma transcripts after HIV-1 infection. PMID- 12374809 TI - Heparin and heparan sulfate disaccharides bind to the exchanger inhibitor peptide region of Na+/Ca2+ exchanger and reduce the cytosolic calcium of smooth muscle cell lines. Requirement of C4-C5 unsaturation and 1--> 4 glycosidic linkage for activity. AB - Heparin and heparan sulfate fragments, obtained by bacterial heparinase and heparitinases, bearing an unsaturation at C4-C5 of the uronic acid moiety, are able to produce up to 80% reduction of the cytosolic calcium of smooth muscle cell lines. Unsaturated disaccharides from chondroitin sulfate, dermatan sulfate, and hyaluronic acid are inactive, indicating that, besides the unsaturation of the uronic acid, a vicinal 1 --> 4 glycosidic linkage is needed. An inverse correlation between the molecular weight and activity is observed. Thus, the ED(50) of the N-acetylated disaccharide derived from heparan sulfate (430 Da) is 88 microm compared with 250 microm of the trisulfated disaccharide (650 Da) derived from heparin. Except for enoxaparin (which contains an unsaturation at the non-reducing end and 1 --> 4 glycosidic linkage), other low molecular weight heparins and native heparin are practically inactive in reducing the cytosolic calcium levels. Thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor), vanadate (cytoplasmic membrane Ca(2+)-ATPase inhibitor), and nifedipine and verapamil (Ca(2+) channel antagonists) do not interfere with the effect of the trisulfated disaccharide upon the decrease of the intracellular calcium. A significant decrease of the activity of the trisulfated disaccharide is observed by reducing extracellular sodium, suggesting that the fragments might act upon the Na(+)/Ca(2+) exchanger promoting the extrusion of Ca(2+). This was further substantiated by binding experiments and circular dichroism analysis with the exchanger inhibitor peptide. PMID- 12374810 TI - Contribution of the Box 1 and Box 2 motifs of cytokine receptors to Jak1 association and activation. AB - Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the beta chain of type I interferon receptor (IFNalphaRbetaL/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1. On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor beta (IL2Rbeta), IFNgammaRalpha/IFNGR1, and IL10Ralpha/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jak1. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFNalphaRbetaL/IFNAR2 and IL2Rbeta subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFNgammaRalpha/IFNGR1 (SILLPKS) and IL10Ralpha/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8-10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38-58 amino acids from the membrane). PMID- 12374811 TI - Isolation and characterization of a novel rat factor H-related protein that is up regulated in glomeruli under complement attack. AB - The factor H family in humans is composed of seven distinct proteins, including factor H-related proteins (FHR) 1-5. All members contain tandemly arranged short consensus repeats (SCR) typical of the regulators of complement activation gene family. FHR-5 is unusual for this group of proteins, as it was initially identified as a component of immune deposits in glomerular diseases. During our cloning of the cDNA for rat factor H from glomerular epithelial cells (GEC), we identified an alternative 2729-bp cDNA transcript. The translated sequence encoded a protein containing 11 SCRs, most similar to SCRs 7-15 and 19-20 in native rat factor H, which is the same basic structure of human FHR-5. As such, this rat protein was termed FHR. Recombinant rat FHR produced in a eukaryotic expression system had a molecular mass of 78 kDa. In functional studies, recombinant FHR bound C3b and inhibited the complement alternative pathway in a dose-dependent fashion. Given the prominent expression of FHR-5 in human membranous nephropathy, a disease in which complement activation occurs in the vicinity of GEC, the expression of FHR in a rat model of this disease was evaluated. In both in vitro and in vivo models of complement activation on the GEC, FHR mRNA was up-regulated by a factor of 3-6-fold compared with controls in which complement could not be activated. Thus, we have identified a novel factor H family member in rats. This FHR protein is analogous to human FHR-5, both in structure and in potential involvement in glomerular immune complex diseases. PMID- 12374812 TI - Activation of an unfolded protein response during differentiation of antibody secreting B cells. AB - The unfolded protein response pathway (UPR) is believed to detect and compensate for excessive protein accumulation in the endoplasmic reticulum (ER). The UPR can be induced by pharmacological agents that perturb ER functions, but may also occur during cellular developmental processes such as the transition of B lymphocytes into antibody-secreting plasma cells. Here we show that major UPR components are activated in B cells stimulated to secrete antibody. Increased expression of UPR targets including the ER chaperones BiP and GRP94 and the transcription factor XBP-1 initiates early in the differentiation program prior to up-regulated synthesis of Ig chains. Furthermore, these same kinetics are observed during differentiation for cleavage of the ER-localized ATF6alpha protein and splicing of XBP-1 mRNA to generate p50ATF6alpha and p54XBP-1, the two known UPR transcriptional activators. All of these UPR events reach maximal levels once Ig synthesis and secretion are markedly induced. Interestingly, these events are not accompanied by expression of CHOP, a transcription factor induced by ER stress agents commonly used to investigate the UPR. These results suggest that a physiological UPR elicited during differentiation of B-lymphocytes into high-rate secretory cells may be distinct from the UPR defined by agents that disrupt protein maturation in the ER. PMID- 12374813 TI - Two-component signal transduction in Enterococcus faecalis. PMID- 12374814 TI - Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence. AB - Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels. In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box. An alignment of 19 operator sites recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence. We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur. The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog. Our analysis of the affinity and stoichiometry of DNA binding by B. subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix. The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR. Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of approximately 30 bp. By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes. PMID- 12374815 TI - Escherichia coli insertion sequence IS150: transposition via circular and linear intermediates. AB - IS150, a member of the widespread IS3 family, contains two consecutive out-of phase open reading frames, orfA and orfB, that partially overlap. These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting. We demonstrate that the InsAB protein represents the IS150 element's transposase. In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules. Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family. Our finding supports the assumption that these products represent true transposition intermediates of members of this family. Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules. Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events. PMID- 12374816 TI - Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli. AB - Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein. Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region. Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function. An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein. E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein. Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341. Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X. Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants. The possible implications of these results for the tertiary structure of the protein are discussed. PMID- 12374817 TI - An ABC transporter from Bacillus thuringiensis is essential for beta-exotoxin I production. AB - beta-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce beta-exotoxin I have suggested that there is a strong correlation between high levels of beta-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry(-))(Pig(+)), with no crystal gene, produced considerable amounts of beta-exotoxin I together with a soluble brown melanin pigment. Therefore, beta-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of beta exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry(-))(Pig(+)) mutants. We screened for nonpigmented mutants with impaired beta-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for beta-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for beta-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers beta-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule. PMID- 12374818 TI - Transcription activation by FNR: evidence for a functional activating region 2. AB - The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters. Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 [AR1 and AR3, respectively]) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription. Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2. Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters. The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted. Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class II promoter. Site-directed mutagenesis of a negatively charged patch ((162)EEDE(165)) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP. Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR. PMID- 12374819 TI - Molecular characterization of the growth phase-dependent expression of the lsrA gene, encoding levansucrase of Rahnella aquatilis. AB - Expression of the lsrA gene from Rahnella aquatilis, encoding levansucrase, is tightly regulated by the growth phase of the host cell; low-level expression was observed in the early phase of cell growth, but expression was significantly stimulated in the late phase. Northern blot analysis revealed that regulation occurred at the level of transcription. The promoter region was identified by primer extension analysis. Two opposite genetic elements that participate in the regulation of lsrA expression were identified upstream of the lsrA gene: the lsrS gene and the lsrR region. The lsrS gene encodes a protein consisting of 70 amino acid residues (M(r), 8,075), which positively activated lsrA expression approximately 20-fold in a growth phase-dependent fashion. The cis-acting lsrR region, which repressed lsrA expression about 10-fold, was further narrowed to two DNA regions by deletion analysis. The concerted action of two opposite regulatory functions resulted in the growth phase-dependent activation of gene expression in Escherichia coli independent of the stationary sigma factor sigma(S). PMID- 12374820 TI - Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation. AB - The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli. C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted. PMID- 12374821 TI - Yersinia enterocolitica type III secretion: yscM1 and yscM2 regulate yop gene expression by a posttranscriptional mechanism that targets the 5' untranslated region of yop mRNA. AB - Pathogenic Yersinia spp. secrete Yops (Yersinia outer proteins) via the type III pathway. The expression of yop genes is regulated in response to environmental cues, which results in a cascade of type III secretion reactions. yscM1 and yscM2 negatively regulate the expression of Yersinia enterocolitica yop genes. It is demonstrated that yopD and lcrH are required for yscM1 and yscM2 function and that all four genes act synergistically at the same regulatory step. Further, SycH binding to the protein products of yscM1 and yscM2 can activate yop gene expression even without promoting type III transport of YscM1 and YscM2. Reverse transcription-PCR analysis of yopQ mRNA as well as yopQ and yopE gene fusion experiments with the npt (neomycin phosphotransferase) reporter suggest that yscM1 and yscM2 regulate expression at a posttranscriptional step. The 178 nucleotide 5' untranslated region (UTR) of yopQ mRNA was sufficient to confer yscM1 and yscM2-mediated regulation on the fused reporter, as was the 28 nucleotide UTR of yopE. The sequence 5'-AUAAA-3' is located in the 5' yop UTRs, and mutations that alter the sequence motif either reduced or abolished yscM1- and yscM2-mediated regulation. A model is proposed whereby YopD, LcrH, YscM1, YscM2, and SycH regulate yop expression in response to specific environmental cues and by a mechanism that may involve binding of some of these factors to a specific target sequence within the UTR of yop mRNAs. PMID- 12374822 TI - Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase. AB - Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity. PMID- 12374823 TI - Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli. AB - The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction. PMID- 12374824 TI - Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough. AB - Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence. PMID- 12374825 TI - A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917. AB - Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli. PMID- 12374826 TI - DNA inversion on conjugative plasmid pVT745. AB - Plasmid pVT745 from Actinobacillus actinomycetemcomitans strain VT745 can be transferred to other A. actinomycetemcomitans strains at a frequency of 10(-6). Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5' end of the invertase gene, inv. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all A. actinomycetemcomitans strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet. PMID- 12374827 TI - Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection. AB - We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes. An L. monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-beta-D thiogalactopyranoside (IPTG)-dependent promoter. Listeriolysin O (LLO) is a pore forming cytolysin that mediates lysis of L. monocytogenes-containing phagosomes. Using hemolytic-activity assays and Western blot analysis, we demonstrated dose dependent IPTG induction of LLO during growth in broth culture. Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG. We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth. Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line. Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent. The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed. PMID- 12374828 TI - Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation. AB - Swarming is an adaptation of many bacteria to growth on surfaces. A search for genes controlling swarmer cell differentiation of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal phosphate-dependent enzyme, an extracellular solute-binding protein, and a membrane-bound GGDEF- and EAL-motif sensory protein. The functions of these motifs, which are named after conserved amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling proteins. Studies with translational fusions supported the predicted localization of the gene products. When the operon was overexpressed, swarmer cell gene transcription was induced in liquid culture. Mutants with defects in any of the three genes exhibited decreased swarming and lateral flagellar (laf) gene expression. Complementation studies confirmed an operon organization and suggested that all three genes participated in laf regulation. The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of the operon inhibited transcription of the CPS gene cpsA. Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS. PMID- 12374829 TI - The first archaeal ATP-dependent glucokinase, from the hyperthermophilic crenarchaeon Aeropyrum pernix, represents a monomeric, extremely thermophilic ROK glucokinase with broad hexose specificity. AB - An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+). The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5 kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses. PMID- 12374830 TI - Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida. AB - Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen. PMID- 12374831 TI - Environmental response and autoregulation of Clostridium difficile TxeR, a sigma factor for toxin gene expression. AB - TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile, Clostridium perfringens, or Escherichia coli, TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli. PMID- 12374832 TI - Real-time imaging of fluorescent flagellar filaments of Rhizobium lupini H13-3: flagellar rotation and pH-induced polymorphic transitions. AB - The soil bacterium Rhizobium lupini H13-3 has complex right-handed flagellar filaments with unusual ridged, grooved surfaces. Clockwise (CW) rotation propels the cells forward, and course changes (tumbling) result from changes in filament speed instead of the more common change in direction of rotation. In view of these novelties, fluorescence labeling was used to analyze the behavior of single flagellar filaments during swimming and tumbling, leading to a model for directional changes in R. lupini. Also, flagellar filaments were investigated for helical conformational changes, which have not been previously shown for complex filaments. During full-speed CW rotation, the flagellar filaments form a propulsive bundle that pushes the cell on a straight path. Tumbling is caused by asynchronous deceleration and stops of individual filaments, resulting in dissociation of the propulsive bundle. R. lupini tumbles were not accompanied by helical conformational changes as are tumbles in other organisms including enteric bacteria. However, when pH was experimentally changed, four different polymorphic forms were observed. At a physiological pH of 7, normal flagellar helices were characterized by a pitch angle of 30 degrees, a pitch of 1.36 micro m, and a helical diameter of 0.50 micro m. As pH increased from 9 to 11, the helices transformed from normal to semicoiled to straight. As pH decreased from 5 to 3, the helices transformed from normal to curly to straight. Transient conformational changes were also noted at high viscosity, suggesting that the R. lupini flagellar filament may adapt to high loads in viscous environments (soil) by assuming hydrodynamically favorable conformations. PMID- 12374833 TI - Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus. AB - The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5' untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5' untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the lambda integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae. PMID- 12374834 TI - L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus, a bifunctional enzyme involved in autotrophic CO(2) fixation. AB - The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA. PMID- 12374835 TI - A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis. AB - The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed. PMID- 12374836 TI - Control by A-factor of a metalloendopeptidase gene involved in aerial mycelium formation in Streptomyces griseus. AB - In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA binding sites. The primary product of sgmA consisted of N-terminal pre, N terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position -60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position -260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation. PMID- 12374837 TI - The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria. AB - The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21 like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages. PMID- 12374839 TI - Conserved filamentous prophage in Escherichia coli O18:K1:H7 and Yersinia pestis biovar orientalis. AB - Microbial virulence is known to emerge by horizontal gene transfer mechanisms. Here we describe the discovery of a novel filamentous prophage, designated CUS-1, which is integrated into the chromosomal dif homologue of the high-virulence clone Escherichia coli O18:K1:H7. An homologous chromosomal element (CUS-2) in Yersinia pestis biovar orientalis is integrated at the same relative location as CUS-1; both lysogenic E. coli and Y. pestis strains produce particles with properties expected of single-stranded DNA virions. CUS(phi) is epidemiologically correlated with the emergence of K1 strains with increased virulence and with the Y. pestis biovar responsible for the current (third) plague pandemic. PMID- 12374838 TI - Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus. AB - The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1 to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle. PMID- 12374840 TI - Tightly regulated gene expression system in Salmonella enterica serovar Typhimurium. AB - A new Salmonella enterica serovar Typhimurium strain has been constructed to facilitate tightly regulated gene expression. Arabinose-inducible and glucose repressible expression of a T7 RNA polymerase gene that has been integrated with an adjacent araC-P(BAD) control element into the bacterial chromosome allows dynamic control of T7 promoter-driven RNA transcription. PMID- 12374841 TI - Roles of PucR, GlnR, and TnrA in regulating expression of the Bacillus subtilis ure P3 promoter. AB - Expression of the P3 promoter of the Bacillus subtilis ureABC operon is activated during nitrogen-limited growth by PucR, the transcriptional regulator of the purine-degradative genes. Addition of allantoic acid, a purine-degradative intermediate, to nitrogen-limited cells stimulated transcription of ure P3 twofold. Since urea is produced during purine degradation in B. subtilis, regulation of ureABC expression by PucR allows purines to be completely degraded to ammonia. The nitrogen transcription factor TnrA was found to indirectly regulate ure P3 expression by activating pucR expression. The two consensus GlnR/TnrA binding sites located in the ure P3 promoter region were shown to be required for negative regulation by GlnR. Mutational analysis indicates that a cooperative interaction occurs between GlnR dimers bound at these two sites. B. subtilis is the first example where urease expression is both nitrogen regulated and coordinately regulated with the enzymes involved in purine transport and degradation. PMID- 12374842 TI - The gene yjfQ encodes the repressor of the yjfR-X regulon (ula), which is involved in L-ascorbate metabolism in Escherichia coli. AB - Mutations in yjfQ allowed us to identify this gene as the regulator of the operon yjfS-X (ula operon), reported to be involved in L-ascorbate metabolism. Inactivation of this gene renders constitutive the expression of the ula operon, indicating that YjfQ acts as a repressor. We also demonstrate that this repressor regulates the nearby yjfR gene, which in this way constitutes a regulon with the ula operon. PMID- 12374843 TI - groEL expression in gyrB mutants of Borrelia burgdorferi. AB - GroEL protein and groEL mRNA transcript were up-regulated in gyrB mutants of Borrelia burgdorferi, a causative agent of Lyme disease. Furthermore, the protein and transcript levels in gyrB mutants were greater than those in experimentally heat-shocked cultures of wild-type B. burgdorferi. Circular DNA in the gyrB mutants was more relaxed than in wild-type cells, although groEL is on the linear chromosome of B. burgdorferi. To our knowledge, this is the first evidence, albeit indirect, for the effect of DNA topology on gene expression from a linear DNA molecule in a bacterium. PMID- 12374844 TI - A green nonsulfur bacterium, Dehalococcoides ethenogenes, with the LexA binding sequence found in gram-positive organisms. AB - Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria. Using a TBLASTN search, the D. ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified. Mobility shift assays revealed that the D. ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter. Our results demonstrate that the D. ethenogenes LexA binding site is GAACN(4)GTTC, which is identical to that found in gram-positive bacteria. In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D. ethenogenes LexA operator. This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B. subtilis. PMID- 12374845 TI - A gene encoding a homologue of dolichol phosphate-beta-D-mannose synthase is required for infection of Streptomyces coelicolor A3(2) by phage (phi)C31. AB - We have shown previously that a gene encoding a homologue to the eukaryotic dolichol-phosphate-D-mannose, protein O-D-mannosyltransferase, was required for (phi)C31 infection of Streptomyces coelicolor. Here we show that a gene encoding the homologue to dolichol-phosphate-mannose synthase is also essential for phage sensitivity. These data confirm the role of glycosylation in the phage receptor for (phi)C31 in S. coelicolor. PMID- 12374846 TI - A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export. AB - The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative in vitro CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways. PMID- 12374847 TI - Neurotoxic effects of thioflavin S-positive amyloid deposits in transgenic mice and Alzheimer's disease. AB - Despite extensive deposition of putatively neurotoxic amyloid-beta (Abeta) protein in the brain, it has not been possible to demonstrate an association of Abeta deposits with neuronal loss in Alzheimer's disease (AD), and neuronal loss is minimal in transgenic mouse models of AD. Using triple immunostaining confocal microscopy and analyzing the images with the cross-correlation density map method from statistical physics, we directly compared Abeta deposition, Abeta morphology, and neuronal architecture. We found dramatic, focal neuronal toxicity associated primarily with thioflavin S-positive fibrillar Abeta deposits in both AD and PSAPP mice. These results, along with computer simulations, suggest that Abeta develops neurotoxic properties in vivo when it adopts a fibrillar beta pleated sheet conformation. PMID- 12374848 TI - Do SNARE proteins confer specificity for vesicle fusion? PMID- 12374849 TI - Chemotherapeutic hope on the horizon for Plasmodium vivax malaria? PMID- 12374850 TI - Cyclooxygenase-3 (COX-3): filling in the gaps toward a COX continuum? PMID- 12374851 TI - Mechanism for saltational shifts in pheromone communication systems. PMID- 12374852 TI - SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria. AB - The SIR2 (silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin SIRT3. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NAD(+)-dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation. PMID- 12374853 TI - The discovery of susceptibility genes for mental disorders. PMID- 12374854 TI - A reassessment of human cranial plasticity: Boas revisited. AB - In 1912, Franz Boas published a study demonstrating the plastic nature of the human body in response to changes in the environment. The results of this study have been cited for the past 90 years as evidence of cranial plasticity. These findings, however, have never been critiqued thoroughly for their statistical and biological validity. This study presents a reassessment of Boas' data within a modern statistical and quantitative genetic framework. The data used here consist of head and face measurements on over 8,000 individuals of various European ethnic groups. By using pedigree information contained in Boas' data, narrow sense heritabilities are estimated by the method of maximum likelihood. In addition, a series of t tests and regression analyses are performed to determine the statistical validity of Boas' original findings on differentiation between American and European-born children and the prolonged effect of the environment on cranial form. Results indicate the relatively high genetic component of the head and face diameters despite the environmental differences during development. Results point to very small and insignificant differences between European- and American-born offspring, and no effect of exposure to the American environment on the cranial index in children. These results contradict Boas' original findings and demonstrate that they may no longer be used to support arguments of plasticity in cranial morphology. PMID- 12374856 TI - The hidden duplication past of Arabidopsis thaliana. AB - Analysis of the genome sequence of Arabidopsis thaliana shows that this genome, like that of many other eukaryotic organisms, has undergone large-scale gene duplications or even duplications of the entire genome. However, the high frequency of gene loss after duplication events reduces colinearity and therefore the chance of finding duplicated regions that, at the extreme, no longer share homologous genes. In this study we show that heavily degenerated block duplications that can no longer be recognized by directly comparing two segments because of differential gene loss, can still be detected through indirect comparison with other segments. When these so-called hidden duplications in Arabidopsis are taken into account, many homologous genomic regions can be found in five to eight copies. This finding strongly implies that Arabidopsis has undergone three, but probably no more, rounds of genome duplications. Therefore, adding such hidden blocks to the duplication landscape of Arabidopsis sheds light on the number of polyploidy events that this model plant genome has undergone in its evolutionary past. PMID- 12374855 TI - Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. AB - Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases. PMID- 12374857 TI - Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer. AB - The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology. PMID- 12374858 TI - Class I negative CD8 T cells reveal the confounding role of peptide-transfer onto CD8 T cells stimulated with soluble H2-Kb molecules. AB - Crosslinking of the T cell receptor has been proposed to be a prerequisite for T cell activation. Although the evidence supports this notion for CD4 T cells, the situation for CD8 T cells is less clear. Soluble class I monomers have been used to determine activation requirements in vitro with contradictory results. The possibility of transfer of peptide from soluble class I molecules onto class I molecules present on the surface of CD8 T cells, with ensuing presentation to other CD8 T cells, has been widely ignored. We show that monomeric and tetrameric class I molecules as well as free peptide can stimulate naive CD8 T cells in vitro. We generate and characterize CD8 T cells that express the OT-I T cell receptor (for K(b)/SIINFEKL) yet lack K(b) and D(b) molecules, and show that their activation requirements differ from their class I positive counterparts when stimulated with soluble K(b) molecules. By eliminating the confounding effect of peptide transfer, we unmask the true activation requirements for naive CD8 T cells and show that multivalent engagement of T cell receptors, as well as costimulation, is required for optimal stimulation. PMID- 12374859 TI - Soluble peptide-MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules. AB - T cell receptor (TCR)-mediated activation of CD4(+) T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide-MHC complexes. In contrast, for CD8(+) T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric L(d) and K(b) peptide-MHC complexes and free peptide as stimulators of CD8(+) T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naive and effector CD8(+) T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (K(b)) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naive T cells. PMID- 12374860 TI - Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis. AB - Bacterial adaptation to nutrient limitation and increased population densities is central to survival and virulence. Surprisingly, <3% of Escherichia coli genes are known to play roles specific to the stationary phase. There is evidence that the leucine-responsive regulatory protein (Lrp) may play an important role in stationary phase, so this study used microarrays representing >98% of E. coli genes to more comprehensively identify those controlled by Lrp. The primary analysis compared isogenic Lrp(+) and Lrp(-) strains in cells growing in steady state in glucose minimal medium, either in the presence or absence of leucine. More than 400 genes were significantly Lrp-responsive under the conditions used. Transcription of 147 genes was lower in Lrp(+) than in Lrp(-) cells whether or not leucine was present; most of these genes were tightly coregulated under several conditions, including a burst of synthesis on transition to stationary phase. This cluster includes 56 of 115 genes already known to play roles in stationary phase. Our results suggest that the actual number of genes induced on entrance into stationary phase is closer to 200 and that Lrp affects nearly three quarters of them, including genes involved in response to nutrient limitation, high concentrations of organic acids, and osmotic stress. PMID- 12374861 TI - PI31 is a modulator of proteasome formation and antigen processing. AB - Regulation of the proteasome system, which is responsible for the generation of most MHC class I-bound peptides, occurs through the interaction of the 20S proteasome with several regulatory proteins. One of these is PI31, which acts in vitro as an inhibitor of proteasome activity. Here, we demonstrate that, rather than inhibiting proteasome function, PI31 acts as a selective modulator of the proteasome-mediated steps in MHC class I antigen processing. Overexpression of PI31 in mouse embryonic cells has no impact on proteasome-mediated proteolysis. Instead, PI31, which localizes at the nuclear envelope/endoplasmic reticulum membrane, selectively interferes with the maturation of immunoproteasome precursor complexes. Consequently, overexpression of PI31 abrogates MHC class I presentation of an immunoproteasome-dependent cytotoxic T lymphocyte epitope and reduces the surface MHC class I levels on IFN-gamma-treated mouse embryonic cells. Thus, PI31 represents a cellular regulator of proteasome formation and of proteasome-mediated antigen processing. PMID- 12374862 TI - A potent analog of 1alpha,25-dihydroxyvitamin D3 selectively induces bone formation. AB - 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is a principal regulator of calcium and phosphorus homeostasis through actions on intestine, kidney, and bone. 1,25(OH)(2)D(3) is not considered to play a significant role in bone formation, except for its role in supporting mineralization. We report here on the properties of 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), a highly potent analog of 1,25(OH)(2)D(3) that induces bone formation both in vitro and in vivo. Selectivity for bone was first demonstrated through the observation that 2MD is at least 30-fold more effective than 1,25(OH)(2)D(3) in stimulating osteoblast-mediated bone calcium mobilization while being only slightly more potent in supporting intestinal calcium transport. 2MD is also highly potent in promoting osteoblast-mediated osteoclast formation in vitro, a process essential to both bone resorption and formation. Most significantly, 2MD at concentrations as low as 10(-12) M causes primary cultures of osteoblasts to produce bone in vitro. This effect is not found with 1,25(OH)(2)D(3) even at 10(-8) M, suggesting that 2MD might be osteogenic in vivo. Indeed, 2MD (7 pmol/day) causes a substantial increase (9%) in total body bone mass in ovariectomized rats over a 23-week period. 1,25(OH)(2)D(3) (500 pmol three times a week) only prevented the bone loss associated with ovariectomy and did not increase bone mass. These results indicate that 2MD is a potent bone-selective analog of 1,25(OH)(2)D(3) potentially effective in treating bone loss diseases. PMID- 12374863 TI - Distributional regimes for the number of k-word matches between two random sequences. AB - When comparing two sequences, a natural approach is to count the number of k letter words the two sequences have in common. No positional information is used in the count, but it has the virtue that the comparison time is linear with sequence length. For this reason this statistic D(2) and certain transformations of D(2) are used for EST sequence database searches. In this paper we begin the rigorous study of the statistical distribution of D(2). Using an independence model of DNA sequences, we derive limiting distributions by means of the Stein and Chen-Stein methods and identify three asymptotic regimes, including compound Poisson and normal. The compound Poisson distribution arises when the word size k is large and word matches are rare. The normal distribution arises when the word size is small and matches are common. Explicit expressions for what is meant by large and small word sizes are given in the paper. However, when word size is small and the letters are uniformly distributed, the anticipated limiting normal distribution does not always occur. In this situation the uniform distribution provides the exception to other letter distributions. Therefore a naive, one distribution fits all, approach to D(2) statistics could easily create serious errors in estimating significance. PMID- 12374864 TI - Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. AB - Hepatitis B virus (HBV) is a prototype for liver-specific pathogens in which the failure of the immune system to mount an effective response leads to chronic infection. Our understanding of the immune response to HBV is incomplete, largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models. We have developed a murine model for studying human HBV replication, immunogenicity, and control. After transfection of hepatocytes in vivo with a replication-competent, over-length, linear HBV genome, viral antigens and replicative intermediates were synthesized and virus was secreted into the blood. Viral antigens disappeared from the blood as early as 7 days after transfection, coincident with the appearance of antiviral antibodies. HBV transcripts and replicative intermediates disappeared from the liver by day 15, after the appearance of antiviral CD8 + T cells. In contrast, the virus persisted for at least 81 days after transfection of NOD/Scid mice, which lack functional T cells, B cells, and natural killer (NK) cells. Thus, the outcome of hydrodynamic transfection of HBV depends on the host immune response, as it is during a natural infection. The methods we describe will allow the examination of viral dynamics in a tightly controlled in vivo system, the application of mutagenesis methods to the study of the HBV life cycle in vivo, and the dissection of the immune response to HBV using genetically modified mice whose immunoregulatory and immune effector functions have been deleted or overexpressed. In addition, this methodology represents a prototype for the study of other known and to-be discovered liver-specific pathogens. PMID- 12374865 TI - HIV-1 infection and AIDS dementia are influenced by a mutant MCP-1 allele linked to increased monocyte infiltration of tissues and MCP-1 levels. AB - Studies in humans and in experimental models of HIV-1 infection indicate an important role for monocyte chemoattractant protein-1 (MCP-1; also known as CC chemokine ligand 2), a potent chemoattractant and activator of mononuclear phagocytes (MP) in the pathogenesis of HIV-associated dementia (HAD). We determined the influence of genetic variation in MCP-1 on HIV-1 pathogenesis in large cohorts of HIV-1-infected adults and children. In adults, homozygosity for the MCP-1 -2578G allele was associated with a 50% reduction in the risk of acquiring HIV-1. However, once HIV-1 infection was established, this same MCP-1 genotype was associated with accelerated disease progression and a 4.5-fold increased risk of HAD. We examined the molecular and cellular basis for these genotype-phenotype associations and found that the mutant MCP-1 -2578G allele conferred greater transcriptional activity via differential DNA-protein interactions, enhanced protein production in vitro, increased serum MCP-1 levels, as well as MP infiltration into tissues. Thus, MCP-1 expression had a two-edged role in HIV-1 infection: it afforded partial protection from viral infection, but during infection, its proinflammatory properties and ability to up-regulate HIV-1 replication collectively may contribute to accelerated disease progression and increased risk of dementia. Our findings suggest that MCP-1 antagonists may be useful in HIV-1 infection, especially for HAD, and that HIV+ individuals possessing the MCP-1 -2578G allele may benefit from early initiation of antiretroviral drugs that effectively cross the blood-brain barrier. In a broader context, the MCP-1 -2578G allele may serve as a genetic determinant of outcome of other disease states in which MP-mediated tissue injury is central to disease pathogenesis. PMID- 12374866 TI - Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering. AB - In vitro "glycorandomization" is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-d-glucopyranosyl phosphate thymidylyltransferase (E(p)). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three "designed" mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization. PMID- 12374867 TI - A two-state kinetic model for the unfolding of single molecules by mechanical force. AB - We investigate the work dissipated during the irreversible unfolding of single molecules by mechanical force, using the simplest model necessary to represent experimental data. The model consists of two levels (folded and unfolded states) separated by an intermediate barrier. We compute the probability distribution for the dissipated work and give analytical expressions for the average and variance of the distribution. To first order, the amount of dissipated work is directly proportional to the rate of application of force (the loading rate) and to the relaxation time of the molecule. The model yields estimates for parameters that characterize the unfolding kinetics under force in agreement with those obtained in recent experimental results. We obtain a general equation for the minimum number of repeated experiments needed to obtain an equilibrium free energy, to within k(B)T, from nonequilibrium experiments by using the Jarzynski formula. The number of irreversible experiments grows exponentially with the ratio of the average dissipated work, W(dis) to k(B)T. PMID- 12374869 TI - The innermost inner core of the earth: evidence for a change in anisotropic behavior at the radius of about 300 km. AB - Since the discovery of the inner core in 1936, no additional spherical subshell of the Earth has been observed. Based on an extensive seismic data set, we propose the existence of an innermost inner core, with a radius of approximately 300 km, that exhibits a distinct transverse isotropy relative to the bulk inner core. Specifically, within the innermost inner core, the slowest direction of wave propagation is approximately 45 degrees from the east-west direction. In contrast, the direction of the slowest wave propagation in the overlying inner core is east-west. The distinct anisotropy at the center of the Earth may represent fossil evidence of a unique early history of inner-core evolution. PMID- 12374868 TI - Cell surface polarization during yeast mating. AB - Exposure to mating pheromone in haploid Saccharomyces cerevisiae cells results in the arrest of the cell cycle, expression of mating-specific genes, and polarized growth toward the mating partner. Proteins involved in signaling, polarization, cell adhesion, and fusion are localized to the tip of the mating cell (shmoo) where fusion will eventually occur. The mechanisms ensuring the correct targeting and retention of these proteins are poorly understood. Here we show that in pheromone-treated cells, a reorganization of the plasma membrane involving lipid rafts results in the retention of proteins at the tip of the mating projection, segregated from the rest of the membrane. Sphingolipid and ergosterol biosynthetic mutants fail to polarize proteins to the tip of the shmoo and are deficient in mating. Our results show that membrane microdomain clustering at the mating projection is involved in the generation and maintenance of polarity during mating. PMID- 12374870 TI - Interactions of climate change with biological invasions and land use in the Hawaiian Islands: Modeling the fate of endemic birds using a geographic information system. AB - The Hawaiian honeycreepers (Drepanidae) represent a superb illustration of evolutionary radiation, with a single colonization event giving rise to 19 extant and at least 10 extinct species [Curnutt, J. & Pimm, S. (2001) Stud. Avian Biol. 22, 15-30]. They also represent a dramatic example of anthropogenic extinction. Crop and pasture land has replaced their forest habitat, and human introductions of predators and diseases, particularly of mosquitoes and avian malaria, has eliminated them from the remaining low- and mid-elevation forests. Landscape analyses of three high-elevation forest refuges show that anthropogenic climate change is likely to combine with past land-use changes and biological invasions to drive several of the remaining species to extinction, especially on the islands of Kauai and Hawaii. PMID- 12374871 TI - Thrombolysis for pulmonary embolism. PMID- 12374872 TI - The new federal medical-privacy rule. PMID- 12374873 TI - Synergistic polymorphisms of beta1- and alpha2C-adrenergic receptors and the risk of congestive heart failure. AB - BACKGROUND: Sustained cardiac adrenergic stimulation has been implicated in the development and progression of heart failure. Release of norepinephrine is controlled by negative feedback from presynaptic alpha2-adrenergic receptors, and the targets of the released norepinephrine on myocytes are beta1-adrenergic receptors. In transfected cells, a polymorphic alpha2C-adrenergic receptor (alpha2CDel322-325) has decreased function, and a variant of the beta1-adrenergic receptor (beta1Arg389) has increased function. We hypothesized that this combination of receptor variants, which results in increased synaptic norepinephrine release and enhanced receptor function at the myocyte, would predispose persons to heart failure. METHODS: Genotyping at these loci was performed in 159 patients with heart failure and 189 controls. Logistic regression methods were used to determine the potential effect of each genotype and the interaction between them on the risk of heart failure. RESULTS: Among black subjects, the adjusted odds ratio for heart failure among persons who were homozygous for alpha2CDel322-325 as compared with those with the other alpha2C adrenergic receptor genotypes was 5.65 (95 percent confidence interval, 2.67 to 11.95; P<0.001). There was no increase in risk with beta1Arg389 alone. However, there was a marked increase in the risk of heart failure among persons who were homozygous for both variants (adjusted odds ratio, 10.11; 95 percent confidence interval, 2.11 to 48.53; P=0.004). The patients with heart failure did not differ from the controls in the frequencies of nine short tandem-repeat alleles. Among white subjects, there were too few who were homozygous for both polymorphisms to allow an adequate assessment of risk. CONCLUSIONS: The alpha2CDel322-325 and beta1Arg389 receptors act synergistically to increase the risk of heart failure in blacks. Genotyping at these two loci may be a useful approach for identification of persons at risk for heart failure or its progression, who may be candidates for early preventive measures. PMID- 12374874 TI - Heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism. AB - BACKGROUND: The use of thrombolytic agents in the treatment of hemodynamically stable patients with acute submassive pulmonary embolism remains controversial. METHODS: We conducted a study of patients with acute pulmonary embolism and pulmonary hypertension or right ventricular dysfunction but without arterial hypotension or shock. The patients were randomly assigned in double-blind fashion to receive heparin plus 100 mg of alteplase or heparin plus placebo over a period of two hours. The primary end point was in-hospital death or clinical deterioration requiring an escalation of treatment, which was defined as catecholamine infusion, secondary thrombolysis, endotracheal intubation, cardiopulmonary resuscitation, or emergency surgical embolectomy or thrombus fragmentation by catheter. RESULTS: Of 256 patients enrolled, 118 were randomly assigned to receive heparin plus alteplase and 138 to receive heparin plus placebo. The incidence of the primary end point was significantly higher in the heparin-plus-placebo group than in the heparin-plus-alteplase group (P=0.006), and the probability of 30-day event-free survival (according to Kaplan-Meier analysis) was higher in the heparin-plus-alteplase group (P=0.005). This difference was due to the higher incidence of treatment escalation in the heparin plus-placebo group (24.6 percent vs. 10.2 percent, P=0.004), since mortality was low in both groups (3.4 percent in the heparin-plus-alteplase group and 2.2 percent in the heparin-plus-placebo group, P=0.71). Treatment with heparin plus placebo was associated with almost three times the risk of death or treatment escalation that was associated with heparin plus alteplase (P=0.006). No fatal bleeding or cerebral bleeding occurred in patients receiving heparin plus alteplase. CONCLUSIONS: When given in conjunction with heparin, alteplase can improve the clinical course of stable patients who have acute submassive pulmonary embolism and can prevent clinical deterioration requiring the escalation of treatment during the hospital stay. PMID- 12374875 TI - Endogenous antimicrobial peptides and skin infections in atopic dermatitis. AB - BACKGROUND: The innate immune system of human skin contains antimicrobial peptides known as cathelicidins (LL-37) and beta-defensins. In normal skin these peptides are negligible, but they accumulate in skin affected by inflammatory diseases such as psoriasis. We compared the levels of expression of LL-37 and human beta-defensin 2 (HBD-2) in inflamed skin from patients with atopic dermatitis and from those with psoriasis. METHODS: The expression of LL-37 and HBD-2 protein in skin-biopsy specimens from patients with psoriasis, patients with atopic dermatitis, and normal subjects was determined by immunohistochemical analysis. The amount of antimicrobial peptides in extracts of skin samples was also analyzed by immunodot blot analysis (for LL-37) and Western blot analysis (for HBD-2). Quantitative, real-time reverse-transcriptase-polymerase-chain reaction (RT-PCR) assays were used to confirm the relative expression of HBD-2 and LL-37 messenger RNA (mRNA) in the skin-biopsy specimens. These peptides were also tested for antimicrobial activity against Staphylococcus aureus with the use of a colony-forming assay. RESULTS: Immunohistochemical analysis confirmed the presence of abundant LL-37 and HBD-2 in the superficial epidermis of all patients with psoriasis. In comparison, immunostaining for these peptides was significantly decreased in acute and chronic lesions from patients with atopic dermatitis (P=0.006 and P=0.03, respectively). These results were confirmed by immunodot blot and Western blot analyses. Real-time RT-PCR showed significantly lower expression of HBD-2 mRNA and LL-37 mRNA in atopic lesions than in psoriatic lesions (P=0.009 and P=0.02, respectively). The combination of LL-37 and HBD-2 showed synergistic antimicrobial activity by effectively killing S. aureus. CONCLUSIONS: A deficiency in the expression of antimicrobial peptides may account for the susceptibility of patients with atopic dermatitis to skin infection with S. aureus. PMID- 12374876 TI - Images in clinical medicine. Thrombolysis of a massive pulmonary embolism. PMID- 12374877 TI - Screening extended families for genetic hemoglobin disorders in Pakistan. AB - BACKGROUND: We have investigated a strategy for identifying and counseling carriers of recessively inherited disorders in developing countries where consanguineous marriage is common. In such communities, gene variants are trapped within extended families, so that an affected child is a marker of a group at high genetic risk. METHODS: Fifteen large Pakistani families, 10 with a history of a hemoglobin disorder and 5 without any such history (controls), were screened for beta-thalassemia and abnormal hemoglobins. All carriers and married couples consisting of two carriers received counseling, and eight families have been followed for two years. RESULTS: In the control families, no carrier was found among 397 members tested. In the 10 families with an index case, 183 of 591 persons tested (31 percent) were carriers; carriers had a 25 percent risk of being in a marriage at risk for producing an affected child, and 17 of 214 married couples (8 percent) consisted of two carriers. No couple at risk was identified among 350 randomly selected pregnant women and their partners. All carriers reported that they have used the information provided in the testing and counseling process: carriers married to carriers with two or more healthy children have avoided further pregnancy, and most such couples with one or no healthy children have used prenatal diagnosis. Seven of eight new marriages and engagements are known not to be at risk. CONCLUSIONS: Testing of extended families is a feasible way of deploying DNA-based genetic screening in communities in which consanguineous marriage is common. PMID- 12374878 TI - Clinical practice. Otitis media. PMID- 12374879 TI - Helicobacter pylori infection. PMID- 12374880 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 31-2002. A 61-year-old man with headache and multiple infarcts. PMID- 12374881 TI - Adrenergic-receptor polymorphisms and heart failure. PMID- 12374882 TI - Antimicrobial peptides in health and disease. PMID- 12374883 TI - Effect of consanguinity on screening for thalassemia. PMID- 12374884 TI - Defying death--HIV mutation to evade cytotoxic T lymphocytes. PMID- 12374885 TI - Screening colonoscopy among persons 40 to 49 years of age. PMID- 12374886 TI - Hereditary amyloidosis. PMID- 12374887 TI - Intranasal mupirocin to prevent postoperative infections. PMID- 12374889 TI - What's ahead for health insurance? PMID- 12374888 TI - Human immunodeficiency virus in pregnancy. PMID- 12374890 TI - Inhaled corticosteroids, growth, and compliance. PMID- 12374891 TI - Kissing and food reactions. PMID- 12374892 TI - A decline in the U.S. share of research articles. PMID- 12374893 TI - Impaired mechanisms of leukocyte adhesion in vitro by the calcium channel antagonist mibefradil. PMID- 12374894 TI - New treatments for myocardial fibrosis. PMID- 12374895 TI - Impaired mechanisms of leukocyte adhesion in vitro by the calcium channel antagonist mibefradil. AB - PURPOSE: Enhanced adhesion to the vascular endothelium and excessive trafficking to extravascular locations can lead to serious tissue injury and destruction. Therefore, interfering with molecular mechanisms of leukocyte adhesion to the vascular endothelium is an important goal to block diseases like chronic inflammations and atherosclerosis. METHODS: We studied the influence of the calcium antagonists mibefradil (T-type channel blocker), amlodipine and verapamil (both L-type channel blockers) on mechanisms related to leukocyte adhesion using isolated peripheral human blood leukocytes. RESULTS: Mibefradil but not amlodipine and verapamil attenuated leukocyte adhesion in vitro. Regarding the mechanisms we found that mibefradil reduced the surface expression of beta2 integrins and L-selectin. The immobilization of the beta2 integrin subunit to the cytoskeleton that was inducible by receptor cross linking was impaired. Mibefradil was able to significantly inhibit the formyl-methionyl-leucyl phenylalanine (fMLP) induced calcium rise, which suggests that mibefradil interfered with integrin signaling through blocking the intracellular calcium rise. SK&F 96365, a blocker of the capacitative calcium entry had no effect on cell adhesion and was less effective to influence integrin mediated mechanisms than mibefradil. CONCLUSION: Our data suggest that mibefradil or chemically related substances are promising to serve as potent drugs to prevent excessive adhesion of leukocytes. PMID- 12374896 TI - Effects of canrenone on myocardial reactive fibrosis in a rat model of postinfarction heart failure. AB - BACKGROUND: Spironolactone reduces overall mortality by 30% in advanced congestive heart failure. Nevertheless, few data are available with regard to the effects of mineral corticoid inhibition in postinfarction heart failure. MATERIALS AND METHODS: Experimental myocardial infarction was induced by left coronary ligation in 70 male rats with body weights ranging from 180 to 200 gr. The day after surgery, animals were randomized to either placebo or canrenone gamma-cyclodestrin 8 mg/kg/die or canrenone-gamma-cyclodestrin 18 mg/kg/die. Twelve animals served as the control group. After two weeks, the rats underwent closed chest left ventricular catheterization. The heart was the rapidly excised for subsequent histological analysis. RESULTS: Compared with controls, infarcted rats had reduced left ventricular systolic pressures (-6%) and higher left ventricular end-diastolic pressures (+600%), associated with a marked increase of mean collagen fraction (+446%) and perivascular fibrosis (+72%). Compared with placebo-infarcted rats, in the group treated with high canrenone dose there was a significant reduction of left ventricular systolic and end-diastolic pressures ( 6.5% and -23%, respectively) and an attenuation of interstitial and perivascular fibrosis (-47% and -34%, respectively). The low-dose canrenone group did not show differences compared with the placebo infarcted rats, except for a slight reduction of mean collagen fraction (-21%). CONCLUSIONS: Canrenone attenuates LV interstitial remodeling and reduces filling pressures in rats with postinfarction heart failure. PMID- 12374897 TI - Antioxidative effects of fluvastatin on superoxide anion activated by angiotensin II in human aortic smooth muscle cells. AB - We examined the antioxidative effects of fluvastatin, a 3-hydroxy-3 methylglutaryl coenzyme A reductase inhibitor (statin), on superoxide anion formation activated by angiotensin II (Ang II) in vitro. The effects of fluvastatin were also compared to simvastatin and a water-soluble analog of alpha tocopherol, trolox. Treatment of human aortic smooth muscle cells (hASMC) with Ang II for 24 hours resulted in a 3.2 +/- 0.5-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. hASMC treated with clinical concentrations of fluvastatin (0-100 nM) showed a dose-dependent decrease in Ang II-activated superoxide anion formation. The addition of similar concentrations of trolox to hASMC inhibited Ang II-activated superoxide anion formation in a dose-dependent manner. However, simvastatin at similar doses failed to inhibit Ang II-activated superoxide anion formation by hASMC. Our results indicate that in addition to its hypocholesterolemic effect, fluvastatin may have direct antioxidative effects, suggesting its possible protective effect on atherosclerotic process. PMID- 12374898 TI - Effects of chronic neutral endopeptidase inhibition on the progression of left ventricular dysfunction and remodeling in dogs with moderate heart failure. AB - BACKGROUND: The diuretic actions of endogenously produced atrial natriuretic factor (ANF) may be beneficial in the treatment of chronic heart failure (CHF). Neutral endopeptidase (NEP) is the primary enzyme responsible for the degradation of ANF. The present study investigates the effects of long-term NEP inhibition on the progression of left ventricular (LV) dysfunction and remodeling in dogs with moderate heart failure. METHODS: LV dysfunction was produced in 12 dogs by multiple sequential intracoronary microembolizations. Embolizations were discontinued when LV ejection fraction (EF) was between 30-40%. Two weeks after the last embolization, dogs were randomized to 3 months of oral therapy with the NEP inhibitor ecadotril (100 mg, once daily, n = 6) or to no therapy at all (control, n = 6). RESULTS: During the 3 months of follow-up, LV EF in control dogs decreased from 37 +/- 1% to 28 +/- 1% (P < 0.01) and LV end-diastolic volume (EDV) and end-systolic volume (ESV) increased (EDV: 72 +/- 3 vs. 84 +/- 5 ml, P < 0.01); ESV: 45 +/- 1 vs. 60 +/- 4 ml, P < 0.01). In dogs treated with ecadotril, LV EF (34 +/- 1% vs. 37 +/- 2%), EDV (79+/- 5 vs. 78+/- 6 ml) and ESV (52 +/- 3 vs. 49 +/- 4) remained essentially unchanged after 3 months of therapy. Histomorphometric measurements at the termination of the study showed that ecadotril was associated with significantly reduced cardiomyocyte hypertrophy compared to control. CONCLUSION: Early, long-term NEP inhibition with ecadotril prevents the progression of LV dysfunction and attenuates progressive LV remodeling in dogs with moderate heart failure. PMID- 12374899 TI - Troglitazone improves cardiac function in patients with congestive heart failure. AB - Troglitazone increased cardiac output and stroke volume, as a result of decreased peripheral resistance, in diabetic patients with normal cardiac function. The cardiovascular effects of troglitazone in patients with heart failure are unknown. The aim of the study was to evaluate the cardiovascular effects of troglitazone in patients with heart failure. Blood pressure and echocardiographic findings were evaluated before and 1, 2, 3 and 4 hours after a single dose of troglitazone (400 mg) or placebo, in eight type II diabetic patients with congestive heart failure. The plasma catecholamines and coefficient of variance of RR intervals (CVRR) were also measured. Neither heart rate nor blood pressure changed after the administration of troglitazone. Left ventricular (LV) end diastolic dimension did not change either, however, the LV end-systolic dimension significantly decreased compared with its baseline value and with that of the placebo group. On the other hand, the % fractional shortening and the E/A ratio increased significantly after troglitazone. The LV end-diastolic volume did not change, whereas the LV end-systolic volume significantly decreased. The stroke volume and the LV ejection fraction significantly increased compared with its baseline value and with that of the placebo group. The peripheral vascular resistance did not change after the administration of troglitazone, whereas plasma catecholamines significantly decreased, and CVRR remained unchanged in both groups. These hemodynamic changes suggest that a single oral dose of troglitazone induced inotropy without activation of the sympathetic nervous system. PMID- 12374900 TI - Assessment of quality of life in a double-blind, randomized clinical trial of imidapril and captopril for hypertensive Chinese in Taiwan. AB - PURPOSE: Although the role of angiotensin-converting enzyme (ACE) inhibitors for the treatment of hypertension has been well established, no data has been generated regarding the influence of ACE inhibitors for health-related quality-of life (QOL) dimensions for Chinese patients. MATERIALS: A double-blind, active control, randomized clinical trial was used to compare the effects of two ACE inhibitors, imidapril and captopril, on quality-of-life dimensions in one outpatient clinic in one tertiary clinical-care facility. After a 2-3 week washout period with placebo, 59 patients with mild-to-moderate hypertension were randomly assigned to receive imidapril (5 to 10 mg per day) or captopril (25 to 50 mg twice per day) for 12 weeks. Patients completed the Short-form 36 (SF 36) health survey questionnaire, which evaluates 8 QOL dimensions, just before treatment, during the 8th week, and at the end of treatment (12th week). ANOVA for repeated measures was used to analyze the QOL-score changes over time and compare treatments, and to assess the interaction of treatment duration and group on these scores. RESULTS: No significant differences were demonstrated for changes in blood-pressure, frequency of adverse effects and withdrawal of patients from the study comparing the two drugs. Significant improvement, however, was demonstrated for mental-component summary scores after 12 weeks of treatment for both drugs (P = 0.029). No significant differences were established for individual QOL dimensions comparing the two drugs. A significantly higher baseline systolic blood pressure was found in the participants who did not complete the questionnaire than in those who did. CONCLUSIONS: Similar and significant improvements were determined for the mental-component QOL summary scores for the two ACE inhibitors, imidapril and captopril, and no significant differences were demonstrated comparing treatments. PMID- 12374901 TI - PERindopril-Function of the Endothelium in Coronary Artery Disease Trial: the PERFECT study--sub study of EUROPA: rationale and design. AB - BACKGROUND: ACE inhibition reduces morbidity and mortality among a variety of patients. Among mechanisms explaining these beneficial effects are the effects on the sympathetic system and on local vasodilating substances such as nitric oxide and bradykinins at the level of the endothelium. The PERFECT study was designed to verify the above mentioned pathophysiological concepts. METHODS: The PERFECT study is a study nested within the EUROPA trial, a three year double-blind, multi centre, placebo-controlled randomised study that aims at studying the effect of the ACE-inhibitor Perindopril on morbidity and mortality in over 12,000 patients with stable coronary artery disease without clinical heart failure. The PERFECT study is designed as a parallel group randomised placebo controlled trial to determine the effect of Perindopril (8 mg) on brachial artery endothelial function in patients with stable coronary artery disease without clinical heart failure. In the PERFECT study, B-mode ultrasonography of the brachial artery is used as a model for changes in the coronary arteries. Endothelial function in response to ischaemia (reactive hyperaemia) and to vasoconstriction (cold pressor test) is assessed. The ischemia test is used a model to assess the effects of ACE inhibition on nitric oxide/bradykinine mediated vasodilatatory response to ischaemia, whereas the cold pressor test is applied to assess the effect of ACE inhibition on the neurohormonal response. The recruitment for the PERFECT study started in May 1998 en was completed in June 1999. 345 patients were recruited in 20 European centers. The Vascular Imaging Center Utrecht, an ultrasound core laboratory, is performing the endothelial function measurements. The primary study outcomes are (1) percentage change in flow-mediated vasodilatation of the brachial artery between the 36 month measurement and the baseline measurement and (2) percentage change in neurohormonal mediated vasoconstriction of the brachial artery between the 36 month measurement and the baseline measurement. The size of the study allows detection of an absolute difference in FMD of 2.0% with a 90% power and a two-sided alpha of 5%. CONCLUSION: The findings of the PERFECT study may help to understand and explain the effects of ACE inhibition, in particular Perindopril, on cardiovascular morbidity and mortality. PMID- 12374902 TI - Inotropic agents and immune modulation. AB - Since depression of myocardial contractility forms the basis for the development of heart failure, many attempts have been made to enhance the inotropic state of the failing heart by cardiotonic agent as the therapeutic modality. However, large scale clinical trials conducted in the Western societies revealed excess mortality in patients with heart failure received long-term treatment with inotropic agent. Therefore, all of these agents are now regarded as unsuitable for chronic heart failure treatment. In contrast, some inotropic agents with phosphodiesterase inhibitory properties exhibited potential benefits in Japanese patients. In Japan mortality due to heart disease is substantially lower than that found in all other Western countries. Thereby, chronic treatment with inotropic agent may be justified as the optimal care in the context of relief of symptoms and an improved quality of life. The salutary effects of these phosphodiesterase inhibitors appear to be related to anticytokine and immunomodulating effects as well as their cardiotonic action. These findings support the recent new concept that immune responses mediated by cytokines play an important role in the pathogenesis of heart failure. PMID- 12374903 TI - Maladaptive growth in the failing heart: the cardiomyopathy of overload. AB - The hypertrophic response to overload plays an important role in the progressive deterioration of the failing heart--the "Cardiomyopathy of Overload"--and so contributes to the poor prognosis in patients with heart failure. Although increased myocyte size reduces the load on individual sarcomeres, hypertrophy also has maladaptive features. The latter include molecular changes that weaken and impair relaxation in the overloaded heart, and accelerate cardiac myocyte death. Different types of overload lead to concentric and eccentric hypertrophy; as the latter tends to progress ("remodeling"), dilatation is associated with an especially poor prognosis. Concentric hypertrophy is due largely to cardiac myocyte thickening, while eccentric hypertrophy is caused by cell elongation. These differences, along with evidence that concentric hypertrophy is initiated by increased diastolic stretch while eccentric hypertrophy results from increased systolic stress, indicate that these growth responses are mediated by different signal transudation pathways. The beneficial effects of neurohumoral blockers in patients with heart failure are due partly to their ability to inhibit maladaptive features of overload-induced proliferative signaling. The molecular complexity of the hypertrophic response now being uncovered offers opportunities for the development of new therapy to inhibit remodeling and cell death in the failing heart. PMID- 12374904 TI - Novel statins: pharmacological and clinical results. AB - Rosuvastatin (ZD4522) and pitavastatin (NK-104) are novel HMG-CoA reductase inhibitors with a peculiar pharmacological profile. In particular, they show a high potency in decreasing LDL-C and their catabolism is not mediated by the cytochrome P-450 3A4, thus reducing the potential for drug-drug interaction and improving the management of blood cholesterol. As the magnitude of LDL-C reduction is directly associated with the decrease in the incidence of myocardial infarction and mortality for CAD, statins with increased LDL-C lowering potency may ensure the achievement of target LDL-C levels and offer a more aggressive cholesterol control, further improving CAD morbidity and mortality. PMID- 12374905 TI - Beta-blocker therapy combined with low-dose pimobendan in patients with idiopathic dilated cardiomyopathy and chronic obstructive pulmonary disease: report on two cases. AB - Pimobendan is an inotropic and vasodilating drug with phosphodiesterase (PDE) III inhibiting and calcium-sensitizing effects. It may also have a bronchodilatory effect by inhibiting PDE III in airway smooth muscle. We tried a beta-blocker combined with low-dose pimobendan in 2 patients who had refractory heart failure of NYHA functional class III or IV with idiopathic dilated cardiomyopathy (DCM) and chronic obstructive pulmonary disease (COPD). Both of them had previously failed to tolerate beta-blocking drugs because of the exacerbation of bronchospasm. After pimobendan was administered at 1.25 to 2.5 mg daily, metoprolol could be successfully introduced from a low dose of 1.25 mg daily without decreasing the peak expiratory flow rate. Over the next 1 to 2 years, they have continued beta-blocker therapy. One is currently receiving 10 mg daily of bisoprolol and another is taking 15 mg daily of metoprolol, and both are in NYHA functional class II without worsening heart failure or COPD. The combination of beta-blocker with low-dose pimobendan may be helpful for patients with DCM and COPD, but further clinical investigation is required. PMID- 12374906 TI - Peptides and Ageing. AB - A technology has been developed for manufacturing of biologically active complex peptide preparations from extracts of different tissues. In particular, the pineal preparation (Epithalamin) augments the in vitro outgrowth of explants from the pineal gland but not from other tissues, the latter being stimulated by peptide preparations from respective tissues. Epithalamin increases melatonin production by the pineal gland of rats, improves immunological parameters in rats and mice, produces anticarcinogenic effects in different experimental models, stimulates antioxidant defenses, and restores the reproductive function in old rats. These effects are combined in the ability of Epithalamin to increase the lifespan in rats, mice, and fruit flies. Many of these effects are reproduced in clinical trials, which have demonstrated the geroprotector activity of Epithalamin in humans. Among the effects of the thymic preparation Thymalin, those related to its ability to stimulate immunity are the most prominent. This ability is associated with anticarcinogenic and geroprotector activities. Clinical trials of the peptide preparations obtained from other organs including the prostate, the cerebral cortex, and the eye retina, have demonstrated beneficial effects reflected by the improvement of the conditions of respective organs. Based on the data about the amino acid compositions of the peptide preparations, novel principles of the design of biologically active short peptides possessing tissue-specific activities has been developed. Dipeptides specific for the thymus and tetrapeptides specific for the heart, liver, brain cortex, and pineal glands stimulate the in vitro outgrowth of explants of respective organs. Interestingly, for eye retina and the pineal gland, a common tetrapeptide Ala-Glu-Asp-Gly (Epitalon) has been designed, probably reflecting the common embryonal origin of these two organs. Epitalon reproduces the effects of Epithalamin including those related to its geroprotector activity. In particular, Epitalon increases the lifespan of mice and fruit flies and restores the circadian rhythms of melatonin and cortisol production in old rhesus monkeys. At the same time, Epitalon prolongs the functional integrity of the eye retina in Campbell rats with hereditary Retinitis Pigmentosa and improves the visual functions in patients with pigmental retinal degeneration. Changes in gene expression were observed to be produced by the short peptide preparations. Therefore, the effects of Epitalon are suggested to be mediated by transcriptional machinery common for the pineal gland and the retina and, probably, for regulation of melatonin production in fruit flies. Based on three decades of studies of the peptide preparations, the peptide theory of ageing has been put forward. According this theory, ageing is an evolutionary determined biological process of changes in gene expression resulting in impaired synthesis of regulatory and tissue-specific peptides in organs and tissues, which provokes their structural and functional changes and the development of diseases. Correspondingly, correction of such disorders by means of stimulation of peptide production in the organism or through their delivery can promote the normalisation of disturbed body functions. PMID- 12374907 TI - Advanced technology in surgery. PMID- 12374908 TI - Early detection of oral premalignant disease and oral cancer: refining the process. PMID- 12374909 TI - Oral Kaposi's sarcoma: biopsy accessions as an indication of declining incidence. PMID- 12374910 TI - Large facial mass of long duration. PMID- 12374911 TI - Late-life depression: psychopathology, medical interventions, and dental implications. AB - BACKGROUND: Late-life depression (LLD) initially occurs after age 65 years and is a major public health concern because the elderly who are at high risk constitute an ever-expanding segment of the population. LLD is a mental illness in which mood, thought content, and behavioral patterns are impaired, causing the individual distress, compromising social function, and impairing self-maintenance skills (eg, bathing, dressing, hygiene). LLD characterized by marked sadness or a loss of interest or pleasure in daily activities and may be accompanied by weight change, sleep disturbance, fatigue, difficulty in concentration, and a high suicide rate. Diagnosis of LLD is sometimes complicated by a denial of mood change and an inability to distinguish symptoms of a concurrent physical illness from those of a depressive etiology. The disorder is most frequently treated with antidepressant medications, and although older individuals have a recovery rate that is comparable with younger adults, they often take longer to recover, have more frequent relapses, and are more sensitive to the side effects of the drugs. CLINICAL IMPLICATIONS: Individuals undergoing treatment for LLD and those whose illness has not been diagnosed or treated often are seen with significant oral disease by the dentist. Dentists need to be cognizant of how to safely and compassionately provide care to those already receiving mental health services. They must also be familiar with the psychiatric symptoms of the disorder to effectuate a timely referral to a physician of those with occult or relapsing disease. LLD is frequently associated with a disinterest in oral hygiene, a cariogenic diet, diminished salivary flow, rampant dental decay, advanced periodontal disease, and oral dysesthesias. Many medications used to treat the disease magnify the xerostomia and increase the incidence of dental disease. Appropriate dental management necessitates a vigorous preventive dental education program, the use of artificial salivary products, antiseptic mouthwash, daily fluoride mouth rinse, and special precautions in administration of local anesthetics with vasoconstrictors and prescription of analgesics. CONCLUSION: Dentists who invoke appropriate precautions can usually provide a full range of services to individuals with LLD, thereby enhancing patient self-esteem and contributing to the psychotherapeutic aspect of management. PMID- 12374912 TI - Grafting mandibular third molar extraction sites: a comparison of bioactive glass to a nongrafted site. AB - OBJECTIVE: The purpose of this study was to evaluate the effectiveness of bioactive glass used as a graft material for regeneration of bone after the removal of impacted third molars. The healing distal to the second molars was followed, with documentation of the level of radiographic osseous fill and measurement of the changes in clinical attachment levels. METHODS: Fourteen (5 male and 9 female) patients completed the study at 1 year. After surgical removal of bilateral impacted third molars, one site was grafted with bioactive glass particles (BioGran, Orthovita, Malvern, Penn). The patients were followed up at 1 and 2 weeks after surgery for signs or symptoms of infection or any other complications that may have been related to the surgical procedures. At 3, 6, and 12 months, the clinical attachment levels distal to the second molars were assessed. At 6 and 12 months, radiographs were obtained to assess the amount of osseous fill distal to the second molars. RESULTS: A split-mouth design was used to test the efficacy of the grafting materials. Clinical attachment levels in the grafted sites were significantly higher than in the nongrafted sites. There was not a significant increase in bone formation when comparing the 2 sides at 1 year. CONCLUSION: This study suggests that treatment of third molar extraction sites with bioactive glass does significantly alter the clinical attachment level but not the level of osseous fill distal to the second molars after 1 year. PMID- 12374913 TI - BIS monitoring during midazolam and midazolam-ketamine conscious intravenous sedation for oral surgery. AB - OBJECTIVE: The purpose of this study was to determine whether the bispectral index scale (BIS) would provide added benefit to established methods of monitoring conscious sedation with midazolam (M group) or midazolam supplemented with ketamine (MK group). STUDY DESIGN: BIS was prospectively and blindly examined in 22 patients receiving outpatient oral surgery with conscious sedation supplemented with local anesthesia. RESULTS: The average midazolam dose in the midazolam group over the treatment period was 0.01 mg/kg/h, and the average midazolam plus ketamine dose was 0.01 and 0.05 mg/kg/h, respectively. Mean BIS values throughout the sedation study period were 90 for the midazolam group and 94 for the midazolam plus ketamine group. The addition of ketamine did not lower BIS. BIS values did not alter significantly over time except for an expected transient drop after the midazolam bolus induction. CONCLUSION: BIS levels remained close to baseline levels, suggesting that BIS would not provided any additional benefit to currently established methods of monitoring patient consciousness during conscious sedation for oral surgery. PMID- 12374914 TI - Assessment of perfusion of facial microvascular transplants and early detection of ischemia by perfusion-CT scan. AB - OBJECTIVE: Perfusion-computed tomography (CT) is a promising new technique to assess ischemic lesions caused by ischemic brain stroke. In this study, the use of perfusion-CT scans to predict ischemia in microvascular transplants of the face was examined. STUDY DESIGN: Thirty-eight patients with microvascular latissimus dorsi transplants after tumor surgery were assessed by perfusion-CT scan 34 to 72 hours after surgery. In these cases, clinical examination of the transplant and examination by means of O(2)-probes were either unsuccessful or impossible. An electron beam tomography of the region of interest was performed by using an intravenous nonionic iodine-containing contrast medium (Ultravist 300, Nycomed, Germany) that was applied with an injector at a flow rate of 5 mL/min. Twenty scans with a scanning time of 300 ms and an interscanning time of 3 seconds were carried out. Changes in the Houndsfield units within the transplant as well as the region of the contralateral erector spinae muscle were measured. RESULTS: Central malperfusion resulting in later complete transplant loss was detected in 2 cases. Peripheral malperfusion was found in 6 cases, resulting in localized resection and secondary wound closure. When no malperfusion was registered, the straightforward healing process took place. CONCLUSION: Perfusion-CT scans are of great aid in the assessment of microvascular transplant perfusion in the face, when adequate perfusion is not verifiable clinically or by O(2)-probe because of removal or malfunction. PMID- 12374915 TI - Salivary albumin and other constituents and their relation to oral and general health in the elderly. AB - OBJECTIVE: Our objective was to study salivary albumin concentrations in the elderly. We expected higher albumin values in the frail and diseased elderly in comparison to the more fit elderly. This was thought to be due to the eventually decreased mucosal integrity in the diseased patients. STUDY DESIGN: Paraffin wax stimulated saliva was collected for 5 minutes from 131 hospitalized elderly, mean age of 82 years, and 252 elderly outpatients, mean age of 77 years. Forty-seven of the hospitalized elderly died during the 2-year follow-up. Albumin was analyzed spectrophotometrically from the saliva specimens, and values were studied statistically between the groups and regarding a number of background variables. RESULTS: The following mean salivary albumin concentrations were observed: outpatients, 200 +/- 157 microg/mL; hospitalized surviving patients, 401 +/- 247 microg/mL; patients who later died, 501 +/- 417 microg/mL. The respective albumin output values were 439.7 +/- 432.8 microg/min in outpatients, 684.3 +/- 396.8 microg/min in hospitalized patients who survived, and 700.0 +/- 481.9 microg/min in the hospitalized patients who died. The differences were significant between the groups. The strongest explanatory factors for higher than median albumin concentrations were the use of analgesics in the hospitalized patients (odds ratio, 4.2; confidence interval, 1.5 to 11.4) and retaining own teeth in the outpatients (odds ratio, 4.3; confidence interval, 1.9 to 4.3). Frequency of mucosal pathologic condition did not appear as an explanatory factor in this respect. CONCLUSIONS: Our study hypothesis was confirmed, showing significantly higher salivary albumin concentrations in the frail elderly. The present results may also be used as reference data for salivary albumin in the elderly. PMID- 12374916 TI - Relationship between bleeding time test and postextraction bleeding in a healthy control population. AB - OBJECTIVE: We sought to determine whether cutaneous bleeding time (BT) is related to bleeding outcome measures after a single tooth extraction. STUDY DESIGN: This was a prospective clinical pilot study of 30 subjects. Cutaneous BT was evaluated before a single tooth extraction. After extraction, an oral BT was determined. Subjects were contacted 3 to 7 hours and 2 days after extraction to assess further postoperative bleeding. RESULTS: The mean cutaneous BT was 5.9 minutes (range 1.5-10.0 minutes). The mean oral BT was 7.5 minutes (range 0-20 minutes). Cutaneous BT did not correlate with oral BT or any of our measures of postoperative bleeding. However, the oral BT correlated with the number of hours of bleeding after surgery (R(s) = 0.54, P =.03). The time necessary to perform the extraction correlated with the extraction site bleeding 3 to 7 hours after surgery (R(s) = 0.67, P =.0006). CONCLUSION: Cutaneous BT did not correlate with measures of postoperative bleeding in the present study, but oral BT immediately after extraction correlated with the duration of subsequent postoperative bleeding. PMID- 12374917 TI - Amyloidosis of the tongue as a paraneoplastic marker of plasma cell dyscrasia. AB - OBJECTIVE: Our objective was to study the results of the medical workup in patients with amyloidosis of the oral cavity. STUDY DESIGN: Patients diagnosed with amyloidosis of the oral cavity during the period from January 1971 to January 2001 at the Departments of Oral and Maxillofacial Surgery/Oral Pathology and Dermatology of the VU University Medical Center, Amsterdam, The Netherlands, were included in this retrospective case study. In total, this series comprised 11 patients, 9 women and 2 men. The patients' medical workup and final diagnoses were traced by means of the medical records. RESULTS: All but one patient presented with amyloidosis of the tongue, most of them manifesting as macroglossia. In 7 of the 11 included patients a diagnosis of myeloma could be established shortly after their referral to the above-stated departments. Three of the 4 remaining patients appeared to have a monoclonal gammopathy of undetermined significance, and 1 patient was diagnosed with a lymphoplasmacytoid non-Hodgkin lymphoma (immunocytoma). CONCLUSIONS: Amyloidosis of the oral cavity predominantly involves the tongue, mainly manifesting as macroglossia. Amyloidosis of the tongue is associated with an occult underlying plasma cell dyscrasia, in particular myeloma, and, therefore, should be regarded as a paraneoplastic phenomenon of these hematologic diseases. PMID- 12374918 TI - Undiagnosed multiple myeloma causing extensive dental bleeding: report of a case and review. AB - A case of multiple myeloma causing profuse bleeding during a minor dental surgical procedure is presented. The value of dental radiography in detection of bone changes associated with an undiagnosed case of multiple myeloma is highlighted. We show that the extensive bleeding during the dental procedure could have been prevented if the panoramic radiograph had been evaluated carefully before initiation of the treatment. In addition, we briefly discuss the etiologic factors responsible for the formation of hemostatic abnormalities in multiple myeloma and the value of imaging methods used in diagnostic assessment of the disease. PMID- 12374919 TI - Regression of HIV parotid swellings after antiviral therapy: case reports with computed tomographic scan evidence. AB - Parotid swellings have been noted in approximately 5% to 10% of patients with HIV 1. These swellings are a result of lymphoproliferative activity or lymphoepithelial cyst formation in response to a viral parotid presence. Two patients with cosmetic concerns regarding parotid swellings are presented before and after antiviral therapy. Regression of the swellings after treatment is substantiated with clinical photographs and computerized tomographic scans. The successful outcome was a reflection of a diminution in viral load and some degree of immune restoration. PMID- 12374920 TI - Anterior lingual mandibular salivary gland defect (Stafne defect) presenting as a residual cyst. AB - Lingual mandibular salivary gland inclusion (Stafne defect) is a developmental anomaly represented by a bone concavity usually containing submandibular gland tissue. The posterior mandible region, particularly at the angle and below the mandibular canal, is the common location, and the anterior mandibular variants occur rather seldom. The latter is usually observed in the premolar and cuspid region, or more rarely in the symphysis, as a round or ovoid radiolucency sometimes appearing superimposed over the teeth's apices, resembling a true cystic lesion or an odontogenic tumor. We report an additional case of anterior lingual mandibular salivary gland defect occurring in a 42-year-old white man. It presented as an asymptomatic radiolucency located on the left side of the mandible, in the region of an absent second premolar and first molar, above the alveolar canal, mimicking a residual cyst. Histopathologic examination of the "cyst" content revealed the absence of a cyst lining and the presence of normal sublingual gland tissue. PMID- 12374921 TI - Aggressive destructive midfacial lesion from cocaine abuse. AB - Since the first reported case in 1912 of cocaine-induced perforation of the palate, an additional 7 cases have been reported describing extensive palatal destruction. The clinical presentation shares similarities with nasal-type natural killer/T-cell lymphoma, Wegener's granulomatosis, and infectious diseases. We describe a 50-year-old woman with a progressively destructive midfacial process that initially appeared as a small, localized palatal defect. Over time, the lesion caused bilateral deformity of the ala, extensive loss of the palate, maxillary and sinonasal complexes, ethmoids, and ulceration of adjacent tissue. Clinical laboratory tests showed elevated cytoplasmic antineutrophil cytoplasmic antibodies, but the histopathology did not support the diagnosis of Wegener's granulomatosis. Special stains and cultures were negative for infectious organisms. Flow cytometry and T-cell gene rearrangement studies ruled out lymphoma. Because of the inability to diagnose this worrisome process, the presence of polarizable foreign material in the original biopsy, and the patient's admission to past cocaine use, a urine drug screen was performed, which was positive for cocaine and marijuana. PMID- 12374922 TI - Tongue necrosis secondary to temporal arteritis: a case report and literature review. AB - We report a case of tongue necrosis secondary to temporal arteritis. Temporal arteritis can have devastating consequences, leading to blindness or death unless recognized and treated appropriately. Initial presentation may be to an oral surgeon, and we discuss the pathogenesis, diagnosis, and management of temporal arteritis. PMID- 12374923 TI - Celiac disease and recurrent aphthous stomatitis: a report and review of the literature. AB - Celiac disease (CD) is a condition related to the small intestine's intolerance to gluten. The diagnosis of CD can be difficult, especially because patients may exhibit a wide spectrum of signs and symptoms. It is important to identify this disease process early because affected individuals have an increased risk for developing lymphoma of the gut. Our objective was to evaluate whether patients with CD have a significantly higher prevalence of recurrent aphthous stomatitis compared with the general population, as some investigators have speculated. Therefore, we screened 61 patients with diagnosed CD for the presence of, or a positive history of, aphthous ulcerations. We then statistically compared this data with a randomly selected control population, matched for age and gender, but without CD. Our results demonstrated no significant differences between groups for age, gender, or prevalence of recurrent aphthous stomatitis. PMID- 12374924 TI - Rothmund-Thomson syndrome: a case report. AB - Rothmund-Thomson syndrome (RTS) is an extremely rare genetic disorder characterized by poikilodermatous skin changes, photosensitivity, and an increased risk of developing skin and bone malignancies. In this case report, the dental and periodontal features of RTS in a 16-year-old female patient are presented. The transmission electron microscopy performed on a gingival biopsy specimen showed structural defects of connective tissue. If the unusual ultrastructural findings of this case are confirmed as being consistent with other RTS patients, it is our opinion that this syndrome can be considered among the systemic diseases associated with early-onset periodontitis. PMID- 12374925 TI - Ciliated epithelium-lined radicular cysts. AB - OBJECTIVE: This report describes 3 cases of ciliated epithelium-lined radicular cysts among 256 apical periodontitis lesions and also illustrates the occurrence of an Actinomyces-infected periapical cyst. STUDY DESIGN: Serial and step serial sections of 256 plastic-embedded root apices with attached apical periodontitis lesions that were prepared for a previous investigation were reviewed for the presence of ciliated epithelium-lined radicular cysts. The lesions that were found to have such epithelial lining were examined in a transmission electron microscope to elaborate the fine structure of the ciliated cells. RESULTS: A total of 3 ciliated columnar epithelium-lined cysts was found among the 256 apical periodontitis lesions examined. Two of the lesions also contained stratified squamous epithelium. All 3 lesions affected maxillary premolars. One of the lesions was a true cyst, and the other 2 were periapical pocket cysts. The lumen of 1 of the latter revealed the presence of typical "ray-fungus" actinomycotic colonies. CONCLUSION: Although the stratified squamous component of the epithelia that lined the radicular cysts reported here may be derived from the cell rests of Malassez, the ciliated epithelial cells may be of sinus origin. Microbial agents from diseased root canals can advance into radicular cysts, particularly in pocket cysts, with the possible threat of such infection in upper posterior teeth spreading into the maxillary sinus. PMID- 12374926 TI - Dialister pneumosintes can be a suspected endodontic pathogen. AB - OBJECTIVE: Dialister pneumosintes is a nonmotile, nonfermentative, non-spore forming, obligately anaerobic, gram-negative bacillus that has been associated with some infections in the human body. A species-specific nested polymerase chain reaction assay was used to investigate the occurrence of D pneumosintes in primary root canal infections. STUDY DESIGN: Samples were collected from 32 teeth with carious lesions, necrotic pulps, and radiographic evidence of periradicular bone destruction. Twenty-two teeth were asymptomatic, and 10 cases were diagnosed as acute apical periodontitis. DNA extracted from the samples was initially amplified with universal 16S ribosomal DNA primers. A second round of amplification used the first polymerase chain reaction products to specifically detect D pneumosintes. RESULTS: This bacterial species was detected in 17 of 22 asymptomatic cases (77.3%) and in 4 of 10 root canals associated with acute apical periodontitis (40%). No relationship was found between the presence of this bacterial species and the occurrence of symptoms. In general, D pneumosintes was detected in 21 of 32 root canal samples (65.6%). CONCLUSION: This study is the first to report a high prevalence of D pneumosintes in root canal infections of humans. Because of this high prevalence and the apparent pathogenicity of the microorganism, we suggest the inclusion of D pneumosintes in the selected group of putative endodontic pathogens. PMID- 12374927 TI - An 18-month longitudinal study on a new silicon-based sealer, RSA RoekoSeal: a leakage study in vitro. AB - OBJECTIVE: Because leakage along root fillings may increase or decrease during the long period after filling, the accumulated leakage data measured repeatedly after filling are clinically more relevant than the data measured just once shortly after filling. The aim of this study was to determine the long-term sealing ability of a recently developed root canal sealer RSA RoekoSeal (Roeko Dental Products, Langenau, Germany). STUDY DESIGN: Eighty extracted human mandibular premolars were equally divided into 4 groups (n = 20) and root canal instrumented. Canals in group 1 were obturated with laterally compacted gutta percha points and RSA, in groups 2 to 4 with vertically compacted warm gutta percha using RSA in group 2, Pulp Canal Sealer (Kerr, Romulus, Mich) in group 3, and no sealer in group 4. With the use of a fluid transport model, leakage along entire root fillings was measured before post space preparation. After post space preparation, leakage along the remaining apical root fillings was measured repeatedly at 1 week, 1, 2, 6, 12, and 18 months respectively and recorded in microliters per day. RESULTS: Before post space preparation, groups 1 and 2 leaked significantly less than groups 3 and 4 (P = .001), whereas no significant difference existed between the groups 1 and 2 (P = .317) or between the groups 3 and 4 (P = .074). For each group, a summation of the leakage at all time intervals after the post space preparation showed that the 2 RSA groups leaked significantly less than the other 2 groups (P =.000). However, no significant difference existed between the 2 RSA groups (P = .993) and between the groups 3 and 4 (P = .149). CONCLUSION: RSA used in combination with either cold laterally compacted or warm vertically compacted gutta-percha provided a consistent seal during a period of 18 months. PMID- 12374928 TI - Comparative study of clinical manifestation, plain-film radiography, and computed tomographic scan in arteriovenous malformations of the jaws. AB - OBJECTIVE: The purpose of this study was to summarize the clinical manifestation, plain-film radiography, and computed tomographic (CT) scan features of arteriovenous malformations (AVMs) of the jaws on the basis of a series of 12 patients. STUDY DESIGN: This study group comprised twelve patients with AVM of the jaws from February 1996 to February 2001. Seven cases were located in the mandible, and 5 in the maxilla. Both plain-film radiography and CT scan were available for all cases. For the patients with lesions in the mandible, panoramic, posterioanterior, and lateral mandibular views were applied. Waters' position view and panoramic radiography were indicated for AVMs of the maxilla. RESULTS: Each patient with AVM of the maxillary bone had involvement of adjacent soft tissues. Various radiographic signs were noted, including erosion, coarse trabeculae, and apparent lack of any radiographic change, and CT scans featured cystic expansion of alveolar process with broken cortex. The radiographic signs and CT scan features of AVMs in the mandible were related to involvement of surrounding soft tissues. If involvement of the adjacent soft tissues was found, "soap bubble" radiolucency was shown radiographically and osteolytic expansion with perforation of cortex was present on CT scan. In cases without surrounding soft tissue involvement, the various radiographic signs included multilocular or unilocular radiolucency or coarse trabeculae; osteolytic expansions with intact cortex were found on CT scan. CONCLUSION: AVMs of the jaws showed intraosseous osteolytic expansion on CT scan but had variable appearance on plain-film radiographs. PMID- 12374929 TI - Carotid calcification on panoramic radiographs: an important marker for vascular risk. AB - OBJECTIVE: The objective of this study was to determine whether carotid calcifications are harbingers of future vascular events. STUDY DESIGN: Between 1986 and 2000, 71 patients were found to have carotid artery calcifications on routine panoramic films. Medical records were reviewed for vascular risk factors existing before and vascular end points subsequent to the radiographs. RESULTS: The mean age of our patients was 68 years. Sixty-one (86%) had preexisting vascular risk factors, 73% with multiple risk factors. Forty-one end points occurred in 29 patients. The average time to an end point was 2.7 years. The end points included myocardial infarction (8, 11%), stroke (5, 7%), death (11, 15%), revascularization procedures (8, 11%), transient ischemic attack (2, 3%), and angina (7, 10%). Twenty-three patients (34%) had major end points of myocardial infarction, stroke, or death. CONCLUSIONS: Carotid calcifications identified on panoramic radiographs are powerful markers for subsequent vascular events. Patients found to have carotid calcification on panoramic radiographs should be referred for cerebrovascular and cardiovascular evaluation and aggressive management of vascular risk factors. PMID- 12374930 TI - Does joint effusion on T2 magnetic resonance images reflect synovitis? Part 2. Comparison of concentration levels of proinflammatory cytokines and total protein in synovial fluid of the temporomandibular joint with internal derangements and osteoarthrosis. AB - OBJECTIVE: We sought to clarify the nature of joint effusion (JE) on T2-weighted magnetic resonance images of the temporomandibular joint (TMJ) by analysis of the synovial fluid in the superior compartment of patients with internal derangement and osteoarthrosis. STUDY DESIGN: One hundred symptomatic TMJs (100 patients) with 65 internal derangements and 35 osteoarthroses were scanned by means of magnetic resonance imaging, and, the synovial fluid was sampled on the same day. The amount of JE was evaluated on a scale of 0 to 3. Grades 0 and 1 indicated absence of JE or a negligible amount of JE, respectively, and grades 2 and 3 indicated the presence of JE. Correlation was evaluated among the amount of JE and the concentrations of the total protein and interleukin-1beta(IL-1beta), IL 6, IL-8, and tumor necrosis factor-alpha in the synovial fluid. RESULTS: Magnetic resonance imaging revealed the absence of JE in 40 joints (grade 0, 17 joints; grade 1, 23 joints) and the presence of JE in 60 joints (grade 2, 31 joints; grade 3, 29 joints). The joints with JE had, on average, significantly higher concentrations of total protein (1,675 microg vs 714 microg; P = .0001) and IL-6 (42.9 pg vs 10.6 pg; P = .009) than did the joints without JE. Furthermore, there were significant correlations between the JE grade and the concentrations of the total protein (P = .0001), IL-6 (P = .001), and IL-8 (P = .004). The detection ratio of cytokines among the presence-absence groups of JE showed a significant difference in tumor necrosis factor-alpha (68.3% vs 47.5%; P = .037) and IL-6 (86.7% vs 67.5%; P = .012). Conclusions. JE may contain the released products when there is pronounced synovitis. It is probably composed of high concentrations of total protein with inflammatory cytokines. Furthermore, IL-6 and IL-8 seem to have an important role in the pathogenesis of JE in TMJ disorders. PMID- 12374931 TI - Somatic cell nuclear transfer. AB - Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term. PMID- 12374933 TI - While industrial agrobiotech R&D falters, opportunities in plant biology in the public sector are growing. PMID- 12374934 TI - Materials for sustainability. PMID- 12374935 TI - China ponders joining fusion project. PMID- 12374937 TI - Discovery of giant asteroid gives Pluto a rocky outlook. PMID- 12374936 TI - Regulators split on gene therapy as patient shows signs of cancer. PMID- 12374938 TI - Call for clinical-trial reform leaves critics unmoved. PMID- 12374939 TI - Swiss law imposes iron rule on biotechnology field trials. PMID- 12374940 TI - Winning universities set out to fulfil Japan's plans for excellence. PMID- 12374941 TI - Universal view nets award for cosmic sleuths. PMID- 12374943 TI - Bubble won't burst for spoof Nobels. PMID- 12374942 TI - Worm cast in starring role for Nobel prize. PMID- 12374945 TI - Nanotechnology. Wired for success. PMID- 12374944 TI - AIDS activist freed by Chinese authorities. PMID- 12374946 TI - New Alexandria Library. A temple of knowledge. PMID- 12374947 TI - Taxonomy needs evolution, not revolution. PMID- 12374948 TI - Metastasis: the role of chance in malignancy. PMID- 12374949 TI - Metastasis: objections to the same-gene model. PMID- 12374951 TI - Chilean decree will save nights for star-gazers. PMID- 12374956 TI - Microbial food webs. The ocean's veil. PMID- 12374957 TI - Archaeology. Life with the artificial Anasazi. PMID- 12374958 TI - Indistinguishable photons from a single-photon device. AB - Single-photon sources have recently been demonstrated using a variety of devices, including molecules, mesoscopic quantum wells, colour centres, trapped ions and semiconductor quantum dots. Compared with a Poisson-distributed source of the same intensity, these sources rarely emit two or more photons in the same pulse. Numerous applications for single-photon sources have been proposed in the field of quantum information, but most--including linear-optical quantum computation- also require consecutive photons to have identical wave packets. For a source based on a single quantum emitter, the emitter must therefore be excited in a rapid or deterministic way, and interact little with its surrounding environment. Here we test the indistinguishability of photons emitted by a semiconductor quantum dot in a microcavity through a Hong-Ou-Mandel-type two-photon interference experiment. We find that consecutive photons are largely indistinguishable, with a mean wave-packet overlap as large as 0.81, making this source useful in a variety of experiments in quantum optics and quantum information. PMID- 12374959 TI - Superconductivity. Putting the squeeze on lithium. PMID- 12374960 TI - Behavioural ecology. Excuses for avian infidelity. PMID- 12374961 TI - Cancer. The silence of the genes. PMID- 12374962 TI - Nanotechnology. Beyond the silicon roadmap. PMID- 12374964 TI - Fluid mechanics. Impact factors. PMID- 12374963 TI - Ecology. Biodiversity in the scales. PMID- 12374965 TI - Quantum physics. Single photons stick together. PMID- 12374966 TI - Obituary: George Porter (1920-2002). PMID- 12374967 TI - Mechanics. Buckling cascades in free sheets. PMID- 12374968 TI - Thin films. Wrinkling of an elastic sheet under tension. PMID- 12374969 TI - Anthropogenic aerosols. Indirect warming effect from dispersion forcing. PMID- 12374970 TI - Palaeoanthropology. Sahelanthropus or 'Sahelpithecus'? PMID- 12374972 TI - Crystal structure of bacterial multidrug efflux transporter AcrB. AB - AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel. PMID- 12374974 TI - Propagation of the polyamorphic transition of ice and the liquid-liquid critical point. AB - Water has a rich metastable phase behaviour that includes transitions between high- and low-density amorphous ices, and between high- and low-density supercooled liquids. Because the transitions occur under conditions where crystalline ice is the stable phase, they are challenging to probe directly. In the case of the liquids, it remains unclear whether their mutual transformation at low temperatures is continuous, or discontinuous and terminating at a postulated second critical point of water that is metastable with respect to crystallization. The amorphous ices are more amenable to experiments, which have shown that their mutual transformation is sharp and reversible. But the non equilibrium conditions of these studies make a firm thermodynamic interpretation of the results difficult. Here we use Raman spectroscopy and visual inspection to show that the transformation of high-density to low-density amorphous ices involves the propagation of a phase boundary-a region containing a mixture of both ices. We find that the boundary region becomes narrower as the transformation progresses, and at higher transformation temperatures. These findings strongly suggest that the polyamorphic ice transition is discontinuous; a continuous transformation should occur uniformly over the entire sample. Because the amorphous ices are structurally similar to their supercooled liquid counterparts, our results also imply that the liquids transform discontinuously at low temperatures and thus support the liquid-liquid critical-point theory. PMID- 12374973 TI - Superconductivity in compressed lithium at 20 K. AB - Superconductivity at high temperatures is expected in elements with low atomic numbers, based in part on conventional BCS (Bardeen-Cooper-Schrieffer) theory. For example, it has been predicted that when hydrogen is compressed to its dense metallic phase (at pressures exceeding 400 GPa), it will become superconducting with a transition temperature above room temperature. Such pressures are difficult to produce in a laboratory setting, so the predictions are not easily confirmed. Under normal conditions lithium is the lightest metal of all the elements, and may become superconducting at lower pressures; a tentative observation of a superconducting transition in Li has been previously reported. Here we show that Li becomes superconducting at pressures greater than 30 GPa, with a pressure-dependent transition temperature (T(c)) of 20 K at 48 GPa. This is the highest observed T(c) of any element; it confirms the expectation that elements with low atomic numbers will have high transition temperatures, and suggests that metallic hydrogen will have a very high T(c). Our results confirm that the earlier tentative claim of superconductivity in Li was correct. PMID- 12374975 TI - Directly measured mid-depth circulation in the northeastern North Atlantic Ocean. AB - The circulation of water masses in the northeastern North Atlantic Ocean has a strong influence on global climate owing to the northward transport of warm subtropical water to high latitudes. But the ocean circulation at depths below the reach of satellite observations is difficult to measure, and only recently have comprehensive, direct observations of whole ocean basins been possible. Here we present quantitative maps of the absolute velocities at two levels in the northeastern North Atlantic as obtained from acoustically tracked floats. We find that most of the mean flow transported northward by the Gulf Stream system at the thermocline level (about 600 m depth) remains within the subpolar region, and only relatively little enters the Rockall trough or the Nordic seas. Contrary to previous work, our data indicate that warm, saline water from the Mediterranean Sea reaches the high latitudes through a combination of narrow slope currents and mixing processes. At both depths under investigation, currents cross the Mid Atlantic Ridge preferentially over deep gaps in the ridge, demonstrating that sea floor topography can constrain even upper-ocean circulation patterns. PMID- 12374976 TI - A correlation between mid-ocean-ridge basalt chemistry and distance to continents. AB - To fully understand the structure and dynamics of the Earth's convecting mantle, the origins of temperature variations within the mantle need to be resolved. Different hypotheses have been proposed to account for these temperature variations: for example, heat coming from the decay of radioactive elements or heat flowing out of the Earth's core. In addition, theoretical studies suggest that the thermal properties of continental masses can affect mantle convection, but quantitative data that could allow us to test these models are scarce. To address this latter problem, we have examined the chemistry of mid-ocean-ridge basalt--which reflects the temperature of the source mantle--as a function of the distance of the ridge from the closest continental margin. No correlation is observed for oceanic ridges close to subduction zones or hotspots; subduction zones probably inhibit thermal transfer between the mantle beneath continents and ocean, whereas hotspots influence the major-element chemistry of ridge basalts, which makes their interpretation with respect to mantle temperature more difficult. However, we do observe a significant correlation for mid-oceanic basalts from the Atlantic and Indian oceans. From this, we conclude that the location of continental masses relative to active ridges influences the large scale thermal structure of the mantle and we estimate that the mantle cools by 0.05 to 0.1 degrees C per kilometre from the continental margins. PMID- 12374977 TI - General patterns of taxonomic and biomass partitioning in extant and fossil plant communities. AB - A central goal of evolutionary ecology is to identify the general features maintaining the diversity of species assemblages. Understanding the taxonomic and ecological characteristics of ecological communities provides a means to develop and test theories about the processes that regulate species coexistence and diversity. Here, using data from woody plant communities from different biogeographic regions, continents and geologic time periods, we show that the number of higher taxa is a general power-function of species richness that is significantly different from randomized assemblages. In general, we find that local communities are characterized by fewer higher taxa than would be expected by chance. The degree of taxonomic diversity is influenced by modes of dispersal and potential biotic interactions. Further, changes in local diversity are accompanied by regular changes in the partitioning of community biomass between taxa that are also described by a power function. Our results indicate that local and regional processes have consistently regulated community diversity and biomass partitioning for millions of years. PMID- 12374978 TI - Genetic similarity between mates and extra-pair parentage in three species of shorebirds. AB - Matings between close relatives often reduce the fitness of offspring, probably because homozygosity leads to the expression of recessive deleterious alleles. Studies of several animals have shown that reproductive success is lower when genetic similarity between parents is high, and that survival and other measures of fitness increase with individual levels of genetic diversity. These studies indicate that natural selection may favour the avoidance of matings with genetically similar individuals. But constraints on social mate choice, such as a lack of alternatives, can lead to pairing with genetically similar mates. In such cases, it has been suggested that females may seek extra-pair copulations with less related males, but the evidence is weak or lacking. Here we report a strong positive relationship between the genetic similarity of social pair members and the occurrence of extra-pair paternity and maternity ('quasi-parasitism') in three species of shorebirds. We propose that extra-pair parentage may represent adaptive behavioural strategies to avoid the negative effects of pairing with a genetically similar mate. PMID- 12374979 TI - Attentional modulation in visual cortex depends on task timing. AB - Paying attention to a stimulus selectively increases the ability to process it. For example, when subjects attend to a specific region of a visual scene, their sensitivity to changes at that location increases. A large number of studies describe the behavioural consequences and neurophysiological correlates of attending to spatial locations. There has, in contrast, been little study of the allocation of attention over time. Because subjects can anticipate predictable events with great temporal precision, it seems probable that they might dynamically shift their attention when performing a familiar perceptual task whose constraints changed over time. We trained monkeys to respond to a stimulus change where the probability of occurrence changed over time. Recording from area V4 of the visual cortex in these animals, we found that the modulation of neuronal responses changed according to the probability of the change occurring at that instant. Thus, we show that the attentional modulation of sensory neurons reflects a subject's anticipation of the timing of behaviourally relevant events. PMID- 12374980 TI - FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. AB - The study of planarian regeneration may help us to understand how we can rebuild organs and tissues after injury, disease or ageing. The robust regenerative abilities of planarians are based upon a population of totipotent stem cells (neoblasts), and among the organs regenerated by these animals is a well organized central nervous system. In recent years, methodologies such as whole mount in situ hybridizations and double-stranded RNA have been extended to planarians with the aim of unravelling the molecular basis of their regenerative capacities. Here we report the identification and characterization of nou-darake (ndk), a gene encoding a fibroblast growth factor receptor (FGFR)-like molecule specifically expressed in the head region of the planarian Dugesia japonica. Loss of function of ndk by RNA interference results in the induction of ectopic brain tissues throughout the body. This ectopic brain formation was suppressed by inhibition of two planarian FGFR homologues (FGFR1 and FGFR2). Additionally, ndk inhibits FGF signalling in Xenopus embryos. The data suggest that ndk may modulate FGF signalling in stem cells to restrict brain tissues to the head region of planarians. PMID- 12374981 TI - The polycomb group protein EZH2 is involved in progression of prostate cancer. AB - Prostate cancer is a leading cause of cancer-related death in males and is second only to lung cancer. Although effective surgical and radiation treatments exist for clinically localized prostate cancer, metastatic prostate cancer remains essentially incurable. Here we show, through gene expression profiling, that the polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in hormone-refractory, metastatic prostate cancer. Small interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells and also inhibit cell proliferation in vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional repression of a specific cohort of genes. Gene silencing mediated by EZH2 requires the SET domain and is attenuated by inhibiting histone deacetylase activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in metastatic prostate cancer; in addition, clinically localized prostate cancers that express higher concentrations of EZH2 show a poorer prognosis. Thus, dysregulated expression of EZH2 may be involved in the progression of prostate cancer, as well as being a marker that distinguishes indolent prostate cancer from those at risk of lethal progression. PMID- 12374982 TI - Class IV semaphorin Sema4A enhances T-cell activation and interacts with Tim-2. AB - Semaphorins are a family of phylogenetically conserved soluble and transmembrane proteins. Although many soluble semaphorins deliver guidance cues to migrating axons during neuronal development, some members are involved in immune responses. For example, CD100 (also known as Sema4D), a class IV transmembrane semaphorin, signals through CD72 to effect nonredundant roles in immune responses in a ligand receptor system that is distinct from any seen previously in the nervous system. Here we report that the class IV semaphorin Sema4A, which is expressed in dendritic cells and B cells, enhances the in vitro activation and differentiation of T cells and the in vivo generation of antigen-specific T cells. Treating mice with monoclonal antibodies against Sema4A blocks the development of an experimental autoimmune encephalomyelitis that is induced by an antigenic peptide derived from myelin oligodendrocyte glycoprotein. In addition, expression cloning shows that the Sema4A receptor is Tim-2, a member of the family of T-cell immunoglobulin domain and mucin domain (Tim) proteins that is expressed on activated T cells. PMID- 12374983 TI - Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. AB - Apoptosis is an evolutionarily conserved cell suicide process executed by cysteine proteases (caspases) and regulated by the opposing factions of the Bcl-2 protein family. Mammalian caspase-9 and its activator Apaf-1 were thought to be essential, because mice lacking either of them display neuronal hyperplasia and their lymphocytes and fibroblasts seem resistant to certain apoptotic stimuli. Because Apaf-1 requires cytochrome c to activate caspase-9, and Bcl-2 prevents mitochondrial cytochrome c release, Bcl-2 is widely believed to inhibit apoptosis by safeguarding mitochondrial membrane integrity. Our results suggest a different, broader role, because Bcl-2 overexpression increased lymphocyte numbers in mice and inhibited many apoptotic stimuli, but the absence of Apaf-1 or caspase-9 did not. Caspase activity was still discernible in cells lacking Apaf-1 or caspase-9, and a potent caspase antagonist both inhibited apoptosis and retarded cytochrome c release. We conclude that Bcl-2 regulates a caspase activation programme independently of the cytochrome c/Apaf-1/caspase-9 'apoptosome', which seems to amplify rather than initiate the caspase cascade. PMID- 12374984 TI - Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. AB - Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions. PMID- 12374985 TI - SATB1 targets chromatin remodelling to regulate genes over long distances. AB - Eukaryotic chromosomes are organized inside the nucleus in such a way that only a subset of the genome is expressed in any given cell type, but the details of this organization are largely unknown. SATB1 ('special AT-rich sequence binding 1'), a protein found predominantly in thymocytes, regulates genes by folding chromatin into loop domains, tethering specialized DNA elements to an SATB1 network structure. Ablation of SATB1 by gene targeting results in temporal and spatial mis-expression of numerous genes and arrested T-cell development, suggesting that SATB1 is a cell-type specific global gene regulator. Here we show that SATB1 targets chromatin remodelling to the IL-2Ralpha ('interleukin-2 receptor alpha') gene, which is ectopically transcribed in SATB1 null thymocytes. SATB1 recruits the histone deacetylase contained in the NURD chromatin remodelling complex to a SATB1-bound site in the IL-2Ralpha locus, and mediates the specific deacetylation of histones in a large domain within the locus. SATB1 also targets ACF1 and ISWI, subunits of CHRAC and ACF nucleosome mobilizing complexes, to this specific site and regulates nucleosome positioning over seven kilobases. SATB1 defines a class of transcriptional regulators that function as a 'landing platform' for several chromatin remodelling enzymes and hence regulate large chromatin domains. PMID- 12374986 TI - Reaction path of protein farnesyltransferase at atomic resolution. AB - Protein farnesyltransferase (FTase) catalyses the attachment of a farnesyl lipid group to numerous essential signal transduction proteins, including members of the Ras superfamily. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. FTase inhibitors are currently in clinical trials for the treatment of cancer. Here we present a complete series of structures representing the major steps along the reaction coordinate of this enzyme. From these observations can be deduced the determinants of substrate specificity and an unusual mechanism in which product release requires binding of substrate, analogous to classically processive enzymes. A structural model for the transition state consistent with previous mechanistic studies was also constructed. The processive nature of the reaction suggests the structural basis for the successive addition of two prenyl groups to Rab proteins by the homologous enzyme geranylgeranyltransferase type-II. Finally, known FTase inhibitors seem to differ in their mechanism of inhibiting the enzyme. PMID- 12374988 TI - Rubber dammit! PMID- 12374989 TI - Ethical selling. PMID- 12374990 TI - Teeth whitening debate I. PMID- 12374991 TI - Teeth whitening debate II. PMID- 12374992 TI - The York report. PMID- 12374993 TI - Aphthous ulcers. PMID- 12374994 TI - A word in your ear. PMID- 12374995 TI - Gingival recession. PMID- 12374996 TI - The use of the QNST-II as a measure for the identification of children with perceptual-motor deficits. AB - This study aimed to examine the ability of the Quick Neurological Screening Test QNST II) (Mutti et al., 1998) to discriminate between children with and without perceptual-motor deficits and to further clarify its psychometric characteristics. Ninety-four children aged six to seven years were tested on the QNST-II. Out of this pool of subjects, 63 children had perceptual-motor deficits and 31 were typical controls. The children with perceptual-motor deficits scored significantly lower than the control children on the total score and on each of the subtest' s scores of the QNST II. Inter-rater reliability indicated a high degree of correlation between both evaluators' total scores of the QNST II. In terms of the test' s sensitivity and specificity, QNST II scores correctly classified 97% of the children with perceptual-motor deficits and 84% of the children from the control group. The findings of this study support the capability of the QNST II to discriminate between children with perceptual-motor deficits and typical children; thereby suggesting its usefulness as a screening measure to identify children at risk for difficulties in school performance. PMID- 12374997 TI - Comparison of Rolyan and Jamar dynamometers for measuring grip strength. AB - This study compared the Jamar and Rolyan hydraulic dynamometers to determine their concurrent validity with known weights as well as their inter-instrument reliability and concurrent validity for measuring grip strength in a clinical setting. Thirty females and 30 males were tested on these two grip strength measurement devices using a repeated measure design. Results showed that the Jamar and Rolyan dynamometers have acceptable concurrent validity with known weights (that is, correlation coefficients were r > or = 0.9994), excellent inter instrument reliability (that is, intraclass correlation coefficients ranged from 0.90 to 0.97) and strong concurrent validity (that is, no significant differences between dynamometers' scores). Data indicate that Jamar and Rolyan dynamometers measure grip strength equivalently and can be used interchangeably. Thus, therapists using the Rolyan dynamometer are justified in using published normative data, which were collected with the Jamar dynamometer. PMID- 12374998 TI - Project placements for undergraduate occupational therapy students: design, implementation and evaluation. AB - This study aimed to document the process undertaken to incorporate project placements as an effective fieldwork option for second- and third-year occupational therapy students, by evaluating the experience of both students and supervisors and identifying areas for improvement. Project placements are full- or part-time placements where a project is completed by a student under the supervision of an occupational therapist. The study is primarily descriptive, and includes a pre-post design using qualitative and quantitative data. The results indicate that the objectives of the study were achieved. Both supervisors and students expressed positive views about the placements. Students also identified changes that could improve the placements. Second- and third-year students gave similar ratings about aspects of the learning experiences during the project placements. The small cohort of third-year students and the low response rate from supervisors limited results. These project placements have shown an applicable model for students in earlier years of the course instead of the usual practice of non-traditional fieldwork being focused on final-year students. The project placements described are presented as one more potential fieldwork model in the range currently offered by curricula worldwide. Future research needs to concentrate on the longitudinal impact of these placements on the developing practice and attitudes of occupational therapy students. PMID- 12374999 TI - Perceived benefits of word prediction intervention on written productivity in children with spina bifida and hydrocephalus. AB - Word prediction has been commonly used as a tool to enhance written productivity. However, the effectiveness of word prediction as a strategy to meet this targeted outcome has not been established. Using a single-subject alternating treatments design, this study evaluated the effect of word prediction on written productivity from the users' perspectives. Three girls and one boy aged 10-12 with spina bifida and hydrocephalus participated in the study over a period of 20 days. The Canadian Occupational Performance Measure (COPM) was used to measure changes in perception of written productivity. Analysis of individual participant data showed that participants perceived word prediction to have the potential to influence written productivity on some writing tasks. Quantitative analysis using a randomization test did not reveal any significant changes in COPM scores after using word prediction. The varied abilities of the participants in the study and the small sample size may be the reasons why statistical analysis did not show any changes. The limitations of this study included use of a copy task, lack of a supporting measure to COPM and limited generalizability. Further studies with a larger sample are necessary to explore the skills required for successful use of word prediction and the impact of word prediction on specific tasks. PMID- 12375000 TI - Functional needs: agreement between perception of rural patients and health professionals in China. AB - This study aimed to investigate the common areas of functional needs of patients with different chronic diseases and to compare the level of agreement between patients and doctors, and patients and an occupational therapist, on perceived priority functional goals. A sample of 113 rural patients from Hebei Province attending outpatient neurology, orthopaedic and cancer clinics completed the COOP/WONCA Charts. These charts are a screening tool that assesses limitations in a set of functional domains. The 80 patients who indicated significant functional difficulty on the charts, 11 doctors and one occupational therapist then responded to questionnaires to elicit the perceived priority functional needs. Respondents remained blind to one another's responses. A consulting doctor and the occupational therapist saw each patient's COOP/WONCA Charts before interviewing the patient. Additional questionnaire items and a focus group interview provided data by professionals on health services thought to be beneficial to improve the function of clinic patients. The difference between the mean percentage of agreement on perceived functional difficulty in therapist/patient matches and doctor/patient matches was 18.5% (95% CI for the difference = 12.4% to 24.6%). The therapist on average agreed or matched with patients significantly more often than did doctors (p<0.0001). The discrepancy between the doctor's and patient's perception of priority functional goals was substantial, indicating a need for initiated effort to narrow this gap. The match rate of doctors with patients in choosing priority goals was significantly lower than for the therapist with patients in this study. Doctors expressed a desire for closer involvement in clinics by appropriate rehabilitation staff. This could expedite the process towards the starting level of a patient-centred approach to health care, within the natural context of teamwork, and with little disruption to clinic routines. Replication of this study using a control group would allow direct comparison of patient incidents when the charts are used and when they are not. PMID- 12375001 TI - Occupation reconsidered. AB - The current article delineates the need for the profession of occupational therapy to maintain relevance and be responsive to current trends. As part of such responsivity, this article proposes a reconsideration of the concept of occupation as an 'adaptive response' to the current societal need for clarification regarding occupational therapy. Reconsideration of what is meant by occupation for general use is discussed and illustrated by the ambiguous use of the term occupation as both a means and an end. Although occupational therapists are comfortable with such ambiguous use because of their apparent ease with complexity, use of the term in an ambiguous manner makes it harder for society to understand what is meant by occupation. Related to this, an annotation of literature on the definitions of occupation is presented in summary form. Furthermore, the political need to reconsider the term occupation is argued in light of the revision of International Classification of Functioning, Disability and Health (ICF), which includes the use of the word activity. Finally, this article proposes that occupation should be considered as the process of doing with meaning, and that activity should be the outcome. Such reconsideration renders us consistent with ICF and paves the way to reduce ambiguity in the use of the term occupation with the general public. PMID- 12375002 TI - The use of silicon gel for treating children's burn scars in Saudi Arabia: a case study. AB - This case study is presented to illustrate the effectiveness of silicon gel as an important option in burn scar treatment and to provide treatment guidelines that address cultural, clinical and patient compliance issues in Saudi Arabia. The case study involves an 18-month-old child whose burn scar was treated for a period of 15 months with silicon gel. The Vancouver Burn Scar Scale assessment (Baryza and Baryza, 1995), used to track progress across the duration of treatment, reflected an improvement in the scar as the composite score changed from 9 to 2. Strategies for problem solving and addressing needs unique to the environment of Saudi Arabia were also developed over the treatment period. The findings of this case study indicate that silicon gel may be a superior treatment option under certain circumstances. Further research with a wider sample is indicated, given the high incidence of childhood burn injuries in Saudi Arabia. PMID- 12375003 TI - Reliability and validity of the Leisure Satisfaction Scale (LSS--short form) and the Adolescent Leisure Interest Profile (ALIP). AB - This study aimed to evaluate the reliability and validity of the Leisure Satisfaction Scale (LSS short form) and the Adolescent Leisure Interest Profile (ALIP). The LSS and the ALIP are instruments that occupational therapists can use to evaluate the leisure activities that clients enjoy. Evaluation of leisure interest and participation will assist in creating goals for therapy to maximize a client's ability to participate in leisure activities. This study examined the test retest reliability and concurrent validity of the LSS and the ALIP using a sample of 37 adolescents between the ages of 13 and 17 with no known impairments. The assessments were administered individually or in small groups 7 to 17 days apart. Cronbach's alpha was used to determine the internal consistency. Pearson product moment correlations were calculated to examine the test retest reliability of the 60 subscales and the six question totals of the ALIP, as well as for the 6 subscales and total score of the LSS. Concurrent validity was evaluated between the 'How often?' question of the ALIP and the LSS (short form). Based on the study results, the ALIP and the LSS seem to have good test retest reliability levels when used with adolescents with no known physical or mental impairments. The concurrent validity between the two instruments was not supported, with many of the scores indicating only weak or no association to each of the subscales, suggesting that the assessments differ in some fundamental way. However, the evidence of some relationships between subscales may indicate some areas where the ALIP and the LSS are similar. PMID- 12375004 TI - Clinical outcomes research from the occupational therapist's perspective. AB - With increasing costs and scarcity of resources, occupational therapists need to embrace outcomes research to demonstrate the effectiveness of its clinical interventions. To explore clinicians' perspectives on clinical outcomes research a qualitative study was undertaken involving three in-depth group interviews with 15 occupational therapists from the South Western Sydney Area Health Service. Five broad themes permeated participants' perception: (a) defining the process, (b) factors that impact on participation in clinical outcomes research, (c) organizational influences, (d) the value of clinical outcomes research, and (e) potential partnerships with academics. Three conceptual categories are identified: knowledge and understanding about clinical outcomes research, clinicians' experience conducting or participating in clinical outcomes research and the relevance of clinical outcomes research to occupational therapy clinical practice. Similarity to findings in the international literature on occupational therapists' engagement in clinical research suggests that the findings from this small sample of Australian therapists are robust. The implications of the findings for continuing professional education programmes and clinical supervision are presented. PMID- 12375005 TI - Occupational therapists' assessments of adults with long-term pain: the Swedish experience. AB - The purposes of this study were to describe the needs for occupational therapy among people of working age with long-term pain, and to describe treatment interventions based on these assessments. Occupational therapists working in primary health care and/or with special interest in pain management (n=109) assessed 113 people aged 18-58 years with long-term pain with the Occupational Therapy Needs Assessment--Pain (OTNA--P) questionnaire. The occupational therapists recommended treatment interventions where appropriate. The results generated categories of needs that have implications for interventions: 1) need for patient education, 2) needs due to limitations in activity performance, 3) needs due to patient's discouragement, 4) need as a result of patient's dependency and 5) needs related to work. The suggested interventions focused on increased knowledge of how to handle daily occupations, mainly categorized as education and stress management' and behavioural' interventions. Significant correlations were found between the assessed needs and the suggested interventions. The results of this study could assist in developing guidelines for practitioners working in occupational therapy pain management programmes. It is recommended that further research is done on the effectiveness of occupational therapy interventions with patients with long-term pain. PMID- 12375006 TI - The role of mentoring on research productivity among occupational therapy faculty. AB - This study surveyed junior and senior occupational therapy faculty in order to further understand the role that mentoring plays in research productivity. Junior faculty with and without mentors were compared in terms of their overall research productivity, and the senior faculty who served as mentors were compared with senior faculty who were not mentors. The role of institutional support factors on research productivity was also examined. The results of this survey suggest that mentoring plays an important role in increasing research productivity in junior faculty in the field of occupational therapy. Also, senior faculty mentors perceived their mentoring experience to enhance their research productivity. A general profile of an occupational therapy mentor and mentee has emerged from the results. Analyses showed a low to moderate positive correlation between faculty research productivity and a number of institutional support factors. Availability of intramural funds, release time, chair and dean support for research, grant writing seminars, and availability of statistical and computing help correlated with research productivity. The authors recommend the need for mentoring in occupational therapy academia. New faculty should have a mentor or mentors to help them succeed in research, teaching and service goals. Developing short-term and long-term goals with the mentor and periodic evaluation of goals will help new faculty to keep pace with the demands and requirements of their academic positions. PMID- 12375007 TI - Intermanual transfer of a new writing occupation in young adults without disability. AB - It has been shown that acquisition of a skill by one hand is facilitated by previous learning of the same skill with the other hand. This is called intermanual transfer of learning, or cross-education. The investigators examined intermanual transfer of occupation of writing in a group of 10 right-handed subjects with no known motor disabilities. Subjects learned to perform a novel occupation of writing a foreign alphabet letter with either their right or left hand. Later, subjects reproduced the skill with the practised and unpractised contralateral hand. Pen movements and surface electromyography of the first dorsal interosseus muscle were recorded to assess the transfer of learning. Analysis revealed an almost full transfer of the learned motor task between hands in either left-to-right or right-to-left direction when movement time and movement size were compared. This indicates that transfer did not depend on hand dominance. These findings suggest that a task already learned by one hand can positively influence the learning of the same task by the other hand. The results have important implications for occupational therapy--namely, that activities comprising tasks previously learned by one hand would be more effective in facilitating improved performance by the other hand than activities comprising previously unlearned tasks in the case of retraining skills in patients with amputation or hemiplegia. Because the participants in this study were a small number of college students, research should be carried out with larger participant pools and participants with disabilities to consolidate the findings. PMID- 12375008 TI - An organizational case study of the case manager's role in a client's return-to work programme in Australia. AB - The purpose of this study was to examine the case manager's role in a return-to work programme in Sydney, Australia. The investigators examined the case manager's role assumed by occupational therapists, physiotherapists, psychologists and rehabilitation counsellors when providing occupational rehabilitation services. Files of closed cases (n=172) were examined to investigate the relationship between the case manager's profession and return-to work outcomes. It was found that the provider of occupational rehabilitation examined in this study achieved above-average return-to-work rates (83%), with no significant difference between case managers. There was, however, a significant relationship between the client's type of injury and the case manager (p<0.001), and case length was significantly different between case managers (p=0.004). The occupational therapist had the largest case management load (43%), followed by the rehabilitation counsellor (23%). There were trends (0.050.05). PMID- 12375016 TI - Economical data and advanced prostate carcinoma: do we need new guidelines for decision making? AB - Based on epidemiological data of incidence, estimated prevalence of advanced prostate carcinoma in Germany, and the cost of androgen deprivation of different regimens were determined in a study model. We analyzed data, published by the Tumor Registry of Munich, which indicate that from 3,838 patients with carcinomas of the prostate, 38% has been treated exclusively with hormone suppression therapy, 14% of patients had undergone a combined radiation therapy and hormone suppression therapy and 9% underwent combined surgical therapy and hormone suppression therapy. The mean survival time of patients treated with medical therapy alone, for patients treated with combined radiation therapy and medical therapy were 60, 24, and 120 months, respectively. The cost for orchiectomy was estimated as $1,072, and for LH-RH therapy as $224/month. We estimated an incidence of 17,700 (per year) and a prevalence of 115,000 patients with advanced prostate cancer for Germany. Provided all patients received LH-RH treatment a total cost of $308,000,000/year would arise. Provided, all patients underwent surgery a total cost of $19,000.000/year would arise. If all patients received LH RH agonists, the treatment would amount to $16,944 per patient, independently of the prognostic group; and for surgery $1,072 per patient would arise. Limited health care budgets mandate critical determination and evaluation of costs to provide a component for the complex decision making process. However, they must be complimented by validated data of quality of life, which can than be a basis for new guidelines of decision making. PMID- 12375017 TI - Mutations of rabphillin-3A-like gene in colorectal cancers. AB - The chromosome region 17p shows frequently allele losses/mutations in colorectal cancers, and such frequent genetic alterations are a hallmark of the presence of tumor-suppressor gene. The RPH3AL, which is located at 17p13, has been identified from a candidate 17p13 medulloblastoma tumor suppressor locus. The aim of this study was to determine whether it is also altered in colorectal cancers. Mutational analyses of the RPH3AL gene were performed on DNA samples from 50 primary colorectal cancer specimens using polymerase chain reaction-single strand conformation polymorphism, and DNA sequencing. Six missense mutations producing amino acid substitution in coding region of the RPH3AL gene were detected in 50 primary colorectal cancer patients. The RPH3AL gene may play a role as a tumor suppressor gene in fraction of colorectal cancers, but a minority show RPH3AL gene mutations. Somatic RPH3AL mutations were identified, providing support for an authentic role as tumor-suppressor gene in colorectal cancer, but only in a minority of cases. PMID- 12375018 TI - Angiostatin expression in endometrial cancer. AB - Angiostatin is a potent inhibitor of neovascularization, tumor growth and metastasis. The expressions of angiostatin and vascular endothelial growth factor (VEGF) were immunohistochemically examined, along with microvessel density, in primary tumors obtained from 57 endometrial carcinoma patients with stage I disease. Angiostatin expression was not related to depth of myometrial invasion, histological grade or lymph-vascular space involvement. VEGF expression also had no relation to depth of myometrial invasion or histological grade, however, it was enhanced in tumors with lymph-vascular space involvement. Angiostatin expression did not correlate with VEGF expression. Microvessel density correlated with VEGF, but not angiostatin expression. Disease-free survival was longer in patients with angiostatin-positive tumors than in patients with angiostatin negative tumors, but did not differ among patients with VEGF-negative and VEGF positive tumors. No patients with angiostatin-positive and VEGF-negative tumors had recurrent disease. We concluded that negative-expression of VEGF and positive expression of angiostatin are significant prognostic factors in endometrial carcinoma patients. PMID- 12375019 TI - Beta-catenin and cyclin D1 expression in human hepatocellular carcinoma. AB - To understand the nature and roles of mutated beta-catenin in human hepatocellular carcinomas (HCCs), 57 cases of surgically resected HCCs were studied. DNAs extracted from each tumor were examined for somatic mutations of exon 3, and the protein expressions of beta-catenin, cyclin D1, and Ki-67 were observed by immunohistochemical staining. beta-catenin mutations in exon 3 were detected in 10 (17.5%) out of 57 HCCs, including nine missense mutations and one deletion mutation. All of the cases with gene alterations had the anti-HCV antibody, and tested negative for the HBs antigen in the sera. All of the mutations occurred at the serine/threonine phosphorylation sites of glycogen synthase kinase-3beta (GSK-3beta) or their neighboring residues. Significant correlation with intracellular expression (p=0.00055) was shown in the HCCs harboring beta-catenin mutations. The intracellular accumulation of beta-catenin showed significant correlation with the cyclin D1 expression (p=0.00858), and with a higher proliferation index (p=0.00072). In addition, the beta-catenin mutations showed significant association with the cyclin D1 expression (p=0.0424). These results suggest that accumulated beta-catenin proteins may bind to the lymphocyte enhancer binding factor-1 (LEF-1), form the beta-catenin/LEF-1 complex, and stimulate such promoters regulating the cell cycle as the cyclin D1 gene. This is the first report to demonstrate a significant correlation between beta-catenin and the cyclin D1 expression in human HCCs. PMID- 12375020 TI - Lack of prognostic significance of nm23 expression in human primary breast cancer. AB - This retrospective study was designed to evaluate the prognostic value of immunohistochemically detected nm23-expression in invasive breast cancer. Cellular expression of nm23 was assessed in 325 primary breast cancer tissues by immunohistochemistry. The data were correlated to established functional factors of prognosis (age, tumor size, nodal status, tumor grade, steroid hormone receptor status), and to clinical follow-up (median observation time, 92 months). 215/325 (66.2%) tumor tissues were considered nm23 positive. nm23 expression did not correlate to any of the investigated markers. Similar results were obtained after subdivision of the patients into node-negative patients (n=153) and node positive cases (n=172). With respect to clinical outcome, nm23 expression displayed no statistical significance to predict the clinical course. In conclusion, the determination of nm23 expression is an independent factor but lacks prognostic or predictive value in breast cancer patients. PMID- 12375021 TI - Metastatic urinary bladder tumor from extragonadal germ cell tumor: a case report. AB - A 37-year old male patient complained of lower back pain. Investigations revealed a retroperitoneal tumor and left-sided cervical lymphadenopathy without abnormal findings in both testicles. The diagnosis of extragonadal germ cell tumor was made based on a lymph node biopsy. Following chemotherapy and retroperitoneal lymphoadenectomy, at outpatient follow-up, beta-HCG elevated again. He complained of gross hematuria, cystoscopy showed a bladder tumor, and abdominal MRI scan showed a right ureteral tumor. Total cystectomy and right nephroureterectomy were performed, which revealed that the bladder and ureteral tumors were metastatic germ cell tumors. Six months later, the patient developed hepatic and mesenteric lymph node metastases that failed to respond to treatment, and died. Germ cell tumors metastasizing to the urinary tract are extremely uncommon, and this is the first report of bladder metastases, other than through direct invasion, from an extratesticular germ cell tumor. PMID- 12375022 TI - Effect of survivin on cell proliferation and apoptosis in gastric cancer. AB - We investigated the effect of expression of survivin, an apoptosis inhibiting protein, on cellular proliferation and apoptosis in gastric cancer and assessed its relation to the pathological characteristics of gastric cancer. Resected gastric cancer specimens from 42 patients (intestinal type; 21 cases and diffuse type; 21 cases) were evaluated. There were no significant differences in the clinicopathologic factors between the two groups. Survivin mRNA expression was measured using real-time reverse transcription-polymerase chain reaction, and survivin protein expression was evaluated by immunohistochemical staining. The Ki 67 index was adopted for cell proliferation scoring, and the ss-DNA positive rate as an apoptotic index. The survivin mRNA expression inversely correlated with the apoptotic index (p<0.05). Survivin mRNA expression (p<0.001) and survivin protein expression (p<0.001) were significantly higher in the diffuse type than the intestinal type. In conclusion, gastric cancer avoids cellular death by survivin expression and this tendency was more conspicuous in diffuse type gastric cancer. PMID- 12375023 TI - Infiltration of tumor-associated macrophages in human oral squamous cell carcinoma. AB - Tumor-associated macrophages (TAMs) have been shown to display both positive and negative effects on tumor growth of various cancer. In this study, TAMs were immunohistochemically labeled using CD68 antibody in 82 oral cancer. Macrophage index (MI) were analyzed in association with clinical and pathological factors, intratumoral microvessel density (IMVD) and angiogenic factors including VEGF and TP. Significantly higher number of CD68-positive cells was noted in oral cancer. TAM expressions were significantly associated with stages of invasion, IMVD, and angiogenic factors. These findings suggest that TAMs possibly play a role in angiogenesis during oral cancer progression. PMID- 12375024 TI - Diagnostic localization of extra-adrenal pheochromocytoma: comparison of (123)I MIBG imaging and (131)I-MIBG imaging. AB - The accurate diagnostic localization of extra-adrenal pheochromocytoma is important in the treatment of primary and metastatic tumors. In this study, we report on a case of extra-adrenal pheochromocytoma in which the tumor could not be diagnosed by 131I-metaiodobenzylguanidine (MIBG) scintigraphy but only by 123I MIBG scintigraphy. Our patient was a 16-year old girl whose chief complaint was headaches. High levels of catecholamines were detected in the blood and urine samples. 131I-MIBG scintigraphy could not detect any abnormal accumulation, but an accumulation of 123I-MIBG was found in the pelvis. Magnetic resonance imaging (MRI) revealed a broad-based bladder tumor at the site of the accumulation. Partial cystectomy was performed, and immunohistochemical staining examination confirmed the diagnosis of extra-adrenal pheochromocytoma in the urinary bladder. In this report, we compare 131I-MIBG scintigraphy with 123I-MIBG scintigraphy and discuss their characteristics. PMID- 12375025 TI - No mutations of the Bub1 gene in human gastric and oral cancer cell lines. AB - Chromosomal instability is one of the most important characteristics underlying tumorigenesis. Certain colorectal cancers with chromosomal instability have been shown to have inactivation of a spindle assembly checkpoint party due to mutation of Bub1, a mitotic checkpoint gene. We performed sequencing analysis on reverse transcriptase-polymerase chain reaction (RT-PCR) product of the Bub1 cDNA (entire coding sequence) from 8 human gastric cancer cell lines. In addition, genomic PCR products of Bub1 exon 8, 10, 12, 15 and kinase domain from 9 oral cancer cell lines were analyzed by polymerase chain reaction-single-stand conformation polymorphism (PCR-SSCP). Although sequencing analysis of the Bub1 cDNA revealed several point mutations in 8 gastric cancer cell lines, we could not confirm the mutations by analyzing genomic DNA. Furthermore, genomic PCR-SSCP analysis revealed no mutations in exon 8, 10, 12, 15 and kinase domain of the Bub1 gene in any oral cancer cell lines examined. These results suggest that mutation of the Bub1 gene might not play a role in human stomach and oral carcinogenesis. PMID- 12375026 TI - Adenoviral p53 gene therapy in head and neck squamous cell carcinoma cell lines. AB - We reconstructed the recombinant p53-expressing adenovirus and examined its infections and effects in head and neck squamous cell carcinoma cell lines. Eight human head and neck squamous cell carcinoma cell lines were infected by the recombinant adenovirus harboring the lacZ gene (AxCAiLacZ) or the wild-type p53 gene (AxCAip53), and the effects were investigated. The eight cell lines were successfully infected by AxCAiLacZ at a level of more than 50%. The survival of all 8 squamous cell lines were inhibited in the range from 8 to 26.7% by only one treatment of the AxCAip53 infection. This result suggested that p53 gene therapy might become a useful tool in head and neck squamous cell carcinoma treatment. PMID- 12375027 TI - Interleukin-18 inhibits lodging and subsequent growth of human multiple myeloma cells in the bone marrow. AB - An intravenous injection of ARH-77 cells (human multiple myeloma cell line) into mice with severe combined immunodeficiency disease (SCID mice) results in lodging of tumor cells in the bone marrow of thoracic and lumbar vertebrae, and in their subsequent growth, the cells destroying bone and invading the spinal cord and surrounding tissues, and the mice show hind leg paralysis. Using this model, we investigated the effects of interleukin (IL)-18 on the lodging and subsequent growth of multiple myeloma cells in the bone marrow. Mouse recombinant IL-18 (mIL 18) at 1 microg/mouse was daily injected according to protocols A and B. In protocol A, mIL-18 was injected from day 6 after tumor cell injection to examine the effect of mIL-18 on tumor growth, and in protocol B, it was injected from day 3 prior to tumor cell injection to day 3 after it to examine the effect of mIL-18 on lodging of tumor cells. The spread of a tumor was monitored as to the appearance of hind leg paralysis and the tumor area in a median longitudinal section of the vertebrae with the surrounding tissues. With protocol A, mIL-18 significantly and markedly decreased the cumulative rate of hind leg paralysis and the tumor area. This antitumor effect of mIL-18 was ascribed to its action on the activation of NK cells because mIL-18 exerted no significant effect when anti asialo GM1 antiserum (a-ASGM1) was simultaneously injected to deplete the NK cell activity. With protocol B, mIL-18 also significantly and markedly decreased the cumulative rate of hind leg paralysis and the tumor area. However, most of this effect was not due to the action of mIL-18 on NK cells because mIL-18 showed a marked and significant effect with the administration of a-ASGM1. The present results indicate that mIL-18 inhibited the lodging and subsequent growth of multiple myeloma cells in the bone marrow, and suggest that IL-18 is worth investigating further as to its usefulness as a therapy for multiple myeloma. PMID- 12375028 TI - Clinicopathologic and immunohistochemical features of surgically resected small cell carcinoma of the esophagus. AB - Esophageal small cell carcinoma (SmC) is considered an aggressive cancer carrying a poor prognosis, although the rarity of this tumor has impeded statistical evaluation. We reviewed records of 457 esophageal cancer patients treated in our department from 1986 to 2000, comparing clinicopathologic factors and post treatment outcomes, for 9 patients with SmC, most undergoing esophagectomy including lymphadenectomy, with data from 128 patients with esophageal squamous cell carcinoma (SqC) invading to the muscular layer or beyond. Immunohistochemical features were compared between the SmC and 12 consecutive SqC. All patients studied had localized disease according to preoperative staging. SmC showed more ulcerative and infiltrative growth, and more aggressive lymphatic spread, than SqC. All SmC patients had lymph node metastasis (thoracic nodes, 9 patients: abdominal 6; cervical 1). All SmC specimens but no SqC were immunoreactive for neuron-specific enolase. Two and three SmC specimens were reactive for epithelial membrane antigen and keratin, respectively. Survival of SmC patients after esophagectomy (median, 11 months) was worse than for SqC patients (p=0.013). However, 1 SmC patient remains alive at 76 months. Survival was not related to any clinicopathologic or immunohistochemical features. While SmC shows aggressive behavior and worse outcomes than SqC, combining esophagectomy with chemotherapy or radiotherapy may prolong survival. PMID- 12375030 TI - Molecular characterization as a target for cancer therapy in relation to orphan status disorders (Review). AB - The long-term effort in investigating chemical methods to eliminate only cancer cells has improved our knowledge and has led to the development of new drugs. The targets for cancer treatment may be large polymeric molecules such as DNA or microtubules as well as regulatory pathways for tumor development and cell survival preservation or tyrosine kinase activity. Examples of new agents are: trastuzumab (Herceptin), a humanized monoclonal antibody that blocks the HER 2/neu proto-oncogene in combination with cytotoxic agents, is used in a percentage of breast cancer patients; signal transduction inhibitor of abl tyrosine kinase STI 571 (Glivec) has been shown to be an active treatment for chronic myeloid leukemia and GISTs; epidermal growth factor receptors in certain tumors have been targeted with agents such as C225 (Cetuximab) and ZD 1839 (IRESSA); an adenosine deaminase analogue of deoxyadenosine, Cladribine (2-chloro 2 deoxy-adenosine) has shown high effectiveness in hairy-cell leukemia and the multitargeted antifolate (Premetrexed) and several vaccines have been studied and are in clinical trials for resistant cancers. These new drug developments represent a promising field for future cancer management. PMID- 12375029 TI - Clinical significance of ST3Gal IV expression in human renal cell carcinoma. AB - Alteration of sialyltransferase expression has been implicated in carcinogenesis. Out of sialyltransferases cloned to date, we focused on ST3Gal IV expression in human renal cell carcinoma (RCC). Levels of ST3Gal IV mRNA were examined in human RCC in comparison with non-tumor kidney. ST3Gal IV cDNA obtained by polymerase chain reaction from cDNA library of human RCC cell line ACHN was identical to STZ in nucleotide sequence. Northern blot analysis was performed for 24 non-tumor kidney and 25 primary RCC tissues, and 5 metastases. ST3Gal IV mRNA level was decreased in 16 cases of 22 primary RCC tissues compared to 21 non-tumor kidney tissues. The mRNA level was low in 4 and equivocal in one, of 5 metastases. The 6 cases that possessed almost the same levels of ST3Gal IV mRNA in primary tumor tissues as those in non-tumor kidneys showed favorable prognoses, as assessed by Kaplan-Meier curve. These results indicate that down-regulation of ST3Gal IV mRNA may be one of the factors associated with the malignant progression of human RCC. PMID- 12375031 TI - Antitumor effect of radioimmunotherapy in a mouse model of testicular tumor with micrometastases defined by polymerase chain reaction. AB - The efficacy of radioimmunotherapy (RIT) was evaluated by using a mouse model of testicular tumor with macro- and micrometastases to various organs. RIT consisting of a single intravenous injection of 5.8 MBq of I-131-labeled anti placental alkaline phosphatase (PLAP) MAb was conducted one day or 7 days after testicular implantation in SCID mice of HeLa Hep2 cells expressing PLAP. RIT antitumor effect was significant for primary tumors as well as micrometastases defined by polymerase chain reaction (PCR) assay. However, ablation of tumor cells could be achieved with this treatment only at the initial stage of tumor growth in the testis, when metastasis could also be prevented. Once micrometastasis had occurred, however, complete elimination of tumor cell(s) was difficult. These findings appear to have implications for the use of RIT in treatment for micrometastasis, especially when detectable only by PCR assay. PMID- 12375032 TI - Correlation between malignancy grade and p53 gene in relation to thymidine phosphorylase activity in colorectal cancer patients. AB - We studied 89 colorectal cancer patients conducting immunohistological staining of thymidine phosphorylase (TP) and investigated correlation between the enzyme activity levels, clinicopathologic factors and patient prognosis. Based on TP expression level, we also assessed p53, an anti-oncogene related to hematogenesis and nucleic acid metabolism. TP staining results were classified into: i) strongly stained group (8 patients) in which 20% or more tumor cell nuclei were stained and ii) weakly stained group (39 patients) in which less than 20% of the nuclei were stained. These 2 groups were defined as TP positive group and those without TP staining (42 patients) were defined as TP negative group. TP positive group showed: a significantly higher incidence of vascular infiltration (p=0.0065) than TP negative group, and slightly higher incidence of lymphatic permeation and tumor progress. Strongly stained group showed significantly higher incidences of vascular infiltration and lymphatic permeation (p=0.0001, p=0.0271). As for 5-year survival rate, TP positive group showed a significantly lower rate (29%) than TP negative group (52%) (p=0.05) indicating that TP expression level was an important prognostic factor in colorectal cancer patients. No differences were found in pathologic factors and patient prognosis between groups with or without point mutation. As for relationship between TP staining and p53, there were significantly more patients with strongly positive TP staining in the p53 negative group. These findings suggest that TP is an important prognostic factor for colorectal cancer patients, and p53, in conjunction with other factors, is also a prognostic indicator. PMID- 12375033 TI - Radiotherapy for elderly non-small cell lung cancer patients. AB - Elderly patients with lung cancer tend to have significant coexisting diseases and less aggressive treatment is often required. To investigate the clinicopathological features of non-small cell lung cancer (NSCLC) in elderly patients who were treated with radiotherapy, we reviewed the medical records at our division. Among the 189 patients 70 years or older between 1992 and 2001, 28 (14.8%) patients were treated with radiotherapy (elderly group). In the elderly group, there was a medical history of chronic obstructive pulmonary disease (COPD) (57.1%), cardiovascular disease (32.1%), diabetes mellitus (18.5%), malignant disease (18.5%), or cerebrovascular disease (10.7%). Moreover, in the elderly group, 17 (60.7%) patients had two or three coexisting diseases. There was statistical difference between the elderly group and the younger group (less than 70 years patients) with regard to COPD (p<0.001). Also there was statistical difference between the elderly group and the younger group with regard to number of coexisting diseases (p=0.002). In the elderly group, forced expiratory volume in one second (FEV1), forced expiratory volume (FVC), FEV1/FVC and diffusing capacity of the lung for carbon monoxide (DLCO) were statistically significant impaired to compare with those in the younger group (p<0.001, p=0.030, p<0.001 and p=0.024, respectively). In the elderly group, 10.7% of patients had concurrent chemoradiotherapy, however, 38.5% of patients of the younger group received concurrent chemoradiotherapy. There was a statistical difference between the two groups (p=0.026). Adequate palliative care to provide prolonged quality survival is an appropriate primary goal of therapy for lung cancer in the elderly and radiotherapy is thought be one of the less invasive treatments. PMID- 12375034 TI - Biological analysis of gastrointestinal stromal tumors. AB - The biological behaviour of a gastrointestinal stromal tumor (GIST) cannot be easily predicted from preoperative clinical examination alone. As a result, there is little standardization in the surgical treatment of GIST. In this study, we analyzed the clinicopathology and immunohistochemistry of 20 cases of GIST to clarify factors associated with tumors showing malignant potential. Immunohistochemical analysis of KIT, CD34, vimentin, alpha-smooth muscle actin (SMA), s-100, p53, ki-67, bcl-2 and bax expression was performed on 20 surgically resected GIST. An apoptotic index (AI) was calculated for each sample using a TdT mediated dUTP-biotin nick end-labeling method. With regard to bcl-2, t(14;18) translocations were also investigated using a polymerase chain reaction based method. Finally, the relationship between these biological results and clinicopathological data was analyzed. Of the 20 cases studied, two patients died due to lung or liver metastasis. All cases stained positive for vimentin, nine cases were positive for alpha-SMA and three cases positive for s-100. All cases were stained for both KIT and CD34, which tended to correlate with malignant potential. There was significant difference in frequency of bcl-2 overexpression (p<0.05) and trend in Ki-67 labeling index (p=0.098) between benign and malignant cases. However, with regard to bcl-2, no chromosomal t(14;18) translocations were detected in four analyzed cases. In GIST, overexpression of bcl-2 may play an important role in increasing malignant potential. Furthermore, Ki-67 L.I. and bcl 2 overexpression may be useful in predicting malignant potential, and therefore help to determine the surgical treatment, follow-up manner, and the necessity of adjuvant therapy. PMID- 12375035 TI - Assessment of carcinoma in the sublingual region based on magnetic resonance imaging. AB - In the present study, we attempted to diagnose and detect the extent of tumors in the sublingual region using magnetic resonance imaging (MRI) and dynamic MRI. MRI with or without gadopentetate dimeglumine (Gd-DTPA)-enhancement in seven lesions of the sublingual regions was performed. The seven lesions included four cases of mucoepidermoid carcinoma (mucoepidermoid Ca), two cases of adenoid cystic carcinoma (ACC), and one case of squamous cell carcinoma (SCC). Whether the tumor was malignant or benign, as well as the differential diagnosis, could not be determined on the basis of the MR signals, even when enhancement was performed. Dynamic MRI was performed in five cases, two cases of ACC, two cases of mucoepidermoid Ca, and one case of SCC. The dynamic MRI showed a rapid enhancement at 30-45 sec in all five cases before the normal sublingual gland began to be enhanced. The early phases at 30-45 sec of the dynamic MRI in five cases showed marked enhancement before the normal sublingual glands were enhanced, and therefore could clearly show the extent of the lesions. In conclusion, the dynamic MRI may be useful in differentiating malignant from benign tumors, and in detecting the extent of the tumors in the sublingual carcinomas. PMID- 12375036 TI - Detecting para-aortic lymph nodal metastasis by positron emission tomography of 18F-fluorodeoxyglucose in advanced cervical cancer with negative magnetic resonance imaging findings. AB - The patient's prognosis is significantly related to para-aortic lymph node metastasis in advanced cervical cancer. Magnetic resonance imaging (MRI) has been widely performed to detect lymph node metastasis with a variable sensitivity. Therefore, in this prospective study, we evaluated FDG-PET to detec para-aortic lymph nodal metastasis in locally advanced cervical carcinoma with negative MRI findings. Forty-two women with advanced cervical cancer and negative abdominal MRI findings were included in this study. Para-aortic lymph node metastases were confirmed according to para-aortic lymph-adenectomy findings. Para-aortic lymphadenectomy findings showed 12 patients with para-aortic lymph nodal metastasis. Two patients had false-negative and 1 patient had false-positive FDG PET findings. The FDG-PET showed sensitivity of 83.3%, specificity of 96.7%, and accuracy of 92.9%. As assessment of para-aortic lymph node metastases may be essential for therapy planning, FDG-PET is an alternative tool to detect para aortic lymph node metastases in advanced cervical cancer with negative MRI findings. PMID- 12375037 TI - The Epstein-Barr virus-associated gastric carcinoma in Southern and Northern China. AB - In the present study, we examined the proportions of Epstein-Barr virus associated gastric carcinomas (EBV-GCs) in Guangzhou, southern China and Shenyang, northern China, two areas differing markedly in nasopharyngeal carcinoma (NPC) incidence. Using in situ hybridization assay, the presence of EBV encoded small RNA (EBER) was examined in 198, and 180 gastric cancer cases in Guangzhou and Shenyang, respectively. The proportion of EBV-GC in Guangzhou (9%) was significantly higher than that in Shenyang (6%), and the odds ratio (OR) for Guangzhou, after adjusting for the effects of age, sex, and tumor subsite, was 2.7 (95% CI = 1.1-6.2) when Shenyang was taken as reference. There was a male predominance of EBV-GC, and the OR for male was 3.0 (95% CI = 1.2-7.3) when female was taken as reference. We observed a weak and negative age dependence in the proportion of EBV-GC (p-values for trend = 0.077). The EBV-GC was most commonly observed in the middle part of stomach in both series. The frequency of EBV-GCs was higher in cases with p53 overexpression than in cases without p53 expression (OR = 2.4, 95% CI = 1.0-5.8). Among p53-positive cases, the frequency of EBV-GC decreased as the proportion of p53-positive carcinoma cells increased (p for trend = 0.021). In conclusion, the present study suggested that the frequency of EBV-GC in Guangzhou, southern China, where NPC is the most common in the world, may be higher than that in other parts of China. PMID- 12375038 TI - Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3,4 dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone). AB - Induction of apoptosis is an attractive strategy in cancer therapy but it clinical practice is not yet sufficient in choriocarcinoma. The quinolinone derivative, vesnarinone, is a novel inotropic agent used for treating congestive heart failure and may also have a potential anticancer activity. It induces apoptosis and differentiation in some tumor cell lines. We examined the antitumor effect of vesnarinone in eight cell lines established from human choriocarcinoma and hydatidiform mole using MTT assay and also analyzed the nuclear fragmentation of tumor cells by DNA electrophoresis assay. Vesnarinone inhibited the proliferation of choriocarcinoma cell lines in a dose-dependent manner and induced DNA fragmentation in cells. However, the BM cell line prepared by subcultivation from hydatidiform mole showed no growth suppression or DNA fragmentation in response to vesnarinone. On the other hand, PCR-SSCP analysis and direct DNA sequencing have shown that a human choriocarcinoma cell line, SCH, has a mutant p53 gene at codon 249. When SCH cells were treated with vesnarinone cellular proliferation was significantly inhibited. Vesnarinone suppressed the proliferation of all choriocarcinoma cell lines and induced apoptosis, regardless of the existence of p53 mutation. In addition, it has been found by RT-PCR that expression of c-Myc mRNA is upregulated by treating choriocarcinoma cells with vesnarinone. The finding suggests that vesnarinone might induce expression of c Myc gene in choriocarcinoma cells, the product of which may be associated with the inhibition of cell growth and induce apoptosis. These results suggest that vesnarinone is a useful reagent for the treatment of choriocarcinoma. PMID- 12375039 TI - Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes. AB - Galectin-3, a multifunctional beta-galactocide binding lectin possibly participates in a variety of biological events including cell proliferation, differentiation, and apoptosis. The implication of galectin-3 during malignancy progression has been suggested in several cancers, including colon, prostate, thyroid, and breast cancer, however, scarce data are available in gastric cancer. We examined the expression of galectin-3 in 86 primary gastric cancers and the 40 metastatic lymph nodes by immunohistochemistry to explore whether it is related to the malignant progression. Positive galectin-3 expression was observed in 84% of the gastric cancer cases. In enhanced cells of cancerous lesions, 48% showed stronger nuclear immunoreactivity than cytoplasmic one, whereas adjacent epithelial cells showed little or weak nuclear immunoreactivity. When galectin-3 expression in gastric carcinoma was compared with that in gastric tissues adjacent to the cancers, there was a significant difference. The degree of enhancement of immunoreactivity was different corresponding to various histopathological subtypes in cancer tissues. A significantly stronger expression of galectin-3 in cancer tissues was only observed in papillary and poorly differentiated adenocarcinoma. When galectin-3 expression and tumor progression (TNM staging) was compared, a significant correlation was observed in overall cases, and only in poorly differentiated adenocarcinoma the galectin-3 expression correlated with tumor progression among various subtypes. Galectin-3 expression was observed significantly stronger in metastatic lymph nodes than in the primary gastric cancers, and also in these cases among histological subtypes, only in poorly differentiated adenocarcinoma, the expression of galectin-3 in metastatic lymph nodes was stronger than the primary cancer. In conclusion galectin-3 might be a useful tumor marker for gastric cancers with respects to tumor progression and potentiality of lymph node metastasis especially in certain histological types of gastric cancer. PMID- 12375040 TI - Analysis of cyclin D1 and CDK expression in colonic polyps containing neoplastic foci: a study of proteins extracted from paraffin sections. AB - Overexpression or amplification of G1 cyclins has been demonstrated in various types of human cancers. Overexpression of cyclin D1, an accelerator of cell cycle, is thought to be an early event in colonic multistage carcinogenesis, but its expression at transformation from benign to neoplastic foci is not clear. We analyzed the expression of cyclin D1 and its catalystic partner CDK4 in colon polyps containing neoplastic foci. For direct comparison of the expression in adenomatous tissues and neoplastic foci, proteins in adequate amounts were extracted from paraffin embedded sections. Western blot and densitometry analyses showed that cyclin D1 expression was 2.3-times and 2.5-times higher in adenomatous tissues and neoplastic foci, compared with normal colonic mucosa. In contrast, the levels of CDK4 and CDK2 expression were only modestly increased in adenomatous tissues but significantly higher in neoplastic foci, relative to normal mucosa. Expression of PCNA, a cell proliferation marker, increased from normal mucosa to adenoma to focal cancer. Our findings suggest that overexpression of cyclin D1 may be associated with high proliferative activity in adenomatous tissues and that concurrent high expression of cyclin D1 and CDK4 may further perturb cell cycle progression and play a pivotal role in colonic carcinogenesis. PMID- 12375041 TI - Potent antitumor effect of 1-(2-deoxy-2-fluoro-4-thio-beta-D arabinofuranosyl)cytosine on peritoneal dissemination models of gastrointestinal cancers. AB - We investigated the antitumor activity of the nucleoside analog 1-(2-deoxy-2 fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (4'-thio-FAC) in peritoneal dissemination models of gastrointestinal cancers. Oral administration of 4'-thio FAC showed a marked effect on the development of ascites and on the survival of nude mice implanted with the highly metastatic MKN-45-P gastric cancer cells into the peritoneal cavity. The toxic effect was observed at a dose of 15 mg/kg, however, no toxicity was observed at a dose of 10 mg/kg with excellent antitumor activity. We established another highly metastatic cell line HCT-15-P by serial intraperitoneal passage of the colon cancer cell line HCT-15. In the HCT-15-P model, 4'-thio-FAC showed an inhibitory effect of peritoneal dissemination and an increase in the life span at a dose of 10 mg/kg. On the other hand, oral administration of 5-fluorouracil (5-FU) and 5'-deoxy-5-fluorouridine (5'-DFUR) did not show an increase in the life span of mice at a dose of 10 mg/kg. In conclusion, 4'-thio-FAC is a promising therapeutic agent for use against peritoneal dissemination of gastrointestinal cancers. PMID- 12375042 TI - Elevated preoperative serum ICTP is a prognostic factor for overall and disease free survival in breast cancer. AB - The aim of this study was to evaluate the prognostic value of preoperative concentrations of the serum markers of type I collagen synthesis (PINP, PICP) and degradation (ICTP) in breast cancer. One hundred and eighty-four breast cancer patients without advanced disease were enrolled. Preoperative serum markers of type I collagen were assessed with specific radioimmunoassays. Elevated preoperative serum concentrations of ICTP (>4.9 microg/l) correlated statistically significantly with a poor prognosis for the breast cancer (P=0.0004) and shorter disease-free survival (P=0.02), and multivariate regression analysis likewise showed an elevated ICTP concentration (P=0.008), high grade (P=0.03) and high pathological stage (P=0.02) to be risk factors for poor survival. Sixty-five out of the 184 patients developed metastatic disease during the follow-up. The median follow-up time was 62 months (range 6-111 months). High ICTP (P=0.02) and large numbers of metastatic nodules (P=0.002) were prognostic factors for shorter disease-free survival in multivariate regression analysis. PINP and PICP had no significant prognostic value with respect to either overall or disease-free survival in this analysis. Our results indicate that preoperatively elevated serum ICTP is a prognostic factor in breast cancer, the measurement of which should improve the accuracy of predictions of the clinical outcome. PMID- 12375043 TI - BRCA1 in non-inherited breast carcinomas (Review). AB - Breast cancer occurs in hereditary and sporadic form. Breast cancer susceptibility gene (BRCA1) is known to be responsible for hereditary breast cancer. BRCA1 mutations are rare in sporadic cancers, but loss of BRCA1 resulting from reduced expression or incorrect subcellular localization is postulated to be important in non-familial breast cancers. More than 300 germline mutations have been identified so far in patients with familial breast and/or ovarian cancer, however, only a few somatic mutations have been identified in sporadic breast cancer. The decreased expression of BRCA1 in sporadic breast cancer is thought to be caused by other mechanisms, such as CpG methylation. BRCA1 expression may play an important role in the pathogenesis and prognosis of sporadic breast carcinoma. PMID- 12375044 TI - Evaluation of tissue harmonic imaging for breast tumors and axillary lymph nodes. AB - We examined whether tissue harmonic imaging (THI) produces higher quality images of the breast and axilla than conventional sonography (CS). We studied 70 breast tumors in 68 consecutive patients and 170 axillary lymph nodes in 19 breast cancer patients. These areas were investigated by THI and CS, then image quality was evaluated by the consensus of three skilled radiologists. THI transmits and receives at 5.0 and 10.0 MHz, respectively, whereas CS uses 9.0 MHz. The evaluation parameters of image quality consisted of contrast and resolution with a visual scoring system. Resolution was individually evaluated on both proximal and distal sides of lesions. The grade of acoustic shadow was also evaluated. The quality in contrast (p<0.001) and proximal resolution (p<0.001) of breast tumors by THI was far superior to those of CS. Intense acoustic shadows degraded the distal resolution of breast lesions in THI compared with CS, but the quality of THI images of the axillary lymph nodes was higher with respect to all parameters (p<0.001). The grade of acoustic shadows did not differ between two modes of gray scale sonography for lymph nodes. THI may improve diagnostic accuracy of breast and axillary lesions because image quality is improved, but acoustic shadows continue to present obstacles. PMID- 12375046 TI - Docetaxel-cisplatin combined chemotherapy in Japanese patients with anthracycline pretreated advanced breast cancer. AB - The aims of this study were to determine the efficacy of docetaxel (DOC) 60 mg/m(2) and cisplatin (CDDP) 80 mg/m(2) combined chemotherapy in Japanese patients with anthracycline-pretreated advanced breast cancer, and to report the side effects of this therapy. Fourteen patients with anthracycline-pretreated advanced breast cancer were enrolled. DOC was administered at 60 mg/m(2) intravenously over at least 1 h, and then CDDP was administered at 80 mg/m(2) intravenously over at least 3 h. Two courses of this regimen were administered at an interval of 3-4 weeks. Two more courses were administered after 6 months if the disease did not progress, or toxicity was acceptable. The overall response (OR) rate was 64.3%. In the patients with metastases, OR was 58.3%, median duration of the response was 31 weeks (range, 6 to 78 weeks), and median overall survival was 85 weeks (range, 35 to >159). Grade 3/4 toxicities were: neutropenia 10/14; anorexia 5/14; nausea/vomiting 3/14; diarrhea 4/14; oral symptoms 1/14. In conclusion, the combination of DOC (60 mg/m(2)) and CDDP (80 mg/m(2)) is an active regimen that can be relatively safely administered to Japanese patients with anthracycline-pretreated advanced breast cancer. PMID- 12375045 TI - Efficacy of combination chemotherapy for stage IV colon cancer with extensive peritoneal dissemination and multiple liver metastases: a case report. AB - A patient was diagnosed as having subacute ileus due to advanced cancer of the descending colon with multiple liver metastases and was treated by palliative left hemicolectomy. He was considered to have Stage IV cancer based on the finding of extensive peritoneal dissemination. Histopathological examination showed that the tumor was moderately differentiated adenocarcinoma. Postoperative palliative chemotherapy was given with 5-FU and LV twice a month as 1 course, and he received a total of 3 courses. As a result, the multiple liver metastases were completely eliminated. However, his liver metastases recurred, so CPT-11 was added to 5-FU and LV for another 3 courses. When bilateral pleural effusions developed about 1 year postoperatively, CPT-11 was changed to CDGP. Jaundice and massive ascites eventually developed, and he died about 1 year and 5 months postoperatively. PMID- 12375047 TI - Intralesional topotecan in advanced ovarian cancer: a clinical report, based on a preclinical study. AB - The aim of this study was to evaluate the response of ovarian cancer to intralesionally administered topotecan. Preliminary experiments were carried out in nude mice subcutaneously grafted with three different human ovarian carcinoma cells (A2780, IGROV/DDP and SKOV-3). Topotecan was administered intravenously (i.v.: 10-15 mg/kg every 4th day for 4 times) or intralesionally (i.t.: single dose of 15-20 mg/kg) and tumor size changes/drug toxicity were evaluated. The results indicate that the sensitivity of the three tumor models was different (rank: A2780 > IGROV/DDP > SKOV-3) but, for each tumor line, the pattern of response was similar after i.v. and i.t. administration. No local toxicity was detected, but appreciable systemic toxicity (animal death rate) was observed in spite of the use of a single i.t. dose. The effects of intralesional topotecan administration were then assessed in a patient with an advanced, epithelial ovarian tumor (endometroid type, poorly differentiated histologic grade), already treated with cisplatin and paclitaxel. The treatment (7.5 mg/m(2)) was repeated three times and, although drug plasma levels were in the range generally reported following i.v. administration and typical systemic toxicity occurred, no tumor regression was observed and the patient died 14 months later. We conclude that the intralesional drug delivery is effective to achieve a rapid tumor shrinkage in large tumor lesions, but in the presence of drug resistance, either intrinsic or acquired, intratumor drug administration can not be recommended. PMID- 12375048 TI - High response rates in patients with pancreatic cancer using the novel oral fluoropyrimidine S-1. AB - S-1 is a newly developed oral fluoropyrimidine derivative with demonstrated activity against several tumor types. The aim of this study was to evaluate the feasibility and efficacy of S-1 administered as a single-agent and in combination with cisplatin, for the treatment of patients with pancreatic cancer. A total of 33 patients with locally advanced or recurrent pancreatic cancer were entered into this study. Seventeen patients were treated with S-1 alone (group A), and 16 patients were treated with S-1 plus weekly cisplatin (group B). Objective tumor responses among the 15 evaluable patients in group A were 1 CR, 2 PR, 9 SD and 3 PD, and among the 14 evaluable patients in group B were 8 PR, 5 SD, and 1 PD. The overall response rates were 20.0% and 57.1% in groups A and B respectively. Seven of the 17 patients in group A with elevated CA19-9 serum concentration and 12 of the 13 patients in group B with elevated CA19-9 level, reduced their CA19-9 by more than 50%. The median follow-up periods/median durations of response were 152/102 days in group A and 105/66 days in group B. Median survivals have not been reached in either group. In group A, no patient developed severe toxicities over grade 3, but in group B, 3 patients developed hematotoxicity over grade 3, and 2 patients experienced grade 3 anorexia. S-1, especially in combination with CDDP, shows promising activity with acceptable toxicities against pancreatic cancer. Further evaluation of this combination in patients with this disease is warranted. PMID- 12375049 TI - Clinicopathological study of chromogranin A, B and BRCA1 expression in node negative breast carcinoma. AB - Chromogranins are representative proteins contained in endocrine cells of various organs including some ductal cells of the breast. Homology between the BRCA1 protein (1214-1223) and the chromogranins has been detected and suggests that chromogranin may play the role of tumor suppressor like BRCA1. To evaluate whether chromogranins function as tumor suppressors in invasive breast carcinoma, we examined the clinicopathological significance and immunohistochemical expression of chromogranin and BRCA1 in 80 patients with lymph node-negative primary invasive ductal carcinomas. Chromogranin A (CgA) and chromogranin B (CgB) were positive in 21 (26%) and 30 cases (38%) respectively. Expression of CgA was correlated with PR, and lower histological grade (p<0.05 respectively). However, the expression of CgB was not correlated with any immunohistological factor. Expression of both CgA and CgB was not correlated with age, tumor size, and treatment modality. With multivariate analysis, expression of BRCA1 significantly influenced the expression of CgA (p=0.01), while no factor significantly influenced to the expression of CgB. The survival curve of the patients with CgA negative tumor indicated a tendency to a poorer prognosis than that seen with CgA positive tumor. The patients with CgB-negative tumors demonstrated a poorer prognosis than seen with patients with CgB-positive tumors (p<0.05). CgA and CgB may mediate tumor progression through some unknown function besides the BRCA1 related function. PMID- 12375051 TI - Gene copy numbers of erbB oncogenes in human pheochromocytoma. AB - ErbB-1, -2, -3 and -4 proteins are growth factor receptors, encoded by the family of respective erbB protooncogenes. These receptor-encoding proto-oncogenes frequently undergo amplification, and less frequently, a deletion, in several human neoplasms. The role of the ErbB family in human endocrine neoplasms, including pheochromocytoma (PHEO), was not extensively tested and not previously established. The expression/overexpression of erbB oncogenes in pheochromocytoma tissue was determined only in a few cases, and to the best of our knowledge, their mutations (amplification or deletion) were not examined in any series of PHEO cases. We, therefore, used a double differential polymerase chain reaction (ddPCR) for determination of the amplification/deletion profiles of erbB-1, -2, 3 and -4 genes in formalin-fixed, paraffin embedded (FFPE) specimens of human PHEOs. We examined the average gene copy number (AGCN) of the genes in 36 samples of pheochromocytomas (2 extra-adrenal and 34 adrenal tumors). We found the mean AGCNs of the oncogenes equal 1.18 for erbB-1 [amplification was found in 11/35 cases (31%) and deletion in 6/35 cases (17%)], 2.00 for erbB-2 [amplification was found in 8/34 cases (24%), no deletion was found], 1.36 for erbB-3 [amplification was found in 4/36 cases (11%) and deletion in 1/36 cases (3%)], and 1.22 for erbB 4 [amplification was found in 5/30 cases (17%) and deletion in 1/30 cases (3%)]. A mutation(s) of any erbB oncogene was found in 25/36 (69%) samples tested. Some abnormalities of the erbB oncogenes showed interesting correlations with one another and with clinical features of the tumors. The frequent occurrence of amplifications and deletions of the erbB oncogenes in human pheochromocytoma implies the importance of the gene family in the development of these tumors. PMID- 12375050 TI - The Das-1 immunostain is useful for discriminating metastatic colon adenocarcinoma from cholangiocarcinoma and hepatocellular carcinoma. AB - The distinction between cholangiocarcinoma, hepatocellular carcinoma and liver metastasis is important but at times difficult solely on microscopic appearances. The Das-1 immunostain exhibits specificity for colonic epithelium. However, its staining of cholangiocarcinomas and hepatocellular carcinomas has not been extensively studied. We evaluated the staining properties of the Das-1 immunostain in cholangiocarcinoma and hepatocellular carcinoma. Forty-four cholangiocarcinomas, 16 hepatocellular carcinomas, 17 colon adenocarcinomas metastatic to the liver and 3 benign biliary tumors were studied. The cases were stained with the Das-1 antibody following deparaffinization and rehydration. The slides were evaluated for membranous and/or cytoplasmic staining in a blinded fashion. The percentage of tumor cells exhibiting strong staining was estimated. Of the intra-hepatic cholangiocarcinomas, 4 of 36 (11%) and 2 of 16 (13%) hepatocellular carcinomas were positive for Das-1, though typically only in rare cells. In comparison, 7 of 8 (88%) extra-hepatic cholangiocarcinomas, 13 of 17 (77%) of metastatic colon carcinomas and 3 of 3 (100%) benign tumors of the biliary tract were strongly positive in a diffuse pattern. The Das-1 immunostain may play a useful role in evaluating liver malignancies. PMID- 12375052 TI - Serum cathepsin D and its density in men with prostate cancer as new predictors of disease progression. AB - We examined whether serum levels of cathepsin D (CatD) and its density (CatD-D), which was determined by dividing the serum levels of CatD by the prostate volume, could be used as predictors of the progression and prognosis in patients with prostate cancer. Serum levels of CatD in 40 healthy controls, 70 patients with benign prostatic hypertrophy (BPH) and 80 patients with prostate cancer were measured by a sandwich enzyme immunoassay, and prostate volume was measured using transrectal ultrasonography. The mean levels of CatD and CatD-D in patients with prostate cancer were significantly higher than those in healthy controls and patients with BPH. Furthermore, the CatD and CatD-D levels in prostate cancer patients with metastasis were significantly elevated compared with those in patients without metastasis. Among patients who underwent radical prostatectomy, the levels of CatD and CatD-D in patients with pathologically organ-confined disease were significantly lower than in those with extraprostatic disease. However, the elevated levels of CatD and CatD-D were not significantly associated with the cause-specific survival in prostate cancer patients. These findings suggest that the elevation of CatD or CatD-D could be used as new predictors of disease progression, but not prognosis, in patients with prostate cancer. PMID- 12375053 TI - Effects of pentosan polysulfate sodium on the estrogen-induced pituitary prolactinoma in Fischer 344 rats. AB - The development of estrogen-induced pituitary prolactinoma in Fischer 344 (F344) rats is associated with enhanced neovascularization. This type of tumor is a rich source of basic fibroblast growth factor (bFGF), which possesses strong mitogenic and angiogenic properties. Pentosan polysulfate sodium (PPS) has been shown to exert antitumor activity by antagonizing the binding of bFGF to cell surface receptors. We have examined the effects of pentosan on tumor growth, hyperprolactinemia and angiogenesis in diethylstilbestrol-induced anterior pituitary adenoma in F344 rats. Chronic treatment with PPS did not cause any changes in the pituitary weight and serum prolactin concentration in comparison with untreated animals. The density of microvessels identified by CD-31 was also not affected by the tested drug. On the other hand, pentosan has been found to decrease cell proliferation evaluated by a number of PCNA-positive stained cell nuclei. Moreover, the TUNEL method has revealed an increased number of apoptotic bodies within the anterior pituitary after treatment with PPS. Despite the antiproliferative and proapoptotic activity of pentosan, the drug failed to inhibit tumor growth. This fact might be due to the lack of antiangiogenic activity of PPS in this experimental design. PMID- 12375054 TI - Prognostic significance of p53 and Bcl-2 in acute lymphoblastic leukemia. AB - p53 and Bcl-2 protein accumulation and mutations have been found in a wide variety of cancers including different types of leukemia, with varying frequencies. The objective of our study is to find out the correlation between p53 and Bcl-2 protein expression with respect to overall survival of patient taking clinical features and hematological parameters into consideration. Acute lymphoblastic leukemia (ALL) patients, 100 de novo and 10 relapse, were investigated taking different percentage of stained lymphocytes of patients into consideration. Immunocytochemistry and western blot analysis were used to investigate protein expression. PCR-SSCP technique was used to detect point mutations in exon 5, 6, 7 and 8 of p53 gene. P53 protein expression is of prognostic significance in univariate analysis and when combine with Bcl-2 expression data it provides additional information about overall survival. p53 and Bcl-2 failed to provide additional prognostic information in multivariate analysis. None of the samples shows mutation in exon 5, 6, 7 and 8 of p53 gene. Contrary to previous reports, p53 negativity was associated with lower relapse free survival. Lower p53 expression was associated with improved disease-free survival. The correlation of p53 and Bcl-2 expression data with respect to clinical outcome may shed new sight into the biological significance of p53/Bcl-2 protein. PMID- 12375055 TI - Rising leukemia rates in Thailand: the possible role of benzene and related compounds in cigarette smoke. AB - Epidemiological studies suggest that cigarette smoking is associated with an increased risk of leukemia and that benzene and related compounds in cigarette smoke may contribute to this elevated risk. This report presents new findings on selected components of cigarette smoke (including benzene and 1,3-butadiene) from major brands of cigarettes sold in Thailand, which represent about 80% of market share. Tested were also two major and popular brands of U.S. cigarettes sold in Thailand, representing about 100% of market share. The cigarettes tested were filter and non-filter, and with high and 'low' tar and nicotine levels. The observed range for benzene, toluene and 1,3-butadiene were found in the range of 25.5-63.7, 36.4-79.8 and 44.6-78.7 microg/cigarette, respectively. The amount of acrolein ranged from 79.9-181 microg/cigarette and for isoprene from 313-694 microg/cigarette. Yields of these substances showed no correlation with tar deliveries in mainstream smoke. Consumption of tobacco products increased in Thailand since 1970. This study also showed increases in leukemia mortality rates in Thailand, and in the relative frequency of leukemia among incident cancers diagnosed at a large hospital in Bangkok. Exposure to benzene and related compounds in cigarette smoke may have contributed to these trends. Analytic epidemiological studies are needed on the relationship between these compounds in smoke from tobacco products used in Thailand. These preliminary findings support the need for voluntary and/or government-regulated reduction in smoke yields of benzene and related compounds in tobacco products, and for expanded smoking prevention and cessation efforts, in Thailand. PMID- 12375056 TI - Free radicals in Parkinson's disease. AB - Although there are a number of hypotheses to explain the pathobiochemistry of Parkinson's disease (PD), the one on oxidative stress (OS) has gained major interest. The evidence for OS participation as a cause of PD can be summarized as follows: 1) OS is involved in physiological aging, 2) there is ample evidence that OS is significantly enhanced in PD compared to age-matched healthy persons, 3) OS is an early feature of PD because OS-dependent aggregation of proteins in the form of advanced glycation end products can be imaged in Lewy bodies at a time in a person's life, when no phenotype of a neurodegenerative disorder is evident, 4) Experimental models of PD show OS and degeneration of dopaminergic neurons. The toxin-induced neurodegeneration can be blocked by antioxidants, and 5) Activated microglia, known to release free radicals and inflammatory cytokines, are present in brains of Parkinsonian patients. In conclusion, a great body of evidence points to the view that OS is a major component underlying the pathobiochemistry of PD. Together a genetic disposition and endogenous/exogenous toxic events of various origins result in a synergistic cascade of toxicity which leads to dysfunction and finally to cell death of dopaminergic neurons. Again, OS plays a significant role in generating cell death signals including apoptosis. PMID- 12375057 TI - Cell death in Parkinson's disease. AB - The cause of neuronal cell death in Parkinson's disease is still an enigma. However, recent results obtained by analyses of postmortem brain suggest that a mitochondria-dependent apoptotic signal was activated. The involvement of dopamine-derived endogenous neurotoxin in the pathogenesis of PD was also indicated. N-Methyl( R)salsolinol was proved to be selectively toxic to dopamine neurons and its level increased in parkinsonian CSF. The enzyme which determines the level of N-methyl( R)salsolinol, ( R)salsolinol N-methyltransferase, was found increased in the lymphocytes prepared from PD patients. The mechanism of dopamine cell death by N-methyl( R)salsolinol was studied in vitro. N-Methyl( R)salsolinol induced apoptosis in human dopaminergic neuroblastoma cells. It was suggested that in the mitochondria there is a molecule which interacts with N methyl( R)salsolinol and initiates an apoptotic signal. PMID- 12375058 TI - Mitochondrial genotypes and cytochrome b variants associated with longevity or Parkinson's disease. AB - We are currently sequencing the entire mitochondrial genome from 600 persons, including centenarians, patients with Parkinson's disease or diabetes, and young adults with or without obesity to search for single nucleotide polymorphisms (SNPs) associated with longevity or diseases. To test the hypothesis that centenarians are free from deleterious mitochondrial variations, we analyzed amino acid variations in cytochrome b of 64 Japanese centenarians. Although the frequencies of some variations, such as N260D and G251S, differed significantly between centenarians and patients with Parkinson's disease, the most striking feature of centenarian cytochrome b was the much greater scarceness of amino acid variations in contrast with the variety of amino acid replacements in patients with Parkinson's disease. Particular deviations from the standard amino acid sequence may be associated with increased production by mitochondria of reactive oxygen species. The absence of certain variations in centenarians and their presence in patients with Parkinson's disease indicate that these variations do not benefit long-term survival but do predispose to adult-onset diseases and that centenarians are genetically hitting the "golden mean." PMID- 12375059 TI - Treatment of Parkinson's disease: levodopa as the first choice. AB - The introduction of levodopa in the 1960s revolutionised the treatment of Parkinson's disease (PD), and it continues to be the most effective symptomatic therapy. The vast majority of PD patients who start treatment with L-dopa experience good to excellent functional benefit. In vitro studies with high doses of L-dopa and absent glia had shown that it may be neurotoxic, but other tissue culture studies show L-dopa to be neuroprotective. Most studies in animal models and clinico-pathological and mortality studies in humans failed to show evidence in favour of accelerated dopaminergic neuronal loss with long-term L-dopa therapy.L-dopa continues to be beneficial throughout the course of PD, although as the disease progresses, escape of some symptoms from adequate control may occur and refractory disabilities such as impaired balance, dysarthria, cognitive decline and hallucinations may emerge. Treatment of advanced PD may also be complicated by the emergence of motor fluctuations and dyskinesias. Studies in animal models and in humans show that these motor complications are not specific to a particular dopaminergic agent, but that they are related both to the extent of the striatal lesion and to the mode of application of dopaminergic agents: Pulsatile administration of L-dopa and of the dopamine agonist apomorphine causes more motor complications than continuous striatal dopaminergic receptor stimulation, and continuous administration can alleviate existing dyskinesias and fluctuations. Several controlled studies comparing levodopa and dopamine agonists as initial treatment have attempted to answer the question whether delaying L dopa therapy can reduce the occurrence of motor complications. Three medium-term (3-5 years) and one 10-year study showed less dyskinesia in the first five years of treatment in patients who had started therapy with a dopamine agonist. However, these studies also consistently showed that levodopa provided better functional improvement in the first years of treatment. Ten-year follow-up data in patients randomised to L-dopa or bromocriptine also showed a slightly lower incidence of motor complications in the bromocriptine arm. However, this difference was not significant for the clinically relevant moderate and severe forms of dyskinesias and fluctuations, and was achieved at the expense of significantly worse disability scores during the first years of therapy. Furthermore, the relative impact of motor disability and dyskinesias on patients' quality of life remains to be established. Concordance is essential in the optimum treatment of PD and patients should be informed of the various therapeutic options available. Treatment should respect individual patients' needs and take into account their particular functional disabilities and specific handicaps. Low-dose L-dopa therapy (up to 400 mg/day), however, remains the most effective initial treatment of choice for the majority of patients. PMID- 12375060 TI - Initial therapy for Parkinson's disease: levodopa vs. dopamine receptor agonists. AB - Levodopa therapy is essential for patients in the advanced stages of Parkinson's disease. However, at early stages, DA agonist therapy has similar efficacy in the treatment of parkinsonism and a lower incidence of motor complications compared to levodopa therapy several years after the initiation of the therapy. The main factors causing motor complications have been speculated to be a severe reduction of dopaminergic nerve terminals because of disease progression, and a pulsatile stimulation of DA receptors using a drug with a short plasma half-life. DA agonists have longer plasma half-lifes than levodopa; therefore, they are expected to have a favorable effect on motor complications. Moreover, two clinical reports confirmed the potential neuroprotection by DA agonists. Although the patient's conditions should be considered in the selsction of a drug, DA agonist therapy is recommended as the initial therapy for Parkinson's disease. PMID- 12375061 TI - New drugs in the future treatment of Parkinson's disease. AB - During the last few decades, there has been a remarkable progress in our understanding of the biology of Parkinson's disease (PD), which has been translated into the development of numerous antiparkinsonian drugs. There are different therapeutic strategies for patients in an early stage versus patients in a late stage of the disease. The current therapeutic arsenal includes levodopa preparations, MAO-B inhibitors, dopamine agonists, COMT inhibitors and several other compounds that target non-dopaminergic systems. Much interest is focused on the potential neuroprotective effect of the already available drugs, as well as on new research approaches for the development of disease-modifying agents. These include mainly anti-glutamategic compounds, anti-apoptotic and antioxidative agents. Future therapy might include targeted delivery of trophic factors or genes involved in the pathogenesis of the disease. Apart from the classic levodopa-associated motor complications, such as dyskinesias and response fluctuations and psychosis, many other problems of advanced disease should be focused upon and solved including fatigue, freezing of gait, postural instability, depression, anxiety and panic attacks, sleep disturbances, autonomic dysfunction and sensory complaints. PMID- 12375062 TI - Recombinant adeno-associated viral vectors bring gene therapy for Parkinson's disease closer to reality. AB - The recombinant adeno-associated viral (rAAV) vector is a powerful tool for delivering therapeutic genes into mammalian brains. In rodents and non-human primates, a substantial number of striatal neurons can be transduced with high titer rAAV vectors by simple stereotaxic injection. Efficient and long-term expression of genes for dopamine (DA)-synthesizing enzymes in the striatum restored local DA production and achieved behavioral recovery in animal models of Parkinson's disease (PD). Moreover, sustained expression of a glial cell line derived neurotrophic factor gene in the striatum rescued nigral neurons and led to functional recovery in a rat model of PD, even when treatment was delayed until after the onset of progressive degeneration. These results suggest that gene therapy using rAAV vectors may become a novel and feasible treatment for PD. PMID- 12375063 TI - Generation of dopaminergic neurons from embryonic stem cells. AB - Neuronal transplantation is considered to be a promising therapeutic approach to neurodegenerative diseases. In addition to fetal tissues and neural stem cells, embryonic stem cells are good candidates for the creation of neurons. We have recently identified a stromal cell-derived inducing activity that promotes neural differentiation of mouse embryoric stem cells. This activity accumulated on the surface of PA6 stromal cells and induced efficient neuronal differentiation of co cultured embryonic stem cells under serum-free conditions without the use of either retinoic acid or embryoid bodies. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained. Induction of neurons with stromal cell-derived inducing activity may be a useful new method for basic neuroscience research and therapeutic applications, including cell transplantation therapy for Parkinson's disease. PMID- 12375064 TI - Viewpoint: unrecognized values of dissection considered. PMID- 12375065 TI - Anatomical basis for a new island axial pattern flap in the perioral region. AB - Soft tissue defects of the perinasal and perioral regions usually result from trauma and tumor resection as well as from congenital diseases. Coverage of facial defects is frequently challenging. The goal of reconstruction is to achieve a functional and esthetically satisfactory result. The most common techniques of wound care, such as full-thickness skin grafts and primary wound closure, are not suitable in all cases and therefore transposition flaps become necessary. Despite the description of numerous flaps, the search for other reconstruction possibilities and the development of additional flaps with good color match and minimal donor site morbidity is continuing. The purpose of our study was to describe the course of the facial artery and the pattern of its branches, because clinical cases have shown that there are branches which are suitable for skin island flaps. During the anatomical dissection of 31 cadavers (62 hemifaces), we analyzed a cutaneous branch of the facial artery, which we named due to its topographical location the "cutaneous zygomatic branch". This vessel shows a highly constant origin and course, as well as a relatively wide area of supplied skin. Based on our anatomical observations, we suggest a new axial pattern skin island flap which awaits clinical application. We feel that this flap has great future potential. PMID- 12375066 TI - Anatomical bases of superior gluteal nerve entrapment syndrome in the suprapiriformis foramen. AB - Observation of a 60 year-old-man with superior gluteal nerve (SGN) entrapment neuropathy in the suprapiriformis foramen encouraged us to explore, through anatomical dissection, the possible morphological etiologies of this condition. Ten SGNs in five embalmed cadavers were dissected via gluteal and pelvic access. The origin, course and distribution of the nervous trunk and its relations were studied. In most cases, the nerve fibers of the SGN arose from ventral branches of L4, L5 and S1 to constitute the nervous trunk in the pelvis, then reached the gluteal area and divided into two branches, cranial and caudal. By running through the suprapiriformis foramen with the cranial gluteal vascular pedicle, the nervous trunk was always up between the superior edge of the piriformis muscle and the greater sciatic notch; rarely some of the nerve fibers went through the muscle. Bone, muscular and vascular morphological factors liable to cause SGN entrapment syndrome, and the circumstances of discovery, were analyzed. The role of hypertrophy of the piriformis muscle, resulting in a narrow suprapiriformis foramen, was confirmed through surgery. PMID- 12375067 TI - Morphology of trachea in benign human tracheal stenosis: a clinicopathological study of 20 patients undergoing surgery. AB - Prolonged tracheal intubation of patients often leads to tracheal stenosis (TS), which may require surgical removal of the narrowed portion of the airway. We studied 20 patients with TS who underwent surgical ablation of the stenotic portion of trachea. The morphology of the tracheal segments was characterized and compared with clinical data and with the prognosis for the disease. We found that TS was usually due to an increase in the width of the mucosa as a result of the fibrosis associated with the chronic inflammation. Plasma cells were the predominant leukocyte type seen in the inflammatory infiltrates of the surgically removed portions of narrowed trachea. In the majority of TS samples, the epithelial surface was intact and presented cilia; in contrast, cilia disappeared when the tracheal lumen was completely obliterated. Mucosal cells and glands were also well preserved in TS samples. The need to remove TS segments was often related to previous tracheal surgery, which was also associated with closing of the tracheal lumen and ossification of cartilage rings. We conclude that (a). chronic inflammation and fibrosis are responsible for the narrowing of trachea in TS patients, (b). metaplastic ossification of cartilage rings only occurs after complete obliteration of the tracheal lumen, and (c). loss of cilia and presence of metaplastic bone tissue are indicators of a poor prognosis for TS. PMID- 12375068 TI - The cubital tunnel: anatomical study of its distal part. AB - Different levels of ulnar nerve compression have been reported (the medial intermuscular septum, the posterior compartment of the arm, soft tissue or bony abnormalities of the cubital tunnel). In some rare cases, compression can lie in a 10-cm long tunnel, distal to Osborne's ligament, between the humeral head of the ulnar flexor muscle of wrist (FCU) and the medial epicondylar muscles. Only few publications mention this fact as a factor of residual or recurrent symptoms after common surgical procedures. However, a distal pathology of the cubital tunnel has proved to be the only factor of nerve entrapment in our clinical practice. Specific anatomical dissection of this area was carried out to find and classify the anatomical structures that may play a role in ulnar nerve distal compression. Twenty-four embalmed limbs from 13 cadavers were dissected. The purpose of this study was to find anatomical fibrous structures at an average of 10 cm from the medial epicondyle. Anatomical structures were classified into five types: no aponeurosis between the FCU and the medial epicondylar muscles (54.2% of cases), a fibrous band taut between the FCU and the fourth- and fifth-finger ulnar insertions of the flexor digitorum superficialis (FDS) (8.3%), a thin (20.8%) or thick (4.2%) partial aponeurosis between the FCU and the medial epicondylar muscles, and total aponeurosis (12.5%). Anatomical variations of the distal cubital tunnel were divided in five types, but their clinical significance remains unclear. PMID- 12375069 TI - Clinical anatomy of the arterial supply of the human patellar ligament. AB - The arterial supply of the human patellar ligament has been systematized on 20 knee joints. After intravascular injection of colored natural latex, the blood supply to the extensor apparatus of the knee was studied by anatomical dissection and tissue transparentation techniques. Three arterial pedicles (superior, middle and inferior) were observed placed on each side of the patellar ligament. Medial pedicles had their origin from the descending and the inferior medial genicular arteries. The lateral pedicles took their origin from the lateral genicular arteries and the recurrent tibial anterior artery. Two main vascular arches anastomosed with these pedicles: the retropatellar and the supratubercular. Both arterial pedicles and anastomotic arches gave rise to a peritendinous network, characterized by a high vascular density next to poles of the patellar ligament. Only the anastomotic arches gave rise to collateral vessels that pierced the tendon, which revealed two vascular segments in the arterial supply of the patellar ligament (bipolar pattern). The upper segment was supplied by deep vessels from the retropatellar arch, whereas the inferior segment received superficial vessels from collaterals of the supratubercular arch. These intratendinous vessels anastomosed in the middle third of the patellar ligament. PMID- 12375070 TI - Skin thickness of Korean adults. AB - Skin thickness varies considerably between different races and age-groups, between men and women, and between different regions of the body surface. A few authors reported the skin thickness of different regions of the body, but no detailed study have been performed on Asian. We performed 452 biopsies on 28 different regions of the normal skin of Korean men and women. The specimens were stained with hematoxylin-eosin and measured microscopically. The thickness of the skin (epidermis plus dermis) ranged from 521 to 1977 microm; the eyelid, prepuce, and inguinal skin was thinnest (521-626 microm), and the back was thickest (1977 microm). The thickness of the epidermis varied from 31 to 637 microm; skin thickness in the prepuce, eyelid, supraclavicular region, postauricular region, and axilla ranged from 31 to 71 microm; the buttock, dorsum of the hand, and dorsum of the foot were relatively thick (138-189 microm); the palm and sole were thickest (601-637 microm). The thickness dermis varied from 469 to 1942 microm; skin thickness in the eyelid, prepuce, inguinal region, and postauricular region ranged from 469 to 645 microm; the buttock, chest, and anterior neck were relatively thick (1318-1586 microm); the back was thickest (1942 microm). The epidermis accounted for 3.7-16.8% of the entire skin in most regions, except in the palm and sole (40.6-44.6%). Thickness data may be useful in harvesting full- or split-thickness skin grafts. PMID- 12375071 TI - Localization of the superior laryngeal nerve during carotid endarterectomy. AB - Knowledge of the topographic anatomy is essential to prevent iatrogenic damage of the superior laryngeal nerve (SLN) in carotid endarterectomy (CEA). The purpose of this study was to analyze the anatomic relationship between the SLN and carotid arteries in order to prevent iatrogenic nerve injury. Anatomic dissections similar to CEA were performed bilaterally in 50 fresh human adult cadavers. The topography of the SLN was analyzed regarding its relationship with the carotid arteries. Furthermore, the distance between the external branch of the SLN and the point of bifurcation of the common carotid artery (dCAB) was analyzed regarding effects of gender, ethnicity, individual stature and side of the neck. The SLN was always located adjacent and posterior to the carotid arteries.The dCAB ranged from 20.3 mm below the point of bifurcation of the common carotid artery to 50.9 mm above this level (average 10.3 mm above). Most dissections (75%) showed the external branch of the SLN emerging from behind the carotid artery above the arterial bifurcation; in only 10% of cases did this nerve emerge from the artery below that anatomic reference. Based on Student's t test, there were no significant differences in the dCAB between genders ( P=0.237), ethnicities ( P=0.410) and sides of the neck ( P=0.872). Moreover, tall stature was not significantly correlated with a shorter dCAB (linear regression: R(2)=0.009, P=0.357). We conclude that most SLNs were located above the carotid artery bifurcation, but anatomic variations occurred in 25% of the dissections. The dCAB was unaffected by gender, ethnicity, individual stature and side of the neck. PMID- 12375072 TI - The human lumbar anterior epidural space: morphological comparison in adult and fetal specimens. AB - To increase our understanding of the clinical anatomy of the epidural space, the human lumbar anterior epidural space was studied morphologically and developmentally. Histological transverse sections of human lumbar spines were taken at the level of the intervertebral disc and the vertebral body in adult specimens and in fetuses aged 13, 15, 21, 32 and 39 weeks (menstrual age). At 13 weeks, connective tissue filled the epidural space. The dura mater was attached anteriorly to the posterior longitudinal ligament (PLL). The PLL was attached to the vertebral body beside the midline, whereas it adhered to the posterior edge of intervertebral disc. The anterior internal vertebral venous plexus was located anterolaterally and anteromedially. The vertebral canal was lined with connective tissue that differentiated in a periosteum in contact with the ossification centers. At 15 weeks, the PLL was composed of deep and superficial layers. At 21 weeks, the attachment between the dura mater and PLL was ligament-like at the level of the vertebral body. At 32 weeks, the dura mater was adherent to the superficial layer of PLL. At 39 weeks, groups of adipocytes were identified, and the dura mater was attached to the PLL by some ligaments. There were many more similarities between the adult and the 39-week fetus. In conclusion, some differences in the anatomy of the epidural space exist at each fetal stage studied. The structures of the epidural space are already formed in the fetus of 13 weeks, but they differentiate progressively within the connective tissue. PMID- 12375074 TI - Optic nerve compression analyzed by using plastination. AB - Plastination is an excellent tool for studying different anatomical and clinical questions. The E12 technique provides transparent slices which show the anatomical structures in their initial position, especially those of the muscular, vascular and interstitial tissue. Here we demonstrate on a plastinated specimen optic nerve compression due to hypertrophy and degeneration of the extraocular muscles near the apex of the orbit. The paper aims to demonstrate the possibilities offered by the E12 plastination technique in analyzing anatomical structures. The plastinated slices obtained were scanned and then analyzed. In a further step the plastinated slices were cut into thin slices (150 microm), stained and finally examined histologically. We measured the intraorbital length (A-B), which had an average value of 25,93+/-0,03 mm. Dividing the optic nerve into uncompressed (UP) and compressed (CP) parts, we obtained the following values for optic nerve width: UP=4192+/-0,455 mm, CP=3215+/- 0,411 mm. Using the E12 plastination method we were able to take morphometric measurements on plastinated orbital slices with coincidentally detected optic nerve compression. This allowed the determination of different parameters of the optic nerve, as well as histological examination by hematoxylin and eosin staining up to a magnification of x40. PMID- 12375073 TI - Renal excretory sectors. AB - One thousand and ninety-four normal human kidneys and 18 abnormal (with duplication of the ureter) were studied by the corrosion method and pyelography followed by topometric and mathematics analyses. It was found that the renal pelvis is a calicopelvic complex built up of renal calices, urine ducts and renal pelvis. Before opening into the renal pelvis, renal calices join together forming urine ducts (superior and inferior; or superior, middle and inferior; or superior, middle anterior, middle posterior and inferior) which transport urine to the container, the renal pelvis. It can be seen that groups of renal calices with pyramids and their surrounding cortical substance form the renal excretory sectors of the kidneys where the processes of uropoiesis and transportation of urine through elements of the nephron and calicopelvic complex take place. These are two (superior and inferior), three (superior, middle and inferior) or four (superior, middle anterior, middle posterior and inferior) renal excretory sectors. The existence of renal excretory sectors is proved by congenital anomalies of the calicopelvic complex such as duplication of the ureter, where urine ducts of the superior and inferior renal excretory sectors do not form a renal pelvis but run separately to the urinary bladder. On the basis of anatomical data obtained, renal excretory sectors may be distinguished, analogous to bronchopulmonary segments in lungs. These data about renal excretory sectors will contribute to further improvement in the operative technique of renal partial resections as well as to anatomical nomenclature. PMID- 12375075 TI - Variation of the ulnar variance with powerful grip. AB - Causal relationships between ulnar variance and wrist disorders are known. Gripping and pronation cause proximal translation of the radius with respect to the ulna, leading to a statistically significant increase in ulnar variance. The purpose of this study was to investigate variation of the ulnar variance with powerful grip. A total of 41 male volunteers aged between 19 and 25 years (mean, 21.2+/-1.7 years) were studied. Posteroanterior X-ray films of all wrists were taken in the standardized position. After neutral posteroanterior X-ray films had been taken, subjects were asked to grip a Takei hand dynamometer with maximum force while repeated standardized posteroanterior X-ray films were obtained. Ulnar variance values were measured using the perpendicular method. Mean maximum grip force was 38.1 kg (range, 26.6-47.9 kg). Mean values of force-free (neutral) and forced ulnar variances were 0.06+/-0.21 mm and 1.87+/-0.23 mm, respectively. The difference in ulnar variance between the two groups was statistically significant ( P<0.001). The increase in ulnar variance with grip observed varied between 0.00 mm (minimum) and 3.97 mm (maximum), with a mean of 1.81 mm. Gaining an understanding of normal limits of ulnar variance modification with grip may be helpful in planning surgical treatment. PMID- 12375076 TI - Anatomical and radiological correlation of Lequesne's "false profile". AB - Lequesne introduced a radiological projection, which is an oblique view of the edge of the acetabulum, to diagnose arthrosis affecting the anterior part of the joint and to measure the anterior coverage of the femoral head. In this study, we attempted to determine the anatomical correlation of his technique. Fifteen in vitro hemipelvises underwent radiography according to Lequesne's description, using metallic markers and wires to mark physical landmarks. According to geometric laws, the points used by Lequesne do not correlate anatomically. Although Lequesne's technique allows a diagnosis of acetabular dysplasia, measurements are on average 5.5 degrees less than those made anatomically. PMID- 12375077 TI - Feasibility of percutaneous transabdominal portosystemic shunt creation. AB - Evaluation of the anatomic feasibility of the percutaneous transabdominal puncture of selected portal and hepatic veins in patients with cirrhosis was performed. This approach would become the framework for an image-guided robot assisted needle drive mechanism for use in transjugular intrahepatic portosystemic shunt (TIPS) creation. Retrospective analysis of 10 CT and 14 MRI axial abdominal studies was carried out to determine whether single simultaneous transabdominal puncture of portal and hepatic veins was possible. A necessary modification of the TIPS procedure was tested in an ex vivo porcine model under fluoroscopy. Eighteen of 24 patients (75%) had intrahepatic vascular anatomy amenable to a single transabdominal puncture. Successful catheterization of portal and hepatic veins using a modified approach for TIPS was accomplished in two ex vivo porcine livers. A suitable anatomic approach for modified TIPS is present in a majority of patients with cirrhosis. Feasibility of the technique using this anatomic approach was confirmed in two ex vivo porcine models. This study serves as an initial step in a novel technical approach to TIPS using a new anatomic approach for this procedure. PMID- 12375078 TI - Anatomical variations in the drainage of the principal adrenal veins: the results of 88 venograms. AB - This study of 88 adrenal venograms looked at the anatomical variations in the drainage of the principal adrenal veins. These were seen with a frequency of 5% on the right and 6% on the left. On the right, we have described ectopic anastomoses with the hepatic veins. On the left, the variations were always linked to an anomaly of the renal vein. PMID- 12375079 TI - Persistent cervical intersegmental artery and aortic arch coarctation. AB - A 15-year-old girl presented with upper extremity hypertension and continuous precordial murmur. Arteriography revealed aortic coarctation proximal to the origin of the left subclavian artery. An anomalous artery originated from the aortic arch, between the left common carotid artery and the stenosis. It ascended cranially and filled an angiomatous vascular formation on the left side of the neck. The "angioma" drained into the left subclavian artery. The embryological explanation of the described anomaly is difficult, but probably related to hemodynamic alterations following the prestenotic increase in blood pressure. This may have impaired the obliteration of cervical intersegmental arteries, resulting in the persistence of one of the first three intersegmental arteries as the anomalous branch of the aortic arch. The angiomatous vascular formation in the neck could be the consequence of altered development of anastomoses between the muscular twigs of both vertebral and deep cervical artery. The vessel draining the vascular formation was probably the thyrocervical trunk. Since there were no overt collateral channels or signs of left ventricular hypertrophy by electrocardiography and echocardiography, it seems that the aberrant collateral flow was hemodynamically significant and reduced the afterload on the myocardium. Although the pattern of collateral flow in our case might be considered extremely rare, it is important in preoperative planning and interpretation of imaging studies. PMID- 12375080 TI - Retro-esophageal subclavian artery: a case report. AB - This case report presents the finding of an aberrant right subclavian artery during the routine dissection of 230 human adults specimens. The case which we describe appeared in an 82-year-old male donor, in whom the right retro esophageal subclavian artery was observed as the last branch of the aortic arch. The rate of occurrence of this variation is stated to be less than 1%. This variation is described in detail; embryology and anatomy are reviewed and we discuss it in relation to other cases. The aortic arch was dissected, and its branches were described as regards their relations, length and diameter. PMID- 12375081 TI - National policies for biosphere greenhouse gas management: issues and opportunities. AB - Biosphere greenhouse gas (GHG) management consists of preserving and enhancing terrestrial carbon pools and producing biomass as a fossil fuel substitute. The discussion of this topic has focused primarily on carbon-accounting and project level issues, particularly relating to carbon sequestration as a source of emissions credits under the Kyoto Protocol. While international consensus on these matters is needed, this paper argues that an important domestic policy agenda also deserves attention. National policies for biosphere GHG management are necessary to bring about large-scale changes in land-use, forestry, and agricultural practices and can address some of the technical and policy issues that have proven to be particularly problematic from carbon-accounting and project-level perspectives. These policies should minimize land-use and resource management conflicts, account for collateral benefits, and ensure institutional compatibility with existing resource-management regimes. Issues relating to project permanence, leakage, and transaction costs should also be addressed. A range of policy instruments should be used and biosphere GHG management should be one component of an integrated approach to environmental and resource management. Countries promoting biosphere GHG management as an important element of their climate change strategies should be developing these domestic policies to complement international negotiations and to demonstrate that carbon sequestration and biomass production can make an effective contribution to the stabilization of atmospheric GHG concentrations. PMID- 12375082 TI - Excreta disposal in Dar-es-Salaam. AB - The sociocultural and socioeconomic situation of sanitation in Dar-es-Salaam (Dsm), Tanzania, was studied with explicit emphasis on pit-latrines. Without considering the sociocultural conditions, the so-called best solution might not be the right one. Therefore, in order to achieve the intended goal, a literature review, a questionnaire survey, and personal visits to the chosen study areas were done. In total, 207 household questionnaires were filled in 16 areas of the city. Interviewers did house-to-house visits and questionnaires were filled out on the spot. Results indicated that the city population is about 3.8 million at present, with over 80% of the dwellers using pit-latrines; some 3% use septic tanks with soakage pits, about 6% are connected to the sewerage system, and 1% have no excreta disposal facility. Difficulties faced include dismal budget allocations, fragmentation of sanitation activities among subsectors, lack of or poor sanitation record keeping, unsatisfactory machinery for septic tank and pit latrine emptying, lack of a clear policy on pit-latrine handling and, in competition for resources, low priority is accorded to an excreta disposal system among the people. City residents will continue to use the pit-latrines for a long time to come. Reusing the fecal sludge is not known by most city dwellers and is influenced by sociocultural habits. To prevent groundwater pollution and to recover useful products in human excreta and urine, ecological sanitation toilets and anaerobic digesters offer a good option. PMID- 12375083 TI - Public-private partnerships for solid waste management services. AB - The increasing cost of municipal solid waste (MSW) management has led local governments in numerous countries to examine if this service is best provided by the public sector or can better be provided by the private sector. Public-private partnerships have emerged as a promising alternative to improve MSW management performance with privately owned enterprises often outperforming publicly owned ones. In Lebanon, several municipalities are transforming waste management services from a public service publicly provided into a public service privately contracted. In this context, a regulated private market for MSW management services is essential. The present study examines a recent experience of the private sector participation in MSW management in the Greater Beirut Area. The results of a field survey concerning public perception of solid waste management are presented. Analysis of alternatives for private sector involvement in waste management is considered and management approaches are outlined. PMID- 12375084 TI - Restoration, stewardship, environmental health, and policy: understanding stakeholders' perceptions. AB - In recent years there has been considerable interest in the health of humans and the environment, restoration of contaminated or otherwise degraded lands, and in long-term stewardship of public lands. Unfortunately, it is unclear whether governmental agencies and the public hold similar views about the meanings of these concepts, making policy decisions about restoration and stewardship difficult. In this paper, I explore how the public conceptualizes restoration and stewardship by examining the relative rating of several attributes of restoration, stewardship, environmental health, ecological health, environmental restoration, and ecological restoration. People were interviewed in Santa Fe, New Mexico, USA, near the Department of Energy's Los Alamos National Laboratory. The ratings of attributes of environmental health and ecological health reported in this paper can be used to understand how the public understands these concepts. The attributes rated most highly by the subjects were more similar to definitions in the scientific literature for these terms than they were to those used by the Department of Energy. For environmental health, the highest rating related to human sanitation, while for ecological health the highest rating was for maintaining functioning ecosystems. Reduction of exposure to hazardous substances was rated the second highest for both environmental and ecological health. The wise use of natural resources, preservation of natural resources, and hazardous waste site cleanup were rated the highest attributes of stewardship. These data suggest that both expert and nonexpert perceptions about restoration and stewardship should be incorporated into environmental management decisions. PMID- 12375085 TI - Setting priorities for research on pollution reduction functions of agricultural buffers. AB - The success of buffer installation initiatives and programs to reduce nonpoint source pollution of streams on agricultural lands will depend the ability of local planners to locate and design buffers for specific circumstances with substantial and predictable results. Current predictive capabilities are inadequate, and major sources of uncertainty remain. An assessment of these uncertainties cautions that there is greater risk of overestimating buffer impact than underestimating it. Priorities for future research are proposed that will lead more quickly to major advances in predictive capabilities. Highest priority is given for work on the surface runoff filtration function, which is almost universally important to the amount of pollution reduction expected from buffer installation and for which there remain major sources of uncertainty for predicting level of impact. Foremost uncertainties surround the extent and consequences of runoff flow concentration and pollutant accumulation. Other buffer functions, including filtration of groundwater nitrate and stabilization of channel erosion sources of sediments, may be important in some regions. However, uncertainty surrounds our ability to identify and quantify the extent of site conditions where buffer installation can substantially reduce stream pollution in these ways. Deficiencies in predictive models reflect gaps in experimental information as well as technology to account for spatial heterogeneity of pollutant sources, pathways, and buffer capabilities across watersheds. Since completion of a comprehensive watershed-scale buffer model is probably far off, immediate needs call for simpler techniques to gage the probable impacts of buffer installation at local scales. PMID- 12375086 TI - Spatial and temporal dynamics of hedgerows in three agricultural landscapes of southern Quebec, Canada. AB - Noncrop areas such as hedgerows in agricultural landscapes can perform several ecological and agronomic functions (e.g., habitat, movement corridors, windbreak, etc.), but their dynamics and drivers of changes are often poorly known. We conducted a study in three agricultural landscapes of southern Quebec, Canada, to assess and compare the spatial and temporal (1958-1997) dynamics of three hedgerow networks in relation to geomorphic conditions (marine, glacial, and mixed deposit) and land-use changes. Hedgerow networks were mapped and described in terms of their structure (density, degree of connectivity, and presence of trees or shrubs) and their relationship to other components of the landscape (connection to woodland). Relationships were assessed in time and space using nonparametric correlation, Mantel test, and principal components analysis (PCA). Results show significant differences between hedgerow structure for the three landscapes and distinct temporal and spatial dynamics that can be related to changes in management practices and agricultural policies. On marine deposits, increases in hedgerow density did not always correspond to an increase in their degree of connectivity, suggesting a possible reduction in network quality. On glacial deposits, hedgerow density declined following abandonment of agricultural land, but rather than disappearing, these linear structures were integrated into adjacent brush or forested areas. Our analysis reveals the complex spatial and temporal dynamics of the hedgerow networks and highlights the need to take into account spatial attributes such as connectivity and connection to woodland to evaluate more accurately overall network quality. PMID- 12375087 TI - Farmers, streams, information, and money: does informing farmers about riparian management have any effect? AB - We assessed relationships between the extent to which farmers reported exposure to relevant information and their attitudes towards, knowledge about, and degree of adoption of riparian management strategies. We also examined associations between knowledge of, or receipt of, financial assistance for riparian fencing/planting and intentions for and extent of adoption of this strategy. A mail survey of 718 pastoral farmers in Otago and Southland in New Zealand [294 surveys returned (41%)] yielded 279 usable questionnaires. Indices were developed to reflect range and frequency of information use and range of practices adopted. Attitudes were measured using Likert-type responses to 11 statements, and knowledge as a score on a ten-question true/false test. Positive relationships between information and the three main response variables (attitude, knowledge, and adoption) were weak but significant and systematic. These associations remained significant when important demographic and farm characteristics were taken into account. Informed farmers were more likely to report intentions to carry out riparian fencing or planting within the next year. Farmers who were aware that funding was available were also more likely to state this intention, independent of information level. The reported extent to which waterways had been fenced to exclude stock was related to receipt of funding, but not to information level. Financial factors were the most influential barrier preventing adoption of permanent fencing. Our research shows a positive correlation between the receipt of information and funding and the adoption of specific riparian management measures. PMID- 12375088 TI - Climatic and stream-flow controls on tree growth in a Western montane riparian forest. AB - Humans have severely impacted riparian ecosystems through water diversions, impoundments, and consumptive uses. Effective management of these important areas is becoming an increasingly high priority of land managers, particularly as municipal, industrial, and recreational demands for water increase. We examined radial tree growth of four riparian tree species ( Pinus jeffreyi, Populus trichocarpa, Betula occidentalis, and Pinus monophylla) along Bishop Creek, California, and developed models relating basal area increment (BAI) and relative basal area increment (RBAI) to climatic and stream flow variables. Between years 1995-1999, univariate regression analysis with stream flow explained 29 to 61% of the variation in BAI and RBAI among all species except P. trichocarpa; growth by P. trichocarpa was not significantly related to stream flows over this period. Stepwise linear regression indicated that species responded differently to climatic variables, and models based on these variables explained between 33 to 86% of variation in BAI and RBAI during the decade of the 1990s. We examined branch growth of P. trichocarpa for sensitivity to differences in stream flow regimes and found that annual branch growth did not vary between a high- and low flow site, but that annual branch growth was significantly higher in wet years with greater stream flows. Our results support the establishment of site-specific management goals by land managers that take into account all of the important tree species present in riparian ecosystems and their differential responses to altered hydrologic condition. Instream flow requirements for maintaining tree growth and vigor are only one of the species-specific responses that need to be evaluated, and these assessments should attempt to separate experimentally stream flow (managed) controls from climatic (unmanaged) controls on growth. PMID- 12375089 TI - Amenity values of public and private forests: examining the value-attitude relationship. AB - Public values toward forests have changed since the late 1980s, from a commodity oriented perspective toward a more inclusive (commodity and non-commodity) orientation. This study examines the influence of four indicators of population diversity (age, ethnic background, place of residence, and gender) on amenity values of forests, environmental attitudes, and forest value-attitude correspondence. Four values of public and private forests were assessed, wood production (utilitarian value), clean air (a life support value), scenic beauty (an aesthetic value), and heritage (a spiritual value). Environmental attitudes were measured using a modified version of the New Environmental Paradigm scale. Five hundred and forty-eight randomly selected residents of households in 13 states of the Southern United States participated in a telephone interview. Age and ethnic background were found to moderate the value-attitude relationship, with the strength of the association being dependent upon the type of forest (i.e., public or private) and the forest value (i.e., utilitarian, life support, spiritual, and aesthetic). Females, younger persons (less than 43 years old), and whites reported lower utilitarian values of forests than their respective counterparts. Results are interpreted within the context of an emerging post material society, in which a biocentric orientation to forests and the natural environment may be favored more by a younger (versus older) generation and increasingly racially diverse U.S. population. Implications for managing forests using a multiple-values (versus multiple-uses) approach are discussed. PMID- 12375090 TI - Multiscale indicators of land degradation in the Patagonian Monte, Argentina. AB - Depletion of vegetation by overgrazing in arid environments has long-lasting effects on the environmental quality over extended geographic areas. An adequate inspection of habitat changes requires scaled up procedures that would allow assessing end-points of environmental status in broad areas that would be based on processes occurring at the plant canopy level. Our purpose was to find indicators of land degradation-conservation status for use in land monitoring programs and in planning management practices that would be amenable to further up-scaling for use with remotely sensed imagery. In several sites of the Patagonian Monte differing in the impact of grazing management, we evaluated vegetation attributes at three spatial scales. At the population scale, we found that the severity of grazing impact was characterized by the reduction of the palatable grass, P. ligularis, outside and inside shrub canopies. At the vegetation patch scale, we found that land degradation by domestic herbivore impact was characterized by changes in attributes of patch shape (radius, height, internal canopy cover) and patch abundance. At the plant community scale, we found that the structure of the plant canopy as described using Fourier analysis of cover data changed after long-term grazing impact consistently with the modifications in plant population and patch structures. We present a conceptual multiscale scenario of structural changes triggered by domestic herbivore impact, and quantitative indicators of plant structure and processes useful to develop management strategies of the Patagonian-Monte that would conserve its natural habitats. The developed end-points are also amenable for use in land conservation assessment through remotely sensed imagery. PMID- 12375091 TI - Assessment of groundwater quality by means of self-organizing maps: application in a semiarid area. AB - The Kohonen neural network was applied to hydrochemical data from the Detritic Aquifer of the Lower Andarax, situated in a semiarid zone in the southeast of Spain. An activation map was obtained for each of the sampling points, in which the spatial distribution of the activated neurons indicated different water qualities. To extract the information contained in the activation maps, they were divided into nine quadrats. Cartesian coordinates were assigned to each quadrant ( x, y), and for each sampling point, three derived variables were selected, which were assigned the values x and y of the corresponding quadrat. A classification was defined based on this simple matrix system which allows an easy and rapid means of evaluating the water quality. This assessment highlights the various processes that affect groundwater quality. The method generates output that is easier to interpret than from traditional statistical methods. The information is extracted from the activation maps without significant loss of information. The method is proposed for assessing water quality in hydrogeochemically complex areas, where large numbers of observations are made. PMID- 12375092 TI - The US Clean Water Act and habitat replacement: evaluation of mitigation sites in Orange County, California, USA. AB - Both permit requirements and ecological assessments have been used to evaluate mitigation success. This analysis combines these two approaches to evaluate mitigation required under Section 404 of the United States Clean Water Act (CWA) and Section 10 of the Rivers and Harbors Act, which allow developers to provide compensatory mitigation for unavoidable impacts to wetlands. This study reviewed permit files and conducted field assessments of mitigation sites to evaluate the effectiveness of mitigation required by the US Army Corps of Engineers for all permits issued in Orange County, California from 1979 through 1993. The 535 permit actions approved during this period allowed 157 ha of impacts. Mitigation was required on 70 of these actions, with 152 ha of enhanced, restored, and created habitat required for 136 ha of impacts. In 15 permit actions, no mitigation project was constructed, but in only two cases was the originally permitted project built; the two cases resulted in an unmitigated loss of 1.6 ha. Of the remaining 55 sites, 55% were successful at meeting the permit conditions while 11% failed to do so. Based on a qualitative assessment of habitat quality, only 16% of the sites could be considered successful and 26% were considered failures. Thus, of the 126 ha of habitat lost due to the 55 projects, only 26 ha of mitigation was considered successful. The low success rate was not due to poor enforcement, although nearly half of the projects did not comply with all permit conditions. Mitigation success could best be improved by requiring mitigation plans to have performance standards based on habitat functions. PMID- 12375093 TI - Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments. AB - Sediments often exhibit low rates of nitrogen fixation, despite the presence of elevated concentrations of inorganic nitrogen. The organisms that potentially fix nitrogen in sediments have not previously been identified. Amplification of nifH genes with degenerate primers was used to assess the diversity of diazotrophs in two distinct sediment systems, anoxic muds of Chesapeake Bay and shallow surficial sediments of the Neuse River. Phylogenetic analysis revealed that sequences obtained from mid-Chesapeake Bay, which receive high organic loading and are highly reducing, clustered closely with each other and with known anaerobic microorganisms, suggesting a low abundance of aerobic or facultative diazotrophs in these sediments. Sulfate reduction dominates in the surface, but methanogenesis becomes more important with depth. A thin (<1 cm) oxidized layer is present only in the spring. No archaeal nifH sequences were obtained from Chesapeake Bay. Sequences of nifH amplified from surficial sediments of the Neuse River were distant from Chesapeake Bay sequences and included nif phylotypes related to sequences previously reported from marine mats and the Spartina rhizosphere. Differences in environmental site characteristics appear to select for different types of sediment diazotrophs, which is reflected in the phylogenetic composition of amplified nifH sequences. PMID- 12375094 TI - Participation of oxygen and role of exogenous and endogenous sensitizers in the photoinactivation of Escherichia coli by photosynthetically active radiation, UV A and UV-B. AB - We studied the mechanisms by which photosynthetically active radiation (PAR) and ultraviolet (UV-A and UV-B) radiation damage Escherichia coli suspended in water. The roles played by oxygen and exogenous and endogenous sensitizers were analyzed by monitoring changes in the physiological state of irradiated cells. Impairment of the cellular functions was more severe in the case of UV radiations. Radiation caused cellular damage in the absence of oxygen. PAR, UV-A, and UV-B radiation induced photobiological and photodynamic reactions mediated by endogenous sensitizers, which significantly shortened the T90 (time needed to reduce a cellular parameter by 90%) based on the growth ability of the cells. In addition, when exogenous sensitizers were present, the photodynamic reactions also had a negative effect on the operation of the electron transport chains. The presence of oxygen might enhance photoinactivation, affecting both the growth ability and the electron transport chains. Endogenous sensitizers were responsible for the noxious action of oxygen. The presence of dissolved organic material played a protective role against the oxygen by absorbing the incident radiation, thereby reducing the energy that reached the endogenous sensitizers. PMID- 12375095 TI - In situ spatial patterns of soil bacterial populations, mapped at multiple scales, in an arable soil. AB - Very little is known about the spatial organization of soil microbes across scales that are relevant both to microbial function and to field-based processes. The spatial distributions of microbes and microbially mediated activity have a high intrinsic variability. This can present problems when trying to quantify the effects of disturbance, management practices, or climate change on soil microbial systems and attendant function. A spatial sampling regime was implemented in an arable field. Cores of undisturbed soil were sampled from a 3 x 3 x 0.9 m volume of soil (topsoil and subsoil) and a biological thin section, in which the in situ distribution of bacteria could be quantified, prepared from each core. Geostatistical analysis was used to quantify the nature of spatial structure from micrometers to meters and spatial point pattern analysis to test for deviations from complete spatial randomness of mapped bacteria. Spatial structure in the topsoil was only found at the microscale (micrometers), whereas evidence for nested scales of spatial structure was found in the subsoil (at the microscale, and at the centimeter to meter scale). Geostatistical ranges of spatial structure at the micro scale were greater in the topsoil and tended to decrease with depth in the subsoil. Evidence for spatial aggregation in bacteria was stronger in the topsoil and also decreased with depth in the subsoil, though extremely high degrees of aggregation were found at very short distances in the deep subsoil. The data suggest that factors that regulate the distribution of bacteria in the subsoil operate at two scales, in contrast to one scale in the topsoil, and that bacterial patches are larger and more prevalent in the topsoil. PMID- 12375096 TI - Estimation and diversity of phylloplane mycobiota on selected plants in a Mediterranean-type ecosystem in Portugal. AB - Mediterranean ecosystems have not been consistently investigated as natural habitats for microbes in general, and fungi in particular. Here we present the results of a survey of epiphytic mycobiota (filamentous fungi and yeasts) on the phylloplane of selected plants in the Arrabida Natural Park, an ecosystem of Mediterranean characteristics in Portugal, using conventional culture-dependent isolation methods. Leaves from the species Acer monspessulanum and Quercus faginea (deciduous trees) and Cistus albidus, Pistacia lentiscus, and Osyris quadripartita (evergreen shrubs) were collected twice a year for two consecutive years, at two distinct locations of Serra da Arrabida: the more humid northern slope and the drier southern slope. A total of 1029 strains of filamentous fungi and 540 strains of yeasts were isolated, which represented at least 36 and 46 distinct species, respectively. Total counts were higher on the plants from the northern slope and there was a general increase from spring to autumn, notably on the deciduous trees for the yeasts. Plant species that had higher numbers of leaf colonists (A. monspessulanum, C. albidus, and Q. faginea) also yielded a wider range of species. Among the filamentous fungi there was a predominance of species of ascomycetous affinity, whereas basidiomycetous species dominated among yeast isolates. Some of the taxa recovered were common to other phylloplane studies (e.g., ubiquitous molds and yeasts such as Cladosporium spp. and Cryptococcus spp., respectively), but less common species were also found, some of which appeared to represent undescribed taxa. Interestingly, a few species seemed to be associated with a particular plant, notably in the case of the evergreen shrub C. albidus. However, for a considerable number of fungi and yeasts the same taxon was recovered throughout the year from more than one plant and at both sites, suggesting that such species might be genuine phylloplane inhabitants (or at least of aerial plant surfaces) even though they appeared not to display host specificity. PMID- 12375097 TI - Comparison of eukaryotic phytobenthic community composition in a polluted river by partial 18S rRNA gene cloning and sequencing. AB - We compared the species composition in phytobenthic communities at different sampling sites in a small French river presenting polluted and unpolluted areas. For each sampling point, the total DNA was extracted and used to construct an 18S rRNA gene clone library after PCR amplification of a ca 400 bp fragment. Phytobenthic community composition was estimated by random sequencing of several clones per library. Most of the sequences corresponded to the Bacillariophyceae and Chlorophyceae groups. By combining phylogenetic and correspondence analyses, we showed that our molecular approach is able to estimate and compare the species composition at different sampling sites in order to assess the environmental impact of xenobiotics on phytobenthic communities. Changes in species composition of these communities were found, but no evident decrease in the diversity. We discuss the significance of these changes with regard to the existing level of pollution and their impact on the functionality of the ecosystem. Our findings suggest that it is now possible to use faster molecular methods (DGGE, ARISA.) to test large numbers of samples in the context of ecotoxicological studies, and thus to assess the impact of pollution in an aquatic ecosystem. PMID- 12375098 TI - Characterization of three spiral-shaped purple nonsulfur bacteria isolated from coastal lagoon sediments, saline sulfur springs, and microbial mats: emended description of the genus Roseospira and description of Roseospira marina sp. nov., Roseospira navarrensis sp. nov., and Roseospira thiosulfatophila sp. nov. AB - Three new spirilloid phototrophic purple nonsulfur bacteria were isolated in pure culture from three different environments: strain CE2105 from a brackish lagoon in the Arcachon Bay (Atlantic coast, France), strain SE3104 from a saline sulfur spring in the Pyrenees (Navarra, Spain), and strain AT2115 a microbial mat (Tetiaroa Atoll, Society Islands). Single cells of the three strains were spiral shaped and highly motile. Their intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were present as photosynthetic pigments. Optimal growth occurred under photoheterotrophic conditions and in the presence of 0.5-4% w/v NaCl. These features are similar to those described for Roseospira mediosalina. Comparative sequence analysis of their 16S rRNA genes placed these strains within the alpha-subclass of Proteobacteria, in a cluster together with Roseospira mediosalina and Rhodospira trueperi. They form a closely related group of slightly to moderately halophilic spiral-shaped purple nonsulfur bacteria.However, the three new isolates exhibited some differences in their physiology and genetic characteristics. Consequently, we propose that they are members of three new species within the genus Roseospira, Roseospira marina sp. nov., Roseospira navarrensis sp. nov., and Roseospira thiosulfatophila sp. nov., with strains CE2105, SE3104, and AT2115 as the type strains, respectively. As a consequence, an emended description of the genus Roseospira is also given. PMID- 12375099 TI - Purification and characterization of an epsilon-poly-L-lysine-degrading enzyme from an epsilon-poly-L-lysine-producing strain of Streptomyces albulus. AB - An epsilon-poly-L-lysine-degrading enzyme of an epsilon-poly-L-lysine-producing strain of Streptomyces albulus was purified and characterized. The enzyme was tightly bound to the cell membrane. After solubilization with NaSCN, the enzyme was purified to homogeneity by phenyl-Sepharose CL-4B column chromatography. The subunit molecular mass of the purified enzyme was 54 kDa. Enzyme activity was inhibited by o-phenanthroline, and could be restored in the presence of 1 mM Mg(2+), Ca(2+), Fe(3+) or Zn(2+). The mode of epsilon-poly-L-lysine degradation was of the exo-type, and the enzyme released N-terminal L-lysines one by one. The enzyme acted on various peptides possessing L-lysine residues at the N-terminus and was classified as an aminopeptidase. Epsilon-Poly-L-lysine-degrading activity was found in the membrane fraction of some other Streptomyces strains as well as that of Streptomyces albulus. Streptomyces virginiae IFO 12827 and Streptomyces norsei IFO 15452 exhibited high epsilon-poly-L-lysine-degrading activity, and both strains could produce epsilon-poly-L-lysine, indicating a correlation between the distribution of membrane-bound epsilon-poly-L-lysine-degrading enzyme and epsilon-poly-L-lysine-producing activity. PMID- 12375100 TI - Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes. AB - Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene. PMID- 12375101 TI - Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures. AB - A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets. PMID- 12375102 TI - The veA gene is necessary for the inducible expression by fructosyl amines of the Aspergillus nidulans faoA gene encoding fructosyl amino acid oxidase (amadoriase, EC 1.5.3). AB - The faoA gene encoding fructosyl amino acid oxidase (FAOD, EC 1.5.3) was isolated from Aspergillus nidulans and characterized. The complete nucleotide sequence of the faoA (fructosyl amino acid oxidase) gene and its cDNA revealed that the faoA gene encodes a 441-amino-acid polypeptide interrupted by five introns. Expression of the A. nidulans faoA gene was inducible by fructosyl propylamine and fructosyl lysine, as is the case for the gene encoding FAOD in other organisms. The faoA gene was not induced by these fructosyl amines in a null mutant of the veA gene, which has been identified as an activator of sexual development and as an inhibitor of asexual development; the faoA gene was induced greatly in a veA(+) wild-type. However, veA gene expression was not affected by fructosyl amines. Even in the absence of fructosyl propylamine, synthesis of the faoA transcript was higher in the veA(+) background than in a veA-null mutation background. These results indicated that faoA gene expression is inducible by fructosyl amines and by the veA gene, and that the veA gene is necessary for full induction of faoA gene expression by fructosyl amines. Thus, the faoA gene is the first gene whose expression is dependent on the veA gene. Furthermore, the faoA gene, present in a single copy, seems to be dispensable for development and growth, since the faoA null mutant grew normally and developed as many conidia and sexual structures as the wild-type. PMID- 12375103 TI - The cbs mutant strain of Rhodococcus erythropolis KA2-5-1 expresses high levels of Dsz enzymes in the presence of sulfate. AB - Two mutants of the dibenzothiophene-desulfurizing Rhodococcus erythropolis KA2-5 1, strains MS51 and MS316, which express a high level of desulfurizing activity in the presence of sulfate, were isolated using the transposome technique. The level of dibenzothiophene-desulfurization by cell-free extracts prepared from mutants MS51 and MS316 grown on sulfate was about five-fold higher than that by cell-free extracts of the wild-type. This result was consistent with results of Western-blot analysis using antisera specific for DszA, DszB and DszC, the enzymes involved in the desulfurization of dibenzothiophene. Gene analysis of the mutants revealed that the same gene was disrupted in mutants MS51 and MS316 and that the transposon-inserted gene in these strains was the gene for cystathionine beta-synthase, cbs. The cbs mutants also expressed high levels of Dsz enzymes when methionine was used as the sole source of sulfur. PMID- 12375104 TI - A novel haem compound accumulated in Escherichia coli overexpressing the cydDC operon, encoding an ABC-type transporter required for cytochrome assembly. AB - cydDC genes encode a heterodimeric ABC transporter required for assembly of the membrane-bound cytochrome bd quinol oxidase and periplasmic cytochromes. Here, we demonstrate that overexpression of functional cydDC genes on a multicopy plasmid results in elevated levels of cytochromes b and d, but most notably formation in anaerobically grown cells of a novel haem-containing component P-574. The pigment has a distinctive absorbance at 574-579 nm and 448 nm in reduced minus oxidised spectra and renders over-producing cells reddish in colour. The highest levels of P-574 were observed in mutants (cydAB) in the structural genes for the polypeptides of cytochrome bd. P-574 is labile; its spectral signal is reduced in cells that are frozen-thawed or subjected to mechanical disruption. P-574 was not detected in cytoplasmic or periplasmic fractions and was predominantly associated with the cell membrane. P-574 did not bind CO or cyanide. Production of P-574 was dependent on haem biosynthesis indicating that it is a haem-containing molecule or derived from haem biosynthesis. These findings suggest that P-574 may result from association of a haem compound with overexpressed transporter subunits, but not with oxidase subunits, and are consistent with an intimate link between the transporter and haem processing during oxidase assembly. PMID- 12375105 TI - Impact of the Mg(2+)-citrate transporter CitM on heavy metal toxicity in Bacillus subtilis. AB - Bacillus subtilis possesses a secondary transporter, CitM, that is specific for the complex of citrate and Mg(2+) but is also capable of transporting citrate in complex with the heavy metal ions Zn(2+), Ni(2+) and Co(2+). We report on the impact of CitM activity on the toxicity of Zn(2+), Ni(2+) and Co(2+) in B. subtilis. In a citM deletion mutant or under conditions in which CitM is not expressed, the toxic effects of the metals were reduced by the presence of citrate in the medium. In contrast, the presence of citrate dramatically enhanced toxicity when the Mg(2+)-citrate transporter was present in the membrane. It is demonstrated that the complex of Ni(2+) and citrate is transported into the cell and that the uptake is responsible for the enhanced toxicity. At toxic concentrations of the metal ions, the cultures adapted by developing tolerance against these ions. Tolerant cells isolated by exposure to one of the metal ions remained tolerant after growth in the absence of toxic metal ions and were cross tolerant against the other two toxic ions. Tolerant strains were shown to contain point mutations in the citM gene, which resulted in premature termination of translation. PMID- 12375106 TI - Molecular and biochemical characterization of a novel two-component signal transduction system, amrA- amkA, involved in rifamycin SV production in Amycolatopsis mediterranei U32. AB - Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Although several model systems, including sporulation in Bacillus subtilis and chemotaxis in Escherichia coli, have been extensively studied, the two-component signal transduction systems in industrially important actinomycetes are not well studied. We report the molecular and biochemical characterization of a novel two component signal system, amrA-amkA,from the rifamycin-SV-producing Amycolatopsis mediterranei U32. The deduced sequences of amkAand amrA contain all the structural features that are highly conserved in the typical bacterial histidine kinases and response regulators, respectively. BLAST analyses showed that AmrA and AmkA displayed high similarities to AfsQ1/AfsQ2 of Streptomyces coelicolor and MtrA/MtrB of Mycobacterium tuberculosis. The amrAand amkA genes were over expressed and the gene products were purified from E. coli. Biochemical studies showed that AmkA is able to autophosphorylate, supporting its functional assignment as a histidine kinase. That AmrA functions as the cognate response regulator for histidine kinase AmkA was demonstrated by in vitro phosphotransfer from [gamma-(32)P]ATP-labeled AmkA to AmrA. Rifamycin SV production was also decreased by 10-20% in amrAor amkA gene disruption mutants under the tested condition. Although the detailed regulatory mechanism is still unknown, this is the first report regarding the involvement of two-component signal systems in rifamycin biosynthesis in the genus Amycolatopsis. PMID- 12375107 TI - Isotopic characterization of six major brands of white basic lead carbonate paint pigments. PMID- 12375108 TI - Chelate-enhanced phytoextraction of lead-contaminated soils using coffeeweed (Sesbania exaltata Raf.). PMID- 12375110 TI - Zinc in the surface soil and cassava crop in the vicinity of an alluvial goldmine at Dunkwa-on-Offin, Ghana. PMID- 12375109 TI - Application of mutant strains of cyanobacteria for Cd(2+) removal. PMID- 12375111 TI - Cadmium, copper, and lead in two species of artemisia (compositae) in southern manitoba, Canada. PMID- 12375112 TI - Accumulation of lead, cadmium, zinc, and copper in the edible aquatic plants Trapa bispinosa Roxb. and Nelumbo nucifera Gaertn. PMID- 12375113 TI - Combined effects of lead and humic acid on growth and lead uptake of Duckweed, Lemna minor. PMID- 12375114 TI - Metal levels in a cuvier's beaked whale (Ziphius cavirostris) found stranded on a Mediterranean Coast, Corsica. PMID- 12375115 TI - Pesticide residue analysis of infant formula in India. PMID- 12375116 TI - Pesticide multiresidue analysis in fresh and canned peaches using solid phase extraction and gas chromatography techniques. PMID- 12375117 TI - Analysis of mutagenic heterocyclic amines in cooked food samples by gas chromatography with nitrogen-phosphorus detector. PMID- 12375118 TI - Air pollution concentrations in haryana subregion, India. PMID- 12375120 TI - Distribution and sources of polycyclic aromatic hydrocarbons in the northern persian gulf as indicated by kinetic and thermodynamic criteria. PMID- 12375119 TI - Fate and toxicity of the algicide irgarol 1051: a marine microcosm study. PMID- 12375121 TI - Airborne exposure concentrations during asbestos abatement of ceiling and wall plaster. PMID- 12375122 TI - Incorporation of toxicity tests to the discharges of pulp paper industry in Turkey. PMID- 12375124 TI - Argemone oil augmented oxidative stress in discrete areas of rat brain. PMID- 12375123 TI - Effects of lanthanum and cerium on the vegetable growth of wheat (Triticum aestivum L.) seedlings. PMID- 12375125 TI - Dicofol formulation induced toxicity on testes and accessory reproductive organs in albino rats. PMID- 12375126 TI - Kinetics of distribution of cypermethrin in blood, brain, and spinal cord after a single administration to rabbits. PMID- 12375127 TI - Structure-affected algicidal activity of triorganotin compounds. PMID- 12375128 TI - Effects of PCB 30 and its hydroxylated metabolites on ecdysteroid-mediated gene expression. PMID- 12375129 TI - Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue. AB - Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase-polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we strongly suggest that the final interpretation of the results from this assay be viewed in light of the other features of the case, including clinical history, histology, and immunohistochemistry, by the diagnostic pathologist. Additional studies such as FISH may be useful in clarifying the nature of equivocal or unexpected results. PMID- 12375130 TI - Heterotopic nephrogenic rests in the colon and multiple congenital anomalies: possibly related association. AB - Heterotopic renal tissue (HRT) in the wall of the colon is a very rare occurrence, with only five cases published. Our patient is only the second patient reported to have this abnormality in the absence of sirenomelia. We describe colonic HRT in a child, associated with multiple congenital anomalies. The congenital abnormalities were of the VACTERL type, accompanied by valvular cardiac anomalies that were clinically diagnosed as Shone syndrome. The HRT was not apparent clinically or grossly. Microscopically, multifocal islands of renal tissue consisting of glomeruli, cystically dilated tubules, and blastema were seen in all layers of the bowel, and simulated "cystic partially differentiated nephroblastoma." Our case provides further support to the belief that VACTERL association and sirenomelia represent related entities. PMID- 12375132 TI - Newborn with anophthalmia and features of Fryns syndrome. AB - We report a newborn female with craniofacial malformations, bilateral anophthalmia, large abnormally shaped ears, short neck, small distal phalanges and nails, left diaphragmatic hernia, hypoplastic optic nerves, severe pulmonary hypoplasia, and an accessory spleen, and describe the autopsy findings. The infant expired at 18 h of life. The features were most consistent with Fryns syndrome although other conditions were considered including Matthew Wood syndrome. Anophthalmia, to our knowledge, has not been reported previously in Fryns syndrome; however, eye findings are common, particularly microphthalmia and cloudy cornea. PMID- 12375133 TI - Diversity and structure of AMF communities as affected by tillage in a temperate soil. AB - Arbuscular mycorrhizal fungi (AMF) were studied in differently tilled soils from a long-term field experiment in Switzerland. Diversity and structure of AMF communities were surveyed either directly on spores isolated from the field soil or on spores isolated from trap cultures, planted with different host plants. Single-spore cultures were established from the AMF spores obtained from trap cultures. Identification of the AMF was made by observation of spore morphology and confirmed by sequencing of ITS rDNA. At least 17 recognised AMF species were identified in samples from field and/or trap cultures, belonging to five genera of AMF--Glomus, Gigaspora, Scutellospora, Acaulospora, and Entrophospora. Tillage had a significant influence on the sporulation of some species and non- Glomus AMF tended to be more abundant in the no-tilled soil. The community structure of AMF in the field soil was significantly affected by tillage treatment. However, no significant differences in AMF diversity were detected among different soil tillage treatments. AMF community composition in trap cultures was affected much more by the species of the trap plant than by the original tillage treatment of the field soil. The use of trap cultures for fungal diversity estimation in comparison with direct observation of field samples is discussed. PMID- 12375134 TI - Effect of two vesicular-arbuscular mycorrhizal fungi on the growth of micropropagated potato plantlets and on the extent of disease caused by Rhizoctonia solani. AB - Two micropropagated potato cultivars, Goldrush and LP89221, were inoculated into sowing trays with either Glomus etunicatum or G. intraradices in a greenhouse. After 2 weeks, plantlets were transplanted into pots and roots were challenged 7 days later with Rhizoctonia solani. At different times after R. solani infection, disease severity, mortality rate, root colonization levels, various growth parameters, and shoot mineral content were evaluated. In Goldrush, only inoculation with G. etunicatum led to a significant reduction in disease severity, ranging between 60.2% and 71.2%, on both shoot and crown. This decrease was not observed in LP89221. Compared with the control plantlets, inoculation of Goldrush with G. etunicatum or G. intraradices reduced significantly the mortality rate by 77% and 26%, respectively, whereas vesicular-arbuscular mycorrhizal (VAM) fungi did not significantly influence the mortality rate in LP89221. In Goldrush, inoculation with G. etunicatum significantly increased shoot fresh weight, root dry weight and the number of tubers produced per plant, whereas G. intraradices only significantly increased the number of tubers. Tuber and root fresh weights of both potato cultivars were significantly reduced by R. solani infection. However, R. solani-infected plantlets of both Goldrush and LP89221, inoculated with G. etunicatum, produced significantly greater tuber fresh weight than non-VAM plantlets. In R. solani-infected plantlets of Goldrush but not LP89221, G. etunicatum and G. intraradices increased root fresh weight by approximately 140.3% and 76.5%, respectively, compared with non-VAM plants. The potato cultivars Goldrush and LP89221 responded differently to VAM fungal inoculation and to R. solani infection in terms of shoot mineral content. PMID- 12375135 TI - Are Sebacinaceae common and widespread ectomycorrhizal associates of Eucalyptus species in Australian forests? AB - A molecular survey of basidiomycete ectomycorrhizal fungi colonising root tips at a site in Eucalyptus marginata (jarrah) forest revealed the presence of many fungal species which could not be identified from a database of ITS-PCR-RFLP profiles from morphologically identified species. Three of these unidentified taxa were among the six most frequently encountered profiles. Phylogenetic analyses of ITS and nuclear LSU sequences revealed a close relationship among the three fungi and that they belong to the family Sebacinaceae (sensu Weiss and Oberwinkler 2001). The possibility that DNA of non-ectomycorrhizal rhizosphere or endophytic fungi had been amplified selectively by the basidiomycete-specific primers was tested by amplification with fungal-specific primers. A single PCR fragment was amplified in all but two of the 24 samples tested and digestion with two restriction enzymes produced RFLP profiles which matched those from the Sebacinoid sequence. We conclude, therefore, that at least three species of Sebacinaceae are common ectomycorrhizal associates of E. marginata. PMID- 12375137 TI - The modelled growth of mycorrhizal and non-mycorrhizal plants under constant versus variable soil nutrient concentration. AB - We studied the response of mycorrhizal and non-mycorrhizal plants to variation in soil nutrient concentration. A model for the relative growth rate (RGR) of plant biomass was constructed with soil nutrients as an explanatory variable. A literature survey was carried out to find the relative magnitudes of parameter values for mycorrhizal and non-mycorrhizal plants. Mycorrhizal plants had higher RGR at low nutrient concentrations and non-mycorrhizal plants at high nutrient concentrations. The RGR of mycorrhizal and non-mycorrhizal plants at constant versus log-normally distributed soil nutrient concentration were compared to see the effect of mycorrhizal status on responses to variation. Variation in nutrient concentration generally reduced RGR, especially in mycorrhizal plants. The RGR of a non-mycorrhizal plant may increase with variation where a growth function threshold exists, i.e. a soil nutrient concentration that must be exceeded to allow growth. Mycorrhizal plants appeared more sensitive to variation in nutrient concentration than non-mycorrhizal plants due to the higher affinity of mycorrhizal roots at low nutrient levels. However, this prediction may be reversed if mycorrhizal symbiosis considerably stabilises flow of nutrients to plant physiological processes, such that mycorrhizal plants experience less variation in soil nutrient concentration than non-mycorrhizal plants. Our results also attain broader significance by suggesting a general trade-off between competitive ability in a constant versus variable resource availability. PMID- 12375136 TI - Low-temperature-induced changes in trehalose, mannitol and arabitol associated with enhanced tolerance to freezing in ectomycorrhizal basidiomycetes (Hebeloma spp.). AB - Ectomycorrhizal fungi have been shown to survive sub-zero temperatures in axenic culture and in the field. However, the physiological basis for resistance to freezing is poorly understood. In order to survive freezing, mycelia must synthesise compounds that protect the cells from frost damage, and certain fungal specific soluble carbohydrates have been implicated in this role. Tissue concentrations of arabitol, mannitol and trehalose were measured in axenic cultures of eight Hebeloma strains of arctic and temperate origin grown at 22, 12, 6 and 2 degrees C. In a separate experiment, mycelia were frozen to -5 degrees C after pre-conditioning at either 2 degrees C or 22 degrees C. For some, especially temperate strains, there was a clear increase in specific soluble carbohydrates at lower growth temperatures. Trehalose and mannitol were present in all strains and the highest concentrations (close to 2.5% and 0.5% dry wt.) were recorded only after a cold period. Arabitol was found in four strains only when grown at low temperature. Cold pre-conditioning enhanced recovery of mycelia following freezing. In four out of eight strains, this was paralleled by increases in mannitol and trehalose concentration at low temperature that presumably contribute towards cryoprotection. The results are discussed in an ecological context with regard to mycelial overwintering in soil. PMID- 12375138 TI - Pisolithus tinctorius promotes germination and forms mycorrhizal structures in Scots pine somatic embryos in vitro. AB - The results of the present study show that inoculation with the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch potentially enhances the germination of Scots pine (Pinus sylvestris L.) somatic embryos in vitro. Stimulation by Pisolithus tinctorius was only observed in the absence of direct contact between the symbionts; mature embryos were not sufficiently robust for balanced interaction with the fungus on half-strength DCR medium. Subsequently, on MMN medium with a reduced sugar concentration, direct contact between somatic embryo-derived plants and the fungus resulted in in vitro formation of mycorrhiza. Ex vitro inoculation also improved adaptation of the somatic embryo derived plants, even though mycorrhizal structures were not observed. The reactions to Pisolithus tinctorius varied between different Scots pine cell lines both in vitro and ex vitro. PMID- 12375139 TI - Glomales rRNA gene diversity--all that glistens is not necessarily glomalean? PMID- 12375140 TI - Barrett's esophagus. PMID- 12375141 TI - Numerical chromosomal abnormality in gastric MALT lymphoma and diffuse large B cell lymphoma. AB - BACKGROUND: We investigated numerical chromosomal abnormalities, using the fluorescence in situ hybridization (FISH) method, in gastric mucosa-associated lymphoid tissue (MALT) lymphoma and diffuse large B-cell lymphoma (DLBL). We also compared the histopathological findings, including the presence or absence of Helicobacter pylori infection, with the analytical results. METHODS: Sixteen patients who underwent operation for malignant gastric lymphoma in our department were divided into three groups: patients with low-grade gastric MALT lymphoma (l MALT; n = 5), those with high-grade gastric MALT lymphoma (h-MALT; n = 8), and those with DLBL (n = 3). Numerical abnormalities of chromosomes 8, 9, 12, and 17 were investigated by the FISH method, and the presence or absence of H. pylori infection was microscopically examined. RESULTS: Numerical abnormality was observed in chromosome 12 in 11 patients (68.8%), in chromosome 8 in 10 (62.5%), and in chromosome 17 in 5 (31.3%), showing a high frequency. H. pylori infection was detected in 80% and 50% of patients with l-MALT and h-MALT, respectively, but no H. pylori infection was observed in patients with DLBL. CONCLUSIONS: A new biological characteristic of gastric MALT lymphoma was obtained, i.e., a high frequency of numerical abnormalities of chromosomes 12, 8, and 17. There was no correlation between the numerical chromosomal abnormalities and the clinicopathological findings. PMID- 12375142 TI - Ambulatory intraesophageal bilirubin monitoring in Japanese patients with gastroesophageal reflux. AB - BACKGROUND: The role of reflux of duodenal contents in gastroesophageal reflux in Japanese patients, which may be different from that in Western patients, was studied. METHODS: Intraesophageal pH and the bilirubin concentration were monitored, using the Bilitec 2000, in 43 patients with reflux symptoms and 10 normal volunteers. The percentage of the time that spectrophotometric absorbance was 0.15 or more and pH was less than 4.0 was defined as the holding times (HTs) of bilirubin and acid, respectively. Severity of esophagitis was classified using the Savary-Miller (S-M) classification. RESULTS: Esophagitis was present in 37 patients; 5, 10, 13, and 9 patients had S-M grades 1, 2, 3, and 4, respectively. Both HTs in the volunteers were less than 5%. Bilirubin HT was more than 5% in 3 of the 6 patients without esophagitis, but the acid HT was less than 5% in these 6 patients. Acid HT was less than 5% in 4, 2, 2, and 2 patients with S-M grades 1, 2, 3, and 4, respectively. Bilirubin HT was less than 5% in 1 patient with S-M grade 2 esophagitis. Bilirubin HT in patients with S-M grades 3 and 4 esophagitis (50.9 +/- 5.8%) was higher than that in grades 1 and 2 (14.9 +/- 2.9%) (P < 0.0001), but this was not so for acid HT. In 32 patients, bilirubin HT exceeded acid HT. Bilirubin HT did not correlate with acid HT. CONCLUSIONS: Duodenogastroesophageal reflux occurred independently of and exceeded acid reflux. The amount of duodenogastroesophageal reflux correlated with the severity of esophagitis. PMID- 12375143 TI - Comparative evaluation of urine-based and other minimally invasive methods for the diagnosis of Helicobacter pylori infection. AB - BACKGROUND: Diagnostic methods have recently been developed for detecting anti Helicobacter pylori antibody in urine and H. pylori antigen in stool samples. Our aim was to evaluate the usefulness of noninvasive urine-based methods for the diagnosis of H. pylori infection. METHODS: The study subjects were 100 asymptomatic Japanese volunteers. We investigated the diagnostic efficacy of various noninvasive diagnostic methods; five serological tests (Immunis anti pylori, HM-CAP, EIAgen Helicobacter pylori IgG, Helico G, and GAP-IgG), one test for antigen in stool (HpSA enzyme immunoassay [EIA]), and two tests for antibody in urine (Urinelisa and Rapirun) by using the urea breath test (UBT) as the gold standard. RESULTS: Fifty subjects were diagnosed as positive for H. pylori infection by the UBT. The serological tests showed good sensitivity, specificity, and accuracy. The diagnostic values of the feces-based test (HpSA EIA) were lower than that of the serological tests. The sensitivities of the two urine-based methods in frozen urine samples were markedly lower than those of the other tests. However, the use of unfrozen samples markedly improved the diagnostic accuracy of these urine-based tests, which was then superior to that of the feces based method. CONCLUSIONS: This study clearly showed that urine-based tests were useful for the diagnosis of H. pylori infection. However, the use of frozen urine samples was not appropriate for the detection of anti-H. pyloriantibody. PMID- 12375144 TI - Absence of specific symptoms in chronic hepatitis C. AB - BACKGROUND: No systematic research has been carried out on the symptoms of chronic hepatitis C, although the disease is believed to induce subjective symptoms such as fatigue or dullness in the legs. METHODS: The Todai Health Index has been developed as a symptom checklist that is used for screening particular diseases or for health management. The index was chosen as the most suitable questionnaire for measuring characteristic symptoms of chronic hepatitis C. Sixty patients with chronic hepatitis C who did not have any severe complications were compared with healthy control subjects who were selected randomly from the residents of Isesaki City, Gunma, Japan. RESULTS: The major findings were as follows: (1) male and female patients with chronic hepatitis C had no characteristic subjective physical symptoms when compared with the healthy controls, except for a significant difference in aggression, and (2) the severity of the hepatitis was not associated with the patients' symptoms after adjusting for age and treatments. CONCLUSIONS: Patients with chronic hepatitis C who did not have any severe complications did not show specific subjective physical symptoms. PMID- 12375145 TI - Long-term cold liver storage induces endothelin-1 release and a time-dependent increase in portal pressure at reperfusion in the rat. AB - BACKGROUND: Long-term cold storage and reperfusion lead to great liver injury involving major microcirculatory disturbance. This study investigated the effect of storage duration on endothelin-1 production, portal pressure, and microcirculation. METHODS: Rat livers were perfused before and after 15 min to 48 h of cold storage in University of Wisconsin (UW) solution. RESULTS: Portal pressure was unchanged for up to 12 h of storage, and increased by 40% and 70% after 24 and 48 h, respectively. Vascular space was increased by a factor of 1.7 following 48 h of storage. Endothelin-1 was released by livers stored in UW solution for 48 h (3.99 +/- 1.85 pg/100 microl). The nonselective endothelin-1 receptor antagonist TAK-044 partially prevented the increase in portal pressure and prevented the increase in vascular space. CONCLUSIONS: Cold storage induces a time-dependent elevated portal pressure at reperfusion. The involvement of endothelin-1 in this process offers opportunities to improve liver graft quality by using endothelin-1 inhibitors. PMID- 12375146 TI - Bacterial translocation and peptidoglycan translocation by acute ethanol administration. AB - BACKGROUND: We examined bacterial translocation (BT) by acute ethanol administration, using a peptidoglycan detecting system. METHODS: Rats were given 20% (v/w) ethanol (10 ml/kg body weight), and heparinized samples of portal blood were collected at specific time points after administration. Plasma peptidoglycan, beta-glucan, and endotoxin concentrations of portal blood were measured. The rats were divided into three groups: a 20% ethanol group (20ET group), a 5% ethanol group (5ET group), and a control group. The groups were given 10 ml/kg body weight of either 20% (v/w) ethanol (20ET group), 5% (v/w) ethanol (5ET group), or distilled water (control group). Femoral arterial blood, portal blood, mesenteric lymph nodes (MLNs), spleen, and liver were cultured to assess BT. Plasma peptidoglycan, beta-glucan, and endotoxin concentrations of femoral arterial blood and portal blood were measured. RESULTS: The plasma peptidoglycan concentration of portal blood was significantly increased 24 h after the administration of 20% ethanol. There was no significant difference in the incidence and magnitude of viable BT to the MLNs, spleen, and liver among any of the groups at this time point. The rate of plasma peptidoglycan positivity and the plasma peptidoglycan concentration were increased significantly in the portal blood of the 20ET group 24 h after administration. CONCLUSIONS: Peptidoglycan was translocated into the portal blood by acute administration of 20% ethanol. Our findings suggest that viable bacterial flora may translocate from the intestine under the influence of ethanol, and BT may be one of the causes of chronic alcoholic liver disease. PMID- 12375147 TI - High rate of sustained response to consensus interferon plus ribavirin in chronic hepatitis C patients resistant to alpha-interferon and ribavirin: a pilot study. AB - BACKGROUND: The aim of this study was to evaluate an alternative treatment (consensus interferon plus ribavirin) for chronic hepatitis C patients resistant to combined therapy. METHODS: Fourteen patients previously resistant to interferon alpha plus ribavirin were consecutively assigned to receive 15 microg of consensus interferon plus ribavirin (1000 mg) daily for 4 weeks, and 9-15 microg every other day plus daily ribavirin for the following 44 weeks. Alanine aminotransferase and hepatitis C virus (HCV) RNA (Amplicor Monitor; Roche) levels were monitored during therapy and for 24 weeks after its completion. RESULTS: A rapid and marked decrease of HCV RNA viremia of more than 2 logs was observed in 10 (71%) of 14 patients at week 2 of treatment. At the end of therapy, 10 (71%) of 14 patients had undetectable HCV RNA. The end-of-treatment response rates were 6 of 9 (67%) patients for genotype 1 and 4 of 5 (80%) for other genotypes. Sustained response was observed in 4 (36%) of 11 patients who completed 24 weeks of follow-up. CONCLUSIONS: A marked and rapid decrease of viral load was observed during therapy with high doses of consensus interferon plus ribavirin in patients previously resistant to combined therapy, even in those infected with genotype 1. Of 11 patients who completed the post-treatment follow-up, 36% presented a sustained response. PMID- 12375148 TI - Extensive hemorrhagic erosive gastritis associated with acute pancreatitis successfully treated with a somatostatin analog. AB - In massive hemorrhage from acute gastric mucosal lesions, it is occasionally difficult to control the bleeding with nonsurgical therapy. We used the somatostatin analog, octreotide, which suppresses gastric and pancreatic function, to treat severe hemorrhagic erosive gastritis in a patient with acute pancreatitis. A 22-year-old man presented with epigastralgia and melena. Blood levels of pancreatitis markers were elevated. Computed tomography revealed diffuse enlargement of the pancreas, without fluid collection around the organ. An endoscopic examination showed extensive hemorrhagic erosions over almost the whole gastric mucosa. We diagnosed extensive hemorrhagic erosive gastritis with acute pancreatitis. A protease inhibitor (nafamostat mesilate 50 mg/day) and an H(2) receptor antagonist (famotidine 40 mg/day) were administered by injection for 6 days; the patient's serum and urine amylase levels fell, but the gastric erosions with hemorrhage were not attenuated. Octreotide was given subcutaneously, at a daily dose of 100 microg for 5 days, without famotidine administration. His melena disappeared, and the gastric erosions were markedly decreased. Administration of the somatostatin analog, octreotide, proved to be effective treatment in a patient with severe hemorrhagic erosive gastritis associated with acute pancreatitis. PMID- 12375149 TI - Unusual type of left paraduodenal hernia caused by a separated peritoneal membrane. AB - Internal hernias are an uncommon cause of intestinal obstruction, with left paraduodenal hernia being the most frequent of these. Computed tomography (CT) with contrast media is advantageous for the clinical workup of suspected paraduodenal hernias. Here, we report a case of an unusual paraduodenal sac formed by a peritoneal membrane between the transverse and the descending colon, which entrapped the proximal jejunum, causing intestinal obstruction. Preoperative CT demonstrated a cluster of jejunal loops between the stomach and pancreas, and showed that the inferior mesenteric vein was laterally displaced. PMID- 12375150 TI - A child with simple ulcer of the colon effectively treated with the combination of prednisolone, azathioprine, and pentoxifylline. AB - A rare case of simple ulcer of the colon in a 7-year-old girl is reported. Simple ulcer is clinically and pathologically recognized as a serious disease linked to intestinal Behcet's disease. Recently, some immunomodulators, such as thalidomide and antitumor necrosis factor monoclonal antibody, have been used to treat Behcet's disease, with varying degrees of success. Pentoxifylline (PTX) is also known to inhibit such inflammatory mediators as tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6. In this present patient combination treatment with prednisolone, azathioprine, and PTX improved corticosteroid dependence palliatively and prevented further relapse during a follow-up period of more than 12 months, without serious side effects. PMID- 12375151 TI - Four immunohistochemically different primary liver cancers in one patient. AB - We present a rare case of four immunohistochemically different primary liver cancers developing in a 54-year-old Japanese man with chronic hepatitis C. In 1989, a liver tumor had been detected at another hospital during follow-up of hepatitis C virus (HCV) infection. He was first admitted to our hospital in July 1991, when a well defined hypervascular tumor, measuring 2.5 cm in diameter was found in the S5 subsegment of the liver on computed tomography (CT); S5 subsegmentectomy was therefore performed, in July 1991. Histopathological examination revealed scirrhous hepatocellular carcinoma (SHCC). Immunohistochemical analysis showed that the tumor was negative for mouse monoclonal anti-human hepatocyte antibody (Hep), but was partially positive for a mouse monoclonal antibody specific for cytokeratin 19 (CK19). Six years after the operation, a large tumor, measuring 10 cm in diameter, was found in the S4 subsegment and a 3-cm tumor was found in the caudate lobe on CT scans. Extended left hepatic lobectomy and partial resection of the caudate lobe were performed in August 1997. Histopathological examination revealed a moderately differentiated hepatocellular carcinoma (HCC) with a trabecular pattern, an SHCC with well differentiated HCC at its periphery, and a small incidental cholangiocellular carcinoma (CCC), measuring 1 cm in diameter. The HCC and CCC showed typical immunostaining for Hep and CK19, respectively. The SHCC was positive for both Hep and CK19, showing characteristics different from those of the previously resected SHCC on immunohistochemical analysis. In conclusion, we experienced four immunohistochemically different primary liver cancers in a patient with chronic hepatitis C. PMID- 12375152 TI - Simple liver cyst with spontaneous regression. AB - We report a-55-year-old woman with spontaneous regression of simple liver cyst. The size of the cyst gradually became reduced without any treatment, and a reduction in diameter from 77 mm to 10 mm was observed after 8 years of follow up. Spontaneous regression of congenital cysts of the liver in an adult seems to be very rare, and its mechanism is discussed. PMID- 12375153 TI - Successful elimination of Ascaris lumbricoides from the gallbladder by conservative medical therapy. AB - Migration of Ascaris lumbricoides into the gallbladder is rare, unlike ascariasis of the bile duct, and, when it does occur, treatment is generally by endoscopic or surgical extraction. We describe a case of the successful treatment of gallbladder ascariasis with conservative therapy. A 44-year-old Korean man was admitted because of nausea and right upper quadrant pain that did not respond to medical control and had worsened 1 day before admission. Abdominal ultrasonography showed a long, linear, moving echogenic structure in the distended lumen of the gallbladder, but no abnormal dilation of the bile duct. Computerized tomography showed a linear soft-tissue density in the dependent portion of the gallbladder. The patient presented with eosinophilia, and abnormal liver function results, but no fever or hepatomegaly. Based on these findings, and presuming a diagnosis of gallbladder ascariasis, we administered antiparasitic medication (albendazole 400 mg/day for 1 day). Seven days later, we obtained one adult female A. lumbricoides from the feces. The symptoms were fully resolved, and no moving structure could be visualized in the gallbladder by ultrasonography. We recommend that initial therapy for gallbladder ascariasis should involve conservative treatment, unless an associated disease is present or a complication arises. PMID- 12375154 TI - A rare association between ulcerative colitis (UC), celiac disease (CD), membranous glomerulonephritis, leg venous thrombosis, and heterozygosity for factor V Leiden. PMID- 12375155 TI - The role of endothelin-1 in hepatic ischemia and reperfusion injury. PMID- 12375156 TI - Aspects of the future of therapy for patients infected with a high viral load and interferon-resistant subtype of HCV. PMID- 12375157 TI - Laparoscopic treatment of ovarian cyst in the newborn. AB - BACKGROUND: Nine cases of persistent or complicated cyst were analyzed in an attempt to answer two questions: whether laparoscopic treatment of ovarian cyst in the newborn is justified and whether a pneumoperitoneum can be used in such infants. METHODS: Laparoscopic treatment was used for nine newborn babies. The children were 4 days to 2.5 months old. Cyst size ranged from 36 to 72 mm in length. RESULTS: The treatment was exclusively laparoscopic in six cases, and the other three cases required conversion. A maximal insufflation of 4 mmHg was used in five cases. Adnexectomy was necessary in three cases. The duration of the laparoscopic procedure was 20 to 75 min, and that of the postoperative stay was 1 to 5 days. There were no intraoperative or postoperative complications. CONCLUSIONS: Laparoscopy in the newborn is justified, but only in the hands of well-trained teams. The use of a pneumoperitoneum is possible, but should be reserved for difficulties with abdominal wall suspension. PMID- 12375158 TI - Changes of sialomolecules during the dimethylsulfoxide-induced differentiation of Herpetomonas samuelpessoai. AB - The expression of sialoglycoconjugates during the dimethylsulfoxide (DMSO) induced differentiation of Herpetomonas samuelpessoai was analyzed by flow cytometry and Western blotting using sialic acid-specific lectins. Parasites reacted strongly with Limax flavus (LFA) and Sambucus nigra (SNA) agglutinins, and only weakly with Maackia amurensis (MAA) lectin. However, analysis of crude protein extracts by Western blotting revealed that bands with molecular masses corresponding to 15 and 40 kDa are recognized by MAA, and that treatment with DMSO induced the expression of two additional polypeptides with molecular masses of 65 and 90 kDa. Profiles of binding to LFA were indistinguishable when protein extracts from control or differentiated cells were analyzed. SNA recognized a major molecule with 25 kDa in extracts from non-differentiated forms and two low molecular-weight bands from differentiated cells. These results indicate that molecules containing alpha2,6 and alpha2,3 sialyl-galactosyl sequences are present in H. samuelpessoai, and that their biosynthesis and expression are influenced by DMSO-induced differentiation. PMID- 12375159 TI - Acquired cell-mediated protection in rainbow trout, Oncorhynchus mykiss, against the haemoflagellate, Cryptobia salmositica. AB - Disease-free rainbow trout, Oncorhynchus mykiss (Walbaum),were inoculated with either the pathogenic Cryptobia salmositica Katz, 1951 or with the attenuated vaccine strain of the parasite. A number of vaccinated fish were then challenged at 6 weeks post-vaccination with pathogenic C. salmositica. Respiratory burst activity in stimulated macrophages (isolated from infected or vaccinated fish) was demonstrated by detection of released super-oxide anions, and the protein contents in these cells were also determined after the cells were digested. At 5 weeks after infection, the respiratory burst activity of macrophages from infected fish was significantly higher than those in naive and vaccinated fish. In addition, macrophages from vaccinated fish had greater activity than naive fish. Macrophage activity in vaccinated and challenged fish was comparable to that of infected fish. Antibodies were detected (ELISA) at 3 weeks after vaccination and at 5 weeks in infected fish. Complement-fixing antibodies (immune lysis test) were detected in infected fish at 5 weeks post-infection but were not detected in vaccinated fish unless they were challenged with the pathogen, in which case the titre rose rapidly. PMID- 12375160 TI - A simple and reproducible method to obtain large numbers of axenic amastigotes of different Leishmania species. AB - This work describes a simple method to yield large amounts of Leishmania amastigote-like forms in axenic cultures using promastigotes as the starting population. The method described induced extracellular amastigote transformation of Leishmania amazonensis (97%), Leishmania braziliensis (98%) and Leishmania chagasi (90%). The rounded parasites obtained in axenic cultures were morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as measured by their ability to revert back to promastigotes and to infect BALB/c mice. L. amazonensis and L. braziliensis promastigotes and axenic amastigotes differed in terms of their Western blot profiles. A 46 kDa protein was recognized by specific antibodies only in axenic and lesion-derived L. amazonensis amastigotes and not in promastigotes. PMID- 12375161 TI - Quinonic derivatives active against Toxoplasma gondii. AB - Quinonic derivatives were tested against a virulent RH strain of Toxoplasma gondii maintained in cell culture in THP-1, a human myelomonocytic cell line. The derivatives were tested at various doses (0.5-4 microg/ml) and compared with the reference molecules clindamycine, sulfadiazine, pyrimethamine and atovaquone. The percentage of parasite growth inhibition was observed after 72 h of incubation. The tested derivatives are bicyclic, tricyclic or tetracyclic quinones. Eight of these compounds exhibit over 70% inhibition of parasite growth; and two were nearly equipotent to pyrimethamine. These data indicate that the most active compounds against the RH strain of T. gondii are bis-heterocyclic quinones. PMID- 12375162 TI - Interaction of different Leishmania mexicana morphotypes with plasminogen. AB - The interaction of plasminogen with Leishmania mexicana promastigotes was found, using immunoperoxidase assays, to occur with a specific morphotype. In in vitro cultured promastigotes, the morphotype that possessed the plasminogen binding capacity had round to ovoid cell bodies. In contrast, neither slender nor metacyclic promastigotes showed this property. In vivo plasminogen immunofluorescence assays showed deposits of plasminogen exclusively on the cell surface of promastigotes. This was observed as intense patches spread around the cell, with higher intensities towards the flagellar pocket. Plasminogen binding capacity detected by plasmin activity increased with the age of the promastigote culture, at pH 7.2 and pH 5.5, as well as after heat shock. The total amastigote population, freshly isolated from a hamster lesion, also bound plasminogen in a lysine-dependent manner as detected by immunoperoxidase studies. PMID- 12375163 TI - The helminth community of Talpa romana (Thomas, 1902) (Insectivora, Talpidae) in southern Italy. AB - The helminth parasite community of Talpa romana in Calabria (southern Italy ) was studied. The helminth fauna comprised six species: Ityogonimus ocreatus (Goeze 1782), Staphylocistis bacillaris (Goeze 1782), Capillaria talpae (Siebold 1850), Parastrongyloides winchesi (Morgan 1928), Spirura talpae (Gmelin 1790), and Tricholinstowia linstowi (Travassos 1918). All species except S. bacillaris were dominant in this community. The helminths are all stenoxenous species of Paleartic Talpaspp. This paper is the first quantitative approach to the helminth community of T. romana and reveals typical characteristics of an isolationist community. This can be explained by genetic and paleogeographic events. PMID- 12375164 TI - Characterization of the immune response induced by a carbohydrate enriched fraction from Echinococcus granulosus protoscoleces in patients with cystic hydatid disease. AB - The serological and cellular immune response against a carbohydrate-enriched fraction of Echinococcus granulosus (E4+) was studied in a group of patients with cystic hydatid disease (CHD). The profile of IgG subclass against E4+ and the native non-enriched extract (protoscolex somatic antigen, PSA) were compared. A relatively higher ratio of IgG2:IgG4 was induced by E4+ than by PSA (12.4 vs 3.6 respectively). Serological data were associated with clinical parameters dealing with the outcome of disease. The expression of IgG1 and IgG4 subclasses against both of the antigens was associated with the progression of the disease. Interestingly, anti-PSA IgG4 was the only variable that was specifically associated with the stage of cysts (P < 0.04). Similar levels of IL-13 were evoked by peripheral blood mononuclear cells from patients with CHD when incubated with either of the antigens, while higher levels of IL-10 were obtained in supernatants from cells stimulated with E4+ (P < 0.029) than from those stimulated by PSA. Considerable amounts of IL-10 (median 60 pg/ml) were obtained in the supernatants of cells from healthy individuals incubated with E4+. Our results suggest a putative role for E4+ in immunoregulation during the course of infection with E. granulosus in humans. PMID- 12375165 TI - Characterization of an ecto-phosphatase activity in the human parasite Trichomonas vaginalis. AB - We have characterized phosphatase activity present on the external surface of Trichomonas vaginalis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at a rate of 134.3+/-14.8 nmol Pi/h per 10(7) cells. This phosphatase activity decreased by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained for at least 1 h. Experiments using classical inhibitors of acid phosphatases, such as ammonium molybdate and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate, [monoperoxo(picolinato)oxovanadate(V)] (mpV-PIC) and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), showed a decrease in this phosphatase activity, with different patterns of inhibition. Cytochemical analysis showed the localization of this enzyme on the parasite surface (cell body and flagellum) and in intracellular vacuoles. Phosphatase reaction products were also observed in exocytosed membrane-bound material. PMID- 12375166 TI - Temporal variability of Cryptosporidium in the Chesapeake Bay. AB - Although Cryptosporidium has been found worldwide in molluscan shellfish from waters contaminated with human and animal feces, little or no related environmental data have been obtained. In the present study, oysters ( Crassostrea virginica) were collected eight times over 3 years from seven sites in the Chesapeake Bay or its tributaries, with accompanying data on water temperature, salinity, rainfall, and streamflow. Oyster gill washings were examined by immunofluorescence microscopy for Cryptosporidium oocysts. Of 1,590 oysters collected, 19.6% had detectable oocysts. Of 53 collections, oocysts were detected 81% of the time. The time when the greatest percentage of oysters at most sites had detectable oocysts coincided with the time of greatest weekly and monthly rainfall, greatest streamflow into the Bay, and lowest water temperatures. In 28% of 53 collections, C. parvum genotypes 1 and 2 and C. baileyi were identified by PCR and gene sequencing. Oocyst infectivity was confirmed from 37.5% of 40 collections by initiating C. parvum genotype 2 infections in mice. PMID- 12375167 TI - Lectin-blot analyses of Trichinella spiralis muscle larvae excretory-secretory components. AB - The rapid recognition of invading pathogen polysaccharides by host lectins might have a significant role in the outcome of the infection. Oligosaccharide structures of the pathogens may provoke an antibody response and serve as a target for specific antibodies. It is well known that Trichinella spiralis antigens, either on the surface or excreted-secreted, are key modulators or targets of the host immune system. In our study of the role of lectins in host defense against T. spiralis infection, an investigation on sugar component of parasite glycoproteins was performed. Affino-blot analyses of T. spiralis muscle larvae excretory-secretory (ES) products by plant lectins revealed that these proteins possess: (1) N-glycans (ConA, PSA, PHA), and probably some O-linked structures (AAA), (2) oligosaccharide structures with mannose residues, especially of the oligomannose type (ConA) and the biantennary complex type with Fuc in the pentasaccharide core (PSA), (3) bisected oligosaccharides, probably some polyantennary glycophorms (PHA), (4) terminally positioned Gal (RCA I, AAA), (5) N-glycans containing oligomers of, or bisected GlcNAc (WGA), that lack alpha2,6 type of linkage (absence of SNA binding). PMID- 12375168 TI - Helminth fauna of the red squirrel (Sciurus vulgaris Linnaeus, 1758) in Belorussian Polesie. PMID- 12375169 TI - Helminth fauna of the striped field mouse (Apodemus agrarius Pallas, 1778) in ecosystems of Belorussian Polesie transformed as a result of reclamation. PMID- 12375170 TI - A study on the helminth fauna of the bats (Mammalia, Chiroptera: Vespertilionidae) in Belarus. PMID- 12375171 TI - The rat glomerular filtration barrier does not show negative charge selectivity. AB - OBJECTIVE: To characterize the effects of size, shape, and negative charge on the transport of macromolecules across the glomerular capillary wall by using the sieving curves (fractional clearance vs. solute molecular radii) of fluorescent polydispersed polysaccharide tracers. METHODS: Glomerular fractional clearances (FC) were measured with fluorescent neutral [isoelectric point (pI) = 7.3 +/- 0.2] and negatively charged (pI = 3.5 +/- 0.4) dextrans (DEX) in comparison with negatively charged (pI = 4.8 +/- 0.3) hydroxy ethyl starch (HES) and (pI = 4.6 +/ 0.1) bovine serum albumin (BSA) in Sprague-Dawley and Fischer 344/Brown Norway rats. FCs (n = 53) were measured by using the urinary clearance of (14)C-inulin to determine the glomerular filtration rate. The relative uptake of each fluorescent probe by endothelial and renal proximal tubule epithelial (LLC-PK(1)) cells, in vitro, was measured microscopically by using a cooled (-25 degrees C) CCD camera. RESULTS: The sieving curves for randomly coiled neutral and negatively charged DEX probes were identical. These FC values were 6-fold greater than those for HES and 200-fold above similarly sized fluorescent BSA. The polysaccharide probes did not show significant binding to serum proteins. The uptake of BSA by LLC-PK(1) cells was 20- to 100-fold greater than that for neutral or negatively charged macromolecules. CONCLUSIONS: These findings indicate that the rat glomerular filtration barrier restricts the transport of polysaccharide macromolecules as a function of their size and configuration but not negative charge. PMID- 12375172 TI - Impaired collateral artery development in spontaneously hypertensive rats. AB - OBJECTIVE: To determine whether collateral artery development is impaired in spontaneously hypertensive (SHR) relative to normotensive (WKY) rats. METHODS: Sequential mesenteric arteries were ligated to create a collateral pathway responsible for the perfusion of approximately 50 first-order arterioles. Collateral development was assessed by measurement of in vivo arterial diameter before and 1 week after ligation. Histological and morphometric measurements were made from cross-sectional preparations of these arteries to evaluate intimal and medial cell numbers and medial area. eNOS expression was evaluated with Western blotting. RESULTS: One week after arterial ligation, collateral diameter was increased more in WKY than SHR both absolutely (137 +/- 9.1 versus 99 +/- 8.6 microm) and relative to same-animal controls (38 +/- 5.5% versus 13 +/- 7.1%). At the time of model creation, blood flow was elevated to comparable levels in both WKY and SHR, and wall shear rate in the SHR collateral was greater than both the SHR control and WKY collateral arteries. Endothelial cell number in arterial cross-section was increased in collaterals by 80% in WKY and only 22% in the SHR. eNOS expression was increased in the WKY (128%) but not in the SHR collateral. CONCLUSIONS: For equivalent arterial occlusion, the data demonstrate that collateral development is suppressed in the SHR as indicated by luminal expansion. This impairment of luminal expansion is associated with a decreased endothelial proliferation and the lack of an increase in eNOS expression. PMID- 12375173 TI - Arteriolar occlusion causes independent cellular responses in endothelium and smooth muscle. AB - OBJECTIVES: To test the hypothesis that arteriolar occlusion causes different cellular changes in endothelial and smooth muscle cells. METHODS: Cheek pouch arterioles (resting diameter 41 +/- 2 microm) of anesthetized hamsters were occluded briefly (<60 seconds) either upstream or downstream from an observation site. Changes in membrane potential and intracellular calcium concentration ([Ca(2+)](i)) of the endothelial or smooth muscle cells were determined by using fluorescence microscopy (ratiometric analysis). RESULTS: The pressure in the occluded segment decreased by 17.4 +/- 2.6 cm H(2)O during upstream occlusion and increased by 16.8 +/- 6 cm H(2)O during downstream occlusion (n = 5). Upstream occlusion caused vasoconstriction of the occluded segment by 2.4 +/- 0.4 microm, whereas downstream occlusion produced brief vasodilatation by 1.1 +/- 0.2 microm. The endothelial cells hyperpolarized during upstream or downstream occlusion (ratio change: 2.26 +/- 0.24% and 2.39 +/- 0.42%, respectively; p < 0.01, n = 5). There were no changes in endothelial [Ca(2+)](i). The smooth muscle cells depolarized (ratio change: -2.08 +/- 0.14%, n = 5) with an increase in [Ca(2+)](i) (ratio change: 2.92 +/- 0.16%, n = 6) during downstream occlusion. However, there was no detectable change in membrane potential or [Ca(2+)](i) of smooth muscle cells during upstream occlusion. All the changes rapidly recovered when occlusion was released. Responses of an in-situ isolated segment on a side branch revealed conducted dilatory signals caused by the occlusions. CONCLUSIONS: Our results show that the endothelial and smooth muscle cells respond independently to arteriolar occlusion. The endothelial and smooth muscle cells do not effectively communicate in [Ca(2+)](i) or membrane potential during acute arteriolar occlusion. Hyperpolarizing signals in endothelium cause conducted dilation. PMID- 12375174 TI - Persistence of impaired hepatic microcirculation after nonarterialized liver transplantation in the rat. AB - OBJECTIVES: The nonarterialized orthotopic rat liver transplant (NOLT) is a frequently used model in transplantation research that was recently used to investigate microcirculatory alterations during acute rejection, which occurs within 7 days. The present study sought to establish whether NOLT represents a reasonable model for the study of the hepatic microcirculation beyond the immediate reperfusion phase. METHODS: Three groups of animals were studied: sham operated control (n = 8), NOLT (n = 7) and arterialized orthotopic liver transplant (AOLT; n = 8). The hepatic microcirculation was investigated by intravital fluorescence microscopy and laser Doppler flowmetry (LDF) on day 7 postoperatively. Liver histology and plasma levels of liver enzymes were also assessed. RESULTS: Plasma levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin were significantly higher in NOLT than in AOLT and control animals. The low LDF signal recorded from the surface of the NOLT liver (92 +/- 25 vs. 210 +/- 25 and 172 +/- 14 PU in AOLT and control liver, respectively; p < 0.05) was associated with heterogeneous perfusion at both the lobular and sinusoidal levels (density of perfused sinusoids (n/40,000 microm(2)): 5.8 +/- 0.8, 8.1 +/- 0.3, 8.0 +/- 0.3, respectively; p < 0.05). The percentage of hyperfluorescent Ho342-stained hepatocytes (apoptotic) ranged between 2 and 5% and was not significantly different between groups. The lack of post-transplant arterial supply was associated with an increased hepatic cord width-to-sinusoid diameter ratio (3.77 +/- 0.3, 2.02 +/- 0.04, and 1.72 +/- 0.06 in NOLT, AOLT, and control animals, respectively; p < 0.001 vs. control and AOLT) and increased temporary leukocyte adherence to the walls of the terminal hepatic venules. Intense vitamin A autofluorescence around shunt- and large-diameter, slow-velocity vessels was occasionally encountered in the NOLT liver, which coincided with mild fibrosis and bile ductular proliferation. In the well perfused areas, both AOLT and NOLT were associated with a significant rise in sinusoidal RBC(vel), which was more marked in the NOLT group. CONCLUSIONS: Our data indicate that NOLT represents an inappropriate model for the long-term study of the hepatic microcirculation. Lack of a post-transplant arterial supply may lead to persistent microvascular perfusion failure, hepatocellular/endothelial cell swelling, and microvascular anomalies related to bile duct injury. Recovery from microcirculatory alterations induced by cold preservation/reperfusion injury appears to depend on an intact hepatic arterial blood supply. PMID- 12375175 TI - Information transfer in microvascular networks. AB - The adequate and efficient functioning of the circulatory system requires coordination of vessel diameters and of vascular responses to local and remote stimuli. Such coordination implies transfer of information about functional status and demands to all parts of the vascular system. In the peripheral circulation, blood flow must be controlled locally to accommodate spatial variations in demand. This requires information transfer from peripheral vessels to the more proximal vessels that feed and drain them. Principal mechanisms available for this information transfer are hemodynamic coupling, diffusive and convective transport of metabolites, and responses conducted along vessel walls. Current knowledge of these mechanisms is reviewed here. Theoretical models provide a framework for examining how information transfer mechanisms and vascular responses are integrated, so as to provide short-term regulation of blood flow and long-term structural adaptation of microvascular networks. PMID- 12375176 TI - Interstitial PO(2) determination by phosphorescence quenching microscopy. AB - OBJECTIVE: This study introduces the technique of microinjection of phosphor probe into skeletal muscle tissue to determine oxygen tension (PO(2)) in the interstitium by phosphorescence quenching microscopy. METHOD: The spinotrapezius muscle of Wistar-Kyoto rats weighing 240-280 g was surgically isolated and underwent the microinjection procedure. We measured the spatial distribution of phosphor probe 10, 30, and 80 minutes after injection; the tissue PO(2) at sites adjacent to arteriolar and venular microvessels; and the decline in tissue PO(2) during a 3-minute period of 8-Hz contraction. RESULTS: The phosphorescence signal from the probe was undetectable outside a 2.5-mm radius from the site of injection at the 10-minute time point, increased to measurable values after 30 minutes, and was double the 30-minute intensity value after 80 minutes. When used to measure periarteriolar PO(2), the tissue microinjection technique demonstrated a nonlinear fall in tissue PO(2) with distance away from secondary arterioles (30 40 microm diameter). Conversely, perivenular tissue PO(2) increased in a nonlinear manner with distance away from secondary venules (60-70 microm diameter). The tissue PO(2) at distances of 16 microm and greater from both types of secondary microvessels was significantly different from values taken directly over the centerline of these microvessels. During muscle contraction, the PO(2) fell from a mean precontraction value of 28.3 +/- 4.9 mmHg to 8.2 +/- 0.9 mmHg at the end of the contraction period. CONCLUSIONS: These observations indicate that the microinjection technique yields values for tissue PO(2) that are in good agreement with previously published results using oxygen microelectrodes. PMID- 12375177 TI - Microvascular cell death in spontaneously hypertensive rats during experimental inflammation. AB - OBJECTIVE: Chronic hypertension is associated with an increased risk for tissue injury that may be mediated in part by endothelium and inflammatory cells. To clarify a possible underlying mechanisms, we examined leukocyte migration in the microcirculation and concomitant parenchymal cell death. METHODS: The mesentery of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats, was examined with digital fluorescence microscopy after topical stimulation with an inflammatory mediator (f-met-leu-phe, 10(-8)M). The migratory pathways of individual leukocytes were traced, and at the same time cell death was detected by use of a life-death indicator (propidium iodide) over a period of 3 hours. RESULTS: Both WKY and SHR had a progressively increasing number of leukocytes migrating across the endothelium in postcapillary venules into the tissue parenchyma. But parenchymal cell death was detected in a random pattern in the mesentery tissue, without correlation to the migratory positions of the leukocytes. Although mature SHR rats (about 17 weeks) exhibited the same level of cell death as age-matched WKY rats, older WKY rats (about 30 weeks) had significantly lower levels of cell death, whereas the SHR rats maintained the same number of parenchymal cell death as mature animals. CONCLUSIONS: These results suggest that in the presence of an inflammatory mediator, the SHR may exhibit a stronger response to an inflammatory mediator than normotensive WKY rats in a fashion that is age, but not blood pressure, dependent. Parenchymal cell death does not correlate with migration of activated leukocytes at the microvascular level. PMID- 12375178 TI - The role of T-lymphocytes in hypercholesterolemia-induced leukocyte-endothelial interactions. AB - OBJECTIVE: Hypercholesterolemia promotes the adhesion of leukocytes to vascular endothelium in large and microscopic blood vessels. Lymphocytes that can modulate endothelial cell adhesion molecule expression have been implicated in the altered structure and function of large arterial vessels associated with chronic hypercholesterolemia. This study assesses the contribution of CD4(+) and CD8(+) T cells to acute inflammatory responses observed in the microcirculation of hypercholesterolemic mice. METHODS: Intravital microscopy was used to quantify baseline leukocyte-endothelial cell interactions in cremasteric postcapillary venules of wild-type (WT) and severe combined immunodeficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. A group of SCID-HC mice received splenocytes from WT-HC mice (WT-->SCID). Separate WT-HC groups were depleted of neutrophils or CD4(+) and/or CD8(+) T-cells. RESULTS: WT-HC mice, compared with WT-ND, exhibited exaggerated leukocyte adherence and emigration. These leukocytes were predominantly granulocytes. These responses were absent in neutropenic WT-HC mice. SCID-HC mice also showed significantly less leukocyte adherence and emigration than WT-HC mice. Elevated leukocyte adherence and emigration were restored in WT-->SCID mice, despite a continued absence of circulating blood lymphocytes. Selective depletion of either CD4(+) or CD8(+) cell populations attenuated HC-induced leukocyte adhesion but not emigration. However, simultaneous depletion of both CD4(+) and CD8(+) cells attenuated both leukocyte adhesion and emigration to ND levels. DISCUSSION: These findings indicate that both CD4(+) and CD8(+) T-cells contribute to granulocyte adhesion and emigration elicited in postcapillary venules by hypercholesterolemia. PMID- 12375179 TI - Contribution of F-actin to barrier properties of the blood-joint pathway. AB - OBJECTIVES: Because fibroblast filamentous actin (F-actin) influences cutaneous interstitial matrix swelling pressure (5), we investigated whether F-actin in fibroblast-derived synoviocytes influences the hydraulic permeability of the trans-synovial interstitial pathway. The study also tested whether F-actin in fenestrated synovial endothelium contributes to the blood-joint barrier in vivo. METHODS: The clearance of Evans blue-albumin (EVA) from plasma into the knee joint cavity was determined in joint infused with F-actin disrupting cytochalasin D (1-200 microM), latrunculin B (100 microM) or vehicle in anesthetized rabbits. The hydraulic permeability of the lining was determined as the slope relating net trans-synovial flow Q(s) to intra-articular pressure P(j). Synovium was examined histologically after i.v. Monastral blue to assess endothelial leakiness. RESULTS: EVA permeation in vivo was increased up to 25-fold by cytochalasin (p = 0.0002, ANOVA), with an EC(50) of 23 microM (95% confidence limits 13-43 microM). Washout quickly reversed the increase. Latrunculin had a similar effect. F-actin disruption switched Q(s) from drainage (control) to filtration into the cavity at low P(j) in vivo and raised the conductance Q(s)/dP(j) by 2.13 (p = 0.001, ANOVA). Circulatory arrest abolished these effects. Monastral blue revealed numerous endothelial leaks. CONCLUSIONS: F-actin is crucial to the barrier function of fenestrated endothelium in situ. No significant effect of synoviocyte F-actin on matrix permeability was detected. PMID- 12375180 TI - Impact of hematocrit value after coronary artery surgery on perioperative myocardial infarction rate. AB - BACKGROUND: The optimal hematocrit (HCT) value after coronary artery bypass grafting on cardiopulmonary bypass (CPB) has not yet been established. The purpose of our retrospective study was to investigate the association between HCr at the time of entry into the ICU and perioperative Ml rate. METHODS: We reviewed the charts of 500 consecutive coronary artery surgery patients with respect to biometric data, operative procedure, aprotinin or tranexamic acid use, perioperative drainage blood loss and transfusion requirements, perioperative Ml, ICU stay and hospital mortality. Perioperative Ml was defined as new Q-wave on ECG and CK-MB 250U/I. Patients were categorized into three groups depending on their HCr value at the time of entry into the ICU: low (HCTcu 27%): medium (HCr,cu 28% to 32%); high(HCTrcu > or =33%). RESULTS: Age, gender distribution, preoperative LV function, and previous Ml rate were similar between the three groups. Low HCT patients (n -133) received 3.1 +/- 1.0 (Mean + SD) grafts during 55 +/- 19 minutes aortic cross clamp time, 98 +/- 31 minutes on CPB (medium HCT: n = 257; 3.2 +/- 1.0 grafts, 51 +/- 20 min cross clamp time, 93 +/- 30 min CPB; p - 0.45 vs. low HCT; high HCT: n = 110: 3.3 +/- 1:0 grafts; 53 +/- 20 min cross clamp time; 104 +/- 38 min CPB; p = 0.02 vs. medium HCT). The perioperative Ml rate was 3.8% in the low, 4.3% in the medium, and 6.4% in the high-HCr group (p =0.59 ). Intraoperative red blood cell and fresh frozen plasma transfusions were similar between the groups. In the low-HCa group, 53.4% of the patients received aprotinin during the procedure (medium HCa: 65.4%; high HCT: 77.3%; p<0.001). Drainage blood loss during the first 24 hours on ICU was 834 +/- 453 ml in the low, 757 +/- 485 ml in the medium (p -0.44 vs. low), and 640 +/- 353 ml in the high-HCr group (p = 0.003 vs. low). Postoperative red blood cell and fresh frozen plasma transfusions were highest in the low-HCa group(p<0.001). ICU stay was similar between the groups. Hospital mortality was 0.75% in the low, 1.9% in the medium, and 4.5%in the high-HCa group (p = 0.12). CONCLUSIONS: In this retrospective analysis of 500 consecutive coronary artery surgery patients, we did not find any association between perioperative Ml rate and HCr value on entry into the ICU. These results do not support the recent suggestion that low HCT at the time of entry into the ICU protects against perioperative Ml. PMID- 12375181 TI - Borderline hypoplastic left heart malformations: Norwood palliation or two ventricle repair? AB - BACKGROUND: Patients with hypoplastic left heart syndrome (HLHS) and associated malformations undergo Norwood palliation or potentially a two-ventricle repair. METHODS: Since 8/99, 8 patients with typical HLHS and two with DILV underwent Norwood/Fontan palliation (group I). Three other patients (group II) had two ventricle repair. Anatomy was: aortic atresia, coarctation, unrestrictive VSD (patient 1), hypoplastic mitral and aortic valve, arch and LV, coarctation (patients 2 and 3). Surgical procedures were Norwood arch reconstruction with either Rastelli operation (patient 1) or ASD-closure (patients 2 and 3). RESULTS: Operative mortality in group I was 1/8 (day 22; RV-failure). Two patients died before Glenn (sepsis, RV-failure). Six patients underwent Glenn procedure successfully. No patient died in group II. Echocardiography after 13 +/- 7.4 months showed mild homograft dysfunction (patient 1) and an LVOT-gradient of 20 mmHg (patient 3). Clinical condition of all survivors in both groups is good. CONCLUSION: Some anatomical subsets of HLHS with borderline mitral valves and small left ventricles may undergo two-ventricle repair despite severe LVOTO. Mortality and morbidity seem to be lower, but selection criteria are so far not defined. PMID- 12375182 TI - Minimally invasive surgery for congenital heart defects in paediatric patients. AB - BACKGROUND: In selected cases, minimally-invasive approaches are favoured for the correction of congenital heart defects with regard to better cosmetic results. METHODS: Between July 1999 and April 2001, 25 children (9 male; mean age 5.8 +/- 4.1 years; mean weight 19.6 +/- 12.6 kg) were operated on using minimally invasive approaches. Diagnoses were: ASD (n = 19), VSD (n = 2), ostium primum defect (n = 3) and Tetralogy of Fallot (n = 1). Female patients with ASD underwent a limited right anterolateral thoracotomy. A ministernotomy was chosen in male patients, in patients under 6 months of age, and in patients with malformations other than ASD. Cannulation was always performed via the chest incision. RESULTS: There were no perioperative complications. Mean operation time was 3.23 +/- 0.89 h. Twelve patients were extubated immediately after surgery, mean ventilation time in the others was 12.1 +/- 11.7 h. Mean ICU stay and hospital stay were 1.5 +/- 0.75 days and 8.3 +/- 2.2 days, respectively. Follow up (mean 4.8 +/- 4.6 months) was uneventful. CONCLUSIONS: Intracardiac repair of some congenital heart defects can be performed safely and effectively via minimally-invasive approaches. Indications are expanding towards more complex defects. Exposure for precise repair is good, additional incisions can be avoided, and cosmetic results have been excellent. PMID- 12375183 TI - Minimal early mortality in CABG--simply a question of surgical quality? AB - BACKGROUND: The increasing number of risk scores and models for the evaluation of the early risk after cardiac surgery reflects the interest in 'calculating' the risk of adverse events. Different time intervals, but also different 'types' of death are generally accepted in the evaluation of early mortality. The aim of this study was to focus on the differences in the calculation of early mortality and to focus on their potentially misleading impact on risk stratification. METHODS: We investigated 7,436 patients who underwent coronary artery bypass grafting from June 30, 1988 through June 30, 2001. A follow-up was performed 180 days after operation (98.7 % complete). RESULTS: According to the definition of 30-day mortality to represent the total time interval between an intervention and the 30th postoperative day, the 30-day mortality was 5.92 % (n = 440 patients). Hospital mortality reflects the number of deaths from the day of intervention through the patient's individual discharge, independent of any fixed time interval. Hospital mortality was 5.86 % (n = 436 patients) in our patient group. 30-day hospital mortality requires the investigation of hospital mortality until the 30th postoperative day; in-hospital and general mortality after the 30th postoperative day remained excluded from the analysis; 30-day hospital mortality was 5.19 % (n = 386 patients). Assuming a maximum hospital stay of 5 days, hospital mortality would decrease to 2.64 % (n = 196 patients). CONCLUSIONS: 30 day mortality, hospital mortality and 30-day hospital mortality are used to determine early outcome. The present data indicate the vulnerability of non standardized time intervals to discharge policy. However, both hospital mortality and 30-day hospital mortality are predominantly used in current risk scores and models. In view of the comparability and meaning of data, the methodology for the evaluation of early risk should be reconsidered. PMID- 12375184 TI - Computer-enhanced telemanipulation enables a variety of totally endoscopic cardiac procedures. AB - BACKGROUND: Since its introduction in the field of cardiac surgery in 1997, computer-enhanced telemanipulation has been used in a number of different specialized areas. In cardiac surgery, various procedures have been successfully completed in totally endoscopic fashion ever since. Between June 1999 and January 2002, 75 closed-chest cardiac procedures have been performed at our institution using the da Vinci telemanipulation system. PATIENTS AND METHODS: In 42 patients, a single-vessel totally endoscopic coronary artery bypass was performed on the arrested heart (left internal thoracic artery (LITA) to left anterior descending artery (LAD), n = 36; right internal thoracic artery (RITA) to right coronary artery (RCA), n = 6). 12 patients had different types of multivessel revascularization using both internal thoracic arteries. 8 patients underwent LITA-to-LAD grafting on the beating heart. 10 patients underwent closure of an atrial septal defect (9 direct, 1 patch). 3 patients received an epicardial left ventricular pacemaker lead, 2 of which were reoperations. RESULTS: Overall conversion rate to any kind of incision was 25 %. The last 26 LITA to LAD patients on the arrested heart had a conversion rate of 4 %. There were no mortalities, 3 patients required reexploration via a median sternotomy, and one patient suffered a hypoxemic brain damage. The first 22 TECAB patients demonstrated excellent graft patency in angiographic control upon discharge. None of the atrial septal defect (ASD) closures showed any residual shunt on the intraoperative transesophageal echocardiogram (TEE). Patients with end-stage heart failure had successful biventricular stimulation. CONCLUSION: Our current experience confirms the feasibility of various totally endoscopic cardiac procedures with good clinical outcomes. After a steep learning curve, the conversion rate could be lowered to an acceptable figure. Some of these procedures at our institution became a reasonable treatment alternative in selected patients. PMID- 12375185 TI - A prospective study on clinical outcome following pleurotomy during cardiac surgery. AB - BACKGROUND: Due to conflicting reports on pleurotomy-associated morbidity following internal mammary artery (IMA) harvesting, we conducted a prospective study to assess the clinical significance and outcome of pleurotomy during cardiac surgery. METHODS: We included patients undergoing cardiac surgery from November 2000 until January 2001. Participants were divided into two groups: one with routine or incidental left pleurotomy and the other with intact left pleurae. RESULTS: Of the 218 patients registered for this study, 12 were excluded (7 deaths occurred, 5 patients were transferred prior to study completion). Of the 206 remaining, 138 had isolated CABG, 39 had valve surgery and 29 had a combined procedure. Although patients with a left pleurotomy (n= 164) had a higher incidence of left lung atelectasis (67.7% vs. 45.2%; p = 0.007), neither radiographic consolidation (7.5% vs. 7.3%; p = 0.96), effusion (42.5%vs. 46.3%; p - 0.66), nor hospital stay (9 days in both groups; p - 0.83) increased. CONCLUSIONS: Left pleurotomy was found to increase the rate of atelectasis. However, this was not associated with an adverse clinical outcome. Pleurotomy during IMA harvesting can be performed according to operator preference. PMID- 12375186 TI - Lung herniation as an anatomic consequence of pneumonectomy. AB - BACKGROUND: Pneumonectomy causes an overdistention of the remaining lung as an adaptive response. Excessive lung herniation occasionally causes serious lung dysfunction. METHODS: Twenty-seven patients were selected from 152 patients who underwent pneumonectomy for lung cancer between 1990 and 1998. Complete resections were accomplished; no recurrence was observed for 3 years in these 27 patients. To evaluate the extent of herniation, the Lung Herniation Index (LHI) was developed and defined as the sum of proportions of the maximal transverse length of the remaining lung divided by the transverse length of the thoracic cavity, measured at the level of the aortic arch and the inferior pulmonary vein on chest computed tomography. Sequential changes in LHI were compared between groups. RESULTS: Changes in LHI did not differ between groups delineated on the basis of an FEV1 of 70 % (p = 0.45) and RV/TLC of 40 % (p = 0.99). Patients with a low body mass index (BMI) (< 20 kg/m(2)), however, showed a significantly greater degree of lung herniation than those with a high BMI (> or = 20 kg/m(2)) (p < 0.05). CONCLUSIONS: Concomitant COPD has no effect on lung herniation. Some preventive procedure should be considered for patients with low BMI. PMID- 12375187 TI - A novel sialyl Lewis(x) analogue attenuates ischemia reperfusion injury in rabbit lung. AB - BACKGROUND: We investigated the effects of OJ-R9545, a novel Sialyl Lewis x analogue, on lung ischemia-reperfusion (IR) injury using an in vivo rabbit model. METHODS: The left hilum of the lung was clamped for 110 minutes; the lung was then reperfused for 90 minutes. Either OJ-R9545 (10 mg/kg) or vehicle solution was administered from 10 minutes before reperfusion to 60 minutes after reperfusion in the OJ-R (+) and OJ-R (-) group (n = 6 in each group), respectively. The sham group (n = 3) underwent an identical procedure without ischemia. RESULTS: Arterial oxygen tensions in the OJ-R (+) group were superior to those in the OJ-R (-) group from 30 to 90 minutes after reperfusion (p < 0.05 and p < 0.01). Lung wet/dry weight ratio and myeloperoxidase activity after reperfusion in the OJ-R (+) group were both significantly lower than the corresponding figures in the OJ-R (-) group (p < 0.05). The intrapulmonary leukocytes were significantly reduced in the OJ-R (+) group compared with those in the OJ-R (-) group (p < 0.01). CONCLUSIONS: OJ-R9545 attenuates lung IR injury by preventing leukocyte infiltration into the lung. PMID- 12375188 TI - Lung transplantation by continuous perfusion in an experimental auto-transplant animal model. AB - OBJECTIVE: The aim of this study was to evaluate the preservation of the lung using the cold flushing technique in association with continuous perfusion of the organ during static hypothermic storage. METHODS: In the first phase, the hearts and lungs of 5 New Zealand rabbits were removed three hours after establishing brain death. The left lungs were each conserved in 200 ml of low-potassium UW solution at 10 degrees C for 3 hours of cold ischemia (control group I). The right lungs were also placed in cold storage but were perfused continuously for three hours with low-potassium UW solution at 10 degrees C (group II). In the second phase, ten rabbits underwent a right lung auto-transplant. Lungs were conserved using two techniques. Histoenzymatic and pathological tests were performed: lung function was evaluated. RESULTS: In the first phase the histopathological examination carried out at the end of storage revealed fewer ischemic alterations in the second group compared to the first. In the second phase a significant hypoxia was observed in group I when both lungs and the right lung only were perfused. The histopathological examination revealed ischaemia/reperfusion lesions in both groups though mainly in group I and a good level of ATPase activity in group II though these results were not significant. CONCLUSIONS: Cold flushing of the pulmonary artery and continuous perfusion during static hypothermic storage appears to guarantee a better partial arterial pressure of oxygen in this model of auto-transplant compared to the classical cold storage method. PMID- 12375189 TI - Right-sided cervical aortic arch with stenosis--treatment with an extra-anatomic bypass graft. AB - Right-sided cervical aortic arch is a very rare vascular anomaly that may lead to stenosis development. Anatomic repair may be impeded by its high course or by abnormal branching of the supraaortic vessels, or both. This report will describe the treatment of a stenotic right-sided cervical aortic arch using an extra anatomic bypass graft without extracorporeal support in an 11-year-old girl. PMID- 12375190 TI - A new combined surgical procedure for severe descending necrotizing mediastinitis with bilateral empyema. AB - In this report, we will describe the treatment of a 38-year-old man with severe descending necrotizing mediastinitis (DNM) with bilateral empyema. DNM is a rare disease with a high mortality rate, and when accompanied by bilateral empyema, this is particularly serious and potentially fatal. To improve the prognosis of such patients, the establishment of an adequate surgical procedure for satisfactory debridement and drainage is essential. This is the first report on a new combined surgical procedure consisting of right standard posterolateral thoracotomy and left video-assisted thoracoscopic surgery (VATS) for severe DNM with bilateral empyema. PMID- 12375191 TI - Use of the intra-aortic balloon pump for dilatation of benign tracheobronchial strictures. AB - Tracheobronchial benign stenoses may cause life-threatening emergencies. Here, we will describe a novel technique for the management of tracheal and bronchial stenoses using an intra-aortic balloon pump. The intra-aortic balloon pump was used for dilatation of a postoperative tracheal stricture in a 43-year-old man and a bronchial stricture in a 29-year-old woman with Wegener's granulematosis. There were no intraoperative or postoperative complications in either patient, and the stenosis was relieved successfully in each patient. The intra-aortic balloon pump can be used safely and effectively for the management of difficult tracheobronchial strictures. PMID- 12375192 TI - Cardiac malformations associated with the Holt-Oram syndrome--report on a family and review of the literature. AB - The Holt-Oram syndrome (HOS) is characterized by mild-to-severe congenital cardiac defects and skeletal abnormalities of the upper limb. The most common cardiac disorder is an ostium secundum atrial septal defect (ASD), followed by ventricular septal defect (VSD) and ostium primum ASD. Electrocardiographic abnormalities, such as various degrees of atrioventricular block, have also been reported. In addition, hypoplastic peripheral vessels of the upper limbs have been observed. Here, we will report about a family with three sons having HOS, and we will detail the cardiac spectrum of HOS as reported in the literature. PMID- 12375193 TI - Evidence-based medicine: lung volume reduction surgery (LVRS). AB - Lung volume reduction surgery (LVRS) was developed as a means of surgical treatment for severe pulmonary emphysema. To date, various studies have been designed to explain the mechanisms involved in pathophysiological changes after treatment, to define criteria for patient selection, to identify the surgical technique of choice and to propose appropriate follow-up care. Preliminary results of follow-up studies (up to five years) have already been published, indicating improved pulmonary function and quality of life after surgical treatment. However, the alarming results from the National Emphysema Treatment Trial (NETT) Research Group indicated a considerable risk for death in patients with homogenous emphysema and low forced expiratory volume in one second (FEV1) undergoing LVRS. This brief review summarizes the results of currently published studies to supply evidence for selection criteria in order to better define the subset of patients for which LVRS offers an effective and safe means of palliation from the symptoms of advanced COPD. Due to acceptable morbidity and mortality rates, stapler device wedge excision and closure has become the standard procedure for removing non-functioning, hyperinflated lung areas in heterogeneously affected organs. LVRS is carried out in two ways - using video assisted thoracoscopic surgery (VATS) as well as thoracotomy/sternotomy-and performed in unilateral and bilateral procedures. In contrast, most clinics have found laser resection of emphysematous parenchyma to be unsuccessful. In some patients, LVRS was carried out as an alternative to lung transplantation, whereas in others, it served as a bridge-to-transplant procedure. LVRS has proven effective in the reduction of dyspnea, especially in patients with recovery options in both the circulatory and pulmonary system. In responders, recovery from labored breathing and O(2) dependency and increased physical capacity are usually accompanied by improved spirometric data. These results are mainly explained by a more regular breathing pattern and an increase in the maximum volume of ventilation in the affected lung. In most cases, functional improvement is maximized during the first six months postoperatively and decreases steadily thereafter indicating the need for a systematic postoperative patient care after surgical treatment. After indicating at-risk patients who should not be considered for LVRS, long-term results from the multicenter NETT research group will hopefully help clarify the impact of this treatment on survival of patients further. PMID- 12375194 TI - [Uterine fibroid embolization - a new therapeutic option for symptomatic leiomyomata of the uterus]. AB - Uterine fibroid embolization (UFE) is a new minimal-invasive therapy for the treatment of symptomatic leiomyomata of the uterus and a uterine-sparing alternative to surgical procedures. Short-term and mid-term results indicate a high clinical success rate with improvement of fibroid-related bleeding symptoms in 80 - 100 % of cases, improvement of bulk symptoms in 60 - 100 % of cases and reduction in fibroid volume at an average of about 36 - 78 % combined with a low rate of complications and side effects. This review discusses indications and contraindications, technique and pathophysiology, choice of material, results and complications of UFE on the basis of the current literature and our own results. PMID- 12375195 TI - [Vertebral manifestation of chronic recurrent multifocal osteomyelitis (CRMO)]. AB - Chronic recurrent multifocal osteomyelitis (CRMO) is a systemic osteo-articular disease that is characterized by a sterile, primarily chronic osteomyelitis with various distribution patterns of the individual lesions. In this article, we describe the "axial type" with predominant involvement of the spine, which represents 13 of our 41 CRMO cases of different age groups. The important element of its diagnosis is the typical lympho-plasmacellular spondylitis that can be detected and staged by scintigraphy, MRI and conventional radiography. Potentially affected are all vertebrae from the mid-cervical spine to the sacrum. One or several segments can be involved, sometimes as transient inflammatory edema, sometimes as "migratory spondylitis" or "saltatory spondylitis", but also as chronic sclerosing type with early radiographically detectable manifestation. Vertebral deformity due to compression and total collapse (vertebra plana) are rare. A complicated course with patulous perivertebral edema can lead to concomitant symptomatic inflammatory changes in adjacent regions and organs. In the course of CRMO, spondylodiscitis only develops as secondary destruction following the spondylitis. This can help to differentiate spondyloarthropathies from CRMO that is initially detected as primary lesion in the spine. While CRMO generally has a good prognosis, its radiological differentiation from rheumatologic conditions plays an important role. PMID- 12375196 TI - [Experiences with phantom measurements in different mammographic systems]. AB - PURPOSE: Determination of image quality between conventional film screen system, digital phosphor storage plate mammography (high resolution) and digital mammography. MATERIALS AND METHODS: Mammograms of the Wisconsin Mammographic Random Phantom, Model 152 A (Radiation Measurements Inc., Wisconsin) were acquired using a conventional film-screen system, a digital storage phosphor plate system and a digital system. RESULTS: Of 225 possible details, 191/a 38.2, 193/a 38.6, and 202/a 40.4 details were detected with conventional film-screen system, digital phosphor storage plate mammography and digital mammography, respectively. There was no significant difference (p < 0,058). The entrance surface air kerma was 9.64 mGy, 7.60 mGy and 7.02 mGy, respectively. CONCLUSIONS: Based on these results, conventional film-screen system can be replaced with both digital phosphor storage plate mammography and digital mammography, to be confirmed with further clinical trials. PMID- 12375197 TI - [Lumbar intraspinal juxtafacet cysts: MR imaging and CT-arthrography]. AB - PURPOSE: To present data on the MR imaging appearance of lumbar intraspinal juxtafacet cysts (JFC) and to assess the importance of additional CT arthrography. MATERIAL AND METHODS: Twenty-eight patients (16 women, 12 men) with a mean age of 64 years (range: 43 - 82), who underwent MR imaging because of radicular pain or spinal claudication, were found to have an intraspinal cyst associated with the facet joint. In 14 patients, additional CT-arthrography was performed to determine whether a communication exists between the cyst and the facet joint and to try to rupture the cyst. RESULTS: In T(2)-weighted images, juxtafacet cysts show a typical pattern consisting of a hyperintense center and hypointense rim. The center is likely to be inhomogeneous because of recurrent hemorrhage in the cyst. In T1-weighted images, the cysts are hypo/isointense. Irregular hyperintensity may indicate subacute hemorrhage, which may aggravate the clinical symptoms. MR allows superior visualization of the cyst in all anatomical planes. It also enables assessment of typical accompanying changes, such as degenerative spondylolisthesis and facet hypertrophy. All patients, who had CT-arthrography, were found to have a direct communication between joint space and cyst. Transarticular rupture of the cyst was possible in five patients. Two of these five patients had good to excellent improvement, and the remaining three patients underwent surgery. CONCLUSION: MR imaging is the method of choice for diagnosing lumbar intraspinal juxtafacet cysts. CT-arthrography of the facet joint is helpful in cases with difficult differential diagnosis, and in the preoperative planning. Furthermore, it assists in the primary interventional treatment. PMID- 12375198 TI - [Percutaneous therapy of inoperable biliary stenoses and occlusions with a new self-expanding nitinol stent (SMART)]. AB - OBJECTIVE: To evaluate the treatment of malignant biliary stenoses and occlusions using a new stent. METHODS: In a prospective study, 25 patients with malignant obstructive jaundice were treated with SMART(R) stents. The handling and the quality of stent expansion were documented. Stent function was assessed 2 - 4 days after intervention by cholangiography and laboratory tests. A follow-up was performed three months, after stent placement. RESULTS: All lesions were treated successfully, with a total of 35 stents implanted. In 14 patients a further balloon dilatation was performed after stent placement (8 - 10 mm diameter/ 40 - 80 mm length). The mean serum bilirubin level decreased significantly from 11.6 mg/dl to 4.6 mg/dl after intervention (p < 0.05). The follow-up showed a mean serum bilirubin level at 4.0 mg/dl. In 4 cases (16 %) a further intervention (PTCD or stent) was performed. Six patients died due to tumor progression. The stents proved to be patent in 79 % (n = 15) of patients alive at the time of follow-up. CONCLUSIONS: Placement of the SMART stent for the therapy of malignant biliary lesions yields good technical and clinical results. PMID- 12375199 TI - [Percutaneous sclerotherapy of a varicocele with atypical retrograde flow using a double-balloon occlusion technique]. AB - OBJECTIVE: To perform a double-balloon occlusion for selective sclerosis of the internal spermatic vein in a varicocele with atypical retrograde flow. METHOD: In a 19-year-old man with a primary varicocele, phlebography demonstrated an 8-mm wide internal spermatic vein with parallel collaterals and marked reflux of the contrast agent in the supine position. A single balloon occlusion could not prevent rapid wash-out of contrast material via collateral veins. Subsequently, two 8-mm balloon-catheters were sequentially inserted into the internal spermatic vein. After inflation of both balloons, the sclerosing agent was administered through the proximal catheter into the balloon-occluded venous segment. RESULT: Percutaneous sclerotherapy using the double-balloon technique achieved complete stasis inside the ectatic and retrograde perfused spermatic vein including its major parallel collaterals. CONCLUSION: In primary varicocele with strong spontaneous reflux and complicated flow pattern of the collateral veins, the double-balloon occlusion technique can achieve high local concentration of the sclerosing agent at optimal position and without wash-out effects. PMID- 12375200 TI - [Intravascular ultrasound thrombolysis for recanalization of peripheral arteries: evaluation of an in vitro model and results of a pilot-study]. AB - OBJECTIVES: To evaluate the effectiveness of ultrasound thrombolysis in vitro in comparison with thrombectomy, and in vivo as a pilot-study for the treatment of thrombotic occlusions of peripheral arteries. METHODS: Under standardized conditions, one-day-old and five-day-old thrombi of whole blood, thrombin-induced thrombi and old organized thrombi of human blood were treated with ultrasound thrombolysis and Amplatz thrombectomy device (ATD). Four patients with arterial occlusive disease of Fontaine stage IIb-III underwent intraarterial ultrasound thrombolysis, applied to long segmental occlusions of the superficial femoral or iliac artery ranging in duration from three days to one year. RESULTS: The weight of the thrombi after ultrasound thrombolysis was 1.5 g +/- 0.53 (ATD: 0 g) compared to 3.65 g +/- 0.34 without treatment, with more weight reduction in five day-old thrombi than in one-day-old thrombi. In vivo, partial recanalization was achieved in a three-day-old femoral occlusion. There was no effect in the other three patients. Urokinase thrombolysis with subsequent PTA and stenting resulted in complete recanalization in three patients. CONCLUSIONS: Ultrasound thrombolysis in vitro was significantly less effective than ATD. The results of ultrasound thrombolysis were influenced by the age of the thrombus and its in vitro formation. Intravascular ultrasound thrombolysis alone was insufficient to treat occluded peripheral arteries in vivo. PMID- 12375202 TI - [Prospective comparison of hydrosonography, endosonography and specimen sonography for TN staging of gastric carcinoma]. AB - PURPOSE: To compare hydrosonography (HUS), endosonography (EUS) and experimental sonography (PUS) with respect to TN-staging accuracy of gastric carcinoma. MATERIAL AND METHODS: Thirty-six patients with gastric carcinoma underwent EUS (7.5/12 MHz transducer, Olympus GF-UM 20) and HUS (3.75 MHz transducer, Toshiba, Sonolayer SSA-270A) for TN-staging according to the UICC-classification. The resected specimens were reexamined (3.75/7.5 MHz transducer) and again TN-staging was performed. Findings were correlated with histopathological results. RESULTS: T- and N-staging accuracies were as follows: EUS 54 % (19/35) and 79 % (27/34); HUS 41 % (15/37) and 61 % (22/36); and PUS 51 % (19/37) and 72 %(26/36). Sensitivities and specificities for the detection of lymph node metastases were as follows: EUS 87 % and 54 %; HUS 57 % and 69 %; and PUS 83 % and 54 %. CONCLUSIONS: The accuracy of sonographic TN- staging is limited in patients with gastric carcinoma. Nevertheless, EUS may contribute to the preoperative management of patients with gastric carcinoma if indications are well defined. HUS is not suited for TN-staging of gastric carcinoma. PMID- 12375201 TI - [MRI-based N-staging in esophageal cancer]. AB - PURPOSE: For planning the therapeutic strategies and estimating the prognosis in esophageal cancer, N-staging is very important. To date, MRI still is of minor importance as imaging modality of the mediastinum despite promising developments in the past, like ECG-gating or "averaging" sequences, e. g., LOTA (Long-term averaging), which facilitate mediastinal and thoracic MR-imaging. In a prospective approach, the value of MRI based N-staging was examined with respect to LOTA-sequences. MATERIAL AND METHODS: Within four weeks prior to esophagectomy, standardized MRI of the esophagus was performed in 15 patients (10 squamous-cell-carcinomas and 5 adenocarcinomas) using a 1.5 T whole body scanner. Imaging quality was classified based on depiction of aortic wall or tracheal wall layers. Criteria for malignant infiltration were a diameter of more than 15 mm or a round appearance of a lymph node together with GD-DTPA enhancement. All data were blinded and separately read by two radiologists. The data of the study were compared with those from the pathological workup of the resected specimen. RESULTS: MRI had a sensitivity of 100 % and a specificity of 78 % for lymph node metastases. Due to incomplete depiction of the celiac trunk (M1), nodal metastasis in a non-enlarged node was missed. CONCLUSION: With modern MRI, N staging is almost as accurate as the gold standard endoscopic ultrasound and should particularly be used in patients not suited for an endoscopic ultrasound examination. PMID- 12375203 TI - [Formation of portal venous collaterals after ligation of the portal vein for induction of liver regeneration]. AB - PURPOSE: Unilateral occlusion of the portal vein induces contralateral lobar hypertrophy - in contrast to complete portal vein occlusion which will result in a cavernous transformation. The impact of the formation of collaterals in partial portal vein occlusion is not sufficiently known. The lobar-hypertrophy- phenomenon is in clinical use for several years to induce iatrogenic liver growth to enable extended resections. After portal vein ligation in patients prior to extended hepatic resections, we noticed a perfusion of the formerly occluded side on CT. Using the well-established mini pig model, we were interested whether portal collaterals are formed as cause of the reperfusion. Ex-situ angiograms of the liver were used for the depiction of collaterals. MATERIALS AND METHODS: Using a median laparotomy as access for preparation of the hepatoduodenal ligament, a proximal left portal vein ligation was performed in eight mini pigs under general anesthesia. The total arrest of the portal blood flow (except in segments VI and VII) was documented by duplex ultrasound. After 4 weeks, all pigs were sacrificed and the weight of the ligated liver segments and non-ligated liver segments was measured and compared to a sham group (n = 5). After insertion of a guiding sheath, an ex-situ DSA of the portal vein was acquired. RESULTS: Compared with the sham group, the liver weight increased by 60 % (23 - 99 %, std. dev. 30 %) in segments VI and VII. Atrophy of the ligated segments was signified by a weight loss of 10 % (standard deviation 15 %). The ex-situ angiograms revealed a uniform pattern of collaterals with subsequent complete total recanalization of the formerly occluded portal vein distal from the ligation. The collaterals reduced the portal venous flow rate. CONCLUSION: After portal vein ligation, uniform collateralization results in recanalization of the occluded portal vein. The extent of the collaterals exceeds the known cavernous transformation. The increase in liver volume is not restrained by the formation of collaterals. PMID- 12375204 TI - [The contribution of MRI to the detection of endovascular aneurysm repair]. AB - PURPOSE: Evaluation of MR-imaging in the follow-up of patients after endovascular repair of abdominal aortic aneurysms concerning detection of endoleaks. MATERIALS AND METHODS: In the postoperative follow-up after endovascular repair of aortic aneurysms, 10 consecutive patients (mean age: 68 years) were suspected to have an endoleak by helical CT and were scheduled for conventional angiography, preceded by supplemental MR-imaging to confirm or refute the diagnosis. The images of helical CT and MRI were evaluated by two independent readers concerning leak, feeding vessel and artifacts. RESULTS: The follow-up MRI was able to detect all endoleaks (type 1 endoleak, n = 7; type 2 endoleak, n = 3) compared to all but one detected by helical CT. Of the 10 patients with an endoleak, MR-angiography visualized the feeding vessel in 7 patients and CT in one patient. MRI did show fewer metal artifacts from the stent wire than CT. For the visualization of feeding vessels and endoleaks, MRA achieved statistically significant superiority. In a single case, helical-CT was not reliable because of strange metal artefacts after previous coil embolization. CONCLUSION: MRI is comparable to helical-CT in detecting endoleaks and superior to CT in demonstrating the anatomy of the feeding vessel after endovascular repair of aortic aneurysms. The major advantages are fewer artifacts after coil embolization and absent radiation exposure. PMID- 12375205 TI - [Contrast-enhanced MR angiography in the routine work-up of the lower extremity arteries]. AB - PURPOSE: Prospective evaluation of the effectiveness of contrast-enhanced moving table magnetic resonance angiography (CE-MRA) as the sole routine tool for the diagnosis of peripheral arterial occlusive disease and determination whether it can replace catheter arteriography. SUBJECTS AND METHODS: In a time period of 23 weeks, 100 consecutive patients were evaluated. A total of 112 contrast-enhanced moving-table MR angiograms were performed at 1.5 Tesla. A dedicated vascular coil system was used. It was evaluated in which cases MR angiography was sufficient to determine the treatment plan and in which cases limited quality required additional examinations. RESULTS: In 93.75 % (105/112) of all examinations, the treatment plan was determined by MRA as the sole diagnostic tool. Twenty-two patients underwent surgery or percutaneous angioplasty based on MRA findings. Additional examinations due to impaired quality were performed in seven (6.25 %) cases: two MR angiographies of the pelvic arteries, one MR angiography of the calf, and four selective arteriographies because of venous overlay at the calf. CONCLUSION: Contrast-enhanced MR angiography can take the place of catheter angiography in the routine work-up of patients with peripheral arterial occlusive disease. Further assessment might be necessary in five to ten percent of the cases when the diagnostic quality is inadequate, mostly due to venous overlay in the lower leg. PMID- 12375206 TI - [Optimized image processing with modified preprocessing of image data sets of a transparent imaging plate by way of the lateral view of the cervical spine]. AB - PURPOSE: To improve the diagnostic quality of lateral radiographs of the cervical spine by pre-processing the image data sets produced by a transparent imaging plate with both-side reading and to evaluate any possible impact on minimizing the number of additional radiographs and supplementary investigations. MATERIAL AND METHODS: One hundred lateral digital radiographs of the cervical spine were processed with two different methods: processing of each data set using the system-imminent parameters and using the manual mode. The difference between the two types of processing is the level of the latitude value. Hard copies of the processed images were judged by five radiologists and three neurosurgeons. The evaluation applied the image criteria score (ICS) without conventional reference images. RESULTS: In 99 % of the lateral radiographs of the cervical spine, all vertebral bodies could be completed delineated using the manual mode, but only 76 % oft the images processed by the system-imminent parameters showed all vertebral bodies. Thus, the manual mode enabled the evaluation of up to two additional more caudal vertebral bodies. The manual mode processing was significantly better concerning object size and processing artifacts. This optimized image processing and the resultant minimization of supplementary investigations was calculated to correspond to a theoretical dose reduction of about 50 %. CONCLUSION: The introduction of optimized organ programs for the upper and lower cervical spine based on the 12-bit data of the images should improve the evaluation of the lateral radiograph of the cervical spine without reducing the latitude value. PMID- 12375207 TI - [Characterization of motion artifacts in multi-slice spiral CT]. AB - PURPOSE: Motion artifacts in multi-slice spiral CT (MSCT) resulting from object motion in and against the table feed direction (z-direction) are examined using a spherical phantom. For image interpretation of complex anatomic structures, qualitative reference points are, also applicable to selected, which are ECG gated cardiac imaging. In this case the motion of the coronary vessels in phase with the cardiac contraction must be considered. METHODS: Measurements are obtained with a multi-slice spiral CT with a rotation time of 500 ms for 4 x 1.0 mm and 2 x 0.5 mm collimation. The phantom consists of an acrylic glass body with imbedded glass beads of 1, 2, and 3 mm diameter. The object motion is sinusoidal with an amplitude of 5 mm and frequencies of 60/min and 90/min. Compensation of the table feed by object motion is examined as a special case. RESULTS: Small parameter changes can induce a strikingly different image quality, and the moving objects emerge in different slices. Depending on the phase of the movement with respect to the CT scan, objects up to a size of 3 mm can vanish completely or appear hyperintense in the image. The model investigated is also applicable to ECG-gated cardiac imaging for the detection of stenosis. It can explain variations in the reproducibility and absolute score values of the calcium scoring. CONCLUSION: The presented considerations and results must be taken into account in image interpretation with possible object motion in the z-direction. Variations in the determination of the degree of stenosis or in calcium score measurements can be explained by different vessel motion during the diastolic heart phase. PMID- 12375208 TI - [Use of a newly developed piezoelectrically driven drilling machine for MR-guided bone biopsies]. AB - PURPOSE: Development and clinical testing of an MR-compatible bone biopsy system, to enable the sample collection from osteosclerotic or subcortical lesions for histological investigation under MR control. MATERIALS AND METHODS: A piezoelectrically driven drilling machine was constructed and tested in connection with an MR-compatible bone biopsy set in a vertical open MR scanner (0.5 T) on a phantom and 10 patients with ambiguous bone lesions. Images were obtained using T(1)-weighted spin-echo sequences and, in case of real-time imaging, a fast spoiled gradient-echo sequence. RESULTS: The influence of the enabled motor (RF-interference) leads to a reduction of the signal to noise ratio of the images, but can be minimised by appropriate measures. The observed slight field distorsions do not affect the image quality during real time acquisition in a substantial manner. No complications occurred. The extracted biopsy material was sufficient and of good quality. CONCLUSIONS: In spite of slightly restricted image quality, the described drilling machine combined with the bone biopsy set is well suited for MR-guided bone biopsies, which require the application of a motor driven drill. Its application within an interventional MR scanner is safe and its handling simple and manageable. PMID- 12375209 TI - [Image quality and detection of pathology by ultrasound: comparison of B-mode ultrasound with photopic imaging and tissue harmonic imaging alone and in combination]. AB - PURPOSE: To determine the accuracy of photopic imaging (PI) in detecting pathology by ultrasound (US) and to assess the image quality in direct comparison with conventional B-mode ultrasound and tissue harmonic imaging (THI). MATERIAL AND METHODS: Fifty-two patients underwent US examination, among them 29 patients for abdominal assessment and 23 for otolaryngological assessment. A total of 208 freeze frames, 52 B-mode scans each with and without THI and 52 B-mode scans each with and without PI, were assessed by three readers, who determined the presence of pathology on a scale of 1 (definitely abnormal) to 5 (definitely normal). All 52 patients underwent US follow-up within six weeks. The results were confirmed by CT in 30 patients and by histology in five cases. Image quality and different color encodings of each technique were rated on a ranking scale of 1 (optimal) to 4 (poor). The different US techniques were compared in terms of image quality, diagnostic accuracy, and color encoding using McNemar's test and ROC analysis. RESULTS: The results for image quality were as follows: B-scan 3.9; THI 1.9; PI 2.8; and THI plus PI 1.5 (each p < 0.05). The following AUCs (Area under Curve, presence of pathology) were calculated: 0.925, 0.990, and 0.990 for B-mode US, THI, and PI, respectively (not significant), and 0.994 for THI plus PI (significant compared to B-mode scan). The different color encodings were rated as follows: reddish brown 1.6, gray 1.9, blue 3.1, and green 3.6 (each p < 0.05). CONCLUSION: For ultrasound examinations, PI in combination with THI improves the image quality and conspicuity of pathology. PMID- 12375210 TI - [Spontaneous closing of cerebral arteriovenous malformations by thrombosis of the nidus and the draining veins]. PMID- 12375211 TI - [Vein sac filling and spiral embolization of pulmonary arteriovenous malformations in Osler's disease]. PMID- 12375212 TI - [The use of randomisation in clinical studies in rehabilitation medicine: basics and practical aspects]. AB - New therapies in rehabilitation medicine have to be evaluated with clinical trials. For drug approval the methodology of clinical trials is standardized world wide and the results of these studies are widely accepted. This standard should be achieved in clinical trials in rehabilitation research, too. One of the standards is the existence of a control group, comparing the effect of the new intervention against controls. In addition, the investigational and control groups must be equal in terms of the structure of possible confounders. Randomisation is the best possibility to distribute the patients to the therapy groups, confounders will be equally distributed by chance. Other procedures for assignment to the study groups can result in confounding and lead into biased results. In spite of these advantages, randomisation is not generally accepted in rehabilitation research up to now. There are some reservations, mostly ethical, organisational and methodological ones. However, randomised clinical trials should be conducted in rehabilitation research in order to obtain more convincing results. Our intention is to bring some input in this debate and to present basics and practical aspects of randomisation. PMID- 12375213 TI - [Women and men after acute myocardial infarction: are there gender differences in participation rates in cardiac rehabilitation?]. AB - In Germany and other countries a possibly lower programme attendance in cardiac rehabilitation of women compared to men has been critically discussed for many years. Up to now however there are only limited data related to gender-specific utilization rates and programme attendance. In a longitudinal study with three points of measurement, 496 men and 172 women after an acute myocardial infarction and their physicians were asked to complete questionnaires referring to different aspects of the medical and psychosocial situation as well as to provision of acute cardiac care and rehabilitation services. The results show that currently in Germany no gender differences exist in the use and provision of cardiac rehabilitation. Whether this apparently "fair" distribution in relation to gender can be seen as an adequate supply however is questionable in the context of different needs - especially with respect to the different psychosocial conditions of women and men. PMID- 12375214 TI - [Psychological implications of medical information for patients - considerations in rehabilitees with spinal cord injuries]. AB - The article describes the necessity of medical information also in patients with severe injury, as a conclusion of subjective illness theory ("lay theory") and other theoretical considerations. Analysing the data of a retrospective questionnaire investigation, 71 persons with spinal cord injuries were explored concerning psychological issues of rehabilitation. The results show empirical evidence of expected positive psychological correlations if patients are satisfied with their medical information. There is a low rate of well-informed patients as well as a high need for this particular information about diagnosis and prognosis; possible reasons are discussed and the lack of information is argued. PMID- 12375215 TI - [Regional networking of medical and vocational rehabilitation-- the bad Krozingen model]. AB - Regional networking facilitates flexible and individual integration of vocational programmes in medical rehabilitation. We present a pilot project of Theresienklinik II in cooperation with the Education centre for occupation and health (Bildungszentrum Beruf und Gesundheit) in Bad Krozingen. Orthopaedic and cardiac patients who are in danger to lose their capacity to work, participate in an integrated vocational reorientation programme during extended medical rehabilitation. The aim of the pilot project is an early assessment of motivation, work hardening, aptitude and interest, in order to accelerate vocational retraining and reintegration. Within one year 30 patients participated in the programme. Presented are the contents and course of the pilot project. First results show a high patient satisfaction with the programme. PMID- 12375216 TI - [SGB IX - Magna Charta of a Functional Participation Philosophy]. AB - Further development of needs-based, efficient health care structures in particular for people with chronic illness, and in conjunction with them, is the paramount objective of SGB IX, book 9 of the German social code. To achieve this it is necessary to define treatment sequences across sectoral boundaries in health care, and to establish cooperation between community-practice physicians, hospitals, rehab and long-term care facilities, cost carriers, and people with disabilities. In this context, the article discusses the current rank of rehabilitation and participation, points out the underlying conflicting issues and interests, and posits the preventive-integrative rehabilitation paradigm at the very centre of health protection networking. PMID- 12375217 TI - [Safeguarding early rehabilitation and hospital treatment of persons with disabilities on implementation of legislation introducing a prospective payment system--Statement of Deutsche Vereinigung fur die Rehabilitation Behinderter]. PMID- 12375219 TI - [Is home respiration effective?]. PMID- 12375220 TI - [Bronchoscopy and rhythmic disorders. Premedication with atropine-sulfate, as a rule?]. AB - Ikeda has introduced flexible bronchoscopy in the seventies of the last century. Since then the over one hundred year old procedure of direct airways inspection has widely spread and enhanced the diagnostic and therapeutic means. Thus the flexible bronchoscopy has become an important part of modern medicine. The close combination of atropine as premedication with bronchoscopy is justified with the terms "cardioprotection" and reduction of mucus secretion. As there is to this date no controlled study to prove this assumption, with the start of bronchoscopy we controlled every patient with a holter-ecg for 24-hours and estimated semiquantitatively the mucus secretion during procedure by a four point scale. Consecutively 55 patients could be randomised, 25 (7 females, 18 males) in the group with and 30 (7 females, 18 males) without atropine. In the records there were no detectable significant differences between the groups with atropine (A) and without atropine (P), as well as for registered bradycardias (A: 0 vs. P: 0, minimum of heart beats A: 63.8 vs. P: 74.1 min -1) as well as for alterations of heart rhythms, e. g. SVES (A: 7.3 % vs. P: 5.5 %), VES (A: 9.0 % vs. P: 9.0 %) or a combination of SVES with VES (A: 12.7 % vs. P: 10.9 %). The same results could be seen for each single of the first twenty minutes, additionally the first and the second recorded hour and the whole registered 24 hours. Moreover the times needed to complete the bronchoscopy showed no significant difference (mean of t A: 16.8 vs. P: 15.6 min, t-minimum 10 vs. 10 min, t-maximum A: 30 vs. P: 35 min). The same absence of differences was seen in estimated endobronchial mucus secretion (mean A: 1.88 vs. P: 2.0). According to these results of our studied group, there are no reasons, why a premedication with atropine in flexible bronchoscopy in local anaesthesia should be used. Even without the administration of atropine, flexible bronchoscopy could be performed as a safe and sophisticated method in direction of not inducing relevant arrhythmia, with low impact on patients. PMID- 12375221 TI - [Ciprofloxacin in the treatment of hospital-acquired pneumonia: a surveillance study in 676 patients]. AB - BACKGROUND: Controlled clinical trials have shown efficacy of high-dose ciprofloxacin for hospital-acquired (HAP) or nosocomial pneumonia. But it has yet to be demonstrated whether this good efficacy also holds true for routine intensive-care patients outside of controlled trials. PATIENTS: In a post marketing surveillance study at 87 intensive-care units in Germany we analyzed 676 cases of nosocomial pneumonia treated with intravenous ciprofloxacin in a daily dosage of at least 400 mg. RESULTS: 538 (80 %) patients were evaluable for efficacy. Cure or improvement was reported in 76 % of the cases. Clinical success rate was higher in previously untreated patients receiving ciprofloxacin as monotherapy (85.3 %) or in combination with other antibiotics (78.4 %) than in those who received ciprofloxacin as monotherapy (73.1 %) or as combination therapy (69.2 %) after an antibiotic pretreatment. In the 66 patients with Pseudomonas aeruginosa as causal pathogen, clinical success rate was 86.4 %. 32 adverse events classified as possibly or probably related to ciprofloxacin occurred in 3.1 % of patients; all of those were reversible. CONCLUSIONS: Due to the high success rate, even in cases with failed antimicrobial pretreatment, and the favourable risk-benefit ratio of high-dose ciprofloxacin, ciprofloxacin appears to be an attractive choice in the empiric treatment of hospital-acquired pneumonia. PMID- 12375222 TI - [beta-lactam-antibiotics in the treatment of community-acquired respiratory tract infections with penicillin-resistant pneumococci]. AB - Streptococcus pneumoniae is still the most important pathogen of community acquired respiratory tract infections. During the last decades in many countries an increase in the spread of antibiotic resistant strains (e. g. against beta lactams, macrolides, tetracyclin) was observed. Resistance against penicillin is often associated with resistance against macrolides and other antibiotic classes. In Germany surveillance studies including isolates from patients with community acquired respiratory tract infections have shown that about 14 % of strains show a reduced susceptibility against penicillin (MIC-values 0.12 - 1 mg/L) and up to 4 % are highly resistant against penicillin (MIC >/= 2 mg/L). Resistance against tetracycline or macrolides was detected in up to 12 and 15 % of strains, respectively. According to the treatment guidelines of the Paul-Ehrlich Gesellschaft fur Chemotherapie and the Deutschen Atemwegsliga penicillins and cephalosporins are recommended as first line antibiotics for the treatment of community-acquired respiratory tract infections. As pneumococcal strains with reduced susceptibility against penicillin show often also a reduced susceptibility against cephalosporins the questions arises which beta-lactam antibiotics should still be used in empirical treatment of such strains. beta Lactam-antibiotics highly differ in their in-vitro-activity against S. pneumoniae and their pharmacokinetic properties. In different models is has been demonstrated for beta-lactams that an adequate clinical and bacteriological efficacy is achievable when the serum levels of the free, i. e. not protein bound fraction of drug exceeds the MIC of the pathogen for at least 40 to 50 % of the dosing interval (T > MIC). In a clinical situation where pneumococci with reduced susceptibility against penicillin cannot be ruled out, only beta-lactam antibiotics with favourable pharmacological properties (good in-vitro activity, high and long lasting serum levels) should be used for treatment. PMID- 12375223 TI - [Health-related auality of life (HRQL) in patients receiving home mechanical ventilation]. AB - Evaluation of health-related quality of life (HRQL) has become steadily more essential during the last two decades in research and health care practice in order to evaluate the human and financial costs and benefits of modern medical techniques. HRQL in its definition is based on different components of health including physical state, psychological well-being, social relations and functional capacities that are influenced by a persons experience, beliefs, expectations, and perceptions. For the purpose of assessment of HRQL several instruments have been developed. Generic instruments are not specific to any particular disease and are therefore most commonly used for general survey research on health allowing comparisons between disease states. In contrast, disease-specific questionnaires which are necessary in order to focus on domains most relevant to a particular disease are thought to be more sensitive than generic instruments following therapeutic interventions. Home mechanical ventilation (HMV) delivered noninvasively by a facial mask is a well established treatment for chronic hypercapnic respiratory failure. It is widely accepted that survival improves following institution of HMV in most patients with chest wall deformities or neuromuscular diseases while this is still controversially discussed in patients with COPD. However, patients receiving HMV usually have severe respiratory insufficiency with a past medical history of several years or decades, and suffer from end stage disease with objectively severe limitations of daily living. In addition, HMV is a time consuming and cost intensive therapy. Therefore, several studies have been conducted in the last decade to evaluate HRQL in patients receiving HMV. Recent studies using generic questionnaires have shown impairments in HRQL in patients receiving HMV compared to normals. This was primarily attributed to severe limitations in physical health, but not in mental health indicating that if severe physical limitation occurs in advanced respiratory disease this will not necessarily lead to mental limitation. In addition, limitations in HRQL in patients with HMV were not substantially higher than in patients with different chronic disease being not dependent on HMV. Improvements in HRQL following the institution of HMV were only mild or even insignificant in patients with COPD, but patients with restrictive ventilatory disorders are suspected to have more benefits. However, well validated disease specific questionnaires which are designed to be more sensitive in the assessment of changes in HRQL than generic instruments have been introduced recently for patients with severe respiratory failure, but the influence of HMV to HRQL remains still unclear, since prospective studies using these questionnaires have yet not been finished. PMID- 12375224 TI - [Idiopathic eosinophilic pneumonias]. PMID- 12375225 TI - [The acute exacerbation of chronic obstructive pulmonary disease. Conference report on an expert workshop]. PMID- 12375227 TI - [Current initiatives in Germany for translating national guidelines into reality a survey]. AB - The role of practice guidelines as a tool for quality management in health care is now widely accepted in Germany- not only by health professionals, but also in politics. The physicians' professional associations as well as health care authorities (physicians' self-governmental bodies) and parliament introduced several incentives and regulations, aiming at a regular use of guidelines in health care. Among these the German guideline clearinghouse with the systematic approach towards identification, dissemination, and implementation of best available evidence-based guidelines, as well as the country-wide implementation of disease management guidelines seem to be effective and efficient in quality management as well as in patient care management in the German health care system. The article gives an overview on background, procedures and barriers to country-wide implementation of clinical practice guidelines within a social security health care system. PMID- 12375228 TI - [Quality of Statutory Inpatient Database Before Introducing DRGs]. AB - According to the legislation of the Federal Republic of Germany (Gesundheitsstrukturgesetz 1993) defined performance figures must be listed for each case of inpatient care. As hospital morbidity data are essential for further development of the health care system and for introducing the DRGs, the corresponding statistical data of a German federal territory of the year 2000 were studied in respect of several aspects of their quality: conformity with the requirements of law, plausibility and ability to transport essential medical information. Notable variations were found between the departments and different medical disciplines without interdependence to variant hospital status. Only about 40 % of departments of surgical disciplines transferred data according to legal requirements. Some disciplines showed higher percentages of unspecific coding (e.g. traumatology). The described deficits impair data reliability. The study offers a feedback to hospital departments with regard to their formal data quality. Periodic investigation may thus help to improve data quality in future. PMID- 12375229 TI - [Validity of care assessment in disabled and mentally retarded children]. AB - 16 children with spastic cerebral palsy and 25 mentally retarded children were assessed via the scales "Self-Care" and "Mobility" of the Pediatric Evaluation of Disability Inventory (PEDI). Age-adjusted PEDI scores were compared with the classification according to the three levels of the German statutory nursing insurance. Good correlations and highly significant dependence were found in children with spastic cerebral palsy but no dependence was seen in mentally retarded children. Apparently, assessment guidelines of the German statutory nursing insurance do not guarantee a valid assessment in all disabled children. In conclusion, future assessments of nursing needs in children should employ standardised assessment methods. PMID- 12375230 TI - [Measures to promote hygiene in geriatric and long-term care]. AB - For hospitals, the Directives for Hospital Hygiene and Prevention of Infection issued by the Robert Koch Institute represent clear and well formulated hygiene guidelines in terms of a set of rules. However, for long-term care facilities there are no standard hygiene procedures, and the above mentioned guideline recommendations are difficult to apply to geriatric and long-term care as well as to rehabilitation. It is left to the institutions themselves to determine the role of hygiene and prevention of infection. Framework guidelines are provided in the Protective Law on Infections and the hygiene requirements contained therein. However, there are no suggestions on how to actually implement the hygiene requirements. This article demonstrates one way in which the protective law might be transferred and used in practice based on the classic procedures of quality management. This is explained as a step-by-step, planned process. The appendix contains one possible structure and excerpts from a control checklist. PMID- 12375231 TI - [Studies on the importance of tick-borne encephalitis in Rhineland-Pfalz]. AB - Within the scope of a prospective clinical study during 2001 in Rhineland Palatinate specimen from sera and cerebrospinal fluids of 163 patients with suspected meningitis were controlled in an enzyme immunoassay concerning a TBE infection. Questionable results were checked via a neutralisation test. In no case such an infection was confirmed. No virus specific nucleic acids could be detected in 998 nymphs and adults of Ixodes ricinus in an additional investigation in 2000. Therefore Rhineland-Palatinate has to be considered as a region with low virus prevalence. A general recommendation for vaccination is not necessary. PMID- 12375232 TI - [Reduction of excessive reactions after tuberculin skin test (mendel-mantoux method)]. AB - In 2.5 % of cases excessive reaction with formation of blisters and local necrosis is seen after tuberculin skin testing with Mendel-Mantoux technique. The objective of this prospective study was to determine whether this rate can be reduced by pretesting. 426 contacts of cases with contagious pulmonary tuberculosis were examined in a public health office. A tuberculin skin test in Mendel-Mantoux technique with 1 unit purified tuberculin Behring (GT 1, bioequivalent to 0.5 units PPD-S) was applied and the results read after 3 to 7 days. Negative results were retested with 10 units (GT 10, bioequivalent to 5 units PPD-S). 422 patients completed the examination course as planned. In two cases forming of blisters was noticed. The difference between this observed rate of 0.5 % and the published rate of 2.5 % is highly significant. The alternative hypothesis that pretesting reduces the rate of excessive reactions can be assumed. PMID- 12375233 TI - [Psychosocial stress preceding drug-related deaths]. AB - This article analyses drug-related deaths in the German Federal States of Bavaria (Munich, Nuremberg and Augsburg counties) during 1999 and Baden-Wurttemberg (Stuttgart and Mannheim counties) during 1999 and in the first half of 2000. The persons who had been in contact with drug care services were studied for psychosocial stress preceding drug-related deaths. Epidemiological data from different sources (police, relatives, counselling centres, detoxification clinics, therapy and substitution treatment) were collated to estimate factors of psychosocial stress preceding drug deaths. The results in both Laender indicate high prevalence rates of a history of at least one non-fatal overdose (approx. 50%) or a suicide attempt (approx. 35%). More than 40% of the deceased had been suffering from at least one additional mental disorder, in most cases from depression. At least one critical life event (in most cases, a relapse) or a period of abstinence (i.e., due to imprisonment, therapy or detoxification) during the past three months before death was reported for more than half of the addicts. The results were discussed in the light of data on opiate users and the general population. Improved specialist training of therapeutic and medical workers as well as of any other co-operating professionals is considered a necessary prerequisite for an early detection of risk factors. PMID- 12375234 TI - [Migration and health: is segregation inevitable?]. AB - In the immigrant-absorbing countries migrants are regularly confronted with manifold forms of segregation and exclusion. This is also true of the health care system, thus impeding it in its humanitarian goals. One must try to understand why there should be a traditional tendency to encourage segregation. Among the reasons are explicit rejection of migration as such, poor understanding of other peoples, fear of infectious diseases and rising health care costs. In addition a misunderstanding of the integration process plays an important role. What is necessary is, firstly, a realistic view of the world-wide process of migration which inevitably affects the rich industrial nations and, secondly, respect for ethnic identity instead of an out-dated concept of assimilation. PMID- 12375235 TI - Genetic proof of unequal meiotic crossovers in reciprocal deletion and duplication of 17p11.2. AB - A number of common contiguous gene syndromes have been shown to result from nonallelic homologous recombination (NAHR) within region-specific low-copy repeats (LCRs). The reciprocal duplications are predicted to occur at the same frequency; however, probably because of ascertainment bias and milder phenotypes, reciprocal events have been identified in only a few cases to date. We previously described seven patients with dup(17)(p11.2p11.2), the reciprocal of the Smith Magenis syndrome (SMS) deletion, del(17)(p11.2p11.2). In >90% of patients with SMS, identical approximately 3.7-Mb deletions in 17p11.2 have been identified. These deletions are flanked by large (approximately 200 kb), highly homologous, directly oriented LCRs (i.e., proximal and distal SMS repeats [SMS-REPs]). The third (middle) SMS-REP is inverted with respect to them and maps inside the commonly deleted genomic region. To investigate the parental origin and to determine whether the common deletion and duplication arise by unequal crossovers mediated through NAHR between the proximal and distal SMS-REPs, we analyzed the haplotypes of 14 families with SMS and six families with dup(17)(p11.2p11.2), using microsatellite markers directly flanking the SMS common deletion breakpoints. Our data indicate that reciprocal deletion and duplication of 17p11.2 result from unequal meiotic crossovers. These rearrangements occur via both interchromosomal and intrachromosomal exchange events between the proximal and distal SMS-REPs, and there appears to be no parental-origin bias associated with common SMS deletions and the reciprocal duplications. PMID- 12375237 TI - Posterior-stabilized versus cruciate-retaining total knee arthroplasty: balancing the gap. AB - A prospective, randomized, double-blind trial was carried out to compare cruciate retaining (CR) and posterior-stabilized (PS) total knee arthroplasties (TKAs). A total of 40 knees were randomized to receive either a NexGen CR (Zimmer, Warsaw, IN) or a Legacy PS (Zimmer, Warsaw, IN) TKA. All knees were implanted with identical surgical technique, making sure to balance precisely the flexion extension gaps before implantation of the components. At 2-year follow-up, there was no difference between the CR and the PS TKAs with respect to their Knee Society clinical, functional, and radiographic scores. These findings suggest that with careful attention to surgical technique and balancing the knee, orthopaedic surgeons should expect similar results whether they use a CR or PS TKA. PMID- 12375236 TI - Maternal genetic effects, exerted by genes involved in homocysteine remethylation, influence the risk of spina bifida. AB - There is currently considerable interest in the relationship between variation in genes that are involved in the folate-homocysteine metabolic axis and the risk of spina bifida. The evaluation of this relationship is, however, complicated by the potential involvement of both the maternal and the embryonic genotype in determination of disease risk. The present study was designed to address questions regarding both maternal and embryonic genetic risk factors for spina bifida by use of the two-step transmission/disequilibrium test. Analysis of data on variants of two genes involved in homocysteine remethylation/methionine biosynthesis--methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G--provided evidence that both variants influence the risk of spina bifida via the maternal rather than the embryonic genotype. For both variants, the risk of having a child with spina bifida appears to increase with the number of high-risk alleles in the maternal genotype: MTR (R1=2.16, 95% CI 0.92-5.06; R2=6.58, 95% CI 0.87-49.67) and MTRR (R1=2.05, 95% CI 1.05-3.99; R2=3.15, 95% CI 0.92-10.85). These findings highlight the importance of considering both the maternal and embryonic genotype when evaluating putative spina bifida susceptibility loci. PMID- 12375238 TI - The effect of partial or full weight bearing ambulation after cementless total hip arthroplasty. AB - The clinical and radiographic results of 46 patients who underwent 50 consecutive primary total hip arthroplasties using a fully porous-coated collared femoral component were determined at a minimum of 2 years' follow-up. Twenty-four patients (25 hips) who were allowed to bear full weight immediately postoperatively were compared with a historical control group of 24 patients (25 hips) who were instructed to bear < or =50 lb of weight for 6 weeks. The average Harris hip score for the partial weight bearing group was 95 compared with 97 for the full weight bearing group. All femoral components in both groups had radiographic evidence of bone ingrowth fixation at the final follow-up. When solid initial fixation is obtained intraoperatively and radiographically using a fully porous-coated (AML) femoral component, it seems that bone ingrowth fixation reliably occurs whether or not a partial or full weight-bearing postoperative protocol is followed. PMID- 12375239 TI - Factors associated with excessive migration in bone impaction hip revision surgery: a radiostereometric analysis study. AB - A prospective radiostereometric analysis (RSA) study of 18 patients with cemented revision hip surgery and impaction grafting with an Exeter stem was done with a follow-up of 2 years for all patients. All factors that could influence migration (ie, micromotion) of the stem were analyzed with a repeated measurements analysis of variance. Two groups could be identified: a stable group and a continuous migrating group. Two factors significantly influenced micromotion during the follow-up measurements. The first factor was the Paprosky classification (the bigger the defect, the higher the micromotion). The second factor was cement mantle defects in > or =1 Gruen zones. The migrating hip stems had more Gruen zones with cement mantle defects (45%) compared with the stable prostheses (21%). The effect of the first factor on micromotion was limited and probably clinically less relevant. Because the cement mantle defects found in this study were caused by poor instrumentation, the second factor stresses the importance of good instrumentation, which is essential to make this technically demanding technique effective in creating a stable stem-allograft construct in the defective femoral canal. PMID- 12375240 TI - Femoral impaction grafting in revision hip arthroplasty with irradiated bone. AB - We evaluated the results of femoral impaction grafting with the Exeter stem (Stryker Howmedica Osteonics, Newbury, UK) and irradiated bone-graft. We followed 57 hips for an average of 27 months. Endo-Klinik grading showed 8 grade 1, 22 grade 2, and 27 grade 3 hips. Radiographic analysis revealed cortical repair in 34% and graft incorporation in 39% but no evidence of trabecular remodeling. Moderate subsidence (5-10 mm) occurred in 7 patients (12.5%), and massive subsidence (>10 mm) occurred in 4 patients (7%). Complications included 6 dislocations, 3 periprosthetic fractures, and 2 stem revisions. Impaction grafting with the Exeter system produces satisfactory results for most patients, but a few hips perform poorly, and the reasons for this are unclear. We have concerns about irradiated bone-graft because the characteristic changes of graft remodeling are not seen. PMID- 12375241 TI - Cementless total hip arthroplasty with a close proximal fit and short tapered distal stem (third-generation) prosthesis. AB - This study presents the results of a prospective, consecutive series of 50 patients (60 hips) who were observed for a minimum of 6 years after they had a primary total hip arthroplasty with a cementless Duraloc 100 series cup (DePuy, Warsaw, IN) with a close proximal fit and short tapered distal stem prosthesis (IPS hip; DePuy, Leeds, UK). There were 37 men and 13 women; the mean age was 46.6 years (range, 26 to 70 years). The mean follow-up was 6.3 years (range, 6 to 7 years). The mean preoperative Harris hip score was 42 points, which improved to 96 points at the final follow-up examination. The prevalence of transitory thigh pain was 2%. All hips had a satisfactory fit of the femoral stem in coronal (average, 88%) and sagittal (average, 94%) planes. There was no aseptic loosening or revision of the components. The wear rate per year was 0.23 mm. Four hips (7%) had osteolysis in the calcar femorale <1 cm in diameter. Although there was no aseptic loosening of the components, a low incidence of osteolysis, and a low incidence of thigh pain, a high rate of polyethylene liner wear in these young patients is a problem. PMID- 12375242 TI - A retrospective clinical and radiographic review of 173 hydroxyapatite-coated screw cups with 5- to 10-year follow-up, showing low revision rates for fixation failure. AB - We reviewed the midterm results of 173 hydroxyapatite-coated screw cups. The average follow-up was 6.5 years (range, 5-9 years). The follow-up rate was 93%. Patients were assessed using the Merle D'Aubigne-Postel clinical scoring scale and by radiographic review. Two patients had revision surgery for recurrent dislocation (1.2%), 3 patients were revised for aseptic loosening (1.7%), 1 patient underwent revision surgery because of deep prosthetic infection (0.6%), and 2 patients were revised for polyethylene wear without loosening (1.2%), which gave a total revision rate of 4.7%. The average postoperative Merle D'Aubigne Postel scores were 5.7 for pain, 5.5 for range of motion, and 5.4 for function. PMID- 12375243 TI - Evaluation of the porous-coated anatomic hip at 12 years. AB - A total of 55 consecutive cementless total hip arthroplasties were done for osteoarthritis by 1 surgeon, who was not a designer of the implant, using 1 surgical technique and Porous-Coated Anatomic (Howmedica, Rutherford, NJ) hip implants. Of hips, 53 were prospectively evaluated, and 39 were followed for an average of 12.3 years (range, 10-16 years). Eight patients (8 hips) died, and 6 of 53 hips (11.3%) required revision for aseptic loosening at an average of 6.5 years. Five of 53 (9.4%) acetabular components were revised at an average of 7.9 years. Four of 53 (7.6%) femoral components were revised at an average of 5.1 years. Survivorship of these total hip arthroplasties was 98 +/- 3.9 at 5 years, 88.9 +/- 9.2 at 10 years, and 86.7 +/- 9.9 at 13 years with revision as the endpoint. PMID- 12375244 TI - Success rate of modular component exchange for the treatment of an unstable total hip arthroplasty. AB - Hip instability is the leading cause of morbidity after total hip arthroplasty. Surgical strategies that have been used to eliminate recurrent instability include component revision, trochanteric advancement, or the use of constrained components. Between 1986 and 1997, 731 revision total hip arthroplasties were performed at our institution. A total of 29 patients underwent modular component exchange to treat hip instability. After revision surgery, 16 of 29 (55%) patients experienced redislocation. Nine (31% overall) patients dislocated repeatedly after modular component exchange. Five of the 9 patients who dislocated repeatedly (17% overall) ultimately required rerevision to obtain stability. Modular component exchange is an unpredictable procedure in definitively solving hip stability problems. The limitations of this procedure in treating this complex multifactorial problem must be understood by patient and surgeon alike. PMID- 12375245 TI - Heterotopic ossification after total hip arthroplasty: a critical analysis of the Brooker classification and proposal of a simplified rating system. AB - We evaluated the reproducibility of the Brooker classification for heterotopic ossification (HO) and, based on the results and weaknesses observed, proposed a simplified system with addition of objective criteria. Six observers classified radiographs of 169 total hip arthroplasties, using the Brooker classification and a modified system consisting of i) absence of HO or islands measuring <1 cm in length, ii) islands >1 cm or spurs leaving at least 1 cm between femur and pelvis, and iii) spurs leaving <1 cm between opposing surfaces or bony ankylosis. Reproducibility was calculated using kappa statistics. For the Brooker classification, interobserver kappa averaged 0.43 (range, 0.74-0.18) (poor). Intraobserver kappa averaged 0.74 (fair). For the modified classification, interobserver kappa averaged 0.59 (range, 0.51-0.76) (fair). Intraobserver kappa averaged 0.78 (good). Interobserver differences were significant (P=.0085). Interobserver consistency to detect severe HO (Brooker 3 and 4, or grade C) improved from 52% to 76% with the modified system. The new classification showed adequate interobserver reproducibility, less variability, and improved consistency for classification of significant HO. PMID- 12375246 TI - Comparison of the LISS and a retrograde-inserted supracondylar intramedullary nail for fixation of a periprosthetic distal femur fracture proximal to a total knee arthroplasty. AB - Simulated supracondylar fractures were created proximal to posterior cruciate ligament-retaining total knee arthroplasty components in paired human cadaver femora and stabilized with either a retrograde-inserted locked supracondylar nail or the Less Invasive Stabilization System (LISS; Synthes USA, Paoli, PA). Loads were applied to create bending and torsional moments on the simulated fracture stabilized with either no gap or a 10-mm gap. The LISS exhibited less torsional stability with anterior (P<.001) and posterior loads (P<.01). When varus loads were applied to 10-mm-gap specimens, the specimens stabilized with a retrograde nail had an 83% reduction in fracture displacement (P<.001) and 80% less medial translation of the distal fragment (P<.001). The samples stabilized with the LISS had a 93% reduction in fracture gap displacement when a valgus load was applied with a 10-mm gap (P<.001). Overall, these results suggest that the retrograde inserted nail may provide greater stability for the management of periprosthetic supracondylar femur fractures in patients with a posterior cruciate ligament retaining femoral total knee arthroplasty component. PMID- 12375247 TI - Effect of total knee arthroplasty on blood flow to the lower limb: a prospective clinical study and review of literature. AB - Ischemic complications after total knee arthroplasty (TKA) are rare. There is a reported association with chronic lower extremity ischemia (CLEI), however. This prospective study examined changes in blood pressure in the lower limb during the early postoperative period. Arterial blood pressure was assessed before and after TKA by the ankle brachial pressure index (ABPI) using Doppler ultrasonography. Seven patients with CLEI (ABPI <0.9) and 33 patients with normal vascular status (ABPI >0.9) were studied. An ABPI of 0.65, correction of minor knee deformity, and a tourniquet time of 2 hours did not alter arterial blood pressure. TKA does not affect the distal arterial blood pressure in the lower limb in normal and CLEI-affected limbs in the immediate postoperative period. PMID- 12375248 TI - An algorithm for the treatment of Vancouver type B2 periprosthetic proximal femoral fractures. AB - The number of individuals with periprosthetic fractures of the proximal part of the femur is increasing. Multiple treatment methods have been described but none that correlate to fracture type and few with mostly good-to-excellent results. This article describes an algorithmic reconstruction tactic for treating patients with periprosthetic fractures associated with a loose femoral component. The stem is approached through the fracture fragments. Reconstructing the tube of the proximal part of the femur with 18G cerclage wires allows for canal preparation and implantation of the new stem. Application of the allograft struts and cables maximizes the biomechanical integrity of the proximal part of the femur to promote fracture repair and implant fixation. No treatment failures have occurred as of this date. PMID- 12375249 TI - Serum levels of cobalt and chromium in a complex modular total hip arthroplasty system. AB - There is concern that modularity in a total hip arthroplasty system increases serum cobalt and chromium ion levels. This study measures the serum cobalt and chromium levels in patients with an Oxford Universal Hip (Corin, Cirencester, United Kingdom), which has a modular sliding mechanism; patients with a similarly manufactured hip with no sliding mechanism; and a control group. Loosening was excluded clinically and radiologically. Arthroplasty patients had statistically higher levels of serum cobalt and chromium than controls, but there was no significant difference in levels between the implanted groups. PMID- 12375250 TI - The uncemented Bi-Contact total hip arthroplasty. AB - We reviewed a consecutive series of 153 uncemented Bi-Contact (Aesculap, Tuttlingen, Germany) total hip arthroplasties (THAs) in 138 patients who had been followed for at least 5 years (mean, 6.8 years; range, 5-9 years). The Bi-Contact uncemented THA consists of a straight femoral stem made of titanium alloy. The proximal portion of the stem is titanium plasma-sprayed. The cup is press-fit with or without hydroxyapatite coating with a facility for anchoring screws with a snap-fit polyethylene liner. The mean age of the patients was 70.8 years (range, 41-94 years). The mean preoperative Harris hip score of 41 (range, 20-80) improved postoperatively to a mean of 92 (range, 56-96). Three acetabular cups were revised for aseptic loosening, and 1 cup was revised for recurrent dislocation. To date, none of the stems have been revised for aseptic loosening. Radiographic evaluation of the remaining 149 hips revealed that the acetabular cup was stable in 146 hips and possibly unstable in the remaining 3 cases with nonprogressive osteolysis behind the cup. None of the stems showed any evidence of instability. Using the recommendation of revision as the endpoint, the cumulative survival for the prosthesis was 97.3% at a mean follow-up of 6.8 years (95% confidence interval, 95.9-99.4), with stem survival of 100%. In the medium term, these results are comparable to cemented primary THA and justify the continued use of this prosthesis. PMID- 12375251 TI - Comparison of hydroxyapatite and hydroxyapatite tricalcium-phosphate coatings. AB - This study compared the effects of hydroxyapatite (HA) coating and biphasic HA/tricalcium-phosphate (HA/TCP) coating on the osseointegration of grit-blasted titanium-alloy implants. Each coated implant was compared with uncoated grit blasted implants as well. The implants were press-fit into the medullary canal of rabbit femora, and their osseointegration was evaluated 3 to 24 weeks after surgery. The coated implants had significantly (P<.05) greater new bone ongrowth than the uncoated implants (HA, 56.1 +/- 3.1%; HA/TCP, 53.8 +/- 2.6%; uncoated, 32.2 +/- 1.4% of the implant perimeter, 12 weeks). Unmineralized tissue (cartilage and osteoid) was seen on the uncoated implants but never on the coated implants. The coated implants had significantly (P<.05) greater interfacial shear strength than the uncoated implants (HA, 4.1 +/- 0.4 MPa; HA/TCP, 4.8 +/- 0.5 MPa; uncoated, 2.6 +/- 0.2 MPa, 12 weeks). There was no difference between HA and HA/TCP coating in regard to new bone growth or interfacial shear strength. These data show a comparable enhancement effect of HA and HA/TCP coatings on the osseointegration of titanium-alloy implants. PMID- 12375252 TI - Precision of EBRA-Digital software for monitoring implant migration after total hip arthroplasty. AB - We assessed the precision of the EBRA-Digital software (EBRA, University of Innsbruck, Innsbruck, Austria) for measuring implant migration after total hip arthroplasty. Study subjects (n = 29) underwent consecutive, standardized, plain radiographic examinations of the hip on the same day after repositioning. The resulting radiograph pairs were digitized and analyzed using EBRA. The precision (95% confidence interval) of the method for measuring migration and wear was <+/ 0.9 mm for both implant components. The 95% confidence intervals for measurement of cup inclination and anteversion and femoral stem/shaft angle were <+/-1.7 degrees. Measurement precision was not strongly related to patient gender, digitization method, or observer. The EBRA-Digital method has sufficient precision to detect clinically relevant migration to allow individual patient monitoring after total hip arthroplasty. The method requires careful patient positioning and radiographic technique to produce consistently images suitable for analysis. PMID- 12375253 TI - Direct plain radiographic methods versus EBRA-Digital for measuring implant migration after total hip arthroplasty. AB - We aimed to determine whether the precision and sensitivity of migration measurements after total hip arthroplasty (THA) using direct plain radiographic techniques could be made comparable to those of digital methods (EBRA-Digital; University of Innsbruck, Innsbruck, Austria) by careful control of radiographic technique and use of modern measuring tools. Precision was examined by analysis of consecutive radiographs taken after repositioning in 20 patients after hybrid THA. The precision (95% confidence interval) of measurements for cup migration using direct methods was +/-1.11 to 3.07 mm (x-axis) and +/-1.28 to 1.92 mm (y axis). The precision of EBRA for cup measurements was +/-1.00 mm (x-axis) and +/ 0.82 mm (y-axis). The precision of stem y-axis migration measurements was +/-1.12 to 6.91 mm using direct methods and +/-0.80 mm using EBRA. Migration of the stem (1.53 mm subsidence; P<.01) and the cup (0.53 mm cranial migration, P<.05) was detected using EBRA in 10 patients followed for 6 months after hybrid THA, but significant migration was not detectable using the most precise of the direct methods. Careful measures to standardize plain radiographs improve precision of direct radiographic measurements; however, their long-term sensitivity remains inferior to methods that employ quality control and measurement algorithms to measure migration from digitized radiographs. PMID- 12375254 TI - Load transfer and fixation mode of press-fit acetabular sockets. AB - Adequate initial fixation is a prerequisite for osseointegration and secondary stability of noncemented cups. Physiologic force transmission between the cup and acetabulum guarantees the best long-term fixation. To study load transfer within the natural hip joint and in the bone-implant interface of 2 different hemispherical noncemented press-fit cups, 10 hips were investigated in an experimental setup simulating single-leg stance. Load distribution and contact area were measured using prescale pressure-sensitive films and digital image analysis. Three dominant locations near the periphery of the acetabulum could be identified. Main load transfer occurs in the cranial region of the acetabulum, where it is buttressed by the iliac bone; the second location is at the posterior inferior region at the ischial facet, and the third location is at the anterior region, where support is provided by the pubic bone. Peripheral rim contact was present in both cups but not completely circumferential. It showed marked loading at the same 3 locations similar to the natural hip joint. The ilioischial diagonal axis produced the highest press-fit. Peak local forces were found at the ischial and iliac facets. Local forces can be grouped into an iliac, an ischial, and a pubic group contributing 55%, 25%, and 20% to the total hip joint force. Pole contact was not present in the natural hip and with the biradial press-fit cup with flattened pole area but was observed with the pure hemispherical cup. Hence, stable fixation of an acetabular cup is achieved best by a 3-point-like bony support at the iliac, ischial, and pubic bone. The acetabular fovea does not provide functional support of the femoral head or endoprosthetic socket. In revision surgery, remaining peripheral bone stock at the iliac, ischial, and pubic locations allows stable implantation of primary cups. PMID- 12375255 TI - Vertical acetabular positioning with an inclinometer in total hip arthroplasty. AB - Vertical acetabular implant positioning is an important technical aspect in total hip arthroplasty. To evaluate the potential benefit of an inclinometer, 50 cup insertions were done on a cadaver pelvis. Acetabular cup vertical angles averaged 44.4 degrees +/- 11.4 degrees by visuospatial perception and 42.2 degrees +/- 3.8 degrees with the inclinometer. All cups were within the safe angle range of 40 degrees to 49 degrees with the inclinometer compared with 64% of cups by visuospatial perception. Use of the inclinometer reduced variability by a factor of 2.0 to 4.5. The addition of an inclinometer for acetabular cup insertion increases the probability of positioning the cup within a vertical safe range during total hip arthroplasty on a cadaver pelvis, suggesting that it could be a useful adjunct in clinical practice. PMID- 12375256 TI - Factors affecting patellar tracking after total knee arthroplasty. AB - This study examined factors that influence patellar tracking after total knee arthroplasty. A total of 62 knees were evaluated radiographically for postoperative patellar tracking. Six factors were examined regarding their influence on postoperative patellar tracking. This study showed the effects of patellar component position, patellar resection angle, and lateral retinacular release on postoperative patellar tracking. There was no significant effect of the remaining 3 factors: the thickness of the patellar resection, preoperative patellar tilt, and rotational alignment of the femoral component. A medialized patellar component and obliquity of resection of the patella are effective for obtaining proper patellar tracking, whereas the evaluation of the influence of the external rotation of the femoral component requires more clinical studies. PMID- 12375257 TI - Acute renal failure after local gentamicin treatment in an infected total knee arthroplasty. AB - Local gentamicin treatment in revision surgery for infected hip and knee prostheses is well established. It is a safe and effective method compared with the systemic use of aminoglycosides. Although nephrotoxic side effects are uncommon, we report a case of acute renal failure after 2-stage revision treatment of an infected knee prosthesis with gentamicin-impregnated beads and block spacers. The combined use of beads and a cement block spacer, both gentamicin impregnated, may have induced this severe complication. Use of this procedure in elderly patients warrants careful follow-up of renal function. PMID- 12375258 TI - Screw migration from total knee prostheses requiring subsequent surgery. AB - Complications in total knee arthroplasty directly related to hardware failure other than polyethylene wear are rare. We report 2 cases of symptomatic screw migration into the joint space from total knee prostheses. In the first case, a screw disengaged from a constrained condylar knee prosthesis. Arthroscopy using standard arthroscopy portals and a small arthrotomy were performed to remove the screw. In the second case, symptomatic screw disengagement and posterior migration from the tibial component of a posterior-stabilized prosthesis occurred. Revision with replacement of the polyethylene insert and locking screw was required. PMID- 12375259 TI - The framework of pathology: good laboratory practice by quantitative and molecular methods. AB - Combined confocal laser scan microscopy (CLSM) and Fourier analysis (FA) by non pathologists of dermal collagen bundle orientation recently gave results superior to subjective evaluation by experts. According to Good Laboratory Practice (GLP) criteria, combined CLSM/FA has not yet been adequately tested to replace current collagen evaluation, but this will not take long. Non-pathologists (clinicians) will then have taken over a laboratory test historically belonging to pathology. A general trend in this direction may develop, because pathologists seem not always to care enough about clinical significance, reproducibility and prognostic value, and new demands for innovative methods. Quantitative image analysis (QIA) and molecular methods are reproducible, inexpensive, and easy to perform; they often have greater value than classical evaluations and their cost-benefit ratio is good. However, their acceptance is not as widespread as one would expect and theoretical reasons which have been advanced do not provide a satisfactory explanation. A formal implementation study was therefore performed, in which an attempt was made to modernize a classical pathology laboratory. An external customer satisfaction investigation showed that 96% of the clinicians were 'very satisfied' (the highest rating possible) with the completed innovations, contrasting with low satisfaction at the beginning. Lack of primary innovative leadership among pathologists was judged to be the dominant cause preventing implementation. Pathologists should focus on carefully reacting to new clinical needs, using GLP criteria. Reproducibility and predictive accuracy should be major themes in any pathology practice. PMID- 12375260 TI - Morphometry of dermal collagen orientation by Fourier analysis is superior to multi-observer assessment. AB - In human dermis, collagen bundle architecture appears randomly organized, whereas in pathological conditions, such as scar tissue and connective tissue disorders, collagen bundle architecture is arranged in a more parallel fashion. Histological examination by one or two observers using polarized light is the most common method to determine collagen orientation. The hypothesis on which this study is based is that an objective image analysis technique, Fourier analysis, would improve the reliability (are the measurements reproducible?) and the accuracy (does the method measure what it is supposed to measure?) of collagen orientation assessment, compared with observer ratings. Fourier analysis was applied to 271 images of scar tissue and normal skin that were acquired by confocal laser scanning microscopy. Observers rated the same areas using polarized light as well as the confocal microscopy images. Computer images consisting of different types of ellipses were generated with a fixed orientation. Observers and Fourier analysis evaluated the images to evaluate accuracy. The inter-observer reliability was acceptable when at least three observers rated polarized light images (r > 0.69), whereas two observers were sufficient for rating confocal microscopy images (r > 0.71). Fourier analysis correlated better with observer ratings of confocal microscopy images (r = 0.69) than with polarized light microscopy images (r = 0.42). Fourier analysis was more accurate than four observers for the evaluation of the 'true' orientation for almost all types of computer-generated images. For the first time it is shown that Fourier image analysis is suitable for the morphometry of dermal collagen orientation and leads to a superior measurement of collagen orientation compared with subjective histological evaluation by several experts. If an evaluation is performed by conventional light microscopy, at least three observers are required to attain an acceptable inter-observer reliability. PMID- 12375261 TI - Inter-laboratory and inter-observer reproducibility of immunohistochemical assessment of the Ki-67 labelling index in a large multi-centre trial. AB - Proliferative activity of tumour cells, as assessed by the Ki-67 labelling index, has been suggested as a potential prognostic indicator in many neoplastic diseases. Meaningful application of the immunohistochemically determined tumour cell growth fraction in clinical decision-making requires information about its inter-laboratory reproducibility. To assess the reproducibility of Ki-67 determined growth fraction, a multi-centre immunohistochemical trial was performed with 172 participating laboratories, each testing 30 different tissue samples. Evaluating 5160 Ki-67 labelling indices with a newly developed tissue microarray, good inter-observer reproducibility but high inter-laboratory variability was found. Reassessment of all stainings revealed considerable inter laboratory differences in the intensity and frequency of labelled nuclei, suggesting that antigen retrieval or staining techniques are predominantly responsible for the inter-laboratory variability found in this trial. Consequently, cut-off levels for Ki-67, suggested to distinguish prognostic subgroups in tumours, appear to have limited reproducibility in a multi-centre approach. It is concluded that there is a need to standardize the immunohistochemical determination of the Ki-67 labelling index when it is used as a prognostic indicator in surgical pathology. PMID- 12375262 TI - Numerical aberrations of chromosome 1 in cervical intraepithelial neoplasia are strongly associated with infection with high-risk human papillomavirus types. AB - The aims of this study were to assess the relationships between numerical aberrations of chromosome 1 and the presence of high-risk human papillomavirus (HPV). Five normal samples, 11 CIN1, 13 CIN2, 18 CIN3, and nine carcinomas were studied by in situ hybridization (ISH), using a DNA probe for the centromere of chromosome 1 (cen#1) and a DNA probe cocktail for HPV types 16 and 18. A short fragment polymerase chain reaction hybridization line probe assay (SPF-PCR-LiPA) technique was used to detect 25 HPV types. The mean number of cen#1 per nucleus (chromosome index, CI) was measured, and the fractional areas of dysplastic epithelium with HPV16/18 infection and with cen#1 aneusomy were estimated. Disomy was found in all normal epithelium and in 36% of CIN1. Tetrasomy was observed in 64% of CIN1, 15% of CIN2, and 17% of CIN3. Hyper-tetrasomy was observed in 77% of CIN2, 83% of CIN3, and 100% of invasive carcinomas. High-risk HPVs were present in 20%, 75%, and 94% of disomic, tetrasomic, and hyper-tetrasomic lesions, respectively. The mean CI value was significantly higher in the lesions infected with high-risk HPV than in the lesions not infected by high-risk HPV (p < 0.001), due to the significantly higher prevalence of hyper-tetrasomy. The ISH study disclosed that HPV16/18 was exclusively found within dysplastically altered epithelium. The area with aneusomy is mostly enclosed within the area infected with HPV. In 83% of the HPV16/18-positive CIN lesions, the fractional area of HPV infected epithelium was equal to, or larger than, the fractional area with aneusomy. In conclusion, aneusomy for chromosome 1 is strongly associated with high-grade CIN lesions and infection with high-risk HPV; it is likely that the occurrence of numerical aberrations of chromosome 1 is preceded by infection with high-risk HPV. PMID- 12375263 TI - Frequent expression of the Epstein-Barr virus (EBV)-induced gene, EBI3, an IL-12 p40-related cytokine, in Hodgkin and Reed-Sternberg cells. AB - Epstein-Barr virus (EBV)-associated Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) usually occur in patients without clinically manifest deficiencies in anti-viral immunity. In spite of expressing viral proteins, both tumours are apparently able to escape EBV-specific immunity in vivo. EBI3 is an EBV-induced cytokine homologous to the interleukin (IL)-12 p40 subunit and can heterodimerize with IL-12 p35. It has been suggested that EBI3 may function to antagonize IL-12 and to inhibit the development of a Th1 immune response. EBI3 expression has been studied in tumour entities frequently associated with EBV infection to examine if EBI3 might contribute to local modulation of the immune response. It is shown that EBI3 is strongly expressed in Hodgkin and Reed Sternberg cells in 32 of 33 HL cases, independently of the EBV status of the tumour cells. Furthermore, EBI3 expression was detected in the epithelial tumour cells of six of 40 NPC biopsies but not in Burkitt lymphomas. The results suggest that EBI3 may be an additional component of the repertoire employed by Hodgkin and Reed-Sternberg cells to inhibit an effective anti-tumour or anti-viral immune response. PMID- 12375264 TI - Regulation of IGFBP-5 expression during tumourigenesis and differentiation of oral keratinocytes. AB - To identify molecular events involved in the pathogenesis of oral squamous cell carcinoma (OSCC), genes differentially expressed in OSCC and non-cancerous matched tissue (NCMT) samples were analysed using a subtractive hybridization strategy. NCMT-enriching clones that have been linked to suppressor pathway in previous studies were subjected to advanced analyses. Complete absence of insulin like growth factor binding protein-5 (IGFBP-5) expression at both the mRNA and the protein level was identified in nearly all (5/6) OSCC cell lines with the exception of the SCC25 cell line, which exhibited high IGFBP-5 expression. However, this protein is consistently present in cultured normal human oral keratinocytes (NHOKs). Immunohistochemistry revealed moderate to strong cytoplasmic immunoreactivity of IGFBP-5 in the stratum spinosum and stratum granulosum in the vast majority of NCMT samples. A remarkable reduction in IGFBP 5 immunoreactivity was detected in 56% (26/46) of OSCC samples, compared with the corresponding NCMT (p < 0.0001). Induction of differentiation in both NHOKs and SCC25 up-regulated IGFBP-5 expression. Administration of a green tea compound with anti-cancer properties, (-)-epigallocatechin 3-gallate, at a concentration of 5-20 micro g/ml also up-regulated IGFBP-5 expression in NHOKs in a dose dependent manner. The findings suggest that IGFBP-5 may be an important factor in the differentiation of oral keratinocytes and that down-regulation of IGFBP-5 may be involved in the neoplastic transformation of oral keratinocytes. PMID- 12375265 TI - Alterations of the INK4a-ARF gene locus in pleomorphic adenoma of the parotid gland. AB - Pleomorphic adenomas of the parotid gland are benign tumours composed of epithelial and mesenchymal cells. The INK4a-ARF (CDKN2A) locus on chromosome 9p21 encodes two tumour suppressor proteins, p16(INK4a) and p14(ARF), which act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in pleomorphic adenomas, this study analysed alterations of p14(ARF), p16(INK4a), p53, and pRb in these tumours. After microdissecting the different histological components, 42 pleomorphic adenomas of the parotid gland were analysed for INK4a-ARF inactivation by DNA sequence analysis, methylation specific PCR (MSP), restriction enzyme-related polymerase chain reaction (RE PCR), mRNA expression, microsatellite analysis, and immunohistochemistry. In addition, microdeletion of p14(ARF) and p16(INK4a) were assessed by differential PCR. The status of p53 and Rb was examined by direct sequencing and immunohistochemistry. Using microdissection, it was possible to examine the tumour components, i.e. epithelial, mesenchymal, and transitional, separately after immunohistochemical identification. Methylation of p14(ARF) was found in 1/42 cases and alterations of p16(INK4a) occurred in 12/42 of pleomorphic adenomas, which correlated with loss of mRNA transcription. Microdeletions or specific mutations of either exon were not detected. Methylation was detected exclusively in the epithelial and transitional components and not within the mesenchymal part of the tumour. p53 mutations were detected in 4/42 adenomas, also occurring solely in the epithelial components of the tumours. pRb was detected immunohistochemically in 40/42 adenomas. In normal, corresponding parotid tissue, p14(ARF), p16(INK4a), p53, and pRb alterations were not observed. The observation that alterations of p14(ARF) and p16(INK4a), and also p53 mutations, occurred exclusively in the epithelial and transitional components of pleomorphic adenoma supports the theory that these areas are prone to malignant transformation to carcinoma in adenoma. PMID- 12375266 TI - Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma. AB - PIK3CA, encoding the catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PI3K), is activated in malignant diseases. However, the role of the PIK3CA gene aberrations for tumourigenesis of head and neck squamous cell carcinoma (HNSCC) is to date unclear. The present study was designed to determine the genomic aberration of PIK3CA in invasive HNSCC and dysplastic precursor lesions by fluorescence in situ hybridization (FISH) with a YAC probe, containing the PIK3CA gene, on isolated interphase nuclei from histomorphologically well-defined regions of formalin-fixed tissue sections and to compare these data with protein and mRNA expression of p110alpha. The mRNA and protein levels of p110alpha were assessed, respectively, by in situ hybridization and immunohistochemistry on consecutive tissue sections. Copy number gains at 3q26 were observed in one of six low-to-moderate dysplasias (17%) and in seven of nine high-grade dysplasias (78%), as well as in 11 carcinomas (100%). In addition, one of seven high-grade dysplasias (14%) and 6 of 11 carcinomas (55%) had amplifications of 3q26. The majority of cases with copy number gain in more than 50% of the cells and/or amplification in more than 10% of cells showed increased p110alpha mRNA and protein expression, whereas only two cases (18%) (one high-grade dysplasia and one carcinoma) with no gain or low-level gain displayed increased p110alpha protein expression. These data suggest that 3q26 copy number gain and amplification represent early genomic aberrations in HNSCC carcinogenesis. In addition, p110alpha mRNA and protein expression in HNSCC may be regulated by these genomic aberrations as well as by epigenetic events. PMID- 12375267 TI - Gains and losses of adhesion molecules (CD44, E-cadherin, and beta-catenin) during oral carcinogenesis and tumour progression. AB - The aim of this study was to define whether or not the impaired expression of CD44, E-cadherin (E-cad), and beta-catenin (beta-cat) correlates with the clinical evolution and prognosis of oral cancer. Ninety-three primary oral squamous cell carcinomas (OSCCs) with tumour-adjacent normal and/or dysplastic mucosa, 30 associated metastases, and 12 recurrences were immunostained for CD44s, -v3, -v4, -v5, -v6, -v7, -v9, E-cad, and beta-cat. In non-neoplastic epithelium, all molecules investigated were constitutively expressed in the basal layers. In the majority of dysplasias, immunoreactivity for all adhesion molecules was increased, but there was restricted loss for CD44s, E-cad, and beta cat in a few cases. In carcinomas, a striking accumulation of CD44s, v3, v4, v9 and a loss of E-cad/beta-cat were observed at the invasive tumour front. In metastases and recurrences, besides a loss of CD44s, v4, v7, and E-cad, a significant increase of v9 was recorded, whereas CD44v5 and v6 remained unchanged. Clinically, reduced expression of CD44v3, E-cad, and changes of CD44v9 phenotype within the primary tumours correlated significantly with poor prognosis; decreased beta-cat expression was a predictive marker for nodal metastases. These findings indicate that there is some perturbed expression of adhesion molecules during the stepwise course of oral carcinogenesis and tumour progression. Distinct phenotypic alterations project poor prognosis, while others predict metastasis. Some of these restricted molecular changes may serve as potential targets for future antibody-based tumour therapy. PMID- 12375268 TI - The random development of LOH on chromosome 9q in superficial bladder cancers. AB - Allelic loss on chromosome 9q is a very frequent event in bladder carcinogenesis. In recent years, efforts have been directed towards identifying the postulated tumour suppressor genes on this chromosome arm by deletion mapping and mutation analysis. However, no convincing candidate genes have been identified. This paper describes the development of chromosome 9q alterations in multiple recurrent superficial bladder cancers of ten patients and shows that loss of heterozygosity (LOH) on this chromosome is almost never the characteristic first step. The regions of loss are multiple and variable in different tumours from the same patient and expand in subsequent tumours. Moreover, the regions of loss vary from patient to patient. It is concluded that even if 9q harbours a bladder cancer gatekeeper gene, it is unlikely that the gene will be identified through LOH analysis alone. PMID- 12375269 TI - Towards defining roles and relationships for tenascin-C and TGFbeta-1 in the normal and neoplastic urinary bladder. AB - Tenascin-C (TN-C) is an extracellular matrix glycoprotein expressed along epithelial/stromal boundaries during tissue remodelling events, such as those that occur during morphogenesis, wound healing, and tumour invasion. Using clinical specimens and a range of in vitro models that simulate homeostasis, wound healing, and malignant progression, this study sought to establish the patterns of TN-C expression in normal and neoplastic bladder and to determine the role of exogenous transforming growth factor beta-1 (TGFbeta-1), interleukin-4 (IL-4), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) in the induction of TN-C expression by bladder uro-epithelial cells. The findings indicate that normal urothelial cells may express TN-C, with both TGFbeta-1 and IL-4 able to induce expression. TN-C was not expressed in neoplastic urothelium, although both TN-C and TGFbeta-1 may be involved in tissue remodelling during papillary tumour formation and invasion. Furthermore, the urothelium of high-grade papillary tumours and carcinoma in situ specimens exhibited little TGFbeta-1 immunoreactivity, compared with the urothelium of low-grade tumours and normal specimens, suggesting an association between TGFbeta-1 expression and urothelial differentiation. A tumour invasion model, in which established bladder cancer cell lines were seeded onto a normal bladder stroma, corroborated the evidence from the clinical specimens and demonstrated that TN-C was strongly expressed around foci of stromal invasion. Thus, TN-C immunoreactivity may provide an additional tool in the assessment of early stromal invasion in bladder cancer. PMID- 12375270 TI - Antimicrobial peptides are expressed and produced in healthy and inflamed human synovial membranes. AB - The objective of this study was to determine the expression and production of antimicrobial peptides by healthy and inflamed human synovial membranes. Deposition of the antimicrobial peptides lysozyme, lactoferrin, secretory phospholipase A(2) (sPA(2)), matrilysin (MMP7), human neutrophil alpha-defensins 1-3 (HNP 1-3), human beta-defensin 1 (HBD-1), and human beta-defensin 2 (HBD-2) was determined by immunohistochemistry. Expression of mRNA for the antimicrobial peptides bactericidal permeability-increasing protein (BPI), heparin binding protein (CAP37), human cationic antimicrobial protein (LL37), human alpha defensin 5 (HD5), human alpha-defensin 6 (HD6), HBD-1, HBD-2, and human beta defensin 3 (HBD-3) was analysed by reverse transcription polymerase chain reaction (RT-PCR). RT-PCR revealed CAP37 and HBD-1 mRNA in samples of healthy synovial membrane. Additionally, HBD-3 and/or LL37 mRNA was detected in synovial membrane samples from patients with pyogenic arthritis (PA), osteoarthritis (OA) or rheumatoid arthritis (RA). BPI, HD5, HD6, and HBD-2 mRNAs were absent from all samples investigated. Immunohistochemistry identified lysozyme, lactoferrin, sPA(2), and MMP7 in type A synoviocytes of all samples. HBD-1 was only present in type B synoviocytes of some of the samples. Immunoreactive HBD-2 peptide was only visible in some inflamed samples. HNP1-3 was detected in both healthy and inflamed synovial membranes. The data suggest that human synovial membranes produce a broad spectrum of antimicrobial peptides. Under inflammatory conditions, the expression pattern changes, with induction of HBD-3 in PA (LL37 in RA; HBD-3 and LL37 in OA) as well as down-regulation of HBD-1. HBD-3 holds therapeutic potential in PA as it has a broad spectrum of antimicrobial activity and accelerates epithelial healing. However, caution is appropriate since defensins also promote fibrin formation and cell proliferation - key elements in joint infection. Clarification of the role of antimicrobial peptides in OA and RA will require further investigation. PMID- 12375271 TI - Multiple symmetric lipomatosis may be the consequence of defective noradrenergic modulation of proliferation and differentiation of brown fat cells. AB - Multiple symmetric lipomatosis (MSL) is an inherited disorder in which enlarging and unencapsulated lipomas symmetrically develop in the subcutaneous tissue of the neck, shoulders, mammary, and truncal regions. In some cases, it is associated with mitochondrial DNA abnormalities. The pathogenesis of MSL is completely unknown, although the fat deposits may be due to a neoplastic-like proliferation of functionally defective brown adipocytes. It has recently been demonstrated that the beta(3)-adrenergic receptor is the functionally relevant adrenergic receptor subtype in brown adipocytes and that its stimulation by noradrenaline (NA) modulates the expression of genes, such as uncoupling protein (UCP)-1 and inducible nitric oxide synthase (iNOS), involved in fat cell proliferation and differentiation. Furthermore, Trp64Arg mutation of the beta(3) adrenoceptor has been implicated in lower NA activity in adipose tissues. The aim of this study was to investigate the molecular and functional characteristics of MSL adipocytes and to analyse the effects of nitric oxide (NO) on the proliferation/differentiation of MSL adipocytes in culture, and the relevance of putative noradrenergic deficit in the development of lipomas in MSL patients. Cultured MSL adipocytes were able to synthesize UCP-1 (the selective marker of brown adipocytes), but unlike that of normally functioning brown fat cells, the expression of the UCP-1 gene was not significantly induced by NA. NA is also defective in inducing iNOS gene expression, thus leading to reduced NO production and a consequent reduction in the anti-proliferative, adipogenic (mitochondrial biogenesis) effects of NA on MSL cells. Furthermore, the transcriptional peroxisome proliferator-activated receptor gamma co-activator-1 (PGC-1), which plays a key role in the sympathetic-stimulated mitochondrial biogenesis of brown adipocytes, is expressed but not induced by NA in MSL cells, as it is in brown adipocytes. The study did not find any association between beta(3)-adrenoceptor gene polymorphism and noradrenergic signalling defects in MSL subjects with or without mitochondrial DNA mutations. PMID- 12375272 TI - MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis. AB - Three major MAP kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase (p38 MAPK), are involved in the regulation of lung inflammation and injury. This study investigated whether MAPKs are activated and associated with lung injury in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The expression of the active ERK, JNK, and p38 MAPK was examined using western blot analysis and immunohistochemistry and apoptosis was also examined by the TUNEL method, in lung tissues from ten patients with IPF obtained by thoracoscopic biopsy and in eight normal lung parenchyma specimens obtained by lobectomy for lung cancer. Activated MAPKs are significantly increased in lung homogenates from patients with IPF compared with controls. Activated ERK in epithelial and endothelial cells, but not in fibroblasts or smooth muscle cells, was decreased, accompanied by the progression of fibrosis. Activated JNK in epithelial and endothelial cells, but not in fibroblasts, was increased, accompanied by the progression of fibrosis. Activated p38 MAPK in epithelial, endothelial, smooth muscle cells, and fibroblasts was increased at the intermediate stage of fibrosis, in which the TUNEL-positive cells were predominantly detected. This is the first study to suggest that MAPKs may be associated with the regulation of inflammation and lung injury in IPF. PMID- 12375273 TI - Transforming growth factor-beta receptors in self-limited vs. chronic progressive nephritis in rats. AB - Increases in transforming growth factor-beta (TGF-beta) expression and extracellular matrix accumulation are transient in acute self-limited mesangial proliferative glomerulonephritis induced by a single injection of anti-thymocyte serum (ATS), while these increases persist following repeated injections that produce chronic progressive sclerosing glomerulonephritis with tubulointerstitial lesions. However, little is known about the expression of TGF-beta receptors (TbetaRs) in cells involved in the proliferative and sclerosing renal lesions. A study of protein and mRNA expression for type I (TbetaRI), type II (TbetaRII), and type III (TbetaRIII) TbetaR in both forms of nephritis was therefore carried out by immunohistochemistry and in situ hybridization. Inhibition of cell proliferation and stimulation of matrix production by TGF-beta1 were assessed in isolated glomeruli using [(3)H]thymidine incorporation and [(3)H]proline metabolic labelling, respectively. In acute self-limited nephritis, expression of TbetaRI, TbetaRII, and TbetaRIII increased in the glomerular and Bowman's capsular epithelial cells comprising the glomerular tuft adhesions to Bowman's capsules. However, TbetaRII expression was not prominent in proliferating mesangial cells. Glomeruli isolated from rats with acute self-limited nephritis at day 7, when mesangial cell proliferation was maximal, were partially resistant to the mitoinhibitory effects of TGF-beta1. In contrast, expression of all three TbetaRs was elevated in glomerular and tubulointerstitial lesions in chronic progressive nephritis, and glomeruli isolated from rats with chronic progressive nephritis 7 days after the second ATS injection were sensitive to TGF-beta1. These data suggest that distinct cellular responses to TGF-beta1 resulting from differential expression of TbetaR underlie the difference between acute self limited mesangial proliferative and chronic progressive sclerosing ATS nephritis in the development of proliferative and sclerotic renal lesions. PMID- 12375274 TI - Therapeutic effect of sulphated hyaluronic acid, a potential selectin-blocking agent, on experimental progressive mesangial proliferative glomerulonephritis. AB - The initial event in the process of leukocyte infiltration is characterized by leukocyte rolling on the surface of the endothelium, which is mediated by selectins. P- and L-selectin bind to the sulphated sugar chains of their natural ligands, including sulphated glycolipids such as sulphatide. Recently, it has been demonstrated that sulphated glycolipids and sulphated oligosaccharides interfere with selectin binding pathways. This study synthesized sulphated hyaluronic acid (SHA), which is a potential selectin-blocking agent, and examined its therapeutic effect on the experimental progressive mesangial proliferative glomerulonephritis induced by anti-Thy-1 monoclonal antibody (1-22-3 MAb) after unilateral nephrectomy. The selectin-inhibitory effect of SHA in vitro was confirmed. SHA inhibited the binding of P- and L-selectin to sulphatide, which is a glycolipid ligand for P- and L-selectin, at a concentration of 1.5 micro g/ml and 100 micro g/ml. Immunohistochemical examination showed that P-selectin was up regulated in the glomeruli in the 1-22-3 MAb nephritis model, while the ligands for L-selectin were not detected in the glomerular tufts. A single administration of SHA ameliorated proteinuria and glomerular leukocyte infiltration in 24 h after the injection of anti-Thy-1 MAb. Anti-P-selectin MAb, but not anti-L selectin MAb, inhibited proteinuria and glomerular leukocyte infiltration. To examine further the therapeutic effect of SHA on chronic glomerulonephritis, SHA was administered daily from day 3 to day 14 in this model. Proteinuria and glomerular leukocyte infiltration were significantly diminished in SHA-treated rats on day 14. These results suggest that SHA ameliorated rat progressive mesangial proliferative glomerulonephritis by inhibiting P-selectin-dependent leukocyte infiltration in glomeruli. Sulphated oligosaccharides may be beneficial for the therapy of mesangial proliferative glomerulonephritis. PMID- 12375275 TI - A capsule review of recent studies on the application of mass spectrometry in the analysis of Chinese medicinal herbs. AB - Chinese herbal medicine is gaining increasing popularity worldwide as an alternative approach to the development of pharmaceuticals in therapeutic applications. Chemical characterization and compositional analysis of Chinese medicines provide the necessary scientific basis for the discovery and development of new drugs of natural origin. Applications of mass spectrometry in the analysis of Chinese herbal medicines have been growing rapidly in recent years owing to the rapid technical advances and increasing availability of the instrumentation. This paper reviews the current status of how different mass spectrometric techniques are being used to support research studies of Chinese medicines. The focus is on crude herbal medicines and their derived products. The review is not meant to be exhaustive, but rather to provide a general overview of the various research activities in this rapidly expanding field. In the discussion of specific herbs, the emphasis is placed on ginseng and Danshen, two of the herbs for which active experimental work is on-going in the authors' laboratories. Other selected herbs will be discussed only briefly, aiming primarily to illustrate the current status of research in the area. PMID- 12375276 TI - Attachment of neutrals during tandem mass spectrometry of sulfonic acid dyes and intermediates in an ion trap. AB - Several positional isomers of 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids prepared as reference materials for development of analytical methods involved in FDA certification of D&C Yellow No. 10 (Quinoline Yellow) were found consistently to show [MH + 14](+) ions when their electrospray- or atmospheric pressure chemical ionization-prepared MH(+) ions were subjected to collisional activation. The source of these ions was found to be the methanol used as solvent in these procedures which combined with their [MH - H(2)O](+) ions under chemical ionization conditions. The reaction was found to be sensitive to their isomeric and chemical structures and other examples of this process are reviewed. PMID- 12375277 TI - Structural characterization of acetylpyridinium-ethyl pyruvate adducts by electrospray ionization mass spectrometry. AB - The reactions of ethyl pyruvate with acetic anhydride and pyridine were studied by electrospray ionization mass spectrometry (ESI-MS). Ethyl 2-acetoxy-2 pyridiniumpropionate (1) (m/z 238) resulting from the reaction of the acetylpyridinium cation with ethyl pyruvate, and the adduct of ethyl 2 acetoxyacrylate with a pyridinium cation (2), bound together by non-covalent interactions (m/z 238), were identified by ESI-MS for the first time. Structures 1 and 2 cannot be distinguished, probably because one may be converted into the other and vice versa. PMID- 12375278 TI - Detection of single nucleotide polymorphisms using electrospray ionization mass spectrometry: validation of a one-well assay and quantitative pooling studies. AB - Single nucleotide polymorphisms (SNPs) are currently being mapped and databased at a remarkable pace, providing a viable means for understanding disease susceptibility, differential drug response and human evolution. Consequently, there is an increasing demand for SNP genotyping technologies that are simple, rapid, cost effective and readily amenable to automation for high-throughput analyses. In this study, we improved the Survivor Assay, a SNP detection method based on electrospray ionization mass spectrometry (ESI-MS), with several developments. One improvement is the development of a one-well assay, requiring no off-line purification of the polymerase chain reaction product, achieved by simple addition of reagent solution into a single well. Another is the on-line separation of magnesium and dideoxynucleotides using an in-house made monolithic metal chelating column, eliminating any off-line sample preparation prior to mass spectrometric analysis. Here the Survivor Assay is extended from a proof-of principle concept to a validated method by genotyping six SNPs from five different regions of human genomic DNA in 55 individual samples with 100% accuracy. This improved Survivor Assay eliminates the tedious and time-consuming steps of sample preparation, minimizes sample handing and offers a high throughput analysis of SNPs by ESI-MS. The current combined preparation and analysis time is 2 min per sample. The simplicity of this method has potential for full automation and parallel chromatography and, thus, reduced analysis time. In addition, we have adapted the Survivor Assay for quantitative SNP analysis in pooled DNA samples. The capabilities and sensitivity of this approach were evaluated. We demonstrate that an allele occurring at a frequency of 2% can consistently be quantitated. PMID- 12375279 TI - Chemical mass shifts in resonance ejection experiments in the quadrupole ion trap. AB - Chemical mass shifts were measured in a Paul ion trap operated in the mass selective instability scan with resonance ejection using a custom-built instrument. These shifts, which can be as much as 2%, decrease with increasing endcap electrode separation owing to changes in the higher order contributions to the electric field. They also decrease with decreasing helium buffer gas pressure. Both of these effects are analogous to those found with boundary ejection. This suggests that the previously proposed chemical mass shift mechanism based on compound-dependent collisional modification of the ejection delay produced by field faults near the endcap electrode apertures holds true also for resonance ejection. The influence of the resonance frequency on chemical mass shifts was also investigated and it is shown that at certain working points (values of the Mathieu parameter q(z) and a(z)) non-linear resonances greatly reduce the ejection delay for all ions, regardless of their chemical structures, and thus reduce the magnitude of the chemical mass shift. Energetic collisions leading to dissociation can take place at an earlier stage during the ejection process in the mass analysis scan when using resonance ejection compared with boundary ejection. This leads to even larger chemical mass shifts of fragile ions in resonance ejection. Increasing the resonance voltage amplitude can enhance this effect. The chemical mass shifts of fragile ions increase with increase in the resonance voltage amplitude, whereas negligible changes occur for structurally stable ions. PMID- 12375280 TI - Fast screening of anabolic steroids and other banned doping substances in human urine by gas chromatography/tandem mass spectrometry. AB - A fast and sensitive method for the comprehensive screening of anabolic agents and other banned doping substances using gas chromatography/tandem mass spectrometry (GC/MS/MS) with an external ionization ion trap mass spectrometer is presented. The method takes advantage of the resolving power of MS/MS to eliminate background interferences, thus speeding up the chromatographic analysis. For each compound, different fragmentation reactions were studied and their collision energies optimized to obtain the best sensitivity in terms of their signal-to-noise ratio (S/N). A dramatic reduction in overall analysis time was achieved compared with other common approaches. More than 50 substances could finally be monitored in less than 7.4 min with detection limits (S/N >3) lower than 0.5 ng ml(-1) for most of the compounds with special sensitivity requirements according to the International Olympic Committee (IOC). A validation procedure for qualitative analysis was performed. The selectivity of the method showed that no interfering peaks were observed at the retention time of the analytes. Good intermediate precision, below 25% for most of the compounds, and robustness were observed. The optimized method was successfully applied to analyse more than 100 real human urine samples with optimum sensitivity and specificity rates. PMID- 12375281 TI - Effects of different alkali metal ions on the cationization of poly(ethylene glycol)s in matrix-assisted laser desorption/ionization mass spectrometry: a new selectivity parameter. AB - The cationization of poly(ethylene glycol)s, PEG 4000 and PEG 6000, under matrix assisted laser desorption/ionization conditions was studied by using different concentration ratios of the sodium ion, as the reference ion, and another alkali metal ion (Li(+), K(+), Rb(+), Cs(+)). A linear correlation was found between the intensity ratio of the sodiated PEGs and PEGs cationized with alkali metal ions versus the initial concentration ratio of sodium and alkali metal ions. The slopes of these straight lines are proposed as a novel selectivity ratio for the ionization process. The intensity distribution of the cationized PEGs was also investigated. It was found that the cationized oligomers follow Poisson statistics. The M(n) and M(w) values were also evaluated. An explanation for the observed effects is given. PMID- 12375282 TI - Probing electrochemical properties of pi-conjugated thienylenevinylenes/fullerene C(60) adducts by ESI/MS: evidence for dimerized cation-radicals. AB - Oligothienylenevinylenes/C(60) dyads n-C and triads n-C(2) are studied by electrospray mass spectrometry. A clear correlation is observed between the nature of the charged species detected by mass spectrometry, i.e. protonated molecule [M + H], (+) cation radical M(+.) and dication M(++), and the oxidation potentials of the molecules. Moreover, under defined solubility conditions, mass spectrometry provides conclusive evidences for the reversible dimerization of cation-radicals of n-C(2) compounds. PMID- 12375284 TI - Current literature in mass spectrometry. PMID- 12375283 TI - Mapping and sequencing of cardiolipins from Geobacillus stearothermophilus NRS 2004/3a by positive and negative ion nanoESI-QTOF-MS and MS/MS. AB - In the course of systematic studies on surface layer (S-layer) glycoproteins of bacilli, the chloroform/methanol extract from whole cells of Geobacillus stearothermophilus NRS 2004/3a has been submitted to MS analysis. Glucosylated cardiolipins were found as minor components of the total lipid and phospholipid mixture by de novo identification. After purification of the crude extract using a combined column chromatography/2D TLC protocol, structural investigations of components in the lipid fraction by high resolution ESI-QTOF MS analysis provided evidence about homologous molecules attributable to the cardiolipin species containing a glycosylated backbone, and about a diversity of ester-linked fatty acid substituents. In comparative studies by positive and negative ion nanoESI QTOF-CID-MS, maps of cardiolipin molecular ions were obtained, followed by MS/MS of the most abundant species, to provide structural details of D glucopyranosylcardiolipin and the fatty acid substituent patterns. Experiments of the parent ion scan type revealed the presence of fatty acid moieties as isobaric combinations, represented in single molecular ion species. PMID- 12375285 TI - Training microsurgeons: a rewarding experience. PMID- 12375286 TI - Acute remote ischemic preconditioning on a rat cremasteric muscle flap model. AB - A previous study showed, in a rat adipocutaneous flap model, that acute ischemic preconditioning (IP) can be achieved not only by preclamping of the flap pedicle, but also by a brief extremity ischemia prior to flap ischemia. The purpose of this study was to determine whether remote IP is also effective in other tissues such as muscle flaps. Twenty male Wistar rats were divided into three experimental groups. The rat cremaster flap in vivo microscopy model was used for assessment of ischemia/reperfusion injury. In the control group (CG, n = 8), a 2 hr flap ischemia was induced after preparation of the cremaster muscle. In the "classic" IP group (cIP, n = 6), a brief flap ischemia of 10 min was induced by preclamping the pedicle, followed by 30 min of reperfusion. A 10-min ischemia of the contralateral hindlimb was induced in the remote IP group (rIP, n = 6). The limb was then reperfused for 30 min. Flap ischemia and the further experiment were performed as in the CG. In vivo microscopy was performed after 1 hr of flap reperfusion in each animal. A significantly higher red blood cell velocity in the first-order arterioles and capillaries, a higher capillary flow, and a decreased number of leukocytes adhering to the endothelium of the postcapillary venules were observed in both preconditioned groups by comparison to the control group (P < 0.05). The differences within the preconditioned groups were not significant for these parameters. Our data show that ischemic preconditioning and improvement of flap microcirculation can be achieved not only by preclamping of the flap pedicle, but also by induction of an ischemia/reperfusion event in a body area distant from the flap prior to elevation. These findings indicate that remote IP is a systemic phenomenon, leading to an enhancement of flap survival. Our data suggest that remote IP could be performed simultaneously with flap elevation in the clinical setting without prolongation of the operation and without invasive means. PMID- 12375288 TI - Discussion of acute remote ischemic preconditioning, parts I and II. PMID- 12375287 TI - Acute remote ischemic preconditioning II: the role of nitric oxide. AB - The purpose of this study was to determine whether nitric oxide (NO) plays a role in the mechanism of acute "classic" as well as acute remote ischemic preconditioning (IP). Thirty-two male Wistar rats were divided into five experimental groups. The rat cremaster flap in vivo microscopy model was used for assessment of ischemia/reperfusion injury. In the control group (CG, n = 8), a 2 hr flap ischemia was induced after preparation of the cremaster muscle. The animals of group NO (n = 6) received 500 nmol/kg of the NO-donor spermine/nitric oxide complex (Sper/NO) intravenously 30 min prior to ischemia. The group LN + P (L-NAME + preclamping, n = 6) received 10 mg/kg Nomega-nitro-L-arginine methyl ester (L-NAME) intravenously before preclamping of the flap pedicle (10-min cycle length, 30-min reperfusion). L-NAME (10 mg/kg) was administered in group LN + T (L-NAME + tourniquet, n = 6) before ischemia of the right hindlimb was induced, using a tourniquet for 10 min after flap elevation. The limb was then reperfused for 30 min. Thereafter, flap ischemia was induced in each group as in group CG. In vivo microscopy was performed after 1 hr of flap reperfusion in each animal. Group NO demonstrated a significantly higher red blood cell velocity (RBV) in the first-order arterioles and capillaries, a higher capillary flow, and a decreased number of leukocytes adhering to the endothelium (stickers) of the postcapillary venules by comparison to all other groups (P < 0.05). The average capillary RBV and capillary flow were still higher in the CG than in the groups receiving L NAME (P < 0.05). The data show that NO plays an important role in the mechanism of both acute "classic" as well as acute remote IP, since the administration of a NO-donor previous to ischemia simulates the effect of IP, whereas the nonspecific blocking of NO synthesis by L-NAME abolishes the protective effect of flap preconditioning. PMID- 12375289 TI - Dehydroepiandrosterone as an enhancer of functional recovery following crush injury to rat sciatic nerve. AB - This study was designed to investigate the effect of dehydroepiandrosterone (DHEA) on the recovery of the rat sciatic nerve following crush injury. A standard hemostat system was used to create the injury, with a length of 1.5 mm in three groups of 18 animals each. In group I, the crush injury was applied without any treatment. In groups II and III, vehicle (ethylene glycol) and DHEA solutions were injected subepineurally 30 min following the crush injury. Sciatic function index (SFI), toe contracture measurement, gastrocinemius muscle weight, total number of myelinated fibers, fiber diameters, myelin thickness, and axon/fiber cross-sectional ratio were measured at 3, 6, and 12 weeks. The SFI values in the DHEA group showed a faster return to normal values confirmed at 3 and 6 weeks (P < 0.05). The number of myelinated fibers and fiber diameters at 6 and 12 weeks were significantly higher in the DHEA group (P < 0.05). In this study, the subepineural injection of DHEA following crush injury was found to enhance functional recovery of the rat sciatic nerve. PMID- 12375290 TI - Superficial or deep implantation of motor nerve after denervation: an experimental study--superficial or deep implantation of motor nerve. AB - Neurorraphy, conventional nerve grafting technique, and artificial nerve conduits are not enough for repair in severe injuries of peripheral nerves, especially when there is separation of motor nerve from muscle tissue. In these nerve injuries, reinnervation is indicated for neurotization. The distal end of a peripheral nerve is divided into fascicles and implanted into the aneural zone of target muscle tissue. It is not known how deeply fascicles should be implanted into muscle tissue. A comparative study of superficial and deep implantation of separated motor nerve into muscle tissue is presented in the gastrocnemius muscle of rabbits. In this experimental study, 30 white New Zealand rabbits were used and divided into 3 groups of 10 rabbits each. In the first group (controls, group I), only surgical exposure of the gastrocnemius muscle and motor nerve (tibial nerve) was done without any injury to nerves. In the superficial implantation group (group II), tibial nerves were separated and divided into their own fascicles. These fascicles were implanted superficially into the lateral head of gastrocnemius muscle-aneural zone. In the deep implantation group (group III), the tibial nerves were separated and divided into their own fascicles. These fascicles were implanted around the center of the muscle mass, into the lateral head of the gastrocnemius muscle-aneural zone. Six months later, histopathological changes and functional recovery of the gastrocnemius muscle were investigated. Both experimental groups had less muscular weight than in the control group. It was found that functional recovery was achieved in both experimental groups, and was better in the superficial implantation group than the deep implantation group. EMG recordings revealed that polyphasic and late potentials were frequently seen in both experimental groups. Degeneration and regeneration of myofibrils were observed in both experimental groups. New motor end-plates were formed in a scattered manner in both experimental groups. However, they were more dense in the superficial implantation group than the deep implantation group. It was concluded that superficial implantation has a more powerful contractile capacity than that of deep implantation. We believe that this might arise from the high activity of glycolytic enzymes in peripheral muscle fibers of gastrocnemius muscle, decrease in insufficient intramuscular guidance apparatus, and intramuscular microneuroma formation at the insufficient neuromuscular junction since the motor nerve had less route to muscle fibers. PMID- 12375291 TI - Repeated upper limb salvage in a case of severe traumatic soft-tissue and brachial artery defect. AB - We present the case of a 9-year-old male patient who suffered a gunshot injury to the right arm. The patient arrived in shock, his right arm severely traumatized, with soft-tissue loss involving the anterior surface and both sides of the right arm. The humerus was exposed. There was brachial artery defect and damage to the lateral fibers of the median nerve. The mangled extremity severity score (MESS) was 8 points. The patient was treated with general resuscitation, blood transfusion, and debridement. A venous graft, 12 cm in length, to bridge the brachial artery defect, and tendon transfer, triceps to the biceps, was performed in one step. Postoperatively, there was a normal radial pulse, normal skin color, normal temperature, and normal movement of the fingers without pain. Unfortunately, the patient then sustained a second trauma to the right arm 3 weeks later, rupturing the graft. This time he lost 1,500 cc of blood. After another blood transfusion, we performed a second reverse saphenous vein graft. The patient stayed at the hospital for 3 weeks. At follow-up 12 months later, the limb has good function and, except for the presence of a scar and skin graft, is equal in appearance to the left side. PMID- 12375292 TI - Neurophysiologic and clinical outcome following medial pectoral to long thoracic nerve transfer for scapular winging: a case report. AB - The use of nerve transfers (neurotization) in the reconstruction of nerve palsy is not new, but its clinical efficacy is still largely based on reports of successful restoration of elbow flexion and shoulder abduction following brachial plexus avulsion. Although its potential application extends beyond the brachial plexus, little has been written about additional indications or associated postoperative outcomes. The case described in this report illustrates yet another indication for which neurotization may be a useful technique. Medial pectoral nerve transfer to the long thoracic nerve was performed via an 11-cm sural nerve graft to treat scapular winging 4 months following nerve injury caused during axillary node dissection. Neurophysiologic and clinical outcome 18 months postoperatively revealed successful reinnervation of the serratus anterior muscle, decreased scapular winging, and symptomatic improvement from the patient's perspective. PMID- 12375293 TI - Aesthetic and functional advantages of the anterolateral thigh flap in reconstruction of tumor-related scalp defects. AB - Eleven patients underwent free-flap reconstruction of tumor-related defects of the scalp, forehead, and temporal region. Flap selection aimed at achieving acceptable functional and aesthetic results combined with negligible donor-site morbidity. Ten males and one female, aged 61.3 +/- 14.3 years, were included in this study. Eight patients presented with tumor recurrences after previous surgery, irradiation, and/or chemotherapy. The average extension of defects was 169.5 (range, 30-600) qcm. Free flaps employed for reconstruction included antero lateral thigh flaps (8), suprafascial radial forearm flap (1), lateral arm flap (1), latissimus dorsi muscle flap (1), and myocutaneous vertical rectus abdominis flap (1). Other procedures included nerve grafts to the facial nerve (2), ectropion correction (2), and fascia lata slings for static procedure in facial palsy (2). There was no pedicle revision and no flap failure. Donor-site morbidity was negligible. Hospitalization averaged 9.2 +/- 1.7 days. The anterolateral thigh perforator flap offers excellent coverage of tumor-related defects of the scalp, which require a thin flap for adequate contouring. The customized harvested myocutaneous anterolateral thigh flap is regarded as an elegant option for covering defects which consist of both deep and superficial areas. Fascia lata and nerve grafts are available at the same donor site. This easily allows additional procedures for cosmetic and functional improvement that are of high benefit for patients. PMID- 12375294 TI - Local heparin is superior to systemic heparin in preventing arterial thrombosis. AB - Thrombosis poses a significant problem in microvascular surgery, despite antithrombotic therapy. The purpose of the present study was to investigate whether a topical application of unfractionated heparin is equally efficient as a systemic bolus in avoiding thrombosis. A rat femoral artery model was used. Three different doses of systemic heparin (50, 100, and 200 U/kg) and one dose of locally administered heparin (100 U/ml) were evaluated and compared to a control group receiving isotonic saline. A thrombogenic injury, simulating poor microsurgical technique, was applied to the artery. The thrombus area was visualized by transillumination, and recorded for 60 min on video, for subsequent measurement. In addition, the level of activated partial thromboplastin time (APTT) and of anti-activated clotting factor X (aXa) was determined. Local heparin significantly reduced thrombus size as compared to isotonic saline and systemic heparin (50 and 100 U/kg). High-dose systemic heparin (200 U/kg) was equally potent, but local heparin had significantly less influence on the hemostatic parameters. PMID- 12375295 TI - Effect of Timolol vs. a postural orthotic on hand tremor during microsurgery. AB - The efficacy of beta blockers to control hand tremor remains equivocal. This study evaluated the effectiveness of Timolol, as well as a postural orthotic, in reducing movement deviation during a laboratory exercise. Eleven volunteers completed three randomized trials involving administration of Timolol, a placebo, or a postural orthotic. Drug administration was blinded. Each trial consisted of a simulated 2-hr clinical procedure, punctuated by video recordings of the subject's hand motion during a standardized pattern of instrument movement. ECRL EMG activity and digital oxygen perfusion were also measured. Recordings were converted into estimates of linear variance at baseline and 1 and 2 hr. A repeated-measures ANOVA, or the Friedman test, were used to assess significant differences. Movement deviation, EMG activity, and oxygen perfusion were unaffected by the duration of exercise or treatment (P > or = 0.454). We conclude that none of the treatments accorded a significant benefit in allaying hand tremor. PMID- 12375296 TI - A simple confidence interval for meta-analysis. AB - In the context of a random effects model for meta-analysis, a number of methods are available to estimate confidence limits for the overall mean effect. A simple and commonly used method is the DerSimonian and Laird approach. This paper discusses an alternative simple approach for constructing the confidence interval, based on the t-distribution. This approach has improved coverage probability compared to the DerSimonian and Laird method. Moreover, it is easy to calculate, and unlike some methods suggested in the statistical literature, no iterative computation is required. PMID- 12375297 TI - Model misspecification sensitivity analysis in estimating causal effects of interventions with non-compliance. AB - Randomized trials often face complications in assessing the effect of treatment because of study participants' non-compliance. If compliance type is observed in both the treatment and control conditions, the causal effect of treatment can be estimated for a targeted subpopulation of interest based on compliance type. However, in practice, compliance type is not observed completely. Given this missing compliance information, the complier average causal effect (CACE) estimation approach provides a way to estimate differential effects of treatments by imposing the exclusion restriction for non-compliers. Under the exclusion restriction, the CACE approach estimates the effect of treatment assignment for compliers, but disallows the effect of treatment assignment for non-compliers. The exclusion restriction plays a key role in separating outcome distributions based on compliance type. However, the CACE estimate can be substantially biased if the assumption is violated. This study examines the bias mechanism in the estimation of CACE when the assumption of the exclusion restriction is violated. How covariate information affects the sensitivity of the CACE estimate to violation of the exclusion restriction assumption is also examined. PMID- 12375298 TI - Cancer-specific survival of patients with multiple cancers: an application to patients with multiple breast cancers. AB - In the analysis of cause-specific survival, the causes of death must be known. For single-cancer patients with a known cause of death, the estimation of the cause-specific survival rate is straightforward. For multiple-cancer patients with two primary cancers, however, the analysis of cause-specific survival rates is more complex, particularly if the cancers are of the same primary site. In these situations, a concept of cancer-specific survival may also be distinguished from cause-specific survival. Cancer-specific survival rates are studied here by introducing two models, the primary one where the death from cancer is attributed to one of the cancers, and an alternative where such an attribution is not necessary. The models are illustrated using data on patients with multiple breast cancers. The model-based survival rates are compared with each other and with the corresponding relative survival rates based on analogous modelling of relative survival. The results show that for the subsequent breast cancer, the cancer specific survival rates based on the alternative, where the distinction between the cancers as a cause of death was not necessary, tended to be higher than those based on that distinction. It is thus possible that the subsequent cancer was too often coded as a cause of death, particularly when being localized at diagnosis. PMID- 12375299 TI - A copula-based model for multivariate non-normal longitudinal data: analysis of a dose titration safety study on a new antidepressant. AB - A new model for multivariate non-normal longitudinal data is proposed. In a first step, each longitudinal series of data corresponding to a given response is modelled separately using a copula to relate the marginal distributions of the response at each time of observation. In a second step, at each observation time, the conditional (on the past) distributions of each response are related using another copula describing the relationship between the corresponding variables. Note that there is no need to consider the same family of distributions for these response variables. The technique is illustrated in a dose titration safety study on a new antidepressant. The haemodynamic effect on diastolic blood pressure, systolic blood pressure and heart rate is studied. These three responses are measured repeatedly over time on ten healthy volunteers during the dose escalation. The available covariates are sex and the concentration of drug in the plasma at time of measurement. PMID- 12375300 TI - Proportional hazards models with frailties and random effects. AB - We discuss some of the fundamental concepts underlying the development of frailty and random effects models in survival. One of these fundamental concepts was the idea of a frailty model where each subject has his or her own disposition to failure, their so-called frailty, additional to any effects we wish to quantify via regression. Although the concept of individual frailty can be of value when thinking about how data arise or when interpreting parameter estimates in the context of a fitted model, we argue that the concept is of limited practical value. Individual random effects (frailties), whenever detected, can be made to disappear by elementary model transformation. In consequence, unless we are to take some model form as unassailable, beyond challenge and carved in stone, and if we are to understand the term 'frailty' as referring to individual random effects, then frailty models have no value. Random effects models on the other hand, in which groups of individuals share some common effect, can be used to advantage. Even in this case however, if we are prepared to sacrifice some efficiency, we can avoid complex modelling by using the considerable power already provided by the stratified proportional hazards model. Stratified models and random effects models can both be seen to be particular cases of partially proportional hazards models, a view that gives further insight. The added structure of a random effects model, viewed as a stratified proportional hazards model with some added distributional constraints, will, for group sizes of five or more, provide no more than modest efficiency gains, even when the additional assumptions are exactly true. On the other hand, for moderate to large numbers of very small groups, of sizes two or three, the study of twins being a well known example, the efficiency gains of the random effects model can be far from negligible. For such applications, the case for using random effects models rather than the stratified model is strong. This is especially so in view of the good robustness properties of random effects models. Nonetheless, the simpler analysis, based upon the stratified model, remains valid, albeit making a less efficient use of resources. PMID- 12375301 TI - A semi-parametric accelerated failure time cure model. AB - A cure model is a useful approach for analysing failure time data in which some subjects could eventually experience, and others never experience, the event of interest. A cure model has two components: incidence which indicates whether the event could eventually occur and latency which denotes when the event will occur given the subject is susceptible to the event. In this paper, we propose a semi parametric cure model in which covariates can affect both the incidence and the latency. A logistic regression model is proposed for the incidence, and the latency is determined by an accelerated failure time regression model with unspecified error distribution. An EM algorithm is developed to fit the model. The procedure is applied to a data set of tonsil cancer patients treated with radiation therapy. PMID- 12375302 TI - A Markov model for sample size calculation and inference in vaccine cost effectiveness studies. AB - Sample size and power formulae are derived for designing trials of vaccine cost effectiveness in healthy working adults. When existing trials of influenza vaccine have presented sample size calculations, these have been based on vaccine effectiveness. A Markov model is introduced to account for non-independence of working days lost. Two effect sizes need to be specified, one for a reduction in the number of episodes of absence and the second for a reduction in the mean duration of episodes. The relative cost of vaccine provision is also incorporated. Applying the resulting formulae to published studies indicates that most were underpowered to detect any financial benefit. Moreover, biased estimates of variance have led to led to incorrect inference. PMID- 12375303 TI - On the potential of measurement error to induce differential bias on odds ratio estimates: an example from radon epidemiology. AB - It is well established that odds ratios estimated by logistic regression are subject to bias if exposure is measured with error. The dependence of this bias on exposure parameter values, particularly for multiplicative measurement error, and its implications in epidemiology are not, however, as fully acknowledged. We have been motivated by a German West case-control study on lung cancer and residential radon, where restriction to a subgroup exhibiting larger mean and variance of exposure than the entire group has shown higher odds ratio estimates as compared to the full analysis. By means of correction formulae and simulations, we show that bias from additive classical type error depends on the exposure variance, not on the exposure mean, and that bias from multiplicative classical type error depends on the geometric standard deviation (in other words on the coefficient of variation of exposure), but not on the geometric mean of exposure. Bias from additive or multiplicative Berkson type error is independent of exposure distribution parameters. This indicates that there is a potential of differential bias between groups where these parameters vary. Such groups are commonly compared in epidemiology: for example when the results of subgroup analyses are contrasted or meta-analyses are performed. For the German West radon study, we show that the difference of measurement error bias between the subgroup and the entire group exhibits the same direction but not the same dimension as the observed results. Regarding meta-analysis of five European radon studies, we find that a study such as this German study will necessarily result in smaller odds ratio estimates than other studies due to the smaller exposure variance and coefficient of variation of exposure. Therefore, disregard of measurement error can not only lead to biased estimates, but also to inconsistent results and wrongly concluded effect differences between groups. PMID- 12375304 TI - Parametric models for accelerated and long-term survival: a comment on proportional hazards. AB - The Cox proportional hazards model (CPH) is routinely used in clinical trials, but it may encounter serious difficulties with departures from the proportional hazards assumption, even when the departures are not readily detected by commonly used diagnostics. We consider the Gamel-Boag (GB) model, a log-normal model for accelerated failure in which a proportion of subjects are long-term survivors. When the CPH model is fit to simulated data generated from this model, the results can range from gross overstatement of the effect size, to a situation where increasing follow-up may cause a decline in power. We implement a fitting algorithm for the GB model that permits separate covariate effects on the rapidity of early failure and the fraction of long-term survivors. When effects are detected by both the CPH and GB methods, the attribution of the effect to long-term or short-term survival may change the interpretation of the data. We believe these examples motivate more frequent use of parametric survival models in conjunction with the semi-parametric Cox proportional hazards model. PMID- 12375305 TI - Multilevel modelling of medical data. AB - This tutorial presents an overview of multilevel or hierarchical data modelling and its applications in medicine. A description of the basic model for nested data is given and it is shown how this can be extended to fit flexible models for repeated measures data and more complex structures involving cross classifications and multiple membership patterns within the software package MLwiN. A variety of response types are covered and both frequentist and Bayesian estimation methods are described. PMID- 12375306 TI - Significance of increased circulating proteasome in autoimmune disease. PMID- 12375307 TI - Methotrexate: vital new information about a middle aged drug. PMID- 12375308 TI - Rheumatoid factors: what do they tell us? PMID- 12375309 TI - Rheumatoid arthritis: time for trials of therapeutic targets. PMID- 12375310 TI - Circulating proteasomes are markers of cell damage and immunologic activity in autoimmune diseases. AB - OBJECTIVE: The 20S proteasome plays a leading immunologic role in the cytosolic generation of MHC class I restricted antigens, and it represents an abundant antigen in several autoimmune diseases. To investigate the effects of autoimmune inflammatory and perioperative traumatic cellular damage, we determined qualitative and quantitative properties of released proteasomes (circulating proteasomes, cProteasomes) from serum samples of patients with a variety of autoimmune diseases. METHODS: cProteasomes were analyzed from serum samples of 314 patients with several systemic and organ-specific autoimmune diseases and 85 healthy controls. The concentrations of cProteasomes were determined by sandwich ELISA using a monoclonal and a polyclonal proteasome-specific antibody. Followup analyses were performed in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) as well as in patients with myasthenia gravis undergoing thoracoscopic thymectomy. RESULTS: Strongly increased levels of cProteasomes (> 1000 ng/ml) were detected in samples obtained from patients with autoimmune myositis, SLE, primary Sjogren's syndrome, RA, and autoimmune hepatitis. Significant differences were observed in the mean values of cProteasomes comparing systemic with organ-specific autoimmune diseases. Followup analyses revealed a close correlation of cProteasome with the autoimmune process as well as cellular damage. Moreover, cProteasomes were isolated in intact and native as well as in degraded or dissociated forms from the serum samples. The immuno-subunit LMP7 was found to be incorporated in the circulating protease complex. CONCLUSION: Levels of cProteasomes are markedly elevated in patients with systemic autoimmune diseases, apparently correlating with disease activity. The cProteasomes represent novel sensitive markers of the autoimmune inflammatory processes and/or reflect the magnitude of cellular damage. PMID- 12375311 TI - Anti-Sa sera from patients with rheumatoid arthritis contain at least 2 different subpopulations of anti-Sa antibodies. AB - OBJECTIVE: Anti-Sa antibodies have been described to be a highly specific marker for rheumatoid arthritis (RA). We demonstrate the existence of 2 different subsets of anti-Sa antibodies, only one of which is specific for RA. Our objective was to purify the Sa antigen, and to achieve partial characterization of these proteins. METHODS: Saline extract and mitochondrial extract from human placenta were used as antigenic sources. Antigens were purified by immunoaffinity chromatography and studied by ELISA and immunoblotting. RESULTS: Three antigenically active bands of 68, 50, and 46 kDa were purified from the saline extract by immunoaffinity chromatography. Two other bands of 29 and 10 kDa that do not react with anti-Sa antibodies were obtained as well. The 68 kDa band was purified from a mitochondrial extract. These bands are not the same as other known mitochondrial autoantigens such as M2, M4, or M9. The amino terminal sequence of the 68 kDa Sa band is DEPKXEVP. The sequence of the 68 kDa Sa band is not compiled in the databases we searched, as either aminoterminal or internal sequence. Antibodies to 50/46 kDa anti-Sa bands detected by immunoblotting were highly specific for RA, while the 68 kDa antigen reacted in ELISA with sera from patients with RA and systemic lupus erythematosus, the latter showing a marked increase in features of RA. Antibodies directed against the 68 and 50/46 kDa Sa bands fluctuated with time, the 50/46 kDa anti-Sa antibodies present during the active period of the disease, and the 68 kDa anti-Sa antibodies during the remission period. CONCLUSION: At least 2 subsets of autoantibodies are present in anti-Sa sera, one directed against a 68 kDa Sa protein and another to the typical 50/46 bands of the Sa system. PMID- 12375312 TI - Enhanced local production of osteopontin in rheumatoid joints. AB - OBJECTIVE: To investigate the involvement of osteopontin (OPN) in the pathogenesis of rheumatoid arthritis (RA), localization and production of OPN were examined in patients with RA. METHODS: Localization of OPN in the rheumatoid synovium was examined by immunohistochemistry. In vitro OPN production by cultured synovial cells from patients with RA (n = 5) and with osteoarthritis (OA) (n = 5) was assessed by ELISA. OPN concentrations in plasma and synovium were quantified in patients with RA (n = 23) by 2 distinct ELISA systems to measure both thrombin cleaved and non-cleaved OPN. The same experiments were done in patients with OA (n = 15) and healthy volunteers (n = 10) as a control. RESULTS: OPN was highly detected by immunohistochemistry predominantly in the RA synovial lining cells, while less and scattered OPN was detected in OA synovial tissues. ELISA revealed that cultured RA synovial cells secreted significantly more OPN than OA cells. ELISA also showed a marked increase of OPN levels in synovial fluid (SF) of patients with RA and with OA compared to the control plasma OPN levels, although OPN levels were not increased in RA and OA plasma compared to healthy controls. SF OPN levels of patients with RA were significantly higher than those of patients with OA, and correlated with serum C reactive protein levels. The ratios of thrombin cleaved versus non-cleaved OPN were significantly increased in RA plasma and SF compared with OA plasma and SF and plasma from healthy controls. CONCLUSION: Our results revealed enhanced local production of OPN in rheumatoid joints, suggesting involvement of OPN in the pathogenesis of RA. PMID- 12375313 TI - Clinical usefulness of genetic information for predicting radiographic damage in rheumatoid arthritis. AB - OBJECTIVE: To determine whether knowledge of genetic information aids the prediction of radiographic damage for patients with rheumatoid arthritis (RA) in whom extensive sociodemographic, family history, clinical, and immunologic information is available. METHODS: Subjects included 146 Caucasian women who were participants in a community based longitudinal study of RA. Our primary outcome measure was the severity of erosive disease. Nongenetic covariates included age at RA onset, disease duration, family history of RA, education level, family income, baseline values of function, painful and swollen joint groups and pain rating, and rheumatoid factor positivity. All women were genotyped for the HLA DRB1 shared epitope (SE) and the tumor necrosis factor a (TNFa) microsatellite. Likelihood ratio tests (LRT) were performed to evaluate the usefulness of genetic information for predicting radiographic damage in RA, after adjusting for nongenetic covariates. Receiver operating characteristic (ROC) curves displaying the sensitivity and specificity of combinations of nongenetic and genetic information were derived, and the areas under the curves (AUC) were compared. RESULTS: Genetic information contributed significantly to the prediction of radiographic damage in RA even after adjusting for all nongenetic covariates (p value for LRT = 0.0019). The odds ratio describing the risk of severe erosive disease among individuals who had inherited both the SE and TNFa allele 11 (TNFa11) was 7.6 compared to individuals who were SE and TNFa11 negative. Analysis of ROC curves confirmed the usefulness of genetic information. CONCLUSION: Genetic information is useful for predicting radiographic damage in RA even for patients in whom extensive sociodemographic, family history, clinical, and immunologic information is available. PMID- 12375314 TI - Rheumatoid factor and antibodies to cyclic citrullinated Peptide differentiate rheumatoid arthritis from undifferentiated polyarthritis in patients with early arthritis. AB - OBJECTIVE: To study the diagnostic value of IgM rheumatoid factor (RF), IgA-RF, antibodies to cyclic citrullinated peptide (anti-CCP), and combinations of these antibodies, measured at baseline, to discriminate rheumatoid arthritis (RA) from undifferentiated polyarthritis (uPA) in patients with recent onset arthritis. METHODS: Patients with early arthritis with peripheral arthritis of 2 or more joints and symptom duration less than 3 years were clinically diagnosed as having RA or uPA by an experienced rheumatologist during the first year. Patients with bacterial, psoriatic, or crystal induced arthritis or spondyloarthropathy were excluded. Optimal cutoff values for serum IgM RF, IgA RF, and anti-CCP were deduced from receiver operating characteristics curves in order to predict the diagnosis of RA in early arthritis. RESULTS: A total of 379 patients (69% female, median age 57 yrs, range 17-86 yrs) were studied; 258 patients were clinically diagnosed as RA and 121 as uPA. Both IgM-RF > 40 IU/ml and anti-CCP > 50 AU/ml showed high specificity, but the sensitivity of these tests was low. In many RA patients the occurrence of IgM-RF and anti-CCP antibodies was independent. Thus the optimal criterion proved to be the combination of IgM-RF > 40 or anti-CCP > 50, which yielded sensitivity of 55.4% and specificity of 96.7%. CONCLUSION: The criterion IgM-RF > 40 or anti-CCP > 50 is able to predict the diagnosis of RA in early arthritis patients with high specificity and acceptable sensitivity. Anti CCP testing combined with IgM-RF testing has additional value over IgM-RF testing alone in patients with early undifferentiated oligo and polyarthritis. PMID- 12375315 TI - Combination therapy with methotrexate and hydroxychloroquine for rheumatoid arthritis increases exposure to methotrexate. AB - OBJECTIVE: To examine the bioavailability of methotrexate (MTX) in the presence of hydroxychloroquine (HCQ), and vice versa, to determine a possible pharmacokinetic explanation for the observation that combination treatment of rheumatoid arthritis with MTX and HCQ has been shown, clinically, to be more potent than MTX used alone. METHODS: In a randomized crossover study, 10 healthy subjects received, on each of 5 dosing occasions, MTX alone as tablets or intravenous solution, HCQ alone as a tablet or oral solution, or a coadministered dose of MTX tablets with an HCQ tablet. The area under the concentration-time curve (AUC) was determined for each subject, on each dosing occasion, for each compound. RESULTS: The mean AUC for MTX was increased (p = 0.005) and the maximum MTX concentration (Cmax) decreased (p = 0.025) when MTX was coadministered with HCQ, compared to MTX administered alone. The time to reach Cmax for MTX administration, tmax, was also increased during the coadministration with HCQ (p = 0.072). The AUC of HCQ showed no significant difference (p = 0.957) between any of the dosing occasions. CONCLUSION: These results may explain the increased potency of the MTX-HCQ combination over MTX as a single agent and also the sustained effects of MTX when administered with HCQ. In addition, the reduced Cmax of MTX observed during the coadministration may explain diminution of acute liver adverse effects. Extra vigilance for MTX adverse effects during combination therapy with HCQ is recommended, especially if renal function is known to be decreased. PMID- 12375316 TI - The methotrexate therapeutic response in rheumatoid arthritis. AB - OBJECTIVE: Methotrexate (MTX) is used frequently as a disease modifying antirheumatic drug (DMARD) for rheumatoid arthritis (RA), and patients tend to continue taking this drug for longer periods than alternative single agents. The shape of the therapeutic response beyond one or 2 years, however, has not been fully studied. We examined the properties of the pure MTX "therapeutic segment," that period that begins with start of MTX and terminates when MTX is discontinued or another DMARD is added, by observational study. METHODS: We studied new MTX starts for the period 1988 through 1996 for 437 patients from a parent cohort of 4253 patients. Patients were drawn from 8 Arthritis, Rheumatism, and Aging Medical Information System (ARAMIS) data centers: 2 community based populations; 2 private rheumatological practices; 2 university referral practices; and 2 university clinics for underserved minority urban populations. Health Assessment Questionnaire (HAQ) Disability Index scores (0-3) were obtained prospectively each 6 months. RESULTS: At MTX start, patients had relatively long average disease duration of 16.7 years, and had moderately severe disability, with an initial HAQ mean disability score of 1.48. Over the 10 year period examined in the parent cohort of 4253 patients (and thus irrespective of therapy), the prevalence of MTX use rose from 19% to 45%, while mean HAQ disability declined from 1.34 to 1.11. This correspondence is consistent with an accrual of benefits from more frequent use of MTX and other DMARD over this period. The MTX therapeutic segment revealed a distinct shape. HAQ-Disability Index values began at 1.48 at baseline and declined to a maximal improvement of 1.23 at 30 months. This long period to maximum benefit may have been partly driven by a slow titration upward to an optimal dosage. After 42 months, disability for this population began to re-progress and reached 1.39 at 84 months, still below the pretreatment baseline. Re-progression to baseline was about 8 or more years. Cumulative disability averted with MTX treatment for this population was roughly 1.30 disability-unit-years. CONCLUSION: MTX treatment of RA in practice differs substantially from common perception and appears suboptimal by being too little, too late, and too long to treatment change. A modification of the "sawtooth strategy" in which the disease is "ratcheted down" by change of MTX therapy at 3 years or when re-progression has proceeded halfway to baseline, rather than waiting for return to baseline, is suggested by these data. Also suggested is the need for more rapid upward dosage titration and longer maintenance of an optimal or highest tolerated dosage. "Therapeutic segment" data provide insights into strategic approaches to management of RA since they allow estimation of population aggregate properties such as time to maximum benefit and the time to return to baseline. PMID- 12375317 TI - Longitudinal measurement of methotrexate liver concentrations does not correlate with liver damage, clinical efficacy, or toxicity during a 3.5 year double blind study in rheumatoid arthritis. AB - OBJECTIVES: In patients with rheumatoid arthritis (RA), we examined whether methotrexate (MTX) and MTX polyglutamate accumulation in the liver correlated with clinical efficacy or clinical/laboratory toxicity. We also began preliminary examination of a new histologic index of liver histology (the Iowa Score) relative to the Roenigk grading system. METHODS: Forty patients with RA participated in a prospective, double blind, 3.5 year study of MTX treatment. Liver biopsies, liver MTX and MTX polyglutamate concentrations, laboratory tests, evaluation of disease activity, and evaluation of adverse events were done prospectively at baseline and at 1, 2, and 3.5 years. Biopsies were examined using the Roenigk grading system and an additional histological scoring system. Radiochemical ligand binding assays and HPLC methods were used to measure MTX and MTX polyglutamates. Statistical analysis included ANOVA, linear regression, and logistic regression modeling. RESULTS: No significant changes in the mean values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, albumin, or hemoglobin occurred. A significant percentage of patients had at least one abnormal alkaline phosphatase, AST, or ALT (25 to 52%), although most abnormalities were small and transient. Histological abnormalities did not progress using either the Roenigk or the Iowa score. The last abnormal AST, the number of abnormal AST and ALT, and female sex correlated with histological liver abnormalities (r2 = 0.41) using a new preliminary histologic scoring system (the Iowa Score). Amount of alcohol use correlated with fatty change, and the MTX dose at biopsy was associated with liver histological abnormalities (p = 0.03 and 0.049, respectively). Total liver MTX concentrations were stable from Year 1 to Year 3.5 and the percentage of higher order polyglutamates was relatively high (38 to 56%) relative to monoglutamates. No correlation of these concentrations with clinical response or toxicity, histology, or liver function tests could be documented. CONCLUSION: This analysis describes the accumulation and stabilization of MTX concentrations in the liver and examined correlations between MTX liver concentrations, patient demographics, liver histology, concomitant medications, and disease activity. No such correlations were found, decreasing the likelihood that MTX concentrations in serum would be useful measures to predict significant hepatotoxicity. PMID- 12375318 TI - "5D" Outcome in 52 patients with rheumatoid arthritis surviving 20 years after initial disease modifying antirheumatic drug therapy. AB - OBJECTIVE: Evaluation of a complex and variable disease such as rheumatoid arthritis (RA) poses a challenge particularly over the medium to long term. A practical framework to evaluate clinically relevant outcomes over the long term is the "5D" approach of Fries, described in 1980. We describe the 20 year outcome in 52 survivors of a 123 patient cohort in terms of change in discomfort, disability, drug side effects, dollar costs, and deaths. METHODS: We studied 123 patients with RA allocated to their first disease modifying antirheumatic drug (DMARD) between 1977 and 1979. All were under the overall care of one physician over the 20 years and were maintained where possible taking a single DMARD. Baseline demographic variables, the Ritchie Articular Index (RAI), Lee functional index, and erythrocyte sedimentation rate (ESR) were initially recorded. The extent to which the demographic and disease variables contributed to need for joint replacement surgery was assessed. Therapies for comorbidity were also documented. RESULTS: At cohort inception mean age was 50 years, RAI was 35, and median disease duration 5.5 years. F:M ratio was 90:33; 96% of patients were positive for rheumatoid factor (RF). Initial median ESR was 55 mm/h. At 20 years, 9 patients (7% of original cohort, 14% of survivors) were lost to followup and 62 (50%) had died. In the 52 survivors RAI, a surrogate for disability, showed a significant improvement (p < 0.0001), but disability measured by Lee functional index showed a deterioration (p = 0.018); 50% underwent joint replacement surgery. Initial ESR and mean ESR over the first 10 years of followup were significantly higher in those who required surgery. Nonsteroidal antiinflammatory drug (NSAID) use declined, but at least 2 deaths and 4 renal deaths that may have been related to therapy were attributed to NSAID use. No unexpected DMARD toxicity or mortality occurred. Concomitant therapy for comorbidity, in particular for cardiovascular disease, osteoporosis, and gastrointestinal disease, increased: more than 60% were on these therapies at 20 year followup. CONCLUSION: Strategies to improve the outcome of RA in all dimensions should include: earlier referral for expert assessment; avoidance of NSAID gastrointestinal and nephrotoxicity; a more intensive effort to identify effective management of comorbidity and those likely to have a poor outcome. Such patients require sustained, intensive therapy to minimize later disability. PMID- 12375319 TI - Production of thromboxane A2 and prostaglandin i2 affected by interaction of heat aggregated IgG, endothelial cells, and platelets in lupus nephritis. AB - OBJECTIVE: To examine the role of immune complexes in the prostanoid metabolism of glomerular capillary endothelial cells (EC) and platelets in lupus nephritis. Heat aggregated IgG (HA-IgG), instead of immune complexes, was incubated using an in vitro coculture system with human umbilical vein EC, instead of glomerular capillary EC, and platelets. The effect of complement component C1q and a novel imidazole-type thromboxane A2 (TXA2) synthetase inhibitor, DP-1904, on this prostanoid metabolism change was also investigated. METHODS: EC monolayers (1.5x10(5) cells/well) were incubated with various concentrations of HA-IgG, monomeric IgG, or medium alone for 1 h at 37 degrees C, and then incubated with platelet suspensions (1x10(8) cells/ml) for various times. Concentrations of TXB2 and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable hydrolysis products of TXA2 and prostaglandin I2 (PGI2), respectively, released in the supernatants were measured by ELISA. RESULTS: HA-IgG bound to EC monolayers produced TXB2 and 6-keto-PGF(1alpha) in a concentration dependent manner and much more than monomeric IgG or medium alone did. However, the production of 6-keto PGF(1alpha) stimulated with HA-IgG was much lower than that of TXB2, indicating a large imbalance between TXA2 and PGI2. Preincubation of HA-IgG with purified C1q partially suppressed the production of TXB2, but not that of 6-keto-PGF(1alpha). DP-1904 suppressed the production of TXB2 completely, but by sharp contrast, it dramatically increased the production of 6-keto-PGF(1alpha) from EC and platelets by HA-IgG. CONCLUSION: The large imbalance of TXA2 and PGI2 produced by the interaction of EC, immune complexes, and platelets may be associated with alterations in glomerular pathological findings and hemodynamics mediated by immune complexes in lupus nephritis. C1q and a TXA2 synthetase inhibitor may improve the abnormal prostanoid metabolism change of lupus nephritis. PMID- 12375320 TI - VH4-34 encoded antibody in systemic lupus erythematosus: effect of isotype. AB - OBJECTIVE: To determine the clinical significance of elevated serum levels of VH4 34 encoded IgM and IgG antibodies with respect to the clinical characteristics of systemic lupus erythematosus (SLE). METHODS: VH4-34 encoded IgM and IgG immunoglobulin was measured in 95 patients with SLE by ELISA using antiidiotype monoclonal antibody (Mab) 9G4. SLE disease activity, severity, and damage were assessed by visual analog scales, Systemic Lupus Activity Measure, Lupus Severity of Disease Index, and Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. Presence of VH4-34 encoded antibodies on patients' B lymphocytes was analyzed by flow cytometry using Mab 9G4. RESULTS: Fifty-two of 95 patients with SLE had elevated levels of VH4-34 encoded antibodies of IgG isotype; 17 patients with VH4-34 IgG had elevated VH4-34 of the IgM isotype. Forty-three of the 95 patients had normal levels of VH4-34 encoded antibodies. When disease severity was correlated to VH4-34 isotype, patients with circulating VH4-34 IgG but without IgM had significantly more severe disease compared to patients who had VH4-34 of both isotypes. Eighty-six percent of patients with SLE nephritis and 100% of those with central nervous system (CNS) lupus had VH4-34 IgG without IgM. In vivo, VH4-34 encoded antibodies were found to bind autologous B lymphocytes. CONCLUSION: Presence of VH4-34 IgG in the absence of VH4-34 IgM was the finding most strongly associated with severe SLE, nephritis, and CNS lupus, suggesting that isotype switching of VH4-34 encoded antibodies or loss of VH4-34 IgM encoded antibodies may influence the progression of disease in SLE. PMID- 12375321 TI - Effect of a culturally sensitive cholesterol lowering diet program on lipid and lipoproteins, body weight, nutrient intakes, and quality of life in patients with systemic lupus erythematosus. AB - OBJECTIVE: To evaluate the effect of a culturally sensitive cholesterol lowering diet program on lipid and lipoproteins, body weight, nutrient intakes, and quality of life (QOL) in patients with systemic lupus erythematosus (SLE). METHOD: Seventeen patients with SLE were randomized to a Step 2 diet intervention group or a control group for 12 weeks. The diet intervention was made up of weekly group sessions during the first 6 weeks followed by telephone counseling every 2 weeks for the last 6 weeks. Data on fasting lipid and lipoproteins, body weight, food intake (3 day food record), and QOL were collected at baseline, 6 weeks, and 12 weeks. Program acceptability was assessed in the diet group at 6 weeks. RESULTS: The intervention was found to be highly acceptable and culturally sensitive. The changes in nutrient intakes at 6 and 12 weeks in the diet group were -49% and -33%, respectively, for cholesterol, -44% and -32%, respectively, for percentage calories from fat, and -46% and -32%, respectively, for percentage calories from saturated fat. The corresponding figures in the control group were +22% and -8% for cholesterol, +9% and +6% for percentage calories from fat, and +5% and +7% for percentage calories from saturated fat. The treatment by time interaction was significant for all the dietary variables (p = 0.0003 to 0.02). QOL was reported to improve by 15-17% in the diet group and decrease by 4-6% in the control group, and the treatment by time interaction was significant (p = 0.05). The changes in the physiological variables at 6 and 12 weeks in the diet group were -10% and -6%, respectively, for total cholesterol, -10% and -2%, respectively, for low density lipoprotein (LDL) cholesterol, -11% and -4%, respectively, for high density lipoprotein (HDL) cholesterol, -25% and -34%, respectively, for very low density lipoprotein (VLDL) cholesterol, -8% and -24%, respectively, for triglycerides, and -2% and -5%, respectively, for body weight. The corresponding figures in the control group were -5% and -3% for total cholesterol, -6% and -5% for LDL cholesterol, 0% and +12% for HDL cholesterol, +4% and -8% for VLDL cholesterol, -6% and -15% for triglycerides, and -5% and -6% for body weight. The treatment by time interaction was significant for HDL cholesterol (p = 0.04). A significant reduction was seen in the diet group for total cholesterol at 6 and 12 weeks, LDL and HDL cholesterol at 6 weeks, and body weight at 12 weeks (p = 0.0002 to 0.01). CONCLUSION: This culturally sensitive cholesterol reducing diet program was highly accepted and effective in changing the diet and QOL of patients with SLE. The effect on serum lipids, lipoproteins, and body weight, however, was modest. A larger randomized study with a longer intervention period is necessary to test the effectiveness of a cholesterol lowering diet on lipids and lipoproteins in patients with SLE. PMID- 12375322 TI - Predictors of sustained amenorrhea from pulsed intravenous cyclophosphamide in premenopausal women with systemic lupus erythematosus. AB - OBJECTIVE: To identify predictors of intravenous cyclophosphamide (IC) induced sustained amenorrhea, especially in young premenopausal women with systemic lupus erythematosus (SLE). METHODS: The cumulative dose resulting in sustained amenorrhea in 50 and 90% of the treated women (D50 and D90) and predictors of sustained amenorrhea at various ages were determined with Kaplan-Meier plots and Cox regressions in a consecutively enrolled cohort of 67 premenopausal women with SLE who received a pulsed IC regimen (monthly doses of 0.75-1.00 g/m2) for nephritis (n = 59) or other indications (n = 8). RESULTS: Twenty-one of 67 women developed sustained amenorrhea of > 12 months' duration. Age was the strongest determinant of this adverse event. For women in the upper age tertile (>or= 32 years old), D50 was 8 g/m2 and D90 was 12 g/m2, and no strong protective or predisposing factors were identified. Conversely, only 5 of 44 women 2 SD below the mean for age. Five (83%) of those with RCL, > 2 SD below the mean for age, had periarticular osteopenia, JSN, or erosion. CONCLUSION: We conclude the frequency of abnormal hand/wrist radiographs is very high very early in the course of polyJRA. More studies are needed to determine to what extent these radiographic abnormalities correlate with clinical outcomes. PMID- 12375337 TI - Expression of melanoma antigen gene by cells from inflamed joints in juvenile rheumatoid arthritis. AB - OBJECTIVE: To determine if synovial fluid (SF) cells from inflamed joints of patients with juvenile rheumatoid arthritis (JRA) express melanoma antigen gene (MAGE) RNA and protein. METHODS: The pattern of MAGE-A1 expression was analyzed in inflammatory synovial tissue and peripheral blood mononuclear cells (PBMC) from patients with JRA by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry. RESULTS: MAGE-A1, previously detected only in tumor cells, is strongly expressed in SF cells from patients with JRA. Immunocytochemistry revealed strong staining of SF cells in all of 22 specimens tested. PBMC from patients (7 of 7) also expressed MAGE-A1, but not as strongly as SF cells. Two-color immunofluorescence showed colocalization with CD4 and CD14. Flow cytometry on 3 samples of SF cells confirmed the presence of MAGE A1 on the cell surface and intracellularly. Five of 5 SF cell samples were positive for MAGE-A1 by RT-PCR. CONCLUSION: Mononuclear cells from inflamed joints and blood from patients with JRA express MAGE-A1. MAGE family proteins were previously thought to be expressed only by certain tumors and presented by HLA Class I, resulting in tumor cell lysis by cytotoxic T cells. The observation of MAGE-A1 expression in JRA suggests an association with autoimmune disease and a possible causal role for MAGE antigen in the chronic inflammation of JRA. PMID- 12375338 TI - Association of low bone mass with vitamin d receptor gene and calcitonin receptor gene polymorphisms in juvenile idiopathic arthritis. AB - OBJECTIVE: To compare bone density with polymorphisms in the calcitonin receptor (CTR) and vitamin D receptor (VDR) genes in 50 patients with juvenile idiopathic arthritis and 80 matched controls. METHODS: Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry at the lumbar spine. Genomic DNA was isolated from EDTA blood samples by standard procedures. Polymerase chain reaction was performed using genomic DNA and 100 pmol of each oligonucleotide primer for VDR and CTR genes. Products from genomic PCR were digested by Alu I enzyme for CTR polymorphism and Fok I enzyme for VDR polymorphism. RESULTS: In the total population, higher prevalence of CC genotype (41.5%) for the CTR gene and FF genotype (59.8%) for the VDR gene was found, in agreement with data for Caucasian populations. No significant differences in distribution of CTR and VDR genotypes were observed between patients and controls. However, patients with TT genotype had lumbar BMD (L-BMD) that was lower in comparison to those with CC genotype (p = 0.04). For VDR gene polymorphism, we observed that patients with ff genotype had lower L-BMD in comparison with FF genotype (p = 0.02). Patients with heterozygosity for the 2 genotypes showed intermediate L-BMD. The differences in L-BMD among these groups did not seem to be related to corticosteroid therapy. CONCLUSION: Our data suggest that patients with particular VDR and CTR genotypes may be at higher risk to lose bone mass. PMID- 12375340 TI - Cystic lung disease in Sjogren's syndrome. PMID- 12375339 TI - Rheumatoid arthritis after 9 years of human immunodeficiency virus infection: possible contribution of tritherapy. AB - Infection by human immunodeficiency virus (HIV) may affect joints in different ways. It usually increases the severity of reactive arthritis. Conversely, when rheumatoid arthritis (RA) is seen in association with HIV infection, remission of RA has been observed in some cases. We describe a patient who developed 3 successive forms of joint manifestations: early HIV related arthralgias followed by drug related arthritis, and more recently by typical RA. The first signs of joint manifestations started one year after HIV seroconversion and resolved when antiviral treatment with AZT was started. The second episode ended when lamivudine dosage was reduced. Finally, after 9 years of infection, the diagnosis of seropositive RA was made in this HLA-DR1 positive patient. The symptoms started when the immune status reached normal CD4 T cell levels, in response to antiviral tritherapy. This observation indicates the complex effect of HIV on joint inflammation. PMID- 12375341 TI - Tribute to Barbara Ansell, 1923-2001. PMID- 12375342 TI - Identification of radiologic healing phenomena in patients with rheumatoid arthritis. PMID- 12375343 TI - How many angels could dance on the head of a pin? PMID- 12375344 TI - Polymorphism in the matrix metalloproteinase-1 promoter gene and severity of rheumatoid arthritis. PMID- 12375345 TI - Dermatomyositis with normal creatine kinase and elevated aldolase levels. PMID- 12375346 TI - Chronic nonmalignant pain. PMID- 12375347 TI - Deflazacort in giant cell arteritis. PMID- 12375348 TI - Manganese superoxide dismutase, glutathione peroxidase, and total radical trapping antioxidant capacity in active rheumatoid arthritis. PMID- 12375349 TI - Influence of work related psychosocial factors and psychological distress on regional musculoskeletal pain. PMID- 12375350 TI - Racing to the finish. House panels pass bills, but it's still a tough battle. PMID- 12375351 TI - A strong contender. Healthcare is likely to take a back seat to economic, national security issues this election season, but it still will be a driver in the debate. PMID- 12375352 TI - Battles at the ballot box. Voters to decide a variety of healthcare referendums. PMID- 12375353 TI - Making room for more. New admission and discharge center at Ill. hospital relieves chronic overcrowding in the emergency room and gets patients care quicker. PMID- 12375354 TI - They've got the blues. Minnesota Blues and hospitals point fingers regarding who is ultimately responsible for the nation's soaring healthcare costs. AB - A battle is escalating between hospitals and Blues plans over who is responsible for soaring healthcare costs, and Blue Cross and Blue Shield of Minnesota has launched the latest volley. The insurer recently irritated hospital officials with its report criticizing Minnesota's hospital construction boom. Colleen Reitan (left), senior vice president of the Minnesota Blues, says a financial reality must be faced. PMID- 12375355 TI - SEC investigates HealthSouth amid reports of shady dealings. PMID- 12375356 TI - Doc bonuses tied to quality. PMID- 12375357 TI - Acquired long QT syndrome: risk assessment, prudent prescribing and monitoring, and patient education. AB - PURPOSE: To inform nurse practitioners (NPs) about risk factors that precipitate the potentially fatal cardiac arrhythmia torsade de pointe (TdP) in patients with long QT syndrome (LQTS), and to recommend preventative strategies and prudent prescribing advice to use in clinical practice. DATA SOURCES: A review of the current literature is used to explain factors that cause prolonged repolarization during phase 2 and phase 3 of the cardiac action potential and relate these to the development of LQTS and TdP. The major risk factors reviewed are drugs, drug drug interactions, electrolyte disturbances, and populations at risk for LQTS. CONCLUSIONS: The LQTS is an increasingly recognized cardiovascular problem. Nurse practitioners should be cognizant of the risk factors and be able to apply them in clinical practice. IMPLICATIONS FOR PRACTICE: Recognition of patients at risk for acquired LQTS is imperative in primary care practice. Currently, there are no practice guidelines that address acquired LQTS. In lieu of practice guidelines, the prudent NP uses physiology to guide treatment decisions, especially those decisions related to the use of drugs. PMID- 12375358 TI - The efficacy of metered-dose inhalers with a spacer device in the pediatric setting. AB - PURPOSE: To systematically review the published research and report on the efficacy of using a metered-dose inhaler with a spacer (MDI-S) device in a pediatric setting to treat acute exacerbations of asthma. DATA SOURCES: A literature search was conducted on the CINAHL, Medline, and Cochrane databases; additional searches were made by hand from the reference lists in each study retrieved from databases and from review articles written on the same topic. CONCLUSION: This critical appraisal of the research demonstrates the MDI-S is as effective as the nebulizer, faster in the delivery of medication, and cost effective. IMPLICATIONS FOR PRACTICE: No significant difference between the MDI-S and nebulizer in delivering medication in an acute exacerbation of asthma was found in this analysis. The practitioner's choice of delivery methods should reflect the family's preference, the practice situation, and economic considerations. PMID- 12375359 TI - Cognitive and motor symptoms in dementia: focus on dementia with Lewy bodies. AB - PURPOSE: To describe the clinical syndrome called dementia with Lewy bodies (DLB) and highlight its common and unique characteristics with respect to diagnosis and management. DATA SOURCES: Review of the scientific literature including psychiatric literature, reports of clinical trials, and clinical practice guidelines. CONCLUSIONS: DLB is a clinical and histopathologic disease, which is second only to Alzheimer's disease (AD) as a cause of dementia in older adults. The clinical syndrome of DLB includes cognitive and motor deterioration reminiscent of symptoms associated with AD and Parkinson's disease (PD) respectively. IMPLICATIONS FOR PRACTICE: The late life intersection of cognitive and motor symptoms can present significant challenges in the primary care setting. Recognizing key features of common neurodegenerative disorders is essential to accurately diagnosing and appropriately treating the growing population of older adults who suffer from AD, PD, and DLB. PMID- 12375360 TI - Workplace empowerment, collaborative work relationships, and job strain in nurse practitioners. AB - PURPOSE: To test a theoretical model linking nurse practitioners' (NPs) perceptions of workplace empowerment, collaboration with physicians and managers, and job strain. DATA SOURCES: A predictive, nonexperimental design was used to test a model in a sample of 63 acute care NPs and 54 primary care NPs working in Ontario, Canada. The Conditions of Work Effectiveness Questionnaire, the Collaborative Behaviour Scale--Parts A (physicians) and B (managers), and the Job Content Questionnaire were used to measure the major study variables. CONCLUSIONS: The results of this study support the proposition that the extent to which NPs have access to information, support, resources, and opportunities in their work environment has an impact on the extent of collaboration with physicians and managers, and ultimately, the degree of job strain experienced in the work setting. Primary care NPs have significantly higher levels of workplace empowerment, collaboration with managers, and lower levels of job strain than acute care NPs. IMPLICATIONS: These findings will benefit NPs and nursing leaders in their efforts to create empowering work environments that enable NPs to provide excellent quality patient care and achieve organizational outcomes. PMID- 12375361 TI - Domestic violence: what do nurse practitioners think? AB - PURPOSE: To examine factors that influence nurse practitioners' (NPs) ability to incorporate universal domestic violence screening practices (e.g., asking, identifying, referring and reporting) into their practices. DATA SOURCES: A stratified random survey of certified NPs in New York state was conducted in 1999. There were 118 family, women's health, OB/GYN, and adult NPs in the survey. Chi-square and ANOVA were used to analyze the data. CONCLUSIONS: There were significant differences in the domestic violence screening practices among women's health, OB/GYN, adult, and family NPs. Women's health and OB/GYN NPs were more likely to ask screening questions and identify victims of domestic violence than their other NP counterparts. IMPLICATIONS FOR PRACTICE: There is a need to identify strategies that encourage all NPs to incorporate universal domestic violence screening behaviors into their practices. PMID- 12375362 TI - Aspects of the life history of Muspicea borreli (Nematoda: Muspiceidae), parasite of the house mouse (Mus domesticus) in Australia. AB - Prevalence of Muspicea borreli (Nematoda) infection in wild populations of Mus domesticus in forests in southeastern New South Wales and in rural Canberra, Australia was variable, relatively low and the parasite occurred predominantly in male mice. Experimental infection of BALB/c mice occurred only via subcutaneous inoculation but was achieved using i) adults containing embryonating eggs, ii) adults containing active larvae and iii) active larvae dissected from the uterus of female worms. Experimental infection was not established using adults containing unembryonated eggs and was not established via intraperitoneal, percutaneous nor oral routes. Evidence indicates that larvae develop to the infective stage in the uterus of the adult worm, suggests that an obligate developmental phase on the host skin does not occur and that autoinfection is possible. Experimental infection predominated in males; females rarely became infected. When male BALB/c mice were inoculated subcutaneously with M. borrelia, immediately paired with an uninoculated female and permitted to breed for 90 days, infection was found in male and female offspring only of the second and subsequent litters or in the breeding female partner. Transmission to the young occurred within 21 days of birth and fifth-stage M. borrelia were found in offspring of the second and subsequent litters only after 35 or more days. However, when a male was inoculated but mating delayed for 23 days, infection was found in progeny of the first and second litters. The life cycle is direct and the prepatent period in BALB/c mice is estimated at 50-60 days. The precise mode of transmission of the parasite in breeding pairs of mice was not determined but larvae remained active for approximately an hour in balanced saline solutions (pH = 7.2) and in human saliva but died under conditions emulating free-living (tap water pH = 7.1) and stomach (pepsin solution pH = 2) environments. Transmission was not effected by transplacental, transmammary nor transseminal routes. Consequently, it is difficult not to conclude that transmission may occur via penetration of skin or mucous membranes, and allogrooming behaviour may be particularly important in this regard. PMID- 12375363 TI - Monogeneans from Pangasiidae (Siluriformes) in Southeast Asia: III. Five new species of Thaparocleidus Jain, 1952 (Ancylodiscoididae) from Pangasius bocourti, P. djambal and P. hypophthalmus. AB - The examination of gill parasites from Pangasius bocourti Sauvage, 1880; P. djambal Bleeker, 1846; P. hypophthalmus (Sauvage, 1878) and P. gigas Chevey, 1930 (Siluriformes, Pangasiidae) revealed the presence of seven species of Monogenea among which five are considered new species. They all belong to Thaparocleidus Jain, 1952 (Ancylodiscoididae) as defined by Lim (1996) and Lim et al. (2001). P. bocourti: T. combesi n. sp., T. komarudini n. sp. and T. vietnamensis n. sp. P. djambal: T. caecus (Mizelle & Kritsky, 1969), T. combesi n. sp., T. euzeti n. sp., T. komarudini n. sp. and T. sadilii n. sp. P. hypophthalmus: T. caecus, T. siamensis (Lim, 1990) and T. vietnamensis n. sp. P. gigas: no Monogenea were found on this host species. PMID- 12375364 TI - [Thelohanellus bicornei n. sp. Myxosporidie (Myxosporea, Bivalvulida) a gill parasite of Labeo coubie Ruppel, 1832 (Osteichthyen, Cyprinidae) from Burkina Faso, West Africa]. AB - Thelohanellus bicornei n. sp. (Myxosporea, Bivalvulida) is described from gill of Labeo coubie (Ruppel, 1832) (Osteichthyen, Cyprinidae) in Burkina Faso, West Africa. The cysts are small and their length is 150 to 350 microns. White, linked together they are rounded shape. The spores are ovoids with smooth valvar surface. Their posterior end is rounded and their anterior part shows two "horns like" expansions. Spores measured 13.5 +/- 0.56 (13-14) microns in length and 8.43 +/- 0.49 (8-9) microns in width. Horns length is 1 to 1.5 microns long. Polar capsule is piriform, it's length is 7.24 +/- 0.45 microns and the width 3.75 +/- 0.32 microns. The polar filament formed 10 turns. PMID- 12375365 TI - Gymnomeropsylla n. gen. (Siphonaptera: Pygiopsyllidae) from Sulawesi, Indonesia, with the description of two new species. AB - Compared to related genera, this new flea genus is characterized by the absence, or presence of very few, bristles on the external surface of femur I and especially by the morphology of the apex of sternite IX in the male, which is hyaline and lacks spiniform bristles. The two new species, G. bunomydis and G. margaretamydis, are distinguished from each other by the structure of the genitalia, and the presence of numerous erect bristles on the thorax and abdominal tergites of the latter species. Both of these new species parasitize murine rodents that are endemic to Sulawesi; G. bunomydis was collected mainly from Bunomys chrysocomus and G. margaretamydis only from Margaretamys parvus. PMID- 12375366 TI - [A new flea species of the genus Allopsylla Beaucournu et Fain, 1982 (Siphonaptera: Ischnopsyllidae) from Central African Republic, found on a little known molossid bat]. AB - A pair of Allopsylla lobayensis Beaucournu & Barriere, new species were collected the first of November 1999 in Ngotto forest, in the south-western part of the Lobaye basin, Central African Republic (CAR). The host specimen, a little known molossid bat (Myopterus whitleyi), constitutes the first record of this species in CAR. The new flea species appears taxonomically close to A. hetera Beaucournu & Fain, 1982, one of the two other Allopsylla species, previously know from other faunal regions (A. alloides in Nigeria and A. hetera in Democratic Republic of the Congo). The parasitism of three distinctly different hosts by the three known Allopsylla species raises questions regarding the host-parasite specificity of this group. PMID- 12375367 TI - [Role of Anopheles melas Theobald (1903) on malaria transmission in a mangrove swamp in Saloum (Senegal)]. AB - From June 1995 to January 1998, entomological studies carried out in five villages located in the Delta's Saloum have allowed to better understand the contribution of An. melas Theobald (1903) to malaria transmission in mangrove swamp. Among the five villages studied, three of them (Simal, Djilor and Marlothie) located along the Saloum river, are colonised by An. arabiensis; the two others (Djifere and Diakhanor) located between the sea and the river, are colonised by An. melas. During the rainy season and at the beginning of the dry season, An. melas and An. arabiensis are sympatric. The ratio of An. melas/An. arabiensis increases when we go closer the coast where An. melas becomes quite exclusive. When An. melas is predominant, endophagy, endophily and anthropophily are very marked. The parturity rates are lower in An. melas than in An. arabiensis. In the predominance area of each species, transmission is on the same level. During the period of sympatry, An. arabiensis is responsible for the transmission and when it is absent, An. melas carries on. Transmission occurs from July to March with a maximum at the beginning of the dry season. In the villages of the mangrove swamp, its prolongation until the middle of the dry season is due to An. melas. PMID- 12375368 TI - Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests. AB - The dissemination of mutant and resistant strains of Plasmodium falciparum makes a considerable contribution to the spread of drug-resistant malaria. Populations around harbours and airports could be particularly exposed to Plasmodium isolates introduced with imported cases of malaria. The use of chloroquine as well as the use of and sulfadoxine/pyrimethamine is currently an effective method for treating uncomplicated cases of malaria in Madagascar. As part of a monitoring programme, in vitro methods were used to assess the sensitivity of P. falciparum isolates in two coastal towns in Madagascar: Mahajanga on the west coast and Toamasina on the east coast. All of the isolates from both sites were sensitive to amodiaquine, quinine, pyrimethamine and cycloguanil. All of the isolates from Mahajanga were sensitive to chloroquine (n = 25; mean IC50 = 22.6 nM, 95% confidence interval: 16.8-28.7 nM), whereas three of the isolates from Toamasina were resistant to chloroquine (n = 18; mean IC50 = 66.3 nM; 95% confidence interval: 42.6-90 nM). The frequency of the Pfcrt Thr-76 and the dhfr Asn-108 mutations was estimated by PCR/RFLP. The 43 P. falciparum isolates examined, including the three in vitro chloroquine-resistant isolates from Toamasina were all wild-type (Lys-76). Phenotyping and genotyping studies suggested that the prevalence of chloroquine- and pyrimethamine-resistant isolates and of mutant strains of P. falciparum is very low. These results showed that in vitro test and genotyping of resistance markers approaches could be successfully used to monitor the emergence of drug-resistant malaria and to try to alleviate the lack of medical teams able to carry out in vivo test. The possible hazard/risk associated with imported cases of malaria is discussed. PMID- 12375369 TI - Insecticide mixtures for mosquito net impregnation against malaria vectors. AB - Insecticides belonging to the pyrethroid family are the only compounds currently available for the treatment of mosquito nets. Unfortunately, some malaria vector species have developed resistance to pyrethroids and the lack of alternative chemical categories is a great concern. One strategy for resistance management would be to treat mosquito nets with a mixture associating two insecticides having different modes of action. This study presents the results obtained with insecticide mixtures containing several proportions of bifenthrin (a pyrethroid insecticide) and carbosulfan (a carbamate insecticide). The mixtures were sprayed on mosquito net samples and their efficacy were tested against a susceptible strain of Anopheles gambiae, the major malaria vector in Africa. A significant synergism was observed with a mixture containing 25 mg/m2 of bifenthrin (half the recommended dosage for treated nets) and 6.25 mg/m2 of carbosulfan (about 2% of the recommended dosage). The observed mortality was significantly more than expected in the absence of any interaction (80% vs 41%) and the knock-down effect was maintained, providing an effective barrier against susceptible mosquitoes. PMID- 12375370 TI - Activity of natural and synthetic naphthoquinones against Toxoplasma gondii, in vitro and in murine models of infection. AB - The effect of 14 natural and synthetic naphthoquinones in the replication of Toxoplasma gondii was evaluated. In vitro studies were accomplished in cultures of 2C4 fibroblasts infected with RH-strain. Enzyme-linked immunosorbent assay was used to quantify parasite growth. For the studies in vivo, mice were infected with tachyzoites of the RH strain or cysts of the T. gondii EGS strain. In vitro, seven naphthoquinones demonstrated significant inhibition of intracellular T. gondii growth at concentrations of 1 and 5 micrograms/ml. Only three compounds were significantly protective when tested in animals: 2-hydroxy-3'-(3'-pentenyl) 1,4-naphthoquinone (PHNQ4), 2-hydroxy-3-(1'-vinylphenyl)-1,4-naphthoquinone (PHNQ5), and 2-hydroxy-3-(1'-propen-3'-phenyl)-1,4-naphthoquinone (PHNQ6). In animals infected with the EGS strain and treated with PHNQ4 (50 mg/kg/day orally), a 7-day prolongation of the time to death was observed. Treatment with 100 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ5 resulted in a 5-day and 16-day prolongation of the time to death, respectively. Treatment with 50 mg/kg/day orally or 50 mg/kg/day i.p. of PHNQ6 resulted in a 4-day prolongation of the time to death or up to 30 days after treatment, respectively. Our results suggest that the naphthoquinones may be important therapeutic agents for the treatment of toxoplasmosis. PMID- 12375371 TI - [Taenia solium taeniasis/cysticercosis in the Menoua division (West Cameroon)]. AB - The present study was carried out between August 1999 and April 2000 with the objective of determining the prevalence of Taenia solium taeniasis in two village communities of Bafou and Bamendou in the Menoua division (West Cameroon). Four (0.13%) out of 3,109 faecal samples were positive for Taenia spp. eggs using the flotation technique. Three of the four worms expelled were T. solium whereas the other one was T. saginata. Two cases of cysticercosis were diagnosed in one of the families with a T. solium carrier. Furthermore, coprological and serological investigations for T. solium taeniasis and cysticercosis were carried out among butchers and/or tongue inspectors (n = 137) of the city of Dschang. The results were compared with those of a control group (n = 198). Taenia spp. eggs were not detected by microscopic examination. The prevalence of cysticercosis in the two groups was relatively similar (3.6 and 4.5% respectively). PMID- 12375372 TI - [Tsetse fly wings, an identity card of the insect?]. AB - The size of tsetse flies is often associated with population dynamics and vectorial capacity parameters. Adult fly size is generally estimated from measurements of wing segments. To take measure of the wing, a semi-automatic software was developed by CIRAD-EMVT and IRD. It was used in wild populations of Glossina tachinoides Westwood and G. palpalis gambiensis Vanderplank (Diptera: Glossinidae) trapped near Bobo-Dioulasso, Burkina Faso. From an numeric picture of the wing, the software calculates the length of vein segments, the ratios between these lengths, the surface of the tsetse characteristic "hatchet cell", and the greyness on the wings. The data were interesting at the level of taxonomy. In addition, they help specify physiological characteristics of the studied populations. PMID- 12375373 TI - An immature filarial worm, probably Wuchereria bancrofti, from the anterior chamber of the eye in a patient from Sri Lanka. PMID- 12375374 TI - Investigation of a varicella outbreak complicated by group A streptococcus in First Nations communities, Sioux Lookout Zone, Ontario. PMID- 12375375 TI - Schemata as moderators of clinical effectiveness of a comprehensive cognitive behavioral program for patients with depression or anxiety disorders. AB - The authors examined the clinical effectiveness of a comprehensive cognitive behavior therapy (CBT) program offered to patients with depression or anxiety disorders. They also tested the prediction, based on Young's schema-focused approach to therapy, that endorsement of maladaptive cognitive schemata predicts poor response to standard CBT. One hundred thirty-four consecutive referrals were assessed on a battery of self-report measures at the commencement of the program, and 121 of these patients (90%) completed the program and provided posttreatment data. Two thirds of the patients showed statistically reliable symptom reduction, and half had large effect size (0.8 standard deviations or more) symptom reduction. Contrary to predictions, greater initial endorsement of schemata did not predict poor therapy response. The CBT program was effective for most patients, including patients with high endorsement of maladaptive schemata. PMID- 12375376 TI - The maintenance of behavior change as an indicator of social validity. AB - This article reviews research pertaining to use of social validity and presents a rationale for expanding the conceptualization and use of this construct. It is proposed that the degree to which obtained treatment gains maintain across time within natural contexts be considered as a primary indicator of social validity. Traditional forms of social validation--subjective evaluation and normative comparison--are presented as measures that, when used within the framework of maintaining behavior change, form an iterative and heuristic process in which behavior change goals, procedures, and outcomes are altered to increase and/or sustain their social value. Procedural guidelines for research using maintenance as the benchmark of social validity are discussed. PMID- 12375377 TI - Self-monitoring and at-risk middle school students. Academic performance improves, maintains, and generalizes. AB - Using a multiple baseline design across six academic settings, we found that teaching 4 at-risk middle school students to self-monitor markedly improved their academic performance as measured by their grades and related academic behaviors. Furthermore, these improvements generalized to settings where self-monitoring was never introduced, and they maintained the following school year. In this charter middle school setting, self-monitoring proved to be an extremely effective intervention. These findings suggest that it would be equally effective in a variety of settings. PMID- 12375378 TI - The parental daily diary. A sensitive measure of the process of change in a child maltreatment prevention program. AB - There is a substantial deficit of sensitive measures of parental discipline in the area of prevention, generally, and in child maltreatment prevention specifically, despite reports that over a million children experience maltreatment in the United States every year. Part of the challenge in locating such measures is the impossibility of obtaining accurate observational measures of the degree of harsh discipline, unless extremely large populations are used. The majority of studies on harsh discipline have dealt with this problem by using self-report instruments or proxy observation tasks (such as observing mother child interactions in a compliance framework). The most well-known self-report instruments, such as the Child Abuse Potential Inventory (Milner, 1986), are constructed to measure parental pathology in maltreating parents rather than to identify parents who might benefit from preventive endeavors. In contrast, there are no well standardized measures of mother-child interaction that document a sensitivity to the presence of harsh discipline, possibly due to the clear pressure of social desirability problems. This paper outlines a daily self observation measure of parental disciplinary behavior in the form of a diary. This self-monitoring instrument offered data on the overall feelings and disciplinary behaviors used daily following each session on parenting group interventions. The study showed a gradual decrease in physical punishment and a gradual increase in planned ignoring across treatment, as these were introduced as part of an ongoing curriculum. The use of an explicit technique, such as time out, increased abruptly rather than gradually and effects were seen only after specific instruction. Advantages and future applications of this kind of ongoing self-observation measure of treatment progress are described. PMID- 12375379 TI - Predicting outcomes of group cognitive behavior therapy for patients with affective and neurotic disorders. AB - An attempt was made to predict outcomes following group Cognitive Behavior Therapy (CBT) for patients with affective and neurotic disorders. A group of 348 patients at a private psychiatric clinic, treated in a group CBT program, completed the Depression, Anxiety, and Stress Scale (DASS) before and after treatment. Prior to treatment, data from the Locus of Control of Behavior (LCB), a Global Assessment of Function (GAF), the Health of the Nation Outcome Scales (HoNOS), and the Rosenberg Self Esteem Scale (RSE) were also collected. Results indicated that posttreatment stress scores of all patients were predicted by pretreatment stress and self-esteem. Among patients with neurotic disorders, posttreatment anxiety was predicted by initial anxiety and self-esteem whereas among patients with affective disorders, posttreatment anxiety scores were predicted by initial anxiety and GAF. For patients with neurotic disorders, self esteem did not predict variance in posttreatment depression in addition to that explained by pretreatment depression. In contrast, for patients with affective disorders, pretreatment depression and Locus of Control predicted posttreatment depression. PMID- 12375380 TI - A descriptive analysis of intervention research in emotional and behavioral disorders from 1980 through 1999. AB - The current study was conducted to examine the trends involved with experimental intervention research designed to modify behaviors of children and youth with emotional and/or behavioral disorders (EBD). Trends are summarized and compared to the intervention research that has been conducted in developmental disabilities (DD). The contents of 10 journals published between 1980 and 1999 were analyzed. Descriptive dimensions of the research including participant demographics, settings, research designs, dependent and independent variables, intervention agents, and measures of ecological validity were investigated. In addition, the databases were examined to determine whether interventions were based on individualized processes of assessment. The results showed strikingly similar trends across interventions with EBD and DD participants. The discussion addresses the general status of intervention research across both populations, as well as the importance of extending the current research to examine additional variables and measures with various populations. PMID- 12375381 TI - A standardized method of diplomatically and effectively reporting child abuse to state authorities. A controlled evaluation. AB - Although many studies have examined issues relevant to reporting child maltreatment to state authorities, empirical evaluation of intervention programs to assist professionals in reporting child abuse is lacking. In the present study, a medical student was taught to perform a standardized behavioral method of reporting child abuse that incorporates nonperpetrating caregivers of child abuse victims in the reporting process. A controlled multiple baseline across behaviors (i.e., initiating child abuse report, responding to upset) experimental design was utilized to evaluate skills acquisition. Improvements in interpersonal skills related to reporting child abuse were demonstrated consequent to intervention. Future directions are discussed in light of these results. PMID- 12375382 TI - Gambling decisions: an early intervention program for problem gamblers. AB - An implementation and one-year follow-up of the Gambling Decisions program attempted to answer several important questions. First, is controlled gambling a viable treatment option for some gamblers? Can earlier stage problem gamblers be separated for treatment from those with more severe problems? Finally, would problem gamblers utilize a community health agency for treatment of their excessive gambling? A pretest/posttest design was chosen where the efficacy of the program was assessed using repeated measures ANOVA analysis. Results showed that an average loss of $608 over a 4-week period was reduced to $113 immediately after the 6-week program and to a loss of $73 at 12 months. The average number of hours spent gambling per 4 weeks was significantly reduced from 23.5 at pretest to 6.5 at the 12 month posttest. Significant decreases were also observed in the number of days per week that clients gambled, and clients reported significant reductions in everyday life problems related to gambling after completing the program. PMID- 12375383 TI - The South Oaks Gambling Screen: a review with reference to Australian use. AB - The South Oaks Gambling Screen (SOGS) is a psychometric instrument widely used internationally to assess the presence of pathological gambling. Developed by Lesieur and Blume (1987) in the United States of America (USA) as a self-rated screening instrument, it is based on DSM-III and DSM-III-R criteria. This paper describes the origins and psychometric development of the SOGS and comments critically in relation to its construct validity and cutoff scores. Reference is made to the use of the SOGS in the Australian setting, where historically gambling has been a widely accepted part of the culture, corresponding to one of the highest rates of legaliZed gambling and gambling expenditure in the world. An alternative approach to the development of an instrument to detect people who have problems in relation to gambling is proposed. PMID- 12375384 TI - The evolving market structures of gambling: case studies modelling the socioeconomic assignment of gaming machines in Melbourne and Sydney, Australia. AB - The expansion of gambling industries worldwide is intertwined with the growing government dependence on gambling revenue for fiscal assignments. In Australia, electronic gaming machines (EGMs) have dominated recent gambling industry growth. As EGMs have proliferated, growing recognition has emerged that EGM distribution closely reflects levels of socioeconomic disadvantage. More machines are located in less advantaged regions. This paper analyses time-series socioeconomic distributions of EGMs in Melbourne, Australia, an immature EGM market, and then compares the findings with the mature market in Sydney. Similar findings in both cities suggest that market assignment of EGMs transcends differences in historical and legislative environments. This indicates that similar underlying structures are evident in both markets. Modelling the spatial structures of gambling markets provides an opportunity to identify regions most at risk of gambling related problems. Subsequently, policies can be formulated which ensure fiscal revenue from gambling can be better targeted towards regions likely to be most afflicted by excessive gambling-related problems. PMID- 12375385 TI - Machine gaming in Sydney clubs: characteristics of the supporting resident populations. AB - This research provided background for surveys and interviews in later stages of a 3 part project. It aimed to identify, from secondary research, sociodemographic characteristics which tend to support registered clubs and their machine gaming activities in the Sydney Statistical Division. Using multiple methods including Pearson's Product Moment correlation, Principal Components factor analysis, and stepwise regression, the study profiled Sydney populations which spend highly on gaming machines. The most important sociodemographic predictors of Sydney statistical local areas where per capita gaming machine expenditure is high are large proportions of the adult resident population who were born in Malta, Greece, Lebanon, China, Italy, Vietnam, Yugoslavia, India or the Philippines; have no vocational or tertiary qualifications; or are unemployed. PMID- 12375386 TI - [Acute abdominal pain... should be relieved rapidly!]. PMID- 12375387 TI - [Thoughts concerning reforms]. PMID- 12375388 TI - [The appropriateness of the indications and diagnostic efficiency of colonoscopy. Results of a survey conducted in all the hospital centers of the Provence-Alpes Cote d'Azur area]. AB - OBJECTIVE: To evaluate the appropriateness of indications and the diagnostic efficiency of colonoscopy taking into account available guidelines. METHODS: This was a retrospective study conducted by the Union Regionale des Caisses d'Assurance Maladie de Provence-Alpes-Cote d'Azur (URCAM-Paca) aimed at assessing the appropriateness of the indications and diagnostic efficiency of colonoscopy. Anonymous data concerning near totality of the colonoscopies carried out during one week in all the establishments (public and private) of the area, were collected by consulting the medical records. RESULTS: The indications for colonoscopy were first analyzed with regard to the guidelines for good clinical practices (references medicales opposables and consensus conference) according to two scales of reading (literal interpretation and broad interpretation). We noted that the available guidelines do not cover the totality of the colonoscopies performed, that their formulation does not make them all controllable, and that the colonoscopies, which respect them, range from 27.5% (strict reading) to 74.1% (broad reading). When taking into account not only the quality of the preparation, but also the result of the examination after re-classification into benign, pathological or normal examinations, the differences in efficiency appear only when one retains the broad interpretation of the reference guidelines. The efficiency analysis makes it possible to highlight a defect of predictability of the recommendations with regard to the pathologies considered as serious. It also appears that approximately 10% of the colonoscopies are, at the same time, out of the reference and have a normal result, and thus can be considered as absolutely not justified. CONCLUSION: Better compliance of the practitioners to colonoscopy recommendations would initially be generated by improvement in the predictability and the legibility of the current reference guidelines. PMID- 12375389 TI - [Larrey or Morgagni hernias treated by laparoscopy]. AB - INTRODUCTION: Diaphragmatic hernia or Morgagni-Larrey hernia is a rare entity. Its treatment is surgical and hence raises the question of the surgical approach. OBSERVATIONS: Two patients underwent laparoscopic surgery for Morgagni-Larrey hernia. The first, aged 17 exhibited a chromosomic abnormality (trisomie 21). The second was 18 years old. Both patients underwent surgery by laparoscopy. In both cases, the surgical act performed was resection of the hernia and closure of the orifice with separate sutures. Their post-surgical courses were uneventful, even two years later. COMMENTS: In the age of mini-invasive surgery, laparoscopy is an excellent alternative to laparotomy in this benign pathology. PMID- 12375390 TI - [Increase of CA-125 in pericardial tamponade]. PMID- 12375391 TI - [Clinical aspects and treatment of osteoarthritis]. AB - ARTHROSIS TODAY: Representing the most frequent articular pathology, the prevalence of which increases with age, arthrosis associates elements of construction and destruction. It is a disease of the cartilage in which genetic, mircotraumas, cytokines and microcrystals intervene, associated with abnormalities (notably alteration in density) of the sub-chondral bone. From a therapeutic point of view, the surgical, medicinal or non-pharmacological treatments that are currently available are of unconfirmed efficacy for some of them. CLINICAL-RADIOLOGICAL PARAMETERS AND BIOCHEMICAL MARKERS: A comparison of the levels of biochemical markers with radiological progress and the algofunctional indices in patients presenting with gonarthrosis has shown that, although there is no clear parallel, there are however certain correlations. The chondroitin sulfates ACS4-ACS6 may intervene not only in radiological progression but also in articular metabolism. RISK FACTORS: Several local and systemic biomechanical factors (notably anklyzing hyperostosis) are significantly related to gonarthrosis, whereas tobacco abuse has a protective role. Compensated soles and internal femorotibial athrosis of the knee A prospective study conducted over 2 years did not show any symptomatic or structural effect of wearing compensated soles in internal gonarthrosis. However the consummation of non-steroid anti inflammatories was reduced and compliance was improved. Following prosthesis of the hip the quality of life of patients having been operated on was similar to that of the control group. However, in nearly one third of the patients, there was no significant improvement in pain and/or articular function. Old age, severe pre-surgical pain, and concomitant muscle-skeleton affections represent factors that are of poor post-surgery prognosis. PMID- 12375392 TI - [Arthrosis of the hands]. AB - A GENETICALLY DETERMINED AFFECTION: For more than two centuries, successive studies have led one to consider arthrosis of the hands as a genetically determined affection, but the localization of the genes responsible is unknown. Other than this genetic factor, various etiological factors have been identified (age-related increase, predominance in women, influence of hormones and obesity, mechanical factors...). Its incidence is of 100 per 100,000 persons/year. IDENTIFICATION OF PATIENTS: The clinical criteria retained for arthrosis of the hands are those of the American College of Rheumatology (ACR). Radiological scores have been established to permit good identification. SELECTION AND EVALUATION: The selection requires the use of classical radiological criteria and the clinical criteria of the ACR or those proposed by Lequesne and Maheu for the study of symptomatic drugs. For drugs with structural effect, an X-ray alone is sufficient. The pain measured on a visual analog scale is the principle criterion of efficacy in clinical trials. It is also possible to measure pain using Maheu's weekly assessment, the daily consumption of analgesics and/or anti inflammatories, or again Drieser's algofunctional index or the AUSCAN. PMID- 12375393 TI - [Measurement of the thickness and volume of the cartilage by nuclear magnetic resonance]. AB - THE CLASSICAL METHODS: The measurement methods of the variations in articular cartilage, essentially "magnifying glass" and semi-automated computerized digitalized radiographic images, permit precise assessment of the maximum pinching point and the mean thickness and surface within a determined area. They do not provide information on the volume of the cartilage, which can be assessed by using nuclear magnetic resonance (NMR). RELIABILITY OF NMR: A study of the images obtained with the NMR of 48 normal and arthritic knees demonstrated the excellent reliability of this imaging method and of a new software for the measurement of the cartilage on these images. FOLLOW-UP OF THE PROGRESSION OF OSTEOARTHRITIS: Nuclear magnetic resonance is a highly sensitive method, more powerful than measurements made on radiographies, to monitor the progression of cartilage destruction, as was demonstrated in a study pursued for 2 years in 30 patients presenting with gonathrosis. PMID- 12375395 TI - [Radiological progression of internal femoro-tibial osteoarthritis in gonarthrosis. Chondro-protective effect of chondroitin sulfates ACS4-ACS6]. AB - OBJECTIVE: To confirm the structure-modulating properties of ACS4-ACS6 in gonathrosis by measuring the modifications in minimum joint space width, the mean thickness and the mean surface of the cartilage in internal femorotibial function. METHOD: This was a double blind prospective study versus placebo including 300 symptomatic patients with internal femorotibial gonathrosis. The measurements were made with a semi-automatic method validated from digitalized radiological images taken at two years in each patient. RESULTS: There was an equal number of dropouts in the 2 groups (40 in the ACS4-ACS6 group and 41 in the placebo group). After 2 years, there was a significant difference, with worsening of the affection in the placebo group. In the group treated with ACS4-ACS6, there was no significant variation in all the radiological parameters, which remained remarkably stable. When comparing the 2 groups, the statistical analysis revealed a significant difference in the ACS4-ACS6 group with maintenance of the cartilage analyzed, not only in the intent-to-treat patients but also in the per protocol population. CONCLUSION: ACS4-ACS6 is superior to the placebo with regard to the stabilization of minimum joint space width of the internal femorotibial articular space, the mean thickness and the surface. PMID- 12375394 TI - [A new mechanism of action of chondroitin sulfates ACS4-ACS6 in osteoarthritic cartilage]. AB - THE MECHANISMS OF ACTION ALREADY KNOWN: In vitro, chondroitin sulfate ACS4-ACS6 have a dose-dependent inhibiting effect on the catabolism of proteoglycanes and collagen. They also stimulate the synthesis of extracellular matrix macromolecules. The existence of a chondrocyte cyto-protector effect and a potentially chondro-protective effect has also been demonstrated. ANOTHER MECHANISM OF ACTION: A new regulation route of chondrocyte metabolism has been proposed in which the Insulin Growth Factor (IGF-1) impedes the catabolic effects of another cytokine, Interleukin 1 (IL-1), by increasing its antagonistic receptor. This control appears essential in the maintenance extracellular matrix homeostasis. CARTILAGE WITH OSTEOARTHRITIS: Chondrocytes from OA cartilage have the capacity to produce more constituting macromolecules of the extracellular matrix. Conversely, these same cells have a greater catabolic capacity. The enhanced activity of IL-1 and the concomitant decrease in its antagonistic receptors leads to the reduction in macromolecule levels in the extracellular matrix, produced in the Cell-associated matrix of OA chondrocytes. ANTI-CATABOLIC EFFECT: It has been demonstrated that ACS4-ACS6 are capable of neutralizing the enhanced catabolic capacity of activated human OA chondrocytes. PMID- 12375396 TI - Warning: your personnel files may follow you forever. PMID- 12375397 TI - Dr. fails to leave post-op. orders: nurses fail to timely call Dr. Case on point: Lupinacci v. Med. Center of Delaware, 2002 WL 31006263 N.E.2d. -DE (2002). PMID- 12375398 TI - CA: nurse's leg injured assisting patient: workers' compensation awarded for 'loss of use'. PMID- 12375399 TI - VA: fall--but no contemporaneous back pain: comp. awarded despite no complaint re back. PMID- 12375400 TI - Supervisors allege discrimination led to termination. Case on point: Garswood v. Henry Ford Hospital, 2002 WL 31013217 N.W.2d -MI. PMID- 12375401 TI - The Branemark Novum implant system: rehabilitating the edentulous mandible. PMID- 12375402 TI - Responsive regulation of Internet pharmacy practice. PMID- 12375403 TI - Are you sorry you went into primary care? PMID- 12375404 TI - Practical approaches for senior patients. PMID- 12375405 TI - M.D. 1. New York Times 0. PMID- 12375406 TI - How a malpractice insurer grew too big & too fast. PMID- 12375407 TI - Who takes call when a doctor leaves the practice? PMID- 12375408 TI - 2002 up & comers. PMID- 12375409 TI - Making IT compute. New initiatives may give guidance to hospitals hoping to reconcile IT projects with their bottom lines. AB - Several groups are intensifying efforts to get out the facts to hospitals to help them determine the benefits of costly healthcare information technology and judge vendors' pitches. Three initiatives are settling into a lineup of guidance on planning for healthcare computerization. These efforts seek to break a pattern of hospital inaction and factually thin conclusions. John Glaser (left) of Partners HealthCare System says the expense of such IT projects is justified. PMID- 12375410 TI - Exec claims HealthSouth will be bigger and better. PMID- 12375411 TI - Paying more for results. CMS tries to enlist Premier to tie hospital reimbursement to quality performance. PMID- 12375412 TI - Gray market for gadgets. Technologies to help the elderly live on their own. PMID- 12375413 TI - The ultimate job: security. The war on terror creates a big need for biometrics. PMID- 12375414 TI - Beyond the lab in biotech. With so many drugs in trial, new jobs are opening up. PMID- 12375415 TI - Brave new job hunt. PMID- 12375416 TI - Able to leap very long odds. PMID- 12375417 TI - Certified organic. PMID- 12375418 TI - A tale of two hogs. PMID- 12375419 TI - Operating on accuracy. PMID- 12375420 TI - The new math of old age. Why the nursing home industry's cries of poverty don't add up. PMID- 12375421 TI - Rx squalor? PMID- 12375422 TI - Sensing danger. Artificial eyes, ears, and noses for stronger, safer troops. PMID- 12375423 TI - The medical significance of Shiga toxin-producing Escherichia coli infections. An overview. PMID- 12375424 TI - Generation of isogenic deletion mutants of STEC. PMID- 12375425 TI - Generation of monoclonal antibodies against secreted proteins of STEC. PMID- 12375426 TI - Microscopic methods to study STEC. Analysis of the attaching and effacing process. PMID- 12375427 TI - Detection and characterization of EHEC-hemolysin. PMID- 12375428 TI - Shiga toxin receptor glycolipid binding. Pathology and utility. PMID- 12375429 TI - Methods for the purification of Shiga toxin 1. PMID- 12375430 TI - Methods for the identification of host receptors for Shiga toxin. PMID- 12375431 TI - Shiga toxin B-subunit as a tool to study retrograde transport. PMID- 12375432 TI - Measuring pH within the Golgi complex and endoplasmic reticulum using Shiga toxin. PMID- 12375433 TI - Detection of Shiga toxin-mediated programmed cell death and delineation of death signaling pathways. PMID- 12375434 TI - Interaction of Shiga toxin with endothelial cells. PMID- 12375435 TI - Shiga toxin interactions with the intestinal epithelium. PMID- 12375436 TI - Serological methods for the detection of STEC infections. PMID- 12375437 TI - Protocols to study effects of Shiga toxin on mononuclear leukocytes. PMID- 12375438 TI - Animal models for STEC-mediated disease. PMID- 12375439 TI - Gnotobiotic piglets as an animal model for oral infection with O157 and non-O157 serotypes of STEC. PMID- 12375440 TI - Bovine Escherichia coli O157:H7 infection model. PMID- 12375441 TI - Detection and characterization of STEC in stool samples using PCR. PMID- 12375442 TI - Molecular typing methods for STEC. PMID- 12375443 TI - STEC in the food chain. Methods for detection of STEC in food samples. PMID- 12375444 TI - STEC as a veterinary problem. Diagnostics and prophylaxis in animals. PMID- 12375445 TI - Methods for detection of STEC in humans. An overview. PMID- 12375446 TI - Cellular microbiology of STEC infections. An overview. PMID- 12375447 TI - Analysis of pathogenicity islands of STEC. PMID- 12375448 TI - [The professional picture of medical physics of the medical division of radiation physics]. PMID- 12375449 TI - [Clinical dosimetry for heavy ion therapy]. AB - Since December 1997, patients are treated with carbon ions at GSI (Gesellschaft fur Schwerionenforschung). Dose delivery is performed with the intensity controlled raster-scanning technique, which allows a highly conformal treatment of the tumor. To meet the special requirements of dosimetry with heavy ion beams, new dosimetric measurement techniques were developed and introduced into clinical application by the DKFZ (Deutsches Krebsforschungszentrum). The techniques comprise calibration of the irradiation monitor, checks of lateral and depth dose profiles, as well as verification of the beam delivery for complex three dimensional dose distributions. The developed dosimetric methods are now integral part of clinical application and enable safe treatment with carbon ion therapy. PMID- 12375450 TI - DICOM--current status and future developments for radiotherapy. AB - Today, DICOM is one of the most successful standards in medicine. The standard was developed based on the increasing need for the transfer of digital images between systems of different vendors. Furthermore these images needed to be archived in a system independent format. DICOM has been continuously evolving since the early 80s and is today the main standard in radiology. After the IEC had started an initiative to develop a communication standard for radiotherapy in the late 80s, they joined forces with the DICOM Committee. This led to the foundation of a new working group (Working Group 7--Radiotherapy Objects) that deals with the radiotherapy extensions of DICOM. In 1997 and 1999 the so-called RT extension of DICOM was published, containing seven new objects and related services. Since then all major vendors of systems for radiotherapy have implemented DICOM interfaces that support the DICOM RT extensions. Working Group 7 is currently working on extending the Workflow support for the RT objects. This extension will increase the available services and will improve the handling of DICOM RT data. The developments so far are encouraging and it is hoped that DICOM will be as indispensable in radiotherapy as in radiology. PMID- 12375451 TI - MRI of the coronary arteries: flip angle train optimization for 3D sequences. AB - Application of contrast agents in MRI of coronary arteries improves contrast-to noise ratio (CNR), but widens the range of T1 relaxation times of the tissues to be imaged. The flip angle train, generated for the measurement of all phase encoding steps in the 3rd spatial dimension of the navigator echo FLASH sequence used, is optimal only for one T1. Computer simulations show that it is not advisable to optimize the sequence on the basis of an extremely short T1 relaxation time (such as in the case of contrast-enhanced vessels) because the imaging of the surrounding tissue would be negatively influenced. A sequence optimization to a T1 of approximately 200 ms seems to allow a CNR improvement of > or = 50%. PMID- 12375452 TI - [Planning for brain surgery in brain tumors]. AB - A precise knowledge of the localization of an intracerebral mass is a basic requirement for the planning of neurosurgical operations. Stereotactic atlases offer the possibility to adapt pre-operative imaging data onto normal anatomical conditions in the CNS. These atlases, however, reflect the standard variants of the CNS and do not allow to draw conclusions on local and secondary changes of the anatomy caused by the presence of pathological processes. The physical model proposed in this paper provides an estimate of the displacement of brain areas by an intracerebral mass. The modeling of brain parenchyma deformation is based on the mechanics of deformed media. The implementation of the model is successful in the group of primary brain tumors and meningiomas, and uses empirically-obtained data of a prospectively-selected patient population. The aim of the proposed model is, as further step, the integration and adaptation in apposite software solutions for the stereotactic orientation in the CNS. PMID- 12375454 TI - [Analysis of the extent of harm from digitally modulated mobile telephone rays]. AB - In the public discussion regarding the health risks of mobile phone system radiation, it is emphasized that the pulse slope of digital modulation, as defined in the GSM-Standard, will cause biological effects. In contrast, the high field strength of broadcasting and television radiation is not considered to be relevant. This paper compares quantitatively the slope of the digital GSM pulses with that of the synchronizing pulse of the television signal. The result shows clearly that the pulse spectrum of the television signal contains that of the GSM signal; in addition, the synchronizing impulse of television exhibits a much steeper slope. Considering the countrywide normal radiation intensities of television and mobile phone systems, it can be stated that the worldwide exposure to the common television signals over more than 50 years can disprove the contention of adverse biological health effects of the pulse slope of digitally modulated radiofrequency. PMID- 12375453 TI - Nonlinear scattering in hard tissue studied with ultrashort laser pulses. AB - The back-scattered spectrum of ultrashort laser pulses (800 nm, 0.2 ps) was studied in human dental and other hard tissues in vitro below the ablation threshold. Frequency doubled radiation (SHG), frequency tripled radiation and two photon fluorescence were detected. The relative yield for these processes was measured for various pulse energies. The dependence of the SHG signal on probe thickness was determined in forward and back scattering geometry. SHG is sensitive to linear polarization of the incident laser radiation. SHG in human teeth was studied in vitro showing larger signals in dentin than in cementum and enamel. In carious areas no SHG signal could be detected. Possible applications of higher harmonic radiation for diagnostics and microscopy are discussed. PMID- 12375455 TI - [Results of the DGMP survey on radiotherapy planning systems]. AB - The present paper presents the results of a survey on radiotherapy-treatment planning systems conducted in the summer of 2000. A total of 263 questionnaires of 108 German, 6 Austrian, and 4 Swiss radiotherapy clinics or practices have been evaluated. Most treatment planning systems are used for teletherapy, with nearly all sites using at least one commercial system. The planning systems are mostly networked with CT, partially also with MRI, simulator and R&V systems of the treatment equipment. For brachytherapy planning, usually orthogonal X-ray images are used, but some systems allow already the use of CT data as well. Stereotactic planning dominates the specialized treatment planning, followed by total body irradiation techniques. Commercial systems are well established for stereotactic planning systems, whereas in-house systems are still predominant for total body irradiation. In-house systems predominate also in the case of simple treatment planning systems and plausibility check programs. These simpler dose calculation programs usually offer variable degrees of correction options. PMID- 12375456 TI - Finite element analysis of deflections in major connectors for maxillary RPDs. AB - PURPOSE: The effect of major connector design on deflection in maxillary removable partial denture (RPD) frameworks under simulated occlusal loading was analyzed by means of three-dimensional finite element models. MATERIALS AND METHODS: Thirteen maxillary major connectors were produced for a Kennedy Class II case. Eleven frameworks consisted of posterior palatal straps with different anteroposterior widths at the midline. Anteroposterior and horseshoe bars were also constructed for comparison. In each framework, the occlusal rest on the abutment adjacent to the edentulous ridge was fixed in a vertical direction, and the rest on the contralateral side was fixed in all directions. A biting force of 20 N was vertically distributed simultaneously on each of the three missing posterior teeth locations. RESULTS: For the posterior palatal straps, the maximum vertical displacement at the saddle and the buccal displacement at both the saddle and the rest adjacent to the saddle decreased as their connector width increased from 6 to 29 mm, whereas maximum distal displacements were insensitive to the connector width. The posterior straps with anteroposterior widths of more than 18 mm revealed comparable rigidity to the anteroposterior bar. The horseshoe bar and the posterior straps with smaller widths demonstrated greater displacements than the other frameworks. CONCLUSION: The rigid connectors proved to be the most effective in transmitting applied occlusal forces to the contralateral side of the framework. PMID- 12375457 TI - An up to 15-year longitudinal study of 515 metal-ceramic FPDs: Part 1. Outcome. AB - PURPOSE: This study reports on the outcome of 515 metal-ceramic fixed partial dentures (FPD) involving 1,209 abutments and 885 pontics placed by one operator in a specialist prosthodontic practice between January 1984 and December 1997. MATERIALS AND METHODS: Clinical and laboratory protocol was kept constant as much as was practical. Each FPD and abutment was given a subjective prognostic rating at the time of cementation. Patients were recalled in 1993 (review 1) and 1998 (review 2). Clinical examination by the author covered 85% of 342 FPDs at review 1 and 82% of 515 FPDs at review 2. At review 2, 37% had been in clinical service for 5 to 10 years (group b), and 34% had been in service for 10 to 15 years (group c). An objective classification protocol was used to assess outcome. RESULTS: At review 2, the FPDs had failure rates of 2%, 7%, and 11% in groups a, b, and c, respectively. There was a significant increase in the failure rate of group c at review 2 (11%) compared with review 1 (5%). Cumulative survival analysis indicated that FPDs have an expected survival rate of 96%, 87%, and 85% at 5, 10, and 15 years, respectively. The applied prognostic rating proved more accurate as clinical service time increased. Outcome was not related to number of units. Cantilevered FPDs, nonvital abutments, and anterior abutments had significantly greater failure rates. Of initially vital abutments, 2% were subsequently endodontically treated. CONCLUSION: Tooth-supported FPDs have an expected survival rate of 85% at 15 years when the described clinical and laboratory protocol is applied. PMID- 12375458 TI - A modified short version of the oral health impact profile for assessing health related quality of life in edentulous adults. AB - PURPOSE: The aim of this study was to develop a shortened version of the Oral Health Impact Profile (OHIP) appropriate for use in edentulous patients and to evaluate its measurement properties. MATERIALS AND METHODS: Data were collected from the Ontario Study of Older Adults and a longitudinal clinical trial of implant-retained prostheses undertaken in Newcastle Dental Hospital, UK. All subjects completed an OHIP at baseline, and UK subjects also completed an OHIP posttreatment. Using an item impact reduction method, a shortened version of the OHIP (called OHIP-EDENT) was derived from both datasets. Discriminant validity and responsiveness properties of this modified version were compared with OHIP-14 and OHIP-49. RESULTS: Using an item impact method of reducing the 49 OHIP items produced very similar subsets in both Canadian and British populations; the modified version had little overlap with the current short version (OHIP-14). Discriminant validity properties of OHIP-EDENT were similar to OHIP-14 and OHIP 49. Using effect sizes to assess sensitivity to change, OHIP-EDENT exhibited less susceptibility to floor effects than OHIP-14 and appeared to measure change as effectively as OHIP-49. CONCLUSION: The modified shortened version of the OHIP derived in this study has measurement properties comparable with the full 49-item version. This modified shortened version may be more appropriate for use in edentulous patients than the current short version. PMID- 12375459 TI - An up to 5-year clinical evaluation of posterior in-ceram CAD/CAM core crowns. AB - PURPOSE: This study evaluated the clinical performance of posterior CAD/CAM generated In-Ceram Alumina and In-Ceram Spinell core crowns. MATERIALS AND METHODS: Nineteen In-Ceram Spinell core crowns (four premolars and 15 molars) and 24 In-Ceram Alumina core crowns (two premolars and 22 molars) in 21 patients were examined using modified USPHS criteria at baseline and after a mean service time of 39 +/- 11 months. The crown copings were machined from Vitablocs In-Ceram Alumina and Vitablocs In-Ceram Spinell using the Cerec 2 CAD/CAM system. RESULTS: Two molar In-Ceram Alumina core crowns fractured after respective service times of 14 and 17 months in the same patient. The Kaplan-Meier survival rate regarding fracture of the ceramic was 92% for In-Ceram Alumina and 100% for In-Ceram Spinell. At the follow-up examination, 80% alpha ratings and 18% beta ratings for In-Ceram Alumina core crowns and 84% alpha ratings and 15% beta ratings for In Ceram Spinell core crowns were recorded. CONCLUSION: Despite the two fractures, the clinical quality of CAD/CAM-generated In-Ceram Alumina and In-Ceram Spinell posterior crowns was excellent. Within the limitations of this study, both types of crowns appeared to be feasible. PMID- 12375460 TI - An in vivo study of the impact of different emergence profiles of procera titanium crowns on quantity and quality of plaque. AB - PURPOSE: The purpose of this study was to evaluate the effect of crowns with different emergence profiles on marginal plaque formation. MATERIALS AND METHODS: Seven crown preparations were performed on premolar teeth in six patients. Four titanium crowns for each tooth--with different marginal emergence angles--were manufactured according to the Procera technique. The three experimental crowns and the final permanent tooth were cemented with phosphate permanent cement. Plaque samples were collected from the marginal area after 1 week with normal oral hygiene, and again after refraining from oral hygiene for 2 days. The contralateral tooth served as a control. The quantity and quality of plaque were registered. The restoration was removed, the next crown version cemented, and the protocol repeated. RESULTS: All experimental crowns, irrespective of emergence profile, showed a significantly lower (P = .01) plaque quantity than controls. No intraindividual differences were found regarding the accumulation of mutans streptococci at the different experimental emergence profiles. No differences in quality between experimental and control sides were found. CONCLUSION: Within the limitations of this study, it was found that titanium crowns with emergence profiles of up to 40 degrees formed less plaque than healthy controls. There was no higher accumulation of mutans streptococci in relation to increasing emergence profiles. PMID- 12375461 TI - Multiple pains and psychosocial functioning/psychologic distress in TMD patients. AB - PURPOSE: This study assessed multiple pain conditions and their association with psychosocial functioning, psychologic distress, and somatization in patients with temporomandibular disorders (TMD) based on RDC/TMD Axis II findings. Nonspecific pain items examined included headaches, heart/chest pain, lower back pain, nausea/abdominal pain, and muscle pain. MATERIALS AND METHODS: In this study, 202 TMD patients (58 men and 144 women) referred to two TMD clinics participated. The mean age of the predominantly Chinese patient population (82%) was 32.6 years (range 13 to 65). The RDC/TMD history questionnaire was input directly into computers by patients. Graded chronic pain and SCL-90 scales were generated online and automatically archived for statistical analysis. Data were subjected to Spearman's rank-order correlation and Kruskal-Wallis and Mann-Whitney tests at a significance level of .05. RESULTS: Of the patients, 43% were moderately to extremely distressed by headaches. The percentage of patients who were distressed by heart/chest pain (7%), lower back pain (26%), nausea/abdominal pain (17%) and soreness of muscles (22%) was lower. Of the TMD patients, 16% experienced more than three pain items. Significant and positive correlations were observed between number of pain items experienced and graded chronic pain severity, depression, and somatization. Correlation coefficients ranged from .27 to .65 for graded chronic pain scales and somatization (without pain items) scores, respectively. CONCLUSION: Results suggest that the number of nonspecific pain conditions reported may be a predictor of psychosocial dysfunction, depression, and somatization. PMID- 12375462 TI - Fracture resistance and marginal adaptation of conventionally cemented fiber reinforced composite three-unit FPDs. AB - PURPOSE: This in vitro study investigated the marginal adaptation and fracture resistance of three-unit fiber-reinforced composite fixed partial dentures (FPD) luted with two different resin-modified glass-ionomers. MATERIALS AND METHODS: A total of 48 FPDs were constructed from the glass fiber-reinforced materials FibreKor/Sculpture, Vectris/Targis, or the polyethylene fiber system BelleGlass/Connect (n = 16 for each brand). The reconstructions were conventionally luted on human molars using resin-modified ProTecCEM or Fuji Plus and then exposed to thermocycling and mechanical loading. RESULTS: During thermocycling and mechanical loading, cementation failed in seven of eight FibreKor or BelleGlass FPDs and in one of eight Vectris/Targis FPDs luted with ProTecCEM. All Fuji Plus-cemented FPDs showed no signs of damage or cementation loss. The fracture resistance of the remaining FPDs was as follows: Vectris/Targis-ProTecCem 1,361 +/- 360 N, Vectis/Targis-Fuji Plus 923 +/- 207 N, BelleGlass/Connect 940 +/- 155 N, and FibreKor/Sculpture 524 +/- 202 N. The marginal adaptation of the cement-tooth interface deteriorated by 13% to 21% for all reconstructions after stress application, which was not statistically significant. The crown-cement interface had a significantly greater marginal gap only with the combination of FibreKor and Fuji Plus after stress simulation (change 33%). CONCLUSION: Conventional cementation of fiber-reinforced FPDs can lead to cementation loss. The marginal adaptation and fracture resistance deteriorated in comparison to adhesively cemented reconstructions. PMID- 12375463 TI - Elements released from dental casting alloys and their cytotoxic effects. AB - PURPOSE: This in vitro study investigated the element release from seven commercially available dental casting alloys and tested their cytotoxic effects. MATERIALS AND METHODS: The casting alloys tested were one high-noble alloy (Bioherador N) and six base-metal alloys, including four Ni-Cr alloys (Remanium CS, Heranium NA, Wiron 99, CB Soft), one Co-Cr alloy (Wirobond C), and one Cu based alloy (Thermobond). Ten specimens from each alloy were prepared in the form of disks, and each of the seven dental casting alloys (10 disks per group) were conditioned in distilled water at 37 degrees C for either 72 or 168 hours. The conditioning media were analyzed for element release, and the cytotoxic effects were assessed on Balb C fibroblasts using MTT assay. RESULTS: Element release was greater at 168 hours of conditioning than at 72 hours. The extract from the high noble alloy showed the least amount of element release (only Zn), with no cytotoxic effects. The greatest amount of element release was detected in the Cu based alloy Thermobond and the Ni-Cr alloy CB Soft; their extracts were significantly more toxic than all the other alloy extracts. The cytotoxic effects of the other Ni-Cr alloy extracts were not statistically significantly different from the high-noble alloy extract. However, the Co-Cr alloy (Wirobond C) extract was significantly more cytotoxic than the high-noble alloy extract. CONCLUSION: Element release from casting alloys is proportional to the conditioning time. The content of Cr and Mo in the alloy protects the alloy from dissolution, while the Cu content makes it more susceptible to corrosion and dissolution, rendering it more cytotoxic. PMID- 12375464 TI - Microleakage of temporary endodontic restorations in overdenture tooth abutments. AB - PURPOSE: The sealing of provisional filling material in overdenture tooth abutments during provisional rehabilitation is of primary importance to the long term success of roots bearing gold casting copies. The aim of this in vivo study was to evaluate the microleakage of four different provisional filling materials after a period of 1 week. MATERIALS AND METHODS: Five patients needing treatment with overdenture prostheses and scheduled for the extraction of at least four teeth were chosen. After performing endodontic treatment on the roots to be extracted, a standardized cavity preparation 3 mm in depth was made using a diamond bur. Each cavity was filled with one of the four provisional materials selected for the evaluation (Cavit-W, IRM Caps, Guttapercha, Fermit-N), and the interim prostheses were delivered to the patients. After 1 week, the roots were extracted and stored for 24 hours in 0.5% basic fuchsin at 37 +/- 1 degrees C for 24 hours. Subsequently, the roots were severed and observed under a stereomicroscope for microleakage evaluation. RESULTS: The materials showed different degrees of microleakage, but none allowed dye penetration to the bottom of the cavity. IRM Caps showed the lowest mean value of dye penetration (168 microns), while Fermit-N showed the highest (1,475 microns). All materials differed from each other (P < .05). CONCLUSION: Within a period of 1 week, the materials provided acceptable to good sealing properties. All of the materials may be considered suitable for provisional fillings if they do not remain in the oral cavity for more than 1 week. PMID- 12375465 TI - Effect of toothbrushing on the material loss, roughness, and color of intrinsically and extrinsically stained porcelain used in metal-ceramic restorations: an in vitro study. AB - PURPOSE: Because of abrasion by toothbrushing, dental materials in the oral cavity are subjected to substance loss to a different extent depending on the hardness of the material. This study investigated the color-change effect of substance loss and change of roughness resulting from toothbrushing of internally and externally stained metal ceramic. MATERIALS AND METHODS: Metal-ceramic specimens 15 mm in diameter and 2 mm thick were made. Blue colorant suspension was applied over the enamel porcelain (enamel-stained), in the body porcelain (dentin-stained), and over the opaque porcelain (opaque-stained). One group was not stained. Each group was made up of seven samples. All specimens were brushed in an experimental device. Material loss, roughness measurements, and spectrophotometric evaluations were made before and after brushing. Overall color change, chroma change, and value change were calculated with the use of the CIE LAB uniform color scale. RESULTS: Significant substance loss as a result of brushing was observed. No significant differences between chroma changes, between value changes of different groups, or between overall color changes of dentin- and enamel-stained groups were found. The difference between overall color changes of opaque-dentin and opaque-enamel stained groups was statistically significant. CONCLUSION: Staining should be done as deeply as possible to obtain durable color appearance. PMID- 12375466 TI - Evolution and use of aluminum oxide single-tooth implant abutments: a short review and presentation of two cases. AB - PURPOSE: This article reviews the development of esthetic implant abutments and illustrates the use of aluminum oxide implant abutments in two cases. MATERIALS AND METHODS: Two patients were restored with single-tooth implants for the replacement of anterior teeth. One patient received a prefabricated aluminum oxide abutment, which was customized by the dental technician. A second patient received a custom aluminum oxide abutment, which was designed and fabricated using CAD/CAM technology. Both cases were restored with all-ceramic crowns. RESULTS: Satisfactory functional and esthetic results were achieved in both cases. The CAD/CAM abutment required no further customization in the dental laboratory. CONCLUSION: The use of aluminum oxide ceramic abutments improves dental and mucogingival esthetics in single-implant restorations. The use of CAD/CAM technology simplifies the design and customization process. Clinical studies are required to confirm the long-term performance of this type of restoration. PMID- 12375467 TI - Changes in translucency and color of particulate filler composite resins. AB - PURPOSE: The purpose of this study was to examine changes in translucency and color of particulate filler composite resins that can be fabricated into metal free crowns. MATERIALS AND METHODS: Eight types of materials were used in this study. The particulate filler composite resins were represented by Artglass, BelleGlass, Estenia, Gradia, and Targis. Herculite XRV and Solidex were the conventional composite resins for crowns and fixed partial denture facings, and Empress was the ceramic material. Disks with a thickness of 1.0 mm were fabricated from each material and subjected to an accelerated test by immersion in 60 degrees C distilled water for up to 8 weeks. Color measurements were made before and after water immersion. Changes in translucency were evaluated by determining the contrast ratio, and changes in color were evaluated by determining color difference. RESULTS: After water immersion, Targis and Solidex demonstrated a significant increase in contrast ratio (6% to 7%) and a decrease in translucency. A color difference of more than 2.0, a visually perceptible value, was found for Targis, Gradia, and Solidex. However, the maximum color difference for these was 3.0, a value that would be considered clinically acceptable. CONCLUSION: Within the limitations of this study, the particulate filler composite resins were stable in both translucency and color. PMID- 12375468 TI - 51st Annual Meeting of the German Society of Prosthetic Dentistry and Materials Sciences. Dresden, 23-26 May 2002. PMID- 12375469 TI - Recreating family practice. PMID- 12375470 TI - Weighing the costs and benefits of family practice. PMID- 12375471 TI - Weighing the costs and benefits of family practice. PMID- 12375472 TI - Pharmaceutical freebies. PMID- 12375473 TI - Using advance beneficiary notices to maximize your Medicare collections. PMID- 12375474 TI - A job-share model for the new millennium. PMID- 12375475 TI - Choosing a practice facility. PMID- 12375476 TI - How many staff members do you need? PMID- 12375477 TI - Getting a grip on your phone calls. PMID- 12375478 TI - 12 tips for increasing your job satisfaction. PMID- 12375479 TI - Heartfelt hurt. Why statins are making some patients really sore. PMID- 12375480 TI - Solving the MS puzzle. PMID- 12375481 TI - Don't drink the water. PMID- 12375482 TI - Smallpox defense. PMID- 12375483 TI - Group therapists' training: what predicts learning? AB - This field study represents the continuing effort to identify the determinants of learning within experiential small study groups. Thirty-seven training groups from the 1996 Institute of the American Group Psychotherapy Association were studied. Three process measures (Group Relationship Questionnaire [GRQ], Leader Adjective Measure [LAM], and Group Climate Questionnaire [GCQ]) were administered to 434 group members after the first two of four group sessions. Process variables were used to predict learning (measured by the Learning Evaluation Form [LEF]) at the end of the training groups. The factors derived from each of the measures showed good to excellent correspondence with previous studies employing the same instruments. Results suggest that perceptions of the leader and the group, rather than perceptions of one's own relationship to other group members, are more robust predictors of learning in these short-term training groups. Specifically, perceptions of an emotionally engaged group willing to confront conflict, and perceptions of a skillful leader, proved to be significant predictors of learning. Implications for the training of group therapists and group therapy research are discussed. PMID- 12375484 TI - Cohesion and outcome in short-term psychodynamic groups for complicated grief. AB - This study used two measures of cohesion for the process analysis of 12 short term, time-limited groups for complicated grief. The measures had similar theoretical definitions but differed in terms of rater source (member vs. observer), measurement score (mean of items vs. global rating), and rating unit (individual vs. group). We examined the relationship between the measures, assessed the development of cohesion over the life of the group, and evaluated each measure's relationship to outcome. A principal components analysis with each measure yielded one cohesion component, which supported a unidimensional model; however, the two cohesion components were independent of each other, which supported a multidimensional model. Repeated measures analyses indicated that observer-rated cohesion developed in a quadratic manner (v pattern) across sessions, while member-rated cohesion developed in a linear manner. The object focus (the group, other members, the therapist) of the members' ratings determined whether cohesion increased or decreased across sessions. No significant relationships between cohesion and outcome were identified. Implications of the findings for the understanding of group cohesion are considered. PMID- 12375485 TI - Interpersonal predictors of group therapy outcome for complicated grief. AB - This study investigated three aspects of patients' interpersonal functioning as predictors of outcome for two forms of group psychotherapy for complicated grief. Patients presented with a variety of death losses and met criteria for complicated grief. The three aspects of interpersonal functioning were the patient's (1) attachment to the lost person, (2) quality of object relations (QOR), and (3) level of recent social role functioning. A more secure attachment to the lost person and better social role functioning were associated with more favorable outcome in both forms of therapy. In addition, patients with higher QOR had more favorable outcome in interpretive therapy while lower QOR patients had more favorable outcome in supportive therapy. The results suggest that each aspect of interpersonal functioning is important to consider when treating patients for complicated grief. PMID- 12375486 TI - Group counseling to enhance adolescents' close friendships. AB - The purpose of this study was to measure the impact of group counseling on adolescents' intimacy with a close friend. The study population was comprised of 174 residential and day students of seven ninth-grade classes in a residential school in Israel. All participants were socially disadvantaged, with a problematic family background. They were randomly divided into experimental and control conditions: group counseling versus an in-class enrichment program. School personnel in the helping professions conducted all counseling groups after receiving training and supervision. Results of the counseling intervention showed a significant late effect in intimacy growth with a close friend. None of the three covariates (gender, residency, divorce) had a significant impact on results. The results support, to some extent, the dual process model of relationship development. PMID- 12375487 TI - A systematic program to enhance clinician group skills in an inpatient psychiatric hospital. AB - This article describes the collaborative effort of a team of discipline directors, administrators, and academicians to create a systematic program to enhance the group competencies of a large clinical staff working at a state hospital. The effects of the program were tested by a quasi-experimental field study. Quantitative measures of group process provided limited support for program effectiveness. Stronger support came from qualitative inquiry. The development and effectiveness of the program is examined within a larger context of group programs housed in large health care organizations. PMID- 12375488 TI - Assessing victim empathy in sexual offenders using the victim letter task. AB - In attempting to enhance victim empathy, it is common to have sexual offenders write an apology letter to their victim. This task is thought to reveal the level of empathy that the sexual offender has for his victim. However, until now there has been no reliable method for judging the quality of empathy revealed in the victim letter. This paper reports the development and evaluation of 2 templates to score letters written to child victims and adult victims, respectively. An acceptable level of interrater reliability was obtained for the templates. Deficits revealed by the templates did not correlate with scores on a generic empathy measure but did correlate with scales measuring minimization and denial. The measures were also sensitive to change following empathy training. PMID- 12375489 TI - Issues concerning the reliability and validity of the diagnosis of sexual sadism applied in prison settings. AB - This study examined limited aspects of the diagnoses of sexual sadism among incarcerated sexual offenders. The diagnoses examined in this study were made by experienced forensic psychiatrists following DSM-III-R or DSM-IV criteria. Archival data was extracted on 51 sexual offenders for whom a psychiatric evaluation had been requested. Analyses of offense history and features, offender self-reports, and phallometric data, indicated few differences between those offenders diagnosed as sadists and those not so diagnosed. In fact, where there were differences, the data indicated that the nonsadists were the most deviant. The results are discussed in terms of their meaning for both forensic practice in prisons and the value of the diagnosis of sexual sadism. PMID- 12375490 TI - Some implications of prenatal alcohol exposure for the treatment of adolescents with sexual offending behaviors. AB - Prenatal alcohol exposure can seriously harm the fetus, resulting in a wide range of physical and central nervous system abnormalities. A follow-up study of persons prenatally exposed to alcohol found that 49% of adolescents and adults had repeatedly displayed inappropriate sexual behavior. While these persons are likely to present to sexual offender treatment programs, they are unlikely to be recognized as neurologically impaired because the sequelae of prenatal alcohol exposure are seldom accurately identified by clinicians. Persistent impairments in response inhibition, memory, and executive functions are common, requiring adaptations to standard sexual offender assessment and treatment. PMID- 12375491 TI - Prediction of recidivism in exhibitionists: psychological, phallometric, and offense factors. AB - Exhibitionists have traditionally been regarded as nuisance offenders. However, empirical studies show that some offenders can be highly recidivistic and can escalate to incidents of Hands-on sexual assault. The objective of this study was to investigate predictors of recidivism in exhibitionists and clarify the differences between Hands-on and Hands-off sexual recidivists. The hundred and twenty-one exhibitionists were assessed at a university teaching hospital between 1983 and 1996. Archival data came from medical files and police files. The Psychopathy Checklist-Revised (PCL-R) was assessed retrospectively. Results indicated that over a mean follow-up period of 6.84 years, 11.7, 16.8, and 32.7% of exhibitionists were charged with or convicted of sexual, violent, or criminal offenses, respectively. Sexual reoffending recidivists were less educated, and had more prior sexual and criminal offenses. Violent, recidivists were also less educated, had lower Derogatis Sexual Functioning Inventory (DSFI) scores, higher PCL-R Totals, and more prior sexual, violent, and criminal offenses. Criminal recidivists were younger, less educated, had lower DSFI scores, higher PCL-R scores, higher Pedophile Indices, and more prior sexual, violent, and criminal offenses. Hands-on sexual recidivists demonstrated higher PCL-R ratings, higher Pedophile and Rape indices, and more prior sexual, violent, and criminal offenses than did Hands-off counterparts. PMID- 12375492 TI - A DSM-IV Axis I comorbidity study of males (n = 120) with paraphilias and paraphilia-related disorders. AB - One hundred and twenty consecutively evaluated outpatient males with paraphilias (PAs; n = 88, including 60 sex offenders) and paraphilia-related disorders (PRDs; n = 32) were systematically assessed for certain developmental variables and DSM IV-defined Axis I comorbidity. In comparison with the PRDs, the PA group was statistically significantly more likely to self-report a higher incidence of physical (but not sexual) abuse, fewer years of completed education, a higher prevalence of school-associated learning and behavioral problems, more psychiatric/substance abuse hospitalizations, and increased employment-related disability as well as more lifetime contact with the criminal justice system. In both groups, the most prevalent Axis I disorders were mood disorders (71.6%), especially early onset dysthymic disorder (55%) and major depression (39%). Anxiety disorders (38.3%), especially social phobia (21.6%), and psychoactive substance abuse (40.8%), especially alcohol abuse (30%), were reported as well. Cocaine abuse was statistically significantly associated with PA males (p = .03). There was a statistically significant correlation between the lifetime prevalence of Axis I nonsexual diagnoses and hypersexual diagnoses (PAs and PRDs). The prevalence of retrospectively diagnosed attention deficit hyperactivity disorder (ADHD) was 35.8%, the third most prevalent Axis I disorder. ADHD (p = .01), especially ADHD-combined subtype (p = .009), was statistically significantly associated with PA status. ADHD was statistically significantly associated with conduct disorder, and both of these Axis I disorders were associated with the propensity for multiple PAs and a higher likelihood of incarceration. When the diagnosis of ADHD was controlled, the differences reported above between PAs and PRDs either became statistically nonsignificant or remained as only statistical trends. Thus, ADHD and its associated developmental sequellae and Axis I comorbidities was the single most common nonsexual Axis I diagnosis that statistically significantly distinguished males with socially deviant sexual arousal (PAs) from a nonparaphilic hypersexual comparison group (PRDs). Sex offender paraphiliacs were more likely to be diagnosed with conduct disorder, alcohol abuse, cocaine abuse, and generalized anxiety disorder. The prevalence of any ADHD in the sex offender paraphiliacs was 43.3%, and nearly 25% of offenders were diagnosed with ADHD-combined subtype. PMID- 12375493 TI - Compensability for psychiatric injury: an opportunity for modernization and reconceptualisation. PMID- 12375495 TI - Law, ethics and the conduct of forensic autopsies. PMID- 12375494 TI - Aspects of legal liability in pain management involving opioid medications. PMID- 12375497 TI - Queensland v Nolan. [2001] QSC 174. PMID- 12375496 TI - So where will the buck stop? Liability and the move for a more diverse health care workforce. PMID- 12375498 TI - Melchior v Cattanach. [2001] QCA 246. PMID- 12375499 TI - Medico-legal knowledge of general practitioners: disjunctions, errors and uncertainties. AB - This article discusses a survey of Victorian general practitioners which investigated doctors' legal knowledge, the impact of law on clinical practice, doctors' current medico-legal information sources and their legal education needs and preferences. Knowledge of legal standards was investigated in relation to three areas: disclosure of risk; ownership of, and access to, medical records; and proxy decision-making. Additionally, the impact of statutory reform in relation to proxy decision-making was explored. Further, doctors' past experience of medico-legal education, current sources of medico-legal information and preferences concerning future medico-legal information were explored. Results indicated that overall, respondents had a very inadequate understanding of relevant law and that relevant statutory standards have had little impact on clinical practice. Professional bulletins and journals were identified as major current legal information sources, whilst printed materials, seminars and conferences were preferred sources of legal information. The authors conclude that there is a significant disjunction between legal standards and doctors' awareness of those standards, thereby creating a significant source of liability for doctors. Results highlight an urgent need to develop legal education programs for general practitioners based on doctors' identified needs and preferences. PMID- 12375500 TI - Compensation: problems with the concept of disability and the use of American Medical Association Guides. AB - The Western Australia Liberal Government made radical changes to the Workers Compensation and Rehabilitation Act 1981 (WA) in 1993. One of the significant changes was the greater application of the American Medical Association Guides to the assessment of permanent injury. In 1999 further amendments to the same legislation required the application of the Guides to workers who wished to proceed with common law claims for negligence against their employers. Recent cases have shown the difficult in reconciling the language of the law with commonly used medical terms. This article surveys the use of the American Medical Association Guides in compensation legislation in Australia with some specific comments on the Western Australian system. It makes some suggestions for reform of the Western Australian system. PMID- 12375501 TI - Loss of a chance in medical malpractice litigation: expanding liability of health professionals versus providing justice to those who have lost. AB - The loss of a chance doctrine in medical malpractice litigation is essentially based on the perceived unfairness of denying recovery to a patient when a health provider's malpractice has reduced the patient's chance of a better outcome. It is the thesis of the article that loss of a chance must the recognised at law, notwithstanding that the chance is less than even or not subject to the benefit of statistical and/or scientific proof and that each lost chance should be assessed according to the value of that chance. Varying approaches to allocating value to the chance lost are examined both historically and internationally. The author contends that the policy arguments--which include potential for increased medical malpractice litigation, tainted reputations and an increase in professional indemnity policies--are insignificant when compared to the value and quality of human life and therefore cannot be supported. PMID- 12375502 TI - Protecting genetic materials and genetic information: a case study of Guthrie Cards in Victoria. AB - The authors are privileged to have been provided with correspondence about a dispute over the ongoing storage of genetic material (as Guthrie Cards) in Victoria. The correspondence details confusion over the roles of government and the private sector service provider in accounting for the storage, use and destruction of these stored genetic materials collected as part of a government public health program. The purpose in publishing this account is to highlight the present inadequacies in current practices and the ongoing potential for a crisis in the management of collected genetic materials through a lack of appropriate regulation, transparency and accountability. The article suggests measures to remedy some of the existing inadequacies in contractual arrangements and recommends that the government retain ownership and control of both the genetic materials and the derived information to ensure some accountability in the present legal environment. PMID- 12375503 TI - Wrongful life actions: the legal and ethical hurdles. AB - This article discusses the various legal and ethical issues arising out of the cause of action for wrongful life. This action involves a claim by a child that but for the negligence of the doctor, hospital or other medical institution, his or her mother would have terminated the pregnancy and he or she would not have been born. The courts have generally rejected this cause of action on the basis of legal, ethical and policy considerations. The author proposes that the legal hurdles can be overcome and that the ethical and policy considerations do not outweigh the desirability of upholding wrongful life claims. PMID- 12375504 TI - Thinking about cloning: a reply to Judith Thomson. AB - Opponents of human cloning typically argue for the prohibition of therapeutic cloning and a permanent prohibition of reproductive cloning, even if a safe cloning technology should become available. In a recent article in this journal, "Legal and Ethical Problems of Human Cloning" (2000) 8 JLM 31, Judith Thomson develops an ethico-legal analysis that would justify prohibitions or restrictions on both therapeutic and reproductive cloning, irrespective of any safety issue. This article criticises Thomson's analysis in detail and suggests, in particular, that it relies upon an intellectually unacceptable understanding of personhood. PMID- 12375506 TI - [Writing, a voyage from there to "psych" Interview by Armel Rivallan]. PMID- 12375507 TI - [Agitation in psychiatry, which benchmarks for which care?]. PMID- 12375508 TI - [Malaise in the institution]. PMID- 12375509 TI - [Distress and pathological agitation in psychotic patients]. PMID- 12375510 TI - [Pathological agitation, therapeutic approach in a closed unit]. PMID- 12375511 TI - [Pathological agitation and nursing care in a unit for difficult patients]. PMID- 12375512 TI - [An unexpected encounter in a medical psychological center]. PMID- 12375513 TI - [The evolution of mental health professions]. PMID- 12375517 TI - [Elements of economic problems in the field of extra-renal dialysis]. AB - The aim of this paper is to enlighten, in an economic perspective, the ongoing debate which has emerged surrounding the question of the respective place of peritoneal dialysis and haemodialysis in the treatment of terminal renal failure. In order to accomplish this, the authors used a cost effectiveness approach aiming to create a link between two kinds of data: data on the effectiveness of treatment methods on the one hand, and data on the costs associated with such treatment modalities on the other. The international literature tends to show that the two dialysis techniques are comparable in terms of effectiveness, except in the case of diabetic patients, for a significantly lower treatment cost in the case of peritoneal dialysis as opposed to haemodialysis, regardless of the geographical location and in spite of certain methodological biases existing due to the sample sizes implemented in the cost studies. All things considered, it seems that in comparison to haemodialysis methods, peritoneal dialysis results in lower cost effectiveness ratios (that is to say, lower cost of treatment to attain a certain level of effectiveness). The conclusion of this report therefore emphasizes the need to develop and promote peritoneal dialysis, in situations where this modality is clinically applicable, in countries where it is currently underused, which is the case in France. PMID- 12375518 TI - [Pharmacists' role in smoking cessation activities in Alsace]. AB - In May 1999, France launched a plan to implement a range of tobacco control measures focusing on reducing tobacco use. This study evaluated two professional assistance measures for smoking cessation included in the plan: the over-the counter sale of nicotine substitutes and replacement therapies and pharmacist training. The article describes the role of pharmacies in smoking cessation and analyses the impact of training on professional practices. The survey was conducted in the year 2000 through a mail poll sent to a random representative sample of 290 pharmacies in the French region of Alsace selected from the Ministry of Health's FINESS database. The cross-sectional survey requested data on the pharmacy and its staff, as well as its role in tobacco prevention and its practices regarding individual smoking cessation interventions (capacity to provide assistance once information is obtained on the patient's smoking status, assessment of the patient's tobacco dependence and their will to quit smoking, initial prescription of a nicotine substitute, and provision of practical advice on maintaining smoking cessation and medical care recourse). Among a total of 82 respondents, 85% proclaimed having provided smoking cessation activities and 37% had undergone training. All of the pharmacies had been declared as tobacco prevention areas. For the pharmacies offering smoking cessation assistance, the methods and activities were in accordance with best practice standards. A comparison between the group of trained pharmacists and those who were untrained did not show any significant difference for the items concerning professional practice. In spite of certain barriers linked to pharmaceutical practice, improvements in the quality of smoking cessation assistance activities provided by pharmacists are possible, such as providing confidential space to advise the client, knowing when referral to a doctor is necessary, and organising personalized follow-up according to the customer's needs. PMID- 12375519 TI - [Humane professional attitudes of doctors in the health region of Sousse (Tunisia)]. AB - The new chronic and multi-factorial morbidity, the limited efficiency of classical medicine, as well as the preponderant position currently occupied by an individual in social life have imposed a patient-oriented approach as an ideal model of medical practice. The objective of this work is to evaluate physicians' listening skills, their empathy and their participation with patients in the health region of Sousse. It is a descriptive and transversal study on a collective of 133 practitioners working in the Sousse region in both the liberal and public sectors of care. Data were collected during the year 1999 through an auto-managed questionnaire. Among the main results: 28% of doctors declared having had difficulties while communicating with their patients and 31% of those asked do not give a great deal of importance to the psycho-social aspects of their patients. Only 31% of practitioners were in favour of patients participating in the decision-making process. This survey demonstrates that the domination of the bio-medical model of care, as much in teaching field as in medical practice, has weakened the basic competencies necessary for humanitarian medical practice based on the respect and the implication of the patient. Reforming the medical teaching curriculum with a focus on the person suffering and orientating the professional environment towards the community are both indispensable in order to reach a reconciliation between the patient's needs and the care available. PMID- 12375520 TI - [External partnerships and prevention relative to psychoactive substances: what should be the position of the school and its partners?]. AB - School health education is one of the primary missions of the educational system which is characterised by the involvement of numerous professional institutions, associations, and others, at the heart of academic establishments. The need for partnership within this complex domain has been recognised and affirmed by the official publications of the national Ministry of Education as it has been in actions. In so far as the move towards the recourse of external intervention, should this be considered as the result of a true partnership or an act of simple delegation (or subcontracting)? Firstly, this article proposes to define the concept of partnership. Secondly, it presents the results of a study on the implementation of drug-use prevention in high schools who underline the importance of the problems associated with partnership in this field and attempt to put into perspective the respective points of view of the educational teams and external actors. On the basis of these results, it proposes a typology of the networks from which the activities are built, then analyses the conditions necessary in order that health education be implemented within a framework of partnership established on the competencies, differences and contributions of each partner. PMID- 12375521 TI - [Mandatory disclosure and infectious disease. From "pestilential" disease to "emerging" diseases]. AB - Due to the recent overhaul of the procedure on mandatory disclosure of infectious diseases (law relating to the reinforcement of sanitation quality control established in July 1998, and the May 1999 and May 2001 decrees on the application of this law), wishing to take advantage of this opportunity, the authors propose a chronological review retracing the history of these legal declarations. For over a century, they have represented the main instrument used for intervention and surveillance allowing for the fight against infectious diseases. The health options kept have varied over the years, as well as the precautions taken to respect secrecy (nominative or anonymous disclosure, modalities of transmission...). Procedures adopted to reconcile the principle of confidentiality along with the necessity to protect public health in the case where it would require an immediate and urgent intervention are examined throughout the chronology (determining the source of contamination, prevention of contagion). PMID- 12375522 TI - [Contaminated blood: the state's renunciation and incompetence in the public health domain, a long tradition]. PMID- 12375523 TI - [Violence, perinatal policy and medical practice]. PMID- 12375524 TI - [Study of the motivation of physicians participating in public health research]. AB - In order to explore practitioners' motivations to participate in epidemiological research, a study was conducted among doctors who had been requested to provide clinical information on their patients included in the group study DESIR in the Inde and Loire regions of France. Six semi-structured individual interviews were carried out by two sociologists, and were then followed-up by 216 questionnaires, 80 of which were completed and analysed. Finally, 18 telephone interviews were conducted to complete the data collected. Reasons given for participating in epidemiological studies are mainly the perceived scientific interest, relevance for public health and the feeling of being a partner in the research. The obstacles seem to be the vague image of the promoter, ignorance of the DESIR study's objectives, lack of direct contact for communicating information, and the mixture and confusion between filling out the study's questionnaire and handling administrative constraints in general. Therefore, better communication, centred on the concept of partnership between research and practical medicine, informing on the usefulness of knowledge from epidemiological studies for the practice of medicine, and clarifying the promoter's role, could improve the level of participation from general practitioners. PMID- 12375525 TI - [1902-2002: one hundred years of public health legislation; and now?]. PMID- 12375527 TI - Proceedings of the 4th Congress of the Spanish Society of Community Nutrition and the 3rd International Workshop of Community Nutrition. Bilbao, Spain, 4-8 October 2000. PMID- 12375526 TI - [Regional evaluation of long-term care facilities for the elderly]. AB - The increase in the total number of very elderly people demands the precision and specification of the needs of long-term care structures in the coming years. A survey conducted in Lower-Normandy is presented and described. Questionnaires were sent to directors of residence homes and nursing homes in order to investigate their operations and their problems. The increase in the number of spaces to foresee seems moderate if the progressive trend remains unchanged (+1% per year until the year 2010), but within the same timeframe, the demographic decline in the number of potential family helpers and home health workers to take care of the elderly in their homes, coupled with the establishment of a new state allowance for dependent people, could alter the situation. Furthermore, more than 50% of nursing homes and residence homes for the elderly are in need of significant improvements: a reduction in the number of shared rooms (52.5% of nursing homes) and the development of equipment to meet the needs of the handicapped and disabled. The means in personnel and staff qualifications are particularly heterogeneous and difficulties in coping with dependency are reported most everywhere. The application of the 1999 decree stipulating the approval of these structures based upon thorough evaluations of available services is urgently needed. PMID- 12375528 TI - Proceedings and abstracts of the 5th International Conference on Lactoferrin. Banff, Alberta, Canada. 4-9 May 2001. PMID- 12375529 TI - [Mexican consensus for prevention and treatment of cerebrovascular disease]. PMID- 12375530 TI - Top 30 assisted living chains. PMID- 12375531 TI - Proceedings of the 3rd National Congress GISCRIS. Radioguided and radioimmunoguided oncology: present and future. 29 November-1 December 2001. PMID- 12375532 TI - Feds sound warning on information technology. PMID- 12375533 TI - At cross purposes. There are widespread claims that the proposal to fine local authorities for delayed discharges is full of loopholes. PMID- 12375534 TI - A bug in the system. As Legionnaire's disease leaves its mark in Cumbria, the government's infectious disease policy is at the centre of controversial change. PMID- 12375536 TI - Proceedings of the 3rd International Conference on Scanning Probe Microscopy, Sensors and Nanostructures. Makuhari, Chiba, Japan, 27-31 May 2001. PMID- 12375535 TI - Simple minds. A few things done properly--and soon--is the short-term strategy for the NHS IT programme. Can it deliver this time? PMID- 12375537 TI - Special issue in honour of the late Mirko D. Grmek. PMID- 12375538 TI - Nicotine patch use in pregnant smokers: smoking abstinence and delivery outcomes. AB - OBJECTIVE: To describe smoking abstinence and fetal effects of pregnant smokers who received 8 weeks of nicotine patch therapy. METHODS: One-sample study of 21 pregnant women smoking > or = 15 cigarettes/day during their third trimester of pregnancy despite physician advice to stop. Nicotine patch therapy (22 mg/24 h) was initiated during the first day of a 4-day in-hospital study and continued for a total of 8 weeks. Subjects returned weekly until delivery, at 4 weeks after delivery, and at 6 and 12 months after patch therapy. Fetal growth and well-being were assessed using ultrasound examinations and non-stress tests. RESULTS: Eight of 21 subjects completed all 8 weeks of patch therapy according to the protocol. Five subjects (24%) discontinued using the nicotine patch, owing to adverse skin reactions. There were eight subjects (38%) who were biochemically confirmed abstinent from smoking at the time of delivery; of these, seven were continuously abstinent from the start of patch therapy. Centile weight for gestational age did not change significantly over time for 12 subjects with serial ultrasound measurements available at baseline, 4 weeks and 8 weeks following initiation of patch therapy. In all cases, non-stress tests remained reactive or became reassuring with observation. No significant preterm deliveries occurred (gestational ages of 36.3-41.1 weeks). Three infants suffered severe neonatal morbidity; however, these problems were unrelated to nicotine patch therapy. CONCLUSION: Nicotine patch therapy has potential benefit for pregnant smokers who continue to smoke despite physician advice to stop. PMID- 12375539 TI - Sonographic estimate of birth weight: relative accuracy of sonographers versus maternal-fetal medicine specialists. AB - OBJECTIVE: To compare the relative accuracy of predicting birth weight among registered diagnostic medical sonographers versus maternal-fetal medicine specialists. STUDY DESIGN: Over 7 months all patients who delivered within 2 weeks and had sonographic measurements of femur length and head and abdominal circumferences by sonographers and physicians were included in the analysis. The exclusion criteria were multiple gestation and anomalous fetuses. Receiver operating-characteristic curves (ROC) were constructed to determine the ability to detect intrauterine growth restriction (IUGR; birth weight < 2,500 g) and macrosomia (birth weight > or = 4,000 g) among term (gestational age > or = 37 weeks) parturients. A level of p < 0.05 was considered significant. RESULTS: Among 365 patients recruited, the mean gestational age was 37.3 +/- 2.4 weeks with a mean birth weight of 3,083 +/- 72.5 g. Among term patients the prevalence of IUGR was 7.5% (18/238) and of macrosomia 12% (29/238). A significantly higher percentage of predictions were within 10% of the birth weight when obtained by sonographers (70%) than physicians (54%; p < 0.0001). Registered sonographers were significantly more likely to detect IUGR than the specialists (area under the ROC curves 0.97 +/- 0.02 vs. 0.92 +/- 0.02, respectively; p = 0.02). Both groups had similar accuracy in detecting macrosomic fetuses (area under the ROC curves 0.92 +/- 0.02 for sonographers and 0.90 +/- 0.02 for physicians; p = 0.40). CONCLUSIONS: Prediction of birth weight is significantly more accurate when sonographers rather than maternal-fetal medicine specialists perform the ultrasonographic examination. PMID- 12375540 TI - Outpatient cervical ripening with prostaglandin E2 and estradiol. AB - OBJECTIVE: To determine whether weekly outpatient administration of prostaglandin gel or estrogen cream initiated labor in women with an unfavorable cervix. METHODS: All uncomplicated pregnancies at term gestation who were candidates for a vaginal delivery with a Bishop score of < or = 6 were randomly assigned to receive on a weekly basis: prostaglandin E2 gel (n = 41); estrogen cream (n = 44); or inert lubricant jelly (n = 43). RESULTS: In the three groups no differences were observed among 128 subjects in the weekly Bishop scores, cervical dilatation or gestational age upon admission to the labor and delivery suite, the percentage of patients presenting with spontaneous labor or ruptured membranes, the number of post-date inductions or neonatal outcome. CONCLUSIONS: Weekly out-patient cervical ripening using either prostaglandin gel or estrogen in women with an unfavorable cervix at 37 weeks' gestation was no more effective than a placebo in Bishop score improvement or in preventing post-date inductions. PMID- 12375541 TI - Cord serum lipid and apolipoprotein levels in preterm infants with the neonatal respiratory distress syndrome. AB - OBJECTIVE: The purpose of this study was to evaluate the effects of birth weight on cord serum lipid and apolipoprotein levels in preterm infants with and without respiratory distress syndrome (RDS). METHODS: Cord serum lipid and apolipoprotein levels were evaluated in preterm infants (39 with RDS and 68 controls without RDS). Based on morbidity and mortality risk, RDS and non-RDS infants were separated into four birth weight groups (2,000-2,499 g, 1,500-1,999 g, 1,000 1,499 g, < 1,000 g) and evaluated for effects of birth weight on cord serum levels. RESULTS: RDS infants with birth weight of 2,000-2,499 g had significantly higher levels of cholesterol, triglyceride, total fatty acids and apolipoprotein A-I, but not arachidonic acid, than controls. RDS infants weighing 1,000-1,999 g had lower total fatty acids and apolipoprotein B levels, including arachidonic acid, than non-RDS infants. Cord serum lipid and apolipoprotein levels were significantly elevated in large (2,000-2,499 g) RDS infants, but lower levels were found in smaller (1,000-1,999 g) RDS infants. CONCLUSIONS: Cord serum arachidonic acid and apolipoprotein levels found in RDS infants suggest that lipid transport across the placenta may be abnormal. Inadequate total fatty acid supplies in utero could interfere with normal fetal growth and maturation, leading to development of neonatal RDS as one manifestation of risk for postnatal morbidity and mortality. PMID- 12375542 TI - Mature adrenomedullin concentrations in plasma during pregnancy. AB - OBJECTIVE: Adrenomedullin is a novel peptide that exerts a potent, dose-dependent and long-lasting hypotensive effect. In human plasma, adrenomedullin consists of two molecular forms: mature and immature. Immature adrenomedullin is much less bioactive than mature adrenomedullin. Although a gradual increase in plasma adrenomedullin has been reportedly observed as pregnancy progressed, mature adrenomedullin has not been examined. The aim of this study was to elucidate the plasma level of mature adrenomedullin in pregnant women. METHODS: We measured the concentrations of mature adrenomedullin in ten pregnant women in the first trimester, ten pregnant women in the third trimester, and ten non-pregnant controls with the immunoradiometric assay. RESULTS: The mean concentration of mature adrenomedullin was significantly increased in pregnant women in the first trimester compared to age-matched non-pregnant subjects (p < 0.05). The mean concentration of mature adrenomedullin was significantly increased in pregnant women in the third trimester compared with pregnant women in the first trimester (p < 0.005). CONCLUSION: Our study demonstrated that concentrations of mature adrenomedullin were elevated in pregnant women compared with non-pregnant women and its concentration in the third trimester was significantly higher than that in the first trimester. PMID- 12375543 TI - The effects of overcoming experimental bladder outflow obstruction in fetal sheep. AB - OBJECTIVE: To develop an ovine model of fetal bladder outflow obstruction and to investigate the effect on the kidney of surgical relief of the obstruction in the prenatal period. METHODS: Ultrasound examination and amniocentesis were performed on 68 date-bred pregnant ewes at day 57 of pregnancy (term = 150 days). Fetal gender was determined using a molecular technique to identify single male fetuses. The urethra and urachus were ligated at hysterotomy on 20 of these fetuses at 75 days' gestation. Comparisons were made with six controls that did not undergo operation. Changes that occurred in fetal urinary tract appearance were detected using serial ultrasound examinations. Seven obstructed cases chosen at random had further prenatal surgery on day 94 to decompress the obstructed urinary tract by vesicostomy. The animals were killed at 110 days' gestation and post-mortem studies were performed. RESULTS: Fourteen days after surgical obstruction, there were increases in the summed renal lengths (33 mm vs. 28 mm, p = 0.003) and renal pelvis anteroposterior (A-P) diameters (8 mm vs. 5.5 mm, p = 0.02). In the group allocated to receive surgical decompression, 9 days' relief of obstruction resulted in significant reductions in summed renal lengths (30 mm vs. 41 mm, p = 0.024; controls 31 mm) and renal pelvis A-P diameters (5.8 mm vs. 8.9 mm, p = 0.012; controls < 2 mm). Postmortem histological examination in the surgical decompression group revealed an estimated number of glomeruli similar to controls and greater than in the obstructed cases. CONCLUSION: Surgical relief of fetal bladder outflow obstruction in ovine mid-pregnancy results in improved renal appearance on ultrasonic and histopathological examinations. PMID- 12375544 TI - Symptomatic hyponatremia following cesarean section. AB - A rare occurrence of the syndrome of inappropriate antidiuretic hormone secretion is described in a 32-year-old previously healthy nulliparous woman who underwent a Cesarean section for non-progressive labor. PMID- 12375545 TI - Regionalization, networks and neonatal transport. PMID- 12375547 TI - Is the correlation between fetal oxygen saturation and blood pH sufficient for the use of fetal pulse oximetry? AB - OBJECTIVES: Fetal pulse oximetry was performed during labor in high-risk cases for fetal distress to determine the diagnostic value of this method. METHODS: The fetal SpO2 values were blinded from the obstetrician so that these values did not influence clinical decisions. Mean and lowest SpO2 measurements for the last 30 min prior to either fetal scalp blood sampling or delivery were correlated with scalp pH or pH from the umbilical artery. RESULTS: No significant correlation was found between pH and mean fetal oxygen saturation (correlation coefficient -0.02, p = 0.9). There was no significant correlation between pH and lowest fetal oxygen saturation (correlation coefficient 0.04, p = 0.84). Concerning the feasibility of the method, we found that only 23 of 65 included patients were suitable for analysis; in 20% of cases, we were not able to perform a SpO2 measurement. CONCLUSIONS: None of three cases with pH below 7.05 would have been detected using mean SpO2 over the last 30 min prior to fetal scalp blood sampling or delivery. Only one case would have been detected using the lowest SpO2 measurement over this period. We conclude that fetal SpO2 measurements during labor are of poor diagnostic value, with a disappointing feasibility and therefore are not ready for implementing into daily clinical practice. PMID- 12375546 TI - Repeat postpartum magnesium sulfate administration for seizure prophylaxis: is there a patient profile predictive of need for additional therapy? AB - OBJECTIVE: To profile patients with hypertensive disorders of pregnancy who require reinstitution of magnesium sulfate therapy for disease exacerbation. STUDY DESIGN: A prospective clinical trial enrolling gravidas with pre-eclampsia. The length of postpartum magnesium sulfate seizure prophylaxis was determined by individual patient characteristics. Patients with exacerbation of their disease after discontinuation of magnesium sulfate received a second course of magnesium sulfate lasting 24 h. RESULTS: Of a total of 503 patients, 38 (7.6%) required reinstitution of postpartum magnesium sulfate therapy for an additional 24-h period. Patients with chronic hypertension complicated by superimposed pre eclampsia were most likely to require further therapy (11/61, 18.0%), when compared with other hypertensive disorders. Additionally, patients who required reinstitution of magnesium therapy had significantly shorter gestations (32.4 +/- 4.2 weeks versus 36.3 +/- 4.2 weeks, respectively; p < 0.001), and higher mean arterial pressure during the initial magnesium course (113.2 +/- 11.2 versus 105.6 +/- 11.3 mmHg; p < 0.001). CONCLUSION: Patients with chronic hypertension complicated by superimposed pre-eclampsia, patients delivered prior to 35 weeks' gestation and patients requiring a longer initial magnesium prophylaxis are at higher risk for the need of reinstitution of seizure prophylaxis postpartum. PMID- 12375548 TI - Chlorhexidine vaginal flushings versus systemic ampicillin in the prevention of vertical transmission of neonatal group B streptococcus, at term. AB - OBJECTIVE: To investigate the efficacy of intrapartum vaginal flushings with chlorhexidine compared with ampicillin in preventing group B streptococcus transmission to neonates. METHODS: This was a randomized controlled study, including singleton pregnancies delivering vaginally. Rupture of membranes, when present, must not have occurred more than 6 h previously. Women with any gestational complication, with a newborn previously affected by group B streptococcus sepsis or whose cervical dilatation was greater than 5 cm were excluded. A total of 244 group B streptococcus-colonized mothers at term (screened at 36-38 weeks) were randomized to receive either 140 ml chlorhexidine 0.2% by vaginal flushings every 6 h or ampicillin 2 g intravenously every 6 h until delivery. Neonatal swabs were taken at birth, at three different sites (nose, ear and gastric juice). RESULTS: A total of 108 women were treated with ampicillin and 109 with chlorhexidine. Their ages and gestational weeks at delivery were similar in the two groups. Nulliparous women were equally distributed between the two groups (ampicillin, 87%; chlorhexidine, 89%). Clinical data such as birth weight (ampicillin, 3,365 +/- 390 g; chlorhexidine, 3,440 +/- 452 g), Apgar scores at 1 min (ampicillin, 8.4 +/- 0.9; chlorhexidine, 8.2 +/- 1.4) and at 5 min (ampicillin, 9.7 +/- 0.6; chlorhexidine, 9.6 +/- 1.1) were similar for the two groups, as was the rate of neonatal group B streptococcus colonization (chlorhexidine, 15.6%; ampicillin, 12%). Escherichia coli, on the other hand, was significantly more prevalent in the ampicillin (7.4%) than in the chlorhexidine group (1.8%, p < 0.05). Six neonates were transferred to the neonatal intensive care unit, including two cases of early onset sepsis (one in each group). CONCLUSIONS: In this carefully screened target population, intrapartum vaginal flushings with chlorhexidine in colonized mothers display the same efficacy as ampicillin in preventing vertical transmission of group B streptococcus. Moreover, the rate of neonatal E. coli colonization was reduced by chlorhexidine. PMID- 12375549 TI - Chronology of neurological manifestations of prenatally diagnosed open neural tube defects. AB - OBJECTIVE: To evaluate the incidence and chronology of sonographic markers of neurological compromise in prenatally diagnosed neural tube defects. METHODS: We reviewed our ultrasound database from 1988 to 1999 to identify all cases of prenatally diagnosed neural tube defects. All patients received an initial detailed targeted ultrasound evaluation with subsequent evaluations every 4-6 weeks. Cases involving multiple congenital anomalies, aneuploidy, or inadequate follow-up were excluded. Specific ultrasound markers assessed included the presence of ventriculomegaly (> 10 mm) and clubfoot. RESULTS: Forty-seven cases of neural tube defects were identified over the study interval. After exclusions, 42 cases were available for evaluation. The overall incidence of ventriculomegaly and clubfoot in the study cohort was 86% and 38%, respectively. In the 33 patients with initial ultrasound examination performed at < 24 weeks' gestation, 76% (25/33) had evidence of ventriculomegaly and 30% (10/33) and clubfoot. Only 9% (1/11) of the patients managed expectantly developed evidence of ventriculomegaly and 3/11 (27%) developed clubfoot from the time of the initial ultrasound examination to delivery. CONCLUSIONS: Ultrasound markers of neurological compromise are early and frequent findings associated with fetal neural tube defects. Development of ventriculomegaly is an uncommon occurrence later in gestation, while the risk for developing clubfoot appears to increase as gestation progresses. PMID- 12375550 TI - Individual longitudinal patterns in biochemical and hematological markers for the early prediction of pre-eclampsia. AB - OBJECTIVE: To analyze the individual longitudinal patterns of maternal biochemical and hematological tests performed throughout gestation in order to predict at the 20th week of pregnancy the later development of pre-eclampsia. STUDY DESIGN: A longitudinal study was conducted on 187 white normotensive pregnant women all with a history of pre-eclampsia. Blood samples were performed at the 8th week of gestation and then every 4 weeks until the 36th week. The longitudinal patterns of urea, creatinine, uric acid, total proteins, hematocrit, red blood cells, hemoglobin, mean red cell volume, ferritin and iron were derived. By means of regression analysis, for each woman and each significant marker, a 'theoretical physiological pattern', from the 8th to the 20th week, was constructed. By comparing the observed values of each marker for each woman with her 'theoretical physiological pattern', variables indicating the match or mismatch to it were derived. Such variables were used, together with other maternal characteristics, in a logit regression for the probability of developing pre-eclampsia later in pregnancy. RESULTS: In 140 cases, pregnancies followed a physiological course, while 47 women developed pre-eclampsia during the third trimester. In the physiological gestations, the weekly mean values of creatinine, hematocrit, total proteins, uric acid and urea showed patterns that were significantly different from those of the pathological group. The logit model was able to classify correctly 96% of the physiological and 87% of the pathological pregnancies, with a negative predictive value of 96% and a positive predictive value of 89% (area under the receiver operator characteristics (ROC) curve 0.98). The ability of the model to predict later complications at the 20th week was confirmed by a validation procedure. CONCLUSION: The simultaneous use of individual longitudinal patterns of parameters, achieved non-invasively as part of the standard methods of antenatal care that provide a global evaluation of plasma volume expansion, showed a high ability to predict, early in pregnancy, the later development of pre-eclampsia. PMID- 12375551 TI - Medical staff standard approved on credentialing volunteer LIPs in emergencies. PMID- 12375552 TI - New and revised standards on medical emergencies approved for assisted living. PMID- 12375553 TI - Can sin taxes and lawsuits make us healthier? PMID- 12375554 TI - NAPH: public hospitals struggling. PMID- 12375555 TI - [37th conference of Japanese Medical Society of Alcohol and Drug Studies. Tokyo, Japan. September 5-8, 2002. Abstracts]. PMID- 12375556 TI - [The 75th congress of the Japanese Biochemical Society. Tyoto, Japan. October 14 17, 2002. Program]. PMID- 12375557 TI - [The 98th annual meeting of the Japanese Society of Psychiatry and Neurology. Yokohama, Japan. August 26, 2002. Abstracts]. PMID- 12375558 TI - [The 46th annual meeting of the Japanese Society for Medical Mycology. Tokyo, Japan. September 28-29, 2002. Program and Abstracts]. PMID- 12375560 TI - Abstracts of the 15th European Colloquium on Animal Cytogenetics and Gene Mapping. Sorrento, Italy, 2-4 June 2002. PMID- 12375559 TI - Ernest Sutton: growing potential. PMID- 12375562 TI - [Abstracts of 50th Scientific Session of the Japanese College of Cardiology. Nagoya, Japan. September 9-11, 2002]. PMID- 12375563 TI - [The 32nd Western/Eastern regional meeting, Japanese Society of Nephrology. Wakayama prefecture, Tokyo, Japan. October 4-5, 2002. Abstracts]. PMID- 12375561 TI - Evolution in action. PMID- 12375564 TI - [The 44th Congress of the Japanese Society of Clinical Hematology. Yokohama, Japan. September 12-15, 2002. Program and Abstracts]. PMID- 12375566 TI - Biochemical Society 677th Meeting, Cardiff University. Abstracts. PMID- 12375565 TI - Helicobacter pylori and nonsteroidal anti-inflammatory drug interactions in bleeding ulcers. PMID- 12375567 TI - Abstracts of the XVIth Congress of the European Association for Cranio Maxillofacial Surgery. 3-7 September 2002. PMID- 12375568 TI - Medicare holds down physician pay. PMID- 12375569 TI - [The concentrations of prolactin and estrogens in women with fibrocystic changes in the breast]. AB - OBJECTIVE AND STUDY DESIGN: To evaluate the concentrations of prolactin and estrogens in blood sera in women with fibrocystic changes in the breast (FCC). MATERIAL AND METHODS: The control group consisted of 32 women without any pathological changes in the breast (mean age 44.9 +/- 4.4 y.). The studied group comprised 81 women having FCC (mean age 45.5 +/- 3.5 y.). Both groups were divided into two subgroups according to age: the first subgroup ranged 40-44 y. and the second 45-51 y. The hormonal profiles, namely: prolactin in basal conditions (PRL I), and after metoclopramide-test (PRL II), follitropin (FSH), lutropin (LH), estradiol (E2), progesterone (P) were assayed by means of "bioMerioux" kits. Estrone (E1) was assayed by means of "DBC Diagnostic kits. RESULTS: In both subgroups of the study group (40-44 and 45-51 y.) a significantly higher (p < 0.001) concentration of prolactin was shown after metoclopramide test, as well as significantly higher percentage increase in prolactin after metoclopramide test (p < 0.001) were shown in comparison with the control group. In age compartment of 40-44 y. in the study group significantly lower (p < 0.05), progesterone-estradiol index (PEL) was found in comparison with the control group. In age compartment of 45-51 y. a progesterone-estradiol index (PEL) in study group was significantly lower (p < 0.05) in comparison with the control group. CONCLUSION: Functional hyperprolactinemia and relative hyperestrogenism are risk factors of the development concerning fibrocystic changes in the breast. PMID- 12375570 TI - 2002 Mangold Award Recipient. Harry E. Grenawitzke, R.s., M.P.H., D.A.A.S. PMID- 12375571 TI - 2002 Walter F. Snyder Award Recipient. Gayle J. Smith. PMID- 12375572 TI - Cytokines and Interferons 2002. Turin, Italy, 6-10 October 2002. Abstracts. PMID- 12375573 TI - Abstracts of the XIX International Complement Workshop. 22-26 September 2002, Palermo, Italy. PMID- 12375574 TI - Environmental risks of applying sewage sludge compost to vineyards: carbon, heavy metals, nitrogen, and phosphorus accumulation. AB - Biosolids are applied to vineyards to supply organic matter. However, there is concern that this practice can increase the concentration of macronutrients and heavy metals in the soil, some of which can leach. We evaluated the environmental hazard of sewage sludge compost applied in March 1999 at 10, 30, and 90 Mg ha-1 fresh weight in a vineyard in southeastern France. Soil organic matter increased in all plots by 3 g kg-1 18 mo after the amendment. Neither total nor available heavy metal concentrations increased in the soil. Mineral nitrogen (N) in the topsoil of amended plots of 10, 30, and 90 Mg ha-1 increased by 5, 14, and 26 kg (NO3(-)-N + NH4(+)-N) ha-1, respectively, the first summer and by 2, 5, and 10 kg (NO3(-)-N + NH4(+)-N) ha-1, respectively, the second summer compared with controls. At the recommended rate, risks of N leaching is very low, but phosphorus (P) appeared to be the limiting factor. Phosphorus significantly increased only in plots amended with the highest rate in the topsoil and subsoil. At lower rates, although no significant differences were observed, P added was greater than the quantities absorbed by vines. In the long run, P will accumulate in the soil and may reach concentrations that will pose a risk to surface waters and ground water. Therefore, although the current recommended rate (10 Mg ha-1) increased soil organic matter without the risk of N leaching, total sewage sludge loading rates on vineyards should be based on P concentrations. PMID- 12375575 TI - [Left ventricular hypertrophy in patients with chronic renal failure treated by hemodialysis]. AB - Left ventricular hypertrophy (LVH) is common and important predictor of risk of death in end-stage renal failure. In the present study we have analysed echocardiographically the left ventricular hypertrophy and some possible risk factors continuing to its development in patients with chronic renal failure (crf) treated by hemodialysis (HD). From a cohort of 85 patients with crf we selected for analysis 59 clinically stable patients. Echocardiography (ECHO), body mass index (BMI), serum creatinine, urea, total protein, albumin, hemoglobin, hematocrit, electrolytes and parathyroid hormone (PTH) concentrations were evaluated in all patients at the next hours after HD session. LVH was common in HD patients: concentric LVH was detected by ECHO in 46 patients and in 13 patients eccentric LVH was observed. Mean serum concentrations of urea, creatinine, PTH and phosphate differed from normal values while hemoglobin, total protein, albumin, sodium kalium, calcium serum concentration were in the normal range. Positive correlation was found between PTH serum concentration and LVM r = 0.704 (p < 0.001), between PTH serum concentration and IVS r = 0.267 (p < 0.04), between PTH serum concentration and PW t = 0.238 (p < 0.04), and negative correlation between BMI and LVMI r = -0.451 (p < 0.05). The correlations between serum PTH concentration and LVH and between BMI and LVH confirmed that both hyperparathyroidism and malnutrition are important factors influencing the development of LVH in HD patients. PMID- 12375576 TI - Abstracts of the 33rd World Conference on Lung Health of the International Union Against Tuberculosis and Lung Disease (IUATLD). Montreal, Canada, 6-10 October 2002. PMID- 12375577 TI - Abstracts of the 3rd International Conference on Early Psychosis. 25-28 September 2002, Copenhagen, Denmark. PMID- 12375578 TI - Role of bovine viral diarrhea virus biotype in the establishment of fetal infections. AB - OBJECTIVE: To examine the role of bovine viral diarrhea virus (BVDV) biotype on the establishment of fetal infection in cattle. ANIMALS: 30 mixed-breed pregnant cows. PROCEDURE: Pregnant cows were inoculated oronasally with either i-WNADL, originating from an infectious BVDV cDNA clone of the National Animal Disease Laboratory (NADL) isolate, or the parental virus stock, termed NADL-A. RESULTS: All cows developed neutralizing antibodies to BVDV, and virus was commonly isolated from peripheral blood mononuclear cells or nasal swab specimens of NADL A inoculated cows; however, virus was rarely isolated from specimens of i-WNADL inoculated cows. i-WNADL did not cause fetal infection, whereas all fetuses harvested from NADL-A inoculated cows at 6 weeks after inoculation had evidence of infection. Immunoblot analysis of fetal virus isolates revealed the absence of NS3, confirming a noncytopathic (NCP) biotype BVDV in the NADL-A stock. The sequence of the NCP contaminant (termed NADL-1102) and the i-WNADL genome were virtually identical, with the exception of a 270 nucleotide-long insert in the i WNADL genome. Phylogenetic analyses revealed that NADL-1102 forms a monophyletic group with 6 other NADL genomes. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the contaminating NCP virus in the NADL-A stock was the ancestral NADL virus, which originally infected a bovine fetus and recombined to produce a cytopathic (CP) variant. Following oronasal infection of pregnant cows, viremia and transplacental transmission of CP BVDV to the fetus is rare, compared with the high occurrence of maternal viremia and fetal infection observed with NCP BVDV. PMID- 12375579 TI - Poultry Science Association 91st Annual Meeting. August 11-14,2002. Newark, Delaware, USA. Abstracts. PMID- 12375580 TI - Early impairment of coronary flow reserve is not associated with Chlamydia pneumoniae antibodies. AB - BACKGROUND: Chlamydia pneumoniae infection has been associated with atherosclerosis by sero-epidemiological, histopathological and interventional studies, and animal experiments. We hypothesized that if chlamydial infection is causative of atherosclerosis, the occurrence of antibodies against C. pneumoniae should be associated with coronary vasomotor dysfunction - an early sign of atherosclerosis. AIM: To study the association between C. pneumoniae infection and coronary vasomotor function in young men without signs of ischemic heart disease. METHODS: Serum IgG and IgA antibody concentrations against C. pneumoniae were determined in 125 clinically healthy subjects undergoing positron emission tomography (PET) studies. Myocardial blood flow was measured at rest and during pharmacologically induced hyperemia using [15O]H2O Coronary flow reserve was calculated as the ratio of hyperemic blood flow to resting blood flow. RESULTS: No association was found between serum C. pneumoniae antibody concentrations and myocardial blood flow parameters. In contrast, more conventional risk factors for coronary artery disease, such as total cholesterol and apolipoprotein B, were inversely associated with hyperemic flow and flow reserve. CONCLUSIONS: We found no association between C. pneumoniae antibodies and coronary vasomotor function in subjects without ischemic heart disease. Thus, these results do not support the role of C. pneumoniae infection as an early phase risk factor for coronary artery disease. PMID- 12375582 TI - Integrating ethics and quality improvement: practical implementation in the transitional/ extended care setting. AB - A major challenge in health care today is to provide high-quality care that results in the best outcomes possible for patients and residents within the limits of available resources. Throughout the past decade, there has been a call from ethicists for health care institutions to integrate the ethics and quality improvement processes. This article describes how a transitional/extended care facility integrated the quality improvement process within an ethical framework to achieve high-quality care while controlling cost. PMID- 12375584 TI - [44th National Congress of Anesthesia and Resuscitation. Paris, 19-22 September 2002. Abstracts]. PMID- 12375583 TI - Correlation of the -3826A >G polymorphism in the promoter of the uncoupling protein 1 gene with obesity and metabolic disorders in obese families from southern Poland. AB - The aim of the study was to examine the allelic frequency of the -3826A > G mutation of UPC1 in patients with familiar obesity and to investigate putative association of this polymorphism with metabolic disorders. One hundred and eighteen overweight /obese patients participated in the study. The UCP1 polymorphism was determined by RFLP. Glucose, lipid, insulin and leptin levels were measured both during OGTT and OLTT. The majority of patients had a homozygous A/A genotype (51,38%), while 14,68% had a G/G genotype. We found no significant association of the G allele with either BMI or glucose tolerance. Patients with the homozygous G/G genotype had significantly higher fasting levels of TG (p < 0.04) and decreased levels of HDL-cholesterol (p = 0,004). They also had an increased concentration of FFA and the rise of TG levels during the OLTT compared to controls was significant (p = 0,058). In addition, the carriers of the G/G genotype had the lowest insulin levels both during OGTT and OLTT. In our study we have demonstrated that the -3826A > G polymorphism of UCP1 does not play a major role in the development of obesity and/or disturbances of glucose metabolism. However, the increased levels of TG and FFA and decreased levels of HDL observed in carriers of the G allele suggest FFA-induced impairment of the HDL turnover and disturbance of the beta-cell function, both of which are risk factors for endothelial injury. PMID- 12375585 TI - [Semen in the works of Hildegard of Bingen]. AB - Hildegard of Bingen (1098-1179), versatile female figure of the late Middle Ages, a passionate lover of music, a mystic, an expert of herbs and medicine, and an abbess in the monastery of Eibingen, besides transmitting the divine word, dedicated her entire life to the study of the natural world. She composed a true encyclopedia of the knowledge of her times, whether it be in regards to natural sciences or medicine, with the conviction that a cure or medical practice could not exist without a theoretical system. The handwritten tradition of her medical achievements relate to the 13th Century; the biographical sources and the protocol of the case for canonization, mention the existence of a medicine handbook entitled Liber subtilitatum diversarum naturarum, handed down from tradition in the form of two distinguished topics: Physica, or Book of medicine for the simple, and Causae et curae, or Composite medicine book. From the reading of her works one gains a vast knowledge of Medieval medicine, basically associated with Galen and Aristotle's philosophy. The analysis of sexuality, which has a very close relationship with astrology, assumes an obvious appearance. If on one hand affliction for the flesh emerges, on the other hand sexuality is seen a divine theorem. Moreover, much space is dedicated to the disorder of sexual life and male impotence, not seen anymore as a remedy to sinful lust, but as a pathology to cure. PMID- 12375586 TI - [Human castration: historical notes]. AB - Human castration has been performed from early times for different reasons: to punish and revenge, to display one's religious fanaticism, to protect or to control women, for eunuchs' trade, for therapeutical purposes. In early modern times men were castrated to obtain sopranos voices, or for eugenic or racial reasons. Nowadays chemical castration is used as a therapeutic treatment or as a way to punish rape and other criminal behaviours. Castration is surgical or chemical act that may obviously cause serious physical and psychological consequences. PMID- 12375587 TI - Abstracts from the 6th Annual Scientific Meeting Heart Failure Society of America. September 22-25, 2002. PMID- 12375588 TI - From the Centers for Disease Control and Prevention. Tetanus--Puerto Rico, 2002. PMID- 12375589 TI - From the Centers for Disease Control and Prevention. Norwalk-like virus associated gastroenteritis in a large, high-density encampment--Virginia, July 201. PMID- 12375591 TI - JAMA patient page. Attention-deficit/hyperactivity disorder. PMID- 12375590 TI - From the Centers for Disease Control and Prevention. National, state, and urban area vaccination levels among children aged 19-35 months--United States, 2001. PMID- 12375592 TI - The Liver Meeting. Abstracts of the 53rd Annual Meeting of the American Association for the Study of Liver Diseases. November 1-5, 2002. Boston, Massachusetts, USA. PMID- 12375593 TI - Abstracts of the American Society for Therapeutic Radiology and Oncology 44th Annual Meeting. October 6-10, 2002. New Orleans, Louisiana, USA. PMID- 12375594 TI - Screening colonoscopy among persons 40 to 49 years of age. PMID- 12375595 TI - Hereditary amyloidosis. PMID- 12375596 TI - Intranasal mupirocin to prevent postoperative infections. PMID- 12375597 TI - Human immunodeficiency virus in pregnancy. PMID- 12375598 TI - What's ahead for health insurance? PMID- 12375599 TI - Kissing and food reactions. PMID- 12375600 TI - An alternative perspective: homeopathic drugs, Royal Copeland, and federal drug regulation. PMID- 12375601 TI - Ethics of health-care systems. PMID- 12375602 TI - Ethics of health-care systems. PMID- 12375603 TI - Ethics of health-care systems. PMID- 12375604 TI - Ethics of health-care systems. PMID- 12375605 TI - [Medicines for towns and medicines for countrysides]. AB - The regulations of the communities of apothecaries, in towns, ruled the conditions of the preparation and of the distribution of medicines to people. The application of these regulations was very strict and many conflicts occurred between apothecaries, grocers and surgeons. But, all that concerned people living in towns. What was it for countrysides? No medicine doctors, no apothecaries, a few surgeons! How could it be possible to cure countrymen? King's medicines, charitable ladies, ecclesiastics and pedlars were the solutions. Many books were redacted, during the XVIIth and the XVIIIth centuries, in order to help charitable persons to prepare easily non-expensive but efficient medicines for poor people. PMID- 12375606 TI - Oxygen, wound healing and the development of infection. Present status. AB - Wounds do not heal in tissue that does not bleed and almost always heal in tissue that bleeds extensively. A continuous supply of oxygen to the tissue is vital for the healing process and to resist infection. External factors may decrease the peripheral oxygen supply, but supplementary perioperative oxygen reduces the surgical wound infection rate to half in patients having colorectal resections. Hyperbaric oxygen may be beneficial when the flow and oxygen supply to the healing tissue are compromised by local injury and particularly if anaerobic infection is present. Assessment of perfusion and oxygenation is essential during and after surgery. PMID- 12375607 TI - Appendiceal abscesses: primary percutaneous drainage and selective interval appendicectomy. AB - OBJECTIVE: To present our results of non-surgical primary management of appendiceal abscesses using ultrasonic percutaneous drainage under local anaesthesia, and selective interval appendicectomy. DESIGN: Retrospective study. SETTING: University hospital, Sweden. SUBJECTS: 24 patients with appendiceal abscesses 3-12 cm in size. INTERVENTIONS: Primary ultrasonic percutaneous drainage under local anaesthesia, antibiotic treatment, and selective surgical treatment. MAIN OUTCOME MEASURES: Long-term follow-up. RESULTS: All patients had their abscesses drained successfully without complications. One patient continued to have fever, but eventually responded to conservative treatment and in one the bowel was perforated by the drain but again this was treated conservatively. Four abscesses recurred. Seven patients underwent planned interval appendicectomy. Another three patients were also operated on-one for caecal adenocarcinoma, and two for persisting symptoms and enterocutaneous fistulas. CONCLUSIONS: Appendiceal abscesses can be effectively drained percutaneously using ultrasound guided drainage under local anaesthesia, without complications. Recurrent appendicitis is common, and malignancy is a substantial risk in elderly patients. Modern laparoscopic appendicectomy and early postoperative discharge makes interval appendicectomy a valid treatment option after primary non-surgical management of appendiceal abscesses. PMID- 12375608 TI - Laparoscopic or open conventional cholecystectomy: clinical and economic considerations. AB - OBJECTIVE: To compare clinical aspects and financial costs of open conventional and laparoscopic cholecystectomy. DESIGN: Retrospective analysis of hospital records of patients who were operated on electively for symptomatic gallstone disease. SETTING: University clinic, Germany. SUBJECTS: 153 consecutive patients who had open conventional (1991-92) and 222 who had laparoscopic cholecystectomy (1993-96). A total of 251 cholecystectomies were done during 1991-92 and 523 cholecystectomies during 1993-96. INTERVENTION: Cholecystectomy. MAIN OUTCOME MEASURES: Clinical aspects: operating time, complications, postoperative stay; financial aspects: total cost of hospital stay after cholecystectomy. RESULTS: When open conventional was compared with laparoscopic cholecystectomy: operating time was 66 and 92 minutes; complications, 9 and 6 cases; postoperative stay, 8 and 3 days; and total cost of hospital stay, US $ 3434 and US $ 2808 respectively. CONCLUSION: The cost of laparoscopic cholecystectomy was 18% less than for open conventional cholecystectomy, principally because of the shorter postoperative stay. PMID- 12375609 TI - Acute pancreatitis: a prospective study of its incidence, aetiology, severity, and mortality in Iceland. AB - OBJECTIVE: To evaluate the incidence, aetiology, severity and mortality of patients with acute pancreatitis. DESIGN: Prospective study. SETTING: University hospital, Iceland. PATIENTS AND METHODS: All 50 patients diagnosed with acute pancreatitis during the one-year period October 1998-September 1999 inclusive. MAIN OUTCOME MEASURES: APACHE II, and Ranson and Imrie scores, and C-reactive protein (CRP) concentrations. The Balthazar-Ranson criteria were used for scoring of computed tomograms (CT). RESULTS: 27 of the 50 patients were male. The median age of the whole series was 60 years (range 19-85). The estimated incidence was 32/100000 for the first attack of acute pancreatitis. The causes were; gallstones 21 (42%), alcohol 16 (32%), miscellaneous 12 (24%), and idiopathic 1 (2%). 15 (33%) of the patients had APACHE II scores > or = 9, 17 (38%) had Ranson scores of > or = 3, 23 (50%) had Imrie scores of > or = 3, and 16 (34%) had CRP concentrations over 210 mg/L during the first 4 days or > 120 mg/L during the first week. Seven patients had severe pancreatitis. 2 patients in the whole group died, and both had clinically severe pancreatitis. CONCLUSIONS: This study indicates that the incidence of less severe acute pancreatitis is rising. Prospective assessment makes it possible to evaluate the aetiological factors more accurately. Measurement of the CRP concentration is an attractive and simple alternative to the severity scoring systems currently in use. PMID- 12375610 TI - Total thyroidectomy for differentiated thyroid cancer: primary compared with completion thyroidectomy. AB - OBJECTIVE: To analyse morbidity after completion total thyroidectomy compared with primary total thyroidectomy in a specialist thyroid surgery centre. DESIGN: Retrospective study. SETTING: Tertiary referral hospital, India. PATIENTS: Medical records of 143 patients who had total thyroidectomy between January 1990 and December 1999. 95 had primary thyroidectomies and 48 were completion thyroidectomies. MAIN OUTCOME MEASURES: Complication rate in both groups. RESULTS: The groups were comparable in respect of clinicopathological variables. Residual tumour was found in 19/48 (40%). After completion thyroidectomy, transient hypoparathyroidism and transient recurrent laryngeal nerve palsy were recorded in 8/48 (17%) and 2/48 (4%), respectively. No permanent hypoparathyroidism or permanent recurrent laryngeal nerve palsy was recorded in the completion thyroidectomy group. CONCLUSIONS: Completion thyroidectomy can be done with acceptable morbidity in a specialist thyroid surgery centre. Fear of increased morbidity after the procedure should not deter surgeon from doing this operation or referring the patients to a specialist centre. PMID- 12375611 TI - Predictors of malignant behaviour in gastrointestinal stromal tumours: a clinicopathological study of 34 cases. AB - OBJECTIVE: The clinicopathological features of gastrointestinal (GI) stromal tumours were analysed to find out the features that influence prognosis in these neoplasms. DESIGN: Retrospective study. SETTING: University Hospital, Spain. SUBJECTS: Review of clinical records and analysis of a series of GI stromal tumours classified in three groups: benign = 7 leiomyomas, malignant or potentially malignant = 16 combined smooth muscle-neural tumours, and 2 miscellaneous, and definitely malignant = 1 leiomyosarcoma and 8 gastrointestinal autonomic nerve tumours. MAIN OUTCOME MEASURES: Electron microscopy and immunohistochemical staining for vimentin, smooth muscle actin, muscle specific actin, desmin, S-100 protein, neuronal specific enolase, chromogranin A, synaptophysin, CD34, CD117 (c-kit), Bcl-2, and Ki-67. RESULTS: We found significant differences (p < 0.0001) between the median (P25, P75) size of tumours in the benign group 2.0 (1.2, 3.5) and in the potentially 7.5 (4.0, 13.0) and definitely 5.0 (4.0,6.5) malignant groups. The percentage of mitoses was lower (p = 0.003) in the benign group 0.4 (0.5) than in the other groups 2.0 (1.0, 5.0). Immunoreactivity for CD117 in leiomyomas was an unexpected finding; this showed a different staining pattern from the diffuse and homogeneous staining in GI stromal tumours. In addition, the MIB-1 proliferation index differentiated (p = 0.002) between the benign group 1.5 (1.0, 2.5) and the other two groups 7.0 (3.0, 11.0). CONCLUSIONS: These findings support the idea that GI stromal tumours form a heterogeneous group of neoplasms in which large size, presence of necrosis, and a high proliferative index (mitotic rate or MIB-1 index, or both) are good predictors of malignant behaviour. PMID- 12375612 TI - Clinical outcomes and quality of life after low anterior resection for rectal cancer. AB - OBJECTIVE: To evaluate clinical outcomes and quality of life in terms of anal, urinary, and sexual function, after low anterior resection for rectal cancer. DESIGN: Retrospective study. SETTING: University hospital, Switzerland. SUBJECTS: 43 patients with low rectal cancers. INTERVENTIONS: 27 were not given adjuvant radiotherapy and 16 had preoperative adjuvant radiotherapy 1.6 Gy twice daily for 13 days. MAIN OUTCOME MEASURES: Anal, urinary, and sexual function postoperatively. RESULTS: 23 patients reported normal defaecation (53%), 9 had incontinence of flatus (21%), 5 had occasional minor soiling (12%), 2 had frequent major soiling (5%), 4 had a total faecal incontinence (9%), and 3 had urinary incontinence (7%). Sexual dysfunction was reported by 9 of the 13 sexually active men and 2 of the 11 sexually active women. CONCLUSION: Despite their reported faecal, urinary and sexual dysfunction most patients were satisfied with their quality of life. Counselling at the time of operation is highly recommended as a means of contributing to personal satisfaction. PMID- 12375613 TI - Gastric necrosis in a patient with bulimia. PMID- 12375614 TI - Differentiated thyroid cancer presenting as distant metastases. PMID- 12375615 TI - Can't get no ... satisfaction! PMID- 12375616 TI - Different photoperiods affect proliferation of lymphocytes but not expression of cellular, humoral, or innate immunity in hamsters. AB - In seasonal mammals, photoperiod change is associated with a suite of alterations in physiology. It has recently been proposed that the immune response is one of the systems regulated by changes in photoperiod, although this hypothesis has not been rigorously challenged by assays of functional immune responses. The aim of this study was to test the hypothesis that photoperiod modulates immune responsiveness in Syrian (Mesocricetus auratus) and Siberian (Phodopus sungorus) hamsters. Consistent with previously reported data, short-day-housed (SD) animals exhibited a significant increase in lymph node cell (LNC) numbers and increased cellular proliferation in response to the polyclonal mitogen concanavalin A compared to long-day-housed (LD) animals. In contrast, LNC numbers from intact or gonadectomized SD animals that had been sensitized with the antigen dinitrofluorobenzene (DNFB) exhibited a reduced ex vivo proliferative response and reduced production of interleukin-6 (IL-6) compared to LD animals. In vivo studies of the contact hypersensitivity response of animals that had previously been sensitized, and subsequently challenged, with DNFB were similar in SD and LD animals, as was the proliferative activity of LNC recovered from these animals. There were also no photoperiodic differences in the antidinitrophenyl antibody response of animals sensitized with DNFB, or the anti-sheep red blood cell (srbc) response of animals immunized with srbc. Furthermore, no differences could be detected in the activity of natural killer cells from spleens of LD and SD Siberian hamsters, or in lipopolysaccharide-induced IL-6 production by LD and SD Syrian hamsters in vivo. Thus, although photoperiod is able to influence factors regulating the gross number and non-antigen-specific proliferation of lymphocytes in seasonally breeding mammals, day length does not directly influence activation of an effective immune response. The authors conclude, therefore, that expression of the immune response is not directly modified or compromised by photoperiod in these seasonally breeding hamster species. PMID- 12375617 TI - Form over function? PMID- 12375618 TI - Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels. AB - The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels. PMID- 12375619 TI - 5-HT1B receptor knockout mice exhibit an enhanced response to constant light. AB - Serotonin (5-HT) can act presynaptically at 5-HT1B receptors on retinal terminals in the suprachiasmatic nucleus (SCN) to inhibit glutamate release, thereby modulating the effects of light on circadian behavior. 5-HT1B receptor agonists (1) inhibit light-induced phase shifts of circadian activity rhythms, (2) attenuate light-induced Fos expression in the SCN, and (3) reduce the amplitude of optic nerve-evoked excitatory postsynaptic currents in SCN neurons in vitro. To determine whether functional disruption of the 5-HT1B presynaptic receptors would result in an amplified response of the SCN to light, the period (tau) of the circadian rhythm of wheel-running activity was estimated under several different conditions in 5-HT1B receptor knockout (KO) mice and genetically matched wild-type animals. Under constant light (LL) conditions, the tau of 5 HT1B receptor KO mice was significantly greater than the tau of wild-type mice. A quantitative analysis of the wheel-running activity revealed no differences between wild-type and KO mice in either total activity or the temporal distribution of activity under LL conditions, suggesting that the observed increase in tau was not a function of reduced activity. Under constant dark conditions, the period of the circadian rhythm of wheel-running activity of wild type and 5-HT1B receptor KO mice was similar. In addition, no differences were noted between wild-type and 5-HT1B receptor KO mice in the rate of reentrainment to a 6 h phase advance in the 12:12 light:dark cycle or in phase shifts in response to a 10 min light pulse presented at circadian time 16. The enhanced response of the SCN circadian clock of the 5-HT1B receptor KO mice to LL conditions is consistent with the hypothesis that the endogenous activation of 5 HT1B presynaptic receptors modulates circadian behavior by attenuating photic input to the SCN. PMID- 12375620 TI - Circadian clock functioning is linked to acute stress reactivity in rats. AB - At least two major physiological systems are involved in the adaptation of the organism to environmental challenges: the circadian system and the stress reaction. This study addressed the possibility that interindividual differences in stress sensitivity and in the functioning of the circadian system are related. At 2 months of age, corticosterone secretion in response to a 20-min restraint stress was assessed in 9 Sprague-Dawley rats for which running wheel activity was recorded as a rhythmic behavioral marker of the circadian clock. Two weeks later, the adaptive response of the circadian system to an abrupt shift in the light:dark (LD) cycle was assessed in those rats using a jet-lag paradigm. Finally, after resynchronization to the new LD cycle, rats were transferred to constant darkness to assess the free-running period of their circadian rhythm of running-wheel activity. Results indicate that stress-induced corticosterone secretion was (1) positively correlated with the number of days to resynchronize the circadian activity rhythm to the new LD cycle, and with the value of its free running period, and (2) negatively correlated with the intensity of daily locomotor activity. Those data, emphasizing the interactions between the stress response of an organism and the functioning of its circadian system, could explain interindividual differences in humans' susceptibility to shift work or other circadian-related disorders. PMID- 12375621 TI - Masking effects of posture and sleep onset on core body temperature have distinct circadian rhythms: results from a 90-min/day protocol. AB - Both recumbency and sleep affect core body temperature (CBT). To characterize their circadian effects and interactions, the authors examined the bedtime temperature drops (TDs) of nine men and eight women (aged 20 to 30) who repeated 90-min sleep-wake cycles over 2.5 days. While awake, subjects were exposed to 50 to 250 lux; while asleep, lights were off. Electroencephalogram-monitored time inbed lasted 30 min during each cycle. Cosinor nonlinear mixed-effects regressions modeled the circadian rhythm of TDs. The circadian maximum of TDs occurred approximately 4 h before the time of circadian CBT minimum, in a model that included the effects of baseline expected CBT, deviations from baseline CBT, time in study, and gender-dependent 24- and 12-h adjustments. Rates of temperature drops were faster during initial periods of lying awake than during periods of initially sleeping. Both rates followed separate circadian rhythms. The circadian maximum of TDs was located near customary nocturnal bedtimes, suggesting its role in fostering sleep during a normal bedtime routine. The apparent deceleration of temperature dropping at sleep onset supports the notion that the sleep onset period has complicated circadian neuroregulatory dynamics. These findings confirm the need for nonlinear models of temperature responses to postural changes and sleep that incorporate circadian variability in these masking effects. PMID- 12375622 TI - A forced desynchrony study of circadian pacemaker characteristics in seasonal affective disorder. AB - The circadian pacemaker is an endogenous clock that regulates oscillations in most physiological and psychological processes with a near 24-h period. In many species, this pacemaker triggers seasonal changes in behavior. The seasonality of symptoms and the efficacy of light therapy suggest involvement of the circadian pacemaker in seasonal affective disorder (SAD), winter type. In this study, circadian pacemaker characteristics of SAD patients were compared with those of controls. Seven SAD patients and matched controls were subjected to a 120-h forced desynchrony protocol, in which core body temperature and melatonin secretion profiles were measured for the characterization of circadian pacemaker parameters. During this protocol, which enables the study of unmasked circadian pacemaker characteristics, subjects were exposed to six 20-h days in time isolation. Patients participated twice in winter (while depressed and while remitted after light therapy) and once in summer. Controls participated once in winter and once in summer. Between the SAD patients and controls, no significant differences were observed in the melatonin-derived period or in the phase of the endogenous circadian temperature rhythm. The amplitude of this rhythm was significantly smaller in depressed and remitted SAD patients than in controls. No abnormalities of the circadian pacemaker were observed in SAD patients. A disturbance in thermoregulatory processes might explain the smaller circadian temperature amplitude in SAD patients during winter. PMID- 12375623 TI - A phase dynamics model of human circadian rhythms. AB - Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms. PMID- 12375624 TI - Vocal characteristics in pre-adolescent and adolescent children: a longitudinal study. AB - A number of cross-sectional studies have been reported for the fundamental frequency and formant frequency data in the voices of a group of 20 pre adolescent children aged between 6 and 10 years (Whiteside, S P, Hodgson, C. Acoustic characteristics in 6-10-year old children's voices: some preliminary findings, Log Phon Vocol 1999; 24: 6-13, Whiteside, SP, Hodgson, C. Some acoustic characteristics in the voices of 6-10-year-old children and adults: a comparative sex and developmental perspective, Log Phon Vocol 2000; 25: 122-132). About 15 of the children who participated in these earlier studies, participated in a follow up study approximately 42 months later. Their speech data were recorded and analysed acoustically to investigate longitudinal patterns of development in their voices by examining data for fundamental frequency and the first three formant frequencies of a phrase final vowel. Data obtained from this follow-up study (Time 2) were compared with earlier corresponding data (Time 1). The findings of this study indicated that there was evidence for maturation in the voice parameters of all subjects between Time 1 and 2, with changes being observed for the majority of comparisons for parameters, across age and sex. In addition there was also evidence of individual differences in the maturational patterns which were observed. These individual differences highlight the degree of variation which occurs in the developmental changes of voice characteristics. PMID- 12375625 TI - Vocal loading among day care center teachers. AB - Day care center teachers suffer from voice disorders more often than nurses do. Several risk factors may increase voice disorder prevalence of day care center teachers. The risk factors can be bound to their job content and manner of working i.e. having to raise their voice to attract the attention of the children and to offer them the possibility to perceive spoken information, or to the environment i.e. poor acoustics and excess background noise. The purpose of this study was to measure some of the risk factors for voice disorders of day care center teachers and of a control group (nurses); these were speaking times and speech levels. The background noise levels during activities and RASTI-values (Rapid Speech Transmission Index), i.e. measures of the acoustics of rooms, were also measured at the day care centers. It was found that day care center teachers use their voices more and with higher levels than nurses do. It was also found that the background noise levels are high, which is partly due to the poor acoustics (lack of sufficient attenuation) of the rooms. Control of excess background noise is of utmost importance both for speakers' speech production as well as children's speech recognition. PMID- 12375626 TI - Cordless amplifying system in classrooms. A descriptive study of teachers' and students' opinions. AB - This study investigated one possible method to diminish teachers' vocal loading and, consequently, voice problems. Thirty-three teachers used electric sound amplification in teaching for at least 1 week. Both the teachers (33) and the students (791) reported their opinions of the amplification in a questionnaire. Ninety-seven percent of the teachers reported easier voice production, 82% found improved vocal endurance. The need for repetition also diminished. Eighty-four percent of the students found listening easier and 63% concentration better when amplification was used. The negative points reported both by teachers and students were technical problems (e.g. incorrect sound level or placement of the amplifier). Electrical amplification, thus, can be recommended in classroom use provided that the technical problems have been solved. PMID- 12375627 TI - Spectral distribution of solo voice and accompaniment in pop music. AB - Singers performing in popular styles of music mostly rely on feedback provided by monitor loudspeakers on the stage. The highest sound level that these loudspeakers can provide without feedback noise is often too low to be heard over the ambient sound level on the stage. Long-term-average spectra of some orchestral accompaniments typically used in pop music are compared with those of classical symphonic orchestras. In loud pop accompaniment the sound level difference between 0.5 and 2.5 kHz is similar to that of a Wagner orchestra. Long term-average spectra of pop singers' voices showed no signs of a singer's formant but a peak near 3.5 kHz. It is suggested that pop singers' difficulties to hear their own voices may be reduced if the frequency range 3-4 kHz is boosted in the monitor sound. PMID- 12375628 TI - Vibratory capacity and voice outcome in patients with scarred vocal folds treated with collagen injections--case studies. AB - This prospective case study evaluates the effect of collagen injections on vibratory capacity and voice outcome in four patients with vocal fold scarring. The minimum follow-up time was 6 months. We studied the effect of the injections on the vibratory capacity in a multidimensional way, using computerized image analysis of videostroboscopy, acoustic and perceptual analysis of voice recordings, as well as patients' self-evaluations. Subjective voice ratings from the patients showed improvement in two patients, while two patients had no change in voice function. None of the patients experienced any side effects from the treatment. The documented acoustic and perceptual changes were marginal, but the videostroboscopic findings suggest some improvement in vibratory capacity. PMID- 12375629 TI - QSAR of human factor Xa inhibitor N2-aroylanthranilamides using principal component factor analysis. AB - Quantitative structure-activity relationship (QSAR) study of human factor Xa inhibitor N2-aroylanthranilamides, recently reported by Yee et al. (J. Med. Chem., 43, 873-882), has been performed using principal component factor analysis as the preprocessing step. The study reveals that presence of electron-donating R2 substituent at the para position (with respect to the amide linkage) is conducive to the binding affinity, whereas a meta R2 substituent decreases the affinity. Again, electron-donating R1 substituents with less bulk and optimum hydrophilic-lipophilic balance (particularly, methyl and methoxy groups) favor the activity. The study further suggests that electron-withdrawing R3 substituents are detrimental for the activity, whereas bulkier R4 substituents (particularly NHSO2Me group) increase the activity. PMID- 12375630 TI - Oxazole- and imidazole-based Ser-Leu dipeptide mimetics in potent inhibitors of antigen presentation by MHC class II DR molecules. AB - Imidazole and oxazole derivatives 1 to 4 were designed and prepared as dipeptide mimetics to replace the Ser-Leu dipeptide sequence of Ro-25-9980 (Ac-(Cha)-RAMA-S L-NH2), a peptidic inhibitor of antigen binding to major histocompatibility complex (MHC) class II DR molecules linked to rheumatoid arthritis (RA). The most potent analog in binding assays (IC50 = 30 nM in DRB1*0401 binding; 1.6 times as potent as Ro 25-9980) was 16, Ac-(Cha)RAMA-(S)S-psi(oxazole)-L-NH2. The SAR of peptide hybrids 10 to 24, prepared by incorporating the dipeptide mimetics 1 to 4 is discussed. Of these hybrids, 23 and 24, analogs that incorporated the imidazole and oxazole mimetics as well as optimized variants at positions 3 to 5, were found to have 70 to 80 nM binding affinity comparable to the parent peptide in DRB 1*0401 binding and were also active in DRB1*0101 binding, while being resistant to proteolysis by cathepsin B. Both of these compounds showed inhibitory activity in an antigen-stimulated T-cell proliferation assay, indicating their potential to suppress autoimmune responses and as leads for therapeutic agents to treat RA. PMID- 12375631 TI - QSAR with electrotopological state atom index: human factor Xa inhibitor N2 aroylanthranilamides. AB - Recently, N2-aroylanthranilamides have been reported as novel series of possible anticoagulant drug candidates and the two aryl rings (A and B) have been suggested to interact with S1 and S4 regions, respectively, of human factor Xa (hfXa). In the present effort, quantitative structure-activity relationship (QSAR) of the hfXa binding affinity of 32 N2-aroylanthranilamides have been attempted, in continuation of our previous report on the QSAR analysis of the data set using linear free energy related (LFER) model, with electrotopological state atom (ETSA) index (Kier and Hall, 1991, Adv. Drug Design., 22, 1-38), to explore the atoms/regions of the compounds that modulate the activity comparatively to the greater extent. The univariate and bivariate relations involving the ETSA values of different common atoms of the compounds show importance of the atom nos. 12, 3 and 17 (arbitrary numbering): B ring carbon bearing meta R2 substituents, C ring carbon bearing R4 substituent, and carbonyl oxygen of A ring amide linkage. The importance of atom 12 is suggested to be due to detrimental effects of meta R2 substituents (B ring) on the hfXa binding affinity, which may be owing to interference in the attainment of the proper orientation of the phenyl ring in the S4 site. Atom 3 signifies the impact of R4 substituents (central C ring) on the binding affinity. Again, atom 17 (carbonyl oxygen of A ring amide linkage) has been suggested to form hydrogen bonding with the NH group of the other amide linkage, producing a pseudo ring and thus stabilizing the structure. The relations were improved further using indicator and physicochemical variables and the present results are in good agreement with the previous findings of the Hansch analysis. PMID- 12375632 TI - Combinatorial solid phase synthesis of multiply substituted 1,4-benzodiazepines and affinity studies on the CCK2 receptor (part 1). AB - One hundred sixty-eight multiply substituted 1,4-benzodiazepines have been prepared by a five-step solid-phase combinatorial approach using syn-phase crowns as a solid support and a hydroxymethyl-phenoxy-acetamido linkage (Wang linker). The substituents of the 1,4-benzodiazepine scaffold have been varied in the -3, 5, -7, and 8-positions and the combinatorial library was evaluated in a chole cys to kinin (CCK) radioligand binding assay. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCK-B (CCK2) receptor have been optimized on the lipophilic side chain, the ketone moiety, and the stereochemistry at the 3 position. Various novel 3-alkylated compounds were synthesized and [S]3-propyl-5 phenyl-1,4-benzodiazepin-2-one, [S]NV-A, has shown a CCK-B selective binding at about 180 nM. Fifty-eight compounds of this combinatorial library were purified by preparative TLC and 25 compounds were isolated and fully characterized by TLC, IR, APCI-MS, and 1H/13C-NMR spectroscopy. PMID- 12375633 TI - Validation of prognostic models for melanoma. PMID- 12375634 TI - Characteristics of educational software use in 106 clinical laboratories. AB - The University of Washington, Seattle, has developed educational software for clinical laboratories. We used a 32-question survey to study software implementation. Of 106 clinical laboratories (response rate, 60%) that purchased the software and completed the survey, 89 laboratories (84%) that reported using the software formed the basis for the study. The most common software users were laboratory personnel, followed by medical technologist or medical laboratory technician students, residents, and medical students; the mean (SD) number of personnel categories using the software per laboratory was 1.8 (0.8). The most common reasons for use were initial instruction, cross-training, and competency assessment. The most frequent setting for software use was an area where laboratory testing occurred, followed by a dedicated training location, a location chosen by the employee, a classroom, and a distance learning mode. On a scale of 1 (poor) to 5 (excellent), the average satisfaction rating as an instructional tool was 4.4 and as a competency assessment tool, 4.2. Compared with laboratories in hospitals with 400 beds or fewer, laboratories in hospitals with more than 400 beds used the software for more categories of users (P = .008), had a higher proportion of laboratories using it for residents (P = .003), and had a higher proportion of laboratories with dedicated training areas (P = .02). PMID- 12375635 TI - Risk assessment in localized primary cutaneous melanoma: a Southwest Oncology Group study evaluating nine factors and a test of the Clark logistic regression prediction model. AB - We studied 9 clinical and pathologic factors in 259 patients using Cox model regression analysis to determine which factors have independent predictive value. Median follow-up time in all patients still alive was 12.3 years (range, 1.7 to 16.7 years). Tumor-infiltrating lymphocytes (P = .005), primary site (P = .006), and thickness (P = .02) had independent predictive value. Ulceration (P = .06) and age (P = .07) had marginal value. We used 6 of those factors to test the Clark logistic regression prediction model, which accurately predicted 8-year survival in 121 (72.9%) of 166 patients and accurately predicted melanoma specific mortality in 32 (43%) of 74 patients. The combined or overall accuracy of the Clark model was only 64%. PMID- 12375636 TI - Chromosome 1p allelic loss by fluorescence in situ hybridization is not observed in dysembryoplastic neuroepithelial tumors. AB - Differentiation of dysembryoplastic neuroepithelial tumor (DNT) from cystic low grade oligodendroglioma, particularly in a limited biopsy orfragmented specimen, may be impossible. Research has shown that allelic loss of chromosome 1p is a relatively common finding in oligodendrogliomas. Little is known about chromosome 1p status in DNT. We retrospectively evaluated 14 DNTs for loss of heterozygosity (LOH) on chromosome 1p by fluorescence in situ hybridization (FISH) and compared the results with 1p FISH analysis in 57 low-grade oligodendrogliomas (World Health Organization grade II). The 14 DNTs arose in 8 females and 6 males (mean age, 20.9 years at the time of surgery). All 14 DNTs were 1p intact by FISH analysis. The 57 low-grade oligodendrogliomas arose in 31 males and 26 females (mean age, 43.2 years). LOH on chromosome 1p was present in 31 (54%) of 57 tumors; the remaining 26 tumors were 1p intact. LOH on chromosome 1p is not a feature of DNTs. LOH on chromosome 1p may be a useful differential diagnostic feature (favoring oligodendroglioma) in a subset of cases in which specimen fragmentation or size raises the differential diagnosis of DNT vs oligodendroglioma. PMID- 12375637 TI - Evidence-based criteria for adequacy in thyroid fine-needle aspiration. AB - To determine whether some thyroid fine-needle aspirates classified as nondiagnostic correlate with benign thyroid nodules and can be distinguished from other nondiagnostic aspirates, I reviewed (from a total of 1,581) 80 nondiagnostic cases, all of which were hypocellular and lacked colloid, and correlated the cytologic findings with the results of pathologic follow-up. Of the 80, 16 had carcinoma at follow-up and 64 were benign. The cellularity of the carcinoma cases ranged from 0 to 100 cells (mean, 20 cells), but every case with epithelial cells had Hurthle cell change or atypia suggestive of papillary carcinoma. The cellularity of the 64 benign cases ranged from 0 to 120 cells (mean, 40 cells), 17 of which had Hurthle cell change. There were 25 cases with at least 10 benign-appearing follicular cells without atypia or Hurthle cell change; all 25 cases were associated with benign follow-up. While these results need to be confirmed by others, the evidence suggests that a proportion of thyroid aspirates that do not meet traditional criteria for adequacy still may be associated strongly with a benign thyroid nodule and can be distinguished from other nondiagnostic aspirates. PMID- 12375639 TI - Improving Pap test turnaround time using external benchmark data and engineering process improvement tools. AB - Turnaround time for Papanicolaou (Pap) tests became an important service quality issue at our institution. We studied Pap test turnaround time using engineering process improvement tools and benchmarked turnaround time against data published as a College of American Pathologists Q-Probes study. An IDEF3 process map revealed the complexity of the Pap test process and the opportunities for process improvement. We used these data and the action-research method to initiate changes in cytopathology laboratory operations with the goal of reducing turnaround time. Before intervention, mean Pap test turnaround time was highly variable; during a 6-month period, monthly means ranged from 2.5 to 10.8 days. A cycle time study conducted over a 2-week period validated these data. After system improvements were implemented, the monthly mean turnaround time decreased and became more consistent, with 11 of 12 months having a mean turnaround time of 3 days or less (range, 1.5-3.9 days). Our study illustrates the value of publishing Q-Probes data for use as external benchmarks and the benefits of using tools from other disciplines to improve laboratory processes. PMID- 12375638 TI - Hormonal receptors expression in epithelial cells of mammary phyllodes tumors correlates with pathologic grade of the tumor: a multicenter study of 143 cases. AB - We used immunohistochemical analysis to detect the presence of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) protein expression in the epithelial and stromal cells of 143 phyllodes tumors (PTs). Expression of epithelial ER and PR proteins was common, occurring in 43% to 84% of PTs. Expression of epithelial AR protein and stromal ER, PR, and AR proteins was low (5% or less) in all tumors. An inverse relationship of epithelial ER and PR protein expression with degree of malignancy in PT was found (P < .05), and ER expression also correlated with mitotic count (P < .05). When considering PT with the expression of ER or PR proteins and the coexpression of both, the inverse relationship with tumor grade also was significant (P < .05). As the hormonal receptor protein expression shows a consistent decrease with increasing malignancy, we infer that the epithelium has a crucial role in the pathogenesis or progression of PT. PMID- 12375640 TI - Delayed diagnosis of osseous blastomycosis in two patients following environmental exposure in nonendemic areas. AB - Blastomycosis generally results from inhalation of Blastomyces dermatitidis conidia following exposure to contaminated soil in an endemic area. Primary infections commonly involve the lungs, although secondary dissemination to other body sites may occur. We describe 2 cases of osseous blastomycosis in people living outside the endemic areas. Both patients reported exposure to soilfollowing injury to the knee from occupational activities. Mold isolated from each case was identified as B dermatitidis by micromorphologic characteristics including yeast conversion testing and by a positive AccuProbe Blastomyces dermatitidis test (GenProbe, San Diego, CA). Retrospective review of histologic slides, initially reported as negative, identified rare poorly staining, broad based budding yeast forms in each case. Both patients were treated successfully with itraconazole with no evidence of recurrent infection after 1 year These cases illustrate the importance of considering blastomycosis in the differential diagnosis of bony lesions, even though the patient may live outside an endemic area for B dermatitidis. PMID- 12375641 TI - Comparison of the Automated Mycobacteria Growth Indicator Tube System (BACTEC 960/MGIT) with Lowenstein-Jensen medium for recovery of mycobacteria from clinical specimens. AB - We examined whether the BACTEC/Mycobacteria Growth Indicator Tube (MGIT) system alone could supplant the use of a supplemental Lowenstein-Jensen (LJ) slant for routine recovery of Mycobacterium species from clinical specimens. A total of 6,062 specimens were included in the study. Of these, 273 specimens were positive for 278 mycobacterial isolates while 15 specimens were smear positive but culture negative using both media. Further analysis showed that 143 (51.4%) of the 278 total isolates were recovered from both the MGIT and LJ media. An additional 106 isolates (38.1%) were recovered from the MGIT only, while 29 (10.4%) isolates grew only on the LJ slant. The overall sensitivities of the MGIT and LJ media were 86.5% and 59.7%, respectively, for the recovery of mycobacteria from clinical materials. This study shows that although the MGIT system demonstrates better sensitivity for the recovery of mycobacteria from clinical specimens, both media types are necessary to maximize the sensitivity of detection. PMID- 12375642 TI - Assessing the usefulness of anticardiolipin antibody assays: a cautious approach is suggested by high variation and limited consensus in multilaboratory testing. AB - An anticardiolipin antibody (ACA) assay has become a laboratory standard for the detection of antiphospholipid antibodies. We evaluated data from a quality assurance program to assess ACA assay usefulness. Cross-laboratory (n = 56) testing of 12 serum samples yielded interlaboratory coefficients of variation (CVs) for lgG ACA and IgM ACA that were higher than 50% in 17 (71%) of 24 cases. The situation for testing consensus was of equal concern. Total consensus occurred in 6 (25%) of 24 cases. General consensus (interlaboratory agreement, 90% or more) was obtained in 10 (42%) of 24 cases. In the majority of test cases, laboratories could not agree on whether a serum sample tested was ACA-positive or ACA-negative. Differing method-related issues also were evident; some methods tended toward higher or lower ACA values. Method-based majority consensus differed from participant majority consensus on many test occasions. Exceedingly high interlaboratory result variation, combined with a general lack of test result grading consensus and method-based variation, indicate that a cautious clinical approach toward laboratory findings is prudent. Laboratory tests should be repeated at least once before making a clinical diagnosis of any anti phospholipid syndrome-like disorder. PMID- 12375643 TI - Differences in CD33 intensity between various myeloid neoplasms. AB - We measured the concentration of CD33 antigen on the surface of cells in 315 bone marrow (BM) samples and 114 corresponding peripheral blood (PB) samples from patients with various leukemias (acute myeloid leukemia [AML], chronic myelogenous leukemia [CML], myeloproliferative disorder [MPD] other than CML, myelodysplastic syndrome [MDS]) and from control subjects. Overall CD33 intensity in total CD33+ cells was significantly higher in BM than in PB. CD33 intensity in total BM CD33+ cells differed significantly with the type of disease. The median number of CD33 molecules per cell was highest in AML, followed by MDS, CML, and control subjects and lowest in MPD. When only CD34+/CD33+ cells were examined, CD33 molecules per cell were highest in CD34+ cells in AML and lowest in MPD (P = .027). Patients with AML or MDS younger than 60 years had significantly higher intensity of CD33 expression on CD34+ cells than patients 60 years or older. Levels of CD33 intensity did not correlate with cytogenetics in patients with AML or MDS. There was no correlation between CD33 intensity and response to therapy or overall survival in 35 patients treated with protocols including Mylotarg. These data demonstrate variation in CD33 intensity between various leukemias. PMID- 12375644 TI - A decision-support system for flow cytometry immunophenotyping. AB - We developed a decision-support system, the flow cytometry workstation (FCW), that provides variable panel definitions, age-adjusted reference ranges, and graphic display of immunologic trends. Automated quality assurance functions include validation of flow cytometry data using user-defined monoclonal antibody sums and delta check computations. We evaluated the FCW to determine whether it would reduce CD4+ technical and clerical errors and to discover patterns or trends within flow cytometric data for research purposes. The FCW reduced the number of technical and clerical errors in its first 2 years of use (P = .003). User-defined quality assurance summation checks such as CD2+ + CD20+ = 95% +/- 5% were applied to a 10-year data set as part of a retrospective analysis. The FCW discovered a relationship between specimen processing and the number of results appearing out of range: 58.11% of reported samples appeared out of range in 1993 compared with 2% in 1996 (P1 < .001). The FCW is a foundation for quality improvement and outcomes-based research for clinical flow cytometry and serves as a platform for state-of-the-art laboratory management. PMID- 12375645 TI - Peripheral T-cell lymphoma arising in the liver. AB - We report 3 cases of primary hepatic peripheral T-cell lymphoma (PTCL). All patients were men, 50 to 57 years of age, who sought care because of systemic symptoms including fever, fatigue, and weight loss. Physical examination revealed hepatomegaly in 2 patients, associated with jaundice in 1. Two patients had abnormal serum liver enzyme levels and coagulation profiles. Imaging studies demonstrated marked hepatomegaly without focal lesions in 1 patient and multiple discrete tumor masses in 2 patients. Tumor infiltrates in biopsy specimens were heterogeneous with a large cell component in 2 cases. An inflammatory background was present in all cases, complicating the histologic recognition of PTCL Immunohistochemical studies showed that all tumors were of T-cell lineage, and 2 cases had monoclonal T-cell receptor gamma chain gene rearrangements. One patient died of disease shortly after diagnosis, and 2 patients treated with multiagent chemotherapy are in clinical remission with 12 and 84 months of clinicalfollow up, respectively. PTCL may rarely arise in the liver. These neoplasms respond to chemotherapy, suggesting that this disease is curable if diagnosed at an early stage. PMID- 12375646 TI - Bone marrow changes in anorexia nervosa are correlated with the amount of weight loss and not with other clinical findings. AB - The clinical history and biochemical and hematologic variables for 44 consecutive patients diagnosed with anorexia nervosa were recorded. Bone marrow aspirates and biopsy specimens were analyzed by standard morphologic procedures, and bone marrow adipocytes were studied morphometrically. The bone marrow of the 44 patients was classified as normal (5 cases [11%]), hypoplastic or aplastic (17 [39%]), with partial or focal gelatinous degeneration (13 [30%]), or with complete gelatinous degeneration of the bone marrow (GDBM; 9 [20%]). These patterns correlated with amount of weight loss (P = .005) but not other clinical findings. WBC counts were lower in patients with GDBM (P = .0189), but this and other peripheral blood variables did not always reflect the severity of bone marrow damage. Hypoplastic or aplastic bone marrow showed an increase in bone marrow fat fraction due to an increase in adipocyte diameters, while in GDBM, fat fraction and adipocyte diameters decreased. Morphologic changes in bone marrow and stereologic alterations in bone marrow adipocytes may be observed in anorexia nervosa. The extent of damage is related to the amount of weight loss, not to other factors. Peripheral blood cell counts may not reflect the extent of damage. In some patients, this process may be reversible with reestablishment of adequate nutritional intake. PMID- 12375647 TI - CD5+ follicular lymphoma: a clinicopathologic study of three cases. AB - Follicular lymphoma (FL) is a low-grade lymphoma that typically lacks CD5 antigen expression. We report 3 cases of FL with unusual expression of CD5. All cases showed histologic features of FL, including effaced nodal architecture, follicular growth pattern, and a spectrum of grades from 1 to 3 using World Health Organization criteria. In flow cytometric studies, all 3 cases showed a light chain-restricted, CD19+, CD20+ B-cell population coexpressing CD10 and low level CD5. Immunohistochemical studies demonstrated an identical B-cell immunophenotype with weak expression of CD5 and coexpression of bcl-2 protein and the germinal center-associated markers, CD10 and bcl-6 protein. None of the cases showed expression of CD43, cyclin D1, or IgD. By molecular analysis, immunoglobulin heavy chain gene rearrangements were demonstrated in all 3 cases, and 2 of 3 cases had a t(14;18). These cases highlight the difficulty classifying these lymphomas by flow cytometric studies alone and emphasize the importance of recognizing FL in the differential diagnosis of CD5+ B-cell lymphomas. PMID- 12375648 TI - Encapsulated follicular variant of papillary thyroid carcinoma. PMID- 12375649 TI - Encapsulated follicular variant of papillary thyroid carcinoma. PMID- 12375650 TI - What's so bad about old platelets? PMID- 12375651 TI - Mortality and morbidity in patients with very low postoperative Hb levels who decline blood transfusion. AB - BACKGROUND: Guidelines for allogeneic transfusion emphasize minimizing use to avoid transmission of serious illness. However, there is little information on the risks associated from withholding transfusion. STUDY DESIGN AND METHODS: A retrospective cohort study of patients who declined RBC transfusions for religious reasons was performed. This analysis was restricted to consecutive patients > or = 18 years old, who underwent surgery in the operating room from 1981 to 1994 and had a postoperative Hb count of 8 g per dL or less. The primary outcome was defined as any inhospital death occurring within 30 days of the surgery. Secondary outcome was 30-day mortality or in-hospital 30-day morbidity. Morbidity was defined as myocardial infarction, arrhythmia, congestive heart failure, or infection. RESULTS: Of 2083 eligible patients, 300 had postoperative Hb counts of 8 g per dL or less. The study population was predominantly female (70.3%) with a mean age of 57 years (SD, +/- 17.7). In patients with a postoperative Hb level of 7.1 to 8.0, 0 died (upper 95% CI, 3.7%), and 9.4 percent (95% CI, 4.4-17.0%) had a morbid event. In patients with a postoperative Hb level of 4.1 to 5.0, 34.4 percent (95% CI, 18.6-53.2%) died and 57.7 percent (95% CI, 36.9-76.6%) had a morbid event or died. After adjusting for age, cardiovascular disease, and Acute Physiology and Chronic Health Evaluation II score, the odds of death in patients with a postoperative Hb level of < or = 8 g per dL increased 2.5 times (95% CI, 1.9-3.2) for each gram decrease in Hb level. CONCLUSIONS: The risk of death was low in patients with postoperative Hb levels of 7.1 to 8.0 g per dL, although morbidity occurred in 9.4 percent. As postoperative blood counts fall the risk of mortality and/or morbidity rises and becomes extremely high below 5 to 6 g per dL. PMID- 12375652 TI - Declining value of preoperative autologous donation. AB - BACKGROUND: Preoperative autologous blood donation (PABD) has been shown to decrease allogeneic blood transfusion requirements in major elective surgery. Changes in transfusion practice motivated an examination of blood use from 1993 to 2000 of patients participating in the Hema-Quebec PABD program. STUDY DESIGN AND METHODS: Blood donation and transfusion, type of surgery, and demographic characteristics were prospectively entered into a computer database for patients participating in the Hema-Quebec PABD program. RESULTS: Autologous donations represented from 0.8 to 2 percent of total blood collections and have declined by 26 percent after peaking in 1995. The mean number of units collected per patient declined, as did the number of units transfused per patient and the utilization rate. For radical prostatectomy, knee replacement surgery, hip replacement surgery, and scoliosis, utilization rates were 72, 60, 83, and 78 percent in 1993 compared with 50, 50, 58, and 58 percent in 2000, respectively. In 2000, 18 percent of patients were receiving a 1-unit autologous transfusion. Depending on the surgical procedure, 85 to 95 percent of patients avoided allogeneic transfusion; this did not change significantly from 1993 to 2000. CONCLUSION: Patients participating in the PABD program successfully avoided allogeneic transfusion in over 85 percent of cases. However, declining utilization rates and frequent 1-unit transfusions demonstrate the decreasing utility of PABD over time. PMID- 12375653 TI - Improved maintenance of 2,3 DPG and ATP in RBCs stored in a modified additive solution. AB - BACKGROUND: All currently used systems for the storage of RBCs result in loss of 2,3 DPG and an associated increase in affinity for oxygen. Previously, it was demonstrated that a hypotonic additive solution for RBC storage (Erythro-Sol) resulted in prolonged maintenance of 2,3 DPG when blood was collected in 0.5 CPD (half-strength CPD), but not when full-strength CPD was used. The present study aims at improving the quality of stored RBCs collected in ordinary CPD. STUDY DESIGN AND METHODS: A new formulation of Erythro-Sol (Erythro-Sol 2) (pH 8.8) in a larger volume (150 mL) was compared with Erythro-Sol (Erythro-Sol 1). In vitro measures during 49 days of storage in the two additives were compared using WBC depleted RBCs after whole-blood collection in CPD and separation in an automated blood separation instrument (Optipress II, Baxter Healthcare). RESULTS: The maintenance of RBC ATP and 2,3 DPG was significantly better in Erythro-Sol 2 than in Erythro-Sol 1. The ATP concentration rose to approximately 30 percent above initial level in both systems; however, the maximum occurred on Day 21 in Erythro Sol 2 as compared with Day 14 in Erythro-Sol 1. In RBCs stored in Erythro-Sol 2, the mean RBC 2,3 DPG concentration increased to 14 percent above initial level on Day 7, then decreased to the initial level on Day 14, whereas in Erythro-Sol 1, the 2,3 DPG had decreased to 85 and 50 percent on Days 7 and 14, respectively. Both intracellular pH and extracellular pH were slightly higher in Erythro-Sol 2 than in Erythro-Sol 1 units but decreased rapidly during the first storage week, which seems to have been the major reason for the limitation in the time of maintenance of 2,3 DPG. Hemolysis was very low in both systems, 0.14 to 0.17 percent on Day 49. The additional amount of inorganic phosphate submitted with Erythro-Sol 2 did not raise concern because the phosphate content in the storage medium, being 1.3 +/- 0.2 mmoL on Day 0, decreased to values below 1 mmoL during most of subsequent storage. CONCLUSION: Erythro-Sol 2 is an improved additive solution for the storage of RBCs. PMID- 12375654 TI - Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol. AB - BACKGROUND: PVC containers are plasticized with di(2-ethyl)hexylphthalate (DEHP) or a related phthalate. The toxicity of DEHP has been questioned. It has been proposed to use butyryltrihexylcitrate (BTHC) as the plasticizer. The purpose of this study was to determine if hexanol, a component of BTHC, plays a role in the preservation of RBCs stored in BTHC-plasticized PVC bags. STUDY DESIGN AND METHODS: WBC-reduced RBCs of ABO- and D-matched blood groups were prepared in 1-L polyolefin (PO) bags (PL732). Six 60-g aliquots were transferred to transfer packs made of PL146 (DEHP-plasticized) and PL2209 (BTHC-plasticized) and four PO (PL732) packs. To the PL146 and PL2209 packs, 30 mL of AS-1 was added. To three of the PO packs, 30 mL of AS-1 with sufficient DEHP, BTHC, or hexanol to achieve a final concentration of 3 mM was added, and to the final PO pack, 30 mL of AS-1 only was added (control). The units were stored for 6 weeks at 1 to 6 degrees C. RBC ATP, hemolysis, morphology, membrane lipids, deformability, and fluidity were measured. RESULTS: ATP levels were not significantly different in any of the systems after 6 weeks. Compared to the PO bags, hemolysis was lowest in the PL146 containers and was also significantly lower (p < 0.006) in the PO bags with added DEHP, BTHC, or hexanol. The accumulation of vesicles was significantly less in the units stored in the PL146 and PL2209 than in the PO plastic with or without added plasticizers or hexanol (p < or = 0.004). There was no significant difference in the formation of vesicles in any of the PO units (p > 0.05). There was no demonstrable change in the membrane fluidity of the RBCs during storage in any of the systems. The decrease in deformability was the same, and the losses of cholesterol and phospholipid during storage were similar in all the studies. CONCLUSIONS: The hexanol component of the BHTC plasticizer in a concentration of 144.6 microg per mL concentration suppresses hemolysis and vesiculation of RBCs during storage. The hexanol and DEHP that are slowly leached during storage have a greater effect in suppressing hemolysis and vesicle formation than when added extraneously to AS-1 in PO containers. PMID- 12375655 TI - Physical and thermal properties of blood storage bags: implications for shipping frozen components on dry ice. AB - BACKGROUND: Frozen blood components are shipped on dry ice. The lower temperature (-70 degrees C in contrast to usual storage at -30 degrees C) and shipping conditions may cause a rent in the storage bag, breaking sterility and rendering the unit useless. The rate of loss can reach 50 to 80 percent. To identify those bags with lower probability of breaking during shipment, the thermal and physical properties of blood storage bags were examined. STUDY DESIGN AND METHODS: Blood storage bags were obtained from several manufacturers and were of the following compositions: PVC with citrate, di-2-ethylhexylphthalate (DEHP), or tri-2 ethylhexyl-tri-mellitate (TEHTM) plasticizer; polyolefin (PO); poly(ethylene-co vinyl acetate) (EVA); or fluorinated polyethylene propylene (FEP). The glass transition temperature (Tg) of each storage bag was determined. Bag thickness and measures of material strength (tensile modulus [MT] and time to achieve 0.5 percent strain [T0.5%]) were evaluated. M(T) and T0.5% measurements were made at 25 and -70 degrees C. Response to applied force at -70 degrees C was measured using an impact testing device and a drop test. RESULTS: The Tg of the bags fell into two groups: 70 to 105 degrees C (PO, FEP) and -50 to -17 degrees C (PVC with plasticizer, EVA). Bag thickness ranged from 0.14 to 0.41 mm. Compared to other materials, the ratios of M(T) and T0.5% for PVC bags were increased (p < or = 0.001) indicating that structural changes for PVC were more pronounced upon cooling from 25 to -70 degrees C. Bags containing EVA were more shock resistant, resulting in the lowest rate of breakage (10% breakage) when compared with PO (60% breakage, p = 0.0573) or PVC (100% breakage, p = 0.0001). CONCLUSIONS: Blood storage bags made of EVA appear better suited for shipping frozen blood components on dry ice and are cost-effective replacements for PVC bags. For the identification of blood storage bags meeting specific storage requirements, physical and thermal analyses of blood storage bags may be useful and remove empiricism from the process. PMID- 12375656 TI - Seven-day storage of single-donor platelets: recovery and survival in an autologous transfusion study. AB - BACKGROUND: Bacterial screening may effectively reduce the morbidity and mortality risk associated with extended storage of platelets. Platelet viability then becomes the primary determinant of acceptable storage time. This study evaluates the effectiveness of platelets stored in plasma for 7 days. STUDY DESIGN AND METHODS: WBC-reduced, single-donor platelets (n = 24) were collected and stored by standard methods at two sites. Standard in vitro platelet biochemical and functional parameters were monitored over the storage period. On Days 5 and 7 of storage, platelets were alternately labeled with 51Cr and (111)In and returned to the subject, and recovery and survival were determined. RESULTS: Component pH(22 degrees C) was maintained in the range 6.2 to 7.61 through 7 days and did not detrimentally affect either in vitro or in vivo outcomes. In vitro platelet characteristics were adequately maintained over 7 days. Day 5 platelets had better recovery (63.0 +/- 4.36 vs. 53.9 +/- 4.36%, p < 0.0001) and survival (161 +/- 8.1 vs. 133 +/- 8.1 hr, p = 0.006) than Day 7 platelets adjusting for radioisotope, center, and donor effects. CONCLUSION: Although declines in recovery and survival were noted, these are less than used previously to gain licensure of 7-day storage and are unlikely to be clinically significant. Extension of storage to 7 days could be implemented with bacterial screening methods to select out contaminated components without a significant effect on the platelet efficacy compared to 5-day components. PMID- 12375657 TI - Experience with universal bacterial culturing to detect contamination of apheresis platelet units in a hospital transfusion service. AB - BACKGROUND: Bacterial contamination of platelet units poses one of the greatest risks of morbidity and mortality to platelet transfusion recipients. A routine culture of all units (WBC-reduced apheresis platelet units) was instituted on Day 2 over a 2-year period to reduce this risk. STUDY DESIGN AND METHODS: A sterile connecting device was used to attach a small transfer pack on the morning of Day 2 after collection, and 10 mL of the unit were transferred to the small bag. After disconnection from the unit, about half of this volume was transferred to an aerobic culture bottle of an automated bacterial detection system. Units were maintained in available inventory until and unless a report was received of growth in the sample. When available, the unit or a retained aliquot was recultured if the initial sample was positive. Units were held up to 2 days beyond their 5-day outdate and used for transfusion if no other suitable units were available to meet the clinical need or were evaluated with in vitro testing on Day 8. RESULTS: Of 2678 units cultured, 16 (0.6%) were positive on initial culture. Thirteen could be recultured, and all of these samples were negative. Shortly after the 2-year period of the study, two units (split from the same collection) were documented as growing coagulase-negative Staphylococci 12 hours after sampling. Units transfused on Day 6 or 7 (n = 40) yielded expected clinical responses, and CCI available on 21 cipients 10 to 60 minutes after transfusion demonstrated acceptable results (mean, 14,400 +/- 8800; median, 12,191; 90% > 7500). More than 96 percent of units tested on Day 8 had pH greater than 6.2 and continued to demonstrate swirling. CONCLUSIONS: Routine culturing of apheresis platelet units is feasible, can be accomplished with a low rate of false positivity, and can detect contaminated units. The cost of such a protocol could be mitigated with extension of the storage period, and clinical experience with units held for 6 or 7 days was satisfactory. PMID- 12375658 TI - Yield of HCV and HIV-1 NAT after screening of 3.6 million blood donations in central Europe. AB - BACKGROUND: HCV and HIV-1 NAT of all blood donations was initiated at our institutions in January 1997 to reduce the residual risk of transfusion transmitted virus infections. The yield of NAT after testing more than 3.6 million donations in central Europe is reported. STUDY DESIGN AND METHODS: Automated pipetting instruments were used to pool up to 96 donor samples including those that were antibody reactive. To compensate for dilution of the individual donor samples by pooling, viruses were enriched from the pools by centrifugation at 48,000 x g. A commercial PCR (Cobas Amplicor, Roche) and an in house PCR were applied for HCV and HIV-1 amplification, respectively. RESULTS: Six HCV and 2 HIV-1 PCR confirmed-positive, antibody-negative donations (yield, 1 in 600,000 and 1 in 1.8 million, respectively) were identified. Thirty-nine and 11 multiple-time donors seroconverted for HCV and HIV, respectively, and look back procedures were initiated. Archived samples from preseroconversion donations were thawed and retested by single-sample PCR and remained negative. The recipients of the blood components were traced and tested. All traced recipients were negative for HCV and HIV antibodies. CONCLUSION: The yield of NAT in central European Red Cross blood donors was less than expected from theoretical calculations for American and German multiple-time donors. Look-back procedures for HCV and HIV indicated that no donation given before seroconversion of the donor was missed by minipool PCR. Sensitivity of minipool PCR testing after virus enrichment seems to be sufficiently high to close the diagnostic window almost completely. PMID- 12375659 TI - NAT for HBV and anti-HBc testing increase blood safety. AB - BACKGROUND: Routine HBV PCR screening of blood donations to our institutes was introduced in January 1997 to complete the NAT screening program for transfusion relevant viruses. Testing was successively extended to customer transfusion services with a total of 1,300,000 samples tested per year. STUDY DESIGN AND METHODS: Minipools of 96 blood donation samples were formed by automatic pipettors. HBsAg-reactive samples were included. HBV particles were enriched from the minipools by centrifugation. Conventional and in-house TaqMan PCRs were successively applied for HBV amplification. Sensitivity reached 1000 genome equivalents per mL for each individual donation. Confirmatory single-sample and single-sample enrichment PCRs were established with sensitivities of 300 and 5 to 10 genome equivalents per mL, respectively. RESULTS: After screening of 3.6 million donor samples, 6 HBV PCR-positive, HBsAg-negative donations were identified. Two samples were from infected donors who had not seroconverted and four were from chronic anti-HBc-positive low-level HBV carriers. Retesting by single-sample PCR of 432 samples confirmed positive for HBsAg identified 37 donations that were negative in minipool PCR. Donor-directed look-back procedures indicated that no infected donor who had not yet seroconverted was missed by minipool PCR. However, recipient-directed look-back procedures revealed two anti HBc-positive recipients of HBsAg-negative minipool PCR-negative, anti-HBc positive and single-sample PCR-positive blood components. After testing randomly selected 729 HBsAg-negative minipool PCR-negative, anti-HBc-positive donors by single-sample enrichment PCR, 7 were identified with < or = 10 HBV particles per mL of donor plasma. CONCLUSION: Minipool PCR testing after virus enrichment was sensitive enough to identify HBsAg-negative donors who had seroconverterd and HBsAg-negative, anti-HBc-positive chronic HBV carriers. HBV NAT in conjunction with anti-HBc screening would reduce the residual risk of transfusion-transmitted HBV infection. PMID- 12375660 TI - Clinical specificity and sensitivity of a blood screening assay for detection of HIV-1 and HCV RNA. AB - BACKGROUND: An HIV-1 and HCV NAT blood screening assay (Procleix HIV-1/HCV, Gen Probe, Inc.) simultaneously detecting HIV-1 and HCV RNA) has been implemented. Donor plasma samples reactive in the Procleix HIV-1/HCV assay are tested with the HIV-1 and HCV discriminatory assays to resolve whether HIV-1 RNA, HCV RNA, or both are present. STUDY DESIGN AND METHODS: To determine the specificity of the Procleix HIV-1/HCV assay, data were analyzed for samples from 192,288 donations, tested in 16-member pools. To determine sensitivity, data were analyzed for 2014 commercial samples known to contain HIV-1, HCV, or both, as well as 10 HIV-1 and 10 HCV commercial seroconversion panels. RESULTS: The specificity of the Procleix HIV-1/HCV assay was 99.7 percent. The HIV-1 and HCV discriminatory assays showed similar specificity. The sensitivity of the Procleix HIV-1/HCV assay was 99.9, 99.6, and 100 percent, respectively, for samples containing HIV-1, HCV, or both. The Procleix discriminatory assays were comparably sensitive. The Procleix discriminatory assays detected all tested samples of known HIV-1 subtype or HCV genotype. Procleix HIV-1/HCV testing of seroconversion panels showed that the median times to a positive reaction for HIV-1 and HCV were reduced by 3 and 25 days, respectively, compared to serologic tests. CONCLUSION: These studies support the use of the Procleix HIV-1/HCV assay for routine blood donor screening. PMID- 12375661 TI - Simian foamy virus infection in a blood donor. AB - BACKGROUND: Infections with simian foamy virus (SFV) are widely prevalent in nonhuman primates. SFV infection was confirmed in a worker, occupationally exposed to nonhuman primates, who donated blood after the retrospectively documented date of infection. Human-to-human transmission of SFV through transfusion and its pathogenicity have not been studied. STUDY DESIGN AND METHODS: Recipients of blood from this donor were identified and blood samples from such recipients were tested for SFV infection by Western blot and PCR assay. RESULTS: One recipient of RBCs and another recipient of FFP had died; retroviral infections were not implicated. One platelet recipient could not be tested. Recipients of RBCs (two), a WBC-reduced RBC unit (one), and a platelet unit (one) tested SFV-negative 19 months to 7 years after transfusion. Tested recipients had transfusions 3 to 35 days after blood donation. Samples of one lot of albumin and three lots of plasma protein fraction (manufactured from recovered plasma from two donations) tested negative both for antibodies and for viral RNA. CONCLUSION: SFV transmission through transfusion was not identified among four recipients of cellular blood components from one SFV-infected donor. Derivatives containing plasma from that donor tested negative for SFV. PMID- 12375662 TI - TT virus infection during childhood. AB - BACKGROUND: TT virus (TTV) is widespread in the general population, however, the mode of its transmission and the mechanism of maintaining it in the general population are unclear. STUDY DESIGN AND METHODS: To determine the possible mother-to-infant route of transmission, 54 infants bom to 50 anti-HCV-positive mothers were assessed longitudinally. Nucleotide sequences amplified by seminested PCR with primers targeting the N22 variable coding region of genotypes 1 through 6 were compared in mothers and their infants. RESULTS: The prevalence of TTV DNA was 30 percent (15/50; 95% CI, 18-45) in mothers and 44 percent (24/54; 95% CI, 31-59) in their infants. TTV DNA was detected during a follow-up period in 13 (87%; 95% CI, 60-98) of 15 infants born to infected mothers and in 11 (28%; 95% CI, 15-45) of 39 infants bom to DNA-negative mothers. None of 38 cord blood samples, but one of 14 blood samples, obtained at 1 month of age had detectable TTV DNA. The lowest infection rate at the earliest ages and the subsequent increasing prevalence of infection (22% at 6 months and 33% [43% cumulative rate] at 2 years) is consistent with an age-dependent acquisition of TTV by nonparenteral routes. In 13-mother-infant pairs positive for TTV DNA, six showed a high degree of nucleotide sequence similarity (99.1-100%), whereas the remaining seven pairs differed more than 10 percent from each other (46.8-89.2%). The viral load of matemal blood was not a plausible risk factor for transmission. Genotype 1, of which pathogenicity failed to be shown by measurement of hepatic enzymes, was more rapidly cleared (88 vs. 8% other genotypes, p < 0.001) among infants. CONCLUSIONS: These observations strongly suggest that the main factor for TTV acquisition in children involves their age-associated increase in environmental interactions with infectious materials. Genotype 1 might be involved in a weak or a limited pathologic role, which can possibly be diluted by other harmless genotypes. PMID- 12375663 TI - QTc prolongation in apheresis platelet donors. AB - BACKGROUND: Citrate infusion during apheresis procedures can cause lowering of the plasma ionized calcium leading to prolongation of the QT interval and hypotension. At the myocardial level, QTc measurement is a sensitive marker of subphysiologic calcium values caused by citrate toxicity. STUDY DESIGN AND METHODS: Seventy-six regular platelet donors were recruited into this study. QTc intervals were measured continuously for 3 minutes at four different points during the apheresis procedures. The blood pressure was recorded every 5 minutes throughout the procedure. RESULTS: The baseline QTc value for men was 406.5 +/- 13.1 milliseconds(1/2) and for women was 417.6 +/- 13.3 milliseconds(1/2) (p = 0.0016). OTc prolongation occurred in all procedures in all donors. Maximum recorded values were significantly higher in women than in men (450.8 +/- 20.8 vs. 424.8 +/- 14.3 ms(1/2); p = 0.0025). All QTc values returned to baseline by 15 minutes after the procedure. Changes in blood pressure were minimal and no donor experienced severe symptoms. CONCLUSIONS: QTc prolongation always occurs during plateletpheresis. This prolongation is greater in women than in men. This study indicates that it may be advisable to assess donors for family or personal history of syncope and family history of sudden death to exclude those at increased risk of arrhythmias because of asymptomatic carriage of a long-QT gene. In addition baseline QTc measurement is a simple noninvasive procedure that could be applied to further studies with a view to enhancing donor safety in apheresis. PMID- 12375664 TI - Change in CD34+ cell concentration during peripheral blood progenitor cell collection: effects on collection efficiency and efficacy. AB - BACKGROUND: An understanding of factors affecting CD34+ cell collection efficacy is essential to minimize donor toxicity and cost. STUDY DESIGN AND METHODS: Peripheral blood CD34+ cell (CD34) measurements were determined at various intervals before, during, and after automated cell collection (Cobe Spectra 6.0). The serial mean of multiple, intraprocedural CD34 levels was calculated for each procedure as an estimate of the mean, inlet-line CD34 level. RESULTS: The CD34+ concentration fell a mean of 30 percent in the first 30 to 70 minutes of collection. The degree of decline was inversely correlated with donor blood volume (BV), but was not due to hemodilution. The mean of the CD34 level before and after collection slightly overestimated the serial mean CD34 level. Cell yields, normalized for the CD34 level before collection, were higher from donors with larger BVs. CONCLUSIONS: The CD34 concentration rapidly decreased to a relative equilibrium level during the collection procedure. The degree of decrease in the CD34 level inversely correlated with the BV of the donor and was consistent with cell pooling in the collection set. The higher equilibrium CD34 levels in donors with larger BVs resulted in increased collection of CD34+ cells, and therefore, large-volume apheresis should be most efficient in these donors. PMID- 12375665 TI - Combined isolation of CD34+ progenitor cells and reduction of B cells from peripheral blood by use of immunomagnetic methods. AB - BACKGROUND: Malignant cells may contribute to relapse after autologous hematopoietic cell transplantation The effectiveness of a double immunomagnetic purging strategy combining CD34-positive with B-negative cell selection to purge peripheral blood progenitor cells (PBPCs) from patients with chronic lymphoproliferative disorders has been analyzed. STUDY DESIGN AND METHODS: Twenty two CD34+ cell selections from patients with follicular lymphoma (n = 14), chronic lymphocytic leukemia (n = 6), mantle cell lymphoma (n = 1), and splenic marginal zone lymphoma (n = 1) were performed by use of a magnetic cell selector followed by a negative cell selection step with anti-CD19 monoclonal antibody bound to immunomagnetic beads. RESULTS: The PBPC components contained median CD34+ cells of 1.24 percent (range, 0.38-3.92%) and CD19+ cells of 1.83 percent (range, 0.06-69.7%). After positive selection (n = 22), 49 percent (range, 16 72%) of CD34+ cells were recovered with a purity of 93 percent (range, 24-99%). The double-positive and -negative selections (n = 20) yielded 57.5 percent of CD34+ cells (range, 33.4-79.4%) with a purity of 95 percent (range, 63-99%). Logarithms of B-cell reduction in the CD34+-cell-enriched B-cell-depleted component had a median value of 3.63 (range, 2.74-4.84 log) and CD19+ and CD5+ cells for chronic lymphocytic leukemia patients with more than 4.56 log (>3.6-5.6 log). Of 13 PBPC components that had a tumor-specific clonal signal, 10 became PCR negative after the double-selection procedure. CONCLUSION: Combined positive and negative magnetic cell selection achieves a high grade of tumor cell reduction with up to 77 percent of the grafts being negative for tumor-specific clonal signal by PCR analysis. This technique preserves an adequate recovery of progenitor cells able to engraft. PMID- 12375666 TI - Bone marrow stromal cells prepared using AB serum and bFGF for hematopoietic stem cells expansion. AB - BACKGROUND: An ex vivo culture system was previously established for stem cell expansion using human marrow stromal cells and serum-free medium. However, the stromal cells were prepared using long-term culture medium containing horse serum and FCS, which may transmit infectious diseases of xenogeneic origin. In this study, therefore, a method was established to prepare stromal cells using an AB serum-based medium. In the case that serum from a transplant recipient or PBPC donor is available, additional infectious diseases would not be transmitted. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with thrombopoietin, stem cell factor, and flt3/flk2 ligand on a monolayer of human marrow primary stromal cells prepared using long-term culture medium or AB serum based medium. After 2 weeks, clonogenic progenitor activity and SCID mouse reconstituting cell activity were assayed. mRNA expression of cytokines and Notch ligand by stromal cells was also examined. RESULTS: There were no remarkable differences in expansion-supporting activity and mRNA expression between stromal cells established by the two methods. CONCLUSION: An ex vivo expansion system completely based on AB serum has been established. PMID- 12375667 TI - Equivalent mobilization and collection of granulocytes for transfusion after administration of glycosylated G-CSF (3 microg/kg) plus dexamethasone versus glycosylated G-CSF (12 microg/kg) alone. AB - BACKGROUND: The aim of this study was to find a regimen for mobilization and collection of granulocytes that combines low-dose G-CSF administration with satisfactory PMN mobilization and apheresis at a low rate of donor adverse reactions. STUDY DESIGN AND METHODS: In a prospective study, 52 healthy unrelated volunteers received a single subcutaneous injection of glycosylated G-CSF (Lenograstim Chugai-Pharma, Frankfurt, Germany) at medians of 3.1 (range, 2.4 3.6) microg per kg plus dexamethasone (8 mg orally; n = 29) or at 11.8 (7.1-18.5) microg of lenograstim per kg (p < or = 0.0001) without dexamethasone (n = 23) and underwent standard apheresis using the PMN program of a cell separator (Spectra, COBE [now Gambro] BCT). WBC and PMN mobilization results and apheresis yields were compared and the severity and clinical significance of donor adverse reactions was evaluated. RESULTS: For the low-dose G-CSF plus dexamethasone versus the high-dose G-CSF alone group, similar mobilization results were observed for WBCs with 31.3 (19.1-44.9) x 10(9) per L versus 27.5 (19.2-44.0) x 10(9) per L (p = 0.21, NS) and PMNs with 29.0 (17.6-42.2) x 10(9) per L versus 25.2 (16.2-39.0) x 10(9) per L (p = 0.08, NS). The PMN apheresis yields were equal with 70 (39-139) x 10(9) per unit with low-dose G-CSF versus 68 (33-120) x 10(9) per unit in the high-dose G-CSF group (p = 0.83, NS). Regarding donor adverse reactions, 7 out of 29 (24%) and 8 out of 23 donors (35%) reported moderate or severe symptoms. The character of these reactions was different; symptoms of greater clinical significance and a higher need for analgesics were observed in the high-dose G-CSF group. CONCLUSIONS: A Lenograstim dose of 3 microg per kg plus DXM assures effective PMN mobilization and acceptable apheresis components. The combination of glycosylated G-CSF with DXM allows a significant dose reduction in G-CSF for PMN mobilization and collection as compared with higher G-CSF doses alone. In the high-dose G-CSF mobilization group, adverse reactions were more severe and required more analgesics. PMID- 12375668 TI - Controlled study of citrate effects and response to i.v. calcium administration during allogeneic peripheral blood progenitor cell donation. AB - BACKGROUND: Leukapheresis procedures are generally performed at citrate anticoagulation rates extrapolated from shorter plateletpheresis procedures. However, neither the metabolic effects nor the management of associated symptoms have been critically evaluated during leukapheresis in healthy donors. STUDY DESIGN AND METHODS: Symptom assessments (n = 315) and laboratory analyses (n = 49) were performed during 244 procedures performed with and 71 without prophylactic calcium (Ca) chloride or Ca gluconate given at a dose linked to the citrate infusion rate (1.0-2.2 mg/kg/min). RESULTS: During leukapheresis of 12 to 25 L processed, ionized Ca and ionized magnesium (Mg) decreased as much as 35 and 56 percent, respectively, each exhibiting a tight negative correlation with marked increases in serum citrate levels. Significant increases in urinary Ca and Mg excretion accompanied the renal excretion of a large citrate load. Serum divalent cation levels remained depressed 24 hours after leukapheresis. Symptoms were more frequent in donors who were women, had low initial total Mg levels, and underwent procedures in which larger volumes were processed at higher citrate infusion rates. Ca infusions reduced clinically significant paresthesias by 96 percent and also attenuated decreases in serum potassium. Ca chloride maintained higher Ca levels than Ca gluconate. CONCLUSIONS: Prophylactic Ca infusions safely attenuate the marked metabolic effects of citrate administration and promote faster, more comfortable, leukapheresis procedures. PMID- 12375669 TI - Quantification of fetomaternal hemorrhage by fluorescence microscopy is equivalent to flow cytometry. AB - BACKGROUND: The quantification of fetal cells in the maternal circulation remains an important goal to determine the amount of anti-D necessary to prevent active immunization of a D- mother giving birth to a D+ baby. Underestimation of fetomaternal hemorrhage (FMH) results in inefficient anti-D prophylaxis and maternal immunization; overestimation of FMH results in higher doses of passively transferred anti-D, higher costs, and the risk of disease transmission. Thus, a reliable method to quantitatively assess FMH is necessary. STUDY DESIGN AND METHODS: Serial dilutions of artificial FMH were quantitatively measured by three different methods: flow cytometry, fluorescence microscopy (each after anti-D staining), and by the Kleihauer-Betke test. The accuracy and precision of the three methods were compared by statistical analysis. RESULTS: Fluorescence microscopy and flow cytometry were comparably accurate and precise in quantifying FMH. In contrast, the accuracy of the Kleihauer-Betke test was poor, resulting in substantial overestimation of FMH in the samples with lower fetal cell concentrations. CONCLUSION: Anti-D flow cytometry and fluorescence microscopy for detection of fetal cells offer equally reliable and precise methods in contrast to the Kleihauer-Betke test. Fluorescence microscopy may be established as standard to quantify FMH in clinical practice because it is comparable to flow cytometry; in addition, it is time saving and is less expensive. PMID- 12375670 TI - Evaluating DNA tests of motherless cases using a Philippine genetic database. AB - BACKGROUND: In 5 percent of paternity determination cases, only DNA samples from the alleged father and child pairs are tested. The absence of the mother's DNA increases the probability of false paternity inclusions, which affects laboratories that use a limited number of DNA markers. The effect of coincidental matches between unrelated individuals on DNA tests of motherless cases was determined using the Philippine population genetic database of the National Capital Region (NCR). STUDY DESIGN AND METHODS: Seven short tandem repeat (STR) markers were used, namely HUMvWA, HUMTH01, HUMCSF1PO, HUMFOLP23, D8S306, HUMFES/FPS, and HUMF13A01. Values of the probability of paternity with (W) and without a mother (W(-mother)) were determined using the equation W = cumulative likelihood ratio/cumulative likelihood ratio + 1). These values were determined for 50 volunteer families and compared with values calculated from randomly matched pairs in a reference NCR population database. RESULTS: The W and W( mother) values of the 50 families range from 96.48 to 99.99 percent and 79.76 to 99.99 percent, respectively. In the NCR database, 195 coincidental matches in seven STR loci out of 5253 possible pairs (3.71%) were detected with W(-mother) values ranging from 12.47 to 99.83 percent. Of these, 53 and 10 random pairs have W(-mother) greater than 95.0 and 99.0 percent, respectively. CONCLUSION: W was higher than W(-mother) in the 50 families. However, the existence of unrelated individuals in the NCR database that randomly matched at seven STR loci and that has W(-mother) values greater than 99.0 percent highlights the need for greater precaution when dealing with motherless cases. PMID- 12375671 TI - Recipient antigen-processing pathways of allogeneic platelet antigens: essential mediators of immunity. PMID- 12375672 TI - Cryopreservation of HPCs with high cell concentration in 5-percent DMSO for transplantation to children. PMID- 12375673 TI - Developing evidence-based medicine for managing diabetes in pregnancy. PMID- 12375674 TI - When diet fails: insulin and oral hypoglycemic agents as alternatives for the management of gestational diabetes mellitus. AB - The principal approach for attaining glycemic control in gestational diabetes mellitus is diet, with the addition of insulin or oral hypoglycemic agents when needed. The purpose of this review is to provide an overview for the understanding of management guidelines that have been validated by appropriately conducted research trials. The similarity between gestational diabetes mellitus and type 2 diabetes is highlighted. Criteria for insulin initiation as well as the role of a hypoglycemic agent as a potential alternative to insulin are discussed. PMID- 12375675 TI - A planned randomized clinical trial of treatment for mild gestational diabetes mellitus. AB - OBJECTIVE: A planned study is described which will determine whether a benefit exists for the treatment of mild carbohydrate intolerance during pregnancy. METHODS: A randomized clinical trial of women with mild gestational diabetes will compare perinatal outcomes in those receiving diet therapy and insulin as required versus those randomized to no specific treatment. RESULTS: The primary outcome of this study will be a composite of neonatal morbidity in the treatment and control groups. CONCLUSIONS: A randomized treatment trial of mild gestational diabetes mellitus will clarify whether identification and treatment of mild gestational diabetes mellitus reduces perinatal morbidity. This information will aid in selecting appropriate thresholds for the treatment of gestational diabetes mellitus. PMID- 12375676 TI - Is the use of insulin lispro safe in pregnancy? AB - OBJECTIVE: To provide a review of the use and safety of insulin lispro during pregnancy. METHODS: This is a review of the available literature on the use of insulin lispro in pregnancy. A MEDLINE search was performed which included published manuscripts and abstracts in the English language to June 2001. RESULTS: The extensive search revealed that data on insulin lispro use during pregnancy are limited. Most of the reports on the use of insulin lispro during pregnancy demonstrated improvement of glycemic control, an increase in patient satisfaction, decreased hypoglycemic episodes, improved maternal and neonatal outcomes, and no deterioration in retinal status. However, there were two reports where it was suggested that there was an association with the use of insulin lispro in pregnancy and increased risk for the development of congenital anomalies and/or development or progression of diabetic retinopathy. CONCLUSIONS: Preliminary data suggest that insulin lispro does not have adverse maternal or fetal effects during pregnancy in women with diabetes. The use of insulin lispro during pregnancy results in improved glycemic control, fewer hypoglycemic episodes, improved patient satisfaction, improved maternal and neonatal outcomes and no deterioration in retinal status. There is no evidence that the use of insulin lispro during pregnancy results in an increased rate of congenital malformations. A prospective randomized clinical trial is imperative for further evaluation of any possible association with the use of insulin lispro during pregnancy and an increased rate of congenital malformations or change in retinal status. PMID- 12375677 TI - Pregnancy outcome and progression of diabetic nephropathy. What's next? AB - OBJECTIVES: The first objective was to assess the association of renal function with maternal and fetal pregnancy outcome in women with diabetic nephropathy. The second objective was to examine the feasibility of a multicenter surveillance program to determine the rates of maternal and fetal pregnancy complications in women with diabetic nephropathy, and to study the effect of pregnancy on the natural history of diabetic renal disease. METHODS: In order to address the first objective, we analyzed data from women with type 1 diabetes and nephropathy enrolled in the Diabetes in Pregnancy Program at our institution. Women were assigned to one of three groups according to enrolment serum creatinine concentration: < or = 1.0 mg/dl, > 1.0 to 1.5 mg/dl and > 1.5 mg/dl. A pilot surveillance program at six centers included women experiencing pregnancy complicated by diabetic nephropathy. In both studies, medical and obstetric history, and maternal and neonatal outcomes, were recorded. Statistical analysis included chi2, logistic regression and analysis of variance. RESULTS: There were 72 pregnancies in 58 women with diabetic nephropathy who enrolled in the pregnancy program. High serum creatinine concentration at enrolment was associated with delivery before 32 weeks' gestation, very low birth weight and increased incidence of neonatal hypoglycemia, independent of quantity of total urinary protein excretion and glycemic control in any trimester. To date, pilot surveillance data have been obtained from six centers on 16 women. Serum creatinine concentrations ranged from 0.4 to 1.1 mg/dl and creatinine clearance from 32 to 317 m/min. Gestational age at delivery ranged from 22 to 39 weeks. CONCLUSIONS: High serum creatinine concentration at enrolment is a risk factor for adverse maternal and neonatal outcome, independent of quantity of total urinary protein excretion and glycemic control during any trimester. A multicenter surveillance program is needed, in order to study less frequent maternal and neonatal outcomes as well as the long-term effects of pregnancy on the natural course of diabetic renal disease. PMID- 12375678 TI - Spontaneous preterm delivery in the type 1 diabetic pregnancy: the role of glycemic control. AB - OBJECTIVE: To determine the role of glycemic control in spontaneous preterm delivery in type 1 diabetic women. METHODS: A secondary analysis of data from women enrolled in the Diabetes in Pregnancy Program prior to 20 weeks was performed. Multiple logistic regression was used to analyze the association between glycohemoglobin A1 in women with spontaneous preterm delivery (n = 53) and women who delivered at term (n = 200). Maternal demographics and obstetric outcomes were also compared between the groups. RESULTS: Glycohemoglobin A1 levels were higher in the spontaneous preterm delivery group than the term group throughout pregnancy, reaching statistical significance after the first trimester. The last glycohemoglobin A1 prior to delivery was 8.1% in the spontaneous preterm delivery group and 7.4% in the term group (p = 0.002). Using multiple logistic regression, each 1% increase in glycohemoglobin A1 increased the risk of spontaneous preterm delivery by 37%. CONCLUSION: Poor glycemic control is associated with an increased risk of spontaneous preterm delivery, suggesting that strict glycemic control may reduce the rate of preterm delivery in these women. PMID- 12375679 TI - Aberrant patterns of cellular communication in diabetes-induced embryopathy. I. Membrane signalling. AB - OBJECTIVE: Our purpose was to investigate the role of membrane signalling in the mechanism of diabetes-induced embryopathy. METHODS: Three groups of 70-90-day-old Sprague-Dawley rats were employed in our study: group 1 was normal control rats receiving a normal diet; group 2 represented experimentally induced diabetic rats with malformed offspring (intravenous injection of 65 mg/kg streptozotocin on pregnancy day 6) and group 3 included streptozotocin-induced diabetic rats with normal offspring. Embryos were examined on day 12 under light microscopy, categorized as morphologically normal or defective, and yolk sac cells were harvested from each group. Activities of ERK1 and 2, Raf-1, JNK1 and 2 in yolk sac cells were analyzed by Western blot with primary antibodies specific to the phosphorylated kinases, respectively. RESULTS: A strong link between hyperglycemia and congenital malformations was confirmed. Key mitogen-activated protein kinases serve as syllabic intermediates: increased activities of Jun amino-terminal kinase (JNK1 and 2) and decreased activities of extracellular signal-regulated kinase (ERK1 and 2) were observed during hyperglycemia-induced embryopathy. CONCLUSIONS: Poorly controlled maternal diabetes results in embryopathy which is mediated via a pattern of aberrant cellular communication manifested by both macroscopic and microscopic membrane injury. PMID- 12375680 TI - Animal models for assessing the consequences of intrauterine growth restriction on subsequent glucose metabolism of the offspring: a review. AB - OBJECTIVE: The aim of this article was to review the most current animal models for intrauterine growth restriction. METHODS: The databases of MEDLINE and Pubmed were searched for articles published between 1969 and 2001. Additional sources included abstract proceedings and relevant reference lists of articles identified by database review. Key words included intrauterine growth retardation, small for gestational age, animal models and glucose metabolism. The inclusion criteria used to select studies included animal models of intrauterine growth retardation in which glucose metabolism was assessed after birth. RESULTS: A variety of physiological and metabolic variables regulate fetal growth, and alteration of these variables can result in fetal growth restriction. Fetal growth restriction is secondary to initiation of fetal adaptation measures in response to inadequate supply of oxygen and nutrients. Several animal models have led to a greater understanding of the pathophysiology and consequences of intrauterine growth restriction. These observations are comparable to those observed in humans born small for gestational age, and are of interest because of the known association between poor fetal growth and development of glucose intolerance and type 2 diabetes mellitus later in life. CONCLUSION: Experimental manipulations have apparently altered a number of metabolic and physiological variables, but the pattern of alterations seems to vary with the procedure employed. This allows only a preliminary conclusion to be drawn at best. Either the laboratory procedures vitiate the natural process under study, or the process itself is variable with respect to the triggering mechanism. As yet, we cannot tell which. PMID- 12375681 TI - Is fetal macrosomia in adequately controlled diabetic women the result of a placental defect?--a hypothesis. AB - Fetal macrosomia may occur even in adequately controlled diabetic mothers. This may reflect the problem of using maternal glycemia as an indicator of fetal glycemia, because the placenta interposed between both compartments has its own glucose metabolism. Here, we propose a model by which the placenta protects the fetus at moderate levels of maternal hyperglycemia. One characteristic feature of the human placenta in diabetes is the increased deposition of glycogen. Neither hyperglycemia nor hyperinsulinemia increase the glycogen content in the trophoblast. Since the glycogen increments in diabetes are predominantly located around fetoplacental vessels, it is tempting to assume a fetal origin of glucose making up the glycogen deposits. In fact, glucose can be transported back from the fetus into the placenta and this reflux is increased in diabetes. Therefore, in conditions of fetal glucose levels exceeding the demand for sustaining fetal growth and metabolism, glucose can be stored in the liver and other fetal tissues. Once these stores are saturated, glucose is extracted from the fetal circulation by the glucose transporters GLUT1 and GLUT3 on cells surrounding the fetoplacental vasculature and stored therein, again in the form of glycogen. These processes might be under the control of fetal insulin, because insulin injected into the fetal circulation increases placental glycogen stores. Fetal macrosomia would then occur only when fetal hyperglycemia exceeds the placental capacity to store excess fetal glucose. Thus, the placental failure to protect the fetus would cause the 'unexplained' phenotypic changes occasionally found in fetuses born to well-controlled diabetic women. PMID- 12375682 TI - Extreme second-trimester serum analyte values in down syndrome pregnancies with hydrops fetalis. AB - OBJECTIVE: To compare second-trimester maternal serum analyte values in Down syndrome pregnancies with, and without, hydrops fetalis. METHODS: Seven hydropic and 85 non-hydropic Down syndrome pregnancies were identified among women with positive second-trimester maternal serum screening results. Values for maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG), unconjugated estriol and inhibin-A, and risks for Down syndrome were compared using the non parametric Mann-Whitney statistical test. RESULTS: Hydropic Down syndrome pregnancies had significantly lower MSAFP and estriol concentrations, while hCG levels were higher. For subgroups of five hydropic and 42 non-hydropic cases, no statistically significant difference in the inhibin-A levels could be demonstrated. CONCLUSION: Second-trimester Down syndrome screening risks are significantly higher in affected pregnancies that are complicated by fetal hydrops. PMID- 12375683 TI - Does nifedipine prevent the tachysystole associated with misoprostol induction? AB - OBJECTIVE: To determine the efficacy of oral nifedipine as prophylaxis against uterine tachysystole associated with misoprostol induction of labor. METHODS: A total of 116 patients undergoing induction with term, singleton pregnancies were enrolled. All patients received 50 microg misoprostol intravaginally every 4 h. Patients were randomly assigned also to receive nifedipine 10 mg orally every 4 h, or no prophylaxis. The primary outcome variable was the incidence of 12 or more contractions in any 20-min interval. RESULTS: Data on 106 patients were available for analysis. There were 55 subjects in the misoprostol-nifedipine group and 51 controls. Nifedipine did not diminish the incidence of tachysystole when added to misoprostol (42% vs. 45% without nifedipine; p = 0.7). CONCLUSION: Prophylactic oral nifedipine was not shown to decrease the uterine tachysystole associated with vaginal misoprostol induction at a 50-microg dose. PMID- 12375684 TI - Maternal-fetal effects of magnesium sulfate on serum osmolality in pre-eclampsia. AB - OBJECTIVE: To determine the effects of magnesium sulfate therapy on maternal and fetal osmolality in pre-eclampsia. METHODS: A total of 34 pre-eclamptic women and 22 normal pregnant women participated in the study. Venous blood was drawn upon admission to the labor and delivery unit. Pre-eclamptic patients received standard magnesium sulfate therapy and had a second sample of venous blood drawn 4 h following the beginning of therapy. Fetal umbilical vein blood was obtained immediately following delivery in both groups. Osmolality was measured using a vapor pressure osmometer. Electrolyte levels were measured with a NOVA 8 biomedical analyzer. Data were analyzed via Student's t test, linear regression and correlation with significance established at p < 0.05. RESULTS: We found no significant difference between the maternal osmolalities of the control and pre eclamptic groups. Interestingly, fetal cord blood osmolality was significantly lower than maternal osmolality in the normal pregnant women. Sodium levels were also lower, while potassium and ionized calcium levels were higher in the fetal blood. In women with pre-eclampsia treated with magnesium sulfate, there was no difference between the osmolality of the maternal and fetal blood, while potassium and ionized calcium levels were still higher in the fetal blood. Finally, we found no correlation between maternal osmolality and blood pressure. CONCLUSIONS: High blood pressure in pre-eclampsia is not associated with altered osmolality. An absence of the normal decrease in fetal osmolality is observed in pre-eclamptic women treated with magnesium sulfate. PMID- 12375685 TI - Total antioxidant capacity of colostrum, and transitional and mature human milk. AB - OBJECTIVE: The relationship between antioxidant (pro-)vitamin content of breast milk and diet of pregnant women has been reported. However, the assessment of a complete and reliable antioxidant index, such as the total antioxidant capacity, has never been performed in human milk. METHODS: In the present study, the total antioxidant capacity of colostrum, and transitional and mature milk was determined by the oxygen radical absorbance capacity (ORAC) assay in 29 lactating women and correlated with their food consumption assessed by dietary history during pregnancy and lactation. In addition, the total antioxidant capacity of 12 infant formulas commonly used in Italy was determined by the same method. RESULTS: Mean ORAC values of mature milk (1.01 +/- 0.37 mmol/l trolox eq.) were slightly lower than those of colostrum and transitional milk (1.17 +/- 0.50 and 1.18 +/- 0.38 mmol/l trolox eq., respectively). Correlations were observed between antioxidant (pro-)vitamin intakes during pregnancy (third trimester) and ORAC values of colostrum and transitional milk (both p < 0.05), but not with ORAC values of mature milk. Correlations were also found between antioxidant (pro )vitamin intakes during lactation and transitional and mature milk ORAC values (both p < 0.05). Term infant formulas (n = 5) showed a total antioxidant capacity of 1.40 +/- 0.23 mmol/l trolox eq., whereas the antioxidant capacity of other formulas (n = 7) oscillated from 1.27 to 2.52 mmol/l trolox eq. CONCLUSIONS: The total antioxidant capacity of breast milk in lactating women was related to a diet in which the food content of antioxidant compounds was inadequate. Efforts should be made to improve women's dietary habits during pregnancy and lactation, in order to optimize the total antioxidant capacity of breast milk. PMID- 12375686 TI - The umbilical coiling index in normal pregnancy. AB - OBJECTIVE: To provide reference values for the umbilical coiling index in uncomplicated pregnancy. METHODS: Umbilical cords were collected from livebom singleton infants born after uncomplicated pregnancies. The umbilical coiling index (UCI) was calculated as the number of coils divided by the cord length in centimeters. The mean value (SD) for the UCI was calculated, and possible correlations of the UCI with maternal age, parity, gestational age at delivery, mode of delivery, sex and birth weight of the infant were examined. RESULTS: A total of 122 umbilical cords were included. The frequency distribution of the UCI was skewed to the right. The mean (SD) UCI was 0.17 (0.009) coils/cm. There were no significant correlations of the UCI with maternal age, parity, gestational age at delivery, mode of delivery, sex or birth weight of the infant. CONCLUSIONS: This is the first study to determine the UCI in a group exclusively consisting of uncomplicated pregnancies. The mean value that we found for the UCI may serve as the standard reference, allowing proper interpretation of umbilical coiling in complicated pregnancy. PMID- 12375687 TI - Possible West Nile virus transmission to an infant through breast-feeding- Michigan, 2002. AB - CDC and the Michigan Department of Community Health (MDCH) continue to investigate West Nile Virus (WNV) infection in a woman, who received a blood product later found with evidence of WNV, and in her child, who was exposed to breast milk later found to be WNV positive by TaqMan. This report updates the findings of this investigation. PMID- 12375688 TI - Update: Investigations of West Nile virus infections in recipients of organ transplantation and blood transfusion--Michigan, 2002. AB - On September 27, 2002, this report was posted on the MMWR website (http://www.cdc.gov/mmwr). CDC, the Food and Drug Administration (FDA), the Health Resources and Services Administration (HRSA), and state and local health departments continue to investigate West Nile virus (WNV) infections in recipients of organ transplantation and blood transfusion. This report summarizes two investigations of Michigan recipients of blood products, one of whom also received a liver transplant. Both persons tested positive for WNV infection after receiving blood products derived from a single blood donation subsequently found to have evidence of WNV. These investigations provide further evidence that WNV is transmitted through blood transfusion. PMID- 12375689 TI - Update: Influenza activity--United States and worldwide, June-September, 2002. AB - During June-September 2002, influenza A (H3N2) and B viruses circulated worldwide and were associated with mild to moderate levels of disease activity. Influenza B viruses predominated in Africa, and both influenza A (H3N2) and B viruses circulated widely in Asia, Oceania, and Latin America, except in Chile and Taiwan, where A (H1) viruses predominated. In North America, sporadic isolates of influenza A (H3N2), A (H1), and B viruses were identified. This report summarizes influenza activity in the United States and worldwide during June-September 2002. Influenza activity in North America typically peaks during December-March, which underscores the need to begin vaccinating against influenza in October and to continue vaccination into December and throughout the influenza season. PMID- 12375690 TI - Increase in African immigrants and refugees with tuberculosis--Seattle-King County, Washington, 1998-2001. AB - The proportion of tuberculosis (TB) cases among foreign-born persons in the United States has increased steadily, accounting for half of reported cases in 2001 for which country-of-origin information was available. During 1998-2001, the annual number of TB cases among African immigrants and refugees in Seattle and all of King County increased approximately threefold to that during 1993-1997. This report summarizes the investigation of cases during 1998-2001 and outlines the public health interventions implemented to prevent TB in this population. The findings indicate that in Seattle-King County, persons at risk for TB who have arrived recently in the United States were primarily from the African-Horn countries of Eritrea, Ethiopia, and Somalia. Primary health-care providers and civil surgeons (i.e., physicians appointed by the Immigration and Naturalization Service to screen for medical conditions as required for changes of immigration status) should be aware of the high TB rate among African immigrants, especially within the first 5 years after immigration, and be alert for severe extrapulmonary forms of TB. PMID- 12375691 TI - West Nile virus activity--United States, September 26-October 2, 2002, and investigations of West Nile virus infections in recipients of blood transfusion and organ transplantation. AB - This report summarizes West Nile virus (WNV) surveillance data reported to CDC through ArboNET and by states and other jurisdictions as of 7 a.m. Mountain Daylight Time, October 2, 2002, and updates preliminary demographic and clinical information on cases of WNV infections inrecipients of blood transfusion and organ transplantation reported to CDC during August 28-October 2, 2002. PMID- 12375692 TI - Alterations to hepatic microcirculation in thioacetamide-induced cirrhotic livers of rats. AB - In liver cirrhosis, increased resistance of intrahepatic microvasculature contribute to the development of portal hypertension. This study aimed to reveal the alterations to hemodynamics in microvasculature of thioacetamide-induced fibrotic and cirrhotic rat livers, using in vivo microscopy. In fibrotic livers, although intrahepatic blood flow remained unaltered, area percentage of sinusoids was significantly decreased. In cirrhotic livers, intrahepatic blood flow was significantly increased concurrently with decrease in area percentage of sinusoids. The flow velocity and volume flow were significantly increased in terminal portal venules (TPVs) without changes in vascular diameters, whereas all these parameters were not altered in terminal hepatic venules (THVs). Intrahepatic shunts which emerged from TPVs and ran toward THVs, and anastomoses between neighboring THVs were formed in cirrhotic livers. These data indicate that the first occurring alteration of microcirculation in liver cirrhosis is decrease in sinusoidal beds. PMID- 12375693 TI - Distant metastatic recurrence after hepatic resection for hepatocellular carcinoma. AB - To identify clinicopathologic features of distant metastatic recurrence (DMR) after hepatic resection for hepatocellular carcinoma, we investigated 352 recurrent patients. Of them, 261 with only intrahepatic recurrence were compared with 91 with DMR. Between two groups, there was significantly difference with respect to preoperative liver function, positive tests for protein induced by vitamin K absence or antagonist-II (PIVKA-II), major resection, tumor-free interval, tumor diameter, extrahepatic progression of the tumor, single nodular configuration, and portal vein invasion. Survival rates at 1 and 5 years after intrahepatic recurrence were 73% and 17%, while these were 57% and 18% when recurrences were DMR (p = .0362). DMR was frequent when a resected tumor was large, extrahepatic, ill defined or multinodular or associated with portal vein invasion, good preoperative liver function, or PIVKA-II positivity. Such patients should receive particularly careful follow-up during the first 2 years after hepatic resection focusing on early detection of DMR. PMID- 12375694 TI - Impact of Prol2Ala variant in the peroxisome proliferator-activated receptor (PPAR) gamma2 on obesity and insulin resistance in Japanese Type 2 diabetic and healthy subjects. AB - The peroxisome proliferator-activated receptor (PPAR) gamma is an important regulator of adipocyte differentiation and a modulator of intracellular insulin signaling events. We examined the roles of the Pro12Ala variant of PPAR gamma2 in obesity and insulin resistance in 402 Japanese patients with type 2 diabetes and 116 control subjects. Among the diabetes subjects, the Pro12Pro homozygotes showed significantly higher body mass index (BMI) than those with the Pro12Ala variant (p = 0.020), while there was no association between genotype and BMI in the controls. Furthermore, diabetic subjects with Pro12Pro showed significantly higher fat body mass index (FBMI) than those with Pro12Ala (p = 0.016), while no association between genotype and lean body mass index (LBMI) was observed. Regarding insulin resistance, there was no difference in the HOMA index or in clamp index between Pro12Ala and Pro12Pro variants. These data suggest that the Pro12Ala polymorphism of PPAR gamma2 does not influence insulin resistance but body composition in Japanese diabetic subjects. PMID- 12375695 TI - Cell swelling as an intermediate signal leading to activation of a Cl- channel in extracellular calcium-sensing of murine osteoclasts. AB - Osteoclasts possess extracellular Ca2+-sensing machinery to regulate bone resorbing activity. In murine osteoclasts, an elevation of the extracellular Ca2+ concentration activated a volume-sensitive outwardly rectifying Cl- (OR(Cl)) channel. Exposure to 40 mM Ca2+ activated the OR(Cl) current in association with an increase in the planar cell area. An actin-destabilizer (cytochalasin D), removal of a major extracellular osmolyte (Na+), and a blocker for the Na+/Ca2+ exchanger (2'4'-dichlorobenzamil hydrochloride) inhibited both swelling and activation of the OR(Cl) channel by extracellular Ca2+. There was a positive correlation between the current density and increment in the cell area. The extracellular Ca2+-induced swelling was confirmed in intact (unclamped) cells by three-dimensional analysis using confocal scanning microscopy with a fluorescent dye (BCECF) in the extracellular medium. These results suggest that swelling is an intermediate signal for extracellular Ca2+ sensing of osteoclasts, which leads to activation of the OR(Cl) channel. Cell volume may be a second-message in the regulation of osteoclast function. PMID- 12375696 TI - Promotion of human blastocyst formation by red cell hemolysate. AB - Recently, blastocyst transfer has been increasingly performed to improve the implantation rate achieved by in vitro fertilization and embryo transfer. This has created the need for new culture methods to improve in vitro blastulation. Culture with erythrocyte hemolysate promotes the development of early mouse embryos due to an antioxidant effect. The present study investigated whether erythrocyte hemolysate could also promote blastulation of human embryos. Eighty surplus embryos were obtained from 15 patients who gave informed consent. These embryos were cultured for 2 to 7 days after oocyte retrieval in medium with or without erythrocyte hemolysate. Blastocyst formation was observed in 53% and 28% of the embryos grown in hemolysate-supplemented and control cultures, respectively. The blastulation rate was significantly higher in hemolysate supplemented culture than in control group (p = 0.02). These results suggest that culture medium supplemented with erythrocyte hemolysate may promote the blastulation of human embryos. PMID- 12375697 TI - Dose-related attenuation of cardiac sympathetic nerve activity and intracardiac conduction with thoracic epidural clonidine in alpha-chloralose-anesthetized cats. AB - Epidural clonidine produces hypotension, bradycardia and sympatholysis. We studied the dose-effects of thoracic epidural and intravenous clonidine (1 to 8 microg/kg) on cardiac sympathetic nerve activity (CSNA), hemodynamics and intracardiac conduction in cats anesthetized with alpha-chloralose. Mean arterial pressure was decreased with epidural clonidine doses above 2 microg/kg, and to a greater extent with 4 microg/kg than 8 microg/kg. Sinus heart rate, Wenckebach atrial rate and CSNA were significantly decreased and corrected sinus node recovery time and Atrium-His interval were significantly prolonged with doses above 2 microg/kg. Vagotomy induced no significant change in these parameters. Thoracic epidural clonidine doses above 2 microg/kg caused a similar extent of sympatholysis. Less of CSNA decrease and hemodynamic changes by intravenous clonidine suggested that sympatholysis caused by epidural clonidine was primarily mediated by spinal mechanism, although hemodynamic changes were influenced by clonidine systemically redistributed from epidural space. Vagal facilitation played no role in suppression of the sinoatrial and AV nodal functions. PMID- 12375698 TI - Brachytherapy for the prevention of neointimal hyperplasia in the canine inferior vena cava after stent placement. AB - The aim of this study was to evaluate the efficacy of brachytherapy for preventing neointimal hyperplasia in the inferior vena cava (IVC) after stent placement. Sixteen beagles underwent Z-stent placement in the IVC and the aorta. For 8 of 16 beagles, irradiation (15 Gy) was delivered endoluminally to the stented segments of each vessel immediately after stent placement using the 192Ir. All animals were sacrificed after 6 weeks for morphometric and histopathologic examination. Morphometrically, neointimal thickness in the IVC of the radiation group was significantly decreased compared with the control group as well as that in the aorta (p < 0.05). Histopathologic findings showed the neointima in the IVC of the control group contained markedly organization of thrombus and neovascularization though that in the IVC of the radiation group consisted mainly of smooth muscle cells without organization of thrombus and neovascularization. From these data intravenous irradiation may prevent clinical restenosis after stent placement. PMID- 12375699 TI - Additive effects of amrubicin with cisplatin on human lung cancer cell lines. AB - Amrubicin (AMR) is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicinol, the C-13 alcohol metabolite of AMR, inhibits purified human topoisomerase II (topo II). We examined the effect of the combination of cisplatin (CDDP) and amrubicinol in vitro using a small cell lung cancer cell line (SBC-3) and an adenocarcinoma cell line (Ma-1), by WST-1 assay and isobologram analysis. When the two drugs were used together either simultaneously or sequentially, their combined effects were additive. A high concentration of CDDP (300 microM) enhanced the topo II inhibitory activity of amrubicinol as determined by kinetoplast-DNA decatenation assay. On the other hand, amrubicinol increased formation of DNA interstrand cross-links (ICL) in the cells, as determined using ethidium bromide fluorescence binding assay (EBFA), for simultaneous exposure to CDDP (0-300 microM) and amrubicinol (2 microM) compared with CDDP alone. These biological interactions might result in additive interaction between amrubicinol and CDDP. PMID- 12375700 TI - Chest wall resection in general thoracic surgery. AB - To clarify the incidence, indications, and efficacy of chest wall resection, a comprehensive review is needed. Chest wall resection was performed in 23 of 162 operations for thoracic disease over a nine-year period. Eight surgeries requiring chest wall resection for benign disease (8/79) were classified as fenestration or thoracoplasty for empyema, or resection of a benign neoplasm. Fifteen patients who underwent chest wall resections for malignant disease (15/83) were classified as contiguous extension of neoplasms of neighboring organs, primary tumor, or local recurrence. The most common procedure in the malignant disease group was resection for contiguous spread of primary lung cancer (n = 7). The survival rate was 50% at 4 years. There were no serious postoperative complications. In some malignant diseases, complete local control with such a procedure may even lead to a long-term survival. This is a safe and an effective procedure for a variety of diseases. PMID- 12375701 TI - The distribution of secretory immunoglobulin A in the liver of patients with hepatolithiasis. AB - Patients with hepatolithiasis are known to be complicated with bile stasis and bacterial infections in the stone-containing ducts. It has been suspected that a decrease in portal venous flow is important in the progression of hepatolithiasis. This study was done to find if the affected liver in hepatolithiasis is inflamed or has lowered local immunity by an examination of the distribution of secretory immunoglobulin A and proliferating cell nuclear antigen in the intrahepatic biliary tracts in operative specimens of 36 patients with hepatolithiasis. The number of biliary epithelia stained for the immunoglobulin was greater when the cholangitis was more severe up to a point; in advanced cholangitis, with severe parenchymal atrophy or proliferating epithelia, there were fewer cells stained for the immunoglobulin than in mild cholangitis. In hepatolithiasis, secretory immunoglobulin A decreases when inflammatory changes become severe and there is parenchymal atrophy caused by stenosis or obstruction of portal branch. PMID- 12375702 TI - Retrospective study of conservation surgery for breast cancer with or without axillary dissection. AB - BACKGROUND: It is already known that for breast cancer patients, an axillary dissection or locoregional radiation has no major effect on survival with a simple mastectomy. We analyzed whether axillary dissection improved the prognosis for breast conservation surgery. METHODS: From 1982 to 1995, 31 patients underwent axillary dissection and 33 did not in association with breast conservation surgery performed at our institution. Median follow up was at 55 months, ranging from 3 to 210. Survival rates of patients were analyzed retrospectively. RESULTS: In the dissection group, 8 patients had recurrence and 5 died within 3 years, yielding a relapse-free survival rate of 71%. In the non dissection group, 7 patients had recurrence, 3 died within 3 years, with a relapse-free survival rate of 79%. Figures for overall survival, relapse-free except breast and relapse-free for breast at 5 years were 93%, 73%, and 96% for the dissection group, and 93%, 89%, and 89% for the non-dissection group, respectively. No difference was found in survival rates between the two groups. Adjusted by Cox's regression analysis, survival rates of overall, relapse-free, relapse-free except breast and relapse-free for breast, at 5 years were 86%, 71%, 77%, and 90% for the dissection group, and 100%, 78%, 85%, and 95% for the non dissection group, respectively. No survival benefit was found in axillary dissection. The first site of recurrence was in the affected breast in 3 patients and in other sites in 5 patients for the dissection group, while for the non dissection group it was in the affected axilla in 1 patient, the skin of the affected breast in 1 patient, the affected breast in 3 patients, and other sites in 2 patients. In the non-dissection group, a patient who had recurrence on the skin of the affected breast, subsequently had an axillary recurrence on the same side with plexus paresis. CONCLUSION: In breast conservation, prophylactic axillary dissection seems to be omitted, but the axilla of the affected side should be examined for metastasis while treatment is possible. PMID- 12375703 TI - Intra-articular corrective osteotomy for the malunited intercondylar humeral fracture: a case report. AB - A 35-year-old patient sustained comminuted intercondylar fracture of the distal humerus. At six months post-open reduction and internal fixation, the malunion was corrected with an intra-articular osteotomy. The patient obtained a painless, functional elbow joint with increased grip strength after corrective osteotomy, although she had complained of severe elbow pain, limited range of motion, and loss of grip strength before osteotomy. A 15 degrees cubitus varus deformity was also corrected . Radiographs of the elbow joint did not show accelerated degenerative changes at over three years follow-up post-corrective osteotomy. Intra-articular corrective osteotomy should be considered as a salvage procedure for the treatment of a malunited intercondylar humeral fracture, especially in patients who are thought to be too young for elbow arthroplasty. PMID- 12375704 TI - Serving the underserved: school-based asthma intervention programs. AB - Asthma is a common and costly disease, which disproportionately affects young, ethnic minority, and impoverished individuals. A heightened public awareness has catalyzed a variety of efforts to reduce the impact of childhood asthma in the United States. Despite these efforts, however, at-risk groups continue to suffer excess morbidity. Strategies are required that can specifically address the needs of underserved communities. We review the underlying rationale and progress of school-based programs designed to assist asthmatic children. PMID- 12375705 TI - Evaluation of allergic bronchopulmonary aspergillosis in patients with and without central bronchiectasis. AB - Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity disorder induced by Aspergillus species colonizing the bronchial tree. Thirty one patients fulfilling criteria of ABPA were evaluated in the present study. Eleven patients were diagnosed as ABPA-S (serological positive) and 20 patients as ABPA-CB (with central bronchiectasis). The two groups of patients were compared on the basis of clinical, serological, and radiographic observations. Serum anti Aspergillus fumigatus IgG was positive in 72% of cases of ABPA-S and 85% of ABPA-CB patients at the time of presentation. Specific IgE against A. fumigatus and total IgE were significantly lower in ABPA-S (specific IgE= 7.42 IU and total IgE= 1127 ng/mL) as compared to ABPA-CB (specific IgE = 44 IU and total IgE = 2874 ng/mL). The spirometric changes in ABPA-S (normal 80%, mild obstruction 10%, and severe obstruction 10%) were milder than in ABPA-CB (normal 40%, mild obstruction 10%, moderate obstruction 20%, and severe obstruction 30%). These patients were monitored closely for seasonal exacerbation with new pulmonary infiltrates which gave lower recordings in ABPA-S patients. No patient in the ABPA-S group progressed to end-stage lung disease. This may be due to early recognition and treatment. The present data suggest that ABPA-S represents the early stage of an apparently less aggressive form of ABPA than ABPA-CB. PMID- 12375706 TI - Quality evaluation of samples obtained by spontaneous or induced sputum: comparison between two methods of processing and relationship with clinical and functional findings. AB - The aim of the study was to assess, on a large group of spontaneous or induced sputum samples, the difference in quality between slides processed by two different methods, and the relationship between quality assessment and some clinical and functional characteristics of the examined subjects. We examined 631 sputum samples obtained from 337 subjects with proven (n = 291) or suspected bronchial asthma. Of these, 467 samples were processed using the whole-sample method (Group I), while 164 samples were processed using the plug method (Group II). Salivary contamination, cell distribution on the slide, and cell borders were evaluated, and samples were classified as inadequate, adequate, or good. Inadequate samples were equally represented in both groups, while good samples were represented more in Group II. No significant difference in most clinical and functional findings was observed between the different quality categories of both groups. A higher proportion of inadequate samples was observed in Group I samples spontaneously collected. Mild intermittent asthmatics produced a better quality of slides in comparison with other groups of asthma severity. In conclusion, sputum quality partially depends on the different methods of sputum collection and/or processing, although the percentage of inadequate samples is similar for the two methods of processing. Sputum quality is only marginally affected by clinical and functional characteristics of asthma, or by asthma severity. PMID- 12375707 TI - Assessment of diurnal variability of peak expiratory flow in stable asthmatics. AB - Five daily readings of peak expiratory flow (PEF) were obtained for three days on 100 patients with chronic stable asthma. The variability of PEF was calculated as the amplitude percent mean (A%M) from the readings obtained on the third day, and compared to previously reported data from 152 healthy Indian adults. Patients with severe asthma exhibited significantly higher A%M than patients with both mild and moderate asthma (p < 0.05), but there was considerable overlap across disease categories. The area under the receiver operating characteristic curve plotted to assess the performance of PEF variability as a discriminator in diagnosing asthma was 0.826, with best discrimination at a value of 12.5 (sensitivity 0.640, specificity 0.941). Using a cut-off value of 16.5 (as proposed earlier by us) improved specificity to 0.987 but reduced sensitivity to 0.510. Using a traditional cut-off of 20, specificity remained almost unchanged (0.993), but sensitivity dropped further to 0.440. Thus A%M>16.5 is a useful marker of bronchial asthma in epidemiological studies in India. However, its use in population screening, clinical diagnosis, or in the assessment of the severity of asthma in individual patients has serious limitations because of poor sensitivity. PMID- 12375708 TI - Education and self-management: a one-year randomized trial in stable adult asthmatic patients. AB - OBJECTIVE: to assess the effects of an educational program in asthmatic patients, following treatment readjustment. METHODS: moderate to severe asthmatic adults underwent a run-in period (up to 45 days) in order to optimize their treatment. Patients were then randomized to an educational or control group over a one-year period. Education consisted of five individual sessions covering: pathophysiology of asthma, role of medication and side-effects, asthma triggers and their avoidance, detection of an asthma flare-up, and self-management plan based on symptoms and peak-flow monitoring. MAIN OUTCOME CRITERION: symptom-free days over the study period (SFD). RESULTS: a total of 72 patients were enrolled (36 in the "education group" and 36 in the "control group"), 54 of whom completed the study. Mean SFD was comparable in the two groups (88% in the education group and 89% in the control group, respectively). When the analysis was restricted to the education group, those patients who complied perfectly with the action plan (n = 5) exhibited a higher SFD, compared to the others (97% vs. 87%, p = 0.009). CONCLUSION: both education and control groups showed high and comparable percentages of SFD. Compliance with self-management plans appears to be an important determining factor in educational programs. PMID- 12375709 TI - A prospective study of the relationship of mood and stress to pulmonary function among patients with asthma. AB - The purpose of this study was to examine the association of psychosocial and other variables to pulmonary function over four months. Thirty-two patients with asthma kept daily records of pulmonary function and psychosocial variables for an average of 140 days. Data on other potential covariates of pulmonary function, as assessed by peak flow meters, were also collected (e.g., allergen exposure). Sixteen subjects (50%) had significant associations between pulmonary function and psychosocial variables. Between-subjects analyses showed small but significant associations between pulmonary function and other variables. These results confirmed previous reports of individual variability in the association of psychosocial variables with pulmonary function. PMID- 12375711 TI - Airway inflammation in premenstrual asthma. AB - Premenstrual asthma (PMA) is a clinical picture with worsening of asthmatic symptoms and pulmonary functions in the late luteal phase of the menstrual cycle. The aim of this study was to evaluate the inflammatory changes in asthmatic women who complain of PMA. Forty asthmatic women attending our outpatient clinic were questioned about worsening of their asthma before menstruation. Eleven women (aged 17-40) who complained of PMA participated in the study. Subjects were asked to record peak expiratory flow rates, symptom scores, and beta-agonist use daily. After the first menses on the seventh day of their cycle, and before the onset of the next menstruation, on the 26+/-3rd day of the cycle, patients were evaluated with pulmonary function tests, methacholine challenge test, and fractionated exhaled nitric oxide (FE(NO)) levels. Eosinophils in peripheral blood and induced sputum were also evaluated. When comparing the two groups of results, the significant changes were in FENO levels, day-time symptom scores, and eosinophils in induced sputum (29.25 ppb/9.16 ppb p < 0.05, 1/0.45 p = 0.05, %6.63/%4.09 p < 0.01, respectively, before and after menstruation). These results show that PMA is not only a clinical picture with a decrease in airway calibre that can be related to the regulation of 2 receptors, but also a complex state with worsening of airway inflammation. PMID- 12375710 TI - Alpha amylase is a major allergenic component in occupational asthma patients caused by porcine pancreatic extract. AB - Porcine pancreatic extracts (PPE) are composed of alpha-amylase and lipase, which are common components of digestive enzymes. They have been known to cause occupational asthma in exposed workers in pharmaceutical and baking industries, as well as in a laboratory technician, but there has been no report of PPE induced occupational asthma in medical personnel and their IgE binding components to each component. Four asthmatic subjects showing positive results on PPE bronchoprovocation testing were enrolled. All of them were nurses working in a university hospital. Their job included grinding and mixing PPE powder for admitted patients. Serum-specific IgE antibodies to PPE, alpha-amylase, and lipase were measured by enzyme linked immunosorbent assay (ELISA). To confirm specificity of IgE binding and cross-allergenicity among the three extracts, ELISA inhibition tests were performed. In order to characterize allergenic components within these three extracts, SDS-PAGE and IgE immunoblot analysis were done. Specific IgE antibodies to PPE, alpha-amylase, and lipase were detectable by ELISA in all study subjects. An alpha-amylase ELISA inhibition test showed significant inhibitions by amylase and PPE, and minimal inhibition by lipase. However, a lipase ELISA inhibition test showed significant inhibitions by alpha amylase and PPE with a lesser degree of inhibition by lipase. Furthermore, IgE immunoblot analysis showed one IgE binding component (55 kDa) within PPE, six components (55 kDa, 43 kDa, 41 kDa, 32 kDa, 31 kDa, 29 kDa) within alpha-amylase and two components (31 kDa, 29 kDa) within lipase extracts. Thesefindings suggest that inhalation of PPE powder can induce IgE-mediated bronchoconstriction in exposed nurses. Alpha-amylase is a major allergenic component within PPE. PMID- 12375712 TI - Sensitization to common food allergens is a risk factor for asthma in young Chinese children in Hong Kong. AB - Sensitization to aeroallergens is a major risk factor for asthma. Although patients frequently consider food ingestion as an asthma trigger, the relationship between serum food-specific IgE antibodies and childhood asthma in China remains unclear. We therefore conducted a case-control study on asthmatic children attending a university hospital-based outpatient clinic to investigate their pattern of food sensitization. Asthmatic patients underwent spirometric assessment, and peripheral blood was collected for serum-specific IgE antibodies to common food and inhalant allergens. Two hundred and thirty-one asthmatics (aged 9.3+/-4.3 years) and 79 age- and sex-matched controls were enrolled. The serum logarithmic total IgE concentrations in patients and controls were 2.49 and 1.92, respectively (p < 0.0001). Subjects with increased serum total IgE level were significantly more likely to have food sensitization than those with normal values (33% vs. 16%; p = 0.001). Twenty-nine (52%) of 56 asthmatics younger than 6 years old and seven (27%) of 26 age-matched controls hadfood-specific IgE in their sera (p = 0.035). Asthmatics with food-specific IgE also used more doses of as-needed bronchodilator weekly (p = 0.005). Nonetheless, no association was found between asthma diagnosis and sensitization to individual food allergens. Significant food sensitization, with food-specific IgE level above 95% predictive values for clinical food allergy as proposed by Sampson, was only found in two patients for peanut and three subjects for egg white. In conclusion, a significant association was found between asthma and the presence of food specific IgE antibodies in young Chinese children. Significant sensitization to common foods is rare in this cohort. PMID- 12375713 TI - The effects of inhaled albuterol and salmeterol in 2- to 5-year-old asthmatic children as measured by impulse oscillometry. AB - The functional assessment of the response to bronchodilators in 2- to 5-year-old asthmatic children is technically difficult. For this reason, there have been no reports on the effects of long-acting bronchodilators, such as salmeterol, in this age group. Of the several techniques available for measuring resistance to airflow, forced oscillation remains the most adaptable to young children and the most practical for research and clinical use. In this stud we used the Jaeger MasterScreen Impulse Oscillometry System to assess the response of 2 to 5 year old asthmatic children to an inhaled long-acting bronchodilator, salmeterol, by comparing it to the effect of a standard dose of the short-acting bronchodilator, albuterol. We performed a placebo-controlled, randomized, crossover study in 10 children aged 2 to 5 years who had a history of physician-diagnosed asthma and who were not on regular controller therapy. At weekly intervals after baseline measurements of reversibility, each child received two inhalations from an albuterol metered-dose inhaler (MDI) with a spacer (200 microg), or placebo MDI with spacer, or two inhalations from a salmeterol MDI (50 microg), or 50 microg from a salmeterol Diskus. Measurements were obtained at 5, 30, 60, 360, and 540 min, the last time interval only on the salmeterol days. Based on previous studies, total respiratory system reactance at 5 Hz (X5), calculated by the MasterScreen computer from mouth pressure and flow data, was used as the primary efficacy variable. The mean intra-individual variability in X5 was 10.5% (range 3.6% to 17.9%). The mean (SE) changes from baseline X5 at each time point were as follows: for placebo, 9.6 (3.0), 10.1 (2.6), 5.1 (2.9), 6.1 (3.5), p=0.36 vs. baseline; after treatment with albuterol, 32.7 (3.8), 53.9 (1.2), 47.3 (5.4), 18.1 (5.8), p<0.01 vs. baseline at all time points; after salmeterol MDI, 16 (6.4), 28.9 (5.2), 32.7 (3.9), 34.6 (4.4), 31.2 (4.8), p<0.05 at 60, 360, and 540 min; and after salmeterol Diskus, 16.4 (4.0), 16.9 (6.6), 27.8 (5.9), 28.6 (5.6), 33.8 (4.0), p<0.05 at 540 min. No significant adverse events or electrocardiographic changes were noted at any time. Impulse oscillometry is an acceptable method of assessing airway responses to bronchoactive drugs in this age group. Compared to albuterol and to its effect in older children and adults, the response to salmeterol Diskus appears to be somewhat blunted in this age group. The MasterScreen system is well suitedfor pharmacodynamic studies and clinical investigations in pre-school-aged children. PMID- 12375714 TI - Relation of airway reactivity and sensitivity with bronchial pathology in asthma. AB - Airway hyperresponsiveness in asthmatics, which may result from inflammation or remodeling, is expressed as the concentration of methacholine that causes a 20% fall in FEV1 in the concentration-response curve (PC20). A decrease in PC20 may be due to a steeper curve (hyperreactivity) and/or a curve shift to the left (hypersensitivity). Our purpose was to analyze the relation of airway sensitivity and reactivity to airway pathological changes. The PC6, as sensitivity parameter, and the slope between PC20 and PC40 as reactivity parameter, were calculated. Total and differential cell counts in the bronchoalveolar lavage fluid, and percentage of epithelial shedding, basement membrane thickness, and submucosal thickness on bronchial biopsy, were measured. The PC6 showed a correlation with the baseline FEV1%. The slope was significantly correlated with the basement membrane thickness, and also demonstrated a strong association with submucosal thickness. The PC20 showed a correlation with the baseline FEV1% and the degree of epithelial shedding. These results suggest that the airway sensitivity and reactivity measurements reflect the degree of airway caliber and remodeling, respectively. PMID- 12375716 TI - The "Maytag repairman": academic medicine and clinical research. PMID- 12375715 TI - Effect of suplatast tosilate on airway hyperresponsiveness and inflammation in asthma patients. AB - Because eosinophilic airway inflammation is a characteristic of bronchial asthma, the treatment of such inflammation is important in the management of this disease. Suplatast tosilate is a novel anti-asthma drug that suppresses eosinophil proliferation and infiltration through selective inhibition of Th2 cytokine synthesis. We investigated the effect of oral suplatast tosilate therapy in patients with mild and moderate asthma. Twenty-eight asthma patients were randomized into two groups with or without suplatast tosilate treatment (100 mg t.i.d. for 28 days). We examined the blood eosinophil counts, eosinophilic cationic protein level, sputum eosinophil count, exhaled nitric oxide level, and airway responsiveness before and after treatment. In patients treated with suplatast tosilate, the eosinophil count in the blood and sputum was significantly decreased after treatment, while there was no such change in the patients without suplatast treatment. The exhaled nitric oxide level and airway responsiveness (measured using an Astograph) were also decreased after treatment with suplatast tosilate, while there were no significant changes in patients without suplatast tosilate. These results strongly suggest that oral administration of suplatast tosilate suppresses airway hyperresponsiveness in asthma patients by reducing eosinophilic inflammation in the airways. PMID- 12375717 TI - The University of Calgary and University of Manitoba MD-PhD exchange initiative. PMID- 12375718 TI - The Institute of Circulatory and Respiratory Health. PMID- 12375719 TI - History of penicillin allergy and referral for skin testing: evaluation of a pediatric penicillin allergy testing program. AB - INTRODUCTION: Penicillin allergy, commonly reported in children, leads to use of more expensive, broad-spectrum drugs. The results and effectiveness of a skin testing program for immediate hypersensitivity to penicillin in children were studied. METHODS: Children seen at the IWK Health Centre in Halifax between 1986 and 2000 with a history of suspected penicillin allergy were referred by their family physician or pediatrician. Two-stage skin testing (scratch, intradermal) of benzylpenicilloyl-polylysin and penicillin G sodium, with histamine and saline as positive and negative controls, was carried out. If the test results were negative, an oral challenge was conducted and the child observed for 60 minutes. If no adverse reaction was noted, a letter was sent to the referring physician and to Health Records at the IWK Health Centre, indicating that warning labels should be removed from the chart. RESULTS: Of 72 children tested, 32% described their past cutaneous eruption as hives and 68% had other rashes; 96% of rashes were generalized. The mean age at the time of the suspected penicillin allergy was 4.4 years; it was 7.4 years at the time of testing. There was no positive response to the scratch testing, but 4% of children had a positive response to intradermal testing. No adverse responses to oral challenge were observed. Letters confirming negative status were not received in 4% (3 of 69) cases, resulting in ongoing avoidance of penicillins and falsely labelling of the child as penicillin allergic. CONCLUSIONS: In this referral setting, true penicillin allergy was uncommon, suggesting that many children are incorrectly labelled as penicillin-allergic. Communication of test results to family and care providers and health records administration must be effective if testing is to affect prescribing behaviour. PMID- 12375720 TI - Outpatient parenteral antibiotic therapy: evolution of the Calgary adult home parenteral therapy program. AB - In the past 25 years, outpatient antibiotic therapy has been recognized as a cost effective, safe and patient-accepted means of managing patients with chronic infections who require prolonged parenteral therapy but otherwise do not need admission to hospital. We describe the home parenteral therapy program in Calgary, which reflects the next generation of outpatient antibiotic therapy in Canada because of its unique inclusion of patients with acute infections. The Calgary home parenteral therapy program has evolved from a few, small, single site programs to a multisite, region-wide program that each year treats thousands of patients who require long- and short-term parenteral therapy. With escalating health care budgets and increased demand on acute-care hospital beds, existing programs in other centres may benefit from the Calgary experience, and this home parenteral therapy model may serve as a template for developing new outpatient antibiotic therapy programs in other regions. PMID- 12375721 TI - Even the "Maytag repairman" must know how to repair appliances. PMID- 12375722 TI - A cognitive-behavioral/psychophysiological model of tic disorders. AB - This article discusses current cognitive behavioral, as well as neurophysiological, accounts of the development and maintenance of tic behavior in chronic (simple or complex) tic disorders. A cognitive psychophysiological model is further elaborated, highlighting the reciprocal interplay of background cognitive and physiological factors preceding tic onset. According to the model, cognitive factors such as perfectionist concerns and heightened sensory awareness and self-attention, as well as physiological factors such as a high level of motor activation and accompanying elevated muscle tension, play a role in tic habits. Negative appraisals of tics and counter-productive coping strategies developed by clients as a means to suppress or to disguise the tic behavior may also locally reinforce tic onset. Neurochemical factors are viewed largely as concomitants of behavioral adaptations or compensations to the tic problem rather than as independent markers or precursors of tic onset. Clinically, the model emphasizes the role of cognitive-behavioral factors in tic onset, and suggests that tic management is best accomplished through cognitive behavioral interventions designed to prevent build up of both tension and pre-monitory urge in tic-affected muscles, rather than reverse the tic at the onset of the premonitory urge. The clinical validity of parts of the model is supported by recent experimental, psychometric and clinical studies. Other parts of the model remain speculative but at least yield testable predictions. A strength of the model is its ability to account for findings over diverse psychological and biological domains. PMID- 12375723 TI - A comparison of flashbacks and ordinary autobiographical memories of trauma: cognitive resources and behavioural observations. AB - We investigated predictions derived from the dual representation theory of posttraumatic stress disorder, which proposes that flashbacks and ordinary memories of traumas are supported by different types of representation. Sixty-two participants, meeting DSM-IV diagnostic criteria for posttraumatic stress disorder, completed a detailed written trauma narrative, and afterwards identified those sections in the narrative that had been written in flashback and ordinary memory periods. Performance on cognitive tasks confirmed predictions that flashback periods would be associated with a specific decrement in visuospatial processing. Contrary to prediction, periods of both flashback and ordinary memory were associated with decrements on a verbal processing task. Independent observer ratings also confirmed that flashback periods were associated with increases in a wide range of autonomic and motor behaviours. PMID- 12375724 TI - Psychological characteristics of aggressive drivers with and without intermittent explosive disorder. AB - We compared two groups of aggressive drivers, those who met criteria for Intermittent Explosive Disorder (IED) (n=10) and comparable aggressive drivers who did not meet IED criteria (n=20), to a group of non-aggressive driving controls (n=20) on measures of psychological distress, anger, hostility, and Type A behavior as well as measures of aggressive driving and driving anger and their driving records. There were few differences between the aggressive drivers with IED and those without IED. The IED positive aggressive drivers endorsed more assaultiveness and resentment as well as more impatience and showed trends to have more hostility and angry temperament. When all aggressive drivers were compared to controls, differences emerged on anxiety, hostility, and anger as well as on measure specific to aggressive driving (competitiveness) and driving anger (at slow drivers and traffic obstructions). PMID- 12375725 TI - Thought-shape fusion in anorexia nervosa: an experimental investigation. AB - Cognitive biases and cognitive distortions have been implicated as important factors in the development and maintenance of many disorders. The concept of thought-shape fusion (TSF) in eating disorders was developed by Shafran, Teachman, Kerry, and Rachman (British Journal of Clinical Psychology 38 (1999) 167) as a variant of thought-action fusion, described by Shafran, Thordarson and Rachman (Journal of Anxiety Disorders 10 (1996) 379). TSF occurs when thinking about eating certain types of food increases a person's estimate of their shape and/or weight, elicits a perception of moral wrongdoing, and/or makes the person feel fat. Shafran et al. (1999) examined both the psychometric and experimental properties of TSF in an undergraduate sample. This paper reports an extension of this work to a clinical group (N=20) of patients with anorexia nervosa. After completing a set of relevant questionnaires, participants were asked to think about a food which they considered extremely fattening. They were then asked to write out the sentence, "I am eating--.", inserting the name of the fattening food in the blank. After being asked to rate their anxiety, guilt, feelings about their weight, morality, etc., participants were given the opportunity to neutralize their statement in any way they chose. The majority of the participants neutralized in ways consistent with the findings of Shafran et al. (1999). The results are discussed in terms of cognitive-behavioural formulations of eating disorders, and of the influence of cognitive biases and cognitive distortions on the processing of information relevant to food, weight and shape in anorexia nervosa. PMID- 12375727 TI - Low fear in childhood is associated with sporting prowess in adolescence and young adulthood. AB - This study sought to establish if low levels of childhood fear were associated with high level sports performance in adolescence and young adulthood. Parent and teacher reports of fearfulness at ages 5, 7, 9 and 11 and self-reports of sporting achievements at age 26 were obtained for members of the longitudinal Dunedin Multidisciplinary Health and Development Study. Findings indicated a dose response relation between levels of childhood fear and later sports achievement such that low levels of fear were associated with the greatest likelihood of playing representative sport. Low levels of fear early in life may be associated with elite sports performance in adulthood. PMID- 12375726 TI - Rumination and social problem-solving in depression. AB - We tested the hypothesis that impaired social problem solving in depression is a consequence of state-oriented rumination, which can be ameliorated by improving awareness of mental processes. 32 currently depressed, 26 recovered depressed, and 26 never depressed participants completed the Means Ends Problem Solving Test while randomly allocated to no questions, state-oriented ruminative questions, (e.g. focusing on why you have a problem) or process-focused questions (e.g. focusing on how you decide to solve a problem). In the no question condition, the currently depressed group was significantly impaired at problem solving compared to the never depressed and recovered depressed groups, which did not differ from each other. As predicted, the process-focused questions significantly improved social problem solving in depressed patients, compared to no questions and state oriented questions, which did not differ from each other. As predicted, compared to the process-focused questions, the state-oriented questions significantly impaired social problem solving in the recovered depressed group. These results are consistent with recent theories and treatment developments which suggest that increased awareness of mental processes can shift people away from ruminative thinking, thereby, reducing depressive relapse. PMID- 12375728 TI - The relationship of thought-action fusion to pathologicial worry and generalized anxiety disorder. AB - Meta-cognitive beliefs associated with pathological worry and generalized anxiety disorder (GAD) may encompass the likelihood subtype of thought-action fusion (TAF), the belief that one's thoughts can influence outside events. In the current study of 494 undergraduate college students, positive correlations between scores on the Penn State Worry Questionnaire (PSWQ) and the two Likelihood subscales of the TAF Scale were found, and participants endorsing at least some DSM-IV diagnostic criteria for GAD scored significantly higher on both TAF-Likelihood subscales than participants reporting no GAD symptoms. However, these TAF scales did not predict GAD diagnostic status with PSWQ included as a predictor. In contrast to previous research, the TAF-Moral scale did not correlate with worry. Relationships between TAF, pathological worry, and meta cognition are discussed in relation to GAD. PMID- 12375729 TI - Obsessive-compulsive disorder and the five-factor model of personality: distinction and overlap with major depressive disorder. AB - Research on individual differences in obsessive-compulsive disorder (OCD) has focused largely on analogue models with participants experiencing sub-clinical obsessions and/or compulsions. Few studies have examined the association between normal, dimensional personality traits and obsessive-compulsive symptomatology in a clinical sample. The purpose of this study was to examine personality differences in patients with a primary diagnosis of OCD (n = 98) or major depression (n = 98) using the domains and facets of the five-factor model of personality (FFM). Patients completed the self-report version of the Revised NEO Personality Inventory (NEO PI-R). When contrasted with community controls (Revised NEO Personality Inventory (NEO-PI-R) and NEO Five-Factor Inventory (NEO FFI) professional manual, Psychological Assessment Resources, Odessa, FL, 1992), participants with OCD were found to differ across the domains (and facets) of neuroticism, extraversion, and conscientiousness and the facets of openness and agreeableness. Further, when compared to depressed participants, those with OCD were found to be more extraverted, agreeable, conscientious and less neurotic. With the exception of the conscientiousness domain (and facets), these significant differences were maintained even after controlling for depression severity. These results highlight the unique associations between trait domains and facets of the FFM and OCD. PMID- 12375730 TI - Perceived health following myocardial infarction: cross-validation of the Health Complaints Scale in Danish patients. AB - With an ageing population and a decline in cardiac mortality rates, the number of patients with cardiac disease is increasing, which in turn poses a major challenge for secondary prevention. For this end, appropriate, sensitive, and validated instruments to assess health complaints and quality of life are required. The objectives of the current study were: (1) to cross-validate the Health Complaints Scale (HCS) in a Danish sample of patients with a first myocardial infarction (MI); and (2) to investigate whether perceived health, as measured by the HCS is related to cardiac disease severity. The HCS was originally developed in Belgian patients with coronary artery disease. One hundred-and-twelve consecutive patients with a first myocardial infarction were assessed by means of a questionnaire four to six weeks post infarction. Clinical measures were sampled from medical records. The factor structure of the HCS and the internal consistency of the Somatic Complaints (alpha = 0.91) and Cognitive Complaints subscales (alpha = 0.94) were confirmed. The construct validity of the scale was confirmed against measures of psychopathology and personality. Patients scored significantly higher on the HCS Somatic and Cognitive scales as compared with self-reports of depression and anxiety (p < 0.0001). Health complaints were unrelated to severity of cardiac disease and rather reflected subjective perception of quality of life. These findings show that the HCS is a valid instrument that is equally applicable in Danish cardiac patients to monitor perceived health as a major component of quality of life. PMID- 12375731 TI - Assessment of dysfunctional beliefs in borderline personality disorder. AB - This study had two aims: to test the hypothesis that borderline personality disorder (BPD) patients hold numerous dysfunctional beliefs associated with a variety of Axis II disorders, and to construct a BPD belief scale which captures these beliefs. Beliefs were measured using the Personality Belief Questionnaire (PBQ) which is designed to assess beliefs associated with various personality disorders, although not specifically BPD. Eighty-four BPD patients and 204 patients with other personality disorders (OPD) were randomly split into two study samples. Fourteen PBQ items were found to discriminate BPD from OPD patients in both samples. These items came from the PBQ Dependent, Paranoid, Avoidant, and Histrionic scales and reflect themes of dependency, helplessness, distrust, fears of rejection/abandonment/losing emotional control, and extreme attention-seeking behavior. A BPD beliefs scale constructed from these items showed good internal consistency and diagnostic validity among the 288 study patients. The scale may be used to assist in diagnosis and cognitive therapy of BPD. PMID- 12375732 TI - Human neutrophils express messenger RNA of vitamin D receptor and respond to 1alpha,25-dihydroxyvitamin D3. AB - 1Alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to modulate the production of various cytokines or the expression of certain differentiation markers in human T cells or monocytes. Its effects on neutrophils, however, are poorly understood. In this paper, we show several lines of evidence indicating that neutrophils express functional vitamin D receptors (VDR). Sort-purified neutrophils from human peripheral blood expressed VDR mRNA at a level comparable to that of monocytes. As reported to occur in monocytes, protein expression of CD14 on the cell surface of neutrophils was augmented when the cells were incubated with 1,25(OH)2D3. To investigate the physiological roles for VDR in neutrophils, we investigated possible modulating effects of 1,25(OH)2D3 on the expression of several genes in lipopolysaccharide-stimulated neutrophils by using differential display analysis. Of the genes we identified, trappin 2/elafin/SKALP, which was originally reported to be an inhibitor of elastase, was induced in neutrophils by lipopolysaccharide, but was suppressed significantly in the presence of 1,25(OH)2D3. Under the same conditions, interleukin-1beta expression was also inhibited. These findings suggest that 1,25(OH)2D3 has a potential to affect the inflammatory process by modulating the expression of neutrophil genes. PMID- 12375733 TI - Treatment of experimental nephrosis by culture filtrate of Cryptococcus neoformans var. gattii (CneF). AB - The therapeutic effect of the culture filtrate of cryptococcus neoformans var. gattii (CneF) was tested in Adriamycin-induced nephropathy. The CneF was administered at different doses (36, 54 and 90 mg/kg based on carbohydrate concentration), one i.p. injection every 72 hours for a total of 10 injections. The treated patient rats showed a significant reduction in proteinuria, plasma cholesterol concentration, BUN and significant increase in urine creatinine levels. Moreover, treatment with CneF significantly reduced number of glomerular leukocytes and decreased the tubular casts. These data suggest that CneF therapy can ameliorate proteinuria, hypercholesterolemia and suppress the progression of glomerular lesions in experimental model of nephrosis. PMID- 12375734 TI - Detection of DNA adducts in developing CD4+ CD8+ thymocytes and splenocytes following in utero exposure to benzo[a]pyrene. AB - Environmental carcinogen exposure may play an important role in the incidence of cancer in children. In addition to environmental pollutants, maternal smoking during pregnancy may be a contributing factor. Major carcinogenic components of cigarette smoke and other combustion by-products in the environment include polycyclic aromatic hydrocarbons (PAH). Mouse offspring exposed during midpregnancy to the PAH, benzo[a]pyrene (B[a]P), show significant deficiencies in their immune functions, observed in late gestation which persist for at least 18 months. Tumor incidences in these progeny are 8 to 10-fold higher than in controls. We have demonstrated a significant reduction in thymocytes (CD4+ CD8+, CD4+ CD8+ Vbeta8+, CD4+ CD8+ Vgamma2+) from newborn and splenocytes (CD4+ CD8+) from 1-week-old mouse progeny exposed to B[a]P in utero. To investigate possible causes of the observed T cell reduction, we analyzed the thymocytes and splenocytes from progeny and maternal tissues for the presence of B[a]P-DNA adducts. Adducts were detected in maternal, placental and offspring lymphoid tissues at day 19 of gestation, at birth and 1-wk after birth. The presence of B[a]P-DNA adducts in immature T cells may, in part, explain the previously observed T cell immunosuppression and tumor susceptibility in mice exposed to B[a]P in utero. The effects of DNA lesions on progeny T cells may include interference with normal T-cell development. These results provide a possible explanation for the relationship between maternal smoking during pregnancy and childhood carcinogenesis. PMID- 12375735 TI - Expression of heat shock proteins in heavy metal-provoked inflamed human skin. AB - The expression of heat shock protein (hsp) 65, 72 and 90 was studied in human inflamed skin after heavy metal salt treatment, by using epicutaneous patch testing procedure and immunohistochemistry. There was a slight immunoreactivity to hsp 65 and hsp 72, but not to hsp 90 in keratinocytes in normal skin. An increased immunoreactivity to hsp 72 was obtained in apically located keratinocytes in a statistically significant (p<0.01) increased number of reactions to cobalt chloride and gold chloride compared to control skin. These metal salts also induced the highest degree of expression of keratinocyte intercellular adhesion molecule (ICAM)-1. There were epidermal dendritic cells as well as macrophage-like cells in the dermis, which expressed hsp 65 and hsp 90, in biopsies from metal salt-treated and control skin, while expression of hsp 72 in macrophage-like cells was found only in the dermis. PMID- 12375736 TI - IL-1beta regulates cellular proliferation, prostaglandin E2 synthesis, plasminogen activator activity, osteocalcin production, and bone resorptive activity of the mouse calvarial bone cells. AB - Interleukin-1beta (IL-1beta) regulates several activities of the osteoblast cells derived from mouse calvarial bone explants in vitro. IL-1beta stimulated cellular proliferation and the synthesis of prostaglandin E2 in the cultured cells in a dose-dependent manner. Furthermore, plasminogen activator activity of the mouse osteoblast was positively affected by IL-1beta in a dose-dependent manner over the dosage range of 0.01 ng-2 ng/mL with a maximal effect being observed at 2 ng/mL. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to vitamin D, two characteristics of the osteoblast phenotype, were significantly antagonized by IL-1beta over a similar dose range. Treatment of mouse calvarial bone cells with IL-1beta resulted in a dose dependent stimulation of bone resorption and the bone resorption induced by IL 1beta was strongly inhibited by calcitonin treatment, indicating osteoclast mediated bone resorption, suggesting that the bone resorption induced by IL-1beta appears to be osteoclast-mediated. This study supports the role of IL-1beta in the pathological modulation of bone cell metabolism, with regard to implication of the pathogenesis of osteoporosis by IL-1beta. PMID- 12375737 TI - Effect of murine recombinant IL-2 on the course of lupus-like disease in (NZBxNZW) F1 female mice. AB - The exacerbation of pre-existing autoimmune diseases is a potential toxic effect of immunoactive drugs. An increase in the incidence of autoimmune thyroiditis has been noted in patients treated with human recombinant interleukin-2 (rIL-2). In contrast, human rIL-2 tends to protect mice from autoimmunity. As the effects of murine rIL-2 on autoimmunity have not been reported in mice, lupus-prone female (NZBxNZW) F1 mice were treated with 20,000 IU murine rIL-2 intraperitoneally, twice weekly for 13 weeks, beginning at 15 weeks of age. No evidence of an exacerbating effect of murine IL-2 on the lupus disease of (NZBxNZW) F1 mice was observed as no change in the following parameters were seen, namely mean survival time, mean body weight, anti-DNA and antinuclear antibody production. These results show that: 1) like human rIL-2, murine rIL-2 does not exacerbate autoimmunity in mice; 2) the biological effects of human as well as murine rIL-2 in mice differ from those seen with human rIL-2 in man. These latter findings suggest that the selection of the relevant animal species for immunotoxicity studies with recombinant cytokines and derivatives may be less straightforward than previously thought. PMID- 12375738 TI - Modulation of antigen-specific immune responses by the oral administration of a traditional medicine, bo-yang-hwan-o-tang. AB - Bo-yang-hwan-o-tang (BHT) has long been used to treat cancer in traditional Korean medicine and is believed to have immune-modulating activity. This study investigated the effect of BHT on the induction of antigen-specific immune responses using hen egg-white lysozyme (HEL) as a model antigen system. Oral administration of BHT enhanced both HEL-specific humoral and lymphocyte proliferative responses in HEL low-responder mice. Feeding BHT to the mice increased INF-gamma levels, but did not change IL-4 levels. Interestingly, however, the oral BHT feeding significantly increased HEL-specific antibodies of the IgG1, IgG2b, and IgG3 subtypes, which are associated with the direct stimulation of B cells. This indicates that BHT treatment enhances anti-HEL specific humoral immune responses via the direct stimulation of B lymphocytes rather than by selective priming of specific subtypes of the helper T-cell population. This conclusion was supported by in vitro experiments, in which the presence of BHT significantly augmented B-cell mitogen-mediated proliferation of mouse splenocytes. This augmentation was closely associated with a glycoprotein with a molecular weight of around 100 kDa. The results suggest that BHT modulates antigen-specific immune responses, and might be used as a therapeutic agent for patients who need enhanced immune function. PMID- 12375739 TI - Inhibitory effect of Artemisia capillaris on ethanol-induced cytokines (TNF alpha, IL-1alpha) secretion in Hep G2 cells. AB - A human hepatoma cell line, Hep G2 cell, is reliable for the study of alcohol induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Artemisia capillaris Thunb (Compositae) plant (AC) on ethanol (EtOH) induced cytotoxicity in Hep G2 cells. AC (0.5-5 microg/mL) inhibited the secretion of EtOH-induced interluekin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). AC also inhibited the EtOH-, IL-1alpha-, and TNF-alpha induced cytotoxicity. Furthermore, we found that AC inhibited the EtOH-induced apoptosis of Hep G2 cells. These results suggest that AC may prevent the EtOH induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells. PMID- 12375740 TI - Anti-allergic effects of Sanguisorba officinalis on animal models of allergic reactions. AB - We investigated the effect of aqueous extract of Sanguisorba officinalis L.(Rosaceae) root (SOAE) on the immediate-type allergic reactions in vivo and in vitro. SOAE (0.01 to 1 g/kg) inhibited systemic allergic reaction induced by compound 48/80. When SOAE was employed in a systemic allergic reaction test, the plasma histamine levels were reduced in a dose-dependent manner. SOAE (0.001 to 1 g/kg) dose-dependently inhibited passive cutaneous anaphylaxis (PCA) activated by anti-dinitrophenyl (DNP) IgE. SOAE (0.001 to 1 mg/mL) also dose-dependently inhibited the histamine release from rat peritoneal mast cells (RPMC) activated by compound 48/80 or anti-DNP IgE. The level of cyclic AMP (cAMP) in RPMC, When SOAE was added, significantly increased compared with that of normal control. Moreover, SOAE (0.01 to 1 mg/mL) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha (TNF-alpha) production. These results suggest that SOAE may be beneficial in the regulation of immediate-type allergic reaction. PMID- 12375741 TI - Immunostimulating effects of acidic polysaccharides extract of Panax ginseng on macrophage function. AB - The root of Panax ginseng C. A. Meyer is one of the most popular natural tonics in oriental countries. In this study, we have isolated polysaccharide fraction of Panax ginseng (ginsan) and examined its effect on the function of murine peritoneal macrophages. When macrophages were treated with ginsan, cytotoxic activity against B16 melanoma cells was significantly induced. In addition, the levels of cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6 and Interferon-gamma (IFN-gamma) were increased and the production of reactive oxygen/nitrogen components such as nitric oxide (NO) and hydrogen peroxide (H2O2) was enhanced. Moreover, phagocytic activity was induced in ginsan-treated macrophages compared to the control. The expression of CD14 and 1-Ab on murine peritoneal macrophages was increased by the treatment with ginsan, while the expression of CD11b was decreased. Taken together, these results suggest that ginsan has an immunopotentiating effects on macrophages and these abilities could be used clinically for the treatment of diseases such as cancer. PMID- 12375742 TI - Effects of Chlorella vulgaris extract on cytokines production in Listeria monocytogenes infected mice. AB - In this study, we have investigated the effects of the unicellular-green-algae Chlorella vulgaris on the production of INF-gamma, IL-2, IL-4 and IL-10 in normal and Listeria monocytogenes infected mice. Our results demonstrated that in normal/non infected mice, CVE administration produced no effects in the levels of all cytokines studied. However, Listeria monocytogenes infection enhanced the production of INF-gamma and IL-2 at 48 and 72 h after the bacteria inoculation. Interestingly, the treatment with five consecutive doses of 50 mg/Kg/day of Chlorella vulgaris given previously to infection, led to further increases in INF gamma and IL-2 levels at 48 and 72 h in relation to the presence of infection alone. No changes in IL-4 and IL-10 production were observed in Listeria monocytogenes and CVE treated/infected mice. These results are in accordance with the literature, which shows that CVE is a biological response modifier that enhances resistance to Listeria monocytogenes through augmentation of IL-2 and IFN-gamma. PMID- 12375743 TI - Cytokines production is altered in mice exposed to airborne suspended matter. AB - The production of IL-2 and IL-4 by thymocytes, spleen and axillary lymph node lymphocytes from female and male mice exposed to airborne suspended matter (ASM) was the scope of our investigations. Cytokines production by activated lymphocytes was determined by the estimation of the percentage of cells positive for intracellular cytokines and by the concentration of both cytokines secreted into the culture medium. Two models of mice exposure to ASM were used: 1/ intraperitoneal injection (acute exposure), and 2/ oral exposure (subacute model). ASM exposure affected both IL-2 and IL-4 production and IL-2R alpha expression on activated lymphoid cells isolated from different lymphoid organs of both female and male mice. The effect was dependent on the route and duration of exposure, ASM dose and the age and sex of mice. A wide panel of changes is discussed. The prolonged exposure to ASM resulted in overproduction of IL-2 in both female and male mice and in overproduction of IL-4 in male mice. Acute exposure to ASM strongly affected IL-2 and IL-4 production, and the effect varied among lymphocytes from different lymphoid organs. Intracellular cytokines expression and the level of secreted cytokines seem to be good tools for the assessment of toxic effects of environmental pollution on the function of the immune system. PMID- 12375744 TI - Dental care considerations for young children. AB - Although the majority of America's children enjoy remarkably good oral health, a significant subset of low-income, minority, medically and developmentally compromised, and socially vulnerable children continue to suffer significant and consequential dental and oral disease. Most of this inequitably distributed disease burden is preventable through early and individualized preventive care. Yet the primary-care medical and dental workforce is ill-prepared to manage the oral health needs of young children. Demographic trends suggest that the problem of disparities in both oral health status and access to competent dental services will continue to worsen for young children. Impediments to improving the oral health of young children include barriers between medical and dental systems of care, paucity of private and safety-net facilities and providers in many areas where vulnerable children reside, and dysfunctional Medicaid insurance programs. Barriers are generated by parents, providers, payers, and systems of care as well as by the age-appropriate behaviors of young children. Vulnerable families often do not access the case management services and disease control information needed to effectively address their young children's needs. Approaches to improving the oral health of young children therefore include enhancing public education about oral health, the appropriateness of early and periodic dental care, and primary prevention. Improvements in workforce numbers, distribution, diversity, and competency are needed. Attention to delivery systems and public insurance capacities are also necessary to effectuate improvements. HRSA's Title VII and VIII health professions training programs could potentially address may of these barriers and shortcomings. Training enhancements for predoctoral, postdoctoral, and graduate dentists and hygienists as well as for primary-care medical providers hold the key to marked improvements in the oral health of young children. Enhanced training of health care providers is the necessary if not sufficient condition to children whose daily life experiences are compromised by dental and oral diseases that are overwhelmingly preventable. PMID- 12375745 TI - Dental care considerations for disabled adults. PMID- 12375746 TI - Dental considerations for the frail elderly. PMID- 12375747 TI - Executive summary: dental care considerations for vulnerable populations. PMID- 12375748 TI - The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable. AB - BACKGROUND: Human polymerized hemoglobin (PolyHeme, Northfield Laboratories, Evanston, IL) is a universally compatible, immediately available, disease-free, oxygen-carrying resuscitative fluid being developed as a red cell substitute for use in urgent blood loss. PolyHeme should be particularly useful when red cells may be temporarily unavailable. This article assesses survival at life threatening RBC hemoglobin concentration ([Hb]) in massively bleeding patients who do not receive red cells. STUDY DESIGN: There were 171 patients who received rapid infusion of 1 to 20 units (1,000 g, 10 L) of PolyHeme in lieu of red cells as initial oxygen-carrying replacement in trauma and urgent surgery. The protocol simulated the unavailability of red cells, and the progressive fall in RBC [Hb] in bleeding patients was quantified. Thirty-day mortality was compared with a historical control group of 300 surgical patients who refused red cells on religious grounds. RESULTS: A total of 171 patients received rapid infusion of 1 to 2 units (n = 45), 3 to 4 units (n = 45), 5 to 9 units (n = 47), or 10 to 20 units (n = 34) of PolyHeme. Forty patients had a nadir RBC [Hb] < or = 3 g/dL (mean, 1.5 +/- 0.7 g/dL). But total [Hb] was adequately maintained (mean, 6.8 +/- 1.2 g/dL) because of plasma [Hb] added by PolyHeme. The 30-day mortality was 25.0% (10/40 patients) compared with 64.5% (20/31 patients) in historical control patients at these RBC [Hb] levels. CONCLUSIONS: PolyHeme increases survival at life-threatening RBC [Hb] by maintaining total [Hb] in the absence of red cell transfusion. PolyHeme should be useful in the early treatment of urgent blood loss and resolve the dilemma of unavailability of red cells. PMID- 12375749 TI - Risk factors for postthyroidectomy hypocalcemia. AB - BACKGROUND: Hypocalcemia is a common complication of thyroidectomy. The aim of this study was to evaluate the incidence of hypocalcemia after thyroid operation and its relation to clinical, biologic, and surgical factors. STUDY DESIGN: A retrospective study of 265 patients who underwent unilateral (n = 50) or bilateral (n = 215) thyroidectomy between 1996 and 2000 was done to determine incidence and risk factors for hypocalcemia. Free thyroxine and thyrotropin levels were obtained before operation in 254 patients, together with preoperative and postoperative calcium and phosphorus levels. All patients were examined for age, gender, extent of thyroidectomy, initial versus reoperative neck operation, pathologic characteristics of resected thyroid tissue, substernal thyroid extension, and parathyroid resection and autotransplantation. RESULTS: Hypocalcemia, defined as a calcium level less than 2 mmol/L, occurred in 42 of 265 patients (16%), including 11 (4%) symptomatic patients who required vitamin D, calcium, or both for 2 to 6 weeks. Factors significantly predictive of postoperative hypocalcemia in univariate analysis included elevated free thyroxine level (p = 0.0064), bilateral thyroidectomy (p = 0.00064), parathyroid autotransplantation (p = 0.0128), and female gender (p = 0.0028). Independent risk factors on multivariate analysis were elevated free thyroxine level (p = 0.0476), bilateral thyroidectomy (p = 0.0338), and parathyroid autotransplantation (p = 0.0003). CONCLUSIONS: Bilateral thyroidectomy, elevated free thyroxine level, and parathyroid autotransplantation are independent risk factors for postthyroidectomy hypocalcemia. Oral calcium supplements may be of value in this group of patients to enhance early hospital discharge. PMID- 12375750 TI - Three- to six-year followup for 379 benign image-guided large-core needle biopsies of nonpalpable breast abnormalities. AB - BACKGROUND: Determining the negative predictive value of benign large-core needle biopsy of nonpalpable mammographically detected breast abnormalities has been difficult because benign results generally preclude surgical excision. Longterm followup of these patients is important to ensure timely diagnosis of new abnormalities and to identify false negatives. STUDY DESIGN: This cohort study comprised 379 patients, all with benign diagnoses following imaging-guided large core needle biopsy of nonpalpable mammographically detected abnormalities. Mammographic, clinical, and laboratory records (when appropriate) were reviewed for all patients followed at our institution. For patients followed elsewhere, these data were provided by each patient's current primary-care physician after obtaining written informed consent from the patient. RESULTS: We obtained followup for 312 patients (82.3% of 379), for whom the mean followup period was 55 months; 67 patients were either lost to followup (44, 11.6%), had no followup by patient choice (18, 4.7%), or died of causes other than breast cancer (5, 1.3%). Of these 312 patients, we found only 1 (0.3%) false negative in which a 4 mm lesion was observed to have grown to approximately 11 mm eight months later, and was found to be an infiltrating ductal cancer at rebiopsy. The negative predictive value was calculated as 0.997 (311/312). Analysis of core histologies indicated the followup group was a representative sample. CONCLUSIONS: These data suggest that benign mammographically detected abnormalities can be diagnosed with a high level of confidence using image-guided large-core needle biopsy, and that mammographic or ultrasonographic screening or both at 6 and 12 months might be sufficient before returning the patient to routine screening mammography. PMID- 12375751 TI - Concordance and validation study of sentinel lymph node biopsy for breast cancer using subareolar injection of blue dye and technetium 99m sulfur colloid. AB - BACKGROUND: We have previously demonstrated the utility, accuracy, and advantages of a subareolar (SA) site of injection for blue dye compared with an intraparenchymal site. In later studies we advocated the additional use of preoperative SA-injected technetium 99m-labeled sulfur colloid as a directional aid in finding blue-stained sentinel lymph nodes (SLNs). Paramount to the usefulness of this dual-tracer, same-site technique is the degree to which SA injected blue dye and SA-injected radiocolloid migrate concordantly and are deposited within the same sentinel nodes. The purpose of this study was to document the correlation and accuracy of SLN biopsy using blue dye and radiocolloid when both nodal markers are injected by the same SA route. STUDY DESIGN: Between September 1999 and February 2002 (29 months), 185 consecutive patients with 187 operable breast cancers underwent 187 attempted SLN biopsies by a dual-tracer, same-site injection technique using the SA approach for both agents. Unfiltered technetium 99m-labeled sulfur colloid (1 mCi [37 MBq]) was SA injected 30 to 45 minutes preoperatively; and just after anesthetic induction, 3 mL of 1% isosulfan blue dye was injected by the same SA route. SLN biopsies or complete axillary dissections were carried out, and SLNs identified during these procedures were classified as containing both blue dye and radioactivity ("blue hot" nodes), radioactivity alone ("hot-only" nodes), or blue dye alone ("blue only" nodes). Cases were categorized and tabulated based on the presence or absence of these three types of SLNs. RESULTS: Of the 187 procedures, a SLN was identified successfully in 184 cases, indicating an SLN identification rate of 98.4% (95% confidence interval, 96.6% to 100.2%). In these 184 cases, a blue-hot node was present in 94.5% (n = 174 of 184). An SLN was positive in 50 cases, or 27.2% of the total group (n = 50 of 184). A blue-hot node was the only positive SLN in 43 of these 50 cases, or 86% of the node-positive cases. There were no false negatives in 20 confirmatory axillary node dissections carried out to document the findings of a negative SLN. A correlation analysis revealed that in 98.9% of cases (174 of 176), blue nodes were also radioactive ("blue-hot" case concordance = 98.9%). In 95.1% of cases (174 of 183), hot nodes had also taken up blue dye ("hot-blue" case concordance = 95.1%). CONCLUSIONS: Using SA injections of both blue dye and radiocolloid, we achieved an SLN identification rate of 98.4% (184 of 187 cases), a false-negative rate of 0% (0 of 20 cases), and an accuracy in predicting the malignant status of the axilla of 100% (70 of 70 cases). The case concordance rate ranged between 98.9% ("blue-hot concordance") and 95.1% ("hot-blue concordance"). The present study is the first to evaluate dual-tracer, same-site SA injections of blue dye and radiocolloid. By demonstrating a high case concordance rate, a high SLN identification rate, and a 0% false-negative rate, this study adds further support to the validity and accuracy of same-site SA injections of both blue dye and radiocolloid during SLN biopsy in breast cancer. PMID- 12375752 TI - Effect of prolonged pneumoperitoneum on intraoperative urine output during laparoscopic gastric bypass. AB - BACKGROUND: Intraoperative oliguria is common during laparoscopic operations. The objective of this study was to evaluate the effects of prolonged pneumoperitoneum during laparoscopic gastric bypass (GBP) on intraoperative urine output and renal function. METHODS: 104 patients with a body mass index between 40 and 60 kg/m2 were randomly assigned to laparoscopic (n = 54) or open (n = 50) GBP. Intraoperative urine output was recorded at 30-min intervals. Blood urea nitrogen and creatinine levels were measured at baseline and on postoperative days 1, 2, and 3. Levels of antidiuretic hormone, aldosterone, and plasma renin activity were also measured in a subset of laparoscopic (n = 22) and open (n = 24) GBP patients at baseline, 2 hours after surgical incision, and in the recovery room. RESULTS: The laparoscopic and open groups were similar in age, gender, and body mass index. There was no significant difference in amount of intraoperative fluid administered between groups (5.4 +/- 1.6 L, laparoscopic versus 5.8 +/- 1.7 L, open), but operative time was longer in the laparoscopic group (232 min versus 200 min, p < 0.01). Urinary output during laparoscopic GBP was 64% lower than during open GBP at 1 hour after surgical incision (19 mL versus 55 mL, p < 0.01) and continued to remain lower than that of the open group by 31-50% throughout the operation. Postoperative blood urea nitrogen and creatinine levels remained within the normal range in both groups. Serum levels of antidiuretic hormone, aldosterone, and plasma renin activity peaked at 2 hours after surgical incision with no significant difference between the two groups. CONCLUSION: Prolonged pneumoperitoneum during laparoscopic gastric bypass significantly reduced intraoperative urine output but did not adversely alter postoperative renal function. PMID- 12375753 TI - Incidence and management of bile leakage after hepatic resection for malignant hepatic tumors. AB - BACKGROUND: Bile leakage is one of the frequent and disturbing complications of hepatic resection. STUDY DESIGN: Clinical records of the 363 patients who underwent hepatic resections without biliary reconstruction for hepatic cancers between January 1994 and June 2001 were reviewed. Postoperative bile leakage was defined as continuous drainage with a bilirubin concentration of 20 mg/dL or 1,500 mg/d lasting 2 days. Leakage that continued longer than 2 weeks or that required surgical intervention was defined as uncontrollable. Differences in incidence and frequency of uncontrollable leakage for the different types of hepatic resection, tumors, and underlying liver disease were investigated. Outcomes after treatment for uncontrollable bile leakage were also reviewed. RESULTS: Postoperative bile leakage occurred in 26 of 363 patients (7.2%). Although the incidence in patients with cholangiocellular carcinoma (3/9 [33%]) was higher (p = 0.03) than in patients with hepatocellular carcinoma, rates of occurrence were similar among the different types of hepatic resection and underlying liver disease. Eight of the 26 patients (31%) had uncontrollable leakage. Two patients required reoperation to control leakage; one of these developed hepatic failure and died 2 months after surgery. Four patients underwent endoscopic nasobiliary drainage 21 to 34 days after hepatectomy, and the leakage resolved within 3 to 21 days. Fibrin glue sealing was effective in two patients whose leaking bile ducts were not connected to the common bile duct. CONCLUSIONS: Although meticulous surgical technique can minimize the risk of postoperative bile leakage, some instances of leakage are unavoidable. Nonsurgical treatments, such as nasobiliary drainage or fibrin glue sealing, are preferable to reoperation. PMID- 12375754 TI - Prevention of pancreatic fistula with a new synthetic, absorbable sealant: evaluation in a dog model. AB - BACKGROUND: Pancreatic fistula complicates up to 15% to 25% of pancreatic resections, especially with soft, normal pancreas, and is most common after distal pancreatectomy. A new synthetic, absorbable hydrogel sealant has recently been developed and tested for sealing of human aorta, bronchi, and dura; it is FDA approved as a lung sealant in humans. Our objective was to test the efficacy of the sealant in preventing pancreatic leaks in a dog model of distal pancreatectomy. STUDY DESIGN: Ten dogs underwent bilateral distal pancreatectomy under general anesthesia. Animals were randomized to receive application of the sealant to the pancreatic stumps (n = 5) or no treatment (n = 5). The transected pancreatic duct was not ligated, and the end of the pancreas was neither oversewn nor stapled; closed-suction drains were placed in proximity to the pancreatic stumps before abdominal closure. All animals received normal chow starting on the second postoperative day. Drainage was collected for volume and amylase determination twice daily for 14 days, after which the animals were sacrificed. Pancreatic tissue was collected from the area of transection and was formalin fixed for histopathology. RESULTS: There was no perioperative mortality. Fluid recovered from closed-suction drains in all animals was uniformly amylase-rich. Over the 14-day study period, daily volume of pancreatic drainage was significantly different between control animals and animals treated with sealant (p < 0.001). By postoperative day 6, the total mean pancreatic drainage in dogs treated with sealant was 25 +/- 5 mL/drain (versus 91 +/- 26 mL/drain in untreated dogs; p < 0.05). This is the point at which we remove the drains in our clinical practice. Examination at 14 days revealed intact sealant at the pancreatic stumps in the treatment group, and histopathology showed a characteristic benign histiocyte reaction to the sealant but no other qualitative differences in the degree of inflammation between control and treatment animals. There were no undrained collections or abscesses. CONCLUSIONS: A new synthetic hydrogel sealant prevents the formation of significant pancreatic fistulae after distal pancreatectomy in the dog and may be suitable for clinical application. PMID- 12375755 TI - Selective inhibition of NF-kappaB attenuates the severity of cerulein-induced acute pancreatitis. AB - BACKGROUND: Acute pancreatitis (AP) is associated with increased cytokine production, which can ultimately produce deleterious local and systemic effects. The transcription factor NF-kappaB is activated by degradation of its inhibitory factor, IkappaB, and can stimulate various cytokines. The purpose of this study was to determine whether the inhibition of NF-kappaB binding activity with a novel peptide that binds to the NF-kappaB essential modifier binding domain (NBD) could attenuate the severity of AP. STUDY DESIGN: AP was induced in Swiss Webster mice by hourly injections of the cholecystokinin analogue cerulein (50 microg/kg). Mice were injected with either the wild-type or control (mutated) NBD peptide at the time of the first cerulein injection; they were then sacrificed over a time course, and pancreata and lungs were harvested for histologic analysis and scoring. Myeloperoxidase activity was measured to assess neutrophil sequestration as an indicator of inflammation. NF-kappaB binding activity and steady-state levels of IkappaB and NF-kappaB subunits were determined by gel shift and Western blot, respectively. RESULTS: AP resulted in increased NF-kappaB DNA-binding activity and decreased steady-state levels of IkappaB. Treatment with NBD peptide decreased inflammation in the pancreas, decreased hemorrhage in the lungs, and decreased myeloperoxidase activity in both pancreas and lung. CONCLUSIONS: The marked induction of NF-kappaB binding activity suggests a role for this transcription factor in the early inflammatory changes associated with AP. Treatment with the NBD peptide attenuated the severity of injury associated with AP. Novel compounds that selectively target NF-kappaB may prove to be useful treatment of AP and AP-associated lung injury. PMID- 12375756 TI - Intraoperative parasympathetic nerve stimulation with tumescence monitoring during total mesorectal excision for rectal cancer. AB - BACKGROUND: Unilateral or bilateral division of the parasympathetic nerves during resection of rectal cancer may result in sexual erectile dysfunction. The purposes of this project were twofold: to determine the ability to demonstrate penile tumescence in response to parasympathetic nerve stimulation after rectal cancer resection and to correlate the nerve stimulation response with clinical sexual function 6 months after operation. STUDY DESIGN: In 21 consecutive male patients with normal erectile function undergoing total mesorectal excision, cavernous nerve identification and integrity before and after pelvic dissection were assessed intraoperatively, both visually by an experienced surgeon and by using the CaverMap nerve stimulator. The minimal effective current necessary to produce a 2% increase in penile tumescence was recorded for both the left- and right-sided nerves, primarily the largest nerve trunk, S3. Postclearance stimulation data were then correlated with sexual function outcomes, specifically erection and orgasm at 6 months after surgery. RESULTS: The operating surgeon's visual assessment of the pelvic autonomic nerve's integrity after pelvic dissection was deemed intact in 20 of the 21 patients (95.2%). Of the 20 patients who were evaluated with CaverMap after completion of total mesorectal excision, 17 (85%) had tumescence response after nerve stimulation on either side, and 3 patients (15%) had unilateral response only. Of the 19 patients evaluated for sexual function 6 months after surgery, 18 (94.7%) had normal function, including the 3 patients with only unilateral nerve stimulation tumescence response. CONCLUSIONS: Intraoperative mapping of the parasympathetic nerve trunks with the CaverMap nerve stimulator may be a valuable aid to less experienced pelvic surgeons and may help in autonomic nerve preservation during total mesorectal excision clearance. PMID- 12375757 TI - Anorectal dysfunction after surgical treatment for cervical cancer. AB - BACKGROUND: Although bowel symptoms and complaints are common after radical hysterectomy, the effects of operation on anorectal function are incompletely understood. In this prospective pilot study we evaluated the incidence of bowel symptoms, changes in anorectal physiology, and quality of life after radical hysterectomy. STUDY DESIGN: Eleven women undergoing radical hysterectomy for early-stage cervical cancer completed bowel function symptom surveys and cancer specific quality-of-life scales before operation and at 6 weeks and 6 months after operation. The bowel function symptom survey was also repeated at 18 months postoperation. Anorectal manometry, balloon defecation, and pudendal nerve latency tests were performed before the operation and 6 months postoperatively. RESULTS: The mean age was 45.3 years (range 34 to 56 years), and four of the patients were postmenopausal. Resting and squeeze sphincter pressures, volume of saline infused at first leak, total volume retained, and threshold volume for maximum tolerable volume were all decreased significantly (p < 0.05) after operation. Pudendal nerve terminal motor latency increased (p < 0.05) bilaterally. There were no significant differences in sensory thresholds. At 18 months, two women reported constipation, six reported flatus incontinence, and two reported fecal incontinence. The total quality-of-life score declined at 6 weeks but then improved significantly by 6 months (p = 0.02). CONCLUSIONS: Bowel dysfunction is common after radical hysterectomy. Many women exhibit manometric and subjective changes compatible with fecal incontinence. PMID- 12375758 TI - The impact of culture on the patient-surgeon relationship. PMID- 12375759 TI - Effects of limited work hours on surgical training. AB - BACKGROUND: Legal mandates to reduce resident work hours have prompted changes in the structure of surgical training programs. Such changes have included modification of on-call schedules and the adoption of "night float" resident coverage. Little is known about the effects of these changes on surgical resident education and perceptions of quality of patient care. STUDY DESIGN: The surgical housestaff and faculty at a single institution completed a 21-point Likert survey. Subjects were asked to compare parameters of resident education, patient care, and resident quality of life before and after institution of a strict 80 hour work week resident training schedule. The number of hours worked per week before and after these changes were reported. American Board of Surgery In Training Examination (ABSITE) scores were compared for the 2 years before and after implementation of this schedule. Total number of surgical cases performed by graduating chief residents were recorded and compared for the 3 years before and after the schedule changes. RESULTS: Resident work hours reduced significantly after schedule changes were implemented. A majority of surgical residents reported an improvement in quality of life, but residents and faculty perceived changes to have a negative impact on continuity of patient care. Mean ABSITE composite percentile scores significantly improved after the reduction of working hours. ABSITE scores for junior residents improved significantly; no significant differences were noted in scores for senior residents. CONCLUSIONS: Reduction in resident work hours has salutary effects on perception of quality of life and basic education for surgical residents. These benefits may come at the expense of patient care, particularly continuity of care. This study did not directly assess patient outcomes but the perceptions of caregivers suggest that patient care may be compromised. Further research is needed to assess the longterm effects of changes on both residents and patients. PMID- 12375760 TI - Medical student contact with patients on a surgery clerkship: is there a chance to learn? AB - BACKGROUND: Earlier studies of medical students on nonsurgical rotations have shown that clinical clerks usually first interact with their patients late in the clinical course. This would seem disadvantageous to the student's learning because they would have less opportunity to generate diagnoses or a management plan. STUDY DESIGN: A questionnaire designed to assess the nature of medical student-patient interactions in all potential clinical sites was administered to third year medical students during their surgical clerkship. Students received questionnaires each day to evaluate their clinical experiences from the previous day. RESULTS: The results from 311 student-patient encounters were collected and analyzed by clinical site as follows: outpatient clinics, outpatient surgery, inpatient surgery, day of surgery admission, inpatient consults, or emergency room consults. Students reported significantly more opportunities to elicit chief complaint, generate potential diagnosis, develop or suggest a management plan, and perform the initial examination when in the clinic setting. CONCLUSIONS: Overall, students were given relatively few opportunities to be the first to interact with any patient in any setting. They infrequently had an opportunity to independently generate a hypothesis or generate a management plan. Currently, the clinic offers the best opportunity for the student to complete these processes. PMID- 12375761 TI - Endovascular therapies: an update on aortic aneurysm repair and carotid endarterectomy. PMID- 12375762 TI - Neurofibromatosis: implications for the general surgeon. PMID- 12375763 TI - The spiritual needs of the dying patient. PMID- 12375764 TI - Hypersegmentation, Klippel-Feil syndrome, and hemivertebra in a scoliotic patient. PMID- 12375765 TI - Plexiform neurofibroma with intraspinal extension. PMID- 12375766 TI - Visualization of the external branch of the superior laryngeal nerve during video assisted thyroidectomy. PMID- 12375767 TI - Force required to elevate the sternum of pectus excavatum patients. PMID- 12375768 TI - Hand-assisted laparoscopic proximal gastrectomy with jejunal interposition and lymphadenectomy. PMID- 12375769 TI - Perceived message sensation value (PMSV) and the dimensions and validation of a PMSV scale. AB - Sensation seeking has been linked to drug abuse and risky behaviors, and is positively associated with preferences for messages high in sensation value (i.e., perceived to be highly novel, arousing, dramatic, or intense). This suggests the utility of valid and reliable measures of perceived message sensation value (PMSV) in research on information processing, persuasion, and reducing risk-related behaviors. Dimensions and construct validity of a 17-item PMSV scale were examined via 2 studies: 1 of 368 high school students' reactions to televised antimarijuana public service announcements (PSAs) and one of 444 college students' responses to televised anticocaine PSAs. Exploratory and confirmatory factor analyses indicated 3-dimensional solutions for the PMSV scale were nearly identical for high sensation seeking (HSS) and low sensation seeking (LSS) respondents in Study 1 and HSS respondents in Study 2. Total scale alphas were .87 for Study 1 and .93 for Study 2. The PMSV scale and its dimensions (Emotional Arousal, Dramatic Impact, Novelty) were positively correlated with affective response measures in both studies for HSS and LSS. Study 1 also examined cognitive, narrative, and sensory PSA processing, which were found to be positively associated with total PMSV and the Arousal and Dramatic Impact dimensions of PSMV for both HSS and LSS. PMID- 12375770 TI - Predicting intentions versus predicting behaviors: domestic violence prevention from a theory of reasoned action perspective. AB - A central assumption of many models of human behavior is that intention to perform a behavior is highly predictive of actual behavior. This article presents evidence that belies this notion. Based on a survey of 1,250 Philadelphia adults, a clear and consistent pattern emerged suggesting that beliefs related to domestic violence correlate with intentions to act with respect to domestic violence but rarely correlate with reported actions (e.g., talking to the abused woman). Numerous methodological and substantive explanations for this finding are offered with emphasis placed on the complexity of the context in which an action to prevent a domestic violence incident occurs. We conclude by arguing that despite the small, insignificant relationships between beliefs and behaviors found, worthwhile aggregate effects on behavior might still exist, thus reaffirming the role of communication campaign efforts. PMID- 12375771 TI - Setting the agenda: an analysis of negotiation strategies in clinical talk. AB - This article analyzes the process whereby physicians and patients set the agenda for medical interviews. Applying a conversation analytic perspective to the analysis of 22 videotapes of primary care interviews at a large, urban, teaching and research hospital, a 3-stage model is developed, consisting of (a) an opening sequence, (b) an initial statement of concerns by the patient, and (c) the negotiation process. The analysis illustrates the critical function of the opening verbal exchanges, showing how patient responses to the physician's first question and subsequent queries and summaries by the physician are intricately interwoven. The interaction at the very beginning of the interview is shown to significantly alter the ensuing interaction. The analysis provides a discursive framework for analyzing problematic communication during the primary care interview. PMID- 12375772 TI - The POWER campaign for promotion of female and male condoms: audience research and campaign development. AB - In this study, we conducted and content analyzed 12 focus groups with women aged 15-25 living in inner city Denver as a process of audience research to develop a male and female condom promotion campaign. We recruited 89 women from school and community sites in central Denver neighborhoods to discuss their knowledge, attitudes, and practices regarding both male and female condoms, then solicited opinions about how to increase knowledge about and familiarity with female condoms, increase positive attitudes toward both male and female condoms, and how to increase access to and use of both male and female condoms. Opinions on these topics drove the development of a targeted media campaign promoting condom use in this population. We report here on the general findings from focus groups and provide details about the campaign the participants helped to develop. PMID- 12375773 TI - Bringing the physician back in: communication predictors of physicians' satisfaction with managed care. AB - Data from a survey of physicians in a west coast city (n = 356) are used to measure physicians' extra-occupational sources of dissatisfaction. Data revealed a significant relationship between physicians' satisfaction and their managed care experience, their communication with managed care organizations, and views of managed care practice. Results suggest that managed care currently plays a large and significant role in predicting physicians' satisfaction. The importance of communication between physicians and managed care organizations is illustrated in the strength of the relationships between communication variables and managed care decisions. Furthermore, in assessing the strength of the relationship, regression analysis reveals that communication with managed care accounts for the largest percentage of variance in physicians' satisfaction. The results of this study suggest that communication with managed care organizations affects physicians' satisfaction with every facet of the organizational environment, including leading physicians who report problematic communication with managed care organizations to say that they would be less likely to choose the same career path again. PMID- 12375774 TI - Cranial mediastinal cysts in nine cats. AB - Nine cats, from 11 to 17 years of age (mean 13.6 years of age), were diagnosed with a cranial mediastinal cyst. Thoracic radiographs in all cats were characterized by an increased soft tissue opacity in the cranial mediastinum confirmed to be a cyst by ultrasonography or necropsy. Ultrasonographically cysts appeared as an anechoic mass. A low-cellularity clear fluid was obtained on aspiration. The majority of the cats (n = 8) presented for unrelated conditions with no signs of respiratory distress. No treatment for the cyst was pursued except for drainage during ultrasonographic-guided aspiration in several cats. On follow-up of eight cats, none were symptomatic for the cyst from 3-45 months after diagnosis. Mediastinal cyst should be considered when a cranial mediastinal mass is evident radiographically in an older cat. The majority of feline cranial mediastinal cysts are benign with no need for treatment. PMID- 12375775 TI - Aortic and cardiac mineralization in the dog. AB - Aortic and cardiac mineralization was found in 21 of 3443 (0.61%) canine thoracic radiographs. In none of 786 feline thoracic radiographs reviewed were such lesions present. Mineralizations were superimposed on the ascending aorta (19 dogs) or on the caudal cardiac silhouette (2 dogs). In 2 of 4 dogs mineralization was identified echocardiographically dorsal to the aortic valve in close proximity to coronary arteries. Computed tomography confirmed mineralization of the aortic arch and root in 2 of 2 dogs. Necropsy and histopathologic examination in 1 dog revealed multiple nodular aortic tunica media calcifications with adjacent areas of degeneration. Lesions were significantly overrepresented in older dogs and in Rottweilers, and regarded as dystrophic calcification, caused either by age-related degenerative changes or chronic disease-related processes. There was no evidence of clinical significance attributed to the mineralization in any dog. Aortic and cardiac mineralization should be recognized as an incidental, non-significant finding in dogs of advanced age and differentiated from pleural and pulmonary structures. PMID- 12375776 TI - Effect of deep digital flexor tendon orientation on magnetic resonance imaging signal intensity in isolated equine limbs-the magic angle effect. AB - Ten normal equine isolated limbs were imaged using a knee coil in a 1.5 Tesla magnetic field, with short echo time sequences (TE < 15 ms). Magnetic resonance imaging was performed on each isolated limb in different positions, with and without extension of the metacarpophalangeal joint. Deep digital flexor tendon orientation ranged from 20 to 60 degrees in relation to the static magnetic field. Increased intratendinous signal intensity was observed when the angle between the deep digital flexor tendon and the constant magnetic field approached 55 degrees ("magic angle"). The increased signal intensity was independent from extension of the metacarpophalangeal joint. Recognition of the magic angle phenomenon is essential for proper evaluation of magnetic resonance imaging studies of the equine foot. PMID- 12375777 TI - Accuracy of magnetic resonance imaging for estimating intramedullary osteosarcoma extent in pre-operative planning of canine limb-salvage procedures. AB - The objective of this work was to compare the accuracy of radiographs and magnetic resonance imaging (MRI) for estimating appendicular osteosarcoma margins. The accuracy of computed tomography (CT) and bone scintigraphy was also assessed when these studies were available. Eight dogs with appendicular osteosarcoma underwent radiographic and MRI of affected limbs. In addition, bone scintigraphy was performed in six dogs and CT examination was performed in five dogs. Two observers jointly measured tumor length on all imaging studies. Correlative gross and histologic evaluation of all affected limbs was performed to determine tumor extent as measured from the nearest articular surface. Results from imaging studies were compared to gross and microscopic morphometry findings to determine the accuracy of each modality for determining tumor boundaries. MRI images were accurate with a mean overestimation of actual tumor length of 3 +/- 13%. T1-weighted non-contrast images were superior in identifying intramedullary tumor margins in most instances whereas contrast-enhanced images provided supplemental information in two dogs. Lateromedial and craniocaudal radiographs overestimated tumor length by 17 +/- 28% and 4 +/- 26%, respectively. Scintigraphy and CT overestimated tumor margins by 14 +/- 28% and 27 +/- 36%, respectively. MRI appears to be an accurate diagnostic imaging modality in determining intramedullary osteosarcoma boundaries. MRI should be considered as part of a pre-operative assessment of appendicular osteosarcoma, particularly when a limb-sparing procedure is contemplated. PMID- 12375778 TI - Assessing diagnostic accuracy in veterinary imaging. AB - With continued specialization in diagnostic radiology and imaging technology, it increasingly becomes important for diagnostic radiologists to focus on the science of diagnosing disease. Imaging modalities provide a means to examine patients; however, it is the detection of imaging signs (Roentgen signs) that constitutes imaging tests. All tests are subject to error, and the degree of error cannot be determined by clinical experience alone. Therefore, measurements of diagnostic accuracy are important. Accuracy estimates are greatly influenced by selection of the decision criterion and sample population. This article reviews some of the principles for assessing diagnostic accuracy in medical imaging, which is helpful for deciding what tests to perform and how to interpret results once a test has been performed. PMID- 12375779 TI - Intracranial intra-arachnoid cyst with intracystic hemorrhage in two dogs. AB - Clinical signs, magnetic resonance imaging (MRI) features, treatment, and outcome of two adult dogs with neurologic dysfunction resulting from hemorrhage into a quadrigeminal intracranial intra-arachnoid cyst are described. In dog 1, the cyst was hyperintense to cerebrospinal fluid (CSF) on T1-weighted MRI and hypointense to CSF on T2-weighted images. In dog 2, the cyst was isointense to CSF on T1- and T2-weighted images. Both dogs were treated with craniotomy and cyst fenestration. A large blood clot was removed from the lumen of the cyst in each dog. Dog 1 is clinically normal 3.5 years post-surgery and has a persistent cyst. Dog 2 had a good initial response to therapy but was euthanized 2.5 years post-operatively due to generalized seizures. The late onset of clinical signs in these dogs most likely resulted from hemorrhage into the cyst. Surgical fenestration and hematoma removal appear to provide a satisfactory treatment for adult dogs with an intracranial intra-arachnoid cyst and intracystic hemorrhage. Persistence of the cyst may occur in some dogs. PMID- 12375780 TI - Comparison of computed radiography and conventional film-screen radiography of the equine stifle. AB - Major advantages of computed radiography are the potential reduction of dose and the possibility of postprocessing. In our study, we compared conventional radiographs to digital radiographs of the equine stifle by subjective evaluation of diagnostic quality when using a decreasing photon flux (mAs). Twelve equine stifle joints from horses of different weight and size were examined. Conventional and digital radiographs were performed identically in a caudocranial projection with the tube angled 15 degrees. A series of four radiographs was performed in each technique with an increasing photon flux starting with 2.5 mAs and going up to 5, 10, and 20 mAs. All radiographs were evaluated subjectively in a blinded fashion by seven readers in terms of contrast, bone structure, and diagnostic value and were graded using a 1-5 scale. Results from conventional and digitized radiographs were compared, and differences between the individual observers were analyzed statistically. Contrast, bone structure, and diagnostic value from digital images were rated significantly better than from conventional images (p < .001). For both techniques, a decrease in ranking was found with a decrease of photon flux. There was only slight interobserver variability. A dose reduction up to a factor of 4 compared to a 100 speed film-screen system seems to be possible without loss of information. Weight and size of the horse are not major influences. PMID- 12375781 TI - Collateral cartilage ossification of the distal phalanx in the Brazilian Jumper horse. AB - Collateral cartilage ossification of the distal phalanx in the Brazilian Jumper horse is a common finding. The objective of this study was to evaluate the prevalence and the degree of ossification of the collateral cartilages of the distal phalanx in Brazilian Jumper horses. In an analysis of 652 collateral cartilages from the front feet of 163 horses, 93% of these cartilages had collateral cartilage ossification (P < 0.005), and 7% of these cartilages did not have any type of ossification. In ossified cartilages, 86.4% had ossification beginning from the base, and 6.6% had a separate center of ossification. PMID- 12375782 TI - Laparoscopic sonography in the urinary system. AB - This work reports the use of laparoscopic-transducer sonography for the examination of the urinary system in a swine model. Animals underwent a two-phase study. In the first phase, the urinary system was examined using laparoscopic sonography. In the second a partial ureteral obstruction was induced, and sonographic changes were recorded and evaluated. Sonography was used to evaluate kidneys, ureters, and bladder. Anatomic structures were evaluated and the following pathological findings were identified: renal cysts, one polycystic kidney, dilation of the renal pelvis, hydronephrosis, and one perirenal pseudocyst. Where necessary, contrast digital fluoroscopy (excretory urography and retrograde ureteropyelography) was also performed. Laparoscopic sonography mainly is used for evaluation prior to laparoscopic surgery to guide decisions relating to surgery. The quality of the images obtained laparoscopically is superior to that of percutaneous or transabdominal images, because artifacts are reduced and the contact surface of the transducer is placed directly over the study area. Laparoscopic sonography proved highly effective for studying renal and ureteral disorders prior to minimally invasive surgery. PMID- 12375783 TI - Preoperative radiotherapy for vaccine associated sarcoma in 92 cats. AB - Medical records for 92 cats with a vaccine associated sarcoma receiving preoperative irradiation, with or without chemotherapy, between December 1985 and September 1998 were reviewed. The purposes were to quantify response to treatment and to attempt identification of factors associated with favorable response. Variables evaluated for a relationship to outcome included signalment, tumor location, presence of gross vs. microscopic tumor, radiation field size, irradiation technique, type of surgical procedure, completeness of excision, and chemotherapy (none, carboplatin alone, and others). Time to first event was calculated for the first day of treatment until local tumor recurrence or metastasis, or the date of euthanasia or death. Median time to first event for all 92 cats was 584 days. Only completeness of surgical excision was related to the time to first event. Median time to first event in cats having complete surgical excision was 986 days compared to 292 days for cats with incomplete excision (P = 0.004). Cats requiring bone removal to effect tumor removal had earlier failure than cats having other types of surgery. There was not a significant relationship between administration of chemotherapy or chemotherapy type and time to first event although outcome in cats receiving carboplatin was better than all other treatment groups. Carboplatin addition to preoperative irradiation appears worthy of further study. Preoperative irradiation is an effective treatment for cats with vaccine associated sarcoma, especially if complete excision can be accomplished following irradiation. PMID- 12375785 TI - Images in medicine. Lymphosarcoma. PMID- 12375784 TI - A comparison of normal tissue complication probability of brain for proton and photon therapy of canine nasal tumors. AB - This study compared the calculated normal tissue complication probability of brain in dogs with a nasal tumor, which had both photon and proton treatment planning. Nine dogs diagnosed with a variety of histologies, but all with large, caudally located nasal tumors were studied. Three-dimensional (3-D) photon dose distribution, and a proton dose distribution was calculated for each dog. To calculate the normal tissue complication probability (NTCP) for brain, the partial brain volume irradiated with the prescribed dose was determined, then a mathematic model relating complications to partial volume and radiation dose was used. The NTCP was always smaller for proton plans as compared to photon plans, indicating conformation of the dose to the target allows a higher dose to be given. If a 5% NTCP were accepted, the mean applicable dose for this group of dogs was 50.2 Gy for photons, but 58.3 Gy for protons. Not all dogs would benefit the same from proton irradiation. If a large partial brain volume has to be irradiated, the advantage becomes minimal. There is also a minimal advantage if the planning target volume (PTV) includes a small, superficial brain volume. However, for a complex PTV shape the degree of conformation is clearly superior for protons and results in smaller calculated NTCPs. PMID- 12375786 TI - Protection of the environment from the effects of ionising radiation. PMID- 12375787 TI - Radiological protection of the environment from the Swedish point of view. AB - The current system of radiological protection is aimed at protecting human health, and largely neglects both the effects of radiation on the environment and the managerial aspects of environmental protection. The Swedish Radiation Protection Act was revised in 1988 and includes environmental protection as one of its aims. In practice, little guidance had been given in the regulations based on the Act until 1998, when the Swedish Radiation Protection Authority (SSI) formulated environmental aims in its regulations concerning protection of human health and the environment in connection to the final management of spent nuclear fuel and waste. These regulations focus on protection of biodiversity and biological resources, based on ecosystem characterisation. In a broader perspective, the Swedish Parliament established 15 national environmental quality objectives in 1999, covering all aspects of protecting the environment, including the effects of radiation. This paper reviews the background for radiological protection of the environment from both an international and a Swedish perspective, describing the aims and current activities in establishing a system for assessing environmental effects and their consequences that can be used in decision-making. Such activities are largely a result of the European Union research project FASSET (Framework for Assessment of Environmental Impact), carried out under the 5th Framework Programme of the Union. This work is complemented at the Swedish national level by government support to initiate a national environmental monitoring and assessment programme for characterising the radiation environment, which will provide the foundation for decision-making. PMID- 12375788 TI - A model for evaluating radiological impacts on organisms other than man for use in post-closure assessments of geological repositories for radioactive wastes. AB - Bioaccumulation and dosimetric models have been developed that allow the computation of dose rates to a wide variety of plants and animals in the context of the deep geological disposal of solid radioactive wastes. These dose rates can be compared with the threshold dose rates at which significant deleterious effects have been observed in field and laboratory observations. This provides a general indication of whether effects on ecosystems could be observable, but does not quantify the level of those effects. To address this latter issue, two indicator organisms were identified and exposure-response relationships were developed for endpoints of potential interest (mortality in conifers and the induction of skeletal malformations in rodents irradiated in utero). The bioaccumulation, dosimetry and exposure-response models were implemented and used to evaluate the potential significance of radionuclide releases from a proposed deep geological repository for radioactive wastes in France. This evaluation was undertaken in the context of a programme of assessment studies being performed by the Agence nationale pour la gestion des dechets radioactifs (ANDRA). PMID- 12375789 TI - The proportion of thyroid cancers in the Japanese atomic bomb survivors associated with natural background radiation. AB - Generalised absolute and relative risk models (with adjustment to the excess absolute risk for time since exposure and age at exposure, and with adjustment to the excess relative risk for age at exposure) are fitted to the Japanese atomic bomb survivor thyroid cancer incidence data followed up over the period 1958-87, taking account of natural background radiation. Thyroid cancers associated with natural background radiation and atomic bomb radiation are overwhelmingly accounted for by exposure at young ages. Over 50% of the excess cases associated with either the atomic bomb radiation or natural background radiation are linked to exposures under the age of 20, irrespective of the assumed risk model or natural background dose rate. The excess risk is overwhelmingly concentrated among females, again irrespective of the assumed model or natural background dose rate. Depending on the assumed natural background dose rate (in the range 0.5-2.0 mSv/year) between 17.3 and 32.0% of the thyroid cancer in this cohort may be associated with natural background radiation if an absolute risk model applies; between 4.2 and 17.1% of the thyroid cancers may be associated with natural background radiation if the relative risk model applies. The proportion of the thyroid tumours attributed to the atomic bomb radiation is between 21.1 and 22.0% for the absolute risk model, and is between 18.7 and 19.1% for the relative risk model, in both cases irrespective of the assumed background radiation dose. In particular, these proportions are not very different from the proportions calculated when fitting models that do not take account of natural background radiation, namely 22.0% for the absolute risk model and 18.6% for the relative risk model. The proportion of thyroid cancers accounted for by natural background radiation progressively increases with attained age, from 0.3% of cancers among those under the age of 15 to 30.5% for those over the age of 60, assuming that the absolute risk model applies. There is a similar increase in this percentage, if to a rather lower level (from 0.2 to 10.2%), assuming that the relative risk model applies. PMID- 12375790 TI - Choice of alpha-probe operating voltage to suit a wide range of conditions. AB - Alpha probes, consisting of a ZnS(Ag) scintillator and a photo-multiplier tube, are commonly used throughout the nuclear industry for radiation protection and clearance of materials during decommissioning. The success in achieving these purposes is dependent on a number of factors including the counting efficiency of the probe, the condition of the material being monitored, the speed of monitoring and the distance between the probe and material. The efficiency of the probe is dependent on the operating voltage and is the only factor that is under the control of the calibration facility. As the calibration laboratory may not be aware of the specific environment in which the probe will be used, an operating voltage to suite a wide range of conditions must be chosen. In the past, it has frequently been assumed that it is necessary to set as high an operating voltage as possible in order to maximise the counting efficiency to low-energy alpha particles. However, the response to gamma rays, particularly those having low energies, also increases with operating voltage and will therefore limit the upper operating voltage that can be set. The efficiency of a scintillation-type probe (NE Technology AP2) in measuring contamination levels on a number of typical surfaces using different operating voltages has been investigated. It has been found that the surface characteristics of the material being monitored have far more effect on the results of alpha monitoring than the choice of operating voltage. Thus the calibration laboratory can set the operating voltage below the level at which there is a risk of response to low-energy gamma rays without significantly affecting the overall counting efficiency for low-energy alpha particles. PMID- 12375791 TI - Response to 'The potential for bias in Cohen's ecological analysis of lung cancer and residential radon'. PMID- 12375792 TI - Comments upon human exposure to depleted uranium during and after the Gulf and Balkans conflicts. PMID- 12375793 TI - Comments on 'Radioiodine therapy: care of the helpless patient and handling of the radioactive corpse'. PMID- 12375794 TI - The 20th L H Gray Conference--Radiation cancer analysis and low dose risk assessment: new developments and perspectives. Ed, The Netherlands, February 2002. PMID- 12375795 TI - NRPG Chilton Seminar: Professor Bryn Bridges--'Genetic effects of radiation: are the paradigms changing?' Chilton, November 2001. PMID- 12375796 TI - Irinotecan in combination with radiation therapy for small-cell and non-small cell lung cancer. AB - Lung cancer is the leading cause of cancer-related death in the United States. There was rapidprogress in the treatment of lung cancer duringpast decades, but local control and survival rates are still poor. Radiation therapy has been an indispensable part of the management of lung cancer, and a recent paradigm is concurrent chemoradiation therapy. Many novel chemotherapeutic agents were recently developed to improve both local and systemic control of cancer, including camptothecin derivatives, which are topoisomerase I inhibitors. Irinotecan (CPT-11, Camptosar) is a semisynthetic water-soluble derivative of camptothecin. Irinotecan is active as a single agent against lung cancer, and is also a potent radiosensitizing agent in human lung cancer cell lines and xenografts. There have been many phase I and II clinical trials demonstrating promising results of single-agent irinotecan and combination with concurrent therapy. This article reviews irinotecan's mechanism of action of cytotoxicity and of radiation-sensitizing effects, as well as recent clinical data regarding combining radiation therapy and irinotecan for both non-small-cell and small-cell lung cancer. PMID- 12375797 TI - Targeted therapy in non-small-cell lung cancer. AB - Although treatment of advanced non-small-cell lung cancer has been improved with the availability of such new agents as the taxanes, topoisomerase inhibitors, vinorelbine (Navelbine), and gemcitabine (Gemzar), platinum-based combination therapy has appeared to reach a threshold of therapeutic effectiveness. A paradigm shift in approach to non-small-cell lung cancer and other tumors may be heralded by the development of agents targeting specific biologic pathways in tumor development. Such new agents include antibody epithelial growth factor receptor (EGFR) inhibitors (eg, the monoclonal antibodies trastuzumab [Herceptin] and cetuximab [IMC-C225, Erbitux]) and EGFR tyrosine kinase inhibitors (eg, ZD1839 [Iressa] and OSI-774), angiogenesis inhibitors (eg, matrix metalloproteinase inhibitors), vascular endothelial growth factor (VEGF) inhibitors (eg, monoclonal antibody to VEGF ligand and small-molecule tyrosine kinase), and signal transduction inhibitors (eg, ISIS-3521, an antisense oligonucleotide to protein kinase C-alpha). A number of these agents have entered advanced-phase clinical investigation. It is likely that targeted therapy will have applications in combination with cytotoxic chemotherapy or radiation therapy at all stages of treatment, including maintenance therapy. It is even possible that these new biologic therapies will be used together as rational combinations (based on pathologic diagnosis) for advanced non-small-cell lung cancer. PMID- 12375798 TI - Topoisomerase I-based nonplatinum combinations in non-small-cell lung cancer. AB - Lung cancer is the leading cause of cancer-related death in males and females in the United States. Most patients have advanced disease at diagnosis. Chemotherapy is the treatment of choice for patients with good performance status. Progress in the management ofpatients with advanced disease has been slow, and platinum-based combinations result in a small survival benefit. The topoisomerase I inhibitors are active as single agents and in combination with platinums in non-small-cell lung cancer. Nonplatinum-based doublet combinations are beginning to be explored in an attempt to reduce toxicity and improve efficacy. Data available on some of the nonplatinum doublets that include topoisomerase I inhibitors suggest that these regimens provide efficacy equal to that achieved with platinum-based doublets. This article reviews the topoisomerase I-inhibitor nonplatinum combinations in the management of advanced non-small-cell lung cancer. PMID- 12375799 TI - Camptothecin and taxane regimens for small-cell lung cancer. AB - For more than 2 decades, combination chemotherapy has been the standard treatment for patients with small-cell lung cancer. Despite high initial response rates in both extensive- and limited-stage disease, long-term survival rates are only 10% to 20%. Camptothecins and taxanes are newer classes of agents that have shown significant activity against small-cell lung cancer. Their incorporation into chemotherapy regimens for small-cell lung cancer is being actively studied. In one randomized multicenter study, patients with advanced small-cell lung cancer treated with irinotecan (CPT-11, Camptosar) and cisplatin had a better survival time than patients receiving standard therapy. The combination of taxanes and irinotecan holds promise as an active regimen that may be tolerated better than cisplatin and irinotecan. PMID- 12375800 TI - Irinotecan plus cisplatin in small-cell lung cancer. AB - The DNA topoisomerase inhibitor irinotecan (CPT-11, Camptosar) is being evaluated as a novel chemotherapeutic agent for small-cell lung cancer that may complement other agents and treatment modalities. Combination chemotherapy is recognized as the most effective means of improving survival in patients with extensive-stage small-cell lung cancer, but until recently, no one combination had emerged as superior. In a recent Japanese phase III study, irinotecan in combination with cisplatin significantly improved survival of previously untreated patients with extensive small-cell lung cancer compared with standard etoposide/cisplatin therapy (median progression-free survival: 6.9 vs 4.8 months, P < .001; median overall survival: 12.8 vs 9.4 months, P = .0021). Two additional phase III trials are planned to confirm these results in the United States, and to investigate how the irinotecan/cisplatin administration schedule may be modified to improve therapeutic index. This article will review the use of irinotecan in combination with cisplatin in patients with small-cell lung cancer. PMID- 12375801 TI - Cardiovascular diabetology--two years later. PMID- 12375802 TI - Anticoagulants for atrial fibrillation: why is the treatment rate so low? AB - The incidence of atrial fibrillation (AF) is increasing in many countries along with aging demographics. Atrial fibrillation is clearly associated with an increased rate of stroke. Numerous large clinical trials have shown that dose adjusted warfarin can reduce the stroke rate in these patients, particularly in the elderly, and clear guidelines for the use of anticoagulants in such patients have been published. However, many studies show that treatment rates remain disappointingly low (< or = 50%). Numerous barriers to the use of dose-adjusted warfarin exist, including practical, patient-, physician-, and healthcare system related barriers. These include the complex pharmacokinetics of warfarin, the need for continuous prothrombin time monitoring and dose adjustments, bleeding events, noncompliance, drug interactions, and increased costs of monitoring and therapy. Possible solutions to this problem are discussed and include improved patient and physician education, the use of anticoagulation clinics, new approaches to AF, and potential treatment improvements through use of newer anticoagulants. PMID- 12375803 TI - C-reactive protein as a marker for active coronary artery disease in patients with chest pain in the emergency room. AB - BACKGROUND: Markers of inflammation, such as C-reactive protein (CRP), were found to be related to risk for cardiovascular disease (CVD) events in patients with angina pectoris. In addition, recent studies have shown that, in the case of atherosclerosis, increased CRP concentration reflects the inflammatory condition of the vascular wall. HYPOTHESIS: The study was undertaken to determine whether CRP levels in individuals with chest pain attending the emergency room (ER) may be used as a marker of active CVD. METHODS: Serum CRP level was measured in 226 of 326 consecutive patients (128 men, 98 women; mean age 61.3 +/- 5.9 years; range 19-87 years) referred to the ER with chest pain. The decision whether to admit orrelease the subjects was determined without taking the CRP level into account. Follow-up was then performed for 1 year. RESULTS: Eighty-four patients were admitted to the hospital. Of these, 9 with acute coronary syndrome (ACS) had very high levels of CRP (25-40 mg/l), 35 had had an acute coronary event within the preceding 3 months, with levels of CRP 14-20 mg/l. Only eight patients with nonsignificant CVD had elevated CRP levels. Twenty-eight subjects who were released from the ER had elevated CRP levels (7-14 mg/l); 8 of these, in addition to 4 subjects with normal CRP levels, had a late coronary event. CONCLUSION: This study indicates that in patients referred to the ER with chest pain and no other indication for hospitalization, a normal level of CRP suggests safe release. Most hospitalized patients with normal CRP will not have acute coronary syndrome. Patients who will develop early coronary events have very high CRP levels. High serum CRP level, after excluding other inflammatory sources, was proven to be a sensitive diagnostic and prognostic marker for significant coronary disease. PMID- 12375804 TI - Inflammatory predictors of mortality in the Scandinavian Simvastatin Survival Study. AB - BACKGROUND AND HYPOTHESIS: The predictive value of specific markers of infection and autoimmunity for coronary events, such as the effects of statins on inflammation, is still controversial. METHODS: A case-control design was used to compare C-reactive protein (CRP) levels, seropositivity for Chlamydia pneumoniae and Helicobacter pylori, and anti-oxidized low-density lipoprotein (oxLDL) antibody levels in prerandomization blood samples from 129 participants in the Scandinavian Simvastatin Survival Study who died (cases), and from 129 matched participants who were alive during 5-year follow-up (controls). RESULTS: Patients with CRP levels in the highest quartile had an increased risk of death compared with those in the first through third quartile (odds ratio [OR] = 2.51, 95% confidence interval [CI] 1.3-4.8). Seropositivity for Chlamydia pneumoniae or Helicobacter pylori and anti-oxLDL antibody levels were similar in cases and controls (p = NS). At a 4-month control, simvastatin reduced CRP levels (p = 0.009) while placebo did not (p = NS). However, the risk of death associated with high baseline CRP levels was similar in patients randomized to placebo (OR = 2.36, 95% CI 1.06-5.26) or simvastatin (OR = 3.13, 95% CI 1.06-9.21). CONCLUSIONS: Elevated CRP levels, but not seropositivity for Chlamydia pneumoniae or Helicobacter pylori, nor levels of anti-oxLDL antibodies, predict the risk of death in patients with stable ischemic heart disease. Simvastatin treatment reduces CRP levels, but without affecting the increased risk conferred by higher CRP levels at baseline. PMID- 12375805 TI - Effects of distension of urinary bladder on coronary conduit and resistance vessels in hyperlipidemic patients. AB - BACKGROUND: Distension of the urinary bladder reflexly causes a change of coronary vasomotor response. The effect of such distension on the coronary circulation in hyperlipidemic patients, a condition with impaired endothelial function, remains unknown. HYPOTHESIS: We tested the hypothesis whether urinary bladder distension caused an exaggerated vasomotor response of epicardial and resistance vasoconstriction in hyperlipidemic patients. METHODS: Thirty patients with early atherosclerosis (< 50% diameter stenosis) were divided into three groups: Group 1 (n = 10): hyperlipidemia without doxazosin administration; Group 2 (n = 10): hyperlipidemia with pretreatment of alpha1-adrenergic receptor blocker (oral doxazosin 2 mg); and Group 3 (n = 10): normolipidemia. A prospective analysis of the results of quantitative angiograms, intracoronary Doppler flow, and lactate concentrations from aortic root and coronary sinus was performed during distension of urinary bladder. RESULTS: Bladder distension significantly decreased coronary diameter at the stenotic segments (p = 0.004), coronary blood flow (p = 0.05), and increased coronary resistance (p = 0.006) compared with baseline values, in Group 1 patients. In Group 2 patients during bladder distension, coronary diameter, coronary blood flow, and coronary resistance showed no significant changes compared with baseline values. There were significant differences of stenotic coronary diameter (p = 0.01) between Groups 1 and 3 during bladder distension despite similar changes in rate-pressure product. No significant differences were noted among the groups in the responses of coronary diameter, coronary blood flow, and coronary resistance after nitroglycerin administration. CONCLUSIONS: The present study showed that urinary bladder distension caused an abnormal vasomotor response of epicardial vasoconstriction and that a concomitant increased coronary resistance involved mechanisms related to alpha1-adrenoceptors. Hyperlipidemia may further impair the response. Pretreated administration of doxazosin had reversed the changes toward baseline. Vasoconstriction during bladder distension can be relieved after nitroglycerin administration, suggesting an unchanged responsiveness of vascular smooth muscle cells to such distension. PMID- 12375806 TI - Triggering effect of physical and mental stress on spontaneous ventricular tachyarrhythmias in patients with implantable cardioverter-defibrillators. AB - BACKGROUND: Physical and mental stress as well as sexual activity are potential triggering factors of acute coronary events and sudden cardiac death. HYPOTHESIS: These factors may also trigger recurrence of spontaneous ventricular tachyarrhythmias in patients with implantable cardioverter-defibrillators (ICDs). METHODS: We performed a case-crossover study in 43 consecutive patients with 95 symptomatic, ICD-documented tachyarrhythmic events and calculated the relative risk of tachyarrhythmia recurrence during physical and mental stress. Physical and mental activity was graded on a 4-step intensity scale, and stress was defined as physical exertion or mental stress with an intensity grade > or = II during or up to 1 h before arrhythmia recurrence. Relative risk was determined taking into account the habitual weekly stress frequency of each patient. RESULTS: Physical stress was present in 26% (n = 25), mental stress in 24% (n = 23), and sexual activity in 2% (n = 2) of analyzed events. The weekly habitual stress frequency was 8 +/- 8 (median 7) for physical stress, 6 +/- 6 (median 4) for mental stress, and 0.5 +/- 0.5 (median 0.25) for sexual activity. Thus, relative risk of arrhythmia recurrence during the presence of stress was 7.5 for physical activity (95% confidence interval [CI] 5.2-11.1), 9.5 (CI 6.3-14.5) for mental activity, and 7.5 (CI 2.3-24.8) for sexual activity. CONCLUSIONS: Physical and mental stress as well as sexual activity are factors that significantly increase relative risk of spontaneous recurrence of sustained ventricular tachyarrhythmias in patients with ICDs. Consideration of this stress-related relative risk increase may contribute to avoidance of harmful shock delivery in ICD recipients. PMID- 12375807 TI - Does racial bias exist in the medical management of heart failure? AB - BACKGROUND: It is suspected that effective therapies are often underutilized in black compared with white patients with coronary artery disease (CAD). HYPOTHESIS: We hypothesized that an unfavorable bias may exist against black patients in the medical management of heart failure. METHODS: In 566 consecutive adult subjects who were discharged alive from the hospital with a principal discharge diagnosis of heart failure, we assessed the effect of patient race on utilization of classes of medications (angiotensin-converting enzyme inhibitors [ACEI], digitalis, diuretic agents) and combinations of medications (effective vasodilators, i.e., ACEI or combined hydralazine and nitrate; effective combination therapy, i.e., effective vasodilator with digitalis and diuretic) known to be beneficial in symptomatic heart failure. RESULTS: Compared with black patients (n = 182), white patients were older, had a higher incidence of coronary artery disease, lower incidence of hypertension, and lower serum creatinine and left ventricular end-diastolic diameter. In crude analyses, the utilization of all medications was similar between white and black patients. After adjustment for clinical differences, black patients were more likely to receive ACEI (adjusted odds ratio [OR] = 1.84; 95% confidence interval [CI] 1.13-3.01), effective vasodilators (OR = 1.97; CI 1.20-3.23), and effective combination therapy (OR = 1.66; CI 1.02-2.69) than white patients at the time of discharge from the hospital. No multivariate association was seen between patient race and use of digoxin or diuretics. In an analysis of subsets of patients with ejection fraction < 45% (n = 260), no association was seen between patient race and utilization of effective medical therapy. CONCLUSION: Our results show no unfavorable bias against black patients with decompensated heart failure. PMID- 12375808 TI - A stratified approach to the treatment of a symptomatic myocardial bridge. AB - A case of symptomatic myocardial bridge requiring intracoronary stent complicated by in-stent restenosis is reported. A stratified approach for the treatment of a symptomatic myocardial bridge is proposed. PMID- 12375809 TI - Giorgio Baglivi. PMID- 12375810 TI - Images in cardiology: Anomalous origin of a diseased left main coronary artery from the right sinus of valsalva. PMID- 12375811 TI - Biocatalyst-adsorbant systems: a viable alternative to proteolytic processes in solution. AB - Proteolytic biocatalysts were adsorbed and stabilized using alumina as a support medium. Two biocatalyst-adsorbant systems were prepared with different physical characteristics of the adsorbant: alumina powder and alumina pearls. Direct adsorption onto the support medium has the main advantage, over other fixation methods, that preliminary steps are not required for a good interaction between the support and the biocatalyst. Proteases were adsorbed and stabilized without modifying or sterically hindering their active sites. Parameters affecting adsorption (pH, temperature, ionic strength) were varied so as to optimize adsorption conditions. Operational viability of the immobilized biocatalysts was demonstrated, taking into account the rate of desorption, resistance to microbial attack, and stability during storage. Desorption in water was studied in batch and continuous-flow processes, at various flow rates. The systems also proved to be resistant to microorganisms. Tests for stability during storage found the systems' activity remained constant after 60 days, and they performed better than biocatalysts in solution. Proteolysis of a solution of g per litre of azocasein was carried out in continuous-flow and batch modes, using our biocatalyst adsorbant systems we prepared. In all cases, free amino group concentrations were around 2.5 times greater after treatment with biocatalyst-adsorbants than they were in the starting solution. PMID- 12375812 TI - Construction and expression of a reshaped VH domain against human CD28 molecules. AB - Compared with the amino acid sequence of a mouse anti-human CD28 VH domain antibody, the two most homologous sequences of human antibodies were pulled out from Genbank. One of them was used as the main template for the framework regions of the reshaped VH domain. While the original mouse antibody CDRs were inserted into the human acceptor FRs, some residues in human acceptor FRs, which were different from those of the original mouse FRs in corresponding positions, were then determined or, alternatively, mutagenized to their conservative properties in kappa classification. Based on the amino acid sequences of the designed VH domain, the nucleotide sequence was deduced by using E. coli bias codons. The sequence was split into ten 30 to 60 nucleotide fragments for synthesizing, then annealed and amplified by overlap PCR. Taq DNA polymerase was used in a buffer with high Mg2+ concentration to induce more random mutations, both in FRs and CDRs. A phage display library was constructed by cloning these PCR products. After three rounds of panning, several reshaped VH with high antigen binding activity were obtained. One of them had the same CDR amino acid sequences as that of the original mouse VH domain. Further study showed that it retained a high antigen binding affinity after being expressed in E. coli BL21 (DE3). PMID- 12375813 TI - Effect of synthetic oligopeptides on osteoporosis. AB - The objective of this study was to establish the solution method of GHRPS, the synthetic oligopeptides Tyr-Gly-Gly-Phe-Met-NH2, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Gly-NH2, and Tyr-Gly-Gly Phe-Gly-OH, to verify their effect on osteoporosis. Male ICR mice (20+/-2 g) were used. The intramuscular injection dose of 6.3 mg/kg prednisone induced a significant decrease of body and femur weight of the animals. The subcutaneous injection dose of 18 microg/kg synthetic peptide was not effective to prevent the decrease of body and femur weight of the animals. The subcutaneous injection dose of 6.3 mg/kg prednisone elicited a decrease in content of femur calcium and in the level of serum calcium of the animals. The subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Leu-NH2, or Tyr-Gly-Gly-Phe-Leu-OH, or Tyr-Gly-Gly-Phe Gly-NH2 significantly increased the content of femur calcium and decreased the level of serum calcium of the animals. It was also observed that the subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Gly-OH, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Met-NH2 significantly increased the content of femur phosphorous and decreased the activity of ALP of the animals. PMID- 12375814 TI - Synthesis and immunological effect of thymic humoral factor-gamma 2 analogues. AB - Nine analogues of thymic humoral factor (THF)-gamma 2 were prepared by the solid phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin(PHA)-stimulated T-lymphocytes of uremic patients with infectious diseases were examined. The results were as follows: [Arg6]-THF gamma 2 exhibited higher restoring activity than that of our synthetic THF-gamma 2. [Sar4]-, [Val1]-, [Arg3]-, [Gly5]-, and [Asn3]-THF-gamma 2 were also active, but less potent than that of our synthetic THF-gamma 2. Three other peptides, [beta Ala4]-, [Arg2]-, and [Gln2]-THF-gamma 2, did not show any restoring activity on the impaired blastogenic response of uremic patients with infectious disease. PMID- 12375815 TI - Screening and simple purification of alanine dehydrogenase in Bacillus strains. AB - Alanine dehydrogenase (EC 1.4.1.1), in the presence of NAD+, catalyzes the reversible deamination of L-alanine. Screening of alanine dehydrogenase in bacillus strains was carried out to develop its utilization as an industrial and analytical catalyst. Eight bacillus strains were used, including Bacillus megaterium LA 199 which abundantly produces enzymes. Alanine dehydrogenase was purified simply from Bacillus megaterium LA 199 by heat treatment at pH 5.4, followed by DEAE-Sepharose CL-6B and Sepharose CL-2B chromotography. The enzyme consisted of six subunits with an identical molecular mass of 42.5 kDa. The Km were 1.17 x 10(-2) mM for NADH and 5.12 x 10(-2) mM for pyruvate. PMID- 12375816 TI - Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes. investigation of some kinetic properties. AB - Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH. PMID- 12375817 TI - The conceptual similarity of intonational tones and its effects on intertranscriber reliability. AB - Tests of intertranscriber agreement in prosodically-labeled corpora have been used as an objective performance measure of reliability. Reasonably high agreements among labelers have been found, but systematic disagreements exist, indicating that some intonational patterns are more difficult for transcribers to label while others are easier. This may be due to differences in the way transcribers distinguish between tonal labels for pitch events. We developed a method to map the subjective similarity space for the categories in an intonational transcription system. From this map, we derived a conceptual tone similarity similarity index indicating the distance between tone categories. This subjective similarity index is used to predict the intertranscriber reliability. It is found that tones which are conceptually similar result in higher transcriptional disagreements while tones which are conceptually dissimilar result in lower disagreement. PMID- 12375818 TI - Assimilation violation and spoken-language processing: a supplementary report. AB - Previous studies have shown that spoken-language processing is inhibited by violation of obligatory regressive assimilation. Weber (2001) replicated this inhibitory effect in a phoneme-monitoring study examining regressive place assimilation of nasals, but found facilitation for violation of progressive assimilation. German listeners detected the velar fricative [x] more quickly when fricative assimilation was violated (e.g., see text) than when no violation occurred (e.g., [baxt] or [see text]). It was argued that a combination of two factors caused facilitation: (1) progressive assimilation creates different restrictions for the monitoring target than regressive assimilation does, and (2) the sequences violating assimilation (e.g., *[ix]) are novel for German listeners and therefore facilitate fricative detection (novel popout). The present study tested progressive assimilation violation in non-novel sequences using the palatal fricative [c]. Stimuli either violated fricative assimilation (e.g., *[see text]) or did not (e.g., [see text]). This manipulation does not create novel sequences: sequences like *[see text] can occur across word boundaries. while *[ix] cannot. No facilitation was found. However, violation also did not significantly inhibit processing. The results confirm that facilitation depends on the combination of progressive assimilation with novelty of the sequence. PMID- 12375819 TI - Prosodic characteristics of skilled reading: fluency and expressiveness in 8-10 year-old readers. AB - Statistical methods of describing prosody were used to study fluency, expressiveness and their relationship among 8-10-year-old readers. 67 children were rated on fluency and expressiveness. The two were partially independent in the full sample: expressiveness rarely occurred without fluency, but fluency occurred without expressiveness. A balanced subsample of 24 was selected for closer instrumental and statistical analysis. There were robust relationships between fluency and measures associated with temporal organization.between expressiveness and variables associated with pitch mobility; and Interactions indicated that the relationships were not simple. Differences between groups depended on sentence content and position-expressive readers distinguished sentences more sharply according to content, and the groups diverged on some measures as the passage progressed. Also, measures associated primarily with either fluency or expression often showed secondary sensitivity to the other: temporal organization was associated with fluency, but worsened over time among inexpressive readers; and readers who were both fluent and expressive were distinctive in several respects. Some measures offer a basis for rules aimed at assigning individuals to skill categories, particularly the magnitude of pitch movements and reading time per syllable. The rules distinguish well among readers who were either at one of the extremes of skill, or fluent but inexpressive; it is harder to discriminate among the other readers (who have mixed skill patterns). The effects suggest psychological hypotheses about the underlying mechanisms. PMID- 12375820 TI - Intermittent cryogen spray cooling for optimal heat extraction during dermatologic laser treatment. AB - Fast heat extraction is critically important to obtain the maximal benefit of cryogen spray cooling (CSC) during laser therapy of shallow skin lesions, such as port wine stain birthmarks. However, a film of liquid cryogen can build up on the skin surface, impairing heat transfer due to the relatively low thermal conductivity and higher temperature of the film as compared to the impinging spray droplets. In an attempt to optimize the cryogen mass flux, while minimally affecting other spray characteristics, we apply a series of 10 ms spurts with variable duty cycles. Heat extraction dynamics during such intermittent cryogen sprays were measured using a custom-made metal-disc detector. The highest cooling rates were observed at moderate duty cycle levels. This confirms the presence, and offers a practical way to eliminate the adverse effect of liquid cryogen build-up on the sprayed surface. On the other hand, lower duty cycles allow a substantial reduction in the average rate of heat extraction, enabling less aggressive and more efficient CSC for treatment of deeper targets, such as hair follicles. PMID- 12375821 TI - Conversion of measured percentage depth dose to tissue maximum ratio values in stereotactic radiotherapy. AB - For many treatment planning systems tissue maximum ratios (TMR) are required as input. These tissue maximum ratios can be measured with a 3D computer-controlled water phantom; however, a TMR measurement option is not always available on such a system. Alternatively TMR values can be measured 'manually' by lowering the detector and raising the water phantom with the same distance, but this makes TMR measurements time consuming. Therefore we have derived TMR values from percentage depth dose (PDD) curves. Existing conversion methods express TMR values in terms of PDD, phantom scatter factor (Sp), and inverse square law. For stereotactic treatments circular fields ranging from 5-50 mm (19 cones) are used with the treatment planning system XKnife (Radionics). The calculation of TMR curves for this range is not possible with existing methods. This is because PDD curves of field sizes smaller than 5 mm (smallest cone size) are needed, but these cones are not provided. Besides, for field sizes smaller than 40 mm, the phantom scatter factor is difficult to determine and will introduce significant errors. To overcome these uncertainties, an alternative method has been developed to obtain TMR values from PDD data, where absolute doses are expressed in terms of PDD, total scatter factor and inverse square law. For each depth, the dose as a function of field size is fitted to a double exponential function. Then the TMR is calculated by taking the ratio of this function at the depth of interest and the reference depth, for the correct field size. For all 19 cones the total scatter factor and PDDs have been measured with a shielded diode in water for a 6 MV photon beam. Calculated TMR curves are compared with TMR values measured with a diode. The agreement is within 2%. Therefore this relatively simple conversion method meets the required accuracy for daily dose calculation in stereotactic radiotherapy. In principle this method could also be applied for other small field sizes such as those formed with a mini multileaf collimator. PMID- 12375822 TI - Electron beam therapy at extended SSDs: an analysis of output correction factors for a Mitsubishi linear accelerator. AB - The effects of extended source-to-surface distance (SSD) on the electron beam dose profiles were evaluated for various electron beam energies--6, 9, 12, 15 and 20 MeV-and the accuracy of various output correction methods was analysed on a Mitsubishi linear accelerator using a radiation field analyser (RFA). The dose fall-off region of the central axis depth-dose curves was nearly independent for SSDs up to 120 cm where as in the build-up region, a marginal reduction of surface dose was observed, particularly for lower energies and for smaller field sizes. Effective SSDs and virtual source distances were evaluated for field sizes ranging from 5 x 5 to 15 x 15 cm2 for various energies. Curve fitting was done with the measured outputs with various equations and coefficients were evaluated. The accuracy of the derived output correction factors by effective SSD, virtual source distance and curve-fit methods was assessed by evaluating correlation coefficients between the calculated and the measured values. The correlation coefficient was best with the linear-quadratic equation followed by the effective SSD method and the virtual source method. The output correction based on the linear-quadratic equation showed the best estimate of electron beam output at extended SSDs with an accuracy well within +/- 1%. The rapid reduction of dose due to the applicator-scattered component at d(max) point with an extended SSD was significant for the 5 x 5 cm2 applicator and lower energies. PMID- 12375823 TI - Two-dimensional pencil beam scaling: an improved proton dose algorithm for heterogeneous media. AB - New dose delivery techniques with proton beams, such as beam spot scanning or raster scanning, require fast and accurate dose algorithms which can be applied for treatment plan optimization in clinically acceptable timescales. The clinically required accuracy is particularly difficult to achieve for the irradiation of complex, heterogeneous regions of the patient's anatomy. Currently applied fast pencil beam dose calculations based on the standard inhomogeneity correction of pathlength scaling often cannot provide the accuracy required for clinically acceptable dose distributions. This could be achieved with sophisticated Monte Carlo simulations which are still unacceptably time consuming for use as dose engines in optimization calculations. We therefore present a new algorithm for proton dose calculations which aims to resolve the inherent problem between calculation speed and required clinical accuracy. First, a detailed derivation of the new concept, which is based on an additional scaling of the lateral proton fluence is provided. Then, the newly devised two-dimensional (2D) scaling method is tested for various geometries of different phantom materials. These include standard biological tissues such as bone, muscle and fat as well as air. A detailed comparison of the new 2D pencil beam scaling with the current standard pencil beam approach and Monte Carlo simulations, performed with GEANT, is presented. It was found that the new concept proposed allows calculation of absorbed dose with an accuracy almost equal to that achievable with Monte Carlo simulations while requiring only modestly increased calculation times in comparison to the standard pencil beam approach. It is believed that this new proton dose algorithm has the potential to significantly improve the treatment planning outcome for many clinical cases encountered in highly conformal proton therapy. PMID- 12375824 TI - Optimization of accelerator target and detector for portal imaging using Monte Carlo simulation and experiment. AB - Megavoltage portal images suffer from poor quality compared to those produced with kilovoltage x-rays. Several authors have shown that the image quality can be improved by modifying the linear accelerator to generate more low-energy photons. This work addresses the problem of using Monte Carlo simulation and experiment to optimize the beam and detector combination to maximize image quality for a given patient thickness. A simple model of the whole imaging chain was developed for investigation of the effect of the target parameters on the quality of the image. The optimum targets (6 mm thick aluminium and 1.6 mm copper) were installed in an Elekta SL25 accelerator. The first beam will be referred to as A16 and the second as Cu1.6. A tissue-equivalent contrast phantom was imaged with the 6 MV standard photon beam and the experimental beams with standard radiotherapy and mammography film/screen systems. The arrangement with a thin Al target/mammography system improved the contrast from 1.4 cm bone in 5 cm water to 19% compared with 2% for the standard arrangement of a thick, high-Z target/radiotherapy verification system. The linac/phantom/detector system was simulated with the BEAM/EGS4 Monte Carlo code. Contrast calculated from the predicted images was in good agreement with the experiment (to within 2.5%). The use of MC techniques to predict images accurately, taking into account the whole imaging system, is a powerful new method for portal imaging system design optimization. PMID- 12375825 TI - A CT image based deterministic approach to dosimetry and radiography simulations. AB - An integral transport equation based deterministic approach has been developed to compute individual specific absorbed dose distributions and photon flux distributions on a detector for image formation studies. An algorithm has been designed to take advantage of parallel processing platforms capable of processing large amounts of data imposed by the small pixel sizes of CT images. CT images have been used to form voxels that would represent both tissue variations and body geometry. Absorbed dose distributions calculated using the Visible Human dataset are provided. PMID- 12375826 TI - Performance of computed tomography for contrast agent concentration measurements with monochromatic x-ray beams: comparison of K-edge versus temporal subtraction. AB - We investigated the performance of monochromatic computed tomography for the quantification of contrast agent concentrations. Two subtraction methods (K-edge subtraction and temporal subtraction) were evaluated and compared theoretically and experimentally in terms of detection limit, precision and accuracy. Measurements were performed using synchrotron x-rays with Lucite phantoms (10 cm and 17.5 cm in diameter) containing iodine or gadolinium solutions ranging from 50 microg ml(-1) to 5 mg ml(-1). The experiments were carried out using monochromators developed at the European Synchrotron Radiation Facility (ESRF) medical beamline. The phantoms were imaged either above and below the contrast agent K-edge, or before and after the addition of the contrast agent. Both methods gave comparable performance for phantoms less than 10 cm in diameter. For large phantoms, equivalent to a human head, the temporal subtraction is more suitable for detecting elements such as iodine, keeping a reasonable x-ray dose delivered to the phantom. A good agreement was obtained between analytical calculations, simulations and measurements. The beam harmonic content was taken into account in the simulations. It explains the performance degradation with high contrast agent concentrations. The temporal subtraction technique has the advantage of energy tunability and is well suited for imaging elements, such as iodine or gadolinium, in highly absorbing samples. For technical reasons, the K edge method is preferable when the imaged organ is moving since the two measurements can be performed simultaneously, which is mandatory for obtaining a good subtraction. PMID- 12375827 TI - Quantitative fluorescence lifetime spectroscopy in turbid media: comparison of theoretical, experimental and computational methods. AB - A Monte Carlo model developed to simulate time-resolved fluorescence propagation in a semi-infinite turbid medium was validated against previously reported theoretical and computational results. Model simulations were compared to experimental measurements of fluorescence spectra and lifetimes on tissue simulating phantoms for single and dual fibre-optic probe geometries. Experiments and simulations using a single probe revealed that scattering-induced artefacts appeared in fluorescence emission spectra, while fluorescence lifetimes were unchanged. Although fluorescence lifetime measurements are generally more robust to scattering artefacts than are measurements of fluorescence spectra, in the dual-probe geometry scattering-induced changes in apparent lifetime were predicted both from diffusion theory and via Monte Carlo simulation, as well as measured experimentally. In all cases, the recovered apparent lifetime increased with increasing scattering and increasing source-detector separation. Diffusion theory consistently underestimated the magnitude of these increases in apparent lifetime (predicting a maximum increase of approximately 15%), while Monte Carlo simulations and experiment were closely matched (showing increases as large as 30%). These results indicate that quantitative simulations of time-resolved fluorescence propagation in turbid media will be important for accurate recovery of fluorophore lifetimes in biological spectroscopy and imaging applications. PMID- 12375828 TI - Anomalous Rayleigh scatter in dilute media. AB - Anomalous Rayleigh scatter is examined for dilute concentrations of the biomedically relevant element iodine in aqueous media including measurements with monochromatic synchrotron radiation in the vicinity of the iodine K-edge. The measurements agree with anomalous scatter-factor corrections to the form-factor approximation which has been shown to have good agreement with higher precision S matrix calculations for small angle scatter over a wide range of energies but has not been adequately tested at the edge. Monte Carlo modelling, including the modelling of polarized Compton and Rayleigh scattered x-rays, is used to determine the relative contributions of the scatter and fluorescent components at the detector as well as the modelling of self-absorption and relative dose in the determination of detection limits. A Rayleigh scatter minimum of 28 barns/sr was observed at an energy 10 +/- 5 eV below the K-edge of iodine at a position predicted from an evaluation of the dispersion integral that includes bound-bound resonance contributions. Minimum detectable concentrations for observation of the anomalous Rayleigh scatter feature at an exposure of 10 mSv, predicted for iodine and iron, are 1 mg ml(-1) and 10 mg ml(-1), respectively. Upper limits to detection of the feature imposed by degradation of the signal by self-absorption are 0.021 g cm(-2) and 0.0029 g cm(-2) radiation lengths, respectively. PMID- 12375829 TI - Effect of marrow on the high frequency ultrasonic properties of cancellous bone. AB - A number of investigators have performed in vitro measurements of cancellous bone to determine how various ultrasonic parameters depend on bone density and trabecular orientation. To facilitate handling and storage of bone specimens, the marrow is often removed prior to ultrasonic measurements. However, the assumption that marrow does not affect ultrasonic measurements at high frequencies (>1 MHz) has not been tested. The goal of this study is to determine the effect of marrow on the ultrasonic properties of bovine cancellous bone at frequencies greater than 1 MHz. Twelve specimens of cancellous bone were obtained from the proximal end of four bovine tibia. Ultrasonic measurements consisting of normalized broadband ultrasonic attenuation (nBUA), speed of sound (SOS) and apparent integrated backscatter (AIB) were measured in each specimen using 2.25 MHz (centre frequency) broadband ultrasonic pulses. These measurements were performed before and after marrow removal either along the superoinferior (SI) or mediolateral (ML) direction. SOS and nBUA showed no significant difference (p > 0.05) for either direction of propagation after marrow removal. AIB showed no significant difference in the SI direction. For the ML direction, a small but statistically significant difference (p = 0.044) was observed after marrow removal. PMID- 12375830 TI - Arranging optical fibres for the spatial resolution improvement of topographical images. AB - Optical topography is a method for visualization of conical activity. Ways of improving the spatial resolution of the topographical image with three arrangements of optical fibres are discussed. A distribution of sensitivity is obtained from the phantom experiment, and used to reconstruct topographical images of an activation area of the brain with the fibres in each arrangement. The correlations between the activated area and the corresponding topographical images are obtained, and the effective arrangement of the optical fibres for improved resolution is discussed. PMID- 12375831 TI - Sampling and reconstruction schemes for biomagnetic sensor arrays. AB - In this paper we generalize the approach of Ahonen et al (1993 IEEE Trans. Biomed. Eng. 40 859-69) to two-dimensional non-uniform sampling. The focus is on two main topics: (1) searching for the optimal sensor configuration on a planar measurement surface; and (2) reconstructing the magnetic field (a continuous function) from a discrete set of data points recorded with a finite number of sensors. A reconstruction formula for Bz is derived in the framework of the multidimensional Papoulis generalized sampling expansion (Papoulis A 1977 IEEE Trans. Circuits Syst. 24 652-4, Cheung K F 1993 Advanced Topics in Shannon Sampling and Interpolation Theory (New York: Springer) pp 85-119) in a particular case. Application of these considerations to the design of biomagnetic sensor arrays is also discussed. PMID- 12375832 TI - A haemodynamic model for the physiological interpretation of in vivo measurements of the concentration and oxygen saturation of haemoglobin. AB - We present a model that describes the effect of physiological parameters such as the speed of blood flow, local oxygen consumption, capillary recruitment, and vascular dilation/constriction on the concentration and oxygen saturation of haemoglobin in tissue. This model can be used to guide the physiological interpretation of haemodynamic and oximetric data collected in vivo with techniques such as optical imaging, near-infrared spectroscopy and functional magnetic resonance imaging. In addition to providing a formal description of well established results (exercise-induced hyperemia, reperfusion hyperoxia, decrease in the concentration of deoxyhaemoglobin induced by brain activity, measurement of arterial saturation by pulse oximetry, etc.), this model suggests that the superposition of asynchronous contributions from the arterial, capillary and venous haemoglobin compartments may be at the origin of observed out-of-phase oscillations of the oxyhaemoglobin and deoxyhaemoglobin concentrations in tissue. PMID- 12375833 TI - Recent advances in biologically sensitive field-effect transistors (BioFETs). PMID- 12375834 TI - A field test for the detection of peroxide-based explosives. AB - A rapid and simple field test for the detection of triacetone-triperoxide (TATP) and hexamethylenetriperoxidediamine (HMTD), two explosives which find significant illegal use, has been developed. Unknown samples are first treated with a catalase solution to remove hydrogen peroxide traces, in order to provide selectivity towards peroxide-based bleaching agents which are contained in commercial laundry detergents. Subsequently, the peroxide-based explosives are decomposed via UV irradiation, thus yielding hydrogen peroxide, which is determined by the horseradish peroxidase (POD) catalysed formation of the green radical cation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS). The limits of detection for this method are 8 x 10(-6) mol dm(-3) for TATP and 8 x 10(-7) mol dm(-3) for HMTD, respectively. As an option, p-hydroxyphenylacetic acid (pHPAA) may be used as peroxidase substrate, resulting in lower limits of detection (8 x 10(-7) mol dm(-3) for TATP and HMTD). The complete method uses a mobile setup to be applied under field conditions. PMID- 12375835 TI - Square wave voltammetric sensing of uric acid using the self-assembly of mercaptobenzimidazole. AB - The self-assembled monolayer of a heterocyclic thiol, mercaptobenzimidazole (MBI) on gold (Au) electrode is successfully utilized for the voltammetric sensing of uric acid (UA). The self-assembly of MBI separates the voltammetric signal of UA from the interfering ascorbate (AA). Selective detection of UA in the presence of a large excess of AA or the simultaneous detection of UA and AA is achieved at the MBI monolayer-modified electrode. This electrode can detect as low as 1 microM of UA in the presence of 100-fold excess of AA with excellent reproducibility. The practical utility of the electrode is demonstrated by measuring the concentration of UA in human serum. PMID- 12375836 TI - Comprehensive two dimensional separation based on coupling micellar electrokinetic chromatography with capillary isoelectric focusing. AB - Concentrating properties of the Capillary Isoelectric Focusing (CIEF) system with continuous whole-column-imaging detection were investigated for application as a second dimension in a comprehensive two-dimensional (2D) separation process. The concentration/separation/detection was completed within 4 min in a 300 microm inner diameter capillary. As the key to the successful coupling of CIEF to a first dimension separation, a novel interface was developed. A 10-port valve with two conditioning loops was used to perform both comprehensive collection and dialysis desalting of the first dimensional effluent, and as an interface coupling Micellar Electrokinetic Chromatography (MEKC) to CIEF. In the loop, salt and other unwanted first dimension effluent components were eliminated by dialysis and carrier ampholytes (CAs) were added. Peak broadening during the dialysis did not have significant impact on the CIEF separation because of its concentrating effect. Protein digests were first separated by MEKC followed by isoelectric point (pI) using whole-column-imaged CIEF. The dialysis interface allows general coupling of the whole-column-imaged CIEF to microscale separations. PMID- 12375837 TI - The electrochemical oxidation of homocysteine at boron-doped diamond electrodes with application to HPLC amperometric detection. AB - The electrochemical oxidation of homocysteine was studied at as-deposited and anodized (oxidized) boron-doped diamond (BDD) thin film electrodes with cyclic voltammetry, flow injection analysis and high-pressure liquid chromatography with amperometric detection. At anodized boron-doped diamond electrodes, highly reproducible, well-defined cyclic voltammograms for homocysteine oxidation were obtained in acidic media, while as-deposited diamond did not provide a detectable signal. In alkaline media, however, the oxidation response was obtained both at as-deposited and anodized diamond electrodes. The potential sweep rate dependence of homocysteine oxidation (peak currents for 1 mM homocysteine linearly proportional to v(1/2), within the range of 0.01 to 0.3 V s(-1)) indicates that the oxidation involves a diffusing species, with negligible adsorption on the BDD surface at this concentration. In the flow system, BDD exhibited a highly reproducible amperometric response, with a peak variation less than 2%. An extremely low detection limit (1 nM) was obtained at 1.6 V vs. Ag/AgCl. In addition, the determination of homocysteine in a standard mixture with aminothiols and disulfide compounds by means of isocratic reverse-phase HPLC with amperometric detection at diamond electrodes has been investigated. The results showed excellent separation, with a detection limit of 1 pmol and a linear range of three orders of magnitude. PMID- 12375838 TI - Frontal analysis on a microchip. AB - Conventional microchip applications involving capillary electrophoresis (CE) typically inject a sample along one channel and use an intersection of two channels to define the sample plug--the portion of sample to be analysed along a second channel. In contrast to this method of zone separation, frontal analysis proceeds by injecting sample continuously into a single channel or column. Frontal analysis is more common in macroscopic procedures but there are benefits in sensitivity and device density to its application to electrophoresis on microchips. This work compares conventional microchip zone analysis with frontal analysis in the separation of PCR products. Although we detect on the order of 5000 fluorophores with a compact instrument using the zone separation CE method, we found a several-fold increase in the effective signal-to-noise ratio by using a frontal analysis method. By removing the need for additional channels and reservoirs the frontal method would allow device densities to be significantly increased, potentially improving the cost-effectiveness of microchip analyses in applications such as medical diagnostics. PMID- 12375839 TI - Chemiluminescence biosensor chip based on a microreactor using carrier air flow for determination of uric acid in human serum. AB - A chemiluminescence biosensor on a chip coupled to a microfluidic system and a microreactor is described in this paper. The chemiluminescence biosensor measured 25 x 75 x 6.5 mm in dimension, and was readily produced in an analytical laboratory. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and luminol in the microreactor, and to immobilize uricase in the enzymatic reactor. The main characteristic of the biosensor was to introduce air as the carrier flow instead of the more common solution carrier for the first time. The uric acid was determined by a chemiluminescent (CL) reaction between the hydrogen peroxide produced from the enzymatic reactor and luminol under the catalysis of HRP in the microreactor. The linear range of the uric acid concentration was 1 to 100 mg L(-1) and the detection limit was 0.1 mg L(-1) (3sigma). PMID- 12375840 TI - Recovery of intact proteins from silver stained gels. AB - Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps. PMID- 12375841 TI - Calculation of electrophoretic mobility in mixed solvent buffers in capillary zone electrophoresis using a mixture response surface method. AB - The electrophoretic mobilities of three beta-blocker drugs, practolol, timolol and propranolol, have been measured in electrolyte systems with mixed binary and ternary water-methanol-ethanol solvents with acetic acid/sodium acetate as buffer using capillary electrophoresis. The highest mobilities for the analytes studied have been observed in pure aqueous, the lowest values in ethanolic buffers. The measured electrophoretic mobilities have been used to evaluate the accuracy of a mathematical model based on a mixture response surface method that expresses the mobility as a function of the solvent composition. Mean percentage error (MPE) has been computed considering experimental and calculated mobilities as an accuracy criterion. The obtained MPE for practolol, timolol and propranolol in the binary mixtures are between 0.9 and 2.6%, in the ternary water-methanol ethanol solvent system the MPE was about 2.7%. The MPE values resulting from the proposed equation lie within the experimental relative standard deviation values and can be considered as an acceptable error. PMID- 12375842 TI - Determination of isotopically labelled monoesterphthalates in urine by high performance liquid chromatography-mass spectrometry. AB - A method of analysis for monoesters of phthalic acid ('monoesterphthalates') in human urine has been developed. The method was needed to determine the hydrolysis and excretion efficiency of isotopically-labelled phthalate diesters ('phthalates') when they were fed to volunteers as part of a biomarker study to estimate total exposure to phthalates. The targeted substances were 13C monobutylphthalate (MBP), 2H4-monobutylphthalate (MBP), 2H4-monobenzylphthalate (MBeP), 13C-monocyclohexylphthalate (MCHP), 13C-monoethylhexylphthalate (MEHP), and 13C-monoisodecylphthalate (MIDP). The monoesters in urine were deconjugated enzymatically, extracted into solvent, and then determined by high performance liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure chemical ionisation in the negative ion mode. The limits of determination were 10 ng ml(-1) for MBP, MCHP, MBeP and MEHP, and 40 ng ml(-1) for MIDP. The recovery from urine spiked at 100 ng ml(-1) was in the range from 70 to 85% except for MIDP which was lower at 55%. The between-batch reproducibility of the analysis was in the range 8 to 17% (n = 6 batches on separate days). PMID- 12375843 TI - Application of central composite design in the optimisation of thermal desorption parameters for the trace level determination of the chemical warfare agent chloropicrin. AB - A quantitative trace-level thermal desorption gas chromatography mass spectrometry method was developed for chloropicrin CCl3NO2 using central composite design. Factors influencing the thermal decomposition were elucidated and optimum conditions for maximum response deduced. Four factors were investigated: desorption time, desorption temperature, valve temperature and line temperature. Only valve and line temperature influenced the response. The storage stability of chloropicrin on Tenax TA was investigated. Only the storage conditions affected recovery: no significant loss of chloropicrin was observed for spiked tubes stored in a refrigerator for up to 30 days. The application of central composite design to study thermal degradation of chloropicrin has not been described in the literature. The benefits in adopting this approach are reflected in the limit of detection, 22 ng on the sorbent tube (equivalent to 3.2 ppbv), the lowest atmospheric detection limit reported to date. PMID- 12375844 TI - One step and highly sensitive headspace solid-phase microextraction sample preparation approach for the analysis of methamphetamine and amphetamine in human urine. AB - A fiber-stable, repeatable and highly sensitive headspace solid-phase microextraction (HS-SPME) method was developed for the analysis of methamphetamine (MA) and amphetamine (AM) in urine using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. For sample preparation, the test specimen was placed in a 7 ml vial along with the additives (KOH and NaCl) and the internal standards (d8-MA and d8-AM), a glass insert containing heptafluorobutyric anhydride (HFBA) and heptafluorobutyric chloride (HFBCl) as derivatizing reagents was inserted into the vial, the vial was then sealed tightly. A SPME device with a 100 microm polydimethylsiloxane fiber was inserted into the vial and the fiber was exposed to the headspace in the insert, then the vial was heated and stirred at 100 degrees C and 600 rpm for 20 min for evaporation/adsorption/derivatization. The vaporized analytes (AM and MA) in the vial diffused into the glass insert though the holes on the insert, they absorbed onto the fiber, and then interacted with the vapor of the derivatizing reagent. Some of the analytes in the headspace of the glass insert may react with the vapor of the derivatizing reagent first, and then adsorb onto the fiber. The needle was finally removed and inserted into the injection port to desorb the analytes with the fiber exposed to the liner of the GC-MS system for analysis. By combining HFBCl and HFBA as derivatizing reagents and placing them in an insert, the HS-SPME method achieves high sensitivity for the analysis of AM and MA. Correlation coefficients derived from typical calibration curves in the 1.0-1700 ng ml(-1) range are 0.998 for MA and 0.994 for AM. The limits of detection and the limits of quantitation using a sample size of 1 ml are 0.3 and 1.0 ng ml(-1), respectively, for both MA and AM in urine specimens. Because the water hydrolysis of derivatizing reagent is much faster than the acylation reaction of the primary and secondary amines with the derivatizing reagent, the amphetamines cannot be acylated effectively over heated aqueous solution, and therefore this study provides a new acylation design in moisture surroundings. The proposed process also simplifies the procedure for urine sample preparation, and makes the automation of SPME possible. PMID- 12375845 TI - Solid phase microextraction with matrix assisted laser desorption/ionization introduction to mass spectrometry and ion mobility spectrometry. AB - Solid phase microextraction (SPME) with matrix assisted laser desorption/ionization (MALDI) introduction was coupled to mass spectrometry and ion mobility spectrometry. Nicotine and myoglobin in matrix 2,5-dihydroxybenzonic acid (DHB), enkephalin and substance P in alpha-cyano-4-hydroxy cinnaminic acid were investigated as the target compounds. The tip of an optical fiber was silanized for extraction of the analytes of interest from solution. The optical fiber thus served as the sample extraction surface, the support for the sample plus matrix, and the optical pipe to transfer the laser energy from the laser to the sample. The MALDI worked under atmospheric pressure, and both an ion mobility spectrometer and a quadrupole/time-of-flight mass spectrometer were used for the detection of the SPME/MALDI signal. The spectra obtained demonstrate the feasibility of the SPME with MALDI introduction to mass spectrometry instrumentation. PMID- 12375847 TI - Quantitation of trace components in liquid process streams by direct liquid sampling mass spectrometry. AB - This paper describes the development of a liquid sampling approach for trace analysis by electron impact ionization magnetic sector mass spectrometry, with no chromatographic separation. Development of a liquid sample introduction interface based on the principle of Programmable Temperature Vaporizing (PTV) GC injection is shown. A univariate procedure for the analysis of trace (mg kg(-1)) propanoic acid in acetic acid was developed. Results from the laboratory-based analysis of acetic acid are presented and compared with conventional GC analysis. The detection limit was 16 mg l(-1) and the speed of analysis was employed to acquire 30 scans per minute thus reducing the confidence intervals of the results and potentially allowing production plants to run much closer to sales specifications. For the analysis of more complex samples where the analytes contained no unique ions, multivariate analysis was employed and up to three scans per minute were acquired. Results from the analysis of an ester for six trace impurities are shown. Calibration was by partial least squares regression. The detection limits for these components were 20-30 mg kg(-1), well within the required product specifications. The system proved to be robust and easy to operate, with analyses being carried out over a period of several months requiring no maintenance of the spectrometer and only cleaning of the injection liner of the PTV injector on a monthly basis. PMID- 12375846 TI - Characterisation of the phosgene response of a membrane inlet 63Ni ion mobility spectrometer. AB - A membrane inlet 63Ni ion mobility spectrometer interfaced to a quadrupole mass spectrometer with permeation, exponential dilution approaches and syringe-based systems were used to characterise the ion mobility spectrometry (IMS) response to phosgene in dry air (water concentration less than 16.5 mg m(-3)). Phosgene produced a principle product ion in the negative mode with a reduced mobility of 2.16 cm2 V(-1) s(-1), with an unresolved artefact at higher concentrations having a reduced mobility of 2.32 cm2 V(-1) s(-1). The limit of detection of the system with a membrane inlet fitted was estimated to be less than 1 mg m(-3), with an upper limit to the dynamic range of 32 mg m(-3). Mass spectrometric data indicated the existence of [(H2O)nCl]-, [(H2O)nCl2]-; [(H2O)n(O2)Cl]-; [(H2O)n(O)Cl]-; and, [(H2O)n(CO2)Cl]-. The existence of two possible mechanisms for product ion formation is proposed: dissociative electron capture, as well as hydrolysis followed by electron capture. The effect of water contamination of the drying media within the ion mobility spectrometer was also investigated, and the effects were similar to those observed previously with studies on chlorine. Reduced mobility of the product ions was observed to decrease with increasing water contamination of the drying media used within the instrument, while limits of detection increased slightly to less than 2.4 mg m(-3), with no significant effect on dynamic ranges of response or resolution. Preliminary results also indicated a positive mode response for phosgene, albeit at significantly higher concentrations to those observed in the negative mode. PMID- 12375848 TI - Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry. AB - Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs. PMID- 12375849 TI - Label dilution method: a novel tool for bioligand interaction studies using bead injection in the lab-on-valve format. AB - This work introduces a novel method, label dilution, which is analogous to the well-established isotope dilution method. The principle is tested on a model system of commercially available antibodies and protein-coated Sepharose beads and implemented using micro-bead injection in the lab-on-valve format. This micro scale method uses a labeled form of the target molecule as an internal standard. Label dilution employs ratiometric measurements using the absorbance signals from the label and the target molecules for quantitative determination of an analyte. The label dilution method is shown to discriminate between selective and non selective binding and provides a means for monitoring bioligand interactions in real time. With a detection limit of 470 ng of IgG, this method provides a sensitive, automated technique for the determination of low-level analytes in complex samples. This technique has been developed with the aim of using it to facilitate diabetes research in which the interactions between autoantibodies and the molecules they target are a central focus. PMID- 12375850 TI - Internally-referenced resonant mirror for chemical and biochemical sensing. AB - The resonant mirror sensor is a planar optical sensor platform that uses frustrated total internal reflection to couple light into and out of a leaky waveguiding layer. The evanescent wave associated with the dielectric structure is very sensitive to changes in surface refractive index caused by the binding of macromolecules to immobilised proteins or other biorecognition species such as antibodies. However, such variations can also be generated by variations in the bulk analyte solution, via changes in the composition or temperature. In the device described here, an additional buried resonant mirror layer is incorporated into the sensor structure generating an internal reference resonant mirror. The efficacy of this internal reference system is demonstrated in both chemical and immunological systems--as a pH sensor monitoring the absorption of an encapsulated sulfonephthalein dye, and as a refractive index sensor measuring the adsorption of anti-protein A and binding of its corresponding antigen. In both cases the internally referenced resonant mirror provides a means by which errors due to fluctuations in light intensity, temperature and bulk composition may be accounted for. PMID- 12375851 TI - Quantitative analysis of NO2 in the presence of CO using a single tungsten oxide semiconductor sensor and dynamic signal processing. AB - We demonstrate that NO2 can be quantitatively analysed in the presence of CO using a single tungsten oxide based resistive gas sensor. The working temperature of the sensor was modulated between 190 and 380 degrees C and its dynamic response to different concentrations of CO, NO2, and CO + NO2 mixtures was monitored. Either the fast Fourier transform (FFT) or the discrete wavelet transform (DWT) was used to extract important features from the sensor response. These features were then input to different (statistical and neural) pattern recognition methods. The species considered can be discriminated with a success rate higher than 90% using a Fuzzy ARTMAP or a radial basis function neural network. The concentrations of the gases studied can be accurately predicted, by using the DWT coupled to partial least squares (PLS) models. The correlation coefficients of the predicted versus real concentrations were 0.923, 0.870 and 0.866 for CO, NO2, and NO2 in CO + NO2 mixtures, respectively. PMID- 12375852 TI - An indirect conductimetric screening method for the detection of antibiotic residues in bovine kidneys. AB - An indirect conductimetric screening method using three test bacterium-medium combinations was developed for rapid detection of antibiotic residues in bovine carcasses. The detection time (DT), i.e. the point when the growth of the test bacterium was detected, was determined by observing the rate of change in the conductance plotted against time. This detection time averaged half of the reference time recorded by the instrument software. Total change in conductance (TC) was used as a further measure of growth. Threshold values for DT and TC were determined with inhibitor-free kidney samples. The presence of a residue was indicated if the DT exceeded the respective threshold value and was confirmed if the TC remained below the TC threshold value. The limits of detection (LODs) determined with fortified samples were at about or below the MRLs for cephalexin, chlortetracycline, ciprofloxacin, dihydrostreptomycin, enrofloxacin, oxytetracycline and penicillin G. The LODs for penicillin G, oxytetracycline and the sum of enrofloxacin and ciprofloxacin were also estimated with incurred samples; these samples were also analysed using liquid chromatography. The LODs determined with fortified and incurred samples were in close agreement. Given its rapid detection, good sensitivity to a wide range of antibiotics and ease of performance, the indirect conductimetric method developed here would seem to offer an appealing alternative to agar diffusion tests. PMID- 12375853 TI - Optimised uncertainty in food analysis: application and comparison between four contrasting 'analyte-commodity' combinations. AB - The optimised uncertainty (OU) methodology is applied across a range of analyte commodity combinations. The commodities and respective analytes under investigation were chosen to encompass a range of input factors: measurement costs (sampling and analytical), sampling uncertainties, analytical uncertainties and potential consequence costs which may be incurred as a result of misclassification. Two types of misclassification are identified-false compliance and false non-compliance. These terms can be used across a wide range of foodstuffs that have regulations requiring either minimum compositional requirements, maximum contaminant allowances or compositional specifications. The latter refers to foodstuffs with regulations that state an allowable tolerance around the compositional specification, i.e. the upper specification limit (USL) and the lower specification limit (LSL). The traditional OU methodology has been adapted so that it is applicable in these cases and has been successfully applied in practice. The Newton-Raphson method has been used to determine the optimal uncertainty value for the two case studies in which analyte concentration is assessed against a 'single threshold' regulatory requirement. This numerical method was shown to give a value of the optimal uncertainty that is practically identical to that given by the previously used method of visual inspection. The expectation of financial loss was reduced by an average of 65% over the four commodities by the application of the OU methodology, showing the benefit of the method. PMID- 12375854 TI - Principal components analysis for the visualisation of multidimensional chemical data acquired by scanning Raman microspectroscopy. AB - Raman microspectroscopy is ideally suited to surface analysis as it allows detailed chemical information to be acquired from surfaces at a relatively high spatial resolution (typically 1 microm). Using a motorised sample table or probe, it is possible to raster scan a surface to obtain spatially resolved chemical information. Visualisation of the acquired data is a problem, however, as the spectrum acquired at each point can contain several hundred individual intensity measurements. Existing visualisation methods are limited to plotting each scanned point with an intensity determined from the measured intensity at a single wavenumber, or the similarly between the point's spectrum and a reference spectrum. Such methods are wasteful as a lot of acquired information is discarded, and results are prone to misinterpretation due to background variance and instrumental noise. In this paper we introduce a new method that uses principal components analysis (PCA) to reduce the spectrum at each point to three factors that are then used to define the red, green and blue components of the corresponding point on a false colour map. To increase the effective resolution, interpolation is used to approximate the colours corresponding to points between those actually scanned. To demonstrate the technique, the internal surface of a beverage can, contaminated with a 40 microm diameter carbonised oven impurity, consisting mainly of sp2- and sp3-hybridised saturated carbon bonds, has been used as a case study. PMID- 12375855 TI - Study on the electrochemiluminescence behavior of ABEI and its application in DNA hybridization analysis. AB - The electrogenerated chemiluminescence (ECL) behavior of N-(4-aminobutyl)-N ethylisoluminol (ABEI) was studied and it was found that ABEI could produce emission light when oxidized at a +1.0 V (vs. Ag/AgCl) potential in alkaline solution. The addition of H2O2 markedly improved the ECL sensitivity. The pH value of the solution as well as the H2O2 concentration and working potential all have influences on the ECL response. Under optimal conditions, ABEI can be detected in the range 1.3 x 10(-6)-6.5 x 10(-12) mol L(-1). A detection limit of 2.2 x 10(-12) mol L(-1) for ABEI was obtained at a signal-to-noise ratio of 3. ABEI was then used as a marker to label a known sequence oligonucleotide, which was used as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were evaluated by the ECL measurements. The results showed that only a complementary sequence could form a double-stranded DNA with the DNA probe and give a strong ECL response. A three-base mismatch sequence and non-complementary sequence have no response. The intensity of the ECL was linearly related to the concentration of the complementary sequence in the range 9.6 x 10(-11)-9.6 x 10( 8) mol L(-1), the detection limit was 3.0 x 10(-11) mol L(-1). PMID- 12375856 TI - Pressure assisted chelating extraction: a novel technique for digesting metals in solid matrices. AB - This work describes a novel technique for the digestion of metals in solid matrices. The technique is called pressure assisted chelating extraction (PACE). In a typical procedure, a solid sample is placed in a stainless steel cell and is mixed with appropriate chelating agents. Using a programmed sequence of temperature, static time, pressure and thermal equilibration available in ASE 200, the metal is removed under moderate temperature (up to 200 degrees C) and pressure (up to 3000 psi). PACE achieves metal recovery that is equivalent to that of wet digestion techniques and also provides for a clean and safe operation by substituting the strong acids commonly used during wet digestion with chelating agents. It uses less solvents and significantly less time (minutes vs. hours) for metal digestion. PACE has been validated using certified standard reference materials (SRMs) including industrial sludge, buffalo river sediments and coal fly ash. The total time required to remove metals was approximately 20 min. Results show that the PACE system provides an ideal platform for efficient, rapid, and safe metal digestion. Good agreement between measured and reference values for Pb, Mn, and Cu were found with recoveries averaging between 80 and 101% and a relative standard deviation of less than 5%. This approach may provide an alternative digestion technique for environmental samples, alloys, biological materials and samples of geological importance. The potential advantage offered lies in non-destruction of the sample, automation and the exclusion of concentrated mineral acids during the digestion procedure. PMID- 12375857 TI - Persistent organic pollutants in Detroit River suspended sediments: polychlorinated dibenzo-p-dioxins and dibenzofurans, dioxin-like polychlorinated biphenyls and polychlorinated naphthalenes. AB - Suspended sediments from the Detroit River were collected in 1999 and 2000 using sediment traps at sites ranging from western Lake Erie to southern Lake St. Clair and analyzed to determine the spatial distributions of contaminants including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs), dioxin-like PCBs (DLPCBs) and polychlorinated naphthalenes (PCNs). Concentrations of all three contaminant classes were clearly elevated at sites in the lower reaches of the river in the Trenton Channel. The potential influence of the Trenton Channel as a source of contamination to western Lake Erie was further evidenced by PCDD/PCDF homologue profiles, which indicated a contribution from chemical manufacturing in addition to the normal background combustion profile. Toxic equivalents (TEQs) for PCDDs/PCDFs generally exceeded those for DLPCBs; combined total TEQs in July 2000 for these two compound classes ranged from 2.30 pg/g in southern Lake St. Clair to 306 pg/g at a station just downstream of the outflow of Monguagon Creek in the Trenton Channel. The spatial distribution of PCN contamination was similar to that of PCDDs/PCDFs and DLPCBs, with the highest level of total PCNs (8200 ng/g) detected at a site in the Trenton Channel near Elizabeth Park; TEQs for PCNs in the Trenton Channel ranged from 73 to 3300 pg/g. The data indicate that PCNs represent a significant contribution to dioxin-like biological activity in Detroit River suspended sediments. PMID- 12375858 TI - Use of the metastable atom bombardment (MAB) ion source for the elimination of PCDE interference in PCDD/PCDF analysis. AB - The analysis of polychlorinated dioxins and furans (PCDDs/PCDFs) can produce erroneous results when polychloro diphenyl ethers (PCDEs) are present because they fragment and rearrange under electron ionization to give isobaric ions with PCDFs. Mass information must be generated to indicate the occurrence of possible PCDEs but uncertainty still exists if a PCDE congener has the same retention time as a PCDF congener. The metastable atom bombardment (MAB) ion source does not transfer enough energy to PCDE upon ionization to fragment the molecular ion, thus eliminating the need for PCDE tracking in PCDF analysis. Experiments were conducted with different gases, representing different ionization energies, to demonstrate that PCDE interference can be eliminated from PCDF analysis. PMID- 12375859 TI - Correlation of PCDD/PCDF and CO values in a MSW incinerator--indication of memory effects in the high temperature/cooling section. AB - The correlation of PCDD/PCDF levels with the CO emissions in a full-scale municipal waste incinerator was assessed during a four-week measurement effort. PCDD/PCDF concentrations in fly ashes-containing more than 99% of the total PCDD/PCDF burden of the fluidized bed incinerator (FBI)-were measured and compared with the emitted CO concentrations. The CO concentration during the sampling time showed no significant correlation to the PCDD/ PCDF amount in fly ash (R2 = 0.078). However, a comparison of the time integrated CO concentration several hours before sampling lead to a correlation with the PCDD/PCDF burden. Maximum correlation was found for the time integrated CO values of 3 and 4 h before sampling (R2 = 0.467 and R2 = 0.457 respectively). This indicates a memory effect in the high temperature cooling section of several hours. Possible mechanisms leading to the memory effect are discussed. The correlation of PCDD/PCDF with CO concentration demonstrate that the combustion conditions play an important role for PCDD/PCDF formation in FBIs. However the variability in the correlation of CO to PCDD/PCDF levels show that other factors have a significant influence on PCDD/PCDF formation. PMID- 12375860 TI - Prediction of physico-chemical properties for PCs/DFs using the UNIFAC model with an alternative approximation for group assignment. AB - The original-type UNIFAC model was used to predict the environmentally important physico-chemical properties of PCDDs/DFs, such as aqueous solubility, Henry's law constant, and 1-octanol/water partition coefficient, through the UNIFAC-derived infinite dilution activity coefficient. In this application, we suggest an alternative approximation that the aromatic ether group AC-O in PCDD/DF molecules is replaced with the aliphatic ether group CH-O, because the AC-O group is not available in the conventional UNIFAC model. With this approximation, the ability of the UNIFAC model to predict those properties was examined by comparing with experimental data. The UNIFAC model provided comparatively good estimation results. From these results, it is shown that the alternative approximation is useful for the UNIFAC estimation of physico-chemical properties for PCDDs/DFs. Furthermore, the predicted solubilities of 2,3,7,8-T4CDD and O8CDD in organic solvents and the co-solvency effect on solubility of PCDDs in methanol/water mixture indicate that the UNIFAC calculation presented here could well predict the physico-chemical properties of PCDDs/DFs in various solution conditions. PMID- 12375861 TI - Differential bioavailability of field-weathered p,p'-DDE to plants of the Cucurbita and Cucumis genera. AB - Field experiments were conducted to assess the bioavailability of weathered p,p' DDE in soil to plants in the Cucurbita (squash, pumpkin) and Cucumis (cucumber, melon) genera. As expected, significant variability exists in the uptake of p,p' DDE between plants of different genera. Root:soil concentration factors, defined as the ratio of p,p'-DDE (ng/g, dry weight) in the roots to that in the soil, approach 1.8 for cucumbers/melons and 16 for squash/pumpkin. However, significant differences were also observed among varieties of squash and pumpkin, with greater than an order of magnitude variation in the root:soil concentration factors and up to two orders of magnitude difference in the absolute amount of contaminant present within the plant. Although root systems routinely contain the highest concentration of p,p'-DDE (ng/g), this compartment comprises less than 2% of the total plant biomass. In all varieties but one, more than 86% of the extracted pollutant was in the shoot system. For two of varieties of Cucurbita pepo, concentrations of p,p'-DDE in the stems reached 1.1-2.2 mg/g and estimations of percent contaminant extraction from the soil ranged from 0.40% to 2.4%. These values approach those observed in the phytoremediation of heavy metals by "hyperaccumulating" species and indicate the potential for a plant based remediation approach to soils contaminated with persistent organic pollutants. PMID- 12375862 TI - PCDD/PCDF congener profiles in soil and herbage samples collected in the vicinity of a municipal waste incinerator before and after pronounced reductions of PCDD/PCDF emissions from the facility. AB - Congener profiles are the fractional distribution of polychlorinated dibenzo-p dioxins (PCDDs) and dibenzofurans (PCDFs) congeners in an environmental release, or in an environmental or biological sample. In 1999, an adaptation to the EU legislation on pollutant emissions from the stack was carried out in an old municipal solid waste incinerator (MSWI) from Montcada (Barcelona, Spain). The main goal of the present study was to determine if the environmental PCDD/F levels in the area under direct influence of the facility were mainly due to PCDD/F emissions from the plant. For this purpose, soil and herbage samples were collected near the MSWI before (1998) and after (2000) the technical improvements were performed. PCDD/F congener profiles were determined and compared with those from samples collected in a suburban area of Constanti (Tarragona, Spain) outside of direct emissions from any MSWI. The results of the present study suggest that the MSWI here assessed is not the main responsible for the environmental PCDD/F concentrations in the area under evaluation. Other PCDD/F emission sources in the same area seem also to have a notable impact on the atmospheric levels of these pollutants. PMID- 12375863 TI - Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and non-ortho, mono-ortho chlorine substituted biphenyls in Japanese human liver and adipose tissue. AB - We measured PCDDs/DFs levels in Japanese human livers and adipose tissues in 1999, and TEQ were calculated with WHO TEF. The mean total levels of PCDDs/DFs in livers and adipose tissues were 57 pg TEQ/g on a lipid basis and 49 pg TEQ/g on a lipid basis, respectively. 1,2,3,6,7,8-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF and 1,2,3,4,6,7,8-HpCDF concentrations in livers considerably differed from those in 1989 (p < 0.05). The mean non-ortho-chlorine substituted biphenyls levels showed 20 pg TEQ/g on a lipid basis and 17 pg TEQ/g on a lipid basis in livers and adipose tissues, respectively. In livers, the mean of 3,3',4,4'-TCB concentrations was 131 pg/g on a lipid basis, and 7.7-fold higher than that in 1989. The mean total mono-ortho-chlorine substituted biphenyls level was 13.0 pg TEQ/g on a lipid basis in livers and 21.6 pg TEQ/g on a lipid basis in adipose tissues. 3,3',4,4',5-PeCB and 3,3',4,4',5,5'-HxCB levels decreased in adipose tissues, and 3,3',4,4',5-PeCB level only decreased in livers. PCDDs, PCDFs, and mono- and non-ortho-chlorine substituted biphenyls levels may have decreased in livers and adipose tissues because of a governmental policy on dioxins discharge for the decade. Then, we estimated the correlations of PCDDs, PCDFs and the related compound levels between livers and adipose tissues. The correlative PCDDs congeners may have had a similar behavior to that between liver and adipose tissue. On the contrary, most PCDFs isomers may have different behavior between liver and adipose tissue, while 2',3,4,4',5-PeCB (IUPAC No. 123) may also have a different behavior between liver and adipose tissue. PMID- 12375864 TI - Long-term fate of the herbicide cinosulfuron in lysimeters planted with rice over four consecutive years. AB - In order to elucidate the long-term fate of the sulfonylurea herbicide cinosulfuron, the 14C-labelled chemical was applied to a clay loam soil, encased in two lysimeters, 22 days after rice (Oryza sativa L.) transplanting, and rice plants were grown for four consecutive years. Throughout the experimental period, leaching through soil profiles, absorption and translocation by rice plants, and distribution of 14C by downward movement in the soil layers were clarified. The total volume of leachates collected through the lysimeter soil over the four years amounted to 168 and 146 L in lysimeters I and II, respectively. The leachates contained 2.43% and 2.99% of the originally applied 14C-radioactivity, corresponding to an average concentration of 0.29 and 0.41 microg/L as the cinosulfuron equivalent in lysimeters I and II, respectively. The total 14C radioactivity translocated to rice plants in the third and fourth year was 0.69% and 0.60% (lysimeter I), and 1.02% and 0.84% (lysimeter II) of the 14C applied, respectively. Larger amounts of cinosulfuron equivalents (0.54-0.75%) remained in the straw in the fourth year than in any other parts. The 14C-radioactivities distributed down to a depth of 70 cm after four years were 56.71-57.52% of the 14C applied, indicating the continuous downward movement and degradation of cinosulfuron in soil. The non-extractable residues were more than 88% of the soil radioactivity and some 45-48% of them was incorporated into the humin fraction. The 14C-radioactivity partitioned into the aqueous phase was nearly 30% of the extractable 14C, suggesting strongly that cinosulfuron was degraded into some polar products during the experimental period. It was found out in a supplemental investigation that flooding and constant higher temperature enhanced mineralization of [14C]cinosulfuron to 14CO2 in soil, indicating the possibility of chemical hydrolysis and microbial degradation of the compound in the flooded lysimeter soil. PMID- 12375865 TI - Analysis of six relevant toxaphene congeners in biological samples using ion trap MS/MS. AB - The quantification of six polychlorinated bornanes (CHBs) was studied using ion trap MS/MS. The significance of the selection of parent ions (Ip) and daughter ions (Id) on the detection of these toxaphene congeners was assessed in standard solution and biological samples. Our results indicate that different Ip and Id, selected at either low or high mass-to-charge (m/z) ratios, influence drastically the response factor of the CHBs and the chemical noise observed. For the octachlorinated toxaphene congeners (Parlar-26 (P-26), Parlar-40/41 (P-40/41), Parlar-44 (P-44)), the detection performance of the ion trap MS/MS is similar whether Ip and Id were chosen at low or high m/z ratios. However, the selection of Ip and Id at high m/z ratios clearly enhances the detection of the nonachlorinated toxaphene congeners (Parlar-50 (P-50), Parlar-62 (P-62)). The improved method, which selects Ip and Id at low m/z ratios for P-26, P-40/41 and P-44 and at high m/z ratios for P-50 and P-62, permitted to obtain low detection limits as well as repeatable and accurate results. PMID- 12375866 TI - Semiconductor-mediated photocatalysed degradation of two selected priority organic pollutants, benzidine and 1,2-diphenylhydrazine, in aqueous suspension. AB - The photocatalysed degradation of two selected priority organic pollutants, namely benzidine (1) and 1,2-diphenylhydrazine (DPH, 2) has been investigated in aqueous suspensions of titanium dioxide (TiO2) under a variety of conditions employing a pH-stat technique. The degradation was studied by monitoring the change in substrate concentration of the model compound employing HPLC analysis and the decrease in total organic carbon content, respectively, as a function of irradiation time. The degradation kinetics were studied under different conditions such as reaction pH, substrate and photocatalyst concentration, type of TiO2 photocatalyst and the presence of alternative additives such as H2O2, KBrO3 and (NH4)2S2O8 besides molecular oxygen. The degradation rates and the photonic efficiencies were found to be strongly influenced by the above parameters. Toxicity tests for the irradiated samples of benzidine measuring the luminescence of bacteria Vibrio fischeri after 30 min of incubation were also performed. 4-amino-biphenyl (7) and hydroquinone (13) were identified as intermediate products by GC/MS technique and probable pathways for the formation of the products are proposed. PMID- 12375867 TI - PCB levels and congener patterns from Korean municipal waste incinerator stack emissions. AB - Congener specific polychlorinated biphenyl (PCB) data from the stack gas of nine Korean municipal waste incinerators was used to determine characteristic congener patterns of emitted PCBs. Principal component analysis revealed three classes of incinerators according to their pattern of PCB congener emissions: those resembling the background sampling material; those producing large quantities of a few tetra-chlorinated congeners; those producing large proportions of mono (MO ) and non-ortho (NO-) congeners relative to di-ortho (DO-) levels. Also, correlations between individual PCB congeners and polychlorinated dibenzo-p dioxins and furans (PCDD/Fs) were discovered for several NO-PCBs and tetra and penta chlorinated PCDFs. Full PCB congener data is presented along with operating conditions for each incinerator. PMID- 12375868 TI - Formation and transport of PCDD/Fs in the packed bed of soil containing organic chloride during a thermal remediation process. AB - The authors previously proposed the concept of a new thermal remediation process for particulate/powder materials contaminated by polychlorinated dibenzo-p dioxins and dibenzofurans (PCDD/Fs) and experimentally verified its validity on the basis of process efficiency. However, contaminees such as soils and fly ashes from waste incinerators often contain a considerable amount of other chlorides, which may act as a main source of chlorine in the formation of PCDD/Fs via thermal processes. The present study aims to examine the formation and transport of PCDD/Fs in the packed bed of soil containing a chloride during the process. Polyvinyl chloride (PVC) polymer was mixed with soil sample as an organic chloride model. A laboratory-scale apparatus was employed as a process simulator. Further, a technique to quench the process was applied to observe the concentration distribution of PCDD/Fs in the solid bed in the vertical direction. The result shows that the PCDFs tend to form dominantly in the high temperature (calcination and/or combustion) zone and are successively trapped in the low temperature (wet) zone. Especially, TeCDF is the most dominant homologue contained in the wet zone and outlet gas. Although PCDD/Fs are once trapped at the wet zone, the concentration of the remediated materials gives fairly low value (1.9 pg/g-dry, 0.04 pg-TEQ/g-dry). It signifies that organic chlorides mingled in the solid contaminee not affect the removal efficiency of PCDD/Fs in the process. Nevertheless, attention should be paid to the potential emission of PCDD/Fs in the outlet gas due to the presence of organic chloride in the soil. PMID- 12375869 TI - Levels of explanation. PMID- 12375870 TI - Amodal completion and the perception of depth without binocular correspondence. AB - Half-occlusions and illusory contours have recently been used to show that depth can be perceived in the absence of binocular correspondence and that there is more to stereopsis than solving the correspondence problem. In the present study we show a new way for depth to be assigned in the absence of binocular correspondence, namely amodal completion. Although an occluder removed all possibility of direct binocular matching, subjects consistently assigned the correct depth (convexity or concavity) to partially occluded 'folded cards' stimuli. Our results highlight the importance of more global, surface-based processes in stereopsis. PMID- 12375871 TI - Propagation of depth information from local regions in 3-D scenes. AB - The effects of regions with local linear perspective on judgments of the depth separation between two objects in a scene were investigated for scenes consisting of a ground plane, a quadrilateral region, and two poles separated in depth. The poles were either inside or outside the region. Two types of displays were used: motion-parallax dot displays, and a still photograph of a real scene on which computer-generated regions and objects were superimposed. Judged depth separations were greater for regions with greater linear perspective, both for objects inside and outside the region. In most cases, the effect of the region's shape was reduced for objects outside the region. Some systematic differences were found between the two types of displays. For example, adding a region with any shape increased judged depth in motion-parallax displays, but only high perspective regions increased judged depth in real-scene displays. We conclude that depth information present in local regions affects perceived depth within the region, and that these effects propagate, to a lesser degree, outside the region. PMID- 12375872 TI - The role of ON- and OFF-channel processing in the detection of bilateral symmetry. AB - We present evidence that grouping for luminance does not take precedence over the detection of bilaterally symmetrical patterns. Using single-axis and double-axis images, we found that element pairs within which luminance is held constant drive symmetry-detection mechanisms more effectively than pairs within which luminance varies. Moreover, the performance decrement observed for patterns defined by element pairs within which luminance varies is not specific to interchannel variation. Luminance variation within the ON and OFF channels has the same effect as variation between the channels on the performance of axis-orientation identification tasks. It is argued that this constitutes possible evidence for subchannels within the ON and OFF channels. One of the characteristics of the subchannels is that each processes only a limited range of luminance steps. The implications of this type of luminance processing for the detection of symmetry in the visual scene are discussed. PMID- 12375873 TI - Flank transparency: the effects of gaps, line spacing, and apparent motion. AB - We analyze the properties of a dynamic color-spreading display created by adding narrow colored flanks to rigidly moving black lines where these lines fall in the interior of a stationary virtual disk. This recently introduced display (Wollschlager et al, 2001 Perception 30 1423-1426) induces the perception of a colored transparent disk bounded by strong illusory contours. It provides a link between the classical neon-color-spreading effect and edge-induced color spreading as discussed by Pinna et al (2001 Vision Research 41 2669-2676). We performed three experiments to quantitatively study (i) the enhancing influence of apparent motion; (ii) the degrading effect of small spatial discontinuities (gaps) between lines and flanks; and (iii) the spatial extent of the color spreading. We interpret the results as due to varying degrees of objecthood of the dynamically specified disk: increased objecthood leads to increased surface visibility in both contour and color. PMID- 12375874 TI - Factors contributing to the implicit dynamic quality of static abstract designs. AB - Participants rated the dynamic quality of a set of twelve-element nonrepresentational or abstract visual designs each composed of one of four types of triangles or four types of quadrilaterals. We investigated the contribution to the perceived or implicit dynamics of the design of the four factors edge alignment of compositional elements, physical weight distribution about the horizontal axis, activity directions within the designs, and type of compositional element. It was found that edge alignment of elements was the most influential factor contributing to the dynamics both of triangle and of quadrilateral designs. In addition, dynamics was found to be positively correlated with the number of perceived activity directions within both types of stimuli. Triangle designs, but not quadrilateral ones, with greater structural weight above the horizontal axis were rated as more dynamic. Results suggest that implicit dynamics of nonrepresentational designs arises from a percept based on a global spatial analysis of the stimulus characteristics studied. PMID- 12375875 TI - Representation of the gender of human faces by infants: a preference for female. AB - Six experiments based on visual preference procedures were conducted to examine gender categorization of female versus male faces by infants aged 3 to 4 months. In experiment 1, infants familiarized with male faces preferred a female face over a novel male face, but infants familiarized with female faces divided their attention between a male face and a novel female face. Experiment 2 demonstrated that these asymmetrical categorization results were likely due to a spontaneous preference for females. Experiments 3 and 4 showed that the preference for females was based on processing of the internal facial features in their upright orientation, and not the result of external hair cues or higher-contrast internal facial features. While experiments 1 through 4 were conducted with infants reared with female primary caregivers, experiment 5 provided evidence that infants reared with male primary caregivers tend to show a spontaneous preference for males. Experiment 6 showed that infants reared with female primary caregivers displayed recognition memory for individual females, but not males. These results suggest that representation of information about human faces by young infants may be influenced by the gender of the primary caregiver. PMID- 12375876 TI - Attention to faces: a change-blindness study. AB - What strategies does human vision use to attend to faces and their features? How are such strategies altered by 2-D inversion or photographic negation? We report two experiments in which these questions were studied with the flicker task of the change-blindness literature. In experiment 1 we studied detection of configural changes to the eyes or mouth, and found that upright faces receive more efficient attention than inverted faces, and that faces shown with normal contrast receive more efficient attention than faces shown in photographic negative. Moreover, eyes receive greater attention than the mouth. In experiment 2 we studied detection of local changes to the eyes or mouth, and found the same results. It is well known that inversion and negation impair the perception and recognition of faces. The experiments presented here extend previous findings by showing that inversion and negation also impair attention to faces. PMID- 12375877 TI - Effects of visual-field inversions on the reverse-perspective illusion. AB - The 'reverse-perspective' illusion entails the apparent motion of a stationary scene painted in relief and containing misleading depth cues. We have found that, using prism goggles to induce horizontal or vertical visual-field reversals, the illusory motion is greatly reduced or eliminated in the direction for which the goggles reverse the visual field. We argue that the illusion is a consequence of the observer's inability to reconcile changes in visual information due to body movement with implicit knowledge concerning anticipated changes. As such, the reverse-perspective illusion may prove to be useful in the study of the integration of linear perspective and motion parallax information. PMID- 12375878 TI - Perceived eye size is larger in happy faces than in surprised faces. AB - We demonstrated that perceived eye size is affected by facial expression. Stimuli were composite photographs generated from happy, surprised, and neutral faces. The top halves of the original photographs were separated from the bottom halves by a horizontal line below the eyes, and were pasted onto the superior areas of the happy and the surprised faces. Thus, the composites were happy faces and surprised faces with happy eyes, neutral eyes, and surprised eyes. When the eyes of the happy and the surprised face composites with the same top halves were compared by 142 naive observers, the happy face composites were judged to have larger eyes than the surprised face composites. PMID- 12375879 TI - Synthesis and anticancer activity of nordihydroguaiaretic acid (NDGA) and analogues. AB - Nordihydroguaiaretic acid (NDGA) 1 is a constituent of the creosote bush Larrea divaricata and is well known to be a selective inhibitor of lipoxygenases. NDGA can also inhibit the platelet derived growth factor receptor and the protein kinase C intracellular signalling family, which both play an important role in proliferation and survival of cancers. Moreover, NDGA induces apoptosis in tumour xenografts. Although it is likely to have several targets of action, NDGA is well tolerated in animals. These encouraging results have prompted interest in the compound for clinical study. However, high concentrations of NDGA are required for efficacy and more potent analogues are required. We have synthesized five analogues of NDGA with different lengths of carbon bridge between the two catechol moieties in order to establish the spacing required for optimum anticancer effect and to compare their activities with NDGA. In order to ascertain if the catechol moieties are essential for anticancer activity, we prepared five analogues of NDGA containing only one hydroxyl group on each aromatic ring. NDGA 1, its racemic form 2, the catechol derivatives 5, 6 with five or six carbon atom bridges and the phenol analogues 8-11 with bridges of three to six carbon atoms all showed similar activity, with IC50 values of approximately 3-5 microM against the H-69 small cell lung cancer cell line. Analogues with shorter (3) or longer bridges (7, 12) were much less active. The most potent analogue was the biscatechol with a four-carbon bridge 4 which was > 10 times more active than NDGA and therefore represents a new lead compound in this area. Surprisingly, the tetramethyl ether 14 of this compound was slightly more active than NDGA, but the trihydroxy analogue 13 was less active than NDGA. The conformationally restricted analogue 15 was also less active than NDGA. In summary, simplification of the structure of NDGA by removal of the methyl groups has produced a new lead compound 4, which is >10 times more potent than NDGA as a proliferative inhibitor of H-69 small cell lung cancer cells. PMID- 12375880 TI - Cyclohexylamino-demethoxy-hypocrellin B and photodynamic therapy decreases human cancer in vitro. AB - 2-Cyclohexylamino-2-demethoxy-hypocrellin B (CHAHB) is a new photosensitizer synthesized by the mild reaction between hypocrellin B (HB) and cyclohexylamine, in which the peri-hydroxylated perylenequinone structure of the parent HB is preserved and the photoresponse is enhanced markedly. Electron paramagnetic resonance (EPR) spin trapping measurements and 9,10-diphenylanthracene (DPA) bleaching studies were employed to investigate the photodynamic action of CHAHB in the presence of oxygen. Singlet oxygen ((1)O2) and superoxide anion radical (O2*-) generated by illuminating CHAHB in aerobic solution have been observed. Compared with HB, CHAHB distinctly enhanced the O2(*-)-generating abilities, although (1)O2-generating abilities were lowered. The photodynamic action of CHAHB in the therapy of cancer was investigated in vitro. The results in vitro revealed a much more significant decrease in cancer cell growth than with HB. Laser or dye alone had no effect, indicating that intratumor CHAHB and laser therapy may prove useful in unresectable cancer. PMID- 12375882 TI - The inhibition of protein phosphatases 1 and 2A: a new target for rational anti cancer drug design? AB - Recent investigations in our laboratories have highlighted that the inhibition of the serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) is an excellent target for the development of novel anti-cancer agents. Using a combination of the known crystal structure of PP1 and the modelled structure of PP2A, we have rationally designed a new class of protein phosphatase inhibitors, cantharimides, which exhibit broad-spectrum anti-cancer activity. Synthetic modifications of the simplest known PP1 and PP2A inhibitor, norcantharidin, has led to the development of potent PP1 and PP2A inhibitors. PMID- 12375881 TI - Photokilling of cultured tumour cells by the porphyrin derivative CF3. AB - We have analysed the photosensitizing properties of the new porphyrin 5-(4-N-(N 2',6'-dinitro-4'-trifluoromethylphenyl)aminophenyl)-10,15,20-tris(2,4,6 trimethoxyphenyl) porphyrin (CF3) on HeLa cells. The fluorescence and singlet oxygen quantum yield for CF3 were, respectively, phiF = 0.032 and phidelta = 0.25. Cell treatments were done with 5 x 10(-6) M CF3 incorporated into liposome vesicles. Under violet-blue exciting light, the red fluorescence of CF3 was mainly detected in lysosome-like granules. No dark cytotoxicity was observed using high concentration (5 x 10(-6) M) and long incubation time (18 h). Cell cultures treated for 18 h with CF3 and exposed to light (360 < lambda < 460 nm; 8 mW/cm2) for 7 min revealed a great amount of apoptotic (75.8%) and detached cells (62%) 8 h later, leading to a cell lethality of 85% (LD85). Apoptosis was identified by chromatin fragmentation and DNA ladder in gel electrophoresis. Necrotic cells were found using 15 min irradiation (LD96) and showed first small and then giant bubbles at the cell surface, with homogeneous nuclear condensation. Incubation with CF3 for 3 h followed by 7 min irradiation (LD38) produced a mitotic arrest 18 h later (mitotic index: 25.1%). Forty-eight hours after this metaphase blockage, cultures showed a great number of apoptotic cells. Taking into account these results, CF3 could be a valuable photosensitizer for the photodynamic therapy of cancer. PMID- 12375883 TI - Linker length in podophyllotoxin-acridine conjugates determines potency in vivo and in vitro as well as specificity against MDR cell lines. AB - We have synthesized two podophyllotoxin-acridine conjugates-pACR6 and pACR8. In these compounds an 9-acridinyl moiety is beta linked to the C4 carbon of the four ring system in 4'-demethylepipodophyllotoxin (epiDPT) via eighter an N-6 aminohexanylamide linker (pACR6) or via an N-8-aminooctanylamide linker containing two more carbon atoms (pACR8). The acridine-linker moiety occupies the position where different glucoside moieties, dispensable for activity, are normally linked to epiDPT in the well known epipodophyllotoxins VP-16 and VM-26. As with VP-16 and VM-26, pACR6 and pACR8 show evidence of being topoisomerase II poisons as they stimulate topoisomerase II mediated DNA cleavage in vitro and induce DNA damage in vivo. This in vivo DNA damage, as well as pACR6/pACR8 mediated cytotoxicity, is antagonized by the catalytic topoisomerase II inhibitors ICRF-187 and aclarubicin, demonstrating that topoisomerase II is a functional biological target for these drugs. Despite their structural similarities, pACR6 was more potent than pACR8 in stimulating topoisomerase II mediated DNA cleavage in vitro as well as DNA damage in vivo and pACR6 was accordingly more cytotoxic towards various human and murine cell lines than pACR8. Further, marked cross-resistance to pACR6 was seen among a panel of multidrug-resistant (MDR) cell lines over-expressing the MDR1 (multidrug resistance protein 1) ABC drug transporter, while these cell lines remained sensitive towards pACR8. pACR8 was also capable of circumventing drug resistance among at-MDR (altered topoisomerase II MDR) cell lines not over-expressing drug transporters, while pACR6 was not. Two resistant cell lines, OC-NYH/pACR6 and OC NYH/pACR8, were developed by exposure of small cell lung cancer (SCLC) OC-NYH cells to gradually increasing concentrations of pACR6 and pACR8, respectively. Here, OC-NYH/pACR6 cells were found to over-express MDR1 and, accordingly, displayed active transport of 3H-labeled vincristine, while OC-NYH/pACR8 cells did not, further suggesting that pACR6, but not pACR8, is a substrate for MDR1. Our results show that the spatial orientation of podophyllotoxin and acridine moieties in hybrid molecules determine target interaction as well as substrate specificity in active drug transport. PMID- 12375884 TI - Topoisomerase I/II selectivity among derivatives of N-[2 (dimethylamino)ethyl]acridine-4-carboxamide (DACA). AB - DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide dihydrochloride) has high experimental antitumor activity and has completed phase I/II clinical trials. It targets both topoisomerase (topo) I and II, but the roles of each of these enzymes in the antitumour action of DACA are not known. We have used a series of DACA analogues (mainly monosubstituted halogen derivatives) to relate in vitro and in vivo biological activity. We measured topo II selectivity by comparing the inhibition of Jurkat human leukaemia cell lines with high and low topo II content. We determined survival curves following exposure of H460 human lung carcinoma cells for 1 h. We used plasmid DNA to compare the effects of DACA analogues on isolated topo I and II, measuring in particular the inhibition of topo I- and II-mediated DNA relaxation. The results indicate that 5-halogen substituted derivatives are the most active in clonogenic cytotoxicity assays and that this activity is related to their selective activity towards Jurkat cells with high topo II activity. In isolated topo assays, 5-halogen substituted derivatives were also the most potent and in each case the concentration required for inhibition of topo II relaxation was greater than that for inhibition of topo I relaxation. The drug concentration providing efficient cytotoxicity corresponded to that which suppressed the activity of topo I but not of topo II. We hypothesize that DACA analogues act both in vitro and in vivo to simultaneously poison topo II and inhibit topo I catalytic activity, and that this combination contributes to the high antitumour activity of DACA analogues. PMID- 12375885 TI - Enantioselective alkylation of aldehydes with chiral organomagnesium amides (COMAs). AB - [reaction: see text] Dialkylmagnesiums react with chiral secondary amines to form chiral organomagnesium amides (COMAs). These reagents alkylate aldehydes to form secondary alcohols with enantioselectivities up to 91:9 er. PMID- 12375886 TI - The NH---FC dipole orientation effect for pendant exocyclic CH(2)F. AB - [structure: see text] DFT and MMFF force field calculations for 2 (R = H) predict that two conformers dominate in water (>/=95%) and both sustain a geometry in which the C-F and H-N dipoles align oppositely in a near-planar arrangement. The (1)H NMR spectra (D(2)O and DMSO-d(6)) and X-ray structure for 2 (R = SO(2)NHEt) confirm the predictions in all essentials. A novel single-conformer system is proposed. PMID- 12375887 TI - Translational isomerism in a [3]catenane and a [3]rotaxane. AB - [structure: see text] Post-assembly covalent modification using Wittig chemistry of [2]rotaxane ylides, wherein NH(2)(+) centers in the dumbbell-shaped components are recognized by dibenzo[24]crown-8 (DB24C8) rings, has afforded a [3]catenane and a [3]rotaxane with a precise and synthetically prescribed shortage of DB24C8 rings. The nondegenerate pairs of translational isomers present in both of these interlocked molecular compounds provide the fundamental platform on which to construct sensory devices and nanochemomechanical systems. PMID- 12375888 TI - Self-assembly of dendrimers by slippage. AB - [reaction: see text] A dendrimer with rotaxane-like characteristics has been assembled under thermodynamic control from complementary wedge-shaped precursors by slippage in CH(2)Cl(2). The driving force for the self-assembly process is the molecular recognition that exists as a result of [N(+)-H.O] and [C-H.O] hydrogen bonds between an NH(2)(+) center in one Frechet-type benzyl ether wedge and a dibenzo[24]crown-8 unit that links the other two such wedges. PMID- 12375889 TI - Carbohydrate-based synthesis of naturally occurring marine metabolites slagenins B and C. AB - [reaction: see text] The first enantioselective syntheses of slagenins B and C, marine metabolites from Agelas nakamurai, starting from L-arabinose have been described. PMID- 12375890 TI - Synthesis of termini-differentiated 6-carbon stereotetrads: an alkylative oxidation strategy for preparation of the c21-c26 segment of apoptolidin(1). AB - [reaction: see text] Two methods have been developed for the synthesis of epoxide 36. The first uses (+)-pulegone 25 as an enantiopure starting material and introduces the requisite intricacy of target 22 in 12 operations. The second method employs an enantiospecific catalytic Jacobsen epoxidation of 1a and is five operations shorter. The second sequence features an oxygen-directed alkylative oxidation reaction that re-establishes the dienyl sulfone functionality with concomitant 1,3-transposition of the sulfone moiety. PMID- 12375891 TI - A novel three-component reaction for the diastereoselective synthesis of 2H pyrimido[2,1-a]isoquinolines via 1,4-dipolar cycloaddition. AB - [reaction: see text] The 1,4-dipole derived from isoquinoline and DMAD has been shown to react readily with N-tosylimines resulting in the diastereoselective synthesis of 2H-pyrimido[2,1-a]isoquinoline derivatives. PMID- 12375892 TI - Simple synthesis of alpha-hydroxyamino carbonyl compounds: new scope of the nitroso aldol reaction. AB - [reaction: see text] The reaction of nitroso compounds with enolates, "the nitroso aldol reaction", occurs in high yield to generate alpha-hydroxyamino carbonyl compounds. Yields range from 42% to 98% with N-selectivity >99:1 from commercially available aromatic or aliphatic nitroso compounds and a variety of alkali metal or tin enolates. PMID- 12375893 TI - N-heterocyclic carbenes as versatile nucleophilic catalysts for transesterification/acylation reactions. AB - [reaction: see text] Imidazol-2-ylidenes, a family of N-heterocyclic carbenes (NHC), are efficient catalysts in the transesterification between esters and alcohols. Low catalyst loadings of aryl- or alkyl-substituted NHC catalysts mediate the acylation of alcohols with vinyl acetate in convenient reaction times at room temperature. Commercially available and more difficult to cleave methyl esters react with numerous alcohols in the presence of alkyl-substituted NHC to form efficiently the corresponding esters in very short reaction times. PMID- 12375894 TI - Expanding the catalytic activity of nucleophilic N-heterocyclic carbenes for transesterification reactions. AB - [reaction: see text] Currently, there is a renewed interest in reactions that are catalyzed by organic compounds. Typical organic catalysts for acylation or transesterification reactions are based on either nucleophilic tertiary amines or phosphines. This communication discusses the use of nucleophilic N-heterocyclic carbenes as efficient transesterification catalysts. These relatively unexplored and highly versatile organic catalysts were found to be mild, selective, and more active than traditional organic nucleophiles. PMID- 12375895 TI - Development of co- and post-translational synthetic strategies to C neoglycopeptides. AB - [reaction: see text] The synthesis of stable, C-linked analogues of glycopeptides is being investigated with two complementary synthetic strategies, co translational and post-translational glycopeptide synthesis. The key feature of the present approach lies in an effective olefin cross-metathesis reaction that allows formation of both glycoamino acids and glycopeptides. PMID- 12375896 TI - Co(III)-alkyl complex- and Co(III)-alkylperoxo complex-catalyzed triethylsilylperoxidation of alkenes with molecular oxygen and triethylsilane. AB - [reaction: see text] Both a Co(III)-alkyl complex and a Co(III)-alkylperoxo complex were found to catalyze triethylsilylperoxidation of alkenes with O(2) and Et(3)SiH. On this basis, together with the nonstereoselectivity in the Co(II) catalyzed peroxidation of 3-phenylindene and the formation of the corresponding 1,2-dioxolane from 2-phenyl-1-vinylcyclopropane (a radical clock), we propose a reasonable mechanism for the Co(II)-catalyzed novel autoxidation of alkenes with Et(3)SiH discovered by Isayama and Mukaiyama. PMID- 12375897 TI - A simple method for electrophilic functionalization of DNA. AB - [reaction: see text] An extremely simple and versatile method for placing an electrophilic functional group (iodide) at the 5' end of oligodeoxyribonucleotides is described. The reaction is carried out while the protected oligodeoxyribonucleotide remains on a solid support and utilizes inexpensive iodination chemistry. We demonstrate that this reaction can be automated on a DNA synthesizer as the last step of DNA synthesis. PMID- 12375898 TI - New insight into the synthesis of a novel azo-based optically active polyamidoamine side chain dendritic polyester architectural photoswitch. AB - [reaction: see text] A novel chiral polyamidoamine side chain dendritic polyester 4 has been synthesized and found to be a photoresponsive system undergoing reversible E/Z photochemical/thermal isomerization of azo units. Thorough characterization (IR,UV-vis, NMR, FAB-MS, elemental analysis, optical rotation, etc.) were performed to ascertain the structures of dendrimers 2, 3, and 4. The intrinsic viscosity of 4 at 36 degrees C in CHCl(3) was found to be 0.48 dl/g. PMID- 12375899 TI - Design and synthesis of activity probes for glycosidases. AB - [structure: see text] A new synthetic route was developed for the preparation of activity probe 1 for beta-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe 1 was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations. PMID- 12375900 TI - Diamine-catalyzed asymmetric Michael additions of aldehydes and ketones to nitrostyrene. AB - [reaction: see text] The direct Michael addition of aldehydes and ketones to nitrostyrene, catalyzed by N-i-Pr-2,2'-bipyrrolidine, is described. The desired 1,4-adducts are obtained in excellent yield with enantioselectivities up to 85% ee and dr up to 95:5 of the syn product. PMID- 12375901 TI - Synthesis of pCpCpA-3'-NH-phenylalanine as a ribosomal substrate. AB - [reaction: see text] The trinucleotide cytidylyl(3'-->5'phosphoryl)cytidylyl(3'- >5'phosphoryl)-3'-deoxy-3'-(L-phenylalanyl) amido adenosine (CpCpA-NH-Phe) was synthesized by phosphoramidite chemistry from 3'-amino-3'-deoxyadenosine as the ribosomal substrate. The 3'-amino-3'-deoxyadenosine was first converted to 3'-(N tert-butyloxycarbonyl-L-phenylalanine)amido-3'-deoxy-6-N,6-N,2'-O-tribenzoyl adenosine and then coupled with cytidine phosphoramidite to produce the fully protected CpCpA-NH-Phe-Boc. The title product was obtained after removing all protection groups and then radiolabeled with (32)P to yield pCpCpA-NH-Phe, which demonstrated high activity for the peptidyl transferase reaction in the ribosome. PMID- 12375902 TI - Novel approach to the synthesis of enantioenriched sulfoxides. Highly diastereoselective alkylation of sulfenate anions with 1,4-asymmetric induction. AB - [reaction: see text] Efficient 1,4-asymmetric induction with an enantiopure aminoalkyl group as the chiral auxiliary has been achieved in the first example of diastereoselective alkylation (up to 98:2) of a sulfenate anion, readily prepared by oxidation of the corresponding thiolate. The stereochemistry of the sulfoxide produced is the opposite of that obtained by the conventional route based on oxidation of the sulfide precursor. PMID- 12375903 TI - Enzymatic formation of an unnatural C(6)-C(5) aromatic polyketide by plant type III polyketide synthases. AB - [reaction: see text] Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural C(6)-C(5) aromatic polyketide, 1-(4 hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In contrast, succinyl-CoA was not accepted as a substrate by the enzymes. PMID- 12375904 TI - Biocatalytic approaches toward the synthesis of both enantiomers of trans cyclopentane-1,2-diamine. AB - [reaction: see text] A lipase-catalyzed double monoaminolysis of dimethyl malonate by (+/-)-trans-cyclopentane-1,2-diamine allows the sequential resolution of the latter compound, affording an enantiopure bis(amidoester), which is subsequently transformed into an optically active polyamine. As an alternative, both enantiomers of the diamine can be obtained from enantiopure (+)- or (-)-2 aminocyclopentanol, prepared by enzymatic resolution. PMID- 12375905 TI - Nanoscale 1,3,5,7-tetrasubstituted adamantanes and p-substituted tetraphenyl methanes for AFM applications. AB - [structure: see text] Tetrahedrally shaped nanoscale molecules 18-20 were synthesized from the corresponding tetraiodide by a series of Sonogashira coupling reactions. Three of the sulfur-containing termini are intended for eventual binding to a gold-coated conventional AFM tip, while the fourth terminus scans the sample. AFM images of 19 demonstrate that the molecule is sufficiently large and rigid to be imaged by a conventional AFM tip. PMID- 12375906 TI - A general and straightforward route toward diarylmethanes. Integrated cross coupling reactions using (2-pyridyl)silylmethylstannane as an air-stable, storable, and versatile coupling platform. AB - [reaction: see text] Pharmacologically important diarylmethane structures have been prepared in a straightforward manner through sequentially integrated Pd catalyzed cross-coupling reactions. (2-Pyridyl)silylmethylstannane was found to be an air-stable, storable, and versatile coupling platform in this synthetic strategy. PMID- 12375907 TI - A cascade cyclization approach to schweinfurthin B. AB - [reaction: see text] A strategy for synthesis of the hexahydroxanthene moiety of the natural products schweinfurthin A, B, and D is described. The relative stereochemistry in the key cationic cyclization step is established through the preference of the phenylselenide substituent for an equatorial orientation. PMID- 12375908 TI - Cycloaromatization of 1,4-pentadiynes: a viable possibility? AB - [structure: see text] The effects of several mostly sigma-withdrawing, pi donating substituents X on the hitherto unknown Bergman-like cyclizations of 3 substituted 1,4-pentadiynes were studied at the BLYP/6-311+G//BLYP/6-31G level of theory. As the cyclization with X = OH(+) has the lowest barrier and is about thermoneutral, we predict that the title reaction is viable, for instance, through activation of derivatives with X = O with Lewis acids. PMID- 12375909 TI - InCl(3)-catalyzed three-component asymmetric Mannich-type reaction in methanol. AB - [reaction: see text] A one-pot InCl(3)-catalyzed Mannich-type reaction was carried out in methanol. High diastereoselectivities and high yields were obtained. In addition, after the reaction was completed, InCl(3) can be recycled and reused without a drop of activity and selectivity. PMID- 12375910 TI - A concise, asymmetric synthesis of tetramic acid derivatives. AB - [reaction: see text] A simple, asymmetric synthesis of tetramic acid derivatives is described in this paper. The key step is a carbonyl transfer from carbonyldiimidazole (CDI) to alpha-diimines (I) to form N-alkyl-4-alkylamino-5 methylenepyrrol-2-ones (II). In turn, these compounds can be easily transformed into tetramic acid derivatives (III) in two additional steps. PMID- 12375911 TI - Synthesis of the C10-C22 bis-spiroacetal domain of spirolides B and D via iterative oxidative radical cyclization. AB - [reaction: see text] A synthetic approach to the novel bis-spiroacetal moiety of spirolides B and D is reported. The strategy hinges upon successive formation of the spirocenters at C15 and C18 by radical oxidative cyclization, followed by base-induced rearrangement of a C19-20 alpha-epoxide to introduce the C19 hydroxyl group. PMID- 12375912 TI - Cycloaddition reactions of alkoxy alkynyl Fischer carbene complexes with o quinodimethanes (oQDMs). AB - [reaction: see text] The reaction of o-quinodimethanes (oQDMs) with alkoxy alkynyl Fischer carbene complexes is highly dependent on the carbene complex. Thus, for arylalkynyl carbene complexes, the initial [4 + 2]-cycloadduct evolves opening a new entry to the benzo[b]fluorene skeleton, which is present in many natural products. However, for alkenylalkynyl carbene complexes, the reaction takes place through the double bond, instead of the triple bond, in an unprecedented fashion, leading to new functionalized alkynyl carbene complexes. PMID- 12375913 TI - Synthesis of substituted cyclohexenyl-based beta-amino acids by ring-closing metathesis. AB - [structure: see text] A versatile ring-closing metathesis (RCM) approach has been developed for the preparation of cis and trans cyclohexenyl-based beta-amino acids that are either unsubstituted (3) or substituted (10 and 12) at the alpha position. PMID- 12375914 TI - Solid-phase synthesis of polysubstituted piperidines by imino-Diels-Alder cycloaddition of 2-amino-1,3-butadienes with solid-supported imines. AB - [reaction: see text] The solid-phase imino-Diels-Alder reaction of 2-amino-1,3 butadienes with solid-supported imines is described. The reaction furnishes 4 piperidones and 4-aminopiperidines with high diastereoselectivity and with very good yields and purity after the release from the solid support. The possibility of introducing variations in both cycloaddition partners gives rise to substituted piperidines with up to five elements of diversity. PMID- 12375915 TI - A simple and effective method for phosphoryl transfer using TiCl(4) catalysis. AB - [reaction: see text] A number of Lewis acids have been evaluated as catalysts for the phosphoryl transfer, the most efficient being TiCl(4). Application of this methodology to the phosphorylation of a number of representative target alcohols is presented PMID- 12375916 TI - Synthesis of alpha-glucuronic acid and amide derivatives in the presence of a participating 2-acyl protecting group. AB - [reaction: see text] Participating acyl groups located at C-2 in glucosyl and related donors generally promote formation of 1,2-trans-glycosides. Reactions of some glucuronic acid donors with TMSN(3)/SnCl(4) or ROH/SnCl(4) gave only the 1,2 cis-glycoside. The stereoselectivity is consistent with participation of the C-6 group. The methodology was used for the synthesis of a Kdn2en mimetic with the alpha-configuration. PMID- 12375917 TI - Synthesis of 2H-1-benzopyrans via palladacycles with a metal-bonded stereogenic carbon. AB - [reaction: see text] Stable oxapalladacycles have been prepared and converted into a series of highly functionalized 2H-1-benzopyrans via regioselective insertion of activated alkynes. PMID- 12375918 TI - A sulfinyl-directed asymmetric [5C + 2C] intramolecular acetoxypyranone-alkene cycloaddition. AB - [reaction: see text] Introduction of a homochiral p-tolylsulfinyl group at the trans terminal position of an alkene induces a total diastereodifferentiation in the intramolecular cycloaddition of the latter to 3-oxidopyrylium ylide precursors. The cycloadducts can be readily desulfinylated to afford enantiomerically pure oxa-bridged bicyclo[5.3.0]decane ring systems. Theoretical calculations confirm that diastereoselectivity stems from the conformational preferences of the alkenylsulfoxide unit in the transition state of the reaction. PMID- 12375919 TI - Intramolecular Baylis-Hillman and morita reactions using unsaturated thiol ester substrates containing enolizable aldehydes. AB - [reaction: see text] Previously unknown intramolecular Baylis-Hillman and Morita reactions involving cyclization of an unsaturated thiol ester onto a pendant aldehyde function are reported. These can be used successfully for the preparation of both cyclopentenols and cyclohexenols, but the results are very sensitive to substrate and precise reaction conditions. PMID- 12375920 TI - A new halo aldol reaction: three-component reaction via 1,4-robust activation of ethynyl alkyl ketones for stereoselective formations of versatile aldol adducts. AB - [reaction: see text] A new three-component halo aldol reaction has been discovered for the tandem formations of I-C/C-C bonds by activating the alpha',beta-positions of alpha,beta-acetylenic ketones. The key intermediates, 1 iodo-3-siloxy-1,3-butadienes, were generated from allenolates and directly monitored by (1)H NMR spectroscopic analysis. Excellent geometric selectivity (>95%) and good yields (65-82%) have been achieved for 10 examples. PMID- 12375921 TI - New stereoselective beta-glycosylation via a vinyl oxirane derived from D-glucal. AB - [reaction: see text] The reaction of the vinyl oxirane 1 derived from D-glucal with a series of O-nucleophiles (alcohols, phenol, and diacetone D-glucose) affords the corresponding 2-unsaturated beta-O-glycosides in a completely stereoselective way (syn 1,4-addition pathway). Epoxide 1 is generated in situ by treatment of the corresponding hydroxy mesylate 2 with t-BuOK in anhydrous benzene. PMID- 12375922 TI - Copper-catalyzed enantioselective conjugate addition of diethylzinc to acyclic enones in the presence of planar-chiral phosphaferrocene-oxazoline ligands. AB - [reaction: see text] A new subclass of chiral phosphaferrocene-oxazoline ligands has been applied to the copper-catalyzed asymmetric conjugate addition of diethylzinc to acyclic enones, furnishing good enantioselectivity. The ligand design readily lends itself to modification, thereby facilitating optimization of ee. Although the dominant stereocontrol element in these 1,4-addition processes is the central chirality of the oxazoline subunit of the ligand, not the planar chirality of the phosphaferrocene, altering the phosphaferrocene subunit can provide useful enhancement of enantioselectivity. PMID- 12375923 TI - Copper-catalyzed arylation of beta-amino alcohols. AB - [reaction: see text] Methods for synthesizing N-aryl beta-amino alcohols and O aryl beta-amino alcohols are described. The presence of a neighboring hydroxyl or amino group, respectively, is thought to activate beta-amino alcohols toward these transformations. These protocols significantly increase access to a variety of arylated beta-amino alcohols. PMID- 12375924 TI - Radical fragmentation of omega-bromoalkyl cyclobutanones. A modular approach to eight-membered carbocycles. AB - [reaction: see text] An eight-membered ring was conveniently appended onto a cycloalkene carboxylate by employing a facile radical cyclization-fragmentation reaction of an omega-bromoalkyl spirocyclobutanone, which was readily accessible by the Kulinkovich cyclopropanation and subsequent electrophilic addition to a 3 bromoalkyl acetal. PMID- 12375925 TI - Synthesis and DNA cross-linking of a phototriggered FR900482 mitosene progenitor. AB - [reaction: see text] The synthesis and biochemical reactivity of the first photoactivated mitosene-based DNA interstrand cross-linking agent is described. PMID- 12375926 TI - Synthesis of cis-solamin using a permanganate-mediated oxidative cyclization. AB - [reaction: see text] cis-Solamin (1) and its diastereoisomer 14 have been synthesized in 13 steps using the diastereoselective permanganate-promoted oxidative cyclization of 1,5-dienes to create the tetrahydrofuran diol core. Notably, no protecting groups are required during the stages of fragment assembly. PMID- 12375927 TI - Diastereoselective synthesis of the C(17)-C(28) fragment (the C-D spiroketal unit) of spongistatin 1 (altohyrtin A) via a kinetically controlled iodo spiroketalization reaction. AB - [reaction: see text] A convergent and stereocontrolled synthesis of spiroketal 15 corresponding to the C-D fragment of spongistatin 1 has been accomplished by a sequence utilizing a kinetically controlled intramolecular iodo-spiroketalization of glycal 2, which in turn was synthesized via a ring-closing metathesis reaction. PMID- 12375928 TI - Synthesis of the C(2)-C(13) fragment (the A-B spiroketal unit) of spongistatin 1 (altohyrtin A): use of a common intermediate for the synthesis of both spongistatin spiroketals. AB - [reaction: see text] A convergent synthesis of 14 corresponding to the A-B spiroketal core of spongistatin 1 has been accomplished via an iodo spiroketalization reaction of glycal 9, which was synthesized in three steps from a late-stage intermediate used in our synthesis of the C-D spiroketal fragment of spongistatin 1. Elaboration of 14 to the A-B spiroketal 15 was accomplished in three steps. PMID- 12375929 TI - Synthesis of 3,4-disubstituted piperidines by carbonyl ene and Prins cyclizations: a switch in diastereoselectivity between Lewis and Bronsted acid catalysts. AB - [reaction: see text] A novel diastereoselective approach to cis and trans 3,4 disubstituted piperidines is described. Carbonyl ene cyclization of aldehydes 6a e catalyzed by the Lewis acid methyl aluminum dichloride in refluxing chloroform affords trans piperidines 8a-e with diastereomeric ratios of up to 93:7. By contrast, Prins cyclization of 6a-e catalyzed by hydrochloric acid at low temperatures affords cis products 7a-e with diastereomeric ratios of up to 98:2. PMID- 12375930 TI - Studies on the biomimetic synthesis of SNF4435 C and D. AB - [reaction: see text] The biomimetic synthesis of the bicyclic core of the novel immunosuppresants SNF4435C and SNF4435D is reported. The core framework was efficiently generated from the all-trans tetraene precursor in one step and in good yield. PMID- 12375931 TI - Anodic modification of proline derivatives using a lithium perchlorate/nitromethane electrolyte solution. AB - [reaction: see text] N-Acyliminium cation of prolines was efficiently generated to accumulate in an undivided cell at 0 degrees C by an anodic oxidation of N acylprolines or alpha'-phenylsulfanylated N-acylproline derivatives in a lithium perchlorate/nitromethane solution. The iminium cation intermediates gave modified prolines by a reaction with nucleophiles under mild conditions. PMID- 12375932 TI - Toward the development of a structurally novel class of chiral auxiliaries: diastereoselective aldol reactions of a (1R,2S)-ephedrine-based 3,4,5,6 tetrahydro-2H-1,3,4-oxadiazin-2-one. AB - [reaction: see text] Asymmetric aldol addition reactions have been conducted with (1R,2S)-ephedrine-derived 3,4,5,6-tetrahydro-2H-1,3,4-oxadiazin-2-one (2). Diastereoselectivities range from 75:25 to 99:1 for the formation of the crude non-Evans syn adducts 8a-h. The facial selectivity of the enolate is directed by the stereogenic N(4)-methyl substituent. Aldol adduct 8a is readily cleaved by acid hydrolysis to afford (2S,3S)-3-hydroxy-2-methyl-3-phenylpropionic acid (9) in >95% ee. PMID- 12375933 TI - An acid-catalyzed macrolactonization protocol. AB - [reaction: see text] An efficient macrolactonization protocol devoid of any base was developed derived from the use of vinyl esters in transesterification. Subjecting a hydroxy acid and ethoxyacetylene to 2 mol % [RuCl(2)(p-cymene)](2) in toluene followed by addition of camphorsulfonic acid or inverse addition provided macrolactones in good yields. PMID- 12375934 TI - An improved catalyst for ring-closing alkyne metathesis based on molybdenum hexacarbonyl/2-fluorophenol. AB - [reaction: see text] An improved "instant" catalyst for ring-closing alkyne metathesis reaction is described. Catalyst formed in situ from molybdenum hexacarbonyl and 2-fluorophenol can be used without exclusion of air and moisture and shows high activity in metathesis of functionalized diynes. This system allows cyclization of substrates which were incompatible with previously known Mo(CO)(6)/phenol catalysts. PMID- 12375935 TI - Functional group diversity in dendrimers. AB - [structure: see text] A methodology for synthesizing dendrons with different peripheral functionalities is described. The benzyl ether-based dendrons reported here were synthesized using allyl and methoxymethyl ether-based protection deprotection strategies. PMID- 12375936 TI - Stereocontrolled synthesis of kelsoene by the homo-favorskii rearrangement. AB - [reaction: see text] (+/-)-Kelsoene (4) has been synthesized from 2,5 dihydroanisole in 16 steps in 12.5% overall yield. The key step involves a base catalyzed reaction of gamma-keto tosylate (5), which effects a homo-Favorskii rearrangement to 16 as well as the corresponding intramolecular S(N)2 product 15 from the enolate of 5. Ketone 15 can efficiently be isomerized to cyclobutanone 17 having the kelsoene carbon skeleton upon acid treatment. PMID- 12375937 TI - Enantioselective additions of diethylzinc and diphenylzinc to aldehydes using 2 dialkyl-aminomethyl-2'-hydroxy- 1,1'-binaphthyls. AB - [reaction: see text] A set of 2-dialkylaminomethyl-2'-hydroxy-1,1'-binaphthyls was prepared and used in the catalytic asymmetric additions of diethylzinc and diphenylzinc to various types of aldehydes. These binaphthyl-based axially chiral amino alcohols show high enantioselectivity in the addition of organozincs to aromatic and aliphatic aldehydes. PMID- 12375938 TI - Two-step electrochemical annulation for the assembly of polycyclic systems. AB - [reaction: see text] A two-step electrochemical annulation has been developed for the preparation of fused furans. The process involves an initial conjugate addition of a furyethyl cuprate and trapping of the enolate as the corresponding silyl enolether. The second step of the annulation involves the anodic coupling of the furan and the silyl enol ether to form a six-membered ring. PMID- 12375939 TI - A novel synthesis of N-methyl asparagine, arginine, histidine, and tryptophan. AB - [reaction: see text] N-Methyl amino acid residues in peptides modify several pharmacologically useful parameters, but synthesis of alkylated peptides is hampered by unavailability of N-methylated monomers. The syntheses of four N methyl amino acids with basic side chains are presented. The side chains of these basic amino acids needed to be specially protected or constructed. This completes the set of 20 common L-amino acid N-methyl derivatives prepared via 5 oxazolidinone intermediates by our group. PMID- 12375940 TI - Cross-coupling reactions of alkenylsilanols with fluoroalkylsulfonates. AB - [reaction: see text] The design and development of an effective protocol for the palladium-catalyzed cross-coupling of (E)- and (Z)-heptenyldimethylsilanols with organo-triflates and nonaflates is described. Optimization of this coupling focused on the issues of both reactivity and stability of the psuedohalides in the presence of the nucleophilic fluoride promoter for the coupling. The crucial role of varying amounts of water to modulate the reactivity of the fluoride ion is highlighted. PMID- 12375942 TI - 1,1',3,3',6,6',8,8'-Octachloro-9,9'-bifluorenylidene and perchloro-9,9' bifluorenylidene, two exceedingly twisted ethylenes. AB - The syntheses of 1,1',3,3',6,6',8,8'-octachloro-9,9'-bifluorenylidene (1), its precursors, and the byproduct 3,3',5,5'-tetrachloro-4-(trichloromethyl)biphenyl (5) are described. Accurate structural X-ray data on 1 and on perchloro-9,9' bifluorenylidene (2) are reported and discussed. Because of chlorine overcrowding, the dihedral angles between their two identical fluorenylidene moieties are abnormally large, the central-ethylene twist angles being 55 and 66 degrees, respectively. Significant out-of-plane carbon-chlorine bond bending is likewise exhibited. Their ESR spectra and magnetic measurements prove that they are singlet species. The exceptionally large bathochromic displacements of their UV-vis absorption spectrum with respect to that of their parent hydrocarbon are mainly attributed to bond bending and molecular warping. PMID- 12375943 TI - Electrostatic interactions between substituents as regioselectivity control elements in Diels-Alder cycloadditions. A DFT study of cycloadditions of 1 methoxy-4-trimethylsiloxy dienes with acrylonitrile. AB - The regioselectivities of Diels-Alder reactions of 1-methoxy-4-trimethylsiloxy 1,3-butadiene and the corresponding o-xylylene with acrylonitrile were explored with density functional theory. The transition state of the reaction of the diene with acrylonitrile was studied in both the gas phase and in a low dielectric (epsilon = 2.247) solvent. The regioselectivities of these reactions are predicted to be controlled by the direct electrostatic interactions between the diene and dienophile substituents. The electron-donating capacities of methoxy and trimethylsiloxy substituents are shown to be very similar and to not contribute to regioselectivity control. A prediction is made that the cycloadditions of the o-xylylene will be unselective, while 1-methoxy-4 trimethylsiloxy-1,3-butadiene will give a small preference ( approximately 5:1) in favor of the proximal methoxy and cyano groups. The thermochromic behavior of trans-disubstituted disiloxy benzocyclobutene was also explored. PMID- 12375944 TI - An exceptional red shift of emission maxima upon fluorine substitution. AB - The effect of perfluorination on photophysical properties was investigated through synthesis and photophysical characterization of two isostructural donor acceptor-donor dye molecules. The synthesis of two versatile fluorinated benzene compounds, 1,4-difluoro-2,5-diperfluorooctylbenzene (1) and 1,4-dibromo-2,5 difluoro-3,6-diperfluorooctylbenzene (2), is presented. The X-ray structure of 2 has been determined and shows that the perfluorinated octyl chains segregate from the benzene rings in the solid state, giving rise to a layered structure. The further synthesis through Suzuki coupling reactions using 4-formylbenzeneboronic acid with (2) and 1,4-dibromo-2,5-dioctylbenzene (3) gave, respectively, 1,4' ' diformyl-2',5'-difluoro-3',6'-diperfluorooctyl-p-terphenylene (4) and 1,4' ' diformyl-2',5'-dioctyl-p-terphenylene (5). The condensation of the dialdehydes 4 and 5 with 9,10-phenanthrenequinone and ammoniumbicarbonate in glacial acetic acid gave the dye molecules 1,4' '-bis(1H-phenanthro[9,10-d]imidazol-2-yl)-2',5' difluoro-3',6'-diperfluorooctyl-p-terphenylene (6) and 1,4' '-bis(1H phenanthro[9,10-d]imidazol-2-yl)-2',5'-dioctyl-p-terphenylene (7), respectively. The UV-vis spectra of the two molecules are nearly identical, whereas the fluorescence spectra are very different. Compound 7 shows blue fluorescence with little solvent dependence (lambda(emission) = 410 nm in THF, CH2Cl2, and hexane), whereas compound 6 shows a highly solvent-dependent emission wavelength (lambda(emission) = 583 nm in THF, lambda(emission) = 560 nm in CH2Cl2, and lambda(emission) = 450 nm in hexane). The fluorescence red shift of compound 6 in a series of solvents with different polarity is discussed using the Lippert Mataga equation. Fluorescence lifetime and quantum yields were also determined. Ultraviolet photoelectron spectroscopy (UPS) was performed on thin films of compound 6 and 7 on a gold substrate. The observed ionization potential was 6.15 eV for 6 and 5.85 eV for 7" [correction]. PMID- 12375945 TI - Free-radical hydroxylation reactions of alkylboronates. AB - The radical hydroxylation of B-alkylcatecholboranes, easily prepared by hydroboration of olefins, has been investigated. When molecular oxygen was used as oxidizing agent, the corresponding alcohols were obtained directly without alkaline treatment. The presence of Lewis base additives such as Et3N or DABCO has a benefic effect on the selectivity and yield. Alternatively, 2,2,6,6 tetramethylpiperidine-N-oxyl (TEMPO) reacts cleanly with B-alkylcatecholboranes to afford alkyl radicals that can be trapped by a second equivalent of TEMPO to give alkoxyamines. Reduction of the alkoxyamines with zinc in acetic acid affords the desired alcohols. The whole procedure is particularly mild and does not require any basic condition. The two approaches presented in this paper are valuable and represent mild alternatives to the classical alkaline oxidation of organoboranes to alcohols. PMID- 12375946 TI - New synthesis of pyrrolidine homoazasugars via aminohomologation of furanoses and their use for the stereoselective synthesis of aza-C-disaccharides. AB - The introduction of a formyl group at the anomeric center of 2,3,5-tri-O-benzyl furanoses and substitution of the ring oxygen with a basic nitrogen atom (aminohomologation) was carried out via stereoselective addition of 2 lithiothiazole to N-benzyl, N-furanosylhydroxylamines (masked N-benzyl sugar nitrones), followed by reductive dehydroxylation of the resulting open-chain adducts, and then ring closure via intramolecular displacement of the free hydroxy group by the amino group and unmasking of the formyl group from the thiazole ring. The resulting formyl aza-C-glycosides were transformed into 2,5 dideoxy-2,5-imino-hexitols (pyrrolidine homoazasugars) by reduction of the formyl to the hydroxymethyl group and removal of the O- and N-benzyl groups by hydrogenolysis. This reaction sequence was applied to four furanoses (D-arabino, D-ribo, D-lyxo, L-xylo) to give the hydroxy- and amino-free homoazasugars, including the natural product 2,5-dideoxy-2,5-imino-D-mannitol, in 17% overall yields (six steps). The formyl aza-C-glycosides proved to be valuable intermediates for the synthesis of more complex derivatives. In fact, these sugar aldehydes were employed in Wittig-type coupling reactions with galactose and ribose phosphoranes to give bis-glycosylated alkenes, which upon reduction of the double bond were transformed into methylene isosteres of (1-->6)- and (1-->5) linked disaccharides in which one of the two sugar moieties was an azasugar (aza (1-->x)-C-disaccharides). PMID- 12375947 TI - Revisiting the Ullmann-ether reaction: a concise and amenable synthesis of novel dibenzoxepino[4,5-d]pyrazoles by intramolecular etheration of 4,5-(o,o' halohydroxy)arylpyrazoles. AB - A concise synthesis of a series of novel dibenzoxepino[4,5-d]pyrazoles was accomplished by implementation of an intramolecular Ullmann-ether reaction on o,o'-halohydroxy-4,5-diarylpyrazoles mediated by CuBr.DMS. An alternative useful approach based on the palladium-catalyzed biaryl-ether linkage formation (Buchwald-Hartwig reaction) was also successfully applied, offering limitations with regard to the steric demand of the substituents. The synthesis of the key o,o'-halohydroxy-4,5-diarylpyrazole intermediates proceeds through the construction of the heterocyclic ring by a tandem amine exchange/heterocyclization sequence of 3-N,N-(dimethylamino)-1,2-diarylpropenones with phenylhydrazine followed by basic hydrolysis for deprotection. The enamino ketone precursors were conveniently prepared from the corresponding O-sulfonyloxy and O-benzoyloxy ortho-substituted 1,2-diarylethanones, starting from inexpensive salicylaldehyde or phenylacetic derivatives. Preliminary binding affinity experiments against peripheral and central nervous system receptors have been done with negative results. PMID- 12375948 TI - A versatile stereocontrolled approach to chiral tetrahydrofuran and tetrahydropyran derivatives by use of sequential asymmetric Horner-Wadsworth Emmons and ring-closure reactions. AB - An approach to chiral tetrahydrofuran and tetrahydropyran derivatives based on the sequential use of an asymmetric Horner-Wadsworth-Emmons reaction and a cyclization step is presented. The approach is both stereochemically and structurally versatile since three different cyclization methods can be employed starting from the same HWE product: (i) palladium-catalyzed substitution, (ii) hetero-Michael addition, or (iii) epoxide opening. The asymmetric HWE reaction controls the absolute configuration of the ultimate product, whereas the relative configuration is controlled by the combined influence of the geometric selectivity in the HWE reaction and the stereochemistry of the respective cyclization method. PMID- 12375949 TI - Uncatalyzed [4 + 2] cycloadditions of 3-nitrocoumarins with vinyl ethers in solventless conditions. A new entry to chromene derivatives. AB - The [4 + 2] cycloadditions of 3-nitrocoumarins 5 with electron-rich dienophiles (ethyl vinyl ether (8), 2,3-dihydrofuran (9), and 3,4-dihydro-2H-pyran (10)) were investigated in water, in neat conditions, and in organic solvents. The cycloadditions do not require the use of catalysts and are highly endo diastereoselective, and in water the cyclic nitronates 13, 18, and 23 are converted into chromene derivatives via hydrolysis, decarboxylation, and acetalation reactions. A one-pot procedure based on consecutive reactions in neat/water conditions allows 3-nitrocoumarins 5 to be used as building blocks for the synthesis of chromanols and tetrahydrofuro- and tetrahydropyranochromenes. For the first time, the hydrolysis of cyclic nitronates having the C-O bond of 1,2-oxazine ring as a part of an acetal was investigated. PMID- 12375950 TI - Catalytic enantioselective synthesis of (-)-prostaglandin E1 methyl ester based on a tandem 1,4-addition-aldol reaction. AB - Catalytic enantioselective 1,4-additions and tandem 1,4-addition-aldol reactions of dialkylzinc reagents to cyclopentene-3,5-dione monoacetals in the presence of an in situ generated Cu(OTf)(2)/chiral phosphoramidite catalyst result in highly functionalized cyclopentane building blocks with ee's up to 97%. A new synthesis of cyclopentene-3,5-dione monoacetals is presented as well as its use in a tandem 1,4-addition-aldol protocol for the catalytic asymmetric total synthesis of (-) PGE(1) methyl ester. This synthesis represents a new approach to this class of natural products. By using only 3 mol % of an enantiomerically pure catalyst in the key step, the absolute configurations at three stereocenters of the basic structure of the PGE(1) are established at once. PMID- 12375951 TI - Synthesis of vinca alkaloids and related compounds. 100. Stereoselective oxidation reactions of compounds with the aspidospermane and quebrachamine ring system. First synthesis of some alkaloids containing the epoxy ring. AB - The first syntheses of the alkaloids (-)-mehranine (3), (+) voaphylline/conoflorine (4), (+)-N(a)-methylvoaphylline/hecubine (5), and (-) lochnericine (2) were achieved by stereoselective epoxidation starting from (-) tabersonine (1), through intermediates with the aspidospermane and quebrachamine skeleton. PMID- 12375952 TI - Mechanism of the aromatic hydroxylation of thiophene by acid-catalyzed peracid oxidation. AB - The oxidation of thiophene (1) with peracids in a strongly acidic environment yielded thiophen-2-one (4) as the product of an apparent direct hydroxylation of the thiophene aromatic ring together with the anticipated thiophene-S-oxide dimers, 2a,b, as the main products. Formation of the latter dimers can be rationalized in a straightforward manner by initial oxidation at the sulfur atom of thiophene (1) to yield thiophene-S-oxide followed by subsequent dimerization in a Diels-Alder type reaction. Trapping experiments in the presence of a competing dienophile indicated that thiophen-2-one (4) did not originate from the monomeric thiophene-S-oxide but was the product of an independent reaction pathway. The extent of thiophen-2-one (4) formation correlated with the acidity of the reaction medium and was suppressed in the presence of water, the latter presumably acting as a competing base. As evidenced by the use of 2,5 dideuterated thiophene (1-D), its mechanism of formation involved a 1,2-hydride shift, a feature commonly described in the peracid-mediated epoxidation of aromatic hydrocarbons and indicative for the occurrence of cationic intermediates. In agreement with all these observations we propose a mechanism involving initial protonation of thiophene followed by nucleophilic attack of the peracid in position 2 of the thiophene ring. Intramolecular epoxidation may lead to the formation of thiophene 2,3-epoxide as a highly reactive intermediate that then undergoes heterolytic ring opening and a 1,2-hydride shift to yield thiophen 2-one (4) after a final, acid-catalyzed, isomerization of the double bond. PMID- 12375953 TI - Oxathiaphospholane approach to N- and O-phosphorothioylation of amino acids. AB - A method of highly efficient synthesis of N- and O-phosphorothioylated amino acids was developed. N- and O-(2-Thiono-1,3,2-oxathiaphospholanyl)amino acid methyl esters (3) were prepared in high yields in reaction of amino acid methyl esters with 2-chloro-1,3,2-oxathiaphospholane in pyridine in the presence of elemental sulfur. Compounds 3 were converted in high yield into the corresponding methyl or benzyl phosphorothioamides 6 and 7 by DBU-assisted treatment with methanol or benzyl alcohol. When 3-hydroxypropionitrile was used instead of methanol or benzyl alcohol, the corresponding 2-cyanoethylphosphorothioamidates 4 were obtained in high yield, from which the 2-cyanoethyl group was removed with concentrated ammonium hydroxide. The oxathiaphospholane methodology was also applied for the phosphorylation of amino acids. Thus, 2-oxo-1,3,2 oxathiaphospholane derivatives 10 were prepared by oxidation of compounds 3 with SeO(2.) Compounds 10 were transformed into the corresponding phosphate diesters or amidoesters upon treatment with 3-hydroxypropionitrile in the presence of DBU. The DBU-assisted oxathiaphospholane ring-opening process in 3 and 10 did not cause any measurable C-racemization of phosphorothioylated/phosphorylated amino acids. PMID- 12375954 TI - Toward a computational tool predicting the stereochemical outcome of asymmetric reactions. 1. Application to Sharpless asymmetric dihydroxylation. AB - A reliable and computationally tractable protocol directed at the study of the stereochemical outcome of asymmetric reactions and its application to the Sharpless asymmetric dihydroxylation reaction are proposed. This method, based on a genetic algorithm and molecular mechanics, effectively provides qualitative as well as semiquantitative results and explains the origin of the observed enantioselectivity. For instance, the method reliably predicts reversal of selectivity between similar substrates, unexpected isomers, and even enantio- and diastereoisomeric excess with good accuracy. Two binding modes, closely related to those proposed by Sharpless and Corey, are favored. After comparison with Sharpless' mnemonic device, we propose two alternative interpretations. PMID- 12375955 TI - Asymmetric synthesis of 2H-azirines derived from phosphine oxides using solid supported amines. Ring opening of azirines with carboxylic acids. AB - A simple and efficient asymmetric synthesis of 2H-azirine-2-phosphine oxides 3 is described. The key step is a solid-phase bound achiral or chiral amine-mediated Neber reaction of beta-ketoxime tosylates derived from phosphine oxides 1. Reaction of 2H-azirines 3 and 11 with carboxylic acids 4 gives phosphorylated ketamides 5 and 12. Ring closure of ketamides 5 and 12 with triphenylphosphine and hexachloroethane in the presence of triethylamine leads to the formation of phosphorylated oxazoles 8 and 13. PMID- 12375956 TI - A straightforward enantioselective route to dialkyl sulfoxides based upon two carbon-for-carbon substitution reactions on the sulfinyl group. AB - Benzyl p-bromophenyl sulfoxide 1 was obtained on a multigram scale and in an enantiomerically pure form by an enantioselective catalytic oxidation, using tert butyl hydroperoxide in the presence of chiral titanium complexes. Some mechanistic and stereochemical features of interest were studied in this process. Compound 1 was then subjected to two different substitution reactions with Grignard reagents, which caused two sequentially stereocontrolled carbon-for carbon displacements, leading to chiral nonracemic dialkyl sulfoxides. PMID- 12375957 TI - Synthesis and two-electron redox behavior of diazuleno[2,1-a:1,2-c]naphthalenes. AB - The Diels-Alder reaction of di-2-azulenylacetylene with tetraphenylcyclopentadienone afforded 7,8,9,10-tetraphenyldiazuleno[2,1-a:1,2 c]naphthalene in one pot via autoxidation of the presumed 1,2-di-2 azulenylbenzene derivative. In contrast, a similar reaction of bis(1 methoxycarbonyl-2-azulenyl)acetylene with tetraphenylcyclopentadienone gave the 1,2-di-2-azulenylbenzene derivative. The following cyclodehydrogenation reaction of the benzene derivative with iron(III) chloride afforded diazuleno[2,1-a:1,2 c]naphthalene 6,11-bismethoxycarbonyl derivative. The redox behavior of these novel diazuleno[2,1-a:1,2-c]naphthalenes was examined by cyclic voltammetry (CV). These compounds exhibited two-step oxidation waves at +0.22 to +0.71 V upon CV, which revealed the formation of a radical cation and dication stabilized by the fused two azulene rings under the electrochemical oxidation conditions. Since the 1,2-di-2-azulenylbenzene derivative was oxidized at higher oxidation potentials (+0.83 and +1.86 V), the fusion of the two azulene rings to naphthalene increased electron-donating properties because of the formation of a closed-shell dicationic structure. Formation of the radical cation was characterized by UV-vis spectroscopy under the electrochemical oxidation conditions, although no evidence was obtained for the presumed dication under the conditions of the UV-vis spectroscopy measurement. PMID- 12375958 TI - The first synthesis of phosphonoacrolein. Application to Diels-Alder reaction as heterodiene. AB - The first synthesis of phosphonoacrolein 3 was made in quantitative yield by acidic treatment of beta-ethoxy-alpha-(methoxymethyl)vinylphosphonate 2, derived from a beta-ethoxy-alpha-phosphonovinyl anion and MOMCl. The phosphonoacrolein 3 easily underwent a hetero-Diels-Alder reaction with electron-rich alkenes 4a-f or alkynes 9a-c under mild conditions, and phosphono-substituted pyrans 5a-d, 6e,f or pyranopyrans 11a-c were obtained in good to excellent yields. The reaction of 3 with cyclopentadiene and cyclohexadiene led to mixtures of [2 + 4] and [4 + 2] cycloadducts 7a, 8a and 7b, 8b in modest yields. The cycloaddition reaction between 3 and pyranopyran 13 or dibromocarbene and 13 resulted in [4 + 2] or [2 + 1] cycloadducts 14 or 15 in good yields. PMID- 12375959 TI - Highly stereoselective addition of stannylcuprates to alkynones. AB - The addition of stannylcuprate reagents such as (Bu3Sn)(PhS)CuLi to alkynones has been found to proceed in high yield and with excellent stereoselectivity for the Z isomer of the product (>95%). The behavior of the stannylcuprates is thus very different from that of their "carbocuprate" counterparts such as Me2CuLi or Me2Cu(CN)Li2 which are nonstereoselective. Furthermore, in contrast to the reactions of (R3Sn)(PhS)CuLi with the corresponding alkynoates, the presence of a proton source in the reaction medium has no effect on the stereoselectivity of the reaction of alkynones. PMID- 12375960 TI - Peroxyoxalate chemiluminescence of N,N'-bistosyl-1H,4H-quinoxaline-2,3-dione and related compounds. Dependence on electronic nature of fluorophores. AB - The title compound N,N'-bistosyl-1H,4H-quinoxaline-2,3-dione (TsQD) provides peroxyoxalate chemiluminescence (PO-CL) when reacted with hydrogen peroxide in the presence of fluorophores. The chemiluminescence (CL) efficiency of TsQD was superior to that of other related compounds such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), a typical oxalate for the peroxyoxalate PO-CL, under an aqueous condition. Factors affecting the PO-CL efficiency are discussed from the viewpoint of the structures of the substrates and the electronic nature of the fluorophores. A linear correlation of the logarithmic values evaluated from the CL quantum yields with the oxidation potentials of the aromatic fluorophores supports the involvement of the chemically initiated electron exchange luminescence (CIEEL) mechanism in both TsQD- and TCPO-CL systems. Also, an excellent Hammett relationship was derived from the correlation between the sigma(+) values and the relative singlet excitation yields in TsQD-CL enhanced by a series of fluorescent para,para'-disubstituted distyrylbenzenes. PMID- 12375961 TI - Novel stereocontrolled addition of allylmetal reagents to alpha-imino esters: efficient synthesis of chiral tetrahydroquinoline derivatives. AB - To prepare in multigram scale new antagonists of the glycine binding site associated to the NMDA receptor, an efficient distereoselective route was set up. The addition of suitable allyltin reagents to chiral N-aryl alpha-imino esters (R (+)-tert-butyl lactate used as chiral auxiliary), gave the corresponding alpha amino acid-type derivative in high chemical yield and optical purity. This allylation reaction represents a novel example of efficient long-range stereodifferentiation process. In the last part of the synthesis, a regioselective Heck-type cyclization reaction enabled preparation of the target tetrasubstituted exocycle and trisubtituted endocycle double bond derivatives. PMID- 12375962 TI - Synthesis and electronic properties of regioisomerically pure oxochlorins. AB - We describe a two-step conversion of C-alkylated zinc chlorins to zinc oxochlorins wherein the keto group is located in the reduced ring (17-position) of the macrocycle. The transformation proceeds by hydroxylation upon exposure to alumina followed by dehydrogenation with DDQ. The reactions are compatible with ethyne, iodo, ester, trimethylsilyl, and pentafluorophenyl groups. A route to a spirohexyl-substituted chlorin/oxochlorin has also been developed. Representative chlorins and oxochlorins were characterized by static and time-resolved absorption spectroscopy and fluorescence spectroscopy, resonance Raman spectroscopy, and electrochemistry. The fluorescence quantum yields of the zinc oxochlorins (Phi(f) = 0.030-0.047) or free base (Fb) oxochlorins (Phi(f) = 0.13 0.16) are comparable to those of zinc tetraphenylporphyrin (ZnTPP) or free base tetraphenylporphyrin (FbTPP), respectively. The excited-state lifetimes of the zinc oxochlorins (tau = 0.5-0.7 ns) are on average 4-fold lower than that of ZnTPP, and the lifetimes of the Fb oxochlorins (tau = 7.4-8.9 ns) are approximately 40% shorter than that of FbTPP. Time-resolved absorption spectroscopy of a zinc oxochlorin indicates the yield of intersystem crossing is >70%. Resonance Raman spectroscopy of copper oxochlorins show strong resonance enhancement of the keto group upon Soret excitation but not with Q(y)()-band excitation, which is attributed to the location of the keto group in the reduced ring (rather than in the isocyclic ring as occurs in chlorophylls). The one electron oxidation potential of the zinc oxochlorins is shifted to more positive potentials by approximately 240 mV compared with that of the zinc chlorin. Collectively, the fluorescence yields, excited-state lifetimes, oxidation potentials, and various spectral characteristics of the chlorin and oxochlorin building blocks provide the foundation for studies of photochemical processes in larger architectures based on these chromophores. PMID- 12375963 TI - Solid-state molecular folding and supramolecular structures of triptycene-derived secondary dicarboxamides. AB - The synthesis and X-ray crystal structures of triptycene-derived secondary dicarboxamides 1 and 4-7 and reference compounds 2, 3, and 8 are reported. For comparison, molecular conformations of 1-8 in the gas phase and those of 1 and 3 6 in CD2Cl2 investigated by AM1 modeling and 1H NMR spectroscopy, respectively, are also included. The solid-state conformations of 1 and 5-8 are folded and compact, resulting from the cooperative effects of intramolecular amide-amide hydrogen bonding and edge-to-face arene-arene interactions between the triptycene and the N-acetylsulfanilyl groups. The sulfonyl ester groups are also essential in the folding of 1 and 5-8 and function as structural turn units. In contrast, the conformations of 2-4 are unfolded due to the lack of one of these three essentials. The extended triptycene ring systems in 6 and 7 provide an arene arene contact mode that is different from that for 1 and 5. While AM1 calculations suggest that the two possible arene-arene contact modes in 6 and 7 have similar conformational energies, the one observed in the solid state is also favored in solutions. To achieve a more regular shape for compact crystal packing, the bulky triptycene groups tend to pack in pairs. As a result, the intermolecular amide-amide hydrogen bonding is perturbed and modified with the participation of either the sulfonyl groups or the methanol solvent molecules, leading to various hydrogen-bonding motifs for these triptycene diamides. PMID- 12375964 TI - Novel synthesis of methylenecyclobutanols and 4-methylenetetrahydrofurans from gamma-oxide ylides. AB - The reaction of delta-halo-gamma-oxide ylide, prepared from methylenetriphenylphosphorane and epichlorohydrin, with aldehydes afforded alkylidenecyclobutanols in moderate yields. The reaction initially proceeded through internal nucleophilic attack on delta-carbon of this ylide. Another novel approach toward the synthesis of 4-methylenetetrahydrofurans was achieved by the reaction of gamma-oxide ylides with paraformaldehyde. PMID- 12375966 TI - Facile synthesis of alpha-substituted acrylate esters. AB - Treatment of 5-monosubstituted Meldrum's acids with dimethylmethyleneimmonium iodide (Eschenmoser's iodide salt) in methanol gives alpha-substituted acrylate methyl esters in good yields. Easy access to 5-monosubstituted Meldrum's acids allowed us to synthesize a wide variety of alpha-substituted acrylate methyl esters. The reaction conditions are mild and tolerate many functional groups commonly used in organic synthesis; thus, this new method has potential as an alternative to conventional preparative methods for alpha-substituted acrylate esters. PMID- 12375965 TI - Total synthesis of anhydrolycorinone utilizing sequential intramolecular Diels Alder reactions of a 1,3,4-oxadiazole. AB - A convergent total synthesis of anhydrolycorinone is detailed, enlisting sequential intramolecular Diels-Alder reactions of a suitably substituted 2-amino 1,3,4-oxadiazole defining a novel oxadiazole --> furan --> benzene Diels-Alder strategy. PMID- 12375967 TI - Micellar catalysis of Diels-Alder reactions: substrate positioning in the micelle. AB - We have studied the kinetics of the Diels-Alder reactions of cyclopentadiene, sorbyl alcohol, and sorbyltrimethylammonium bromide with a series of N substituted maleimides in micellar media. Micellar rate constants have been determined and were found to be 20-40 times lower than the respective aqueous rate constants. Nevertheless, it was found that upon addition of sodium dodecyl sulfate the observed rate constants could be enhanced up to a factor of about 4.5. The low micellar rate constants can be attributed to the relatively apolar (water-poor) region of the micelle, in which the reactions take place. NMR experiments indicate that the reactants usually reside near the alpha- or beta CH2 groups of the surfactant molecules in the micelle. Comparison of the micellar rate constants with rate constants in water/1-propanol mixtures suggests a concentration of water of 10-15 M in the micellar region where the diene and dienophile react. PMID- 12375968 TI - Quantum chemical calculations on the Peterson olefination with alpha-silyl ester enolates. AB - The reaction of stabilized Peterson reagents (alpha-silyl ester enolates) with ketones has been studied theoretically and experimentally. Enolate geometry was studied by trapping experiments and NMR spectroscopy and was found to differ markedly with the nature of the base (LiHMDS vs LDA vs KHMDS). The chelating effect of the lithium counterion was found to be critical for the reaction. For the two ketones studied, the combined weight of experimental and computational data assigns geometrical selectivity to the initial addition transition state, though in general there appears to be a fine balance between three possible choices for the rate-determining step. PMID- 12375969 TI - Structures and relative energies of polylithiated benzenes. AB - The geometries of dilithiobenzene, trilithiobenzene, tetralithiobenzene, pentalithiobenzene, and hexalithiobenzene were optimized at the B3LYP/6-311+G** level. The lowest energy structures can be understood in terms of maximizing the electrostatic interactions between carbanions and lithium cations. In particular, in-plane lithium cations bridging ortho dianions is a geometric arrangement that is repeatedly found, as epitomized in the star-shaped D6h hexalithiobenzene structure 11a. Disproportionation reactions involving phenyllithium leading to polylithiated benzenes are exothermic, suggesting that it may be posible to prepare highly lithiated aromatic species. PMID- 12375970 TI - Application of the NT solvent nucleophilicity scale to attack at phosphorus: solvolyses of N,N,N',N'-tetramethyldiamidophosphorochloridate. AB - The specific rates of solvolysis of N,N,N',N' tetramethyldiamidophosphorochloridate have been measured at 25.0 degrees C in 31 solvents. Analysis with the extended Grunwald-Winstein equation leads to sensitivities toward changes in solvent nucleophilicity (l) of 1.20 +/- 0.07 and toward changes in solvent ionizing power (m) of 0.69 +/- 0.04. The correlation is improved by omission of the four data points for 2,2,2-trifluoroethanol-ethanol mixtures (F-test value from 155 to 320) with very small reductions in both l and m values. Activation parameters are reported for eight of the solvolyses. The l and m values are very similar to those previously reported for solvolyses of several arenesulfonyl chlorides, consistent with a concerted substitution process. This assignment is supported by a large k(Cl)/k(F) ratio for hydrolysis and a corresponding ratio for hydroxide-assisted hydrolysis of 178. The stereochemistry of nucleophilic attack at tetracoordinate phosphorus(V) is discussed. PMID- 12375971 TI - Study of interhalogens/silver trifluoromethanesulfonate as promoter systems for high-yielding sialylations. AB - We have studied interhalogen/silver trifluoromethanesulfonate (IX/AgOTf) promoted glycosylations and found differences in the sensitivity of the formed oxocarbenium ions (e.g. from compounds with or without participating groups) toward halide nucleophiles. These differences can be explained using the HSAB theory. By applying this theory on sialylations, we increased the yield for a model reaction from a highly unpredictable 35-46% using ICl to 74% using IBr. We have also showed that the most prominent role of the silver ions is lowering the concentration of the halide nucleophile rather than activating the interhalogen compound and, by increasing the amount of AgOTf from 1 to 1.5 equiv (with respect to IBr), the yield in the model reaction improved from 74% to 89%. A comparison of two different anomeric leaving groups showed that glycal formation can be minimized using a thiophenyl donor instead of xanthate. By combining these observations, we were able to increase the yield of the model reaction to 97%. PMID- 12375972 TI - Palladium-catalyzed selective synthesis of 2-allyltetrazoles. AB - The palladium-catalyzed three-component coupling (TCC) reaction of cyano compounds, allyl methyl carbonate, and trimethylsilyl azide under a catalytic amount of Pd2(dba)3.CHCl3 (2.5 mol %) and tri(2-furyl)phosphine (10 mol %) gave various kinds of 2-allyltetrazoles in good to excellent yields. A pi allylpalladium azide complex is proposed as a key intermediate in the TCC reaction. PMID- 12375973 TI - Pd-catalyzed asymmetric allylic alkylation of glycine imino ester using a chiral phase-transfer catalyst. AB - Pd-catalyzed asymmetric allylic alkylation of the glycine imino ester 1a has been developed using a chiral quaternary ammonium salt 3d without chiral phosphine ligands. The proper choice of the achiral Pd ligand, P(OPh)3, is important to achieve high enantioselectivity. By this method with the dual catalysts, numerous enantiomerically enriched alpha-allylic amino acids 4a-h could be prepared with comparable to higher enantioselectivity than that of the conventional asymmetric alkylation of 1a. In addition, the Pd-catalyzed reaction of 1a with 1-phenyl-2 propenyl acetate 2i afforded the branch product 6 with high enantio- and diastereoselectivity (>95% de, 85% ee). PMID- 12375974 TI - Introduction of a hydroxy group at the para position and N-iodophenylation of N arylamides using phenyliodine(III) bis(trifluoroacetate). AB - The reaction of anilides with phenyliodine(III) bis(trifluoroacetate) (PIFA) in trifluoroacetic acid (TFA), TFA-CHCl3, or hexafluoroisopropyl alcohol (HFIP) is described. When the acyl group of the anilide is highly electronegative, such as trifluoroacetyl, or the phenyl group is substituted with an electron-withdrawing group, the 4-iodophenyl group is transferred from PIFA to the amide nitrogen to afford acetyldiarylamines. On the other hand, when the acyl group contains an electron-donating function, such as 4-methoxyphenyl, or the phenyl group is substituted with an electron-donating group, a trifluoroacetoxy group is transferred to the para position of the anilide aromatic ring. This group is hydrolyzed during workup to produce the corresponding phenol. PMID- 12375975 TI - Mild alpha-halogenation reactions of 1,3-dicarbonyl compounds catalyzed by Lewis acids. AB - Lewis acid Mg(ClO4)2, combined with NBS, in CH3CN or EtOAc provided mild and fast bromination of 1,3-dicarbonyl compounds. In particular, this protocol could be applied to the alpha-monobromination of alpha-unsubstituted beta-keto esters. Similar Lewis acid catalysis was also extended to the alpha-chlorination and iodination of 1,3-dicarbonyl compounds with NCS and NIS, respectively. PMID- 12375976 TI - Theoretical studies on cycloaddition reactions between 1-aza-2-azoniaallene cation and olefins. AB - Density functional (B3LYP) calculations using the 6-31++g basis set have been employed to study the title reaction between the cationic 1,3-dipolar 1-aza-2 azoniaallene ion (H2C=N(+)=NH) and ethene. Our calculations confirmed that [3 + 2] cycloaddition reaction takes place via a three-membered ring intermediate. In addition, solvent effects and substituent effects were also studied. For the reactions involving tetrachloroethene, there are two attacking sites. One is on the NH group in the 1-aza-2-azoniaallene ion, another is on its terminal CH2 group, and they are competitive for both approaching positions. Electron releasing methyl substituents on ethene favor the reaction, and the potential energy surface is quite different from the previous one. PMID- 12375977 TI - Intramolecular hydrogen abstraction reaction promoted by alkoxy radicals in carbohydrates. Synthesis of chiral 2,7-dioxabicyclo[2.2.1]heptane and 6,8 dioxabicyclo[3.2.1]octane ring systems. AB - The reaction of specifically protected anhydroalditols with (diacetoxyiodo)benzene or iodosylbenzene and iodine is a mild and selective procedure for the synthesis of chiral 6,8-dioxabicyclo[3.2.1]octane and 2,7 dioxabicyclo[2.2.1]heptane ring systems under neutral conditions. This reaction can be considered to be an intramolecular glycosidation that goes through an intramolecular hydrogen abstraction promoted by an alkoxy radical followed by oxidation of the transient C-radical intermediate to an oxycarbenium ion. This methodology is useful not only for the preparation of chiral synthons but also for the selective oxidation of specific carbons of the carbohydrate skeleton, constituting a good procedure for the synthesis of protected uloses. PMID- 12375978 TI - Rhodium complex-catalyzed Pauson-Khand-type reaction with aldehydes as a CO source. AB - With aldehydes as a CO source under solvent-free conditions, rhodium complex efficiently catalyzed an intramolecular carbonylative alkene-alkyne coupling (Pauson-Khand-type reaction) and various bicyclic enones were obtained in high yield. Experiments under argon flow and a 13C-labeling experiment suggested that almost no free carbon monoxide was generated in this reaction. When noncationic rhodium complex with chiral phosphine was used as a chiral catalyst, the reaction proceeded enantioselectively to give various chiral cyclopentenones in up to 90% ee under solvent-free conditions. PMID- 12375979 TI - Alkynylzirconation of alkynes and application to one-pot bisalkynylation of alkynes. AB - Stereocontrolled alkynylzirconation of unactivated alkynes was achieved by the reaction of an alkyne with Cp2ZrEt2 and alkynyl halide in this order. After hydrolysis of the alkynylzirconation product, trisubstituted enyne derivatives were obtained in good yields. Functionalized enynes were also prepared by the reaction of the alkynylzirconation products with a variety of electrophiles. Subsequent addition of the second alkynyl halide to the alkynylzirconation products provided an in situ protocol for bisalkynylation of alkynes into (Z) enediynes in good yields. PMID- 12375980 TI - Selective synthesis of meso-naphthylporphyrins. AB - A series of novel meso-(8-substituted naphth-1-yl)porphyrins has been synthesized creating derivatives with a tight recognition environment above the porphyrin plane. The selective synthesis of single atropisomers is discussed. Condensation of bisnaphthaldehyde 12 with phenyldipyrromethane unexpectedly led to selective synthesis of the alpha,alpha-5,10-bridged isomer 14. A mechanism is proposed for this unusual scrambling, and alternative syntheses of alpha,alpha-5,15 bisnaphthylporphyrins are described. Synthesis of 5,15-analogues can be achieved by employing (pentafluorophenyl)dipyrromethane or via presynthesis of a bis(dipyrromethane) derivative 22 (from bisnaphthaldehyde 12) and subsequent condensation with benzaldehyde. PMID- 12375982 TI - Site of nucleophilic attack and ring opening of five-membered heterocyclic 2,3 diones: a density functional theory study. AB - The site of nucleophilic addition to five-membered heterocyclic 2,3-diones (4 iminomethylfuran-2,3-dione A1 and 4-formyl-pyrrole-2,3-dione B1) is studied by density functional theory calculations (B3LYP/6-31G) with water as the nucleophile. Both uncatalyzed and water-assisted 1,2-addition to the lactone (lactam) and the keto carbonyl group, and conjugate addition to C5 of the heterocycle and the heteroatom of the 4-iminomethyl (formyl) moiety are considered. In addition, concerted and stepwise ring fission of the lactone (lactam) ring is also treated. The effect of solvation (aqueous solution) is taken into account by the polarizable continuum model (PCM) and the Poisson Boltzmann SCRF method (PB-SCRF), as well as explicit water molecules. Only this latter approach yields meaningful activation free energies. Barriers for addition of H2O increase in the order 1,4-addition to C5 < addition to the lactone (lactam) carbonyl < hydration of the 3-keto group. No reaction path for concerted water-assisted ring opening could be found. PMID- 12375981 TI - Addition of ester enolates to N-alkyl-2-fluoropyridinium salts: total synthesis of (+/-)-20-deoxycamptothecin and (+)-camptothecin. AB - Several 4-substituted dihydropyridones or 2-pyridones have been prepared by nucleophilic addition of alpha-(methylsulfanyl)ester enolates to N-alkyl-2 fluoropyridinium salts, followed by acid hydrolysis or oxidation with concomitant hydrolysis, of the intermediate 2-fluoro-1,4-dihydropyridine adducts, respectively. Addition of the enolate derived from isopropyl alpha (methylsulfanyl)butyrate to N-(quinolylmethyl)-2-fluoropyridinium triflate 21 followed by DDQ treatment gave pyridone 29, from which (+/-)-20-deoxycamptothecin (31), a known precursor of camptothecin, was synthesized by a radical cyclization desulfurization, with subsequent elaboration of the lactone E ring by chemoselective reduction. A similar sequence starting from the enolate of a chiral 2-hydroxybutyric acid derivative (33) provides access to natural (+) camptothecin (37). PMID- 12375983 TI - Synthesis and conformational analysis of small peptides containing 6-endo-BT(t)L scaffolds as reverse turn mimetics. AB - Two new dipeptide isosteres derived from L-leucine and meso-tartaric acid derivatives, named 6-endo-BTL and 6-endo-BtL, were inserted in a small peptide by means of SPPS, and the conformational features of the resulting peptides 3 and 4 were studied by NMR, IR, and molecular modeling techniques. The presence of a reverse turn conformation was observed in all the structures, suggesting the key role of the scaffolds as reverse turn promoters. Peptides 3 and 4 did not adopt a preferred conformation as indicated by the presence of equilibria between open turn and intramolecular hydrogen-bonded structures. 6-endo-BTL-peptide 3 showed a 3:1 mixture of conformers. The major conformer adopted mainly an open turn structure in equilibrium with hydrogen-bonded structures. The minor conformer displayed a better organized structure with a 14-membered ring hydrogen-bond typical of a beta-hairpin-like structure, in equilibrium with a gamma-turn, too. 6-endo-BtL-peptide 4 showed a unique conformer, and did not adopt as good a conformation as 3, due to the bulky equatorial substituent at C-2. Thus, marked structural differences between peptides containing 6-endo-BTL and 6-endo-BtL scaffolds as reverse turn inducers exist. PMID- 12375984 TI - A highly selective aerobic oxidation process catalyzed by electron-deficient nitroarenes via single electron transfer processes. AB - A versatile and simple method for aerobic oxidation of various aromatics containing an oxygen-functionalized benzylic carbon is reported. Results from oxidation experiments with methyl aryl ketones, benzaldehydes, benzylic alcohols, and mandelic acid are reported; all provide high yields of the corresponding aromatic carboxylic acids. By means of response surface methodology and mechanistic interpretation it is revealed that the oxidation process operates catalytically using electron-deficient nitroarenes, such as 1,3-dinitrobenzene, 1,2,3-trifluoro-4-nitrobenzene, and 2,4-difluoro-1-nitrobenzene, with molecular oxygen as the terminal oxidant. The reaction is carried out in a basic solution of potassium tert-butoxide in tert-butyl alcohol. Since the terminal oxidant is molecular oxygen and only 5-10 mol % of, e.g., 1,3-dinitrobenzene as catalyst is used, the new method represents an environmentally benign alternative to, for example, the well-known haloform reaction. The method is also a convincing alternative when transition metal free conditions are required. PMID- 12375986 TI - Synthesis and structural characterization of new stiff rod oligomeric domains by X-ray crystallography and NMR. AB - The monomers N,N'-dibenzylbenzene-1,4-diamine (1), N,N'-dibenzylnaphthalene-1,5 diamine (2), and N,N'-dibenzylanthracene-1,9-diamine (3) were reacted with phosgene in the presence of a base to produce the corresponding N,N'-dibenzyl-1,4 bis(chlorocarbonylamino)benzene (4), N,N'-dibenzyl-1,5 bis(chlorocarbonylamino)naphthalene (5), and N,N'-dibenzyl-9,10 bis(chlorocarbonylamino)anthracene (6). These monomers were used to create zigzag type stacks, in a stepwise fashion, of trimers and 9-mers of either 1,4 diureidobenzenes ((Phe)K(3) and (Phe)K(9)) or 1,5-diureidonaphthalenes ((Nap)K(3) and (Nap)K(9)). A byproduct in the formation of (Phe)K(9) was a cyclic hexamer (Phe)K(6). NMR gave evidence of the structure in solution while X-ray crystallographic information was obtained for 5, 6, (Nap)K(3), and the cyclic (Phe)K(6). PMID- 12375985 TI - Tuning the redox chemistry of 4-benzoyl-N-methylpyridinium cations through para substitution. Hammett linear free energy relationships and the relative aptitude of the two-electron reduced forms for H-bonding. AB - In anhydrous CH3CN a series of nine 4-(4-substituted-benzoyl)-N-methylpyridinium cations (substituent: -OCH3, -CH3, -H, -SCH3, -Br, -Ctbd1;CH, -CHO, -NO2, and (+)S(CH3)2) demonstrate two chemically reversible, well-separated one-electron (1 e) reductions in the same potential range as other main stream redox catalysts such as quinones and viologens. Hammett linear free energy plots yield excellent correlation between the E(1/2) values of both waves and the substituent constants sigma(p)(-)(X). The reaction constants for the two 1-e reductions are rho(1) = 2.60 and rho(2) = 3.31. The lower rho(1) value is associated with neutralization of the pyridinium ring, and the higher rho(2) value with the negative charge developing during the 2nd-e reduction. Structure-function correlations point to a purely inductive role for substitution in both 1-e reductions. The case of the 4 (4-nitrobenzoyl)-N-methylpyridinium cation is particularly noteworthy, because the 4-nitrobenzoyl moiety undergoes reduction before the 2nd reduction of the 4 benzoyl-N-methylpyridinium system. Correlation of the third wave of this compound with the 2nd-e reduction of the others yields sigma(p)(-NO)2*- = -0.97 +/- 0.02, thus placing the -NO2-* group among the strongest electron donors. Solvent deuterium isotope effects and maps of the electrostatic potential (via PM3 calculations) as a function of substitution support that 2-e reduced forms develop H-bonding with proton donors (e.g., CH3OH) via the O-atom. The average number of CH3OH molecules entering the H-bonding association increases with e donating substituents. H-bonding shifts the 2nd reduction wave closer to the first one. This has important practical implications, because it increases the equilibrium concentration of the 2-e reduced form from disproportionation of the 1-e reduced form. PMID- 12375987 TI - Syntheses of chiral homoazacalix[4]arenes incorporating amino acid residues: molecular recognition for racemic quaternary ammonium ions. AB - Chiral calixarene analogues incorporating amino acid residues into the macrocyclic rings were prepared from the cyclization reactions of bis(chloromethyl)phenol-formaldehyde tetramer with amino acid methyl ester in moderate yields. The macrocycles form a chiral concavity, which is induced by the chiral transmission from the point chirality of the amino acid residues to the phenol-formaldehyde tetramer unit. The macrocycles have the cavity pi-basic enough to include the quaternary ammonium ion due to the cation-pi interaction and can serve as a shift reagent for racemic ammonium ions during 1H NMR analysis. PMID- 12375988 TI - A novel approach to a one-pot synthesis of unsubstituted oligo(alpha-thiophenes). AB - Oligo(alpha-thiophenes) alpha-4T and alpha-8T were prepared by the following one pot sequential conversions: thiophene (T) --> alpha-2T --> alpha-4T --> alpha-8T. PdCl(2)-induced coupling of a mono-alpha-mercuration derivative of each of the n mers T, alpha-2T, and alpha-4T was applied in these conversions. PMID- 12375989 TI - A novel access to disubstituted acetylenes. AB - Reactions of organometallic reagents with 1-(substituted ethynyl)-1H-1,2,3 benzotriazoles 5 derived from a variety of benzotriazolylmethyl ketones 3 afforded disubstituted acetylenes in synthetically useful yields. PMID- 12375990 TI - Easy access to N,N-bis(but-3-enyl)-, N-allyl-N-(but-3-enyl)-, and N-(but-3-ynyl) N-(but-3-enyl)-amines. AB - N,N-Bis(but-3-enyl)amines 5a-i were prepared in overall 74% yield from 1 (triphenylphosphoroylideneaminoalkyl)benzotriazole using an aza-Wittig reaction with aldehydes followed by a double Grignard reaction with allylmagnesium bromide. Use of vinyl or 1-propynylmagnesium bromide and allylmagnesium bromide in a sequential fashion also formed the expected doubly unsaturated amines 9a,b and 12, respectively. PMID- 12375991 TI - SnCl4-catalyzed reaction of o-benzoquinones and aryl acetylenes: an unprecedented one-pot synthesis of tropone derivatives. AB - Highly substituted tropone derivatives were obtained as a result of SnCl4 catalyzed cycloaddition of 3-methoxy-substituted o-benzoquinones with aryl acetylenes and subsequent rearrangement of the adducts with concomitant decarbonylation. PMID- 12375992 TI - The Pd3(dppm)3(CO)2+ cluster: an efficient electrochemically assisted Lewis acid catalyst for the fluorination and alcoholysis of acyl chlorides. AB - The dicationic palladium cluster Pd3(dppm)3(CO)2+ (dppm = bis(diphenylphosphino)methane) reacts with acid chlorides RCOCl (R = n-C6H13, t Bu, Ph) to afford quantitatively the chloride adduct Pd3(dppm)3(CO)(Cl)+ and the acyl cation RCO+ as the organic counterpart. The dicationic reactive cluster can be reformed by electrolyzing the chloride complex with a copper anode leaving CuCl as a byproduct. The combination of these two reactions provides an electrocatalytic way to form the acylium from the acid chloride. Indeed, in CH2Cl2, 0.2 M NBu4PF6, or NBu4BF4, the electrolysis of the acid chloride in the presence of a catalytic amount of the cluster (1%) gives in good yields the acid fluoride RCOF, arising from the coupling of the acylium with a F(-) issued from the fluorinated supporting electrolyte. Alternatively, in CH2Cl2 or 0.2 M NBu4ClO4, by operating with an alcohol R'OH as the nucleophile, the electrolysis gives the ester RC(O)OR' as the only final product. PMID- 12375993 TI - Functionalized pyridylboronic acids and their Suzuki cross-coupling reactions to yield novel heteroarylpyridines. AB - 2-Bromo-5-pyridylboronic acid 2a, 2-chloro-5-pyridylboronic acid 2b, 2-methoxy-5 pyridylboronic acid 2c, and 5-chloro-2-methoxy-4-pyridylboronic acid 4 have been synthesized and shown to undergo palladium-catalyzed cross-coupling reactions with heteroaryl bromides to yield novel heteroarylpyridine derivatives. The X-ray crystal structures of 2a and 2b have been obtained. PMID- 12375994 TI - Synthesis of R-(-)-imperanene from the natural lignan hydroxymatairesinol. AB - A convenient and high yielding method for the synthesis of R-(-)-imperanene, starting from the readily available natural lignan hydroxymatairesinol from Norway spruce, was developed. Hydroxymatairesinol was degraded in strongly basic aqueous conditions to (E)-4-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-3 methoxyphenylmethyl)but-3-enoic acid, which was esterified and then reduced by LiAlH(4) to afford R-(-)-imperanene. The configuration at the crucial stereocenter was preserved in the synthesis, and the obtained product was identified by optical rotation measurements and chiral HPLC analyses as the R-(-) enantiomer (ee 86-92%). PMID- 12375995 TI - Stereoselective syntheses of (+)-goniodiol, (-)-8-epigoniodiol, and (+)-9 deoxygoniopypyrone via alkoxyallylboration and ring-closing metathesis. AB - A convenient synthesis of (+)-goniodiol, (-)-8-epigoniodiol, and (+)-9 deoxygoniopypyrone has been developed via asymmetric alkoxyallylboration and ring closing metathesis pathways. PMID- 12375996 TI - A non-cryogenic method for the preparation of 2-(indolyl) borates, silanes, and silanols. AB - 2-Indolyl borates are prepared via addition of LDA to a mixture of N-Boc-indole and triisopropyl borate at 0-5 degrees C. Following acidic hydrolysis, the boronic acids are isolated by crystallization in good to excellent yield (73 99%). The method is quite general, tolerating a wide range of functional groups, and also provides access to 2-silyl derivatives (80-91%). PMID- 12375997 TI - Direct synthesis of guanidines using di(imidazole-1-yl)methanimine. AB - A direct synthetic approach to guanidine compounds is reported here using di(imidazole-1-yl)methanimine and di(imidazole-1-yl)cyanomethanimine as guanylating reagents. PMID- 12375998 TI - Ru3(CO)12-catalyzed C-H/CO/olefin coupling of N-pyridylindolines. Direct carbonylation at a C-H bond delta to the pyridine nitrogen. AB - The reaction of N-pyridylindolines with CO and ethylene in the presence of Ru3(CO)12 results in direct carbonylation at a C-H bond delta to the pyridine sp2 nitrogen, which represents a new type of C-H/CO/olefin coupling. The presence of a pyridine ring as a directing group on the substrates is essential for the reaction to proceed. The choice of N,N-dimethylacetamide (DMA) as the solvent is crucial for the reaction to proceed efficiently. PMID- 12375999 TI - Synthesis of a carbon analogue of N-acetylmannosamine via acetolysis on a relatively stable ozonide. AB - After a 2-methylpropenyl group was added to a carbohydrate framework through regioselective opening of a glucose-derived epoxide, hydrolysis or acetolysis of the methyl glycoside in various derivatives was problematic. Ozonolysis of the olefin followed by brief treatment with dimethyl sulfide gave a mixture of diastereomeric ozonides that proved to be stable for weeks at room temperature. Acetolysis of these ozonides at low temperature allowed selective cleavage of the methyl glycoside in the presence of the 1,2,4-trioxolane as well as selective formation of the alpha-acetate. PMID- 12376000 TI - Stereoselective synthesis of Neu5Acalpha(2-->5)Neu5Gc: the building block of oligo/poly(-->5-O(g)(lycolyl)Neu5Gcalpha2-->) chains in sea urchin egg cell surface glycoprotein. AB - The synthesis of a sialic acid dimer derivative, Neu5Acalpha(2-->5)Neu5Gc, is described. The synthetic strategy is based on the use of allyl alcohol to achieve an exclusive alpha-sialylation product. The allyloxy group is also a latent glycolic acid that provides the subsequent coupling with neuraminate with minimal protection-deprotection manipulations. PMID- 12376001 TI - Calix[4]- and calix[5]arene-based multicavity macrocycles. AB - Synthesis and single-crystal X-ray structures of mixed triple and double calixarenes 6 and 7, obtained from the base-catalyzed condensation of calix[5]arene 1 with cone pertosylated calix[4]arene 2, are reported. VT-NMR studies on 7 are consistent with a molecular motion arising from the anti-gauche conformational interconversion of its ethylene linkages. PMID- 12376002 TI - Synthesis of enantiopure cis- and trans-2,3-disubstituted piperidines. AB - The synthesis of enantiopure cis- and trans-2,3-disubstituted piperidines 4 is described. The key step of the synthesis involves the stereoselective reduction of chiral nonracemic lactams 2 by using BH3.Me2S. A rationalization of the stereoselectivity is presented. PMID- 12376004 TI - Cavitation versus degassing: in vitro study of the microbubble phenomenon observed during echocardiography in patients with mechanical prosthetic cardiac valves. AB - BACKGROUND: With the advent of second harmonic imaging in echocardiography, microbubbles have been observed during opening and closure of mechanical prosthetic valves. The single phenomenon of cavitation, an extremely short event described in the literature, cannot explain the persistence of microbubbles during several hundred milliseconds. Therefore, in vitro we reproduced two distinct phenomena created by a local depression occurring during the closure and/or opening of prosthetic valves: Cavitation and degassing. METHODS: We used a water circuit system enriched with CO(2) that passes through a Venturi tube in order to create variable pressure gradients. Three types of observations were performed: (1). the dimensions of the bubbles as a function of pressure, (2). calibration of the echocardiograph, and (3). comparison and illustrations of the difference between bubble formation by cavitation (vaporization) and degassing (liberation of CO(2)). RESULTS: According to the different pressures exerted, the dimensions of the bubbles only vary by several microns, not measurable in practice. Second, the calibration of the echocardiograph reveals that the dimensions of the bubbles measured by ultrasound are greater by a factor of 1.75. Finally, the observed cavitation is a short phenomenon (several milliseconds) and happens under a great local pressure gradient. The degassing produces microbubbles lasting up to as long as > 1 second under much lower pressure. CONCLUSION: This in vitro study suggests that microbubbles observed during several hundred milliseconds after the opening of prosthetic cardiac valves are the result of degassing of CO(2) in blood rather than a cavitation phenomenon as suggested in the literature. PMID- 12376003 TI - D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro. AB - Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25 kDa (SNAP-25) are the main components of the soluble N -ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction. PMID- 12376006 TI - Prognostic value of dobutamine echocardiography in patients with intermediate coronary lesions at angiography. AB - The prognostic value of dobutamine echocardiography (DOBU-ECHO) in patients with intermediate coronary lesions has not been described in the literature. The aim of this study was to determine the prognostic value of DOBU-ECHO in patients presenting with coronary lesions smaller than 50% at angiography. Ninety-four consecutive patients were analyzed and followed-up for 64 +/- 7 months (range: 12 to 75 months). All patients presented with coronary lesions between >or= 30% and < 50% of the luminal diameter of at least one major epicardial vessel. The patient population was divided into two groups: Those with a positive DOBU-ECHO (n = 23) and those with a negative DOBU-ECHO (n = 71). The number of coronary lesions did not differ between the two groups. The patients with a positive DOBU ECHO result were more likely than those in the negative group to have a family history of coronary artery disease or suffer from hypertension or a dyslipidemia. During the follow-up period, 13 cardiac events occurred (1 cardiac death, 5 myocardial infarctions, 2 unstable anginas, and 5 myocardial revascularizations), 11 (47.8%) of which occurred in patients with positive DOBU-ECHO. The annual incidence for a cardiac event was 7.9% per year in the positive DOBU-ECHO group and 0.5% per year in the negative DOBU-ECHO group (P < 0.001). This incidence remained significant for spontaneous cardiac events, such as cardiac death, myocardial infarction, and unstable angina (5.8% per year vs 0.2% per year; P < 0.001). CONCLUSIONS: In patients with angiographically confirmed intermediate coronary lesions, a positive DOBU-ECHO is an additional risk factor for the onset of a cardiac event, whereas a negative DOBU-ECHO can be used to define patients with a low cardiac risk. PMID- 12376005 TI - Clinical utility of low dose dobutamine echocardiography in regional myocardial viability detection before and after surgical revascularization. AB - Seventy-eight consecutive patients (mean 63 +/- 10 years, 79.5% men) with a history of myocardial infarction and indication of coronary artery bypass grafting (CABG) were studied with low dose dobutamine stress echocardiography (DSE) before (DSE 1) and at 3-month follow-up (DSE 2) to evaluate its clinical utility in the detection of viable myocardium. We determined the expected utilities of global patients (P; n = 67) and coronary territories (T; n = 126) with the classic strategy: DSE 1 and results of a rest follow-up echocardiogram (REST 2) and applying them to a complementary strategy that submitted false positives (Fp) and false negatives (Fn) results to DSE 2. Assigned utilities in each node of the decision tree were maximal (1.0), submaximal ( 0.75), and intermedium (0.50) using the folding method as a mathematical model. RESULTS: Global P and T expected utilities when performing DSE 1 were 0.84 and 0.89, respectively for positive viability; 0.85 and 0.82, respectively; and for negative viability 0.83 and 0.82, respectively. The expected utilities with the decision of performing a DSE 2 to Fp were 0.74 and 0.76, respectively (viability was detected in 66% of P and in 58% of T) and 0.47 and 0.45, respectively, as applied to Fn. CONCLUSIONS: Low dose DSE results in high clinical utility by finding viable or scar myocardium before CABG as well as when discordant results are found in follow-up, particularly with Fp. PMID- 12376007 TI - An early predictor of left ventricular remodeling after reperfused anterior acute myocardial infarction: ratio of peak E wave velocity/flow propagation velocity and mitral E wave deceleration time. AB - Several studies have demonstrated that the ratio of peak E wave velocity/flow propagation velocity (E/FPV) using color M-mode Doppler echocardiography and the mitral E wave deceleration time make it possible to estimate left ventricular filling pressure. Recent studies have indicated that deceleration time can predict left ventricular dilation after acute myocardial infarction. The purpose of this study was to determine whether the early assessment of deceleration time and E/FPV could predict left ventricular dilation after acute myocardial infarction. We studied 55 patients with first anterior acute myocardial infarction who underwent successful coronary angioplasty by two-dimensional (2-D) Doppler echocardiography within 12 hours and at 1 and 6 months after reperfusion. Patients were divided into three groups according to deceleration time and E/FPV immediately after reperfusion: (1). restrictive filling (deceleration time < 140 msec and E/FPV > or= 2.0), (2). elevated filling pressure (deceleration time >or= 140 msec and E/FPV >or= 2.0), (3). and normal filling pressure (deceleration time >or= 140 msec and E/FPV < 2.0). The end-diastolic volume index (EDVI) was similar in the three groups immediately after reperfusion. EDVI in the groups with restrictive filling and elevated filling pressure was significantly greater than that in the group with normal filling pressure at 6 months (93 +/- 11 and 89 +/- 16 vs 59 +/- 11 ml/m(2), respectively; P < 0.0001). E/FPV shows a better correlation with the change in EDVI at 6 months than deceleration time (r = 0.77; P < 0.0001 and r = - 0.46; P < 0.001, respectively). The early measurement of E/FPV provides a simple and accurate means for predicting left ventricular dilation after acute myocardial infarction. PMID- 12376008 TI - One-year follow-up of a patient with reversible tricuspid valve stenosis due to lymphomatic mass into the right atrioventricular wall. AB - We present the case of a 66-year-old man with a history of coronary artery disease and chronic lymphocytic leukemia (CLL) who was admitted to the hospital complaining of chest discomfort and shortness of breath on exertion. The echocardiogram revealed a severe pericardial effusion and a large echogenic mass that infiltrated the lateral wall of the right atrium and ventricle and created a moderate tricuspid valve stenosis. B cell intracardiac non-Hodgkin lymphoma/CLL was diagnosed, and the patient was treated with six courses of CHOP chemotherapy. After the third course, the mass disappeared and the patient's general condition was substantially improved. PMID- 12376009 TI - Periprosthetic aortic valve ring abscess. PMID- 12376010 TI - Benign metastasizing leiomyomatosis diagnosed by echocardiography. PMID- 12376011 TI - Transesophageal color Doppler three-dimensional echocardiographic assessment of left circumflex coronary artery fistula. AB - We describe an adult patient with a left circumflex coronary artery fistula in whom color Doppler three-dimensional transesophageal echocardiography demonstrated clearly the exact site of the communication with the coronary sinus near the left atrial appendage. This could not be delineated definitively by multiplane two-dimensional transesophageal echocardiography. PMID- 12376012 TI - Update on intraoperative echocardiography, part I. PMID- 12376013 TI - Intraoperative echocardiography and minimally invasive cardiac surgery. AB - The term minimally invasive cardiac surgery encompasses a number of different techniques, each with its own rationale, origin, and development, but all focusing on limiting the physiologic trespass of cardiac surgery on the patient. In this article, we discuss the application of intraoperative echocardiography to three types of these procedures: Offpump coronary artery bypass graft (OPCABG) surgery, valvular surgery through limited thoracic incisions, and port-access heart surgery. PMID- 12376014 TI - Intraoperative transesophageal echocardiography with a special focus on a patient undergoing advanced robotic-assisted procedures. AB - Intraoperative echocardiography (IOE) has earned a major role in surgical decision-making and helps the cardiac surgeon decide and proceed with appropriate intervention based on the visualized pathology. With the advent of minimally invasive surgical techniques and robotic-assisted operations, more emphasis and dependence has been placed on IOE. We describe our experience with IOE during mitral valve repair at our center, which is one of the pioneer centers for these telemanipulation techniques. PMID- 12376015 TI - Thoracic aortic disease: endovascular stents. AB - Aortic disease is a serious, often life-threatening condition. The keys to instituting appropriate therapy in diseases of the aorta include accurate and rapid diagnosis and anatomical assessment. Endovascular aortic repair is a new alternative to conventional surgical approaches. Because arterial rupture is a risk of this procedure, appropriate facilities for resuscitation must be present during the procedure. This paper reviews the important aspects of aortic anatomy, echocardiographic imaging of the thoracic aorta, aortic pathology, endovascular surgery, and the role of echocardiography in the evaluation of the surgical outcome. PMID- 12376016 TI - The role of intraoperative transesophageal echocardiography in heart transplantation. AB - The number of centers that perform heart transplants has increased rapidly in recent years. Although transthoracic and transesophageal echocardiography (TTE and TEE) are utilized frequently to diagnose and manage cardiac complications commonly found in this population postoperatively, little has been written about the routine use of intraoperative TEE. Intraoperative echo is ideally suited to identify acute complications during cardiac transplantation. This can include immediate signs of rejection, valvular abnormalities, and mechanical complications related to the surgical procedure. Many of these patients might require ventricular assist devices (VAD) to provide circulatory support, and intraoperative TEE can be used to verify correct positioning of the VAD hardware. In addition, many of the chronic complications that patients with heart transplants are at risk for may be serious yet asymptomatic. Therefore, a high quality, complete intraoperative echocardiographic study might serve as an important baseline to compare postoperative changes. PMID- 12376017 TI - Volume reduction surgery for end-stage ischemic heart disease. AB - The Dor procedure, or infarction excision surgery, was first used in 1984. It is a surgical treatment option for patients with end-stage ischemic heart failure. In a recently published multicenter study that included a total of 439 patients, average ejection fraction increased from 29 +/- 10% to 39 +/- 12% after surgery. In our experience, the overall survival rate 18 months after surgery is 89%, and the preoperative mortality rate is 6.6%. These results are similar to the previous reports from Dor's group, which confirmed the certain value of the surgery. Echocardiography, including intraoperative transesophageal echocardiography, plays an important role in clarifying cardiac anatomies, absolute left ventricular (LV) volumes, ejection fraction, and mitral regurgitation in patients with ischemic heart failure undergoing this surgery. With the development of ultrasound and computer technology, three-dimensional echocardiography may be preferred when evaluating the surgical results, including determination of absolute LV volumes. Communication between experienced cardiac surgeons and echocardiographers in the operating room is essential for successful outcomes and reliable evaluation of the surgery. PMID- 12376018 TI - Comparison of different ligand densities for the manufacture of CB Hep-1 immunosorbents. AB - Different ligand densities of monoclonal antibody (Mab) CB.Hep-1 were studied during covalent coupling on Sepharose CL-4B for recombinant hepatitis B surface antigen (rHBsAg) immunoaffinity purification. Ligand densities of 2.2, 3.2, 4.2 and 5.2 mg Mab/ml immunosorbents, respectively, were assayed during five cycles of immunoaffinity chromatography (IAC). Adsorption capacities averaged either 3.2 mg/ml (0.57 mg rHBsAg/ml immunosorbent/5.42 mg of total purified protein) or 5.2 mg/ml (0.56 mg rHBsAg/ml immunosorbent/5.05 mg total purified protein). Immunosorbents showed ligand leakage levels below 3 ng Mab/microg rHBsAg. Antigen purity was higher than 95% in all cases. The results suggest that a ligand density (LD) of 3.2 mg Mab/ml immunosorbent should be used for immunoaffinity chromatography because no significant differences were found in the ligand densities studied (P-value=0.012), which saves 40% of CB.Hep-1 immunosorbent manufacturing cost in comparison with 5 mg Mab/ml immunosorbent, which is currently used in large-scale production. PMID- 12376019 TI - A comparison between SDS-PAGE and size exclusion chromatography as analytical methods for determining product composition in protein conjugation reactions. AB - Horseradish peroxidase (HRP) was conjugated with bovine serum albumin (BSA) or human alpha(1)-proteinase inhibitor (alpha(1)-PI). The enzyme was maleimidylated using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and then allowed to react with thiolated BSA or reduced alpha(1)-PI. The conjugation products were analysed both by SDS-PAGE and size exclusion chromatography (SEC) on Sephadex G200. The two methods of evaluating conjugative processes were compared with respect to information provided in relation to the behaviour of the products in solution. The results showed that neither SDS-PAGE nor SEC alone provides sufficient information about conjugate structure. The basic conjugate units observed in electrophoresis tend to form dimeric or higher-order aggregates under gel chromatographic conditions. PMID- 12376020 TI - High efficient encapsulation of plasmid DNA in PLGA microparticles by organic phase self-emulsification. AB - To overcome the drawbacks of encapsulating plasmid DNA (pDNA) in poly (D,L-lactic co-glycolic acid) (PLGA) by water-in-oil-in-water double-emulsion solvent evaporation method, we have developed a novel procedure for encapsulating pDNA in PLGA microparticles called DNA organic phase self-emulsification (DOPSM). This method was based on both the extraction plasmid DNA from aqueous phase into organic phase and the spontaneous emulsification DNA in organic phase by solvent diffusion method. The efficiency of extraction plasmid DNA into organic phase is 99% and the concentration of pDNA in organic phase is up to 2.4 mg/ml. The efficiency of microencapsulation of plasmid DNA in PLGA is up to 76% and can be enhanced by lowering the pH of aqueous solution of emulsion. The microparticles size of PLGA of pDNA is in a narrow range of 1-2 microm. This procedure does not involve the high mechanical energy to emulsify which may damage the integrity of pDNA. This method can be applied to encapsulate the pDNA into microparticles of other biocompatible polymers with high efficiency. PMID- 12376021 TI - A nonradioactive assay method for determination of enzymatic activity of D ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). AB - A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3 phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference. PMID- 12376022 TI - Voltammetric studies of the interaction of transition-metal complexes with DNA. AB - The interaction of the two new synthesized transition-metal complexes, ML(2) (M=Co, Cu, L=1,8-dihydroxyethyl-1, 3,8,10,13-hexa-azacyclotetradecane) with calf thymus DNA was probed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Adding deoxyribonucleic acid (DNA) into [CoL](2+) and [CuL](2+) solution, the i(p) value of all the peaks of [CoL](2+) and [CuL](2+) significantly decreased in proportion to concentration of DNA. Glassy carbon electrodes (GCEs) were modified with DNA by adsorption, and it was electrochemically characterized with transition-metal complexes, [ML](2+). The DNA modification layer on the GCE is unstable to alkali and to heat, but stable to acid solutions and very stable in long stock in a dry state. It could be seen that peak potential shifted positively and the peak current increased significantly. The electrochemical parameters, binding constant (k(n+)) and binding sites(s) were calculated by a nonlinear regression method. PMID- 12376023 TI - Serological evidence of Mycoplasma pneumoniae infection in acute exacerbation of COPD. AB - A prospective study was conducted to identify and characterize hospitalizations for acute exacerbation of chronic obstructive pulmonary disease (AECOPD) with serologic evidence of infection with Mycoplasma pneumoniae (Mp). Two hundred forty hospitalizations for AECOPD were included in a 17-month prospective study. Paired sera were obtained for each of the hospitalizations and were tested serologically for Mp using a commercial enzyme immunoassay (EIA) kit. Only significant changes, according to the formula in the manufacturer's instructions, in antibody titers for IgM and/or IgG and/or IgA were considered diagnostic for Mp infection. In 34 hospitalizations (14.2%) the serologic tests for Mp were positive (MpH). In 29 of these hospitalizations (85%) a significant change in IgA was found. In 11 of these hospitalizations (32%) the only change identified was in IgA. In 24 MpH (71%) there was serologic evidence for infection with at least one other respiratory pathogen. In comparison to the 206 hospitalizations without serologic evidence of infection with Mp, MpH had higher rates of inhaled steroid therapy (41% vs. 24%, p = 0.033) and a longer time interval between the appearance of dyspnea and hospitalization (6.6 +/- 3.8 days vs. 5.0 +/- 3.5 days, p = 0.012). There were no significant differences between these two groups in a broad spectrum of patient- and exacerbation-related clinical variables. Specific antibiotic therapy for Mp in the MpH group did not shorten the hospital stay. Serologic evidence of Mp infection is common in patients hospitalized for AECOPD, and is usually based on changes in specific IgA antibody titers. In most MpH another respiratory pathogen can be identified. The vast majority of clinical characteristics are the same in patients with and without serologic evidence of infection with Mp. The practical implications of these findings should be clarified in further studies. PMID- 12376024 TI - Evaluation of the Coral UTI Screen system for rapid automated screening of significant bacteriuria in a regional centralized laboratory. AB - An evaluation of the Coral UTI screen system (Coral Biotechnology, San Diego, CA) compared to urinalysis/urine culture was done to assess its performance for rapidly screening a high volume of urine samples for significant bacteriuria in a regional central microbiology laboratory. A total of 1094 urine samples from ambulatory patients were evaluated. 670 (61.2%) urine samples were negative or positive [178 (16.3%)] by both methods. 217 (19.8%) other samples were UTI screen positive but had either no growth or no uropathogens on culture; 9 of these samples were possibly false negative by culture because of the presence of pyuria, indicating the presence of either a urinary tract infection or another inflammatory process. Another 29 (2.7%) samples had false negative screens because the urine culture was positive, but only 5 of these patients were treated with antibiotics after urine specimen collection. Overall, the Coral UTI screen has a sensitivity of 86.0%, a specificity of 75.5% and a positive and negative predictive value of 45.0% and 95.9% respectively. Routine use of the UTI screen would allow same day reporting of 65% of all urine culture results without having to proceed to culture. PMID- 12376026 TI - Identification of the first isolates of Trichosporon asahii var asahii from disseminated trichosporonosis in China. AB - Infection with Trichosporon asahii is a major cause of deep-seated and disseminated trichosporonosis, which is associated with a high mortality rate. Disseminated trichosporonosis in individuals with no underlying disease has not been reported. In this study, we report the identification of the first isolate of Trichosporon asahii var. asahii in China. Two isolates were obtained from the liver and skin of a patient with disseminated trichosporonosis who displayed no evidence of underlying disease. The morphologic and physiologic characteristics of the two isolates differed slightly from those of usual strains of T. asahii var. asahii, including the type strain CBS 2479. Ubiquinone-9 was identified as the major ubiquinone in both isolates. Sequence analysis of the LSUrDNA D1/D2, ITS, and IGS1 regions from the two isolates showed them to be T. asahii var. asahii, and random amplified polymorphic DNA analysis strongly suggested that they were the same strain. PMID- 12376025 TI - Hematogenous infections due to Candida parapsilosis: changing trends in fungemic patients at a comprehensive cancer center during the last four decades. AB - This study was performed to evaluate trends in species distribution in patients' with hematogenous candidiasis at a comprehensive cancer center. The results of a retrospective analysis from January 1, 1993 to December 31, 1998 were compared with prior reports from Memorial Sloan-Kettering Cancer Center in the last forty years. In 570 total episodes since 1974, 43.9% were due to Candida albicans. During 1990's, C. parapsilosis emerged as the most frequent yeast species in the non-C. albicans group (36.1% during 1993-1998 from 20.9% 1974-1982; p < 0.01). An increase in C. krusei from 5.9% (1974-1982) to 10.5% during the recent six years (1993-1998) was also noticed. The proportion of C. tropicalis among non-albicans fungemia during 1974-1982 was 42.8%, whereas in 1993 to 1998 a marked decline in C. tropicalis hematogenous infection was observed (27.8%; p < 0.01). During 1998, the incidence of candidemia declined from 7.1% (1972-1973) and 6.5% (1982) to 3.4% (p < 0.01), and improved survival among fungemic patients (33% mortality in 1998; 77.3% during 1974-1982; p < 0.001) was encouraging. The increase in C. parapsilosis bloodstream invasion during 1990's was associated with a significant reduction in the endogenous non-albicans Candida tropicalis infection that probably resulted in part due to the common prophylaxis, and/or preemptive fluconazole given routinely in high-risk patients undergoing treatment for cancer. The widespread use of extraneous implantable and/or semi-implantable indwelling intra-vascular devices may also have played an important role in promoting (exogenous) C. parapsilosis infection. This study emphasizes the importance of periodic evaluation of candidemia, especially at centers caring for patients at risk. PMID- 12376027 TI - Candida peritonitis due to peptic ulcer perforation: incidence rate, risk factors, prognosis and susceptibility to fluconazole and amphotericin B. AB - Sixty-two cases of peritonitis due to peptic ulcer perforation were diagnosed between January 2000 and December 2000. Of these 62 cases, 23 isolates of Candida in 23 cases (CP) were cultured from peritoneal fluid. Cultures of peritoneal fluid of 10 (BP) of the remaining 39 cases was positive for bacteria only. Cultures of peritoneal fluid of the remaining 29 cases was negative. Comparison of CP, BP and culture-negative cases did not reveal any significant risk factor. Of the 23 Candida isolates, the Candida species and 48-h MICs of fluconazole and amphotericin B (mean, range ug/ml) were C. albicans 18 (0.688, 0.125-1.0; 0.297, 0.031-0.5), C. glabrata 3 (0.542, 0.125-1.0; 0.25, 0.125-0.5), C. tropicalis 1 (0.25; 0.5), C. intermedia 1 (1.0; 0.125) respectively. Mortality rates of CP, BP and culture-negative peritonitis due to infection were 5/23(21.7%), 0/10 and 1/29(3.4%) respectively. Without effective antifungal therapy, the mortality rate of CP was not low. PMID- 12376028 TI - Identification of Echinococcus granulosus eggs. AB - The eggs from Echinococcus granulosus contaminate the environment spreading out the disease among the herbivorous. The differential diagnosis of the embriophores recovered from the soil is very difficult by morphologic and immunologic methods. In this paper we evaluate the EgO/DNA-IM1 for identification of E. granulosus oncosphere DNA and differentiation of eggs from other Taeniid. The positive result of the PCR technique shows an amplification fragment of the expected size (285 bp) corresponding to the partial sequence of the mitochondrial gene of the cytochrome oxidase CO1 from E. granulosus (391 bp). The fragment is not present in the DNA from Echinococcus multilocularis, Taenia hydatigena, Taenia saginata, Diphyll-obothrium latum, and Hymenolepis nana. It could be useful to rule out Taenia taeniformis, Taenia solium, Taenia pisiformis, and Taenia crassiceps, which sequences do not belong to the primer. We concluded that the PCR amplification employing the EgO/DNA-IM1 primer set showed high sensitivity and specificity for the identification of Echinococcus granulosus eggs. PMID- 12376029 TI - Cefepime, piperacillin/tazobactam, gentamicin, ciprofloxacin, and levofloxacin alone and in combination against Pseudomonas aeruginosa. AB - A beta-lactam plus an aminoglycoside is the standard for treating severe Pseudomonas aeruginosa infections. However, the fluoroquinolones are safer and have been widely used as an alternative to the aminoglycosides in this setting. In this study we compared the synergistic activities of piperacillin/tazobactam and cefepime when either drug was combined with gentamicin, ciprofloxacin, or levofloxacin against P. aeruginosa. Susceptibility testing and time-kill curves were performed against 12 clinical isolates of P. aeruginosa. All combinations were bactericidal and retained this activity over the 24 hr period except for piperacillin/tazobactam in combination with levofloxacin or ciprofloxacin against 2 isolates and cefepime in combination with levofloxacin against 1 isolate. None of the combinations were antagonistic. No statistical difference in the frequency of synergy exists between the beta-lactam plus gentamicin (79%) and the beta lactams plus either ciprofloxacin or levofloxacin combinations (58%, 67%). Furthermore, no differences in synergistic activity were noted between ciprofloxacin combinations (58%) and levofloxacin combinations (67%). In conclusion, the degree of synergy between a beta-lactam plus aminoglycoside and a beta-lactam plus fluoroquinolone seem to be comparable. Furthermore, there is a similar rate of synergy among different fluoroquinolone-based combinations. However, faster killing, less regrowth, and decrease in the development of resistance were seen with the beta-lactam plus aminoglycoside combination. PMID- 12376030 TI - Pharmacodynamics of 750 mg and 500 mg doses of levofloxacin against ciprofloxacin resistant strains of Streptococcus pneumoniae. AB - An in vitro pharmacokinetic model (IVPM) was used to evaluate the pharmacodynamics of the 750 mg and 500 mg doses of levofloxacin against 4 ciprofloxacin-nonsusceptible Streptococcus pneumoniae. Levofloxacin MICs ranged from 1.4 to 3.2 micro g/ml. Log-phase cultures (5 x 10(7) cfu/ml) were inoculated into the IVPM and exposed to the peak free-drug concentrations of levofloxacin achieved in human serum with each dose. Levofloxacin was dosed at 0 and 24 h, elimination pharmacokinetics were simulated, and viable counts were measured over 30 h. The 750 mg dose was rapidly bactericidal against all 4 strains, achieving eradication within 30 h. Against strains with levofloxacin MICs of 1.4 and 1.8 micro g/ml, the 500 mg dose exhibited pharmacodynamics similar to the 750 mg dose. In contrast, against strains with levofloxacin MICs of 2.6 and 3.2 micro g/ml, viable counts never fell below 10(4) cfu/ml. The rapid killing and eradication of these pneumococci by the 750 mg dose warrant the clinical evaluation of this new dose in the treatment of pneumococcal infections. PMID- 12376031 TI - Pharmacodynamic profiling of continuously infused piperacillin/tazobactam against Pseudomonas aeruginosa using Monte Carlo analysis. AB - Standard doses of piperacillin/tazobactam (9-13.5 g over 24 h) administered by continuous infusion (CI) routinely provide serum concentrations in excess of the susceptibility breakpoint (< or =16/4 micro g/ml) for most Enterobacteriaceae. Since the breakpoint of this agent for Pseudomonas aeruginosa is considerably higher (< or=64/4 micro g/ml), the likelihood of obtaining adequate drug exposures with these doses against this bacterium is currently unknown. Monte Carlo simulation was utilized to determine the probability of obtaining adequate piperacillin concentrations above its MICs for P. aeruginosa in patients receiving CI. MICs of 557 P. aeruginosa isolates were determined by E-test and a distribution was constructed for the 496 susceptible isolates. Using a previously validated population pharmacokinetic equation, steady-state serum concentrations were estimated for 210 patients who received piperacillin/tazobactam via CI. A Monte Carlo simulation was performed to predict the probability of obtaining concentrations at the MIC, 2 x MIC, 4 x MIC, 5 x MIC, and 6 x MIC for patients infected with susceptible P. aeruginosa isolates. MICs ranged from 0.09 to 64 micro g/ml with modal and median values of 3 and 4 micro g/ml, respectively. Steady-state concentrations of 51.14 +/- 17.52 micro g/ml were estimated in our patient population. The simulation resulted in a median level of exposure 12.62 times the MIC. The level of certainty of obtaining concentrations at the MIC, 2 x MIC, 4 x MIC, 5 x MIC, and 6 x MIC for piperacillin administered by CI was 97, 93, 85, 81, and 77%, respectively. Despite concern for the place of CI piperacillin/tazobactam in the management of P. aeruginosa infections due to the higher established breakpoint, these data suggest a high probability of achieving adequate drug exposure against susceptible isolates with this dosing regimen. PMID- 12376032 TI - Comparative killing rates of gatifloxacin and ciprofloxacin against 14 clinical isolates: impact of bacterial strain and antibiotic concentration. AB - The influence of bacterial strain and antibiotic concentration on the time to achieve in vitro bactericidal activity was determined for gatifloxacin and ciprofloxacin using time-kill methodology. Killing rates were significantly affected by bacterial strain, antibiotic concentration, and type of fluoroquinolone. The most rapid bactericidal activity was seen against members of the Enterobacteriaceae and with fluoroquinolone concentrations of 8-16 X MIC. In general, gatifloxacin demonstrated faster killing against Acinetobacter baumanii, Legionella pneumophila, Staphylococcus aureus, and Streptococcus pneumoniae. PMID- 12376034 TI - Safety and efficacy of gatifloxacin in community-acquired pneumonia: rationale for the Tequin Clinical Experience Study (TeqCES). PMID- 12376035 TI - Fluoroquinolones for the treatment of outpatient community-acquired pneumonia. AB - The increasing prevalence of beta-lactam and macrolide resistance in bacteria that cause respiratory infections has underscored the need for effective antimicrobial agents. The broad spectrum, excellent oral bioavailability, and once-daily dosing of fluoroquinolones contributed to the introduction of several new agents in the past decade. This class is among the world's most used antimicrobial therapies in community and hospital settings. Fluoroquinolones are generally well tolerated, but safety profiles differ widely among agents. Knowledge of in vitro activity, local microbiologic susceptibility and resistance patterns, adverse effects, and potential drug interactions should influence the selection of the best agent for individual patients. This overview of the fluoroquinolones directs particular attention to use in community-acquired pneumonia and safety. PMID- 12376036 TI - Gatifloxacin phase IV surveillance trial (TeqCES study) utilizing 5000 primary care physician practices: report of pathogens isolated and susceptibility patterns in community-acquired respiratory tract infections. AB - Recently FDA-approved fluoroquinolones like gatifloxacin possess enhanced activity against Gram-positive pathogens such as Streptococcus pneumoniae. However, experience with adverse events among previously used fluoroquinolones has led to expanded post-marketing investigations of clinical efficacy and safety. An open-label gatifloxacin trial was initiated in early 2000, using 2795 (>15000 enrolled cases) primary care providers for treatment of community acquired respiratory tract infections (CARTI) such as community-acquired pneumonia (CAP), acute bacterial exacerbation of chronic bronchitis (ABECB), acute sinusitis. Microbiology specimens and sputum slides were referred to a reference laboratory, pathogens identified and reference antimicrobial susceptibility tests performed. Results were classified by infection site, geographic census region and patient profile/demographics. The most frequent pathogens were: for CAP (n = 384)-S. pneumoniae (37%) > Hemophilus influenzae (31%) > Moraxella catarrhalis (13%); for ABECB (528)-H. influenzae (37%) > M. catarrhalis (26%) > S. pneumoniae (17%); and for sinusitis (2691)-M. catarrhalis (29%) > H. influenzae (24%) > S. pneumoniae (17%). H. parainfluenzae (ABECB) and S. aureus (sinusitis) were also commonly isolated. CAP S. pneumoniae isolates had significantly less high-level resistance (5% at > or =2 micro g/ml) than those isolates from ABECB or sinusitis (13-15%). United States census zone differences in S. pneumoniae resistance were identified (greatest in West or East South Central, South Atlantic). S. pneumoniae macrolide resistance was high (23-33%) and H. influenzae clarithromycin susceptibility was only 56-62%. beta-lactamase rates in H. influenzae and M. catarrhalis were 21-29% and 88-92%, respectively. Only one S. pneumoniae was not susceptible to gatifloxacin, and this new fluoroquinolone was fourfold more potent than levofloxacin (MIC(50,) 0.25 vs. 1 micro g/ml). This Phase IV surveillance trial (TeqCES) confirmed the clinical importance of S. pneumoniae, H. influenzae and M. catarrhalis in CARTI, and high fluoroquinolone potency/spectrum (>97% susceptible). beta-lactams and macrolides continue to be compromised by increasing resistances in pathogens isolated in these monitored primary care settings. PMID- 12376037 TI - Oral gatifloxacin in outpatient community-acquired pneumonia: results from TeqCES, a community-based, open-label, multicenter study. AB - Gatifloxacin is an 8-methoxy fluoroquinolone with broad activity against respiratory tract pathogens, including those commonly associated with community acquired pneumonia (CAP). To evaluate the efficacy and safety of oral gatifloxacin 400 mg once daily for seven to 14 days, community-based physicians enrolled adult outpatients with confirmed or suspected CAP in a prospective, single-arm, open-label, noncomparative study. Of 1488 clinically evaluable patients with radiographically confirmed or clinically suspected CAP, 1417 (95.2%) were cured. All strains of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, the most commonly isolated pathogens, were susceptible to gatifloxacin. Penicillin nonsusceptibility was seen in 32.6% of S. pneumoniae isolates, and beta-lactamase production was detected in H. influenzae (26.9%) and M. catarrhalis (88%) isolates. Clinical cure rates of 91%, 94%, and 92% were achieved in patients with S. pneumoniae, H. influenzae, and M. catarrhalis, respectively. All seven patients with fully penicillin-resistant S. pneumoniae (MIC > or =2 micro g/ml) were cured. Gatifloxacin was well tolerated, with the most common drug-related adverse events being nausea (2.8%) and diarrhea (1.7%). Gatifloxacin is effective and well tolerated as empiric therapy for CAP in the outpatient community setting. PMID- 12376038 TI - Gatifloxacin used for therapy of outpatient community-acquired pneumonia caused by Streptococcus pneumoniae. AB - Gatifloxacin is an advanced-generation fluoroquinolone with demonstrated efficacy and safety as therapy for community-acquired pneumonia (CAP). As part of a phase IV postmarketing surveillance program (TeqCES), 136 outpatients with CAP whose sputum was culture-positive for Streptococcus pneumoniae were enrolled in an open label trial of oral gatifloxacin 400 mg daily for 7 to 14 days. An antibiogram of isolates showed 100% susceptibility to gatifloxacin (MIC(90) 0.5 micro g/mL) and respective susceptibilities of 67%, 70%, and 80% to penicillin, erythromycin, and tetracycline. Clinical cure was achieved in 95.3% of evaluable patients, including seven patients infected with penicillin-resistant S. pneumoniae (MIC > or =2 micro g/mL). The bacteriologic eradication rate for S. pneumoniae was 94.5%. Diarrhea, nausea, and dizziness, the most common adverse events in CAP patients (<3%), were generally mild to moderate; no serious adverse events were recorded. These results support recommendations to treat CAP, particularly due to S. pneumoniae including multidrug-resistant strains, with the newer 8-methoxy fluoroquinolone, gatifloxacin. PMID- 12376039 TI - Haemophilus influenzae in respiratory tract infections in community-based clinical practice: therapy with gatifloxacin. AB - In this community-based safety surveillance study, the advanced-generation fluoroquinolone gatifloxacin was administered empirically to 15625 adults with community-acquired respiratory tract infections (RTIs), including 1562 clinically evaluable patients with community-acquired pneumonia (CAP) and 2391 with acute exacerbations of chronic bronchitis (AECB). Haemophilus influenzae was the most common pathogen isolated in AECB (40.1%) and the second most common in CAP (36.8%). In vitro susceptibility to gatifloxacin and other fluoroquinolones, amoxicillin/clavulanate, ceftriaxone, cefuroxime axetil, tetracycline, and azithromycin ranged from 95.8% to 100%. In comparison, a significant percentage of the isolates were not susceptible to clarithromycin ( approximately 41%), ampicillin (22% to 28%), and trimethoprim/sulfamethoxazole (14% to 18%). The susceptibility pattern was generally independent of exposure to another antimicrobial in the previous 30 days. CAP and AECB patients infected with H. influenzae had signs and symptoms similar to those infected with Streptococcus pneumoniae. Among clinically evaluable patients with H. influenzae, gatifloxacin cured 159 of 166 (95.8%) with AECB and 112 of 118 (94.9%) with CAP. The cure rate was independent of the beta-lactamase status or serotype of the H. influenzae strain. H. influenzae is not a more benign pathogen in community-acquired RTIs but causes signs and symptoms that are indistinguishable from those caused by other pathogens, notably S. pneumoniae. PMID- 12376040 TI - Gatifloxacin in community-based treatment of acute respiratory tract infections in the elderly. AB - The elderly are at increased risk for respiratory tract infections. To evaluate the safety and efficacy of gatifloxacin in adults of any age with community acquired respiratory tract infections, this open-label, multicenter, noncomparative study in community-based practices enrolled male and female outpatients at least 18 years old with a clinical diagnosis of community-acquired pneumonia (CAP), acute-bacterial exacerbation of chronic bronchitis (AECB), or acute uncomplicated maxillary sinusitis. Gatifloxacin 400 mg was administered once daily for seven to 14 days. Of 14781 clinically evaluable patients, 2505 were at least 65 years old, 499, at lest 80. Cure rates for CAP, AECB, and sinusitis ranged from 91.6% to 95.5% for patients less than 65 years old, 91.1% to 96.2% for those 65 to 79 years of age, and 89.5% to 94.8% for those at least 80 years old. Each age group, including patients with concomitant cardiovascular or diabetic conditions, tolerated treatment well. Gatifloxacin is efficacious and well tolerated in adult outpatients of any age with respiratory tract infections and is an important therapeutic option, particularly in communities with a high prevalence of resistant pathogens. PMID- 12376041 TI - Efficacy and safety of gatifloxacin in elderly outpatients with community acquired pneumonia. AB - To evaluate the safety and efficacy of gatifloxacin in adults <65, 65 to 79, or > or =80 years old with community-acquired pneumonia, adult male and female outpatients from general community-based practices were enrolled in an open label, multicenter, noncomparative study. Gatifloxacin 400 mg once daily was administered for seven to 14 days. Medical history, physical examination, signs and symptoms of infection, Gram stain and culture if specimen available, clinical response, and safety were determined. Of 1655 treated patients, 1103 were at least 65 years old, 405 were 65 to 79, and 147 were at least 80. Patients > or =80 years old presented with chills, chest pain, fever, or headache less often than younger patients. Cure rates were 95.5% for patients <65 years old, 96.2% for those 65 to 79, and 90.2% for those at least 80 years old. Neither the frequency nor susceptibility of isolated pathogens appeared to differ with age. Between 93.7% and 100% of subsets of the two younger groups with verified Streptococcus pneumoniae or Hemophilus influenzae were cured. All oldest-group patients in the subset with verified S. pneumoniae and 71.4% (7) of patients with H. influenzae were cured. Each age group, including current or past smokers and patients receiving medications for concomitant conditions, tolerated treatment well. Gatifloxacin is safe and efficacious in adults of any age with community acquired pneumonia, including the elderly up to 100 years old and patients with S. pneumoniae including penicillin-resistant strains. PMID- 12376043 TI - Mathematical modelling for the new millenium: medicine by numbers. AB - Physicists, engineers and mathematicians are accustomed to the combination of elegance, rigour and utility that characterise mathematical models. They are familiar with the need to dip into their mathematical toolbox to select the technique of choice. However, medicine and biology have not been characterised, in general, by a mathematical formalism. The relative paucity of mathematical models in biology and medicine reflects in part the difficulty in making accurate and appropriate experimental measurements in the field. Signal noise, the lack of appropriate sensors, and uncertainty as to what constitutes the significant measurements are largely to blame for this. The purpose of this paper is to characterise a 'good' model, encourage the development and application of such models to new areas, and outline future developments in the field. It is proposed that a good model will be accurate, predictive, economical, unique and elegant. These principles will be illustrated with reference to four models: radiosensitisation of tumours, modelling solute clearance in haemodialysis, the myogenic response in reactive hyperaemia and cardiac electrical activity. It is suggested that, in the immediate future, the mathematical model will become a useful adjunct to laboratory experiment (and possibly clinical trial), and the provision of 'in silico' models will become routine. PMID- 12376042 TI - Implications of TeqCES: efficacy and safety of gatifloxacin in community-acquired pneumonia. PMID- 12376044 TI - Modelling of flow and wall behaviour in a mildly stenosed tube. AB - In the present computational analysis, pulsatile flow and vessel wall behaviour in a simplified model of a stenosed vessel were investigated. Geometry of a 45% axisymmetrically stenosed (by area) cylindrical tube and a sinusoidal inflow waveform were simulated, with the fluid being assumed to be incompressible and Newtonian. The vessel wall was treated as a thick-walled, incompressible and isotropic material with uniform mechanical properties across the normal as well as the constricted segment. The study of fluid flow and wall motion was initially carried out separately using two commercial codes CFX4.2 and ABAQUS7 respectively. Their combined effects and interactions were later investigated through an iteratively coupled algorithm. Model validations on the rigid-wall fluid and static no-flow solid models were satisfactory, with Root Mean Square deviations of around 7% in centreline axial velocity between the prediction and measurement values for the rigid wall stenosis model, and 5% in circumferential stress for a cylindrical tube model under static loading when compared with the analytical solution. Results on velocity profiles, wall shear stress, intramural strain and stress for the rigid and compliant cases were all presented. Comparison between the rigid and compliant models revealed that, the flow separation layer distal to the stenosis was thicker and longer, and wall shear stress was slightly lower in the compliant model by less than 7.2%. Results obtained from the static wall model (with uniform pressure loading) and coupled fluid/wall interaction modelling of pulsatile flow showed qualitatively similar wall strain and stress patterns but considerable differences in magnitude. The radial and axial stresses were reduced by 31 and 8%, while the circumferential stress was increased by 13% due to the presence of pulsatile flow. Under the flow and structural conditions investigated, the effects of wall compliance were small, and did not change the flow and solid behaviours qualitatively in this case. PMID- 12376045 TI - Dynamic model of the role of platelets in the blood coagulation system. AB - In order to confirm which process is the most important in the blood coagulation cascade, a dynamic model of the function of platelets in blood coagulation is provided based on biochemical experiments. A series of conclusions based on qualitative analysis and mathematical simulation are drawn about the influence of the activation rate of factor VIII and factor IX on the generation of thrombin (IIa). It is evident that the pro-coagulation stimulus must exceed a threshold value to initiate the coagulation cascade. The value is related to the rate of platelet activation, the binding constant d2. The stability of the fixed value is also related to the pro-coagulation stimulus. This article also evaluates the influence of the stimulus strength and the activated rate parameter of platelets on thrombin. The proportion of platelets activated at any given time is designated c. To each c, we obtain a maximum concentration of thrombin. It is evident that when the level of factor IX is below 1% of normal levels, the rate of thrombin generation reduces dramatically resulting in severe bleeding tendency. PMID- 12376046 TI - An advanced computer-aided geometric modeling and fabrication method for human middle ear. AB - This paper presents a practical and systematic method for reconstructing accurate computer and physical models of the entire human middle ear. The proposed method starts with the histological section preparation of human temporal bone. Through tracing outlines of the middle ear components on the sections, a set of discrete points is obtained and employed to construct B-spline curves that represent the exterior contours of the components using a curve-fitting technique. The surface skinning technique is then employed to quilt the B-spline curves for smooth boundary surfaces of the middle ear components using B-spline surfaces. The solid models of the middle ear components are constructed using these surfaces and then assembled to create the entire middle ear in a computer-aided design environment. This method not only provides an effective way to visualize and measure the three dimensional structure of the middle ear, but also provides a detailed knowledge of middle ear geometry that is required for finite element analysis or multibody dynamic analysis of the human middle ear. In addition, the geometric model constructed using the proposed method is smooth and can be fabricated in various scales using solid freeform fabrication technology. The physical model of the human middle ear is extremely effective in realizing the middle ear anatomy and enhancing discussion and collaboration among researchers and physicians. PMID- 12376047 TI - Does low output laser stimulation enhance the healing of crural ulceration? Some critical remarks. AB - The objective of the experiment was to evaluate the impact of laser stimulation on crural ulceration healing. Three groups were established at random from patients with crural ulceration: A, B and C. Group A included 21 patients, group B included 22 patients and group C, 22 patients. Patients in all groups were treated with pharmaceuticals and with compressive therapy. The ulcers in group A were additionally irradiated with laser light of wavelength 810 nm, so that a dose of 4 J/cm2 was applied in each procedure. Patients in group B were additionally subjected to a blind test (with placebo in the form of quasi-laser therapy). At the end of the treatment a statistically significant reduction of the area and volume of the ulcers was found in all groups. No statistically significant difference was found between the groups in terms of average rate of change per week of the relative area of ulceration and average rate of change per week of the relative volume of ulceration. Reduction of infected area was observed in all groups, but a significant change was only observed in group C. No significant impact of laser light (lambda=810 nm, P=65 mW, p=4 J/cm2) on any of the stages of ulceration healing was observed. PMID- 12376048 TI - Morphological study of the proximal femur: a new method of geometrical assessment using 3-dimensional reverse engineering. AB - This study presents a new method of using computerized tomography images combined with the reverse engineering technique to obtain and analyse the three dimensional inner and outer geometry of the proximal cadaveric femur. Three dimensional models were reconstructed from the computerized tomography images and approximated with 2D and 3D fitting algorithms based on reverse engineering methods. The following parameters were calculated for each femur: femoral head diameter, femoral neck axis, femoral shaft axis, anteversion angle and neck-shaft angle. These data represent the geometry of the studied proximal femur, and can be used for the design of proper size and shape of femoral prostheses and trochanteric nail systems. PMID- 12376049 TI - The sensitivity of posturographic parameters to acquisition settings. AB - The objective of this study was to evaluate the sensitivity of posturographic parameters (PP) to changes in acquisition settings. A group of eight young adults underwent a set of typical orthostatic posture trials, and selected PP were then calculated from a set of centre of pressure (CoP) displacement time series obtained by applying different cut-off frequencies to the same set of raw data. Four PP out of 11 showed significant changes with respect to cut-off frequency. Statistical mechanics parameters exhibited smaller sensitivity than summary measures. On the basis of the results obtained, a proposal for a standard cut-off frequency and a sampling rate value is embodied in the paper together with some suggestions on measurement settings, with a view to standardized use of instrumentation for quantitative analysis in orthostatic posturography. PMID- 12376050 TI - Precision test apparatus for evaluating the heating pattern of radiofrequency ablation devices. AB - Radiofrequency has established itself as a useful technique for managing cardiac arrhythmias and treating soft tissue tumors. However, despite its pervasive use, many of the biophysical principals needed to fully understand and optimize the radiofrequency ablation technique have not been explored. We have designed a test rig that is useful for studying the heat transfer mechanisms that affect the outcome of radiofrequency ablation devices. Using both solid and liquid phantom materials, which simulate body tissues and blood, the test rig is designed for systematic testing of the effects of predictable flow patterns on the temperature profiles generated within the solid phantom. The test rig consists of a custom built thermistor array, a linear test chamber, and a radiofrequency generator. We calibrate the flow of a liquid phantom material to demonstrate that predictable laminar flow profiles are generated. To demonstrate the performance of the ablation system, we present preliminary data attained using a commercially available cardiac ablation catheter. The advantages of this test system are its flexibility, its reproducibility, its precision, and its low cost. Thus, it is ideally suited for studying a variety of complex ablation problems involving multiple tissues types and complex blood flow geometries. PMID- 12376052 TI - How should a birth cohort study be organised? Experience from the German MAS cohort study. AB - Birth cohort studies offer the opportunity to study average risks, rates and occurrence times of disease longitudinally from birth. The effect of genetic and environmental factors and their interactions can be studied. Furthermore, quantity and duration of exposure to environmental agents can be evaluated prospectively. However, prospective birth cohort studies are expensive, labour intensive and take many years to complete. Loss of subjects over time as well as recall bias complicate the interpretation of observations. This paper summarises the potential pitfalls of such studies and discusses the experience of the German Multicentre Allergy Study (MAS), which began in 1990 in five German cities and included 1314 newborns for the study of the natural course of atopic diseases. PMID- 12376053 TI - A cohort of children hospitalised with acute RSV bronchiolitis: impact on later respiratory disease. AB - This paper reviews the results from a cohort study in which 47 children hospitalised with respiratory syncytial virus (RSV) bronchiolitis and their 93 controls, matched for age, sex and place of living, were prospectively followed up at the mean ages of 1, 3 and 7.5. Asthma was significantly more common in the RSV bronchiolitis group at all times. Asthma during the year prior to follow-up at age 7.5 was seen in 23% of the RSV children and in 2% of the controls (P < 0.001). Allergic sensitisation was found in 41% of the RSV children and in 22% of the controls (P = 0.039). When comparing these results with findings from other studies it is obvious that the rate of asthma and other bronchial obstructive symptoms are increased after RSV bronchiolitis but the various results concerning allergic sensitisation are not conclusive. Prospective studies are needed with some kind of randomised intervention against RSV before the mechanisms behind the post-bronchiolitic symptoms and the possibly increased risk for IgE mediated allergy can be settled. PMID- 12376054 TI - Follow-up studies of asthma from childhood to adulthood. AB - One of the main questions from parents of children with asthma is whether the child will outgrow the disease and what is the role of treatment. All outcome studies show that in the transition period from childhood to adulthood asthma symptoms decrease and thereby asthma seems to be cured. However, the reality is that more than 50% of children with asthma suffer from asthma in adult life. The role of treatment is uncertain, since all studies began in a period when inhaled corticosteroids and other novel medications were not available. The recent findings from a study of bronchial biopsies in subjects in remission from asthma for more than 3 years suggests that asthma might persist throughout life. PMID- 12376055 TI - What have we learned from the Tucson Children's Respiratory Study? AB - The Tucson Children's Respiratory Study was the first longitudinal assessment of the natural history of asthma in which children were enrolled at birth. Over 1200 children were originally included and over 800 were still participating at age 13. The study has provided general indications about the most important risk factors for and the prognosis of different phenotypes associated with recurrent airway obstruction during childhood. The most important conclusion from the study is that asthma is a heterogeneous disease, with different predominant expressions at different ages. The form of the disease that is associated with atopy is not very frequent in early life, but becomes preponderant during the school years. However, this form is more persistent and is associated with significant deficits in lung function growth up to age 11. Up to two-thirds of infants who wheeze have a transient form of recurrent airway obstruction associated with low premorbid lung function. Many children who wheeze during the preschool years do so only during viral infections. These children usually have a history of wheezing due to respiratory syncytial virus during early life and low levels of lung function during the school years. Understanding the different asthma phenotypes of childhood will provide new clues for strategies for the primary prevention of the disease. PMID- 12376056 TI - Analysis of epidemiological studies: facts and artifacts. AB - Cohort studies have provided the foundation for much of our knowledge of childhood asthma. Four important lessons have been learned from these longitudinal studies: that asthma is a complex disease, encompassing many phenotypes; that it is linked to the development of the immune system and respiratory tract in the first years of life; that early life events strongly affect the development of asthma risk and that relationships between certain exposures and asthma risk are age dependent. The Tucson Children's Respiratory Study is used to exemplify these lessons and to illustrate the advantages of cohort studies in investigating a complex disease. PMID- 12376057 TI - Preventive measures and their effects. Results from cohort studies. AB - The prevalence of asthma and atopic diseases continues to rise. Genetic factors alone cannot explain this rapid rise and the immunological mechanisms involved are insufficiently explained to allow direct intervention on a population-wide scale. Long-term observational birth cohort studies have provided data on which primary prevention studies are based. This review discusses the "who", "how", "when" and "what" of primary prevention and the experiences to date in prospective intervention cohort studies. PMID- 12376058 TI - Angiogenesis in paediatric airway disease. AB - A number of characteristic changes occur in the bronchial wall in paediatric airway diseases. The process of remodelling is usually associated with specific changes to the vasculature, resulting in an increase in vessel numbers, vasodilatation, vessel leakage and cellular margination with transmigration to target tissues. This combined action in conditions such as asthma, cystic fibrosis and bronchiolitis lead to airway wall thickening and reduced airflow. Each component of the vascular response has been shown to be controlled by a range of inflammatory mediators and growth factors. These factors are regulated by a complex process involving gene expression, transcription and translation at the molecular level, protein release, binding to matrix elements and receptors on endothelial cells, then the endothelial response itself. A number of commonly used airway medications are potentially capable of modulating the vascular response to inflammatory stimuli. New therapies may be able to improve airflow through better regulation of vessel growth, dilatation and leakage in the airway wall. PMID- 12376059 TI - Advances in Burkholderia cepacia complex. AB - Burkholderia cepacia is an important opportunistic pathogen in certain compromised hosts, particularly those with either cystic fibrosis (CF) or chronic granulomatous disease. The "family" of bacteria known as B. cepacia is highly heterogeneous and is composed of at least nine discrete species or genomovars, constituting the B. cepacia complex. Bacteria from the B. cepacia complex are particularly virulent in susceptible hosts, often causing necrotising invasive infection and death. Whereas the microbial determinants of virulence in B. cepacia complex are currently not defined, the bacteria appear to have features facilitating survival within host cells. Burkholderia cepacia is highly resistant to antibiotics and to neutrophil-mediated non-oxidative killing; infection should be treated with combination antimicrobial therapy. Burkholderia cepacia can spread from one CF patient to another. Transmission appears to be facilitated by close personal contact and by certain bacterial factors. PMID- 12376060 TI - Fertility issues in cystic fibrosis. AB - With increasing survival in cystic fibrosis (CF) there is an increasing need to deal with the desires of CF patients to become parents. In the context of 98% male infertility in CF, new techniques offer the prospect of successful parenthood. For females, successful pregnancy is possible but careful planning is required. The practical and ethical aspects of reproductive health in CF are discussed. PMID- 12376061 TI - The imaging of paediatric thoracic trauma. AB - Trauma is a significant cause of morbidity and mortality in children and radiological investigations are almost always used in its management. This article reviews the radiological findings in thoracic trauma, in relation to their effects on lung parenchyma, the airways, the pleura, the great vessels, the diaphragms and the bony skeleton. Particular emphasis is given to plain radiographical and computed tomography findings, since these are the commonest imaging modalities used. PMID- 12376062 TI - Environmental factors relevant to difficult asthma. AB - Symptom persistence in difficult asthmatics may be related to their home environment. If sensitised asthmatics are to benefit from indoor allergen avoidance measures, these must be rigorous and drug adherence satisfactory. This is difficult for many families. The relationship between traffic pollution, asthma diagnosis and symptom severity is persuasive but requires objective validation. Overall, it seems that house dust mite control and tobacco smoke avoidance are important for asthmatics and advice about how to avoid these adverse factors must be given. Whether these measures are effective in difficult asthmatics and whether moving house makes any difference is unknown. PMID- 12376063 TI - Bronchoscopy--how and when? AB - At the Royal Brompton Hospital, we perform over 200 flexible bronchoscopies per year in children. We will be presenting a series of five articles describing various aspects of our clinical and research practice. This first article is about the basics of how and when to perform flexible bronchoscopy. It highlights aspects of training, preparation of the patient and the equipment required. It discusses anaesthesia and gives practical details on how to actually use the bronchoscope, as well as detailing techniques of lavage, brushings and biopsy. Particular attention is paid to indications and contraindications, as well as potential complications. PMID- 12376064 TI - The development of childhood asthma: lessons from the German Multicentre Allergy Study (MAS). AB - Epidemiological surveys have indicated that there has been a notable increase in the prevalence of both asthma and other allergic symptoms in children and young adults. Since it seems unlikely that genetic factors would contribute to the rising trend, environmental factors might play a major part in the development of childhood asthma. In a prospective birth-cohort study, we assessed the relevance of different exposures such as mite and cat allergen exposure, environmental tobacco smoke (ETS) exposure, early infectious diseases and vaccinations for the development of childhood asthma up to the age of 10 years. Data up to 7 years of age have been evaluated. Of 1314 newborn infants enrolled in five German cities in 1990, follow-up data at age 7 years were available for 939 children (72%). Assessments included repeated measurements of specific IgE to food and inhalant allergens, measurement of indoor allergen exposure at 6 months, 18 months and 3 years of age and yearly interviews by a paediatrician. At age 7 years, pulmonary function was tested and bronchial responsiveness was determined in 645 children. At age 7, the prevalence of wheezing in the past 12 months was 10% (94 out of 938), and 6.1% (57 out of 939) parents reported a doctor's diagnosis of asthma in their children. Sensitisation to indoor allergens was associated with asthma, wheeze and increased bronchial responsiveness. However, no relationship between early indoor allergen exposure and the prevalence of asthma, wheeze and bronchial responsiveness was seen. During the first 3 years of life, intra-uterine tobacco and consistent ETS exposure have an adjuvant effect on allergic sensitisation that is transient and restricted to children with a genetic predisposition for allergy. Children sensitised to any allergen early in life and sensitised to inhalant allergens by the age of 7 years were at a significantly increased risk of being asthmatic at this age (odds ratio (OR) = 10.12; 95% confidence interval (CI) = 3.81-26.88). Children with repeated episodes (> or =2) of runny nose before the age of 1 year were less likely to develop asthma by the age of 7 years (OR = 0.52; 95% CI = 0.29-0.92). Our data do not support the hypothesis that exposure to environmental allergens directly causes asthma in childhood but that induction of specific IgE responses and the development of childhood asthma are determined by independent factors. Indoor allergen avoidance is recommended as first line treatment in secondary and tertiary prevention; however, conclusions should be drawn with caution about the possible effect of primary preventative measures. Since allergic asthma seems to be a Th2-disease, immunomodulating factors such as early childhood infections, LPS-exposure or other factors influencing gene-environment interaction and individual susceptibility seem to be relevant for the development of childhood asthma. PMID- 12376065 TI - Case 2: assessment. A 7.5-year-old boy with respiratory syncytial virus. PMID- 12376068 TI - Case 1: assessment. Burkholderia cepacia. PMID- 12376069 TI - Adipokinin, a proopiomelanocortin-derived lipid-mobilizing anterior pituitary hormone. AB - Hormonal control of lipid metabolism during prolonged fasting is unclear. The involvement of the classical, lipid-mobilizing hormone from the anterior pituitary, i.e., beta-lipotropin (beta-LPH), is suspect. It and adrenocorticotropin (ACTH) are formed concurrently in the pituitary during the processing of the prohormone, proopiomelanocortin (POMC), and are secreted together. During prolonged fasting the control of metabolism requires minimal participation by ACTH and maximal lipid-mobilizing activity which is inconsistent with the present concept of ACTH and beta-LPH secretion. Hypothetically, the needed control can be satisfied by the alteration of the accepted processing of POMC so as to form beta-LPH and a new lipid-mobilizing hormone which also has modest ACTH-like activity. It is proposed that this hormone be named 'adipokinin'. An analog of this proposed hormone appears to have been isolated previously from porcine pituitaries. PMID- 12376070 TI - Electrical resection: new concept in management of focal epilepsy. AB - Focal epilepsy secondary esp to scar or injured cortical tissue forms a source of constant depolarisation site with or without emitting negative charges (current of injury) into the surrounding area. This focal site is localised by EEG, MRI and if required, by PET and/or SPECT studies. The author postulates to implant electrical source of opposite charge overlying the focal site to nullify the constant depolarisation site (electrical resection) or short-circuit the current of injury to an inert site to alleviate focal epileptic attack. Preliminary trials in the form of scalp application of positivity have markedly improved EEG picture in terms of occurrence of epileptiform activity. PMID- 12376071 TI - Immunostimulation of the chemical-induced carcinogenesis in the phase of initiation MH-G 0166. AB - It is postulated that the immune reaction on chemical carcinogens can inhibit or stimulate the chemical-induced carcinogenesis depending on the individual peculiarities of the synthesis of antibodies. Critical events take place on the barriers between external and internal media. Antibodies to chemical carcinogens, which are secreted into the digestive or bronchial tract, bind the environmental carcinogens and prevent them from penetrating into the blood through the epithelium and thereby inhibit the beginning of the tumour growth in any organs. On the contrary, the serum antibodies promote the penetration of carcinogens through the digestive and bronchial epithelia and thereby stimulate carcinogenesis in the proper organs. The other events take place in the organs such as breast and prostate where carcinogens are transported from the blood. Secretory antibodies bind carcinogens in the blood and transport them through the epithelium of these glands and thereby stimulate the tumour origin in these organs. Antibodies, which are not secreted into the ducts of breast and prostate, hold carcinogens in the blood and thereby inhibit carcinogenesis in these organs. Antibodies to steroid hormones function in the same way, i.e., secretory antibodies stimulate, while the serum antibodies inhibit the genetoxic action of the hormones on breast and prostate. The stimulation of carcinogenesis in the lymphoid cells is realized owing to hapten-specific binding of carcinogens by the membrane receptors of the corresponding clones. Antibodies to the natural inhibitors of carcinogenesis (retinoids, tocopherole, etc.) stimulate the beginning of the tumour growth. Antibodies to carcinogens and antiidiotypic antibodies to the cytochromes p-450 may act as abzymes, i.e., as cytochromes p 450 and thereby increase the level of the carcinogen metabolites. PMID- 12376073 TI - The hydraulic influence in androgen-related hair growth: implications in autoimmune disease. AB - Androgen-related changes in hair growth represent something of a mystery. Through the action of dihydrotestosterone (DHT), hair growth is increased in specific areas of the body. Elevated levels of DHT produce a general increase over the larger part of the body, often accompanied by hair loss in specific areas of the scalp. Because of this 'opposite' effect, a genetic difference in the hair follicles is proposed. This view is supported through the success of the 'plug graft' transplantation technique. However, this is unsatisfactory, because transplantation procedures that should work well according to this theory, ultimately fail. There is an alternative 'mechanism', that demonstrates its origins in the prime function of hair as an insulator. This simple mechanism makes sense of all the recognized effects of DHT in the dermal system, and throughout the body. In DHT-related hair growth it can be directly observed. The implication is that DHT achieves its effects through a primary physiological action that can be easily tested given the necessary expertise. Given existing knowledge, such a proven action of DHT would have serious implications for further understanding of female susceptibility to autoimmune disease. PMID- 12376072 TI - Thalamic neuron theory: meridians=DNA. The genetic and embryological basis of traditional Chinese medicine including acupuncture. AB - This hypothesis proposes a mechanism by which the genetic information contained in the one-dimensional genome may be converted into a three-dimensional body plan for development. Prior to mitosis of the fertilized egg, the chromatids, after being unpackaged from the chromosomes, link up to form a giant circular loop which is then folded upon itself into a wired-frame structure that embodies the architectural embryological developmental scheme. This intranuclear spatial body design is then translated into a three-dimensional cellar plan surrounding the fertilized egg with the positional value of each surrounding daughter cell preferentially activated by specific spatially oriented gene products diffused through the neatly arranged nuclear pores of the cell nucleus of the fertilized egg. This group of cells of the primitive embryo then leads to the formation of the Spemann Organizer, which directs embryological development of the brain as well as the rest of the body. The Spemann Organizer thus retains control over the CNS which in turn controls the development and functions of the peripheral tissues. The chains of cells that compose the Spemann Organizer, forming a homunculus in the image of the wired frame formed by the chromatids are believed to be the equivalents of acupuncture meridians. To support the hypothesis, evidence is also presented to substantiate the intimate relationships between the acupuncture meridians and embryological development, evolution, the central nervous system as well as the genome. This theoretical model is capable of dispelling the mystery of acupuncture, traditional Chinese medicine and myriads of modern clinical observations, and may have the potential to usher in a multitude of innovative therapeutic methods for many difficult to treat medical conditions. PMID- 12376074 TI - Malignant tumor disease as a sub-chronic, progressive intoxication on the basis of the perpetuation of the release of amino acids, initiated by a retrograde differentiated muscle degradation protease. AB - The proliferation of malignant tumor cells in the tissue of origin, as well as their invasion and survival in other tissues, is based on a pathogenicity mechanism, which, via the perpetuation of the release of amino acids, leads to a toxic, progressively acting impairment of normal cell functions. Consequently, a malignant tumor disease can be considered as a progressive intoxication exhibiting a sub-chronic course. The initialization is effected by means of a protease which, when related to the evolution scale of proteases, had experienced a downgrading. This corresponds to the degree of differentiation of the malignant tumor cells. As a faulty protein, it cannot, or at least not effectively, be degraded by the host organism. The aim of this faulty protein is the degradation of the proteins of the skeletal muscles. The amino acid lysine seems to be of particular importance for the synthesis of the retrograde-differentiated muscle degradation protease (RMDP), since the reduction of the availability of lysine exhibits a correlation with an anti-tumor effect. PMID- 12376075 TI - Factors governing speed of societal responses to threats: precision of definition of the threat may be more important than its anticipated likely severity. AB - Responses to two recent international public health threats, AIDS and BSE, both exemplify missed opportunities for early intervention. They are in contrast to the response to the Y2K computer issue, a threat of lesser-anticipated consequence but one that was more precisely defined. Lessons from these experiences should inform the public health response to new threats, such as the emergence of multiple drug resistance in human pathogens. PMID- 12376076 TI - Insulin resistance: a metabolic link between depressive disorder and atherosclerotic vascular diseases. AB - The association of depression with insulin resistance (IR) and athersclerotic vascular diseases has been well documented. This review examines the relevance of IR as a link between depressive disorder and atherosclerotic vascular diseases. Relevant articles collected from Medline database over the period of 1966-2001 were reviewed. Studies have shown that IR is a state-dependent abnormality in depression and depression increases the risk of vascular morbidity and mortality. Given that IR is a central component of cardiovascular risk factors, depression related IR might play a role in the development and progression of coronary and cerebral atherosclerosis in chronic-resistant depression. Further, IR may contribute to the pathophysiology of depressive disorder. In conclusion IR could account for the linkage between depression and atherosclerotic vascular diseases. More studies are needed to examine the importance of improving insulin sensitivity in the treatment of chronic-resistant depression and prevention of depression-related vascular morbidity and mortality. PMID- 12376077 TI - Melatonin deficiency and fibrous dysplasia: might a relation exist? AB - Fibrous dysplasia of bone might be monostotic, polystotic, or occurs as a part of McCune-Albright syndrome and Jaffe-Lichtenstein syndrome. Activating mutations of GNAS1 gene was identified in patients with fibrous dysplasia. However, fibrous dysplasia might occur in the absence of these mutations and fibrous dysplastic tissue was produced in vitro by the effects of excess exogenous cAMP on human osteogenic cells. It was proved that the fibrous dysplastic tissue is deficient in bone sialoprotein. Melatonin deficiency might be hypothesized in syndromes associated with fibrous dysplasia or formation of fibrous dysplasia-like tissue. The receptor RZR/ROR is the nuclear receptor of melatonin and the human bone sialoprotein gene contains a RZR/ROR response element. It was supposed that binding of melatonin to its membrane receptors results in changes in the levels of activity of nuclear cAMP that lead to alteration of expression of bone sialoprotein. Also, melatonin deficiency might increase cAMP in bone through its effect on prostaglandins of the E group. Further, melatonin deficiency might explain precocious puberty in cases of McCune-Albright syndrome. We might hypothesize that melatonin deficiency might play a role in development of fibrous dysplasia in some cases. PMID- 12376078 TI - Are neuronal activity-associated magnetic fields the physical base for memory? AB - Despite intensive investigation into the mechanisms underlying the memory process, the physical bases for this superior cognitive function remain elusive. Recall of past events and actions depends on the generation of complex memory carriers that would have to integrate many items of information. Some human memory processes, like contextual recall, work at such high speed and integrate such a large number of cortical neurons and neuronal networks that molecular mechanisms of information storage and synaptic transmission seem insufficient. This limitation argues against molecular information storage mechanisms as being truly effective carriers for the memory process. In this paper, I propose that any type of information can be stored in the form of 'neuronal activity associated magnetic fields' that would record information in much the same way as the magnetic tape of a tape recorder. Integration and/or combination of the neuronal activity-associated magnetic fields throughout the complex three dimensional structure of the human cortex could provide a storage medium for high speed processing and discrimination that would support the complexity of the human memory process. PMID- 12376079 TI - Is cancer really a 'local' cellular clonal disease? AB - Cancer is not simply the result of specific genetic alterations in key regulatory genes, but rather a complex multistep process involving selection of a clonal population of cells. To accumulate three, or often as many as seven, specific mutations in a single cell without incurring a significant number of additional mutations that might lead to cell lethality requires a large number of target cells, some mutagenic activity acting on those target cells for a variable period of time, and efficient selection strategies, which may be to some extent tissue specific. A number of 'protective' intracellular regulatory circuits might be present in proliferating cells deliberately to protect against carcinogenesis. If it does require some seven sequential carcinogenic 'genetic hits' in a single cellular clone for a malignant tumor to develop, it is mathematically more likely to occur in a tissue with a high background of genetic alterations in neighboring cellular clones, than in a tissue with a low background of such alterations, or with no detectable carcinogenic mutations at all. In this context, the old 'field cancerization' theory by Slaughter and the more recent 'multistep carcinogenesis' model by Fearon and Vogelstein can come together in a single model: 'multistep field cancerization'. This simple conclusion, and our ability to measure 'background carcinogenesis' in different parts of the body, might allow early detection of cancer risk, and eventually help us to develop suitable therapeutic strategies to delay or suppress the carcinogenic process. Molecular technologies are just beginning to be sufficiently sensitive to start testing the hypothesis. PMID- 12376080 TI - Euchromatinization of mammalian nuclei. AB - This paper discusses a mechanism for converting heterochromatin into the more relaxed state of euchromatin. Actuation of such state conversion stems from disassembly of internal vesicles that are incapable of surviving when trapped within the inner chamber of mammalian nuclei. PMID- 12376081 TI - Hypothesis - the J-shaped follow-up relation between mortality risk and disease risk-factor is due to statistical confounding. AB - There is currently conflicting evidence from longitudinal follow-up studies concerning the relation between mortality risk and disease risk factor at low levels or the risk factor. This applies to risk factors such as blood pressure, cholesterol, and body-mass index. In some studies, this relation was found to be positive. In others it was negative. This is an issue of importance to clinical and public-health policy, because a negative relation between mortality risk and blood pressure, for instance, implies that anithypertensive medication which lowers blood pressure below a critical threshold could be dangerous. It seemed likely that the conflict could be due to statistical confounding that artifactually elevates mortality risk at low risk-factor levels in survival analyses of longitudinal data. The present paper describes a crude analysis using data from the Framingham Offspring Study to test the idea that such statistical confounding could be caused by the decrease in risk factor with age among subjects near the end of the lifespan (referred to as late-life subjects). The analysis yielded evidence supporting this idea. on the basis of the findings it is hypothesized that: (1). the decrease in risk factor with age during late life causes the late-life bias. This bias distorts a positive relation between mortality risk and risk factor to appear U- or J-shaped in mixed-age adult follow up cohorts; and (2). removal of the late-life and reverse-causation biases will show that this relation is monotonically positive. PMID- 12376082 TI - An etiologic model proposing that non-insulin-dependent diabetes mellitus is chronic hypoxic stress hyperglycemia. AB - This etiologic model equates non-insulin-dependent diabetes mellitus (NIDDM) to chronic hypoxic stress hyperglycemia produced by increased stimulation of the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. In the initial stages of the disease, hypoxia is believed to result from hemodilutional anemia precipitated by a reduction in vascular smooth muscle tone. The reduction increases lumen diameter necessitating an increased blood volume to maintain pressure. Increased lumen diameter may also trigger atherosclerotic changes that characterize the later stages of NIDDM. The increased diameter decreases the shear stress experienced by endothelial cells and they respond by releasing endothelin, a smooth muscle constrictor and mitogen. The constricting action is hypothesized to be relatively ineffective in NIDDM leading to long-term endothelin release and activation of its mitogenic properties. The resulting increase in the number of smooth muscle cells may explain the intimal thickening of atherosclerosis. Restoration of vascular muscle tone is proposed as a treatment strategy for mild NIDDM. PMID- 12376083 TI - BCR-ABL insufficiency for the transformation of human stem cells into CML. AB - On the basis of numerical and kinetic data, the paper argues against the tenet that BCR-Abl is sufficient for transforming into Chronic Myelogenous leukemia a human hemopoietic stem cell, regardless of degree of pluripotency. PMID- 12376084 TI - A note on the meaning of stochasticity. PMID- 12376085 TI - Viral cirrhosis with chronic right heart failure and cardiac liver sclerosis: a hypothesis on the differentiation between the two diseases through pulsed Doppler sonography examination. AB - Chronic right heart failure determines cardiac liver sclerosis as a consequence of the hepatic venous congestion that is easily detectable with pulsed Doppler sonography measuring hepatic venous pulsatility. These Doppler parameters profoundly change on the basis of the causative agent of the liver sclerosis. In fact, on pulsed Doppler examination, we detected a flat waveform in hepatic veins of subjects with viral liver cirrhosis whereas we only observed biphasic waveforms in subjects with cardiac liver sclerosis. On the contrary, a pulsed Doppler sonography pattern in subjects with viral cirrhosis and associated chronic right heart failure is unknown. We suppose that splanchnic pulsed Doppler sonography has an important role both in differentiating cardiac liver sclerosis from viral cirrhosis with chronic right heart failure and in the follow-up of the seriousness of the right ventricular failure. PMID- 12376086 TI - Treatment of multiple sclerosis with lofepramine, L-phenylalanine and vitamin B(12): mechanism of action and clinical importance: roles of the locus coeruleus and central noradrenergic systems. AB - In a randomized, placebo-controlled double-blind trial a combination of lofepramine, phenylalanine and vitamin B(12) was found to be effective in relieving the symptoms of multiple sclerosis (MS). The effect occurred within 2-4 weeks, and improved all types of symptoms in all types of MS. The combination was also effective in relieving symptoms in patients with chronic pain and chronic fatigue. We hypothesize that the action of this combined therapy may relate to activation of the noradrenergic locus coeruleus/lateral tegmentum (LC/LT) system which has the potential to influence the functioning of large areas of the brain and spinal cord. PMID- 12376087 TI - Are latent, immediate-early genes of herpes simplex virus-1 essential in causing trigeminal neuralgia? AB - The etiology and pathogenesis of major trigeminal neuralgia remain largely unknown, but are believed to result from an irritative lesion near the semilunar ganglion. We suggest that its primary cause is a single, active DNA sequence in the persistent but non-integrated genome of latent herpes simplex virus type 1 commonly observed in a few infected A-delta nerve fibers of the cheek. Facial pain occurs as a result of herpes virus reactivation and when supplies of neurotrophins controlling normal transport functions of axolemmal ion channels become depleted. PMID- 12376088 TI - Does increased leukotriene B4 in type 1 diabetes result from elevated cholesteryl ester transfer protein activity? AB - Elevated cholesteryl ester transfer protein (CETP) activity has been reported in type 1 diabetic subjects and may be one cause of the high incidence of macrovascular complications in these patients. LDL delivers arachidonic acid (AA), in the form of cholesteryl ester (CE), to cells such as monocytes and fibroblasts, as precursor for eicosanoid synthesis. We discovered that AA content in LDL CE was significantly correlated with CETP activity, even after controlling for CETP concentration, in type 1 diabetic children. The production of LTB(4), a potent chemotactic and pro-inflammatory factor which plays a role in atherogenesis, has been shown to be increased in type 1 diabetic patients. We hypothesized that in these subjects, increased AA content in LDL CE, resulting from increased CETP activity and transient hyperinsulinemia, may lead to enhanced synthesis of LTB(4) and subsequently the higher incidence of cardiovascular disease. PMID- 12376089 TI - A simple conceptual model of primary pulmonary blast injury. AB - Primary pulmonary blast injury arises from direct exposure to blast overpressure, and may lead to severe lung injury and systemic air embolism. The phenomena of spallation and implosion can be explained by a simple conceptual model without invoking complex physical principles. PMID- 12376090 TI - The imprinted oedematous-small mutation on mouse chromosome 2 identifies new roles for Gnas and Gnasxl in development. AB - The Gnas locus is highly complex and encodes several oppositely imprinted and alternatively spliced transcripts. Gnas itself encodes Gsalpha, which is involved in endocrine function and bone development, but the roles for the other transcripts have not been established. Here we describe a mouse mutation that provides further biological functions for the Gnas locus. The mutation Oed-Sml, induced by ethylnitrosourea (ENU), has been mapped to the distal chromosome 2 imprinting region that includes Gnas. The mutation displays two distinct phenotypes dependent on parental origin. When the mutation is maternally transmitted, a microcardia with gross edema (Oed) results. By contrast, when the mutation is paternally transmitted, a growth retardation (Sml) is seen that becomes evident within 5 days of birth. Here we show Oed-Sml to be a point mutation in Gnas exon 6, resulting in a valine to glutamate substitution at residue 159 (V159E). Both maternal- and paternal-specific transcripts derive from this missense mutation. The maternally expressed mutant Gnas transcript is the candidate for Oed and the paternally expressed mutant Gnasxl transcript is the candidate for Sml. We propose a new role for Gnas in heart growth and a role for Gnasxl in postnatal growth. These findings potentially have implications for human Albright hereditary osteodystrophy, a condition caused by mutations in GNAS. PMID- 12376091 TI - Assaying DNA methylation based on high-throughput melting curve approaches. AB - Here we describe two high-throughput methods to assay DNA methylation, melting curve methylation specific PCR (McMSP) and melting curve combined bisulfite restriction analysis (McCOBRA), which adapt standard MSP and COBRA methods to a melting curve analysis based platform. We show that McMSP and McCOBRA can accurately determine methylation status in a high-throughput and gel-free manner. Moreover, McCOBRA can be used to quantitatively estimate the percent of methylated DNA at a specific CpG site within a heterogeneous sample. The accuracy of McMSP and McCOBRA was initially tested using the 5'-CpG site of the tumor suppressor gene CDKN2A as a model system in homogeneous and heterogeneous controls, and cancer cell line samples. Furthermore, the robustness of McMSP and McCOBRA was validated in four additional loci. We demonstrate that McCOBRA and McMSP provide several advantages over existing methods, as they are simple, accurate, and high-throughput, which makes them widely applicable to large-scale methylation studies. PMID- 12376092 TI - Quality indicators increase the reliability of microarray data. AB - Large-scale gene expression profiling with DNA microarrays opens new dimensions to molecular biology but still lacks the overall precision of traditional low scale techniques. We developed a novel strategy of data processing linking search stringency to quality indicators for efficient detection of low-level, regulated genes. Using retinoid-induced differentiation of NB-4 promyelocytic cells, the variation of expression profiles between biological duplicates was studied and compared with the changes induced by all-trans retinoic acid (atRA) treatment. An analysis of 4320 genes showed that retinoic acid has mainly geneactivating function in NB-4 cells. Treatment with atRA for 18 hours induced metabolic genes that may be associated with cell differentiation and signaling factors triggering later events leading to apoptosis; cytokine genes were among the highest stimulated by atRA. Notably, we identified a regulatory loop inhibiting MYC action: as MYC was downregulated, a cognate repressor of MYC was upregulated. PMID- 12376093 TI - Synteny comparison between apes and human using fine-mapping of the genome. AB - Comparing the genomes of the great apes and human should provide novel information concerning the origins of humankind. Relative to the great apes, the human karyotype has one fewer chromosome pair, as human chromosome 2 derived from the telomeric fusion of two ancestral primate chromosomes. To identify the genomic rearrangements that accompanied human speciation, we initiated a comparative study between human, chimpanzee, and gorilla. Using the HAPPY mapping method, an acellular adaptation of the radiation hybrid method, we mapped a few hundred markers on the human, chimpanzee, and gorilla genomes. This allowed us to identify several chromosome rearrangements, in particular a pericentric inversion and a translocation. We precisely localized the synteny breakpoint that led to the formation of human chromosome 2. This breakpoint was confirmed by FISH mapping. PMID- 12376094 TI - A new family of chimeric retrotranscripts formed by a full copy of U6 small nuclear RNA fused to the 3' terminus of l1. AB - Long interspersed nuclear elements (LINE-1, L1) constitute a large family of mammalian retrotransposons that have been replicating and evolving in mammals for more than 100 million years and now compose 17% of the human genome. They have an important creative role in human genomic evolution through mechanisms such as new integrations, generation of processed pseudogenes, and transfer of non-L1 DNA flanking their 3' ends to new genomic locations. Here we present evidence that the L1 integration machinery was used for the creation of a new family of chimeric retrotranscripts, which contain a full copy of U6 small nuclear RNA and a 3' part of L1 at their 5' and 3' ends, respectively. There are at least 56 members of this family in the human genome. The integrations of such fused retrotranscripts into the human genome took place until recently. Here we report one U6-L1 insertion that is polymorphic in humans. We also propose a mechanism used to generate chimeric retrotranscripts. PMID- 12376095 TI - Characterization of the gene encoding mouse retinoblastoma binding protein-7, a component of chromatin-remodeling complexes. AB - RBBP7 is a highly conserved WD-repeat protein that interacts with histone deacetylases and is a component of several co-repressor complexes. The mouse gene Rbbp7 spans approximately 20 kb, consists of at least 12 exons, and contains a C/T polymorphism in the 3' splice acceptor region of intron 3. We found that Rbbp7 contains a TATA-less promoter with multiple transcription initiation sites. In transient transfection assays, we identified potential positive regulatory elements upstream of the proximal promoter at -668 to -1710. RBBP7 protein is detectable from at least day 9.5 of embryogenesis and is strongly expressed in the developing kidney and brain. Consistent with its association with co repressor complexes, we demonstrate that RBBP7 represses the c-FOS transactivation domain in response to mitogen stimulation. We have also excluded human RBBP7 as a candidate gene in six patients that exhibit X-linked mental retardation, a heterogeneous developmental disorder that has been linked in some cases to mutations in genes involved in chromatin remodeling. PMID- 12376096 TI - Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13. AB - We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine human-mouse comparative map of this region was constructed. PMID- 12376097 TI - Instability of a premutation-sized CGG repeat in FMR1 YAC transgenic mice. AB - Fragile X syndrome results from the massive expansion of a CGG repeat in the 5' untranslated region of the gene FMR1. Data suggest that the hyperexpansion properties of FMR1 CGG repeats may depend on flanking cis-acting elements. We have therefore used homologous recombination in yeast to introduce an in situ CGG expansion corresponding to a premutation-sized allele into a human YAC carrying the FMR1 locus. Several transgenic lines were generated that carried repeats of varying lengths and amounts of flanking sequence. Length-dependent instability in the form of small expansions and contractions was observed in both male and female transmissions over five generations. No parent-of-origin effect or somatic instability was observed. Alterations in tract length were found to occur exclusively in the 3' uninterrupted CGG tract. Large expansion events indicative of a transition from a premutation to a full mutation were not observed. Overall, our results indicate both similarities and differences between the behavior of a premutation-sized repeat in mouse and that in human. PMID- 12376098 TI - A novel brain-expressed protein related to carnitine palmitoyltransferase I. AB - Malonyl-CoenzymeA acts as a fuel sensor, being both an intermediate of fatty acid synthesis and an inhibitor of the two known isoforms of carnitine palmitoyltransferase I (CPT I), which control mitochondrial fatty acid oxidation. We describe here a novel CPT1 family member whose mRNA is present predominantly in brain and testis. Chromosomal locations and genome organization are reported for the mouse and human genes. The protein sequence contains all the residues known to be important for both carnitine acyltransferase activity and malonyl-CoA binding in other family members. Yeast expressed protein has no detectable catalytic activity with several different acyl-CoA esters that are good substrates for other carnitine acyltransferases, including the liver isoform of CPT I, which is also expressed in brain; however, it displays high-affinity malonyl-CoA binding. Thus this new CPT I related protein may be specialized for the metabolism of a distinct class of fatty acids involved in brain function. PMID- 12376099 TI - Localization and analysis of the principal promoter for human tenascin-X. AB - Tenascin-X is a large extracellular matrix protein expressed in connective tissues. Mutations in TNXB are a cause of Ehlers-Danlos syndrome. Comparison of 25 kb of human and mouse DNA near the TNXB untranslated exon identified eight regions of >80% identity. Of 17 cell types and lines screened, TNXB expression was abundant only in fibroblasts and HT1080 human skin fibrosarcoma cells. Expression of TNXB promoter/reporter constructs in HT1080 cells showed that region E, near the untranslated exon, had the greatest activity, and the two regions of greatest identity, 5.0 and 3.3 kb upstream, had no activity. Mobility shift assays identified six protein-binding regions. Regions I, II, and IV bound Sp1 and Sp3, but only I and IV were functional in HT1080 cells. Regions III and V bound unknown proteins and exerted strong enhancer-like activity. Mutation of regions III and V in promoter/reporter constructs decreased TNXB transcription and identified functionally important Sp1 and Sp3 sites. These experiments provide an essential foundation for understanding the regulation of this vital protein. PMID- 12376100 TI - Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. AB - The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors which all share common sequence elements and structural motifs. These proteins are known to exert a wide spectrum of biological responses on a large variety of cell types in both vertebrates and invertebrates. Many of them have important functions during embryonic development in pattern formation and tissue specification, and in adult tissues, they are involved in processes such as wound healing, bone repair, and bone remodeling. The family is divided into two general branches: the BMP/GDF and the TGF-beta/Activin/Nodal branches, whose members have diverse, often complementary effects. It is obvious that an orchestered regulation of different actions of these proteins is necessary for proper functioning. The TGF-beta family members act by binding extracellularly to a complex of serine/threonine kinase receptors, which consequently activate Smad molecules by phosphorylation. These Smads translocate to the nucleus, where they modulate transcription of specific genes. Three levels by which this signaling pathway is regulated could be distinguished. First, a control mechanism exists in the intracellular space, where inhibitory Smads and Smurfs prevent further signaling and activation of target genes. Second, at the membrane site, the pseudoreceptor BAMBI/Nma is able to inhibit further signaling within the cells. Finally, a range of extracellular mediators are identified which modulate the functioning of members of the TGF-beta superfamily. Here, we review the insights in the extracellular regulation of members of the BMP subfamily of secreted growth factors with a major emphasis on vertebrate BMP modulation. PMID- 12376101 TI - Regulation of Tbx3 expression by anteroposterior signalling in vertebrate limb development. AB - Tbx3, a T-box gene family member related to the Drosophila gene optomotor blind (omb) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar-mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarising region which controls anteroposterior pattern. Here we carried out tissue transplantations, grafted beads soaked in Shh, Bmps, and Noggin in chick limb buds, and analysed Tbx3 expression. We also investigated Tbx3 expression in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. We show that Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centred on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. Our results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number. PMID- 12376103 TI - Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number. AB - Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development. PMID- 12376102 TI - Maternally supplied Smad5 is required for ventral specification in zebrafish embryos prior to zygotic Bmp signaling. AB - We have previously shown that the maternal effect dorsalization of zebrafish embryos from sbn(dtc24) heterozygous mothers is caused by a dominant negative mutation in Smad5, a transducer of ventralizing signaling by the bone morphogenetic proteins Bmp2b and Bmp7. Since sbn(dtc24) mutant Smad5 protein not only blocks wild-type Smad5, but also other family members like Smad1, it remained open to what extent Smad5 itself is required for dorsoventral patterning. Here, we report the identification of novelsmad5 alleles: three new isolates coming from a dominant enhancer screen, and four former isolates initially assigned to the cpt and pgy complementation groups. Overexpression analyses demonstrate that three of the new alleles, m169, fr5, and tc227, are true nulls (amorphs), whereas the initial dtc24 allele is both antimorphic and hypomorphic. We rescued m169 mutant embryos by smad5 mRNA injection. Although adult mutants are smaller than their siblings, the eggs laid by m169(-/-) females are larger than normal eggs. Embryos lacking maternal Smad5 function (Mm169(-/-) embryos) are even more strongly dorsalized thanbmp2b or bmp7 null mutants. They do not respond to injected bmp2b mRNA, indicating that Smad5 is absolutely essential for ventral development and Bmp2/7 signaling. Most importantly, Mm169( /-) embryos display reducedbmp7 mRNA levels during blastula stages, when bmp2b and bmp7 mutants are still normal. This indicates that maternally supplied Smad5 is already required to mediate ventral specification prior to zygotic Bmp2/7 signaling to establish the initial dorsoventral asymmetry. PMID- 12376104 TI - Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds. AB - To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth. PMID- 12376105 TI - Affinity regulates spatial range of EGF receptor autocrine ligand binding. AB - Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. We employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF(L47M). The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. Our experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling. PMID- 12376106 TI - Adult corneal limbal epithelium: a model for studying neural potential of non neural stem cells/progenitors. AB - Recent studies suggest that tissue-specific stem cells possess much wider potential for differentiation than previously thought and can, in some instances, even cross germ layer boundaries. However, information is lacking regarding the efficiency and the fidelity of their differentiation along heterologous lineages. To address these issues of transdifferentiation, we have analyzed the heterologous potential of stem cells within the same germ layer. We report the neural potential of cells isolated from the limbal epithelium of the adult cornea. Limbal epithelium, which, like the neuroepithelium, is ectodermally derived, participates in the regeneration of cornea throughout life. We have observed that limbal epithelial cells, when removed from their niche and cultured in the presence of mitogens, begin to express neural progenitor markers. Based on the self-renewal property, it is likely that the nestin-positive progenitors are derived from limbal stem cells rather than transit-amplifying (TA) cells that have limited proliferating potential. In differentiation conditions, a subset of these cells acquire neural morphology and express transcripts and proteins specific to neurons and glia, suggesting their differentiation along neural lineage. The acquisition of neural properties is regulated by BMP signaling. Neural differentiation of these cells is also observed upon heterotopic transplantation. Investigation of functional differentiation of cells by electrophysiological analysis reveals properties consistent with the presence of glia that are influenced by extracellular cues. However, similar analyses coupled with Ca(2+) imaging suggest an incomplete differentiation of limbal epithelial derived neural progenitors into neurons in the condition studied. Our study, therefore, draws attention toward the necessity for rigorous characterization of transdifferentiation and offers a model for characterizing neural potential of heterologous stem cells/progenitors. PMID- 12376107 TI - SEK1/MKK4-mediated SAPK/JNK signaling participates in embryonic hepatoblast proliferation via a pathway different from NF-kappaB-induced anti-apoptosis. AB - Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction ofsek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additionaltnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-Jun. PMID- 12376108 TI - Sperm from the calmegin-deficient mouse have normal abilities for binding and fusion to the egg plasma membrane. AB - Calmegin is a putative testis-specific molecular chaperone required for the heterodimerization of fertilin alpha/beta and the appearance of fertilin beta on the sperm surface. Calmegin-deficient mice are almost completely sterile. The cause of the sterility initially was considered to be impaired abilities in sperm/zona pellucida (ZP) and sperm/egg plasma membrane (EPM) binding, and in the ascension of sperm to the oviduct, phenotypes similar to those seen in sperm from fertilin beta-deficient animals. We have developed a new method in which eggs were prepared without any detectable ZP3 on their surfaces by using a piezo driven micromanipulator. Using these eggs and sperm containing the green fluorescent protein in their acrosomes, which can distinguish acrosome-intact from acrosome-reacted sperm, the binding and fusing abilities of calmegin deficient sperm were reexamined. Under these conditions, acrosome-reacted sperm retained their ability to bind to and fuse with the EPM. The reduction in EPM binding of sperm from the calmegin(-/-) animals was apparently due to the artifactual binding of large numbers of acrosome-intact sperm from calmegin(+/-) mice to ZP remnants remaining on the EPM prepared with acidic Tyrode's solution. Thus, the sperm defect in calmegin-null animals is not at the level of sperm-EPM binding but rather may involve either sperm-ZP binding and/or sperm transit to the oviduct. Because fertilin beta is absent from calmegin-deficient mice, these results also suggest that the role of fertilin beta in sperm-EPM interaction needs to be reevaluated. PMID- 12376109 TI - Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta. AB - Mammalian embryos have an intimate relationship with their mothers, particularly with the placental vasculature from which embryos obtain nutrients essential for growth. It is an interesting vascular bed because maternal vessel number and diameter change dramatically during gestation and, in rodents and primates, the terminal blood space becomes lined by placental trophoblast cells rather than endothelial cells. Molecular genetic studies in mice aimed at identifying potential regulators of these processes have been hampered by lack of understanding of the anatomy of the vascular spaces in the placenta and the general nature of maternal-fetal vascular interactions. To address this problem, we examined the anatomy of the mouse placenta by preparing plastic vascular casts and serial histological sections of implantation sites from embryonic day (E) 10.5 to term. We found that each radial artery carrying maternal blood into the uterus branched into 5-10 dilated spiral arteries located within the metrial triangle, populated by uterine natural killer (uNK) cells, and the decidua basalis. The endothelial-lined spiral arteries converged together at the trophoblast giant cell layer and emptied into a few straight, trophoblast-lined "canals" that carried maternal blood to the base of the placenta. Maternal blood then percolated back through the intervillous space of the labyrinth toward the maternal side of the placenta in a direction that is countercurrent to the direction of the fetal capillary blood flow. Trophoblast cells were found invading the uterus in two patterns. Large cells that expressed the trophoblast giant cell-specific gene Plf (encoding Proliferin) invaded during the early postimplantation period in a pattern tightly associated with spiral arteries. These peri/endovascular trophoblast were detected only approximately 150-300 microm upstream of the main giant cell layer. A second type of widespread interstitial invasion in the decidua basalis by glycogen trophoblast cells was detected after E12.5. These cells did not express Plf, but rather expressed the spongiotrophoblast-specific gene Tpbp. Dilation of the spiral arteries was obvious between E10.5 and E14.5 and was associated with a lack of elastic lamina and smooth muscle cells. These features were apparent even in the metrial triangle, a site far away from the invading trophoblast cells. By contrast, the transition from endothelium-lined artery to trophoblast-lined (hemochorial) blood space was associated with trophoblast giant cells. Moreover, the shaping of the maternal blood spaces within the labyrinth was dependent on chorioallantoic morphogenesis and therefore disrupted in Gcm1 mutants. These studies provide important insights into how the fetoplacental unit interacts with the maternal intrauterine vascular system during pregnancy in mice. PMID- 12376110 TI - spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning. AB - Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains. PMID- 12376111 TI - Different regulation of T-box genes Tbx4 and Tbx5 during limb development and limb regeneration. AB - The T-domain transcription factors Tbx4 and Tbx5 have been implicated, by virtue of their limb-type specific expression, in controlling the identity of vertebrate legs and arms, respectively. To study the roles of these genes in developing and regenerating limbs, we cloned Tbx4 and Tbx5 cDNAs from the newt, and generated antisera that recognize Tbx4 or Tbx5 proteins. We show here that, in two urodele amphibians, newts and axolotls, the regulation of Tbx4 and Tbx5 differs from higher vertebrates. At the mRNA and protein level, both Tbx4 and Tbx5 are expressed in developing hindlimbs as well as in developing forelimbs. The coexpression of these genes argues that additional factors are involved in the control of limb type-specific patterns. In addition, newt and axolotl Tbx4 and Tbx5 expression is regulated differently during embryogenesis and regenerative morphogenesis. During regeneration, Tbx5 is exclusively upregulated in the forelimbs, whereas Tbx4 is exclusively upregulated in the hindlimbs. This indicates that, on a molecular level, different regulatory mechanisms control the shaping of identical limb structures and that regeneration is not simply a reiteration of developmental gene programs. PMID- 12376112 TI - TGF-beta(3)-induced chondroitin sulphate proteoglycan mediates palatal shelf adhesion. AB - In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by beta-D-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-beta(3)) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-beta(3) is added to TGF-beta(3) null mutant palatal shelves in culture. When chick palatal shelves (that do not adherein vivo nor express TGF-beta(3), nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-beta(3) is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-beta(3). PMID- 12376114 TI - Analytical methods for biological monitoring of exposure to pesticides: a review. AB - Synthetic pesticides have been used since in the early to mid twentieth century. In the US alone, over 800 pesticide active ingredients are formulated in about 21,000 different commercial products. Although many public health benefits have been realized by the use of pesticides, their potential impact on the environment and public health is substantial. For risk assessment studies, exposure assessment is an integral component, which has unfortunately, often been weak or missing. In the past several decades, researchers have proposed to fill these missing data gaps using biological monitoring of specific markers related to exposures. In this paper, we present a review of existing analytical methodology for the biological monitoring of exposure to pesticides. We also present a critical assessment of the existing methodology and explore areas in which more research is needed. PMID- 12376115 TI - Biomonitoring of polycyclic aromatic hydrocarbons in human urine. AB - Measurement of polycyclic aromatic hydrocarbons (PAH) metabolites in human urine is the method of choice to determine occupational and/or environmental exposure of an individual to PAH, in particular, when multiple routes of exposure have to be taken into account. Requirements for methods of biomonitoring PAH metabolites in urine are presented. Studies using 1-hydroxypyrene or phenanthrene metabolites including its phenols and dihydrodiols are summarized. The role of these PAH metabolites as established biomarkers and also more recent developments of PAH biomonitoring are discussed. PMID- 12376116 TI - Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans. AB - Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given. PMID- 12376117 TI - Elements in environmental and occupational medicine. AB - Occupational and environmental medicine traditionally dealt with elements, particularly with heavy metals. The interest was justified by the wide exposure in the workplace and in the general environment and by the evidence of their specific biological and toxicological effects. During the last 2 decades of 20th century the availability of indicators of exposure or of internal dose has substantially increased thanks to improvement in AAS-ETAAS techniques and to the entrance of ICP-MS into the field of biological monitoring. There are now more and more demands for controlling pre-analytical and analytical factors, for analysing biological matrices in addition to blood and urine and for setting up methods for elements not yet extensively studied in respect to their possible biological or toxicological role. Finally, deeper knowledge has to be reached in order to evaluate the significance of elements and, possibly, of their species in biological fluids at current doses and in order to face their effects, especially those in the first portion of the dose-response curve, which is going to be the main field of interest of occupational and environmental toxicology for the next few years. PMID- 12376119 TI - Estimating occupational exposure to the pyrethroid termiticide bifenthrin by measuring metabolites in urine. AB - The control of subterranean termites in Australia is predominantly through the application of chemical barriers in the soil beneath and surrounding buildings. The chemicals used to repel or kill termites are the organophosphorus insecticide, chlorpyrifos, and the synthetic pyrethroid, bifenthrin. These are applied through surface sprays and subfloor injection by licensed pest control operators. To determine the exposure of these personnel to these pesticides it is most usual to measure airborne concentrations or dermal deposition rates. However, to support information obtained from these methods it is often appropriate to determine the amount of the chemicals absorbed, using biological monitoring techniques including measurement of the chemicals or their metabolites in urine. While there are effective techniques for the monitoring of chlorpyrifos exposure by measuring either the alkyl phosphate or trichloropyridinol metabolites, there have been no published reports of suitable methods to measure bifenthrin metabolites in urine. This paper describes an extraction and HPLC-UV method used to simultaneously measure the urinary excretion of 2-methyl-3 phenylbenzoic acid (MPA), a metabolite of bifenthrin, and 3-phenoxybenzoic acid (PBA), a metabolite of several other common pyrethroid insecticides, with a detection limit for each of 2.5 ng/ml. The paper also describes the pilot application of this method to a study of South Australian pest control operators handling bifenthrin. MPA ranged from 1.8 to 31.9 microg/g creatinine and PBA from 1.3 to 30.0 microg/g in the urine of pest control workers. MPA was detected in urine of control workers without bifenthrin exposure only at low levels (1.0-1.4 microg/g creatinine), but PBA was found in both at higher levels (1.2-61.1 microg/g creatinine). PMID- 12376118 TI - A multi-analyte method for the quantification of contemporary pesticides in human serum and plasma using high-resolution mass spectrometry. AB - We have developed a sensitive and accurate analytical method for quantifying 29 contemporary pesticides in human serum or plasma. These pesticides include organophosphates, carbamates, chloroacetanilides, and synthetic pyrethroids among others and include pesticides used in agricultural and residential settings. Our method employs a simple solid-phase extraction followed by a highly selective analysis using isotope dilution gas chromatography-high-resolution mass spectrometry. Our method is very accurate, has limits of detection in the low pg/g range and coefficients of variation of typically less than 20% at the low pg/g end of the method linear range. We have used this method to measure plasma pesticide concentrations in females living in an urban area. We found detectable concentrations of carbaryl/naphthalene, propoxur, bendiocarb, chlorpyrifos, diazinon, dicloran, captan and folpet or their metabolites in more than 20% of the plasma samples tested. PMID- 12376120 TI - New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine. AB - We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1 carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3 phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean up, NaOH was added to the organic phase and the carboxylic acids were re extracted into the aqueous phase. After acidification and extraction into n hexane again, the metabolites were then derivatised to volatile esters using N tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples. PMID- 12376121 TI - Evaluation of respiratory and cutaneous doses of chlorothalonil during re-entry in greenhouses. AB - Five female workers were monitored for 5 consecutive days during re-entry into a greenhouse containing ornamental plants. Skin contamination (excluding hands) was evaluated with nine pads of filter paper placed on the skin. Hand contamination was assessed by washing with 95% ethanol. Respiratory exposure was evaluated by personal air sampling. The respiratory dose was based on a lung ventilation of 15 l/min. The doses absorbed were estimated assuming 10% skin absorption and 100% lung retention. Dislodgeable foliar residue was determined on days of re-entry to evaluate the decay of chlorothalonil. Chlorothalonil was analysed in the different matrices by GC-MS. Respiratory exposure was less than skin contamination, being 11.4+/-5.1% (mean+/-SD) of total exposure. The estimated total absorbed dose did not exceed the acceptable daily intake of 0.03 mg/kg body mass. The hands and unexposed skin of all workers were always found to be contaminated. Greater precautions are therefore needed to reduce skin exposure (clean gloves and suitable clean clothing every day). PMID- 12376122 TI - Determination of polychlorinated biphenyl congeners in human milk by gas chromatography-negative chemical ionization mass spectrometry after sample clean up by solid-phase extraction. AB - This paper describes a simple and efficient procedure for measuring 25 congeners of polychlorinated biphenyls in human milk. The limit of quantitation was 0.1 ng/ml for five less chlorinated congeners (PCB 70, 74, 87, 99,101), and 0.01 ng/ml for the remaining 20 congeners (PCB 77, 105, 118, 126, 128, 138, 151, 153, 156, 169, 170, 180, 183, 187, 191, 194, 205, 206, 208 and 209). Solid phase extraction technology was applied to extract the analytes from the matrix and to remove lipids. Three columns were used sequentially, and they were a Bond Elut C(18), a Sep-Pak Plus NH2 and a Bond Elut PCB cartridge. The instrumental method was gas chromatography-mass spectrometry with negative chemical ionization, and selected ion monitoring mode was used. PMID- 12376123 TI - Determination of selected monohydroxy metabolites of 2-, 3- and 4-ring polycyclic aromatic hydrocarbons in urine by solid-phase microextraction and isotope dilution gas chromatography-mass spectrometry. AB - Eighteen monohydroxy polycyclic aromatic hydrocarbon metabolites (OH-PAHs) representing polycyclic aromatic hydrocarbons (PAHs) containing up to four rings in human urine have been measured. The method includes the addition of carbon-13 labeled internal standards, enzymatic hydrolysis, and solid-phase microextraction followed by gas chromatography with high-resolution mass spectrometry. By using response factors calculated with the carbon-13 labeled standards, results are presented for calibration, relative standard deviations and analyte levels from an unspiked human urine pool. The method detection limits ranged from 0.78 ng/l for hydroxyphenanthrenes to 15.8 ng/l for 1-hydroxynaphthalene, and the recoveries ranged between 6% for hydroxychrysene and 47% for 1-hydroxypyrene. The relative standard deviation was lowest for 3-hydroxyphenanthrene at 2.4% and went up to 18.7% for 6-hydroxychrysene. The method was calibrated from 10 to 1200 ng/l. Eleven of the 18 metabolites were found in background pooled urine samples. This validated method is a convenient and reliable tool for determining urinary OH-PAHs as biomarkers of exposure to eight PAHs. PMID- 12376124 TI - Is 1-hydroxypyrene a reliable bioindicator of measured dietary polycyclic aromatic hydrocarbon under normal conditions? AB - Five healthy volunteers consumed similar amounts of identical foods for 5 consecutive days. The concentration of pyrene and of benzo(a)pyrene was determined in each of the 15 meals by a short analytical method that included sample saponification, solvent extraction, and HPLC analysis. The volunteers also provided three daily total volume 8-h urine samples for the duration of the study for the assessment of 1-hydroxypyrene, a biomarker of pyrene and polycyclic aromatic hydrocarbon (PAH) exposure. Mean recoveries were 83 and 75%, respectively, for pyrene and benzo(a)pyrene in food. Daily dietary pyrene doses varied from 0.7 to 3 microg. Excluding two outliers consisting of meals containing charbroiled pork and beef, pyrene content in the meals estimated from the published literature data was correlated to the measured pyrene, but overestimated the actual concentration by ca. 70%. Despite the identical ingested doses of pyrene, there was a 50-76% (coefficient of variation) interindividual variability in the daily-excreted amount of 1-hydroxypyrene. Urinary excretion of this metabolite was not correlated with ingested dose of pyrene under the normal feeding conditions used in this study. Bioavailability, enzymatic polymorphism, and differences in enterohepatic cycling of the metabolite may contribute to the observed variability. It was calculated that dietary pyrene intake accounts for between 87.5 and 99.8% of the sum of dietary and inhalation intake. From the presented data, unless the above-mentioned factors are taken into account, 1 hydroxypyrene might not be a reliable bioindicator of ingested pyrene (PAHs) under normal feeding conditions. PMID- 12376125 TI - Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry. AB - A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4 aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6 dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4 aminobiphenyl or metabolites of explosives in the samples. PMID- 12376126 TI - Determination of nitroglycerin and its dinitrate metabolites in urine by gas chromatography-mass spectrometry as potential biomarkers for occupational exposure. AB - This paper describes a method for the quantitative analysis of nitroglycerin and its dinitrate metabolites (1,2- and 1,3-glycerol dinitrate) in urine. After liquid-liquid extraction the analytes were separated and quantified using gas chromatography-mass spectrometry with negative ion chemical ionisation. The method can detect above 0.3 nmol/l for all analytes and is linear over the range of at least 0-44 nmol/l. The method was then used to determine metabolite levels in a subject using nitroglycerin therapeutically for the treatment of angina. Metabolites were stable in urine for at least 6 days at room temperature, approximately 4 degrees C and -20 degrees C. PMID- 12376127 TI - Analysis of benzene, toluene, ethylbenzene and m-xylene in biological samples from the general population. AB - A method for the determination of benzene, toluene, ethylbenzene and xylene in blood and urine of people not occupationally exposed to solvents is described. The headspace technique combined with gas chromatography with a mass spectrometer detector is used. The sensitivity of recent mass spectrometers is good enough to furnish reliable results also in biological samples collected from the general population. No treatment for concentrating solvents present in the blood or urine is necessary. The main features of the method are easy preparation of biological samples, small volumes (7 ml), good repeatability and linearity in the range of interest. The limits of detection in blood were 16, 43, 22 and 52 ng/l for benzene, toluene, ethylbenzene and m-xylene respectively. Slightly greater sensitivity was found for urine samples. The results obtained in biological samples from 25 woodworkers not occupationally exposed to BTEX (15 non-smokers and 10 smokers) are comparable to those obtained by other investigators. PMID- 12376128 TI - Personal exposure to different levels of benzene and its relationships to the urinary metabolites S-phenylmercapturic acid and trans,trans-muconic acid. AB - This report is part of an extensive study to verify the validity, specificity, and sensitivity of biomarkers of benzene at low exposures and assess their relationships with personal exposure and genetic damage. The study population was selected from benzene-exposed workers in Tianjin, China, based on historical exposure data. The recruitment of 130 exposed workers from glue-making or shoe making plants and 51 unexposed subjects from nearby food factories was based on personal exposure measurements conducted for 3-4 weeks prior to collection of biological samples. In this report we investigated correlation of urinary benzene metabolites, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t MA) with personal exposure levels on the day of urine collection and studied the effect of dose on the biotransformation of benzene to these key metabolites. Urinary S-PMA and t,t-MA were determined simultaneously by liquid chromatography tandem mass spectrometry analyses. Both S-PMA and t,t-MA, but specifically the former, correlated well with personal benzene exposure over a broad range of exposure (0.06-122 ppm). There was good correlation in the subgroup that had been exposed to <1 ppm benzene with both metabolites (P-trend <0.0001 for S-PMA and 0.006 for t,t-MA). Furthermore, the levels of S-PMA were significantly higher in the subgroup exposed to <0.25 ppm than that in unexposed subjects (n=17; P=0.001). There is inter-individual variation in the rate of conversion of benzene into urinary metabolites. The percentage of biotransformation of benzene to urinary S-PMA ranged from 0.005 to 0.3% and that to urinary t,t-MA ranged from 0.6 to approximately 20%. The percentage of benzene biotransformed into S-PMA and t,t-MA decreased with increasing concentration of benzene, especially conversion of benzene into t,t-MA. It appears that women excreted more metabolites than men for the same levels of benzene exposures. Our data suggest that S-PMA is superior to t,t-MA as a biomarker for low levels of benzene exposure. PMID- 12376129 TI - Quantitative determination of 5-hydroxy-N-methylpyrrolidone in urine for biological monitoring of N-methylpyrrolidone exposure. AB - The aim of this work was to validate a sensitive method for quantitative analysis of 5-hydroxy-N-methylpyrrolidone (5-HNMP) in urine. This compound has been recommended as a marker for biological monitoring of N-methylpyrrolidone (NMP) exposure. Different solvents and alternative methods of extraction including liquid-liquid extraction (LLE) on Chem Elut and solid-phase extraction (SPE) on Oasis HLB columns were tested. The most efficient extraction of 5-HNMP in urine was LLE with Chem Elut columns and dichloromethane as a solvent (consistently 22% of recovery). The urinary extracts were derivatized by bis(trimethylsilyl)trifluoroacetamide and analysed by gas chromatography-mass spectrometry (GC-MS) with tetradeutered 5-HNMP as an internal standard. The detection limit of this method is 0.017 mg/l urine with an intraassay precision of 1.6-2.6%. The proposed method of extraction is simple and reproducible. Four different m/z signal ratios of TMS-5-HNMP and tetralabelled TMS-5-HNMP have been validated and could be indifferently used in case of unexpected impurities from urine matrix. PMID- 12376130 TI - Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection. AB - A new high-performance liquid chromatographic (HPLC) method is described for the determination of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), the final product of the conjugation reaction between a metabolic intermediate of N,N dimethylformamide (DMF) and glutathione. Urine samples were purified by C(18) solid-phase extraction and then directly analysed by HPLC with an Aminex Ion Exclusion HPX-87H column maintained at 25 degrees C and a UV detector set at 196 nm. Under isocratic conditions (2.4 mM sulphuric acid, flow-rate=0.6 ml/min) AMCC eluted at 20.2 min. The reproducibility (C.V.%) was 1.3-2.7% (intra- and inter assay, N = 5); the accuracy was 98.0+/-1.7% at 10 mg/l and 101.9+/-1.5% at 800 mg/l (mean+/-SD, N = 3). AMCC was measured in urine from 22 exposed subjects. A strong correlation was found between AMCC and environmental DMF [AMCC (mg/g creatinine)=3.40xDMF (mg/m(3)) + 3.07; r=0.95], while in the urine of 20 unexposed subjects the concentration of AMCC was constantly below the detection limit of the method (0.9 mg/l in urine). The method described appears to be useful for the biological monitoring of DMF exposure. PMID- 12376131 TI - Evaluation of 2,5-hexanedione in urine of workers exposed to n-hexane in Brazilian shoe factories. AB - Urinary 2,5-hexanedione (2,5-HD) is used as a biomarker for biological monitoring of workers exposed to n-hexane. The purpose of this study was to compare two types of treatment of urine samples during clean-up (with and without acidic hydrolysis) and to study the exposure situation of workers exposed to n-hexane during shoe manufacturing. There, various glues containing n-hexane are used. Quantification of 2,5-HD was carried out by gas chromatography and flame ionization detection (GC-FID). Fifty-two urine samples taken from workers of seven shoe factories were analyzed. Thirty-four persons from the administrative staff of the same factories served as controls. They were not known to be exposed to n-hexane. The samples treated with acidic hydrolysis showed levels (average 0.94 mg/l) approximately 10 times higher than samples without acidic hydrolysis (0.09 mg/l). The difference is predominantly caused by the conversion of other metabolites of n-hexane (e.g. 4,5-dihydroxy-2-hexanone) to 2,5-HD in the presence of acids. Our results also show, that exposure to n-hexane is different between various industries. Levels of 2,5-HD in urine are predominantly dependent on the type of operation (how the glue is applied on the leather during shoe manufacturing). Simple measures, e.g. using a glue handgun instead of a paintbrush significantly decreased exposure to n-hexane. PMID- 12376132 TI - Biological monitoring of workers exposed to dichloromethane, using head-space gas chromatography. AB - A biological monitoring method for urinary dichloromethane (DCM) has been developed by using head-space gas chromatography with FID detection. The calibration curve is linear in a wide range of DCM levels between 0.01 and 2 mg/l. The recovery rate is almost 100% and within-run coefficients of variation are 2.9-3.7%. A highly significant correlation is found between exposure levels and urinary concentrations of DCM. Determination of urine DCM by this method has many advantages such as sample storage, no need for correction of urine concentration, absence of gender difference and also no confounding effect of glutathione S-transferase T1 polymorphism. PMID- 12376133 TI - Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry. AB - A method based on liquid chromatography tandem mass spectrometry (LC-MSMS) applying atmospheric pressure chemical ionisation (APCI) in the positive ion mode was developed for the direct determination of nicotine, cotinine, trans-3' hydroxycotinine, their corresponding glucuronide conjugates as well as cotinine-N oxide, norcotinine, and nicotine-N'-oxide in the urine of smokers. The assay involves filtration of crude urine, fast liquid chromatography on a reversed phase column and mass-specific detection using MSMS transitions. Deuterium labeled nicotine, cotinine, and trans-3'-hydroxycotinine were used as internal standards. Glucuronides used as reference material were either chemically (cotinine-N-glucuronide) or enzymatically synthesized (nicotine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide). Precision for the major nicotine analytes at levels observable in urine of smokers was better than 10%. Accuracy expressed in recovery rates in urine matrix for nicotine, cotinine, trans-3' hydroxycotinine, and cotinine-N-glucuronide ranged from 87 to 113%. Quantitative results for the three glucuronides in urine samples of 15 smokers were compared to an indirect method in which the aglycons were determined with gas chromatography and nitrogen-selective detection (GC-NPD) before and after enzymatic splitting of the conjugates. Good agreement was found for cotinine-N glucuronide (coefficient of variation, CV: 9%) and trans-3'-hydroxycotinine-O glucuronide (CV: 20%), whereas the accordance between both methods was moderate for nicotine-N-glucuronide (CV: 33%). The described LC-MSMS method allows the simultaneous determination of nicotine and eight of its major metabolites in urine of smokers with good precision and accuracy. Since the method requires a minimum of sample clean-up and a very short time for chromatography (3 min), it is suitable for determining the nicotine dose in large-scale human biomonitoring studies. PMID- 12376134 TI - Arsenic species excretion after controlled seafood consumption. AB - Influence of controlled consumption of marine fish on the urinary excretion of arsenite, arsenate, dimethylarsinic and monomethylarsonic acid (DMA, MMA) was investigated in two experiments. Arsenic species were separated by anion-exchange chromatography and detected with hydride-technique atomic absorption spectrometry (detection limit 1, 10, 2, 2 microg/l). Firstly, 13 probands ate different types of seafood after having refrained from any seafood for 1 week. DMA levels rose from 3.4+/-1.3 microg/g creatinine (n=12; a day before seafood) to a mean peak level of 28.2+/-20.6 microg/g (n=13; 10-23 h after; P<0.001; max. 77.7 microg/g). No other species were excreted before the meal, but small amounts of arsenite (8.5% positive; max. 1.7 microg/g) and MMA (1.2%; 1.6 microg/g) within 2 days after it (n=82). Consumption of white herring caused the highest DMA levels. Secondly, eight probands ingested white herring (dose 3.5 g/kg; DMA content 32.1+/-15.3 ng/g wet weight; n=36). No arsenite, arsenate and MMA was found in the urine or in the herring tissues. The mean DMA mass excreted after the meal (65.3+/-22.0 microg/24 h) was about 6-fold higher than the sum of base DMA excretion (3.0+/-1.7 microg/24 h) and the ingested DMA mass (7.9+/-2.7 microg). This indicates that the elevated DMA excretion after herring consumption is not caused by the metabolism of inorganic arsenic but of other arsenic species present in the fish tissue, e.g. arsenobetaine or fat-soluble arsenic species. PMID- 12376135 TI - Determination of 235U and 238U in urine samples using sector field inductively coupled plasma mass spectrometry. AB - The high sensitivity of SF-ICP-MS (sector field inductively coupled plasma mass spectrometry) using a torch with the "guard-electrode" (capacitive decoupled plasma) allows the determination of 238U (isotope abundance 99.2%) and 235U (0.8%) and their isotope ratio in human urine samples down to the physiological level of <10 ng/l total uranium. For sample preparation UV photolysis was used. Some quality criteria like for the detection limit, the reproducibility, recovery and the isotope ratio are given. The method can be applied in occupational as well as in environmental medicine because of its outstanding detection power. PMID- 12376136 TI - Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications. PMID- 12376137 TI - Use of haemoglobin adducts in exposure monitoring and risk assessment. AB - Many industrial bulk chemicals are oxiranes or alkenes that are easily metabolised to oxiranes in mammalian systems. Many oxiranes may react with DNA and are therefore mutagenic in vitro. Some oxiranes have been shown to be carcinogenic in rodents in vivo as well. Despite the very limited evidence of the carcinogenicity of oxiranes in humans, they should be considered potential human carcinogens. As a consequence, exposure to these compounds should be minimised and controlled. Twenty-five years ago, Ehrenberg and co-workers suggested that exposure to oxiranes might be determined through the measurement of the adducts they form with haemoglobin (Hb). Ten years later, a modification of the Edman degradation was developed at Stockholm University that allowed determination of adducts with the N-terminal valine of Hb by GC-MS. In our laboratory, this methodology was modified and adapted for analysis on an industrial scale. Since 1987, exposure of operators in our facilities to ethylene oxide (EO) has been routinely monitored by determination of N-(2-hydroxyethyl)valine in Hb. Biological monitoring programmes for propylene oxide (PO) and 1,3-butadiene (BD) were developed later. In this review, the methodology and its results are discussed as a tool in human risk assessment of industrial chemicals. Two major advantages of Hb adduct determinations in risk assessment are (1) the qualitative information on the structure of reactive intermediates that may be obtained through the mass spectrometry, which may provide insight in the molecular toxicology of compounds such as BD, and (2) the possibility of reliable determination of exposure over periods of several months with limited number of samples for compounds such as ethylene oxide (EO), propylene oxide (PO) and BD which form stable adducts with Hb. Since good correlations between the airborne concentrations of these chemicals with their respective adducts have been established, Hb adducts can also be used to quantitate airborne exposure which is of paramount importance as exposure assessment is usually one of the weaker parameters in risk assessment. PMID- 12376138 TI - Applications of mass spectrometry for quantitation of DNA adducts. AB - DNA adducts are formed when electrophilic molecules or free radicals attack DNA. 32P-postlabeling has been the most commonly used assay for quantitation of DNA adducts due mainly to its excellent sensitivity that allows quantitation at concentrations as low as approximately 1 adduct per 10(9) normal bases. Such methods, however, do not have the specificity desired for accurate and reliable quantitation, and are prone to produce false positives and artifacts. In the last decade, mass spectrometry in combination with liquid and gas chromatography has presented itself as a good alternative to these techniques since it can satisfy the need for specificity and reliability through the use of stable isotope labeled internal standards and highly specific detection modes such as selected reaction monitoring and high-resolution mass spectrometry. In this article, the contribution of mass spectrometry to the quantitation of DNA adducts is reviewed with special emphasis on unique applications of mass spectrometry in the area of DNA adduct quantitation and recent applications with improvements in sensitivity. PMID- 12376139 TI - Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures. AB - It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines. PMID- 12376140 TI - Improved gas chromatographic-mass spectrometric determination of the N methylcarbamoyl adduct at the N-terminal valine of globin, a metabolic product of the solvent N,N-dimethylformamide. AB - A sensitive method for determination of the N-methylcarbamoyl adduct at the N terminal valine of globin, a new metabolic product of the industrial solvent N,N dimethylformamide (DMF), has been developed and validated. The method includes conversion of the adduct by the Edman degradation to 3-methyl-5 isopropylhydantoin (MVH), which is followed by optimized gas chromatographic analysis with mass spectrometric detection at m/z 114. The recovery of MVH from terminal N-methylcarbamoylvaline was determined using a model dipeptide to be 90%. Calibration of the method is done with MVH, employing 3-methyl-5 isobutylhydantoin as the internal standard. The limit of detection is 0.2 nmol MVH/g globin when a 100-mg sample is used. Within- and between-day precision is 4 10%. The method has been used to determine the background levels of MVH in unexposed subjects. Further, toxicokinetic studies in volunteers laid the grounds for setting the reference value for biological monitoring of occupational exposure to DMF. PMID- 12376141 TI - Non-linear production of benzene oxide-albumin adducts with human exposure to benzene. AB - Benzene is initially metabolized to benzene oxide, which either undergoes further metabolism or reacts with macromolecules including proteins. Previously reported levels of benzene oxide-albumin adducts (BO-Alb) are analyzed from 30 workers exposed to 0.2-302 ppm benzene and 43 controls from Shanghai, China. Although both exposed workers and controls had significant levels of BO-Alb in their blood, exposed subjects' adduct levels (GM=378 pmol/g protein) were much greater than those of controls (GM=115 pmol/g protein). When the natural logarithm of the BO-Alb level was regressed upon the natural logarithm of exposure among the 30 exposed subjects, a strong effect of benzene exposure was observed (R(2)=0.612; p<0.0001). Because the slope of the relationship between BO-Alb and benzene exposure was significantly less than one in log-space, we infer that production of benzene oxide was less than proportional to benzene exposure. Since benzene is a substrate for CYP2E1, these results are consistent with saturation of CYP450 metabolism. They indicate that deviations from linear metabolism began at or below benzene exposures of 10 ppm and that pronounced saturation was apparent at 40-50 ppm. To our knowledge, this is the first study to investigate the linearity of human metabolism of a carcinogen based upon protein adducts. PMID- 12376142 TI - Precision and sensitivity of aminobiphenyl hemoglobin adduct assays in a long term population study. AB - Exploratory statistical analysis of aminobiphenyl hemoglobin adduct data obtained in a large-scale population study was performed to assess precision and sensitivity over the 7 years required to conduct the analyses. A time-dependent trend toward higher values was observed that may be attributable to aging of the internal standard used throughout the study. A several-fold improvement in sensitivity from the beginning to end of the study was also noted. Repeated analysis of duplicate blood specimens provided a worst-case estimate of the coefficient of variation to be 0.31, attributable almost entirely to sample preparation rather than instrumental analysis. Substantial variability in calibration curves for the deuterated internal standard (standard deviation was +/-15% of the mean) was observed. The results obtained here will be used in support of further analyses of the data with respect to factors of epidemiological interest. PMID- 12376143 TI - Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route. AB - The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N HPVal measurements in humans, similar binding was found. PMID- 12376144 TI - Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection. AB - There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8 OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used. PMID- 12376145 TI - Quality assurance of biological monitoring in occupational and environmental medicine. AB - Biological monitoring of chemical exposure in the workplace has become increasingly important in the assessment of health risk as an integral part of the overall occupational health and safety strategy. In environmental medicine biological monitoring plays also an important role in the assessment of excessive, acute or chronic exposure to chemical agents. To guarantee that the results obtained in biological monitoring are comparable with threshold limit values and results from other laboratories, the analysis must be carried out with tested and reliable analytical methods and accompanied by a quality assurance scheme. Confounding influences and interferences during the pre-analytical phase can be minimised by recommendations from experienced laboratories. For internal quality control commercially available control samples with an assigned concentration are used. External quality control programs for biological monitoring are offered by several institutions. The external quality control program of the German Society of Occupational and Environmental Medicine has been organised since 1982. In the meantime the 27th program has been carried out offering 96 analytes in urine, blood and plasma for 47 substances. This program covers most of the parameters relevant to occupational and environmental medicine. About 350 laboratories take part in these intercomparison programs. At present, ten German and 14 international laboratories are commissioned to determine the assigned values. The data evaluated from the results of the intercomparison programs give a good overview of the current quality of the determination of analytes assessed in occupational and environmental toxicological laboratories. For the analysis of inorganic substances in blood and urine the tolerable variation ranges from 7.5 to 43.5%. For organic substances in urine the tolerable variation ranges from 12 to 48%. The highest variations (36 60%) were found for the analysis of organochlorine compounds in plasma. The tolerable variations for the determination of solvents in blood by head space gas chromatography range from 26 to 57%. If the recommendations for the pre analytical phase, the selection of reliable analytical methods by the laboratory and the carrying out of adequate quality control are observed, the pre-requisites for reliable findings during biological monitoring are fulfilled PMID- 12376146 TI - Finnish quality assurance programme for biological monitoring of organic solvents. AB - The FIOH quality assurance programme for organic solvents and their metabolites consists of analyses for 2,5-hexadione, phenol, mandelic acid, methylenedianiline, methylhippuric acid, trans,trans-muconic acid and trichloroacetic acid in urine, and creatinine and relative density for standardisation. Four times a year two levels of spiked urine or urine specimens collected from occupationally exposed workers are distributed to the participants in 22 countries. RSD and recovery were studied during 1997-2000. Average RSDs of all participants varied between 23 and 56% and were clearly dependent on the analytical method used and the concentration level of the samples. Since 1997 the target values have been determined in reference laboratories for five of the analytes. Lower RSDs (9-21%) and good recoveries were obtained for all analytes in these laboratories, indicating that good performance can be achieved even in the complex analyses performed in biological monitoring of exposure to industrial chemicals. PMID- 12376147 TI - Dopaminergic system modulation of nociceptive response in long-term diabetic rats. AB - The present study examines the effects of dopaminergic system modulation on nociceptive response time in male diabetic rats. In this study, diabetes was induced by streptozotocin (STZ, 45 mg/kg) in adult male Sprague-Dawley rats. Insulin replacement therapy was initiated 6 weeks after the induction of diabetes for one-half of the diabetic group (1.5-2.5 IU/12 h/rat) and was continued throughout the duration of the study (up to 14 weeks). After 6 weeks of daily insulin replacement therapy, eight rats from each experimental group (STZ diabetic, STZ-diabetic+insulin and nondiabetic control) were injected with either bromocriptine (BROM, 3 mg/kg/12 h), haloperidol (HALO, 1.5 mg/kg/12 h) or vehicle. Nociceptive response was measured by the hot plate (HP) latency test before the induction of diabetes (baseline), every 3 weeks for the first 12 weeks and then on days 5, 9 and 14 of treatment with dopaminergic agents. Animals were sacrificed 3 or 4 days after the last HP test and the brain, blood, spinal cord (SC), pituitary and adrenal glands (AD) were dissected for Met-enkephalin (ME) assay. The results show that nociceptive response of untreated diabetic animals increased gradually and significantly over the duration of this study. Administration of BROM and HALO significantly decreased and increased the nociceptive response, respectively, in all groups. However, the response of the diabetic group was more pronounced than that of the other two groups, especially for those treated with BROM. Daily insulin administration normalized nociceptive response to that of the nondiabetic controls. Diabetic animals receiving insulin replacement+BROM also showed normalized nociceptive response while the diabetic animals+HALO did not. Moreover, the administration of HALO and BROM resulted in an increase and decrease ME concentrations, respectively, in most tissues and brain regions examined. The effect of these dopaminergic agents on ME levels was greater in brain regions and tissues of the diabetic rats than in the diabetic groups receiving vehicle or in the nondiabetic control receiving these two agents. These data suggest that diabetes alters the sensitivity of the dopaminergic receptors and that altered response of the dopaminergic system could be indirectly involved in the modulation of nociception in diabetic rats possibly through the enhancement and/or deactivation of the endogenous Met-enkephalinergic system. PMID- 12376148 TI - Effects of dopamine receptor antagonism with haloperidol on nurturing behavior in the biparental prairie vole. AB - Dopamine (DA) receptor activity in lactating rats is critical for retrieval and licking of pups, whereas its inactivity facilitates quiescent nursing. The role of DA in the maternal behavior of other species and its role in paternal behavior are unknown. This experiment examined the effects of the DA antagonist haloperidol (HAL) on parental behavior in the biparental prairie vole (Microtus ochrogaster). Three days after birth of pups, parental behavior of male and female voles was observed for 30 min beginning 1 h after intraperitoneal injection of 0.1, 0.5, or 2.5 mg/kg of HAL. Controls received the propylene glycol vehicle. Control males were slower to contact pups, licked them more, and quiescently huddled/nursed less than control females. Even at the lowest dose of HAL that had no effect on general activity, pup licking was decreased in both sexes and the latency to contact pups increased in males. The latency to contact pups was most increased in females by the highest HAL dose. Retrieval of pups was not often displayed by any group. HAL dose-dependently decreased the latency and increased the duration of huddling/nursing in both sexes, but did not affect litter weight gains. These data indicate some subtle species differences in the dopaminergic regulation of parenting, as well as sex differences in the sensitivity of some vole parental behaviors to HAL. PMID- 12376149 TI - Effects of ethanol on the accumbal output of dopamine, GABA and glutamate in alcohol-tolerant and alcohol-nontolerant rats. AB - Effects of ethanol on the accumbal extracellular concentrations of dopamine, as well as of the amino acid transmitters gamma-amino butyric acid (GABA), glutamate and taurine, were studied in the alcohol-insensitive (alcohol-tolerant, AT) and alcohol-sensitive (alcohol-nontolerant, ANT) rats selected for low and high sensitivity to ethanol-induced motor impairment. Ethanol (2 or 3 g/kg ip) enhanced the output of dopamine and its metabolites in freely moving rats of both lines as measured by in vivo microdialysis. The effect of ethanol on the metabolites of dopamine tended to be stronger in the ANT rats. The smaller dose of ethanol decreased the output of GABA only in the AT rats, whereas the larger dose of ethanol decreased the output of GABA in rats of both lines to a similar degree. Ethanol at the dose of 2 g/kg slightly, but statistically, significantly decreased the output of glutamate in rats of both lines, but the larger dose of ethanol decreased the output of glutamate only in the AT rats. Ethanol at the dose of 2 g/kg induced a small transient increase in the output of taurine within 2 h after its administration in rats of both lines, but the larger dose of ethanol was without significant effect. These results confirm the previous findings that ethanol suppresses the release of GABA more in the AT than ANT rats. Thus, among the neurotransmitter systems we studied, the effects of ethanol might be the most relevant on GABAergic transmission regarding the sensitivity towards ethanol. However, our findings suggest that glutamate is also involved in this respect. PMID- 12376150 TI - SR-141716A-induced stimulation of locomotor activity. A structure-activity relationship study. AB - The central cannabinoid receptor (CB(1)) antagonist, SR-141716A, has been used extensively to ascertain that cannabinoids interact with the CB(1) receptor. SR 141716A has been shown to produce effects opposite of cannabinoids when administered alone. It has been theorized that SR-141716A may act as an inverse agonist at the CB(1) receptor or by disinhibiting an endogenous cannabinoid tone. In an effort to ascertain the exact interaction between SR-141716A and the CB(1) receptor, we have conducted a structure-activity relationship study to compare CB(1) receptor affinity of SR-141716A analogs with their ability to produce an increase in locomotor activity. SR-141716A produced a significant increase in locomotor activity in mice within the first hour of administration. Twenty SR 141716A analogs from five different chemical series were also tested. Our data implicate particular regions of the SR-141716A molecule that may be involved in stimulation and depression of locomotor activity. When the K(I) of the analogs was plotted against the percent stimulation that each analog produced, it is evident that there is no correlation between the ability of the analogs to stimulate locomotor activity and their affinity for the CB(1) receptor. [35S]GTPgammaS binding data indicate that SR-141716A and five of the analogs are inverse agonists. However, none of the analogs demonstrating inverse agonism produce stimulation of locomotor activity. It is therefore concluded that the SR 141716A-induced stimulation in locomotor activity is not the result of inverse agonist activity at the CB(1) receptor or by disinhibition of an endogenous tone. PMID- 12376151 TI - Ethanol flavor preference conditioned by intragastric carbohydrate in rats. AB - The unpalatable flavor of ethanol solutions greater than approximately 6% may limit their consumption by rats. We determined if ethanol flavor avoidance, like bitter or sour taste avoidance, can be reversed by intragastric (IG) carbohydrate conditioning. Ad lib fed rats drank 5% ethanol and a matched flavor (0.05% citric acid+0.5% maltodextrin, CM) on alternate days. For control rats, postingestive effects were equated: when they drank one solution they were infused IG with the other. Conditioned rats were also infused with 5% ethanol when they drank CM, but when they drank 5% ethanol they were infused with CM + 16% maltodextrin, a potent reward in flavor preference learning. In choice tests, only the conditioned rats preferred ethanol to CM; both groups preferred 5% ethanol to water. Conditioned rats but not controls preferred ethanol to water when the concentration was raised to 10%, and sustained their preference when the infusate carbohydrate was gradually removed. When ethanol concentration was gradually raised to 25%, ethanol preference declined from 48% to 30% in the control rats and from 84% to 50% in the conditioned rats. Thus, ethanol flavor avoidance can be reversed or reduced by postingestive nutritive conditioning, which may combine with the pharmacological effects of ethanol to produce the acquired appetite for the flavor of alcoholic beverages. PMID- 12376152 TI - Intracerebroventricular injection of the antibiotic cefoselis produces convulsion in mice via inhibition of GABA receptors. AB - A majority of beta-lactam antibiotics (e.g., cephalosporins and penicillins) have convulsive activity to a greater or lesser extent. (6R,7R)-3-[[3-Amino-2-(2 hydroxyethyl)-2H-pyrazol-1-ium-1-yl]methyl]-7-[(Z)-2-(2-aminothiazol-4-yl)-2 methoxyiminoacetylamino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate monosulfate (cefoselis), a newly developed injectable beta-lactam antibiotic with activity against methicillin-resistant Staphylococcus aureus (MRSA), might induce convulsions if cerebral concentrations become highly elevated. In the present study, we examined whether or not cefoselis had convulsive activity after direct brain administration, and we attempted to clarify the pharmacological mechanism of action. When cefoselis was injected into the lateral ventricle of the mouse brain at doses higher than 20 microg/animal, it produced convulsions dose dependently. Cefoselis (50 microg/animal)-induced convulsions were prevented by pretreatment with 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), diazepam and phenobarbital (ED(50) values (mg/kg) of 0.78, 1.59 and 33.0, respectively), but not by carbamazepine or phenytoin. When the effects of these anticonvulsants on the convulsions induced by intracerebral injection of bicuculline methiodide (BMI) or N-methyl-D-aspartate (NMDA) were investigated, the inhibitory profile of anticonvulsants on cefoselis-induced convulsions was similar to those induced by BMI (125 ng/animal) but differed markedly in their inhibitory activity on NMDA (100 ng/animal)-induced convulsions, which were not inhibited by diazepam. These results suggest that cefoselis may be convulsive at higher concentrations through a mechanism involving inhibition of gamma aminobutyric acid (GABA)(A) receptors. PMID- 12376153 TI - Fixed ratio discrimination training increases in vivo striatal dopamine in neonatal 6-OHDA-lesioned rats. AB - Massed training in the conditional discrimination task, the fixed ratio discrimination (FRD) task led to elevated extracellular dopamine (DA) concentrations in the neonatal 6-hydroxydopamine (6-OHDA)-treated rat, a model of Lesch-Nyhan disease (LND). Rats neonatally treated with 6-OHDA or its vehicle were, as adults, implanted with microdialysis probes and assessed for basal pretraining concentrations of DA and its major metabolites. Subsequently, microdialysis samples were collected each day following three separate FRD training periods (trained group) or three separate periods of noncontingent food presentations (untrained group). The present study found that there were significant increases in extracellular DA in the caudate-putamen from basal pretraining concentrations in the repeated sample collections of trained 6-OHDA lesioned animals but not in the samples of untrained 6-OHDA-lesioned animals. Consistent with previous studies [Brain Res. 508 (1990) 30.], there was an increase in the extracellular concentrations as compared to tissue concentrations of DA and 3,4-dihydroxyphenylacetic acid (DOPAC). Similar to our previous studies with long-term FRD training [Pharmacol. Biochem. Behav. 51 (1995) 861; Brain Res. 713 (1996) 246.], there was also an indication of an increase in cortical and striatal tissue concentration of DA in the trained 6-OHDA-lesioned animals as compared to the untrained 6-OHDA-lesioned animals. The elevations in striatal DA concentrations following operant performance in the present study illustrate how operant procedures of the behavior therapy used with individuals with LND and other mental retardation syndromes may interact with the modulation of dopaminergic function by the pharmaceutical application of DA antagonists to suppress aberrant behaviors. PMID- 12376155 TI - Locomotor stimulant effects of nornicotine: role of dopamine. AB - Nornicotine (NORNIC) is a tobacco alkaloid and behaviorally active nicotine metabolite in vivo. Previous behavioral research has shown that NORNIC has locomotor stimulant and reinforcing effects in rats similar to that of nicotine. Results from the current study showed that a bilateral lesion of the nucleus accumbens decreased the locomotor stimulant effect of NORNIC across repeated injections. Pretreatment with the dopamine (DA) D1 receptor antagonist SCH23390 did not block the locomotor stimulant effect of NORNIC or the initiation of sensitization following repeated NORNIC administration. The D2 receptor antagonist eticlopride, however, blocked both the stimulant effect and the initiation of sensitization following repeated NORNIC. Additionally, NORNIC was found to increase synthesis and metabolism of DA, with a greater effect in the mesolimbic pathway compared to the nigrostriatal pathway. Taken together, these results suggest that expression of NORNIC-induced locomotor activity is dependent upon ascending dopaminergic mesolimbic projections, providing additional evidence that NORNIC plays a contributory role in tobacco dependence. PMID- 12376154 TI - Dextromethorphan and ketamine potentiate the antinociceptive effects of mu- but not delta- or kappa-opioid agonists in a mouse model of acute pain. AB - Animal and clinical studies have reported potentiation of opioid antinociception by NMDA receptor antagonists such as ketamine and dextromethorphan. The aim of this study was to compare these clinically available NMDA antagonists in combination with classical morphine, mu-selective fentanyl-like opioids, the delta-opioid agonist SNC80 and the kappa-opioid agonist U50,488H. Using a mouse hot-plate test, dose-response relationships were first determined for all compounds individually and then for opioids co-administered with fixed doses of ketamine or dextromethorphan. All compounds were administered intraperitoneally ED(50) values were calculated from the proportion of animals failing to exhibit any response within a fixed cut-off criterion of 30 s. To varying degrees, all compounds produced increases in response latencies over time. Dextromethorphan produced lower ED(50) values for morphine, fentanyl and sufentanil but exerted no effect on the potency of SNC80 or U50,488H. Similarly, ketamine potentiated the antinociceptive potency of morphine, fentanyl and sufentanil but not SNC80 or U50,488H. In summary, these results support the use of mu-opioid agonists in combination with NMDA antagonists, but suggest that there may be no advantage in combining dextromethorphan or ketamine with delta- or kappa-opioids in the management of acute pain. PMID- 12376157 TI - Effects of ovariectomy and estradiol on acoustic startle responses in rats. AB - Long-term (3 months) ovariectomized (OVX) rats were used to model hormone withdrawal as occurring in menopause. We previously reported alterations in brain dopamine (DA), GABA and serotonin receptors following ovariectomy in this model. To assess the functional effect of these biochemical changes, we compared rats that were intact, OVX and OVX-treated with 17beta-estradiol (E(2); OVX+E(2)) for 2 weeks on measures of their acoustic startle responses (ASR) and prepulse inhibition (PPI) of acoustic startle. The effects of a mixed D(1)/D(2) dopaminergic agonist, apomorphine (APO; 0.25, 0.5 and 0.75 mg/kg sc) were tested on ASR and PPI of acoustic startle. Without APO, all groups of rats showed no difference in baseline ASR or PPI of acoustic startle. Following administration of APO (0.25, 0.5 and 0.75 mg/kg), ASR was significantly increased in OVX rats compared to intact rats and this was corrected with E(2) treatment. In all groups of animals, APO decreased PPI of acoustic startle. APO disrupted PPI to a lesser extent in OVX animals with or without E(2) treatment compared to intact rats. However, when group differences in APO-induced ASR were statistically controlled for, there were no longer any differences in APO disruption of PPI among the three treatment groups. These results indicate that long-term ovariectomy has persistent effects on the modulation of ASR, and these effects can be at least partly corrected with E(2) replacement therapy. PMID- 12376156 TI - The effects of acute and subchronic treatment with fluoxetine and citalopram on stimulus control by DOM. AB - Previous reports from our laboratory have provided evidence that acute, i.e., concurrent, treatment with selective serotonin reuptake inhibitors (SSRIs) augments the stimulus effects of indoleamine and phenethylamine hallucinogens in the rat. In the present investigation, the acute effects of fluoxetine and citalopram on stimulus control induced by (-)-2,5-dimethoxy-4-methylamphetamine (DOM) were compared with those following subchronic, i.e., 10-day treatment with the SSRIs. Stimulus control was established using DOM (0.56 mg/kg; 75-min pretreatment time) in a group of 11 rats. A two-lever, fixed ratio 10, positively reinforced task with saline controls was employed. The effects of a range of doses of DOM when given alone were compared with those following both acute and subchronic pretreatment with fluoxetine and citalopram in combination with DOM. It was found that acute administration of fluoxetine and citalopram potentiated the stimulus effects of DOM. Furthermore, it was observed that the degree of potentiation was not diminished by treatment with either fluoxetine or citalopram for a period of 10 days. It is concluded that whatever adaptive changes may take place in response to a 10-day period of treatment with either citalopram or fluoxetine, these adaptations are independent of the mechanisms responsible for the potentiation of the stimulus effects of DOM by the SSRIs. PMID- 12376158 TI - Broad spectrum anticonvulsant activity of BW534U87: possible role of an adenosine dependent mechanism. AB - The novel putative anticonvulsant drug 1-[2,6-difluorophenyl)-methyl]-1H-1,2,3 triazolo[4,5-c]) pyridine-4-amine monohydrochloride (BW534U87) effectively reduced seizures induced in rodents by threshold maximal and supramaximal electroshock, electrical kindling, pentylenetetrazole (PTZ) infusion and by vestibular stimulation in the genetically seizure-prone epilepsy-like (EL) mouse. The range of animal seizure models in which BW534U87 was effective is consistent with a broad spectrum anticonvulsant profile. In the EL mouse, the activity of BW534U87 was partially reversed by predosing with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), suggesting that an adenosine-dependent mechanism contributed to the antiseizure activity of the drug. BW534U87 inhibited rat brain homogenate adenosine deaminase activity, thus, raising the possibility that, by blocking the metabolism of endogenous adenosine by this route, BW534U87 limited seizure activity by promoting the inhibitory tone mediated by endogenous adenosine in the brain. The seizure protection conferred by the selective adenosine deaminase inhibitor erythro-9-(2-hydroxy-3 nonyl)adenine (EHNA) in EL mice and mice infused with PTZ confirms that inhibition of adenosine metabolism by deamination is an effective antiseizure strategy in these models. PMID- 12376159 TI - The nucleus accumbens as a site of action for rewarding properties of testosterone and its 5alpha-reduced metabolites. AB - Testosterone (T)'s positive hedonic effects may be mediated by actions of its metabolites, dihydrotestosterone (DHT) or 3alpha-androstanediol (3alpha-diol), in the nucleus accumbens (NA). In Experiment 1, adult, intact, male rats were systemically administered 1 mg of T, DHT, 3alpha-diol or vehicle, at different time points to examine concentrations of androgens in the NA. Rats administered 3alpha-diol had significantly increased concentrations of 3alpha-diol in the region of the brain encompassing the NA. These data are consistent with previous data from our laboratory demonstrating that 3alpha-diol elicits a conditioned place preference (CPP) more effectively than either T or DHT, when administered systemically. In Experiment 2, rats received implants of T, DHT or 3alpha-diol to the NA immediately prior to placement in the CPP apparatus on conditioning days. Implants of T, DHT or 3alpha-diol, but not vehicle, significantly increased time spent on the non-preferred side of the chamber on the test day. This effect was only produced by androgenic stimulation of the shell of the NA and not the core of the NA. Thus, androgen regimens we have previously found to enhance CPP produced the greatest increases in 3alpha-diol concentrations in the NA region and direct implants of T, DHT or 3alpha-diol to the shell, but not the core, of the NA enhanced CPP. These data are consistent with the hypothesis that the hedonic effects of T may be due to actions of its metabolites in the NA. PMID- 12376160 TI - Tolerance to the disruptive effects of Delta(9)-THC on learning in rats. AB - Tolerance to the effects of the cannabinoid agonist Delta(9)-tetrahydrocannabinol (Delta(9)-THC) was characterized in rats responding under a multiple schedule of repeated acquisition and performance. During the acquisition component, subjects acquired a different three-response sequence each session, whereas in the performance component the sequence was the same each session. Responding was maintained under a second-order fixed-ratio 2 (FR2) schedule of food reinforcement. Acute doses of Delta(9)-THC (1-10 mg/kg) decreased rate and accuracy in both components, whereas doses of the cannabinoid (CB1) receptor antagonist N-(piperidin-1-yn-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl 1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 0.32 and 1 mg/kg) were ineffective. While 5.6 mg/kg of Delta(9)-THC disrupted responding when administered acutely, tolerance to the rate-decreasing and error-increasing effects of this dose developed in both components after daily administration. When 1 mg/kg of SR141716A was substituted for Delta(9)-THC during chronic administration, this previously ineffective dose selectively increased within session errors in the acquisition component of the multiple schedule. During the postchronic phase, subjects were generally less sensitive to the disruptive effects of Delta(9)-THC. In summary, these data demonstrated that tolerance to Delta(9)-THC developed across two different behavioral tasks and that learning was generally more sensitive than performance to the effects of SR141716A during chronic treatment with Delta(9)-THC. PMID- 12376161 TI - Characteristics of flurothyl-induced seizures and the effect of antiepileptic drugs on flurothyl-induced seizures in Mongolian gerbils. AB - We investigated the characteristics of the flurothyl-induced seizures and the effects of antiepileptic drugs on the flurothyl-induced seizure model in a previously untested Mongolian gerbil species. Mongolian gerbils demonstrated tonic extension immediately after or within 1 min after the appearance of clonic convulsion. Very high amplitude spike waves appeared in these regions concurrent with the appearance of clonic convulsion. When the tonic extension appeared immediately after the clonic convulsion, the high amplitude spike waves continued during tonic convulsion. When the tonic extension occurred, high amplitude spike waves appeared in these three regions within a very short time, and afterward Mongolian gerbils died. Administration of valproic acid-Na (200 mg/kg), ethosuximide (100 and 200 mg/kg), clonazepam (2 mg/kg) and diazepam (0.5, 1 and 2 mg/kg) significantly prolonged the latency of clonic convulsion. Zonisamide-Na, phenytoin and carbamazepine, however, had no such effect. In Mongolian gerbils, tonic extension was demonstrated immediately after the appearance of clonic convulsion, yet, this effect was inhibited by all these drugs in a dose-dependent manner. Diazepam completely blocked the appearance of any behavioral changes in animals. These findings suggest that diazepam has a significant effect on flurothyl-induced seizures. Flurothyl-induced convulsions are associated with GABA receptors; hence, benzodiazepine (BDP) suppression may result from the strong relation between BDP and GABAnergic neurons. PMID- 12376162 TI - Delayed stress-induced antinociceptive effect of nitric oxide synthase inhibition in the dentate gyrus of rats. AB - Stimulation of the hippocampal formation can modulate nociceptive mechanisms, whereas painful stimuli can activate this structure. Stress exposure can produce plastic changes in the hippocampus. Nitric oxide (NO) is an important neuroregulatory agent present in the hippocampus. The objective of the present study was to investigate the effects of intrahippocampal administration of N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NO synthase (NOS), on nociceptive processes in stressed and nonstressed rats. Male Wistar rats (n=6-11/group) received unilateral microinjection of L-NAME (50-300 nmol/0.2 microl) into the dentate gyrus (DG) of the dorsal hippocampus. Immediately after the injection tail-flick reflex latency was measured. Stressed animals were submitted to 2 h of restraint and tested immediately or 1, 2, 5 or 10 days later. L-NAME failed to modify nociception in nonstressed rats. However, 5 days after, restraint L-NAME, at all doses tested, produced an antinociceptive effect (ANOVA, P<.05). The dose-response curve had an inverted U shape. L-NAME antinociceptive effect was antagonized by previous treatment with L-arginine (150 nmol/0.2 microl, P<.05). The results suggest that the modulation of nociceptive processes by NO in the dorsal hippocampus is dependent on previous stress exposure and on poststress interval. PMID- 12376163 TI - Central stimulants as discriminative stimuli. Asymmetric generalization between ( )ephedrine and S(+)methamphetamine. AB - Central stimulants readily serve as training stimuli in drug discrimination studies and typically substitute for one another in tests of stimulus generalization regardless of which is used as training drug. We have previously found that, although substitution occurs between (+)amphetamine and (-)ephedrine, substitution did not occur upon administration of S(+)methamphetamine to ( )ephedrine-trained animals. In the present investigation, rats were trained to discriminate S(+)methamphetamine (1 mg/kg) from saline vehicle and tests of stimulus generalization were performed with several stimulants, including ( )ephedrine. The S(+)methamphetamine stimulus (ED(50)=0.06 mg/kg) generalized to R(-)methamphetamine (ED(50)=1.61 mg/kg), S(+)amphetamine (ED(50)=0.28 mg/kg), S( )methcathinone (ED(50)=0.21 mg/kg), methylphenidate (ED(50)=0.28 mg/kg), cocaine (ED(50)=3.68 mg/kg) and (-)ephedrine (ED(50)=13.1 mg/kg). Hence, stimulus generalization between S(+)methamphetamine and (-)ephedrine is apparently asymmetrical. In a companion study, R(-)methamphetamine was administered to rats trained to discriminate (-)ephedrine (4 mg/kg); substitution occurred and R( )methamphetamine (ED(50)=0.92 mg/kg) was found to be nearly equipotent with ( )ephedrine (ED(50)=0.8 mg/kg). Although the exact basis for the observed results are unclear, they are discussed in terms of the different effects of (-)ephedrine and the methamphetamine optical isomers on neurotransmitter release and reuptake. PMID- 12376164 TI - Naltrexone suppresses the late but not early licking response to a palatable sweet solution: opioid hedonic hypothesis reconsidered. AB - Opioid antagonists suppress the intake of sweet solutions, but typically have little effect on the initial rate of drinking. The lack of an early drug response was investigated in the present study because it questions the general idea that opioid antagonists reduce the hedonic response to sweets. The first experiment, which measured the rat's licking response to a sucrose+saccharin (S+s) solution, revealed that naltrexone suppressed S+s intake but not initial lick rates. Experiment 2A indicated that the drug's delayed behavioral effect was not due to the 10-min injection-test interval used. Increasing the interval to 20 min did not reduce the latency of drug action. Experiment 2B tested the idea that rats require several minutes to detect that naltrexone has reduced the hedonic value of the S+s solution. The S+s solution was presented either for 30 min without interruption or for 3 min followed, after a 6-min delay, by another 27-min access. In both test conditions, naltrexone did not suppress S+s licking until 7 9 min of drinking had occurred. However, the drug blocked an "appetizer effect"; a post-delay increase in licking rate produced by the split-session test procedure. Microstructure analysis indicated that in all cases, naltrexone reduced S+s licking by reducing the number of lick clusters rather than lick cluster size. In contrast to these drug effects, Experiment 2C showed that reducing the concentration of the S+s solution decreased initial lick rates. Together, these findings suggest that opioid antagonists do not affect all aspects of flavor hedonics, but may primarily alter the intake-maintaining action of palatable flavors. PMID- 12376165 TI - Effects of oral contraceptives on acute cocaine response in female volunteers. AB - A growing number of recent reports have demonstrated sex and menstrual cycle differences in the subjective, physiological and pharmacokinetic effects of stimulant drugs in humans. The present study was conducted to further investigate the relationship between gonadal hormones and cocaine effects by examining whether oral contraceptives (OCs) alter the acute effects of cocaine. Seven female volunteers, who were taking triphasic OCs and who were occasional users of cocaine, provided informed consent and participated in this placebo-controlled, four-visit study. Subjects were studied twice during days 6-10 of the menstrual cycle (equivalent to the follicular phase) and twice during days 21-28 of the menstrual cycle (equivalent to the luteal phase) and were challenged with an acute dose of intranasal (in) cocaine (0.9 mg/kg or placebo). There were no differences in cocaine-induced subjective, physiologic or plasma cocaine and metabolite levels during the times equivalent to the follicular and luteal phases of the menstrual cycle. Our findings provide evidence that OCs do not present an added risk of cocaine-induced cardiovascular effects and that exogenous administration of estrogen and progesterone at the physiologic doses found in OCs do not alter the subjective responses to acute cocaine. PMID- 12376166 TI - Chronic stress effects on adenine nucleotide hydrolysis in the blood serum and brain structures of rats. AB - We have previously observed that adenosine 5'-diphosphate (ADP) hydrolysis was decreased 25% in spinal cord synaptosomes of chronically stressed male rats, while no changes were observed in ATPase activity. In the present study, we investigated the effect of chronic stress on the hydrolysis of adenine nucleotides in two cerebral structures (frontal cortex and hypothalamus) and in the blood serum of male rats. Adult male Wistar rats were submitted to 1-h restraint stress/day for 45 days (chronic) and were sacrificed 24 h after the last session of stress. Adenosine 5'-triphosphate (ATP) or ADP hydrolysis was assayed in the synaptosomal fraction obtained from the frontal cortex and hypothalamus of control and chronically stressed animals. No effects on ADP or ATP hydrolysis were observed in any of the cerebral structures analyzed after chronic stress. On the other hand, reduced ADP hydrolysis was observed in the blood serum of chronic stressed rats. It is possible that the effects observed in the blood serum may represent an adaptation to chronic stress and may reflect different functions of nucleotides and/or enzymes in these tissues. It is possible that altered levels of ADPase activity in the serum may be a biochemical marker for chronic stress situations. PMID- 12376167 TI - Involvement of dopamine D2 receptors of the central amygdala on the acquisition and expression of morphine-induced place preference in rat. AB - In the present study, the effects of intra-central amygdala (CeA) injections of dopamine (DA) D2-like receptor agonist and antagonist on the acquisition and expression of morphine-induced place preference in male Wistar rats have been investigated. Subcutaneous administration of different doses of morphine sulphate (0.5-10 mg/kg) produced a dose-dependent conditioned place preference (CPP). Using a 3-day schedule of conditioning, it was found that the DA D2/D3 receptor agonist, quinpirole (0.3-3 microg/rat), or the DA D2 receptor antagonist, sulpiride (0.04-5 microg/rat), did not produce a significant place preference or place aversion. Intra-CeA administration of quinpirole (0.3 and 1 microg/rat) with an ineffective dose of morphine (0.5 mg/kg) elicited a significant CPP. On the other hand, quinpirole (0.3 microg/rat) injection into the CeA induced CPP in combination with the lower doses of morphine (0.5 and 2.5 mg/kg), but decreased the response of higher dose (7.5 mg/kg) of morphine. This response of quinpirole was attenuated by sulpiride (0.2 microg/rat). Sulpiride by itself (0.04-5 microg/rat) reduced the acquisition of morphine (7.5 mg/kg)-induced place preference. The administration of the higher dose of sulpiride (1 and 5 microg/rat) or the higher dose of quinpirole (3 microg/rat) during acquisition decreased the locomotor activity of the animals on the testing days. The injection of the low dose of quinpirole (0.3 microg/rat) on the test day reduced the expression of morphine-induced CPP, but the high dose of quinpirole (3 microg/rat) potentiated this expression. The administration of sulpiride (5 microg/rat) attenuated the quinpirole response. The injection of sulpiride (1 and 5 microg/rat) abolished the expression of morphine-induced CPP. It is concluded that the CeA DA D2-like receptors may play an active role in morphine reward. PMID- 12376168 TI - Increase in testosterone metabolism in the rat central nervous system by formalin induced tonic pain. AB - The effects of formalin-induced tonic pain (FITP) on testosterone (T) concentrations in the central nervous system (CNS) and serum were investigated in rats. T was nearly eliminated from the brain and spinal cord 1.5 and 24 h after a single subcutaneous injection (100 microl/rat, sc) of 5% formalin and its levels were similar to that seen following castration. In serum, T concentrations were decreased significantly 1.5 h following formalin injection, but after 24 h, the serum level of T was within normal range. T concentrations in the brain, spinal cord, and serum were not modified 20 min after formalin injection. Pretreatment of rats with finasteride, a 5alpha-reductase (5alpha-R) inhibitor (5 mg/kg, sc) blocked T elimination from the brain and spinal cord by FITP, but it failed to prevent decrease in serum T. However, 3 h after administration of exogenous T (5 mg/kg, sc), FITP did not cause a significant decrease in T levels in the CNS and serum. These results suggest that FITP eliminates endogenous T in the brain and spinal cord by increasing 5alpha-R activity in the CNS. PMID- 12376169 TI - Acute or chronic effects of cannabinoids on spontaneous or pharmacologically induced yawning in rats. AB - Yawning is a reflex or event that is not fully understood. It is controlled by many neurotransmitters and neuropeptides and can be induced pharmacologically by cholinergic or dopaminergic agonists. Amongst their many actions, cannabinoids acting on cannabinoid (CB(1) or CB(2)) receptors can alter cholinergic and/or dopaminergic activity. This study examined the effects of Delta(8) tetrahydrocannabinol (Delta(8)-THC) administered acutely (2.5 mg/kg intraperitoneally [ip], 15 min before test) or chronically (5 mg/kg for 30 days followed by 24 h or 7 days of discontinuation) on yawning induced by pilocarpine, a cholinergic agonist (0, 1, 2, 4 or 8 mg/kg ip), or apomorphine, a dopaminergic agonist (0, 20, 40 or 80 microg/kg subcutaneously [sc]). Acute effects of different doses of Delta(9)-tetrahydrocannabinol (Delta(9)-THC: 0, 0.5, 1.25 or 2.5 mg/kg ip) on yawning induced by pilocarpine (2 mg/kg ip) or apomorphine (40 microg/kg sc) were also investigated. Both pilocarpine and apomorphine produced yawning in a dose-related manner. Acute administration of Delta(8)-THC and Delta(9)-THC significantly reduced yawning induced by both pilocarpine and apomorphine. Chronic administration of Delta(8)-THC did not change yawning induced by either agonist 24 h or 7 days after discontinuation of Delta(8)-THC. However, a high frequency of spontaneous yawning was observed 7 days after Delta(8)-THC discontinuation. These results suggest that cannabinoid agonists inhibited yawning induced by cholinergic or dopaminergic agonists. In addition, the increased frequency of spontaneous yawning following cessation of chronic administration of a cannabinoid agonist may be of importance as a withdrawal sign for these drugs. PMID- 12376170 TI - Chromaproline and Chromaperidine, nicotine agonists, and Donepezil, cholinesterase inhibitor, enhance performance of memory tasks in ovariectomized rats. AB - Chromaproline and Chromaperidine, two recently synthesized and pharmacologically characterized nicotinic agonists, and Donepezil (Aricept), an acetylcholinesterase inhibitor approved for the treatment of memory loss, were evaluated for effects on performance of a visual recognition memory task (object recognition) and a spatial memory task (object placement). Ovariectomized female rats received the drugs chronically via subcutaneous Alzet minipumps. None of the drugs altered activity in the open field or the time spent exploring objects in the field. One week following initiation of treatment, all three drugs enhanced performance of the visual recognition task, but only Donepezil enhanced performance of the spatial memory task. With a longer period of treatment (3 weeks), the nicotinic agonist Chromaproline also enhanced object placement performance. Current results show the memory-enhancing efficacy of Donepezil in two additional memory tasks in rats and suggest that the novel nicotinic agonists, Chromaproline and Chromaperidine, may also be useful new drugs for the treatment of memory impairments/loss. PMID- 12376171 TI - Differential effects of carbamazepine on negatively versus positively reinforced responding. AB - To assess its effects on negatively versus positively reinforced operant behavior, carbamazepine (CBZ) or vehicle was acutely administered to rats. Negative reinforcement baselines consisted of a free-operant avoidance task with 5-s shock-shock and 20-s response-shock intervals. Positive reinforcement baselines consisted of responding for food pellets on a variable interval 30-s schedule. Ascending dose-effect functions were established using CBZ for negatively reinforced responding (vehicle, 25, 50, 100 mg/kg ip) and positively reinforced responding (vehicle, 12.5, 25, 50, 100 mg/kg ip). Negatively reinforced responses and avoided shocks were significantly reduced by CBZ injections at 100 mg/kg. Positively reinforced responses and food pellet deliveries were significantly reduced by CBZ injections at 25, 50, and 100 mg/kg. The results show that CBZ has differential, dose-dependent effects on negatively versus positively reinforced responding. PMID- 12376172 TI - Flavor quality and ethanol concentration affect ethanol-conditioned flavor preferences. AB - A previous report showed that outbred rats acquired preferences for a sweetened conditioned stimulus (CS) flavor paired with intragastric ethanol. To evaluate the role of sweet taste in ethanol conditioning, this study compared training with sweetened and unsweetened flavors. In Experiment 1, nondeprived rats were trained to drink one flavored solution (CS+, e.g., grape) paired with intragastric infusion of 5% ethanol and another (CS-, e.g., cherry) paired with intragastric water on alternate days. The volume of ethanol solution infused was matched to the volume of flavored solution the rats consumed. The sweet group's flavors initially contained 0.2% saccharin, reduced to 0.1%, 0.05%, and 0% over days; the plain group's flavors were unsweetened. The sweet group drank more and self-infused more ethanol during training and its preference for the CS+ over the CS- (without saccharin) exceeded that of the plain group (75% versus 62%). Experiment 2 equated total ethanol intake in rats trained with two combinations of flavor quality and ethanol concentration. The Sweet5 group drank flavors with 0.2% saccharin throughout training and tests and received 5% ethanol when they drank CS+, while the Plain10 group drank unsweetened flavors and the CS+ was paired with 10% ethanol. Despite equal daily ethanol doses, the Sweet5 group strongly preferred the CS+ (89%) while the Plain10 group avoided it (31%). The two groups continued to show opposite CS+ preference profiles even when both were tested with sweet CS flavors and 10% ethanol infusions. Thus, sweet taste contributes to the development of ethanol-conditioned flavor preferences, and this effect is not explained by a simple enhancement of ethanol intake. PMID- 12376173 TI - Focal kappa-opioid receptor-mediated dependence and withdrawal in the nucleus paragigantocellularis. AB - The nucleus paragigantocellularis (PGi) has been hypothesized to play an important role in the development of physical dependence on opioids, including the prototype mu-opioid receptor agonist, morphine, and the mixed agonist/antagonist, butorphanol, which shows selective kappa-opioid receptor agonist activity, in rats. In confirmation of previous work, electrical stimulation of the PGi in opioid-nai;ve rats induced stimulus-intensity-related, withdrawal-like behaviors similar to those observed during naloxone-precipitated withdrawal from dependence upon butorphanol. Novel findings were made in rats surgically implanted with cannulae aimed at the lateral ventricle and the right PGi and made physically dependent by intracerebroventricular infusion of either morphine (26 nmol/microl/h) or butorphanol (26 nmol/microl/h) through an osmotic minipump for 3 days. Two hours following termination of the opioid infusion, microinjections of naloxone (11 nmol/400 nl), a nonselective opioid receptor antagonist, or nor-binaltorphimine (nor-BNI) (3.84 nmol/400 nl), a selective kappa-opioid receptor antagonist, were made into the PGi of morphine-dependent and butorphanol-dependent rats. Discrete PGi injections precipitated withdrawal behaviors, with significant (P<.05) increases noted in the incidence of teeth chattering, wet-dog shakes, and scratching. Composite scores for behavioral withdrawal were significantly higher in nor-BNI-precipitated, butorphanol dependent rats (score=6.8+/-0.6), in naloxone-precipitated, butorphanol-dependent rats (8.9+/-0.8), and in naloxone-precipitated, morphine-dependent rats (11.5+/ 0.9) than in all other groups. Both kappa- and mu-opioid receptor mediated dependence can be demonstrated at the level of a discrete medullary site, the PGi, which further supports a specific role for this nucleus in elicitation of behavioral responses during opioid withdrawal. PMID- 12376174 TI - Metabotropic glutamate receptors. PMID- 12376175 TI - Improvement in neuronal survival after ischemic preconditioning in hippocampal slice cultures. AB - The main goals of the current study were to assess: (a) whether a sublethal ischemic insult could protect the CA1 subregion of the hippocampus in organotypic slices against a lethal ischemic insult; and (b) whether this protection is long lasting as determined with an accurate immunohistochemical neuronal marker, NeuN. Hippocampal slice cultures were grown for 12-14 days in vitro. Slices were exposed either to oxygen/glucose deprivation (OGD) for 45 min (ischemia), or OGD for 15 min (ischemic preconditioning), 48 h prior to 45 min OGD, or were untreated (sham). Cell death was estimated by propidium iodide fluorescence 1 day after OGD and by NeuN immunohistochemistry 7 days after OGD. Image analysis was employed to measure the relative optical density of the NeuN-signal in all groups. After ischemia, damaged neurons were shrunken or lost and NeuN immunoreactivity was reduced. Relative optical density of NeuN (ROD [NeuN]) was 0.193+/-0.015 in control (sham) (n=9). In slices that underwent ischemia, ROD [NeuN] declined to 0.108+/-0.018 (n=5) in CA1 (*P<0.05 ROD [NeuN] in preconditioned slice cultures was 0.190+/-0.037 (76% higher than the ischemia group). Similar results were found after measuring PI fluorescence. In the CA1 sub-region, PI fluorescence was about 13, 47 and 17% in the sham, ischemic and IPC groups, respectively. We suggest that the immunohistochemical approach validates the dye uptake method used in slice cultures and yields quantitative data specific for neurons. We also conclude that the organotypic hippocampal slice model is useful for studying delayed ischemic preconditioning that is maintained for hours or days after the preconditioning event. PMID- 12376176 TI - Inflammatory reactions in human medial temporal lobe epilepsy with hippocampal sclerosis. AB - Many experimental studies suggest that NFkappaB, a transcription factor involved in acute inflammation, and cytokines participate in neuronal excitability and/or glial scar formation in epilepsy. In this report, we looked for the expression of NFkappaB in hippocampi surgically removed in patients with medial temporal lobe epilepsy (MTLE) and hippocampal sclerosis (HS) who had an history of febrile convulsions. We analyzed 18 hippocampi from epileptic patients with MTLE and HS, and we used as control specimens three hippocampi from non-epileptic patients and four hippocampi from patients with cryptogenic MTLE without HS. We used antibodies raised against the NFkappaB-p65 subunit and we identified glial cells with specific antibodies. Hippocampi from patients with MTLE and HS displayed severe neuronal loss surrounded by gliosis in CA1 area and more or less in CA3/CA4 areas. Double immunolabeling showed that reactive astrocytes of lesioned areas over-expressed NFkappaB-p65 (significantly when compared to control specimens). Moreover, some surviving pyramidal neurons in these areas and numerous dentate granule cells were strongly positive for NFkappaB-p65 in cytoplasm and nucleus, whereas control hippocampi showed a faint basal cytoplasmic staining in neurons. These results suggest that in epileptic hippocampi with typical sclerosis, inflammatory processes are chronically active or transiently re-induced by recurrent seizures. Whether NFkappaB over-expression reflects protective or deleterious mechanisms in the epileptic focus remains to be elucidated. PMID- 12376177 TI - FOS and FOSB expression in the medial preoptic nucleus pars compacta of maternally active C57BL/6J and DBA/2J mice. AB - C57BL/6J and DBA/2J inbred mice differ in aspects of maternal behavior and in the morphology of the medial preoptic nucleus (MPO), suggesting a possible association. DBA/2J mice have a compact subnucleus in the MPO, the MPOpc, that is sexually dimorphic and absent in C57BL/6J mice. To determine whether MPOpc cells are activated by maternal behavior, FOS and FOSB immunohistochemistry was performed on brain sections of C57BL/6J and DBA/2J mothers following the return of their pups after a separation of 2 days. In both light and dark phases of the daily cycle, stimulation of DBA/2J mothers evoked an increase in FOS- and FOSB immunoreactivity in the MPOpc. Stimulated C57BL/6J mice, which lack the MPOpc, did not show an increase in cellular activity in the corresponding MPO region. Cells immediately lateral to the MPOpc were activated by pup stimulation, in both strains. These results suggest that MPOpc cells are active during maternal behavior, and that strain differences in maternal behavior are related to anatomical differences in the MPO. PMID- 12376178 TI - Cardiovascular responses and neurotransmission in the ventrolateral medulla during skeletal muscle contraction following transient middle cerebral artery occlusion and reperfusion. AB - We hypothesized that static skeletal muscle contraction-induced systemic cardiovascular responses, and central glutamate/GABA release in rostral (RVLM) and caudal ventrolateral medulla (CVLM), would be modulated by cerebral ischemia. In sham-operated rats, a 2-min tibial nerve stimulation induced static contraction of the triceps surae, evoked pressor responses, increased glutamate in both the RVLM and CVLM, decreased GABA in the CVLM, and increased GABA in the RVLM. In rats with a temporary 90-min left middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion, pressor responses during muscle contractions were attenuated, as were glutamate within the left RVLM and left CVLM. Glutamate within the right RVLM and right CVLM were unaltered and similar to those in sham rats. In contrast, GABA increases during muscle contractions were enhanced in the left RVLM and CVLM but changes within the right CVLM and RVLM were similar to those in sham rats. These results indicate that unilateral ischemia increases ipsilateral GABA/glutamate ratios during muscle contraction in the RVLM. In contrast, opposite changes in ipsilateral glutamate and GABA release within the RVLM and CVLM were observed following a 90-min right-sided MCAO followed by 24 h reperfusion. However, cardiovascular responses during muscle contraction were depressed following such an ischemic brain injury. These data suggest that transient ischemic brain injury attenuates cardiovascular responses to static exercise via modulating neurotransmission within the ventrolateral medulla. PMID- 12376179 TI - Effects of antalarmin, a CRF type 1 receptor antagonist, on anxiety-like behavior and motor activation in the rat. AB - Molecular studies point to a role for the type 1 corticotropin-releasing factor receptor (CRF(1)) in anxiogenic-like and activating effects of CRF and stress. However, CP-154,526, a selective CRF(1) antagonist, has yielded mixed results in such tests. Few studies have examined the behavioral effects of other CRF(1) antagonists. Therefore, we examined the effects of antalarmin, a structurally related analog of CP-154,526, on anxiety-like behavior and motor activation. Antalarmin blocked the anxiogenic-like effect of CRF in the elevated plus maze, without affecting anxiety-like behavior in vehicle-treated animals. Antalarmin decreased spontaneous defensive withdrawal behavior in a novel, brightly illuminated open field. Finally, antalarmin blocked the activating effects of CRF, but not D-amphetamine, without producing motor sedation. These findings indicate that the CRF(1) receptor mediates anxiogenic-like effects of novelty stress and the anxiogenic-like and activating effects of CRF and support the hypothesis that CRF(1) antagonists may be useful for the pharmacotherapy of pathological anxiety. PMID- 12376181 TI - Increment of in vivo binding of [3H]SCH 23390, a dopamine D1 receptor ligand, induced by cyclic AMP-dependent protein kinase in rat brain. AB - The effects of cyclic AMP (cAMP)-related compounds on in vivo [(3)H]SCH 23390 binding to striatal dopamine D(1) receptors were investigated using autoradiography in order to clarify the possible regulation of the cAMP-dependent mechanisms in the in vivo ligand-receptor bindings in the living brain. Intrastriatal infusion of the cAMP analogue, N6,2'-O-dibutyryl-cyclic AMP (db cAMP; 5, 25 and 100 nmol/side) produced a dose-dependent increase of in vivo [(3)H]SCH 23390 binding in conscious rats. This increasing effect of [(3)H]SCH 23390 binding completely disappeared by 6 h after the infusion of db-cAMP. A similar increase of in vivo [(3)H]SCH 23390 binding to striatal D(1) receptors was also observed by intrastriatal injection of 8-bromo-cyclic AMP (8Br-cAMP, 100 nmol/side). Pretreatment with Rp-cyclic AMP triethylamine (Rp-cAMPS, 100 nmol/side), an inhibitor of the cAMP-dependent protein kinase (PKA), completely blocked the increasing effect of [(3)H]SCH 23390 binding induced by db-cAMP. In contrast, in vitro [(3)H]SCH 23390 binding was not significantly altered by intrastriatal infusion of db-cAMP, which indicated that the maximum number of binding sites (B(max)) for D(1) receptors was not changed. The kinetic analysis employed the graphical method indicated that a db-cAMP-induced increase of in vivo [(3)H]SCH 23390 binding was mainly due to an increase in the bimolecular association rate constant (k(on)). These results strongly indicate that the PKA mediated phosphorylation may play a pivotal role in the regulating the in vivo [(3)H]SCH 23390 dopamine D(1) receptor binding in intact rat brain. PMID- 12376180 TI - Urocortin shares the memory modulating effects of corticotropin-releasing factor (CRF): mediation by CRF1 receptors. AB - Intracerebroventricular (i.c.v.) administration of corticotropin-releasing factor (CRF) biphasically affects performance in tests of learning and memory. In the present study, we used CRF, urocortin (Ucn), a recently cloned CRF homologue, and CRF receptor antagonists, to determine which CRF receptor subtype(s) mediate the memory modulating effects of CRF receptor agonists in male Wistar rats. Under difficult learning conditions (massed trials), i.c.v. pretreatment with CRF or Ucn facilitated the acquisition of spatial navigation in the Morris water maze in a non-dose-dependent fashion (optimal doses of 0.1 and 0.03 microg, respectively). Under less difficult learning conditions (spaced trials), both peptides impaired water maze performance. In addition, with i.c.v. posttraining treatment, the peptides were equipotent (1.0 microg) in facilitating the consolidation of passive avoidance learning. The performance-enhancing effects of Ucn in both water maze and passive avoidance paradigms were reversed by i.c.v. pretreatment with D-Phe CRF(12-41) (2.5, 5 microg), a broad CRF(1)/CRF(2) receptor antagonist, or antalarmin (10 microg), a potent, nonpeptide, CRF(1) selective receptor antagonist. Thus, Ucn shares CRF's memory-modulating effects, and these effects appear to be mediated via the CRF(1) receptor. These findings are consistent with the hypothesis that CRF receptor agonists affect performance in tests of learning and memory by increasing arousal. PMID- 12376182 TI - Blockade by magnesium of sodium currents in acutely isolated hippocampal CA1 neurons of rat. AB - The effects of magnesium (MgSO(4)) on sodium currents (Na(+) currents) in freshly dissociated rat hippocampal neurons were studied using the whole-cell patch clamp techniques. MgSO(4) caused a concentration-dependent and voltage-dependent reversible decrease of Na(+) currents. The half-blocking concentration (IC(50)) of MgSO(4) on Na(+) currents was 4.05 mM. But the action was frequency independent. In addition, 4 mM MgSO(4) shifted the steady state activation curve of Na(+) currents toward positive potential (control V(h)=-55.83+/-6.79 mV, MgSO(4)V(h)=-34.15+/-6.18 mV, n=8, P6 cm (P=0.03), lymphatic or vascular invasion (P=0.04), tumour invasion of rete testis (P=0.05), and lower TIL count (P=0.02). On multivariate analysis, statistically significant predictors of risk of relapse were age < or =33 years (hazard ratio (HR) 4.6 (95% confidence intervals (CI): 1.7-12.2)) and tumour diameter >6 cm (HR 2.8 (CI: 1.2-6.5)). Lower TIL count was of borderline statistical significance (HR 1.8 (CI: 0.96-3.44)). The functional role of the lymphocytic infiltrate in testicular seminoma warrants further study. PMID- 12376207 TI - Capecitabine named-patient programme for patients with advanced breast cancer. the UK experience. AB - Following the encouraging results achieved with the oral fluoropyrimidine capecitabine in clinical trials, a named patient programme was initiated in the UK, through which patients with advanced breast cancer were prescribed capecitabine monotherapy. In this programme, patients were treated with the standard dose of oral capecitabine (1250 mg/m(2) twice daily on days 1-14 of a 21 day treatment cycle). Efficacy and safety data were collected and analysed from 102 patients receiving outpatient treatment with capecitabine. All patients had previously received chemotherapy and for the majority (75%) this was in the metastatic setting. In total, 482 treatment cycles were administered, with a median of 4.5 treatment cycles (range 1-22) per patient. Tumour responses were observed in 20 patients (20%), with an additional 47 patients (46%) achieving disease stabilisation. The median time to disease progression was 4.1 months and median overall survival was 7.7 months. The most common treatment-related adverse events were palmar-plantar erythrodysaesthesia (PPE) (36%) and gastrointestinal toxicities (diarrhoea (33%) and nausea (24%)). Dose reductions due to adverse events were required in 33% of patients, but capecitabine was administered without a dose reduction for 90% of cycles. The results achieved with capecitabine in this named-patient programme confirm that, under 'real practice' conditions, capecitabine is active and well tolerated in patients with advanced breast cancer. PMID- 12376208 TI - Psychological impact of genetic testing in women from high-risk breast cancer families. AB - Psychological adjustment in 90 women (30 carriers and 60 non-carriers) who had undergone genetic testing for mutations in BRCA1 and BRCA2 breast/ovarian cancer susceptibility genes was compared with that of 53 women who were not offered genetic testing. Women were assessed prior to genetic testing and 7-10 days, 4 and 12 months after carrier status disclosure using self-administered questionnaires. Compared with women not offered testing, mutation carriers had significantly higher breast cancer distress 7-10 days (t=2.80, P=0.005) and 12 months (t=2.01, P=0.045) post-notification. Non-carriers showed a significant decrease in state anxiety 7-10 days post-notification (t=2.27, P=0.024) and in depression 4 months post-notification (t=2.26, P=0.024), compared with women not offered testing. These data show that non-carriers derive psychological benefits from genetic testing. Women testing positive may anticipate a sustained increase in breast cancer distress following disclosure, although no other adverse psychological outcomes were observed in this group. PMID- 12376209 TI - Population mixing, childhood leukaemia, CNS tumours and other childhood cancers in Yorkshire. AB - We tested the hypothesis that variation in population mixing attributable to the diversity of migrants moving to an area is associated with the incidence of childhood leukaemia and other childhood cancers. An ecological analysis was performed on 954 children (<15 years) diagnosed with a malignancy between 1986 and 1996 in 532 electoral wards in Yorkshire, UK. Incidence rate ratios (IRR) were calculated for all childhood leukaemias (n=325), acute lymphoblastic leukaemia (ALL) (n=248), central nervous system (CNS) tumours (n=236) and other solid tumours (n=393) Incidence of all childhood leukaemias was significantly lower in areas of high (top decile) population mixing (IRR 0.72, 95% Confidence Interval (CI) 0.54-0.97) and higher in areas of low (bottom decile) population mixing (IRR 1.56, 95% CI 0.73-3.34), but similar patterns of incidence were not observed for central nervous system or other solid tumours. Population mixing may be a proxy for the range of infections circulating in a community and these results are consistent with the hypothesis that greater exposure to infections reduces the risk of developing childhood leukaemia by conferring efficient modulation of the immune system. PMID- 12376210 TI - Age-specific differences in treatment and survival of patients with cervical cancer in the southeast of The Netherlands, 1986-1996. AB - Age at diagnosis has been proven to be an important determinant of the choice of initial treatment for several sites of cancer. Elderly patients are more likely to receive no treatment or less intensive treatment modalities. This study analysed the influence of age on treatment choice and survival in patients diagnosed with cervical cancer. This population-based study used data on 1176 new cases of invasive cervical cancer diagnosed in the period of 1986-1996 from three regional cancer registries in the Netherlands. All available information on treatment and survival (on 1 January 1998) was recorded. Relative survival rates were calculated according to the Hakulinen method. Relative risks (RR) for excess mortality due to the diagnosis of cervical cancer were calculated with a regression model for relative survival rates. Only 5% of the patients aged 70 years and older (n=224) were diagnosed with stage IA disease, compared with 11 and 30% of the patients aged 50-69 years and 49 years and younger, respectively. Almost 50% of the 70+ patients with stage IB-IIA were treated with radiotherapy as a single treatment modality, whereas 64% of the patients aged < or =49 years were treated with surgery alone. In all age groups, treatment for advanced stage disease (stage > or =IIB) was radiotherapy alone. No treatment was given to 10% of the patients aged 70 years and older, 5% of those aged 50-69 years and 1% of those aged 49 years and younger. Five-year relative survival was 69% (95% Confidence Interval (CI): 66-72%) and differed significantly (P=0.001) with age (70+ years: 49%; 50-69 years 58%; < or =49 years: 81%). Multivariate analyses on a subset of patients showed that age was not an independent prognostic factor, whereas stage and treatment modality were very important prognostic factors. Although elderly cancer patients were sometimes treated differently from younger patients, this was in accordance with the guidelines. Relative survival rates differed significantly by age. The multivariate analyses on the subset of patients also revealed that excess mortality increased with age. However, when adjustment was made for stage and treatment, this difference disappeared. The influence of treatment on survival is likely to be due to the selection of patients based on other characteristics, such as tumour volume, comorbidity and performance status. PMID- 12376211 TI - Expression and mutation analyses of MKK4, a candidate tumour suppressor gene encoded by chromosome 17p, in human gastric adenocarcinoma. AB - Homozygous deletion or somatic mutations of mitogen-activated protein kinase kinase 4 (MKK4), a candidate tumour suppressor gene located at 17p11, have been observed in many types of human tumours. To explore the likelihood that MKK4 acts as a suppressor in gastric tumorigenesis, we examined the expression and mutation status of MKK4 in 144 gastric tissues and cell line specimens. Expression of the MKK4 transcript was easily detectable in all normal and benign tumour tissues and none of 102 primary carcinomas and cell lines showed an abnormal reduction in MKK4 expression. Expression levels of MKK4 transcript showed no cancer-specific reduction in 43 matched sets and did not correlate with stage, grade and histopathological types of the tumours. Western blot analysis also revealed that MKK4 protein expression in carcinoma tissues and cell lines is comparable to non cancerous tissues. A significant loss of heterozygosity (LOH) was detected at telomeric markers of the MKK4, locus. However, no allelic deletion of the MKK4 gene or at the centromeric loci was identified. Moreover, no evidences for somatic mutations leading to amino acid substitutions or frameshifts of MKK4 were identified in the carcinoma tissues and cell lines, whereas a substantial fraction of the same set showed allelic loss or mutations of the TP53 gene located at 17p13, suggesting that LOH at telomeric loci or the TP53 locus might not extend into the MKK4 gene in gastric cancers. In this study, we also report the identification of a highly conserved MKK4 processed pseudogene, which shares 95% homology with the coding region of the functional MKK4 transcript. Collectively, our data demonstrate that genomic deletion or somatic mutation of MKK4 is infrequent in gastric cancers, suggesting that MKK4 might not be a critical target of genetic inactivation in gastric tumorigenesis. PMID- 12376212 TI - Insulin-like growth factor binding protein-6 inhibits neuroblastoma cell proliferation and tumour development. AB - In neuroblastoma cells, survival and proliferation are dependent upon the insulin like growth factor (IGF) system. IGFs actively participate in cell growth, whereas IGFBP-6, is associated with the arrest of growth. With a view to blocking IGF-II action, we produced recombinant human IGFBP-6 capable of binding IGFs with affinities between 1.23 and 6.36 x 10(9) M(-1). Ex vivo mitogenic activities were tested on two human neuroblastoma cell lines, in which 100 ng/ml IGFBP-6 completely abolished the effects of both endogenous and exogenous IGF-II. In vivo, nude mice previously injected with neuroblastoma cells were submitted to either 15 daily injections of 4-20 microg IGFBP-6 or implantation of mini-pumps diffusing 20-100 microg IGFBP-6 over 2 weeks. The result was an average 18% reduction in the incidence and development of tumours. Delivery of the IGFBP-6 via mini-pumps also delayed tumour appearance by 6-15 days. Our results therefore show the involvement of IGFBP-6 in neuroblastoma cell growth, both ex vivo in terms of proliferation and in vivo in terms of tumour development. PMID- 12376213 TI - Are sentinel lymph node mapping and resection applicable to chinese breast cancer patients? PMID- 12376214 TI - Paediatric renal transplantation--a 15 year experience. AB - OBJECTIVE: To review the outcome of paediatric renal transplantation over a period of 15 years in a developing country. METHODS: This is a retrospective study of 63 children, less than 15 years of age, who underwent living-related renal transplantation in Christian Medical College and Hospital Vellore between 1984 and 1996. RESULTS: The records of 12 patients were not adequate for detailed analysis. Parents were the donors for these children in 84.3% of cases. The most common known cause of end-stage renal disease in these children was reflux nephropathy. Combinations of cyclosporine, azathioprine and prednisolone were used as immunosuppressive drugs. Complications occurred in 16 patients. During the follow-up period, eight patients died and two returned to receiving haemodialysis. Patient survival was 92% at the end of 1 year and 90% at the end of 3 years. Graft survival was 88% and 86% at 1 and 3 years, respectively. CONCLUSION: Our study validates the concept of renal transplantation as optimal therapy with adequate medical, social and functional rehabilitation for children with end-stage renal disease. Our study also indicates that vesicoureteric reflux appears to be underdiagnosed and should be actively pursued to prevent complications. PMID- 12376215 TI - Induction of higher expression of IL-beta and TNF-alpha, lower expression of IL 10 and cyclic guanosine monophosphate by pulmonary arterial hypertension following cardiopulmonary bypass. AB - OBJECTIVE: Pulmonary arterial hypertension [PAH] and cardiopulmonary bypass [CPB] induce systemic inflammatory cytokines that are critical factors related to postoperative mortality of open heart surgery. We studied the expression of proinflammatory cytokines and cyclic guanosine monophosphate [cGMP] in patients suffering from PAH after CPB. METHODS: Seventy-six patients who underwent valve replacement surgery were recruited and divided into two groups according to their pulmonary arterial pressure [< 50 mmHg for Group A and > or = 50 mmHg for Group B]. Blood samples were taken to measure the concentrations of interleukin-1 beta [IL-1 beta], tumour necrosis factor alpha [TNF-alpha], interleukin-10 [IL-10] and cGMP. RESULTS: IL-1 beta and TNF-alpha were significantly higher in Group B [28.6 +/- 9.1 mmHg] than in Group A [65.8 +/- 10.2 mmHg] at baseline. After CPB, IL-1 beta of both groups rose significantly, while only TNF-alpha of Group B rose significantly higher. There were significant differences between the two groups after CPB. IL-10 and cGMP in Group B were lower than in Group A at baseline. They all decreased significantly after CPB. Significant differences were seen between the groups after CPB. CONCLUSION: Patients suffering from PAH had different levels of proinflammatory and anti-inflammatory cytokines compared to normal patients. PAH aggravates the production of IL-1 beta and TNF-alpha, while it decreases the production of IL-10 and cGMP after CPB. PMID- 12376216 TI - Rapid diagnosis of fungal infection in patients with acute necrotizing pancreatitis by polymerase chain reaction. AB - OBJECTIVE: This study was conducted to assess the rapid diagnosis of fungal infections in patients with acute necrotizing pancreatitis by polymerase chain reaction [PCR] using universal primers targeting the 18S rRNA gene. METHODS: In this study, a PCR assay was developed to identify clinically isolated fungi, and both PCR technique and conventional culture were used to detect fungi in 37 samples from patients with acute necrotizing pancreatitis. RESULTS: A 197-bp fragment was amplified by PCR from all the clinically isolated fungal strains. This fragment was not isolated from gram-positive, gram-negative bacteria or human leucocytes. Thirty-seven samples of necrotic tissue or peripancreatic fluid from 11 patients were also analyzed, and eight samples were positive for fungi by PCR, six of which were also positive by conventional culture. The whole PCR procedure was completed within 7 hours. CONCLUSION: PCR can be used to diagnose fungal infection secondary to acute necrotizing pancreatitis rapidly and sensitively. PMID- 12376217 TI - Rapid diagnosis of fungal infection in patients with acute necrotizing pancreatitis by polymerase chain reaction. PMID- 12376218 TI - Outcome of percutaneous nephrostomy for the management of pyonephrosis. AB - OBJECTIVE: The aim of this study was to evaluate the efficacy of percutaneous nephrostomy (PCN) drainage for the interim management of pyonephrosis. METHODS: Ninety-two consecutive patients [29 men, 63 women; mean age, 57 years; range, 23 to 88] who underwent PCN for the treatment of pyonephrosis from 1996 to 1999 were evaluated retrospectively. The clinical presentation, bacteriology and patient outcomes were analyzed. RESULTS: The majority [77%] of patients had underlying obstructing urinary calculi. Other causes of obstruction included strictures [9%], papillary necrosis [7%], pelvi-ureteric junction obstruction [4%] and malignant stricture [3%]. The microorganisms cultured were Escherichia coli [30%], Klebsiella [19%], Proteus [8%], Pseudomonas [5%], Enterococcus [5%], and Candida spp [5%]. The microorganisms were sensitive to gentamicin [79%], ceftriaxone [71%], cephalexin [54%], nitrofurantoin [40%], cotrimoxazole [35%], nalidixic acid [32%] and ampicillin [29%]. Only 30% of bladder urine cultures were positive for microorganisms; the addition of PCN cultures improved this yield to 58%. The antibiotic regimen was revised according to the PCN culture whenever there was a discrepancy. After PCN, 69% of patients underwent minimally invasive procedures as definitive treatment of the obstructing lesion. Only 14% of patients required open surgery. There was low procedure-related morbidity [14%] and low overall mortality [2%]. CONCLUSIONS: PCN cultures yield important bacteriological information. The procedure is associated with minimal morbidity, facilitates definitive treatment and provides therapeutic benefit. PMID- 12376219 TI - Mucin histochemical analysis of the ileocaecal valve and lymphoid tissues of the terminal ileum: role against tumour invasion. AB - OBJECTIVES: We previously reported clinicopathological data on 78 patients who underwent a right hemicolectomy from 1990 to 1997. Our results indicated that the ileocaecal valve [ICV] and lymphoid tissue of the terminal ileum might, together, play a protective and local immune role against carcinoma invasion. Furthermore, we previously reported that mucin histochemical features of the transitional zone [TZ] might also play a role in predicting metastasis and, thus, prognosis. The aim of this study was to examine the clinicopathological correlation between lymphoid infiltration and mucin secretion in the terminal ileum with carcinoma of the right colon. METHODS: According to the proximity of the tumour to the ICV, a total of 16 specimens with lymphoid infiltration to and around [< 1 cm] the ICV were studied in order to identify the mucin expression and histochemical features of the TZ as a prognostic indicator. RESULTS: Patients with sulphomucin-staining tumours in the terminal ileum and ICV had a relatively favourable course. Even when the clinical staging was the same for different tumours, greater lymphoid infiltration in the ICV, greater staining for sulphomucin in the ICV and a relatively favourable course were observed in nine patients. The sulphomucin-type TZ showed a favourable course as well. On the other hand, patients with sialomucin staining of the ICV and the TZ tended to have low-grade lymphoid infiltration and a very poor course, although two patients with moderately high grade lymphoid infiltration had a favourable course. Overall survival was significantly associated with the mucin type of the ICV [p < 0.01]. CONCLUSIONS: Our results indicated that lymphoid infiltration of the terminal ileum may lead to an alteration in mucin secretion and, thus, play a protective role in the invasive and metastatic process of advanced right colon carcinoma. PMID- 12376220 TI - The cricothyroid space: a guide for successful thyroidectomy. AB - OBJECTIVE: The frequent complications of thyroid surgery are mostly related to the anatomy of the region. This stimulated us to look for a starting point that makes exploration of the region easier and consequently reduces complications. We aimed to explore and define the anatomy of the cricothyroid [CT] region from cadaveric dissection and to present the outcome of 73 consecutive thyroidectomies starting from a space in the CT region. METHODS: Dissection in the thyroid gland region and creating a space in the CT region was performed on five cadavers [10 spaces], followed by 73 consecutive thyroidectomies through a standard approach beginning from the CT space. RESULTS: In all cadavers, a space was easily created in the CT region. Vessels, nerves and the parathyroid glands were identified. Standard thyroidectomy starting from the CT space was performed on 73 patients. The external laryngeal nerve was seen in 40% of the cases. The recurrent laryngeal nerve was identified and preserved in all patients. Six patients had temporary hypocalcaemia and eight had a temporary voice change. None of the patients had permanent hypoparathyroidism or recurrent laryngeal nerve palsy. CONCLUSION: The CT space is an avascular space medial to the thyroid lobe and is a good starting point for thyroidectomy that allows easy and safe exploration of the region. PMID- 12376221 TI - Intravesicle formalin instillation with a modified technique for controlling haemorrhage secondary to radiation cystitis. AB - OBJECTIVE: Intractable haemorrhage, secondary to radiation cystitis, is a serious complication of radiotherapy for pelvic malignancies. Formalin instillation is often effective for intractable haemorrhage unresponsive to other agents, but carries the risk of significant morbidity. The placement of formalin-soaked pledgets is a modified technique for the treatment of this complication. We compare the effectiveness and complications of both techniques. METHODS: Eleven patients with intractable haemorrhage secondary to radiation cystitis were treated by intravesicle 4% formalin instillation [Group I] and eight were treated by the endoscopic placement of 10% formalin-soaked pledgets on the bleeding points for 15 minutes [Group II]. RESULTS: Cessation of bleeding was 9 of 11 [82%] and 6 of 8 [75%] in Group I and Group II, respectively. One patient in Group II required two treatments, due to recurrent haemorrhage. Four major and several minor complications were found in Group I, and only three minor complications were found in Group II. CONCLUSION: Formalin instillation is effective in controlling severe bladder haemorrhage after radiation of the pelvis, but the complications secondary to the fixative properties are severe. Topical application of formalin-soaked pledgets is as effective in controlling the haemorrhage as conventional intravesicle formalin instillation, with fewer complications. This technique should be the initial treatment for this complication. PMID- 12376222 TI - Purse-string closure of a mucous fistula in loop colostomy. AB - The technique of purse-string closure of a mucous fistula to prevent faecal contamination during loop colostomy is described in six patients who underwent the procedure. Complete faecal diversion was achieved in all six patients without complications. PMID- 12376223 TI - Improved detection rate of prostate cancer using the 10-core biopsy strategy in Singapore. AB - OBJECTIVES: To evaluate if changing the biopsy regime to 10 cores might improve the positive predictive value (PPV) of elevated prostate-specific antigen [PSA, elevated range, 4 to 20 ng per ml, normal range, < 4 ng per ml] for the diagnosis of prostate carcinoma. METHODS: From February 2000 to April 2001, 191 patients, mean age 64 years [range, 38 to 85 yr], underwent transrectal ultrasound [TRUS] for either elevated PSA [elevated range, 4 to 20 ng per ml] and/or abnormal digital rectal examination [DRE]. A 10-core TRUS-guided biopsy of the prostate was performed. This included the standard sextant biopsy and two additional cores for each far lateral zone. RESULTS: Using this technique, 47 out of 191 patients [24.6%] had prostate cancer. The PPV for PSA levels of 4.1 to 10.0 ng per ml and 10.1 to 20.0 ng per ml were 19.3% and 35.4%, respectively. The lateral cores contributed 21.3% of the cancer cases, which would have been missed if only sextant biopsies were performed. CONCLUSIONS: With the 10-core biopsy method, the PPV for prostate cancer for patients with a PSA in the range of 4 to 20 ng per ml was in the range of 25%. This is significantly different from previous reports. The reason for this may be due to the adoption of a better, more uniform and systematic biopsy strategy for patients with elevated PSA, or it may be a true reflection of the current population incidence. Hence, this biopsy strategy is highly recommended. PMID- 12376224 TI - Medical management of abdominal compartment syndrome: case report and a caution. AB - We report the case of a 55 year old woman who developed abdominal compartment syndrome [ACS] following total gastrectomy for caustic ingestion. Contributing factors for the development of ACS included peritonitis and massive fluid resuscitation for cardiovascular support of septic shock. The adverse cardiovascular and pulmonary effects of intra-abdominal hypertension [IAH] were reversed with pharmacological neuromuscular blockade [NMB]. Surgical decompression of ACS was, therefore, postponed, but the patient required re operation for intra-abdominal sepsis several days later and subsequently died. Although medical management of ACS with NMB may lower IAH and reverse its negative cardiopulmonary effects, surgical decompression may still be required for definitive treatment. PMID- 12376225 TI - Vascular complications of central venous line insertion. PMID- 12376226 TI - Hepatic angiomyolipoma mimicking hepatocellular carcinoma. AB - Angiomyolipoma [AML] is a rare benign lipomatous tumour of the liver. It is typically echogenic on ultrasound, hypodense on computed tomography and hyperintense on magnetic resonance imaging. Its varied imaging appearance is due to the different proportion of the three cell types which make up the tumour. This is a case report of a hepatic AML mimicking hepatocellular carcinoma [HCC] with fatty change in a hepatitis B carrier. Diagnostic difficulty and implications on subsequent management are discussed in the context of an endemic region for HCC. PMID- 12376228 TI - Current status of liver transplantation--an Asian perspective. PMID- 12376227 TI - Strategies in the management of mid and distal rectal cancer with total mesorectal excision. AB - In the last two decades, dramatic improvement in outcome has been made in the management of rectal cancer. This has been brought about mainly by advancements in surgical technique for radical resection. With the recognition of the importance of the circumferential margin and presence of spread in the lymphovascular tissues in the mesorectum, total mesorectal excision is now commonly recognized as the optimal surgical technique for cancer of the mid and distal rectum. Not only have local control and disease-specific survival improved with the practice of total mesorectal excision, but various bodily functions have also been preserved following surgery for rectal cancer. New issues have arisen with the practice of total mesorectal excision and the strategies for management of rectal cancer require re-evaluation. In this article, the rationale and the outcomes of total mesorectal excision are reviewed. Issues such as the high anastomotic leakage rate following sphincter-preserving surgery, the poor results of abdominoperineal resection, the role of adjuvant therapy and bowel function disturbances will be addressed. Lastly, the status of the laparoscopic approach to rectal cancer with the principle of total mesorectal excision are discussed. PMID- 12376229 TI - How teaching should be conducted in an IT era: back to the future. PMID- 12376230 TI - Open tension-free mesh repair for adult inguinal hernia: eight years of experience in a community hospital. AB - OBJECTIVE: The aim of this study was to determine the feasibility of using open tension-free mesh repair for adult inguinal hernias performed by resident surgeons under the supervision of a chief surgeon in a community hospital. METHODS: From May, 1992 through April, 2000, we performed 314 open tension-free mesh repairs on 289 patients (234 men, 55 women) with a mean age of 65.7 years. There were 173 right and 141 left hernias, and 25 were bilateral; while 220 were indirect, 77 were direct and 17 were of the femoral type. There were 281 primary and 33 recurrent lesions. Resident surgeons under the supervision of the first author (SY) performed all hernioplasties. Three types of open tension-free mesh repairs were performed; the Lichtenstein repair (n = 72), the mesh-plug repair (n = 134), and the Hernia System repair (n = 108). RESULTS: The duration of surgery averaged 73.0 minutes. There was no perioperative mortality. Five patients developed subcutaneous wound infections; no case required mesh removal. Hematoma occurred in eight patients, and seroma developed in 25. All haematomas and seromas subsided with repeated aspiration. The average duration of hospitalization was 6.5 days. The length of follow-up rose from 1 to 8 years, with a mean of 3.7 years. No patients in any group had a recurrence during the follow-up period. CONCLUSIONS: Under the close supervision of the staff surgeon, tension-free hernioplasties can be performed on adult inguinal hernias by surgeons-in-training in non-specialist centres with excellent outcomes, low postoperative complications and no recurrence. PMID- 12376231 TI - Clinical significance of 99mTc-MIBI breast imaging in the diagnosis of early breast cancer. AB - OBJECTIVE: To find an effective, sensitive, specific and noninvasive diagnostic method for cancer. METHODS: 109 masses from 102 patients with breast lesions smaller than 2 cm in diameter were divided into three groups to undergo 99mtechnetium-methoxyisobutylisonitrile (99mTc-MIBI) imaging. The results were compared with their pathology. Twenty cases without breast lesions were selected as a control group. Abnormal density of 99mTc-MIBI in the breast and a threshold level 10% higher than that in the counterpart of the healthy breast was regarded as positive. RESULTS: Of 32 breast cancers, positive imaging appeared in 25. Negative imaging was found in 31 of 38 benign breast lesions. Of 39 nonpalpable breast lesions, five cases were breast cancers and 34 cases benign. Positive MIBI imaging appeared in all of the breast cancers, while in the benign lesions, four were positive and 30 negative. No positive imaging was found in the control group. The diagnostic accuracy, sensitivity, specificity, positive predictive value and negative predictive value of 99mTc-MIBI were 88.4%, 89.2%, 88.0%, 75.0% and 95.3%, respectively. CONCLUSIONS: 99mTc-MIBI imaging had high sensitivity and accuracy in the diagnosis of breast cancer, as well as in the differentiation between benign and malignant breast lesions. It could provide reliable information in confirming the diagnosis in patients with clinically suspected breast cancer. PMID- 12376232 TI - Endoscopic treatment of benign ureteral strictures. AB - BACKGROUND: The traditional choice of procedure for treatment of ureteral stricture is open surgical repair. Advances in endourology have provided the urological surgeon with an alternative to open surgery for the treatment of benign ureteral stricture. METHODS: Twenty-seven benign ureteral strictures in 24 patients were treated by the endourological method. Twelve endoureterotomies were performed using a cold knife via a 9.5Fr Storz ureteroscope and 15 high pressure balloon dilations were performed. The ureters were stented with 7 Fr double-J stents for 6 weeks. RESULTS: The success rate was 9/12 (75%) in the endoureterotomy group and 9/15 (60%) in the balloon dilation group after follow up for more than 6 months. CONCLUSIONS: Endoscopic treatment of ureteral strictures appeared to be a safe and reasonably effective modality for the treatment of ureteral strictures, especially for the short type that are non ischaemic in origin and not associated with radiation therapy. Endourological treatment of ureteral strictures is the procedure of choice for initial management of benign ureteral strictures and has high success rates and fewer complications. PMID- 12376233 TI - Transperineal end-to-end anastomotic urethroplasty for traumatic posterior urethral disruption and strictures in children. AB - OBJECTIVE: To report the long-term results of transperineal end-to-end anastomotic urethroplasty for post-traumatic posterior urethral stenosis in children. METHODS: From 1975 to 1996, 25 boys [aged 3 to 12 years] with post traumatic posterior urethral stenosis or obliteration, and one boy [aged 7 years] with disrupted posterior urethra were treated with transperineal end-to-end anastomotic urethroplasty. Final follow-up assessments including voiding status, urinary continence and erectile function were performed in June 1999. RESULTS: Smooth voiding was restored in 25 boys postoperatively. one child failed an ill prepared repair and was waiting for further intervention. Among the 25 patients, seven were lost to the final follow-up. All seven boys had a single urethroplasty for simple urethral stenosis and had been followed for 3 to 5 years postoperatively with smooth voiding. The other 18 boys, including seven with complex urethral stenosis [three with a history of failed previous urethroplasties, three with urethrorectal fistula and one with urethroperineal fistula], underwent a total of 22 end-to-end anastomotic urethroplasties [one successful primary repair, 17 successful delayed repairs and four failed repairs]. Of the 17 patients with successful delayed repair, 14 succeeded with one repair, two with two repairs and one with three repairs. The success rate per repair for simple urethral strictures was 94.7% [18 of 19], and for complex strictures 63.6% [7 of 11]. Stress incontinence was found in three cases, impotence in two. Concomitant impotence and stress incontinence were found in one of the five patients. CONCLUSION: Transperineal end-to-end anastomotic urethroplasty can achieve good long-term outcomes in children with simple post traumatic posterior urethral stenosis. In experienced hands, good results can also be achieved for complex urethral strictures. PMID- 12376234 TI - Surgical management of phaeochromocytoma. AB - OBJECTIVES: Phaeochromocytoma is a rare tumour, which is benign but metabolically functional, with a potential for malignancy. Surgical resection is the primary treatment. The purpose of this study was to identify the presenting features, diagnostic tests and appropriate surgical approaches for phaeochromocytoma. METHODS: Retrospective analysis was performed on 26 patients with phaeochromocytoma who were admitted to General Surgery, Department of Postgraduate Institute of Medical Education and Research, Chandigarh, India from 1993 to 1999. RESULTS: Of the 26 patients, 15 were males and 11 were females. Their ages ranged from 13 to 60 years, with a peak phaeochromocytoma incidence in the second decade of life. Two patients had phaeochromocytoma as a manifestation of multiple endocrine neoplasia type 2 syndrome (MEN-2). Hypertension was present in 92% of patients. Six (23%) patients had extra-adrenal phaeochromocytoma. The incidence of bilateral tumour was 25% of adrenal tumour and 11.5% had malignant phaeochromocytoma. The diagnostic measurements were: 1) plasma catecholamines, 2) 24-hour urinary vanillylmandelic acid and 3) fractionated urinary catecholamines. Computed tomography of the abdomen was extremely accurate in diagnosing phaeochromocytoma (92% accuracy). Magnetic resonance imaging was performed in 2 patients with 100% accuracy. Preoperatively, all the patients were put on alpha adrenergic blocking agents to control blood pressure for varying periods of time. The transabdominal midline approach was used in all cases with excellent results. There was no surgical mortality. All the resected specimens were subjected to histopathological examination and phaeochromocytoma was subsequently confirmed. CONCLUSION: If preoperative diagnosis and localization of phaeochromocytoma is accurate, surgical resection can be done with minimal morbidity and mortality. PMID- 12376235 TI - Intestinal obstruction due to tuberculosis. AB - OBJECTIVES: Intestinal obstruction due to tuberculosis is a rare form of mechanical bowel obstruction. The objectives of this study were to determine the clinical features, to evaluate the role of surgery and to choose procedures in management of this disease. METHODS: In this 7-year retrospective study (from 1992 to 1998), 23 patients (20 males, three females) were included, accounting for 4.5% of all mechanical intestinal obstructions. More than 80% of the patients had a clinical picture of lower small bowel obstruction, while 90.5% of patients had advanced pulmonary tuberculosis. RESULTS: In 54.6% of cases, obstruction occurred in the ileocaecal region. The main lesion causing obstruction was intestinal tuberculosis in the hypertrophic form (86.4%). Diagnosis of intestinal tuberculosis as a cause of obstruction was not easy because it has no specific symptoms and signs. CONCLUSION: In terms of management, ileocolostomy was often used (68.2%) but long-term results were not very good. Blind loop syndrome was one of its disadvantages. Resection may be the safe and effective procedure. PMID- 12376236 TI - Allopurinol plus pentoxifilline in hepatic ischaemia/reperfusion injury. AB - OBJECTIVES: Ischaemia/reperfusion injury of the liver is the major cause of liver dysfunction and cellular death in transplantation and in liver resection with hepatic pedicle clamping. Many agents are used to prevent this phenomenon, which occurs following interaction of different mediators during both ischaemia and reperfusion. In this study, we aimed to assess the effects of allopurinol, a xanthine oxidase inhibitor, and pentoxifilline, on liver ischaemia/reperfusion injury when used together and to compare these with the effects of using these agents singly. METHODS: Thirty-two rats were divided into four groups consisting of eight rats: Group C, control; Group P, pentoxifilline; Group A, allopurinol; and Group PA, pentoxifilline + allopurinol. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels were measured before hepatic pedicle clamping, on the 45th minute of ischaemia and 15 and 45 minutes after reperfusion. Group P rats were injected with 50 mg/kg pentoxifilline, Group A rats 50 mg/kg allopurinol and Group PA rats were injected with both agents 15 minutes before hepatic pedicle clamping. RESULTS: Ischaemia/reperfusion injury was produced by hepatic pedicle clamping, as demonstrated by AST, ALT and LDH increase. Injury prevention occurred in Groups P, A and PA. No significantly different (better) prevention was provided by giving allopurinol plus pentoxifilline to the rats. Furthermore, no difference was observed between the allopurinol and pentoxifilline injected groups in terms of preventing ischaemia/reperfusion injury. CONCLUSIONS: Pretreatment with allopurinol or pentoxifilline resulted in significantly lower hepatic enzyme elevation than that in controls in the rat liver ischaemia/reperfusion model. Using both agents does not provide better protection than using either agent alone. PMID- 12376237 TI - Experience in the treatment of patients with burns covering more than 90% TBSA and full-thickness burns exceeding 70% TBSA. AB - OBJECTIVE: The objective of this study was to explore our experience in the treatment of serious burn patients (total burn surface area [TBSA] > 90% and full thickness burns > 70% TBSA). METHODS: Thirty patients who were admitted to our unit over a period of 12 years were analyzed retrospectively; 23 cases (76.7%) were successfully treated. RESULTS: There were seven out of 12 cases (58.3%) in the first 5 years and 16 out of 18 cases (88.9%) in the latter 7 years of the study period. CONCLUSIONS: It is concluded that: 1) giving electrolyte-free fluids (around 3700 ml) and the maintenance of hourly urine output at 70 ml or more appear to be beneficial in resuscitation therapy; 2) the first operative procedure should be undertaken early, at about the third day after injury; 3) a higher percentage area of eschar to be excised in the first operation is encouraged and eschar excision of up to 40% or more is preferable; 4) controlling the area of exposed wound under 5% in the entire therapeutic course is essential in the prevention of burn infection; 5) emphasis should be placed on the vital role of a dehumidifier in reducing the incidence of fungal infection; and 6) early enteral nutrition with the use of growth hormone in correcting the nutritional state of the patient is also emphasized. PMID- 12376238 TI - Ultrastructural study of the vascular endothelium of patients with spontaneous rupture of hepatocellular carcinoma. AB - BACKGROUND: Spontaneous rupture of hepatocellular carcinoma (HCC) is common in Asia and Africa, although the mechanism is unclear. In our previous study, we found that vascular injury exemplified by collagenase synthesis and collagen degradation in small arteries was related to the HCC rupture. METHODS: In this study, transmission electron microscopy was used to study 22 specimens from ruptured HCC and non-ruptured HCC. RESULTS: In nine specimens of ruptured HCC, there was evidence of vascular injury with fewer cell junctions and larger fenestrae in vascular endothelial cells. The phenomenon of increased endothelial protein synthesis was also present. In the specimens of non-ruptured HCC, evidence of vascular injury was found in only two cases (p < 0.01). Fewer cell junctions and larger fenestrae could increase the permeability of the vascular wall. Increased protein synthesis in endothelial cells correlates with the phenomenon that more collagenase is expressed in these cells. The resulting breakdown of collagen could render the blood vessels weak; hence, these blood vessels are more prone to splitting/breakage. CONCLUSION: We conclude from our study that this vascular injury may result in HCC rupture. PMID- 12376239 TI - Pathological spectrum of nephrectomies in a general hospital. AB - OBJECTIVES: To first categorize a series of nephrectomies according to underlying pathology, as practised in a major general hospital in the north of Jordan, and then compare the results with published figures for Western countries. Also, to create standards for future evaluation of nephrectomies performed by laparoscopy. METHODS: The hospital and pathological records of 423 consecutive nephrectomies performed at Princess Basma Teaching Hospital in the north of Jordan during the period of 1991 to 2000 were reviewed. RESULTS: Benign disease led to surgery in 298 cases, of which 161 were secondary to infection-related conditions. Malignancy resulted in the removal of 125 kidneys. The rate of nephrectomy for benign conditions has declined during the last few years in comparison with that for malignant conditions. Patients operated on for benign diseases were younger [mean age, 38.4 years] than those with malignant tumours [mean age, 46.7 years]. CONCLUSIONS: The mean age of patients undergoing surgery for benign and malignant disease was lower than in publications from Western countries. The frequency of nephrectomy performed for tuberculosis, hydatid disease, and xanthogranulomatous pyelonephritis is still higher than the rates published in Western countries. There is a remarkably low frequency of upper urothelial carcinoma compared with Western countries, probably due to environmental differences and genetic susceptibilities. Malignant renal tumours tend to affect people at a remarkably young age in Jordan, which is thought to be a reflection of the high proportion of young people. Nephrectomy for malignant disease had a higher rate of complications (16.8%) than for benign conditions [9.4%; p less than 0.0228]. The re-operation rate was 3.1% for all patients who underwent nephrectomy. The overall 30-day mortality rate was 0.9%. Both screening and education programmes are needed to decrease the rate of nephrectomy for preventable conditions. PMID- 12376240 TI - Complications of otitis media requiring surgical intervention. AB - BACKGROUND: Although the incidence of complications of otitis media that require surgical interventions has decreased substantially over the past few years, it is a prevailing condition for which clinicians should remain vigilant. METHODS: We conducted a 3-year review [June 1998 to June 2001] in our hospital of surgical records of patients with complications of otitis media that were treated surgically. RESULTS: There were 16 patients with complications of otitis media, of which nine [56%] were intracranial; brain abscess and lateral sinus thrombosis were the most common intracranial complications. Extracranial complications were present in 15 [94%] of the patients; mastoid abscess [40%] was the most common extracranial complication. Seven [44%] patients had two or more concomitant complications. All patients with intracranial complications recovered well with no neurological deficits after aggressive antibiotic therapy and initial surgical treatment by neurosurgeons. Modified radical mastoidectomy was the most common surgical otological procedure that was performed in these cases. CONCLUSIONS: Aggressive antibiotic therapy and combined management of cases by otologists and neurosurgeons are the key to reducing the morbidity and mortality of the serious complications of otitis media. PMID- 12376241 TI - Gallbladder torsion: report of four cases and review of the literature. AB - Gallbladder torsion is a rare cause of acute acalculous cholecystitis. Preoperative diagnosis is difficult. The treatment of choice remains immediate cholecystectomy. We present four cases of gallbladder torsion and review the literature. PMID- 12376243 TI - Liver transplantation for hepatolithiasis. AB - Hepatolithiasis is frequently encountered in Asia, but is relatively uncommon in Western societies. The improved surgical and stone fragmentation techniques that have evolved over the past decade have reduced the incidence of retained or recurrent stones with a consequent reduction in progressive liver damage and cirrhosis. Nonetheless, disease-related mortality from liver failure, bleeding oesophageal varices and cholangiocarcinoma still exists and a proportion of patients are cirrhotic at their initial presentation. There have been good long term results following liver transplantation for a variety of cholestatic liver diseases, but transplantation for hepatolithiasis has seldom been reported. This paper reports four patients who underwent successful liver transplantation for hepatolithiasis with secondary biliary cirrhosis. PMID- 12376244 TI - New operative strategies in primary hyperparathyroidism. AB - More than 95% of patients with primary hyperparathyroidism have been treated with bilateral neck exploration by experienced surgeons. This procedure has been performed without employing preoperative localization tests or specialized techniques of intraoperative measurement. A renewed interest in unilateral neck exploration for primary hyperparathyroidism emerged (in three developments), in an attempt to maintain the excellent cure rate and to minimize the invasiveness of the procedure. The first development was the introduction of sestamibi scintigrams as a new preoperative localization technique and intraoperative nuclear mapping with a hand-held gamma probe. The localization of adenomas using this technique was much more accurate than that of previous localization studies, allowing unilateral procedures to become feasible. Sestamibi guidance enables parathyroidectomies to be performed much more rapidly through a significantly less invasive dissection. Secondly, the intraoperative quick parathyroid hormone assay allows the confirmation of removal of the parathyroid mass. The third development was endoscopic parathyroidectomy. Various approaches have been shown to be technically feasible, including endoscopic procedures that rely on CO2 insufflation to create a working space or video-assisted procedures in which the working space is maintained through conventional external retraction. Given the safety and high success rate of the standard exploration, the potential advantages of these new strategies include decreased operating time, local or regional anaesthesia rather then general anaesthesia, and smaller incisions. PMID- 12376245 TI - Unknown primary squamous cell carcinoma of the head and neck: a review of diagnosis, treatment and outcomes. PMID- 12376246 TI - [Establishment of transgenic Xenopus laevis by intracytoplasmic sperm injection]. AB - OBJECTIVE: To study the feasibility of establishing transgenic laevis by intracytoplasmic sperm injection (ICSI). METHODS: The testes of mature Xenopus laevis were taken for the purification of their sperms, which was subsequently incubated with digitonin to prepare concentrate of the sperms. Treatment of the concentrate with linearized reporter vector pCMV-EGFP-N1 was performed, and the sperms were then injected into unfertilized ova harvested from female laevis, followed by culture and observation of the development of the ova. RESULTS: The condensed sperm we obtained were of high quality and after intracytoplasmic injection into the ova, a fertilization rate of 10% was achieved and 20% of the zygotes survived the neurula stages and developed into tadpoles, but all of which were slightly deformed. The integration ratio of green fluorescent protein (GFP) reporter gene was 81%, but GFP expression was not observed in the laevis. CONCLUSION: ICSI is a simple and practicable method for establishing transgenic Xenopus laevis. PMID- 12376247 TI - [Expression and identification of phage display library for Fab fragments of colorectal cancer-related antibodies]. AB - OBJECTIVE: To express the original human Fab antibody phage display library with positive recombined bacterium XL1-blue-Pcomb3 and identify its specific binding activity with colorectal cancer cells in vitro after screening with human colorectal cancer-related antigens. METHOD: The recombination rate of Fd fragment of the heavy chain and insertion of kappa chain of the antibodies was determined with PCR, and the original Fab library was expressed. The antigens were extracted from 3 sensitized colorectal cancer tissues previously used for construction of the original Fab library and from 13 non-sensitized colorectal cancer tissues, along with the antigens from LoVo, HT-29 and LS-174T cells cultured in vitro. The original Fab antibody library was screened with the 3 groups of mixed antigens derived in preceding procedure and 3 tertiary Fab antibody libraries were obtained, which were then mixed in equal volume for subsequent tests of binding activity with human colorectal cancer tissues and cells in vitro using enzyme linked immunosorbent assay (ELISA) and immunohistochemical staining. Specimens of gastric and esophageal carcinomas and normal intestinal mucosa, together with liver cancer cells and gastric cancer cells were utilized as control. RESULT: The recombination rate of Fd and kappa chain were 40 % and 70 % respectively, and the rate of their simultaneous insertion into Pcomb3 vector was 28%. The capacity of library for Fab fragment genes was 2.1x10(6), and the original antibody libraries screened with the 3 groups of mixed antigens were enriched to varied degrees, which all displayed relatively specific binding activity with human colorectal cancer tissue and cells in vitro. CONCLUSION: Colorectal cancer-related antibody Fab fragments are obtained through screening phage display library, which show relatively specific binding activity with human colorectal cancer tissues and cells. PMID- 12376248 TI - [Functional connection between the marginal division and hippocampus in rats]. AB - OBJECTIVE: To investigate the functional connection between the marginal division of the striatum and hippocampus, a brain region that play a vital role in learning and memory. METHODS: Morphological localization of functional activity of the nervous system was employed. Kainic acid (0.01%) was stereotaxically injected into the hippocampus as a chemical stimulus, and immunohistochemistry method was used to show the expression of c-Fos in rat brain. RESULTS: c-Fos was intensely expressed in the hippocampus, amygdaloid nucleus, the bed nucleus of the stria terminals and cerebral cortex; in the striatum, a stretch in the marginal division where c-Fos-positive nuclei congregated was observed, while c Fos expression was scarcely detectable in the caudate putamen and globus pallidus. CONCLUSION: Functional connection exists between the marginal division and hippocampus in rats. PMID- 12376249 TI - [Ultrastructural features of cultured rat cortical astrocytes with stretch induced injury]. AB - OBJECTIVE: To investigate the changes of ultrastructural features of cultured rat cortical astrocytes after stretch-induced injury. METHODS: Rat cortical astrocytes isolated from 1- to 2-day-old rats were cultured till confluency, and then plated in tissue culture wells with flexible silastic bottom after purification. A computer-controlled device was used to produce stretch-induced injury in the astrocytes with the imposed pressure of 50, 150, and 250 kPa respectively, followed by observation of the ultrastructural changes in the astrocytes with light and electron microscopy. RESULTS: Obvious ultrastructural destruction of the astrocytes occurred when the imposed stretch pressure was 50 kPa, and scanning electron microscopy demonstrated increased intercellular space and laceration of the cell body and its processes. Transmission electron microscopy revealed mitochondria swelling 1 h after stretch-induced injury and 6 h after the injury, vacuolar degeneration of the mitochondria occurred. Increased stretch pressure caused further decrease in the amount of glial filaments and densification of astrocytes. CONCLUSIONS: Stress, even at a relatively small scale, can cause disruption of intercellular juncture and ultrastructural change of the cultured astrocyte, which may be related with extensive brain edema after traumatic brain injury. PMID- 12376250 TI - [Cloning and sequence analysis of a novel TT virus varian]. AB - OBJECTIVE: To clone the DNA of a TT virus (TTV) variant isolated from a patient with elevated alanine transaminase (ALT) of unknown etiology, and conduct sequence analysis. METHODS: The long fragment of TTV DNA was amplified by nested PCR and then cloned into pGEM-T vector. A clone named 56-B containing 3.2 kb TTV DNA was selected for sequence analysis besides homology analysis with other 5 TTV variants retrieved from GenBank, and phylogenetic analysis was carried out by maximum likelihood method. RESULTS: The nucleotide identities of 56-B with the other 5 TTV strains TA278, JA10, US35, SANBAN and TUS01 were 42.4%, 48.2%, 47.9%, 49.8 % and 61.7 % respectively, and the corresponding amino acid identities were even lower. Phylogenetic analysis showed that 56-B was far from other TTV strains in genetic distance that ranged from 0.344 to 0.458. However, the sequences in the 5'- and 3'-end were still much conservative. CONCLUSION: The isolated 56-B showed high heterogeneity in genetic background and was therefore quite distinct from other TTV strains as a novel TTV variant that represents a new TTV genotype. PMID- 12376251 TI - [Identification and cloning of adenylate kinase gene, a novel gene of Schistosoma japonicum]. AB - OBJECTIVE: To subclone a novel gene of Schistosoma japonicum (Sj), adenylate kinase (AK) cDNA, which was identified through expressed sequence tag (EST) strategy and homology search, so as to prepare for further functional study of this gene. METHOD: The inserted cDNA fragment was sequenced and searched with BLASTn program. Two PCR primers were designed according to the sequence of this Sj AK cDNA and the cloning sites in pET32a (+) plasmid, with the product purified before linkage with pMD 18-T vector. The recombinant T-vector was digested with EcoRI /XhoI to obtain Sj AK cDNA, which was then introduced into the expression plasmid pET32a (+). RESULTS: The novel gene possessed 86% homology with Sm AK cDNA, and the PCR product is of expected length. Double digestion with EcoR I and Xho I proved that the recombinant T-vector and the expression plasmid had the insert with length identical to that of the target fragment. CONCLUSION: The novel cDNA codes for adenylate kinase of Schistosoma japonicum, and the recombinant expression plasmid pET32a (+)-Sj AK have been successfully constructed. PMID- 12376252 TI - [Preliminary study of cercaria antigen of Schistosoma japonicum before and after ultraviolet irradiation]. AB - OBJECTIVE: To study the changes in the constituents of the cercaria antigen of Schistosoma japonicum before and after ultraviolet irradiation. METHODS: The cercaria of Schistosoma japonicum were exposed to ultraviolet light (UV) irradiation at a dose of 400 mgrW/cm2 for 1 min, and the UV-irradiated cercaria antigen (UVCA) and normal cercaria antigen (NCA) were simultaneously analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: At least 2 antigens with relative molecular mass (Mr) of 212 000 and 82 000 were identified in UVCA but not in NCA by SDS-PAGE analysis, and the concentrations of the antigens with Mr of 116 000, 26 000 and 16 000 in UVCA were significant higher than those in NCA. On the other hand, the antigenic molecule with Mr of 67 000 in NCA was recognized by serum from pigs vaccinated with UV-attenuated cercariae, but not by serum from pigs with Schistosoma japonicum infection. Antigens with Mr of 79 000 and 94 000 were apparently more strongly reactive with the former porcine serum than with the latter. CONCLUSION: The results suggest that all the novel antigens arising from or increased by UV exposure, or antigens specifically recognized by serum from pigs vaccinated by UV attenuated carcariae may be the principal factors in the highly protective immunity provoked by irradiated cercariae. PMID- 12376253 TI - [Expression of Fas genes transduced into colorectal cancer cells]. AB - OBJECTIVE: To construct colorectal cancer cells expressing exogenous Fas gene and observe the expression level of its mRNA and protein before and after transduction. METHODS: Fas cDNA was inserted into the multiple cloning site of the expression vector pBK-CMV with molecular cloning technique, and the resultant recombinant plasmid was transduced into colorectal cancer LoVo cells via lipofectamine. G418 was utilized to screen the positive clones containing the recombinant plasmid, where Fas mRNA and protein expression was determined with Western blotting and dot blotting. RESULTS: pBK-CMV Fas cDNA plasmid was successfully constructed. The transduced colorectal cancer cells were screened by G418 and a resistant cell line (LoVo Fas cells) was obtained. Fas expression was detected in both transduced and non-transducted cell lines, but the expression level of both Fas mRNA and protein was much higher in the former, which showed lowered proliferation rate and lengthened doubling time and logarithm growth period than the non-transducted cells, but the difference was not significant. Treatment of the transduced cells with Fas antibody produced significant difference (P<0.05), manifested by apparently inhibited cell growth. CONCLUSIONS: LoVo cells normally has only very low expression level of Fas gene, while transduction with pBK-Fas cDNA can enhance the efficiency of Fas mRNA and protein expressions. Fas antibody significantly inhibits the growth and proliferation of in vitro cultured Fas-expressing LoVo cells. PMID- 12376254 TI - [Association between aldosterone synthase gene polymorphism and hypertrophic cardiomyopathy]. AB - OBJECTIVE: To investigate the relationship between aldosterone synthase (CYP11B2) gene polymorphism and hypertrophic cardiomyopathy (HCM). METHODS: Fifteen HCM patients and 18 healthy subjects were enrolled in this study. Peripheral blood samples were collected from these subjects to exact genome DNA. PCR and Hae III restriction endonuclease digestion were employed to study -344C/T polymorphism of CYP11B2 gene. RESULTS: CYP11B2 gene showed a significant difference in CT genotype distribution in HCM groups as compared with that in the control groups (P<0.05). CONCLUSION: CT genotype of CYP11B2 gene may be one of factors responsible for the pathogenesis of HCM in a proportion of patients. PMID- 12376255 TI - [Application of multiplex PCR in genotyping of hepatitis B virus prevailing in Guangdong Province of China]. AB - OBJECTIVE: To establish a convenient method for the genotyping of hepatitis B virus (HBV) using multiplex PCR. METHOD: Based on the alignment of 114 complete nucleotide sequences of HBV DNA belonging to different genotypes, acquired from the GenBank, genotype-specific sequences were identified according to which 6 pairs of primers were designed corresponding to each genotype. Subsequent genotyping of HBV was performed using these primers that were added, either alone or in conjunction with others, into a multiplex PCR reaction tube, and HBV genotype was determined according to the length of amplified DNA. RESULT: The genotyping result of multiplex PCR was consistent with that produced by PCR- restriction fragment length polymorphism as established by Lindh. We found in this study that among the HBV carriers in the vicinities Guangzhou of City, about 45% belonged to B genotype, 38.75% to C genotype and 16.75% to D genotype. CONCLUSION: This multiplex PCR method is simple, convenient and more differential. PMID- 12376256 TI - [Effects of Buyanghuanwu decoction (BYHWT) on proliferation of cultured rat cortical neurons]. AB - OBJECTIVE: To observe the effect of rat serum containing Buyanghuanwu decoction (BYHWT) on the proliferation of cultured rat cortical neurons, so as to understand the mechanism of BYHWT in the treatment of hypoxia brain damage. METHODS: The growth of cultured rat cortical neurons were observed by MTT assay to evaluate the effect of the serum containing BYHWT on the neurons cultured in both normal and hypoxia conditions. RESULTS: BYHWT significantly promoted proliferation of the neurons cultured under both normal and hypoxia conditions, in comparison with the response of the cells to drug-free serum (P<0.05). CONCLUSION: Some of the constituents of BYHWT in rat serum can promote the proliferation of rat cortical neurons cultured in both normal and hypoxia conditions. PMID- 12376257 TI - [Establishment of rabbit model of renal allograft transplantation using microsurgical technique]. AB - OBJECTIVE: To establish rabbit model of renal allograft transplantation with reduced complications and high survival rate using microsurgical technique. METHODS: Twelve healthy adult rabbits were randomly divided into 2 groups of equal number, one as donor group and the other recipient. The left kidneys of the donor rabbits were removed followed by immediate reperfusion with 4 degrees celsius H-CA solution, before they were transplanted into the recipient rabbits with their left kidneys excised and end-to-end anastomosis of the renal arteries, veins and ureter respectively performed with microsurgical technique. Another 12 normal rabbits received operations to temporarily block the right renal arteries and veins, serving as control group, in which 11 completed the experiment. RESULTS: No thrombosis or stricture occurred at the site of anastomosis in rabbits with renal allograft transplantation, and the survival rate reached 91.7% (11/12). CONCLUSION: This rabbit model of renal allograft transplantation has markedly fewer complications with improved survival rate, thus providing a more practical and reliable model for experimental and clinical studies of renal transplantation. PMID- 12376258 TI - [Seizure inhibition by vagal nerve stimulation in rats]. AB - OBJECTIVE: To study the effect of vagal nerve stimulation in seizure inhibition in rats. METHODS: In rat epileptic models induced by penicillin, electromyogram (EMG), electroencephalogram (EEG) and extracellular electric activity of the cortex were recorded to study the inhibiting effect of vagal nerve stimulation on epilepsy. Results Inhibiting effect of vagal nerve stimulation on epilepsy was observed from the changes in behavior, EMG, EEG and extracellular electric activity of the rats, and this inhibiting effect was enhanced as the frequency for stimulation increased from 5 Hz to 20 Hz. Conclusion Vagal nerve stimulation can inhibit the epileptic activity in rats, the effect of which depends on the stimulation conditions imposed on the rats. PMID- 12376259 TI - [Purification of PCR products with cetyltrimethylammonium bromide]. AB - OBJECTIVE: To establish a method for purifying PCR products with cetyltrimethylammonium bromide (CTAB). METHOD: Selective precipitation of the PCR product was performed using CTAB, which forms compound with DNA fragment in salt solution of appropriate concentration, but not with single strain oligonucleotide or dNTPs. The precipitation could be dissolved in 1.2 mol/L NaCl while the addition of ethanol caused the desired PCR product to precipitate so as to be recovered. RESULT: The primers and small-molecule dNTP could be effectively eliminated after this procedure. Although the output of the purification process reached only 80% that by current reagent kit, it reduces the cost to as low as 1/8 of that normally required by the kit. CONCLUSION: CTAB is applicable for purification of the PCR product. PMID- 12376260 TI - [Effect of heparin lithium as anticoagulant in assay of FT3, FT4 and TSH]. AB - OBJECTIVE: To evaluate the application of heparin lithium as anticoagulant in in vitro tests of thyroid function. METHODS: The blood samples obtained by venipuncture from 32 subjects (including 10 normal subjects and 22 patients with thyroid disorder) were collected in parallel dry vacuum tubes with one of them containing heparin lithium, for assay of TSH, FT3, FT4 by chemoluminescence immunoassay. RESULTS: No difference was found in TSH and FT4 levels determined separately from the parallel tubes (TSH: t=1.846, P=0.075; FT4: t=1.649, P=0.110) with closely correlated results (TSH: r=1.000, P=0.000; FT4: r=0.999, P=0.000), and the clinical diagnoses therefrom derived were perfectly matched. FT3 level in ordinary dry vacuum tubes, however, was significantly higher than that in the tube containing heparin lithium (t=-6.253, P=0.000), but still close correlation was observed between them (r=0.999, P=0.000 0). Inconsistent clinical decisions occurred in 7 of the 32 subjects in respect of FT3 levels respectively assayed in the 2 tubes, but when the normal ranges of FT3 level were established for the 2 tubes separately, the same clinical diagnoses were reached. CONCLUSION: TSH and FT4 levels as determined in the 2 tubes are comparable, and even though FT3 levels does not present this feature, close relation is obvious and therefore the 2 values can be equivalent after establishment of their respective normal limits or after linear regression processing. PMID- 12376261 TI - [Preliminary study of development of gene chips for HIV diagnosis]. AB - OBJECTIVE: To study the technology for establishing DNA chips for the diagnosis of HIV. METHODS: HIV 1U26942 DNA fragments were isolated by restriction display PCR (RD-PCR) and printed onto aminosilane-coated glass slides by Pixsys 5500 arrayer as probes to prepare the gene chips. HIV samples, after labeled with Cy3, were hybridized with the microarray followed by scanning for analysis of hybridization kinetics of the RD fragments. RESULTS: The experimental condition for preparing the gene chips was investigated and 12 RD fragments were screened as probes for further study. CONCLUSION: The technique established in this study for preparing DNA chips is specific and applicable. PMID- 12376262 TI - [Analysis of the clinical manifestations and imaging features of Budd-Chiari syndrome: report of 81 cases]. AB - A comprehensive analysis of the clinical manifestations and imaging features in 81 cases of Budd-Chiari syndrome (BCS) was conducted, and criteria for classification of this disease was proposed. Diagnostic modalities included digital subtraction angiography, ultrasound, computed tomography and percutaneous transhepatic cholangiography. The main clinical manifestations of BCS was inferior vena cave obstruction and portal hypertension. Wall thickening of the gallbladder was indicative of BCS during imaging diagnosis. PMID- 12376263 TI - [Quantitative analysis of Sokal's risk index in relation to 2 therapy protocols: their respective impact on clinical remission of chronic myeloid leukemia]. AB - OBJECTIVE: To quantitatively evaluate the impact of Sokal's risk index and that of 2 therapy protocols on the clinical outcome of patients with chronic myeloid leukemia (CML). METHODS: With the assistance of Access 2000 database of CML, 94 patients with CML were grouped on the basis of either different therapy protocols utilizing harringtonine plus Ara-C (HA) vs hydroxyurea (Hu) or Sokal scores, and the impact of therapy protocol and risk profile were quantitatively evaluated respectively. RESULT: Treatment protocol utilizing HA was incapable of lengthening the duration of chronic phase (DCP) of CML, regardless of its better short-term effect than that of Hu. The impact of risk profile of the patients on clinical remission rate and DCP was more significant than that of the therapy protocols. CONCLUSION: HA should not be used as the first-line protocol in the treatment of CML patients in chronic phase who have not received any previous medical intervention. Patients should be categorized according to the risk profile for choosing appropriate treatment protocol and making better clinical judgement. PMID- 12376264 TI - [Application of PCR technique in genetic diagnosis of Duchenne/Becker muscular dystrophy]. AB - OBJECTIVE: To study the application of PCR technique in genetic detection of Duchenne/Becker muscular dystrophy (DMD/BMD). METHODS: A multiple PCR system is established according to the multiple sites of DMD/BMD exon deletion. Under different PCR conditions, multiple exon deletions, single-strand conformation polymorphism, allopolyploid, chain labeling, restriction fragment length polymorphism and microsatellite phenomenon were examined in 23 DMD/BMD patients and 57 suspected carriers of these genes. RESULTS: Fourteen of the 23 DMD/BMD patients were identified as having gene deletion, with another 2 carried gene duplicates. Forty female relatives of these 23 DMD/BMD patients were diagnosed as carriers of the genes. CONCLUSION: This PCR system can be applied in detecting gene mutation of DMD/BMD, screening the carriers and in appropriate genealogical analysis of the patients with DMD/BMD. PMID- 12376265 TI - [Superselective intra-arterial infusion chemotherapy for recurrent cancer in the remnant stomach after partial gastrectomy]. AB - OBJECTIVE: To investigate the effect of superselective intra-arterial infusion chemotherapy in the treatment of advanced recurrent cancer in the remnant stomach after previous partial gastrectomy. METHODS: Eighteen patients with advanced recurrent cancer in the remnant stomach that were non-resectable as confirmed in the operations were included in this study, who subsequently received superselective intra-arterial infusion chemotherapy. RESULTS: Improvement of the symptoms to various degrees were achieved in all patients after the therapy, with the total rate of tumor reduction of 77.8% and pathologically confirmed improvement rate of 83.3%. The 0.5-, 1.0-, 1.5- and 2.0-year survival rates were 94.4%, 66.7%, 50.0% and 27.8% respectively. CONCLUSION: Superselective catheterization is effective in treatment of advanced recurrent cancer in the remnant stomach, which can significantly prolong the tumor-bearing survival period of the patients. PMID- 12376266 TI - [Construction of plant expression vectors containing the gene encoding cholera toxin B subunit]. AB - OBJECTIVE: To construct the plant expression vector containing the nucleotide sequence encoding cholera toxin B (CTB) subunits. METHOD: Using high-fidelity PCR, we amplified CTB genes that were then subcloned into the transition vector pRTL2. Following confirmation of the CTB nucleotide sequence, the vector was subcloned into the plant vector pBI121 that was subsequently transferred into Agrobacterium tumefaciens LBA4404 by electroporation. RESULTS: CTB DNA that was ligated into the transition vectors resulted in the 2 vectors designated as pRCTB and pRCTBK. After the 2 vectors were ligated into the plant binary vector pBI121 respectively, new plant binary vectors, namely pBI-CTB and pBI-CTBK, were produced. Analysis with restriction endonucleases confirmed successful transfer of pBI-CTB and pBI-CTBK into Agrobacterium tumefaciens LBA4404. CONCLUSION: With appropriate technological strategy, the plant binary expression vectors encoding CTB have been constructed, which facilitates further investigation of CTB protein expressions in transgenic plant. PMID- 12376267 TI - [Role of magnetic resonance imaging in the diagnosis of intracranial germinoma]. AB - OBJECTIVE: To investigate the value of magnetic resonance imaging (MRI) in the diagnosis of intracranial germinoma. METHODS: A retrospective analysis of the MRI features was conducted in 19 cases of pathologically confirmed intracranial germinoma. RESULTS: The lesion was located in the sellar region in 10 cases, in the pineal region in 6 and in the thalamus and basal ganglia in 3. The characteristic MRI of intracranial germinoma included the following features: (1) In T1-weighted images (T1WI), the lesions were isointense or slightly hypointense, which appeared isointense or slightly hyperintense in T2-weighted images (T2WI). The germinoma in the sellar region and pineal region showed no edema, but those in the thalamus and basal ganglia showed minimal or moderate edema with space-occupying effect. (2) Homogeneous or inhomogeneous Gd-DTPA enhancement was observed in most of the tumors. CONCLUSION: Multiplanar imaging and Gd-DTPA enhancement in MRI are helpful in the diagnosis and differential diagnosis of intracranial germinoma, which presents features characteristic of the gender and age of the patients with the disease, location, size, form and image intensity of the lesion, and therefore, preoperative MRI diagnosis of the tumor can be possible. PMID- 12376268 TI - Effect of seizures and antiepileptic drugs on prolactin secretions. AB - OBJECTIVE: To study the effect of seizures and antiepileptic drugs on prolactin (PRL) secretions. METHODS: Serum PRL level was measured by radioimmunoassay in 110 epileptic patients who received different treatment protocols with antiepileptic drugs (AEDs), and the test was also performed in 64 of these patients before and after seizures. Another 21 untreated epileptic patients and 42 healthy subjects served as the control groups. RESULTS: Serum PRL level was significantly increased after seizures, which peaked at 15 min postictal and attained the levels more than 5-fold the baseline in 59 patients. At 90-minute postictal, PRL levels decreased in 57 patients and dropped within normal range in 38 patients. The changes of hormone levels correlated significantly with the types of seizures. The basal PRL levels in patients with exclusive phenytion (PHT) or valproate (VPA), and in those with combined ministration of carbamazpine (CBZ+VPA+PHT), were significantly lower than the control levels (P<0.05). Patients receiving treatment with traditional Chinese medicine had comparable serum PRL levels with the normal control group (P>0.05). CONCLUSION: Seizures of epilepsy and medication with AEDs given as either monotherapy or polytherapy affect the secretion of PRL in the pituitary, but traditional Chinese medicine therapies does not. PMID- 12376269 TI - [Cryopreserved viable pedicled aortic homografts for complex congenital heart diseases: report of 2 cases]. AB - OBJECTIVE: To report the authors' experience in treating complex congenital heart diseases with cryopreserved viable pedicled aortic homografts (CVAHs). METHODS: CVAHs were obtained within 3 h after death of donors younger than 35 years old due to non-cardiovascular and non-infectious diseases. After routine treatment, the homografts were cryopreserved in liquid nitrogen and thawed in 0.9% sodium chloride solution at 37 to 42 degrees celsius before use. A CVAH was used as a valved conduit to connect the pulmonary artery and right ventricle in one case of double-outlet right ventricle, pulmonary stenosis and coronary artery deformation. In another case of severe tetralogy of Fallot, a CVAH was used as a valved patch to enlarge the right ventricle outlet. RESULTS: The homografts were preserved satisfactorily. The surgical results of the 2 cases with congenital heart diseases were satisfactory in perioperative and follow-up periods. CONCLUSION: CVAH is a good substitute to rebuild right ventricle-pulmonary artery connection in treatment of complex congenital heart diseases. PMID- 12376270 TI - [Experimental study of monoclonal antibody to intercellular adhesion molecule-1 for incipient acute tubular necrosis]. AB - OBJECTIVE: To explore the role of leukocyte adhesion in the pathophysiology of glycerol-induced acute renal tubular necrosis (ATN). METHODS: Rat models of ATN were established by intramuscular injection of glycerol in 24 Wistar rats, which were divided randomly into 3 groups of equal number according to the agents coinjected with glycerol for the prevention of ATN, with another 8 rats serving as normal control. One of the 3 groups received a monoclonal antibody (mAb) against intercellualr adhesion molecule-1 (anti-ICAM-1) and another received CD3 mAb, leaving one group untreated. Both functional impairment and histological changes in the rats were observed. RESULTS: Plasma creatinine measured 24 h after the injection of glycerol was 412.31+/-94.42 micromol/L in rats treated with anti ICAM-1, significantly lower than that in CD3 mAb-treated rats (990.21 +/-171.25 micromol/L, P<0.05). Moderate to severe necrosis in the outer renal medulla with frequent mitoses was present in rats with ATN receiving CD3 mAB or nothing, but only mild necrosis with few mitoses occurred in merely 2 of those rats with anti ICAM-1 treatment. CONCLUSION: Leukocytes and adhesion molecules play critical roles in the pathophysiology of glycerol-induced ATN, and anti-ICAM-1 which blocks the adhesion of the leukocytes may alleviate the pathological changes in the kidney. PMID- 12376271 TI - Plasma exchange for thrombotic thrombocytopenic purpura: report of 2 cases. AB - Two patients with thrombotic thrombocytopenic purpura (TTP) were treated with daily plasma exchange with fresh frozen plasma of the plasma volume of the body (40 mg/kg . b.w.), in combination with red blood cell transfusion. The clinical symptoms resolved immediately and the parameters of laboratory examination improved promptly after the treatment, and follow-up of the 2 patients revealed no recurrent TTP episodes. This result indicates that plasma exchange is obviously superior to traditional treatment modalities and has significantly changed the course of TTP. PMID- 12376272 TI - Management of acute rejection of kidney allograft. AB - OBJECTIVE: To evaluate the management of acute rejection (AR) after kidney transplantation and investigate the factors influencing the clinical outcome of the patients. METHODS: A retrospective study was conducted in 86 cases of AR developed after primary kidney transplantation in the light of therapeutic measures, clinical outcome and prognosis. RESULTS: Among these patients, 81 survived AR after treatment. In patients with pulse treatment with methylprednisolone (MP), 48 out of 68 managed to survive the crises, while in those who received ATG as the first line drug therapy 10 out of 11 patients survived and in other cases, 6 out of 7 did due to first-line OKT3 administration. All the 20 patients who did not respond to MP received ATG or OKT3 instead, with 14 recovered. Of the 8 patients who failed to be cured by the management above, 6 with previous CSA treatment took FK506 and 3 were consequently cured. Five patients lost the allografts because of uncontrollable infection, allograft rupture or thrombosis. CONCLUSIONS: MP therapy is still the most commonly used primary treatment for acute rejection episodes. Increase of SCr by more than 10% on days 2 and 3 of MP therapy indicates poor prognosis. ATG or OKT3 can be effective against acute rejection not only as first-line but also as second-line drug. In condition of steroid-resistant rejection when ATG and OKT3 fail to manage, a change to baseline immunosuppression may be considered as the replacement of CSA with FK506. PMID- 12376273 TI - [Echocardiographical features during the rejection free period after heart transplantation: report of one case]. AB - OBJECTIVE: To study the echocardiographic features during the period free of acute allograft rejection after heart transplantation by monitoring one patient within one year after heart transplantation. METHODS: The dimension of atrial and ventricular, interventricular septal and ventricular wall thickness, blood flow pattern through the mitral and tricuspid valves, left ventricular muscle weight (LVMW) and LVMW index during different periods were measured by echocardiography, and all these data were compared with those of the donor before operation. RESULTS: The patient recovered well without any signs of acute rejection. Echocardiography revealed that the dimension of right atrial, left and right ventriculars decreased significantly while left atrial showed significant increase, with obvious increase in the interventricular septal and ventricular wall thickness. LVMW and LVMW index were also significantly increased. The peaks E-and A.wave velocifies of the mitral and tricuspid E peak flow velocity decreased significantly, while the A peak flow velocity of the tricuspid did not undergo any significant changes. Mitral back flow occurred 4 months after the operation and tricuspid back flow persisted after operation. CONCLUSIONS: The changes of morphology, structure and function of the transplanted heart during periods without rejection are to some extent specific to this special phase, when some of these changes are similar to those during early acute rejection, and correct diagnosis relies on endo-myocardial biopsy. PMID- 12376274 TI - [Effects of dexamethasone on epidural morphine-related nausea and vomiting]. AB - OBJECTIVE: To observe the effect of intravenous dexamethasone injections in preventing nausea and vomiting resulted from epidural morphine for post-operation pain relief. METHODS: Eighty-four adult patients (ASA class I to II ) requiring epidural anesthesia for low abdominal surgical procedures were randomly divided into 2 groups, of which Group 1 (n=42) received intravenous dexamethasone injections at 10 mg and Group 2 (n=42) intravenous injection of 2 ml normal saline before administration of 2 mg epidural morphine for post-operation pain relief. The incidence of nausea and vomiting were recorded within 24 h after surgery. RESULTS: The incidence of nausea and vomiting were 12% and 7% in Group 1, while 31% and 21% in Group 2 respectively, showing significant difference between the 2 groups (P<0.05). The total incidence of nausea and vomiting were also significantly different (19% vs 52%, P<0.01). CONCLUSION: Intravenous dexamethasone injections at 10 mg can significantly decrease the incidence of epidural morphine-related nausea and vomiting. PMID- 12376275 TI - [Surgical correction of congenital alveolar cleft: report of 10 cases]. AB - The paper reports our experience in surgical correction of unilateral maxillary alveolar cleft complicated with oronasal fistula by iliac spongy bone marrow autografts in cases. Primary healing of the surgical wounds occurred in all the 10 cases and obvious improvement in external appearance was achieved 3 to 6 months after the surgery. Clear evidence of osteogenesis was found by X-ray examination, and the density of the newly generated bone was comparable to that of normal bones, without visible bounds between the autografts and the normal bones. PMID- 12376276 TI - [Clinical value of emergency bedside ultrasonocardiogram in cardiac care unit]. AB - OBJECTIVE: To evaluate the clinical value of application of emergency bedside ultrasound cardiogram (UCG) in cardiac intensive care unit. METHODS: We conducted a retrospective analysis of 116 cases who received examination with emergency bedside UCG within the period from April 2000 to March 2001. RESULTS: The positivity rate of examination with emergency bedside UCG was 100% among these patients. The diseases identified were as follows: acute myocardial infarction in 41 cases, angina pectoris in 16, ventricular aneurysm in 5, perforation of ventricular septum in 2, rheumatic valvular disease in 9, dilated cardiomyopathy in 9 and aortic dissecting aneurysm in 7. CONCLUSION: Emergency bedside UCG has important value in clinical diagnosis of cardiac emergencies. PMID- 12376277 TI - [English representation of tables and figure legends in the section of results in medical papers]. AB - In this article, the author provides a sketchy description of English representation of the tables and figure legends mostly seen in the Results section of a medical paper, with some examples which might be helpful for potential medical journal contributors. PMID- 12376278 TI - Detection of an unkown peak at 1.2 ppm in proton magnetic resonance spectra of cats with cerebral ischemic necrosis. AB - OBJECTIVE: To confirm an unknown peak (Pu) in proton magnetic resonance spectra ((1)H-MRS) in cats with permanent focal cerebral ischemia, and surmise its potential value of application. METHODS: After focal cerebral ischemia, diffusion weighted imaging (DWI) was used to identify regions of ischemia for (1)H-MRS voxel localization with the assistance of stereotaxic atlas of cat brain. (1)H MRS was used to monitor the progression of focal cerebral ischemia in 6 cats over a period of 7 d following middle cerebral artery occlusion (MCAO). The changes in lactate, N-acetyl-aspartate (NAA), choline, creatine, and Pu were observed. RESULTS: In the involved regions, lactate was elevated almost immediately after the onset of cerebral ischemia, and NAA declined within several hours of acute infarction. The Pu at 1.2 ppm was persistently detected in the affected cerebral areas 2 to 7 d after MCAO. CONCLUSION: Pu has a close relationship with cerebral ischemia necrosis and may be a intrinsic production of the necrotic tissue, which can be utilized as a specific marker and therefore has important clinical diagnostic value. PMID- 12376279 TI - Detection of the high-pathogenicity island of Yersinia enterocolitica in enterotoxigenic and enteropathogenic E.coli strains. AB - OBJECTIVE: To describe the distribution of high-pathogenicity island (HPI) of Yersinia enterocolitica in enterotoxingenic E.coli (ETEC) and enteropathogenic E.coli (EPEC), and to understand the structure and function of HPI. METHODS: PCR was used to detect irp2, fyua and asn-intB genes with subsequent sequence analysis of these genes. Nucleic acid in situ hybridization was employed to identify the specificity of irp2 and fyua. RESULTS: Thirty irp2-positive strains were isolated from 93 ETEC strains and 3 from 10 EPEC strains, making a positivity rate of 32.25% and 30% respectively, and the positivity rates of fyua gene in ETEC and EPEC were 21.51% and 30% respectively. In most of these positive isolates, HPI was bordered by an asn tRNA locus, as in Yersinia sp. CONCLUSIONS: This study demonstrates that the high positivity rate of HPI of Yersinia enterocolitica in ETEC and EPEC strains may be crucial to the virulence changes, virulence evolution and virulence regulation in E.coli. PMID- 12376280 TI - Elastic registration of medical images through multiquadric method. AB - OBJECTIVE: To improve the precision and reliability of elastic registration of the medical images and to simplify the registration process. METHODS: Previous study concerning elastic registration mostly focused on manual selection of the landmarks and then use of adequate interpolating for elastic transformation. The landmarks extraction, however, was prone to error that often showed impact on the registration results, besides the difficulty and time consumption of manual identification of the landmarks. On the basis of Multiquadric method that allowed smooth adjustment of the parameters, we utilized a semi-automatic method to extract the landmarks by combining these 2 steps, and proposed a novel registration method. RESULTS: Using this method for medical image elastic registration, rapid and accurate registration between standard and deformed images was achieved. CONCLUSION: The method proposed presently is accurate, convenient and reliable. PMID- 12376281 TI - Effect of conditioned media from decidual cell culture on the expression of genes regulating the invasion of trophoblastic cells. AB - OBJECTIVE: To investigate the effect of the conditioned media from decidual cell cultures (DCM) on the expression of genes regulating the invasion of trophoblastic cells. METHODS: In vitro culture of decidual cells obtained from healthy women of both early (within the first trimester) and full-term pregnancy respectively was performed to prepare conditioned media of decidual cells (DCM). Trophoblastic cells were also obtained in these subjects and treated with DCM for 24 h in in vitro culture, to observe the effect of DCM, with the help of semi quantitative reverse transcriptase-PCR, on the expression of genes in these cells that regulate their invasion. RESULTS: Expression of matrix metallsoproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1 and urokinase type plasminogen activator (u-PA), other than that of TIMP-2 and plasminogen activator inhibitor type (PAI)-1, was observed in normal trophoblastic cells in in vitro culture. DCM derived from wemon of early and full-term pregnancy down regulated the expression of MMP-2, MMP-9, u-PA while up-regulated the expression of TIMP-1, PAI-1. CONCLUSION: DCM may exhibit its anti-invasive activity by regulating the expression of genes involved in the regulation of trophoblastic cell invasion, such as MMP-2, MMP-9, TIMP-1, u-PA and PAI-1. PMID- 12376282 TI - Construction of pGFP-FL plasmid and its application in preparing FL-secreting tumor vaccine. AB - OBJECTIVE: To construct a plasmid containing a green fluorescent protein (GFP) reporter gene as the effective vector for preparing fms-like tyrosine kinase receptor-3 ligand (FL)-secreting tumor vaccines. METHODS: A pGFP-FL plasmid, harboring a FL gene and a GFP gene, was designed and constructed by routine molecular cloning techniques. In this plasmid, FL gene was under the control of cytomegalovirus promoter, while EF-1a promoter acted to drive GFP gene. A prokaryotic/eukaryotic selective gene Kan(R)/neo was also introduced into the plasmid. After structure identification by restriction analysis, pGFP-FL plasmid was further transferred into Hepa1-6 cells, and the expression of GFP and FL genes was examined by way of fluorescent microscopy and reverse transcriptase-PCR respectively. RESULTS: Restriction analysis showed that the structure of pGFP-FL plasmid was exactly the same as anticipated. Further results indicated that both GFP and FL genes were simultaneously expressed in Hepa1-6 cells. CONCLUSION: A new plasmid has been established as the vector for studying the FL-secreting tumor vaccines, in which GFP gene can serve as a reporter gene reflecting the expression of FL gene. PMID- 12376283 TI - MRI diagnosis of embryonal tumors in the spinal canal: analysis of 6 cases. AB - OBJECTIVE: To evaluate the magnetic resonance imaging (MRI) features characterizing embryonal tumors in the spinal canal. METHODS: Retrospective analyses of the MR images were conducted in 6 patients with surgically and pathologically confirmed embryonal tumors in the spinal canal. RESULTS: Epidermoid cysts in 3 cases displayed a variety of non-characteristic signal intensity patterns on T1- and T2-weighted images (T1WI and T2WI). Lipomas in 2 cases showed high signal intensity on T1WI and attenuated to intermediate signal intensity on T2WI. An intramedullary neurenteric cyst in 1 case was illustrated as low signal intensity on T1WI and high signal intensity on T2WI. CONCLUSION: The MRI presentation of embryonal tumors in the spinal canal with the exception of lipomas is devoid of typical features, and their MRI diagnosis should be complemented by history of developmental anomaly and pathological and etiological findings. PMID- 12376284 TI - Screening of tumor necrosis factor-alpha-binding peptides by phage display peptide library. AB - OBJECTIVE: To identify and characterize tumor necrosis factor (TNF)-alpha-binding peptides from c7c phage display peptide library, in an attempt to find short peptides that can be used as antagonist of TNF-alpha. METHODS: The TNF-alpha binding peptides were screened from c7c phage display peptide library by using rhTNF-alpha as target protein and identified by sandwich ELISA. RESULTS: After 3 rounds of screening, 11 of 23 phage clones were identified as positive clones which can bind to rhTNF-alpha. The amino acid sequence in two of these 11 clones is c-ALWHWWH-c, and that in the others is c-(T/S)WLHWWA-c. CONCLUSION: These phage display peptides are TNFalpha-binding peptides. PMID- 12376285 TI - Clinical anatomy of the fibrous capsule of human lumbar facet joint. AB - OBJECTIVE: To describe the anatomical and histological characteristics of the fibrous capsule of human lumbar facet joints. METHODS: Specimens of the facet joint capsules were obtained from 5 embalmed cadavers for macroscopic investigation, and microscopic observation of L5 facet joint capsules obtained from 2 fresh cadavers was performed after the specimens were stained by way of resorzinoroseine-van Gieson method. RESULTS: The outer layer of the fibrous capsule was constituted by dense regular connective tissue that was in turn composed of parallel bundles of collagenous fibers. In the superior part of the joint capsules, the fibers were arranged in the direction different from that taken by the fibers in the inferior part. In the middle layer of the joint capsules, large quantities of elastic fibers were identified in the roots of the capsule. CONCLUSIONS: The anatomical and histological complexities adapt the lumbar facet joint to better withstand loads from various directions. Immoderate rotatory manipulations may result in capsule injuries, which may aggravate low back pain in some cases. PMID- 12376286 TI - Screening of single nucleotide polymorphisms in nasopharyngeal carcinoma associated genes by denaturing high-performance liquid chromatography. AB - OBJECTIVE: To screen single nucleotide polymorphisms (SNPs) of 4 human genes at 6p21.3 by way of denaturing high-performance liquid chromatography (DHPLC). METHOD: Four exons and 1 intron fragments in the PPP1R11, PPP1R10, FLOT1 and KIAA0170 genes at 6p21.3 isolated from the blood samples from 30 patients with nasopharyngeal carcinoma (NPC) and 28 healthy subjects were amplified by PCR, and the products analyzed by DHPLC. Some fragments of interest were sequenced and compared with the sequences available in National Center for Biotechnology Information (NCBI) database. RESULTS: In the 5 fragments, 4 new SNPs were identified and 4 known SNP loci and genotypes were confirmed. CONCLUSION: DHPLC is an effective, economical, and simple method with reliability for SNPs screening. PMID- 12376288 TI - Establishment of dextran sulfate sodium-induced ulcerative colitis model in mice. AB - OBJECTIVE: To establish a model of ulcerative colitis in mice. METHODS: The mice were given 5% dextran sulfate sodium (DSS) solution freely for 7 consecutive days after which distilled water was given in stead for another 10 d, to complete one cycle of whole treatment plan that consisted of 4 such cycles. The symptoms were observed daily. At the end of the first and the fourth cycle respectively, the mice were killed for examining the whole colon under anatomic microscopy and serial tissue sections were prepared for histological observation. RESULTS: The symptoms observed in the mice including hematochezia diarrhea and loss of the body weight were similar to those of ulcerative colitis patients. Histological examination revealed infiltration of neutrocytophilia and lymphocythemia, with loss of integrity of the colon mucosa in the gland. CONCLUSION: This mouse model of ulcerative colitis can be easily induced and readily applied in various studies of ulcerative colitis. PMID- 12376287 TI - Effect of high temperature on gastrin, somatostatin and motilin production in ulcerous gastric antral mucosa of rats. AB - OBJECTIVE: To investigate the changes of gastrin, somatostatin and motilin production in the gastric antral mucosa of rats with experimental gastric ulcer. METHODS: Rat models of gastric ulcer model were induced successfully by injection of acetic acid into the gastric antral wall of 2 groups of Wistar rats (7 in each group) that were subjected to environment of either high or normal temperature. Another 2 groups of rats (n=7) receiving normal saline injection in the same manner, along with still another 2 groups (n=7) without any treatment, all of which were kept under conditions with different temperatures accordingly, constituted the control groups. The levels of gastrin, somatostatin and motilin in the gastric antral mucosa of the rats were measured with radioimmunoassay. RESULTS: In rats with gastric ulcer, the levels of gastrin and motilin in the antral mucosa increased, but in a lesser scale in rats with ulcer kept in high temperature than in normal temperature group, while that of somatostatin was reduced. The level of somatostatin declined less in the high temperature group with ulcer than in the normal temperature group with ulcer. CONCLUSION: High temperature can affect gastrin, somatostatin and motilin production in the gastric antral mucosa of rats with gastric ulcer. PMID- 12376289 TI - Effects of simulated weightlessness on bone metabolism in rats at different ages. AB - OBJECTIVE: To compare the effects of simulated weightlessness on age-related bone metabolism and on the mechanical parameters of the weight-bearing bones of rats at different ages. METHODS: Two-month-old and 6-month-old rats (8 in each group) were both subjected to tail suspension test for up to 4 weeks, with 2 groups of rats of corresponding ages (n=8) serving as control. The bone metabolism markers, biomechanical parameters of the femurs, along with the growth and mineral contents of the tibia, were respectively measured and compared with those of the controls. RESULTS: The bone formation markers, alkaline phosphatase and osteocalcin levels, dropped drastically in both groups of rats undergoing tail suspension test (P<0.01), which also induced significant hypocalcaemia (P<0.01). Bone material loss of the tibia occurred in both groups of rats receiving the test (P<0.01) whose effect on the volume and mass of fresh tibia was age-related (P<0.05), but the degree of mineral loss was not consistent with calcium loss in rats at different ages. Except for elastic deformation (P>0.05), both structural and biomechanical properties altered significantly after tail suspension test (P<0.01), and the changes of maximum deformation and maximum load were related to the age of the rats (P<0.05). CONCLUSION: The result is an age-related difference in the response of bone metabolism to simulated weightlessness. PMID- 12376290 TI - Establishment of computer-aided acquisition system of complete blood pressure wave parameters in rats. AB - OBJECTIVE: To establish an accurate and efficient system for acquiring the complete blood pressure wave parameters in rats. METHODS: With the help of Powlab, the equipment for physiological data recording, and Chart (soft ware), the basal parameters of the the blood pressure waves were acquired in rats, including the parameters of a single wave and the average parameters recorded in 1 min, which were then processed according to mathematical formulas or calculated approximately to acquire other parameters. RESULTS: Through the system the complete parameters of the blood pressure waves were acquired accurately in rats. Compared with the actual values, the results from some approximate calculation showed little difference. CONCLUSION: A precise and automated system for acquiring complete parameters of the blood pressure waves is successfully established in rats, which makes possible more comprehensive and convincing analysis of the blood pressure waves in rats. PMID- 12376291 TI - Effect of pretreatment with adenosine, diazoxide or ischemic preconditioning on ischemia- reperfusion injury in the limbs of rats. AB - OBJECTIVE: To study the effect of ischemic preconditioning (PC) and pharmacological preconditioning with adenosine or diazoxide on ischemia reperfusion (IR) injury in the limbs of rats. METHODS: According to different treatment received before ischemic-reperfusion injury, 66 SD rats were divided into 6 groups including a normal control and a ischemia-reperfusion control group, IP10 group in which the rats received 10-min ischemia followed by 10-min interval for reperfusion for 3 times before IR, IP5 group in which the rats were subjected to 5-min ischemia with 5-min reperfusion intervals for 3 times before IR, adenosine (Ade) pretreatment group and diazoxide (Dia) pretreatment group. Except the normal control group, which consisted of 6 rats, each group contained 12 rats, and IR injury was induced by blocking the blood flow in bilateral limbs for 4 h, followed by reperfusion for 2 or 24 h when twitch and spastic contractility of the tibialis anterior muscle and serum creatine phosphokinase (CPK) were measured. RESULTS: In IP5 and Ade pretreatment group, the twitch tension of the tibialis anterior muscle of the rats was significantly enhanced after 2 and 24 h of reperfusion, achieving the level of the normal control. The twitch tension was also enhanced in rats of IP10 group at 24 h, but at 2 h, though with less effectiveness than that in IP5 and Ade group. Dia pretreatment reduced twitch and tetanic contraction forces of the tibialis anterior muscle after 2 h of reperfusion, but obviously improved twitch tension at 24 h. Improvement in the tetanic tension, however, was seen in none of the groups. Serum CPK of IP5, IP10 and Ade groups after 2 and 24 h while Dia at only 24 h of reperfusion was obviously lower than that of IR group. CONCLUSIONS: Ischemic and Ade preconditioning can protect the limbs of rats from ischemia-reperfusion injury, and Dia has delayed protective effect. IP5 is superior than IP10 and Ade PC can produce similar effect to that of ischemic PC. PMID- 12376292 TI - In vitro amplification of Toxoplasma gondii rhoptry protein 2 gene and construction of its eukaryotic expression plasmid. AB - OBJECTIVE: To construct a recombinant eukaryotic expression plasmid containing rhoptry protein 2 (ROP2) gene of Toxoplasma gondii. METHOD: The truncated ROP2 gene was amplified from the genomic DNA of Toxoplasma gondii RH strain and cloned into plasmid pGEX-4T-1. The PCR product was subcloned into an eukaryotic expression plasmid pcDNA3 which was identified by enzyme digestion and PCR amplification. Sequence analysis of the insert in the recombinant plasmid was performed by Sanger's method. RESULTS: The amplified DNA fragment was about 1043 bp in length, with identical sequence with the published ROP2 gene sequence. Enzyme digestion and PCR analysis showed that the truncated ROP2 gene had been successfully cloned into plasmid pGEX-4T-1 and pcDNA3. CONCLUSION: The truncated ROP2 gene of Toxoplasma gondii has been amplified and cloned into the eukaryotic expression plasmid pcDNA3. PMID- 12376293 TI - In vitro culture and study of the biological characteristics of rabbit keratocytes. AB - OBJECTIVE: To improve the technique for culturing rabbit keratocytes in vitro and investigate the biological characteristics of these cells. METHODS: Fresh rabbit corneas were obtained and the epithelial and endothelial cells were removed after digestion with trypsin. The stroma was rinsed, minced, and incubated in DMEM/F12 medium supplemented with 10% fetal bovine serum and the biological characteristics of the keratocytes were observed with MTT assay and compared with those of the cells in serum-free culture media. RESULT: On about the third day of incubation, some keratocytes germinated from the stromal tissues and migrated onto the flask surface presenting fibroblast-like arrangement with spindle-shaped appearance. The keratocytes became confluent after 10 day's incubation and the peak of cell proliferation occurred on day 8 in the presence of serum in the media, while in the absence of serum, the peak took place on day 10. CONCLUSION: The method improved for in vitro keratocyte culture is convenient and effective, and the presence of serum in the media may to some degree affect the growth of the keratocytes. PMID- 12376294 TI - Effect of protein kinase C on multidrug resistance of multidrug-resistant colorectal cancer LoVo/Adr cells. AB - OBJECTIVE: To observe the effect of protein kinase C (PKC) on the multidrug resistance of multidrug-resistant colorectal cancer LoVo/Adr cells and explore the mechanism. METHODS: The changes of PKC activity in LoVo/Adr cells in response to treatment with staurosporine (SP) and phorbol-12-myristate-13-acetate (PMA) were detected by way of 32P incorporation. The effect of PKC on adriamycin uptake in LoVo/Adr cells was detected by flow cytometry. Reverse transcriptase-PCR was utilized to observe the effect of PKC on mdr1 gene expression. RESULTS: PMA evinced bi-directional regulation of PKC activity in LoVo/Adr cells, and SP significantly inhibited membrane and cytosol fraction of PKC activity. Preincubation with PMA for 30 min caused the uptake of adriamycin to decrease significantly, but when the preincubation was prolonged to 24 h, significant increase occurred in adriamycin uptake. Neither PMA nor SP, however, could affect the expression of mdr1 gene. CONCLUSION: PKC regulates multidrug resistance of the cells through mechanisms other than regulation of mRNA level of mdr1 gene. PMID- 12376295 TI - Endothelial NO synthase gene expression in experimental vasospasm in rabbits. AB - OBJECTIVE: To investigate the relationship between endothelial NO synthase (eNOS) gene expression and experimental vasospasm. METHODS: Sixteen rabbits were randomly divided into 2 groups, which were subjected to balloon endothelial denudation with normal diet (n=8) or with hypercholesterol diet group (n=8). Angiography was performed to detect the vasospasm induced by ergonovine before and after denudation and 8 weeks after hypercholesterol feeding. In situ hybridization and Northern blotting were performed to localize and quantitate respectively the expression of eNOS mRNA. RESULTS: Visible vasospasm was induced at the denuded sites in rabbits with hypercholesterol diet, in which the expression of eNOS mRNA was detected in the endothelium by in situ hybridization at a lower level than that of rabbits with normal diet, as demonstrated by Northern blotting. CONCLUSION: The decrement of eNOS mRNA expression resulted from balloon endothelial denudation and hypercholesterolemia may play an important role in the pathogenesis of experimental vasospasm. PMID- 12376296 TI - Changes of plasma adrenomedullin and proadrenomedullin N-terminal 20 peptide concentrations in patients with heart failure. AB - OBJECTIVE: To study the changes in plasma adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) concentrations and their clinical significance in the pathological process of congestive heart failure (CHF). METHODS: Plasma ADM and PAMP concentrations in 45 patients with CHF (according to the functional classification of New York Heart Association, NYHA) and 20 control subjects were measured by specific radioimmunoassay. RESULTS: Plasma ADM concentrations were 51.464+/-.52 pg/ml and 70.39+/-3.22 pg/ml respectively in patients of NYHA class II and class III, which were significantly higher than those in control subjects (24.12+/-1.59 pg/ml, P<0.05 for both comparisons), while significant differences in plasma PAMP concentrations were not identified in the 2 groups of patients (6.24+/-1.71 pg/ml and 7.38+/-1.28 pg/ml, respectively) in comparison with the control level(8.56+/-2.44 pg/ml, P>0.05 for both comparisons). Patients of NYHA class IV, when compared with the 2 groups of patients mentioned above, had significantly decreased plasma ADM and PAMP concentrations (36.33+/-2.17 pg/ml and 2.79+/-0.89 pg/ml respectively, P<0.05 in both cases), but had higher plasma ADM and lower PAMP concentrations when compared with the control subjects, (P<0.05 respectively). CONCLUSION: The changes of plasma ADM and PAMP concentrations at different stages of CHF indicate intramolecular regulation disturbances of vasodilator peptides of proadrenomedullin, and ADM may play a more important role in the development of CHF. PMID- 12376297 TI - Serum leptin levels of menopausal women before and after hormone replacement therapy. AB - OBJECTIVE: To measure serum leptin levels of menopausal women before and after hormone replacement therapy, and to evaluate the action of hormone replacement therapy in regulating serum leptin levels. METHODS: Serum leptin levels of 27 menopausal women were measured before and after hormone replacement therapy by double-antibody enzyme-linked immunosorbent assay (ELISA) and compared with those measured in 35 women with regular menstrual cycle. RESULTS: Compared with normal control (17.11+/-1.24 ng/ml), menopausal women showed significant higher mean serum leptin levels (20.75+/-2.80 ng/ml, P<0.01). After hormone replacement therapy for 6 months, serum leptin levels in these women declined to (17.46+/ 1.71 ng/ml), similar to the leptin levels of normal control (P>0.05). CONCLUSION: Serum leptin levels are significantly higher in menopausal women than in women with regular menstrual cycle, and can be down-regulated by hormone replacement therapy. It is suggested that serum leptin levels might be used as an indicator to evaluate the effect of hormone replacement therapy in menopausal women. PMID- 12376298 TI - Expression of endogenous heme oxygenase on surface of placental trophoblasts of pregnant women with intrauterine growth retardation of the fetus. AB - OBJECTIVE: To examine endogenous heme oxygenase (HO) expression on the surface of placental trophoblasts of pregnant women who had a fetus with intrauterine growth retardation (IUGR), so as to explore the pathogenesis of IUGR. METHODS: Immunohistochemical method were used to detect the expression of HO-1 in idiopathic IUGR patients (IUGR group), patients with pregnancy induced hypertension (PIH) complicated by IUGR (PIH+IUGR group) and normal pregnant women (control group) with specific HO antibody. Quantitative analysis was conducted to determine the quantity of HO. RESULT: The staining areas of HO-1 in IUGR group (64.27+/-10.59 micrometer(2)) and PIH+IUGR group (61.54+/-10.59 micrometer(2)) were significantly lower than that of control group (96.56+/-15.23 micrometer(2), P<0.01), but there was no significant differences between the former 2 groups. CONCLUSION: Abnormal reduction in endogenous HO on the surface of placental trophoblasts may be one of the important mechanisms for the onset of IUGR. PMID- 12376299 TI - Therapeutic approaches for chronic gastralgia based on differentiation of symptoms and signs. AB - OBJECTIVE: To report our clinical experience with the treatment of chronic gastralgia on the basis of the differentiation of symptoms and signs. METHOD: A total of 160 cases of chronic gastralgia we treated in recent years were reviewed. RESULTS: Among the 160 cases, 120 falls in the category characterized by the predominant clinical symptoms such as depression of liver-energy and spleen-asthenia. For the other 40 cases, their clinical symptoms are of deficiency of stomach-yin, and retention of damp-heat in the interior or accumulation of blood stasis constituted the major clinical presentations, but more or less featured by depression of liver-energy and spleen-asthenia. Through analysis by differentiation of the symptoms and signs, we were convinced that the major symptoms of chronic gastralgia and some accompanying minor symptoms were all the representation and extension of the underlying cause, disorder of stomach energy. The treatment was therefore targeted at dispersing the depression of liver-energy and invigorating the spleen, smoothing and regulating the stomach energy. The treatment adopts primary prescription and good effects were achieved. CONCLUSION: The pathogenesis of chronic gastralgia predominantly lies in the depression of liver-energy and spleen-asthenia, together with disordered stomach energy. The treatment should be implemented to disperse the depression of liver energy and invigorate the spleen, with attention to smoothing and regulating the stomach-energy throughout the whole treatment course. PMID- 12376300 TI - Detection of soluble tumor necrosis factor-p55 levels in the serum and ascitic fluid of patients with hepatocellular carcinoma. AB - OBJECTIVE: To examine soluble tumor necrosis factor receptor-p55 (sTNFR-p55) levels in the serum and ascitic fluid and investigate the significance of this examination in assessment of the clinical status of patients with primary hepatocellular carcinoma (HCC). METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to examine sTNFR-p55 levels in the serum and ascitic fluid in 25 patients with HCC and 25 with liver cirrhosis (LC). RESULTS: sTNFR-p55 levels in the serum and ascitic fluid in patients with HCC were significantly higher than those in patients with LC and controls (P=0.001). No significant difference was found between LC and the control in terms of serum sTNFR-p55 levels (P=0.19). Positive correlation was observed between sTNFR-p55 levels in the serum and in ascitic fluid of patients with HCC and LC (r=1.000, P<0.001). Logistic regression revealed that in patients with HCC, serum sTNFR-p55 levels were positively correlated with TBil and AFP in the peripheral blood (r=0.524, P=0.01 and r=0.234, P=0.03, respectively). CONCLUSIONS: Increased sTNFRs-p55 levels in the serum and ascitic fluid reflect abnormal immune status of the patients with HCC and help predict the development of tumor. PMID- 12376301 TI - Radiosurgical treatment of intractable epilepsy with low radiation dose. AB - OBJECTIVE: To localize the epileptic foci with positron emission tomography (PET), and study the principles of target definition and method to determine the optimal range of exposure in radiosurgery for intractable epilepsy. METHODS: This study included 176 patients with intractable epilepsy, who received linear accelerator radiosurgery after (18)F-FDG PET for epileptic foci localization. The patients were divided according to different peripheral doses used in the treatment into Group A in which radiation dose of 9 to 11 Gy was used, Group B with 11 to 13 Gy and Group C with exposure to over 13 Gy. Follow-up study was conducted in all the patients for a period ranging from 3 to 16 months, during which the frequency of seizure after treatment was recorded to evaluate the therapeutic effect. RESULTS: The seizure frequency significantly decreased after radiosurgical treatment in all the groups, but between the groups, the decrement evinced no significant difference. According to Wieser's classification of the effect after operation, 46.9% cases belonged to grade I to II and 41.5% to grade III to IV. Obvious complications were not observed, nor did disability or mortality occurred in these cases. CONCLUSIONS: Stereotactic radiosurgery with low radiation dose under the guidance of PET provides a safe, effective and minimally invasive surgical approach for patients with intractable epilepsy, and peripheral radiation doses of 9 to 11 Gy for the epileptic foci localized by PET is sufficient to ensure good clinical outcome. PMID- 12376302 TI - Application of polyacrylamide hydrogel in augmentation mammoplasty. AB - OBJECTIVE: To review our experience with the application of polyacylamide hydrogel (PaH) as an injectable filling material in augmentation mammoplasty. METHODS: From Oct. 1997 to Mar. 2002, PaH was used in 800 patients with breasts of insufficient size to be commensurate with the body. PaH was injected into the cavity under the breast tissue at a chosen point at the level of submammary crease. RESULTS: The reconstructed beast by injection of PaH was satisfactorily improved in terms of the shape and feel in comparison with the state before the operations. The total incidence of complications was 12%, but all of them were corrected. CONCLUSION: PaH is an ideal filling material in augmentation mammoplasty with such merits as safety, operability, good tolerability and absence of postoperative scars, to ensure excellent effect of the operation. PMID- 12376303 TI - Glycyrrhizic acid content in Gegenqinlian decoctions prepared according to different prescriptions. AB - OBJECTIVE: To compare the differences in glycyrrhizic acid contents of Gegenqinlian decoctions (a traditional Chinese herbal preparation) prepared according to varied prescription, so as to establish a referential standard for controlling the acid level in such preparations. METHODS: Reverse phase-high performance liquid chromatography (RP-HPLC) was employed to determine the content of glycyrrhizic acid in different Gegenqinlian decoctions. RESULTS: Glycyrrhizic acid contents in the decoction of exclusive liquorice was 0.89%, and in Gegenqinlian decoctions composed of 2 or 3 ingredients, the acid contents varied from 0.24%, to 0.59%,. CONCLUSION: The presence of the constituents in Gegenqinlian decoction such as radices puerarire, radices scutellariae and coptis may significantly reduce the release of glycyrrhizic acid content from within liquorice into the decoction. PMID- 12376304 TI - Analysis with gas chromatography and mass spectrography of volatile components of Ligusticum wallichii Franch extracted by CO2 supercritical fluid extraction in combination with molecular distillation. AB - OBJECTIVE: To evaluate the possibility of using the technique of CO2 supercritical fluid extraction (SFE) combined with molecular distillation (MD) to extract, separate and purify the volatile components of Ligusticum wallichii Franch, a Chinese herbal medicine. METHODS: SFE was employed to extract the volatile components of Ligusticum wallichii Franch, followed by MD of the product. Analysis of the chemical constituents of the extract both before and after MD was performed with gas chromatography (GC) in conjunction with mass spectrography (MS). RESULTS: There were 45 kinds of chemical constituents in the extract of Ligusticum wallichii Franch yielded from SFE, among which 39 were left after distillation by MD. Changes also took place in the content of the constituents in the extract after distillation. CONCLUSIONS: SFE combined with MD, a technique better than simple SFE, can be used to extract, separate and purify the volatile components of Chinese herbs. PMID- 12376305 TI - Determination of L-sesamin and L-asarinin in Zanthoxylum(Roxb.) DC. by high performance liquid chromatography. AB - OBJECTIVE: To establish a method for determining the content of L-sesamin and L asarinin indifferent parts of Zanthoxylum(Roxb.)DC. METHOD: High-performance liquid chromatography (HPLC) was utilized for this purpose with the column of HYPERSIL BDS C18 and the mobile phase containing acetonitrile and water (50:50), the detection wavelength being 287 nm. RESULTS: The average recovery rates for L sesamin and L-asarinin were 100.13% and 1.61% and their relative standard deviation (RSD) were 101.24% and 1.46% respectively. CONCLUSION: The method can be used for the quality control of Zanthoxylum(Roxb.)DC, and the roots of this herb contains more L-sesamin and L-asarinin than its other parts do. PMID- 12376306 TI - Sources and elimination of interference in patch clamp electrophysiological experiment. AB - OBJECTIVE: The sources of the interference in the patch clamp electrophysiological experiments were investigated, and the methods for eliminating the interference, along with the authors experience, are introduced. PMID- 12376307 TI - Treatment of stroke with "five-center needling": clinical observation of 78 cases. AB - OBJECTIVE: To observe the efficacy of "five center needling" in the management of stroke. METHOD: Seventy-eight patients suffering stroke were divided into 2 groups to receive either routine acupuncture (control group, n=38) treatment or "five-center needing" therapy besides the routine treatment (treatment group, n=40). The curative effect between the 2 groups was compared after 30 days treatment. RESULT: No significant differences were noted between the 2 groups in terms of the curative effect in general (P>0.05). But "five-center needling" showed better effect in treating the syndromes of wind-fire stirring up the orifices and phlegm stagnation leading to mental disorder, and was more effective in the management of hemorrhagic stroke than in ischemic stroke, and in treating the left-side cerebral injuries than in treating those on right side (P<0.05). PMID- 12376308 TI - Single photon emission computerized tomography of spongiform leukoencephalopathy heroin addicts: analysis of 10 cases. AB - OBJECTIVE: To investigate the changes in cerebral circulation in heroin addicts with pongiform leukoencephalopathy. METHODS: Single photon emission computerized tomography (SPECT) was performed in 10 such patients. RESULTS: Regional cerebral blood flow (rCBF) in the involved white matter of bilateral cerebral and cerebellar hemispheres was obviously reduced. rCBF of temporal lobes, parietal lobes, cerebellar hemispheres and basal ganglion were reduced in varying degrees. CONCLUSION: As demonstrated by the result of SPECT, rCBF in the white matter of bilateral cerebral and cerebellar hemispheres was reduced with partial involvement of the gray matter of heroin addicts with spongiform leukoencephalopathy. PMID- 12376309 TI - Application of electromagnetic navigation in surgical treatment of intracranial tumors: analysis of 12 cases. AB - OBJECTIVE: To explore the application and characteristics of electromagnetic navigation in neurosurgical operation. METHODS: Neurosurgical operations with the assistance of electromagnetic navigation were performed in 12 patients with intracranial tumors. RESULTS: Total removal of the tumor was achieved in 8 cases, subtotal removal in 3 and removal of the majority of the tumor in 1 case. The error in the navigation averaged 1.9+/-0.9 mm and the time consumed by preoperative preparation was 19+/-2 min with the exception in 1 case. CONCLUSION: In comparison with optic navigation, electromagnetic navigation offers better convenience and absence of signal blockage, and with a head frame, automatic registration can be achieved. PMID- 12376310 TI - Ultrasound ablation of intravascular plaque for treating atherosclerosis obliterans of bilateral iliofemoral arteries: report of one case. AB - OBJECTIVE: A patient with atherosclerosis obliterans of bilateral iliofemoral arteries was successfully treated by ultrasound ablation of intravascular plaque, who had an uneventful postoperative recovery. PMID- 12376311 TI - Fibrous dysplasia of the skull complicated with meningioma: report of 2 cases. AB - OBJECTIVE: Two cases of fibrous dysplasia of the skull complicated with meningioma that are seldom seen were reported. PMID- 12376312 TI - General considerations in describing materials and methods in English medical papers. AB - OBJECTIVE: On the basis of a review of the requirements by some famous medical journals, along with personal experiences, the authors summarize the general guidelines for writing Materials and Methods section in English medical papers, which could be of value for potential contributors of English research papers. PMID- 12376313 TI - Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment. AB - We previously demonstrated that C75, a specific and potent inhibitor of fatty acid synthase (FAS), reduced food intake and decreased body weight in mice. In the present study, we determined that these effects were not due to conditioned taste aversion. To investigate the mechanism of C75 action, we examined FAS brain expression. FAS was expressed in a number of brain regions, including arcuate and paraventricular nuclei (PVN) within regions that comprise the arcuate-PVN pathway in mouse and human. Although C75 and fasting significantly downregulated liver FAS, FAS levels remained high in hypothalamus, indicating that FAS levels were regulated differently in brain from those in liver. Double fluorescence in situ for FAS and neuropeptide Y (NPY) showed that FAS co-localized with NPY in neurons in the arcuate nucleus. NPY immnuoreactivity after C75 treatment was decreased in axon terminals that innervate the PVN and lateral hypothalamus. Collectively, these results demonstrate that FAS is present and active in neurons and suggests that C75 may alter food intake via interactions within the arcuate-PVN pathway mediated by NPY. PMID- 12376314 TI - Potentiation of insulin secretion by phorbol esters is mediated by PKC-alpha and nPKC isoforms. AB - Culturing clonal beta-cells (HIT-T15) overnight in the presence of phorbol ester [phorbol myristate acetate (PMA)] enhanced insulin secretion while causing downregulation of some protein kinase C (PKC) isoforms and most PKC activity. We show here that this enhanced secretion required the retention of PMA in the cell. Hence, it could not be because of long-lived phosphorylation of cellular substrates by the isoforms that were downregulated, namely PKC-alpha, -betaII, and -epsilon, but could be because of the continued activation of the two remaining diacylglycerol-sensitive isoforms delta and mu. The enhanced secretion did not involve changes in glucose metabolism, cell membrane potential, or intracellular Ca2+ handling, suggesting a distal effect. PMA washout caused the loss of the enhanced response, but secretion was then stimulated by acute readdition of PMA or bombesin. The magnitude of this restimulation appeared dependent on the mass of PKC-alpha, which was rapidly resynthesized during PMA washout. Therefore, stimulation of insulin secretion by PMA, and presumably by endogenous diacylglycerol, involves the activation of PKC isoforms delta and/or mu, and also PKC-alpha. PMID- 12376315 TI - Metabolic and cardiorespiratory responses to "the lactate clamp". AB - To evaluate the hypothesis that precursor supply limits gluconeogenesis (GNG) during exercise, we examined training-induced changes in glucose kinetics [rates of appearance (R(a)) and disappearance (R(d))], oxidation (R(ox)), and recycling (R(r)) with an exogenous lactate infusion to 3.5-4.0 mM during rest and to pretraining 65% peak O(2) consumption (VO(2 peak)) levels during exercise. Control and clamped trials (LC) were performed at rest pre- (P(R)R, P(R)R-LC) and posttraining (P(O)R, P(O)R-LC) and during exercise pre- (P(R)E(X)) and posttraining at absolute (P(O)A(B), P(O)A(B)-LC) and relative (P(O)R(L), P(O)R(L) LC) intensities. Glucose R(r) was not different in any rest or exercise condition. Glucose R(a) did not differ as a result of LC. Glucose R(ox) was significantly decreased with LC at P(O)R (0.38 +/- 0.03 vs. 0.56 +/- 0.04 mg. kg( 1). min(-1)) and P(O)A(B) (3.82 +/- 0.51 vs. 5.0 +/- 0.62 mg. kg(-1). min(-1)). Percent glucose R(d) oxidized decreased with all LC except P(O)R(L)-LC (P(R)R, 32%; P(R)R-LC, 22%; P(O)R, 27%; P(O)R-LC, 20%; P(O)A(B), 95%; P(O)A(B)-LC, 77%), which resulted in a significant increase in oxidation from alternative carbohydrate (CHO) sources at rest and P(O)A(B). We conclude that 1) increased arterial [lactate] did not increase glucose R(r) measured during rest or exercise after training, 2) glucose disposal or production did not change with increased precursor supply, and 3) infusion of exogenous CHO in the form of lactate resulted in the decrease of glucose R(ox). PMID- 12376316 TI - Effect of endotoxin on expression of TNF receptors and transport of TNF-alpha at the blood-brain barrier of the rat. AB - The transport mechanism mediating brain uptake of tumor necrosis factor (TNF) alpha has been studied. When (125)I-labeled rat TNF-alpha was used in internal carotid artery perfusions in rats, the cytokine showed transcytosis through the blood-brain barrier in intact form (permeability-surface area product 0.34 +/- 0.13 microl. min(-1). g(-1)). Uptake was inhibited by low nanomolar concentrations of unlabeled rat TNF-alpha. Human TNF-alpha, which does not interact with the p80 TNF receptor in rodents, showed no brain uptake. mRNA expression of both p60 and p80 receptors could be demonstrated in native brain microvessel preparations. These transcripts increased to 149% (p60) and 127% (p80) of control 4 h after a systemic immune stimulation (2 mg/kg bacterial endotoxin ip). Lipopolysaccharide treatment did not alter the rate of brain uptake of TNF-alpha measured between 4 and 24 h later. In conclusion, a receptor mediated mechanism is responsible for the transcytosis of TNF-alpha. Saturable transport, requiring the p80 receptor, occurs at concentrations encountered under pathophysiological conditions and therefore constitutes a relevant mechanism of communication between the immune system and the brain. PMID- 12376317 TI - Endotoxemia reduces skeletal muscle protein synthesis in neonates. AB - Protein synthesis in skeletal muscle is reduced by as much as 50% as early as 4 h after a septic challenge in adults. However, the effect of sepsis on muscle protein synthesis has not been determined in neonates, a highly anabolic population whose muscle protein synthesis rates are elevated and uniquely sensitive to insulin and amino acid stimulation. Neonatal piglets (n = 10/group) were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 microg. kg(-1). h(-1)]. Plasma amino acid and glucose concentrations were kept at the fed level by infusion of dextrose and a balanced amino acid mixture. Fractional protein synthesis rates were determined by use of a flooding dose of [(3)H]phenylalanine. LPS infusion produced a septic-like state, as indicated by an early and sustained elevation in body temperature, heart rate, and plasma tumor necrosis factor-alpha, interleukin-1, cortisol, and lactate concentrations. Plasma levels of insulin increased, whereas glucose and amino acids decreased, suggesting the absence of insulin resistance. LPS significantly reduced protein synthesis in longissimus dorsi muscle by only 11% and in gastrocnemius by only 15%, but it had no significant effect in masseter and cardiac muscles. LPS increased protein synthesis in the liver (22%), spleen (28%), kidney (53%), jejunum (19%), diaphragm (21%), lung (50%), and skin (13%), but not in the stomach, pancreas, or brain. These findings suggest that, when substrate supply is maintained, skeletal muscle protein synthesis in neonates compared with adults is relatively resistant to the catabolic effects of sepsis. PMID- 12376318 TI - Alcohol impairs insulin and IGF-I stimulation of S6K1 but not 4E-BP1 in skeletal muscle. AB - The present study determined whether acute alcohol (ethanol; EtOH) intoxication in rats impaired components of the insulin- and IGF-I-signaling pathway in skeletal muscle. Rats were administered EtOH, and 2.5 h thereafter either insulin, IGF-I, or saline was injected and the gastrocnemius removed. EtOH did not alter the total amount or tyrosine phosphorylation of the insulin receptor, IGF-I receptor, insulin receptor substrate (IRS)-1, or protein kinase B (PKB)/Akt under basal or hormone-stimulated conditions. In contrast, the ability of insulin or IGF-I to phosphorylate T389 and T421/S424 on S6K-1 was markedly diminished by EtOH, and these changes were associated with a reduction in the phosphorylation of the ribosomal protein S6. Under basal conditions, EtOH altered the distribution of eukaryotic initiation factor (eIF)4E, as evidenced by a decreased amount of active eIF4E. eIF4G complex, an increased amount of inactive eIF4E. 4E binding protein (BP)1 complex, and decreased 4E-BP1 phosphorylation. In contrast, EtOH did not impair the ability of either hormone to reverse the changes in eIF4E distribution or 4E-BP1 phosphorylation. Pretreatment with a glucocorticoid receptor antagonist was unable to attenuate either the basal EtOH-induced changes in eIF4E distribution or the impaired ability of IGF-I to stimulate S6K1 and S6 phosphorylation. Hence, acute alcohol intoxication alters selected aspects of translational control under both basal and anabolic hormone-stimulated conditions in skeletal muscle in a glucocorticoid-independent manner. PMID- 12376319 TI - Effect of training on the GH/IGF-I axis during exercise in middle-aged men: relationship to glucose homeostasis. AB - The aim of this study was to compare circulating levels of growth hormone (GH), IGF-I, and IGF-binding protein (IGFBP)-1 and IGFBP-3 in response to a long duration endurance exercise in trained vs. sedentary middle-aged males and to determine whether a relationship with glucose homeostasis exists. Seven trained men (Tr) were compared with seven age-matched sedentary men (Sed) during two trials of 60 min of cycling exercise performed below (-VT) and above (+VT) the ventilatory threshold. Insulin sensitivity (S(I)) was higher in Tr than in Sed (P < 0.001). Basal GH, IGF-I, and IGFBP-1 and -3 were higher in Tr (P < 0.05). During +VT, Tr had a threefold higher GH response, whereas their blood glucose level was better maintained (P < 0.05). Basal IGFBP-1 was correlated with S(I) (P < 0.01). These data indicate that endurance training in middle-aged men increased the activity of the GH/IGF-I system and improved glucoregulation both at rest and during high-intensity endurance exercise. PMID- 12376320 TI - Impaired glucose homeostasis in insulin-like growth factor-binding protein-3 transgenic mice. AB - Glucose homeostasis was examined in male transgenic (Tg) mice that overexpressed the human insulin-like growth factor (IGF)-binding protein (IGFBP)-3 cDNA, driven by either the cytomegalovirus (CMV) or the phosphoglycerate kinase (PGK) promoter. The Tg mice of both lineages demonstrated increased serum levels of human (h) IGFBP-3 and total IGF-I compared with wild-type (Wt) mice. Fasting blood glucose levels were significantly elevated in 8-wk-old CMV-binding protein (CMVBP)-3- and PGK binding protein (PGKBP)-3-Tg mice compared with Wt mice: 6.35 +/- 0.22 and 5.22 +/- 0.39 vs. 3.99 +/- 0.26 mmol/l, respectively. Plasma insulin was significantly elevated only in CMVBP-3-Tg mice. The responses to a glucose challenge were significantly increased in both Tg strains: area under the glucose curve = 1,824 +/- 65 and 1,910 +/- 115 vs. 1,590 +/- 67 mmol. l(-1). min for CMVBP-3, PGKBP-3, and Wt mice, respectively. The hypoglycemic effects of insulin and IGF-I were significantly attenuated in Tg mice compared with Wt mice. There were no differences in adipose tissue resistin, retinoid X receptor-alpha, or peroxisome proliferator-activated receptor-gamma mRNA levels between Tg and Wt mice. Uptake of 2-deoxyglucose was reduced in muscle and adipose tissue from Tg mice compared with Wt mice. These data demonstrate that overexpression of hIGFBP 3 results in fasting hyperglycemia, impaired glucose tolerance, and insulin resistance. PMID- 12376321 TI - Effects of fasting and glucocorticoids on hepatic gluconeogenesis assessed using two independent methods in vivo. AB - The purpose of this study was to compare the assessment of gluconeogenesis (GNG) in the overnight- and prolonged-fasted states and during chronic hypercortisolemia using the arteriovenous difference and [14C]phosphoenolpyruvate liver biopsy techniques as well as a combination of the two. Two weeks before a study, catheters and flow probes were implanted in the hepatic and portal veins and femoral artery of dogs. Animals were studied after an 18-h fast (n = 8), a 42 or 66-h fast (n = 7), and an 18-h fast plus a continuous infusion of cortisol (3.0 microg. kg(-1). min(-1)) for 72 h (n = 7). Each experiment consisted of an 80-min tracer ([3-(3)H]glucose and [U-(14)C]alanine) and dye equilibration period (-80 to 0 min) and a 45-min sampling period. In the cortisol-treated group, plasma cortisol increased fivefold. In the overnight-fasted group, total GNG flux rate (GNG(flux)), conversion of glucose 6-phosphate to glucose (GNG(G-6-P- >Glc)), glucose cycling, and maximal GNG flux rate (GNG(max)) were 0.95 +/- 0.14, 0.65 +/- 0.06, 0.62 +/- 0.06, and 0.70 +/- 0.09 mg. kg(-1). min(-1), respectively. In the prolonged-fasted group, they were 1.50 +/- 0.18, 1.18 +/- 0.13, 0.40 +/- 0.07, and 1.28 +/- 0.10 mg. kg(-1). min(-1), whereas in the cortisol-treated group they were 1.64 +/- 0.33, 0.99 +/- 0.29, 1.32 +/- 0.24, and 0.91 +/- 0.13 mg. kg(-1). min(-1). These results demonstrate that GNG(G-6-P- >Glc) and GNG(max) were almost identical. However, these rates were 15-38% lower than GNG(flux) generated by a combination of the two methods. This difference was most apparent in the steroid-treated group, where the combination of the two methods (GNG(flux)) detected a significant increase in gluconeogenic flux. PMID- 12376322 TI - Involvement of the vagus nerves in the regulation of basal hepatic glucose production in conscious dogs. AB - We determined if blocking transmission in the fibers of the vagus nerves would affect basal hepatic glucose metabolism in the 18-h-fasted conscious dog. A pancreatic clamp (somatostatin, basal portal insulin, and glucagon) was employed. A 40-min control period was followed by a 90-min test period. In one group, stainless steel cooling coils (Sham, n = 5) were perfused with a 37 degrees C solution, while in the other (Cool, n = 6), the coils were perfused with -20 degrees C solution. Vagal blockade was verified by heart rate change (80 +/- 9 to 84 +/- 14 beats/min in Sham; 98 +/- 12 to 193 +/- 22 beats/min in Cool). The arterial glucose level was kept euglycemic by glucose infusion. No change in tracer-determined glucose production occurred in Sham, whereas in Cool it dropped significantly (2.4 +/- 0.4 to 1.9 +/- 0.4 mg. kg(-1). min(-1)). Net hepatic glucose output did not change in Sham but decreased from 1.9 +/- 0.3 to 1.3 +/- 0.3 mg. kg(-1). min(-1) in the Cool group. Hepatic gluconeogenesis did not change in either group. These data suggest that vagal blockade acutely modulates hepatic glucose production by inhibiting glycogenolysis. PMID- 12376323 TI - AMP kinase activation ameliorates insulin resistance induced by free fatty acids in rat skeletal muscle. AB - We examined whether acute activation of 5'-AMP-activated protein kinase (AMPK) by 5'-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) ameliorates insulin resistance in isolated rat skeletal muscle. Insulin resistance was induced in extensor digitorum longus (EDL) muscles by prolonged exposure to 1.6 mM palmitate, which inhibited insulin-stimulated glycogen synthesis to 51% of control after 5 h of incubation. Insulin-stimulated glucose transport was less affected (22% of control). The decrease in glycogen synthesis was accompanied by decreased glycogen synthase (GS) activity and increased GS phosphorylation. When including 2 mM AICAR in the last hour of the 5-h incubation with palmitate, the inhibitory effect of palmitate on insulin-stimulated glycogen synthesis and glucose transport was eliminated. This effect of AICAR was accompanied by activation of AMPK. Importantly, AMPK inhibition was able to prevent this effect. Neither treatment affected total glycogen content. However, glucose 6-phosphate was increased after inclusion of AICAR, indicating increased influx of glucose. No effect of AICAR on the inhibited insulin-stimulated GS activity or increased GS phosphorylation by palmitate could be detected. Thus the mechanism by which AMPK activation ameliorates the lipid-induced insulin resistance probably involves induction of compensatory mechanisms overriding the insulin resistance. Our results emphasize AMPK as a promising molecular target for treatment of insulin resistance. PMID- 12376324 TI - Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5'-flanking region. AB - In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5'-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5'-flanking region of sgk1. PMID- 12376325 TI - Central stimulatory effect of leptin on T3 production is mediated by brown adipose tissue type II deiodinase. AB - To assess whether intracerebroventricular leptin administration affects monodeiodinase type II (D2) activity in the tissues where it is expressed [cerebral cortex, hypothalamus, pituitary, and brown adipose tissue (BAT)], hepatic monodeiodinase type I (D1) activity was inhibited with propylthiouracil (PTU), and small doses of thyroxine (T4; 0.6 nmol. 100 g body wt(-1). day(-1)) were supplemented to compensate for the PTU-induced hypothyroidism. Two groups of rats were infused with leptin for 6 days, one of them being additionally treated with reverse triiodothyronine (rT3), an inhibitor of D2. Control rats were infused with vehicle and pair-fed the amount of food consumed by leptin-infused animals. Central leptin administration produced marked increases in D2 mRNA expression and activity in BAT, changes that were likely responsible for increased plasma T3 and decreased plasma T4 levels. Indeed, plasma T3 and T4 concentrations were unaltered by central leptin administration in the presence of rT3. The additional observation of a leptin-induced increased mRNA expression of BAT uncoupling protein-1 suggested that the effect on BAT D2 may be mediated by the sympathetic nervous system. PMID- 12376326 TI - Ethnic differences in in vitro glyceride synthesis in subcutaneous and omental adipose tissue. AB - Considerable evidence suggests that there are ethnic differences in lipid metabolism between African American and Caucasian women, which may result in increased synthesis of fat in adipose tissue. The purpose of this study was to measure the in vitro rates of [14C]glucose incorporation into the glyceride glycerol backbone of triglycerides (TG) and diglycerides (DG) in abdominal subcutaneous (SAT) and omental adipose tissue (OAT). Morbidly obese [African American (n = 15): body mass index (BMI) = 45 +/- 2.3; Caucasian (n = 18): BMI = 51 +/- 2.3] and preobese [African American (n = 7): BMI = 27 +/- 1.0; Caucasian (n = 7): BMI = 25 +/- 1.0] women were examined in this study. There were no significant differences in the rates of synthesis of either TG or DG in SAT of either preobese or obese women. On the other hand, both preobese and obese African American women had higher rates of synthesis of TG in OAT compared with their Caucasian counterparts. This increase in TG synthesis in OAT was not due to differences in cell size or rates of reesterification. Thus African American woman have an increased capacity to synthesize TG in OAT compared with Caucasian women, which may contribute to the higher prevalence of obesity in African American women. PMID- 12376327 TI - Protection against diet-induced obesity and obesity- related insulin resistance in Group 1B PLA2-deficient mice. AB - Group 1B phospholipase A2 (PLA2) is an abundant lipolytic enzyme that is well characterized biochemically and structurally. Because of its high level of expression in the pancreas, it has been presumed that PLA2 plays a role in the digestion of dietary lipids, but in vivo data have been lacking to support this theory. Our initial study on mice lacking PLA2 demonstrated no abnormalities in dietary lipid absorption in mice consuming a chow diet. However, the effects of PLA2 deficiency on animals consuming a high-fat diet have not been studied. To investigate this, PLA2(+/+) and PLA2(-/-) mice were fed a western diet for 16 wk. The results showed that PLA2(-/-) mice were resistant to high-fat diet-induced obesity. This observed weight difference was due to decreased adiposity present in the PLA2(-/-) mice. Compared with PLA2(+/+) mice, the PLA2(-/-) mice had 60% lower plasma insulin and 72% lower plasma leptin levels after high-fat diet feeding. The PLA2(-/-) mice also did not exhibit impaired glucose tolerance associated with the development of obesity-related insulin resistance as observed in the PLA2(+/+) mice. To investigate the mechanism by which PLA(2)(-/-) mice exhibit decreased weight gain while on a high-fat diet, fat absorption studies were performed. The PLA(2)(-/-) mice displayed 50 and 35% decreased plasma [(3)H]triglyceride concentrations 4 and 6 h, respectively, after feeding on a lipid-rich meal containing [(3)H]triolein. The PLA(2)(-/-) mice also displayed increased lipid content in the stool, thus indicating decreased fat absorption in these animals. These results suggest a novel role for PLA(2) in the protection against diet-induced obesity and obesity-related insulin resistance, thereby offering a new target for treatment of obesity and diabetes. PMID- 12376328 TI - Insulin inhibits glucose production by a direct effect in diabetic depancreatized dogs during euglycemia. AB - In our previous studies in nondiabetic dogs and humans, insulin suppressed glucose production (GP) by both an indirect extrahepatic and a direct hepatic effect. However, insulin had no direct effect on GP in diabetic depancreatized dogs under conditions of moderate hyperglycemia. The present study was designed to investigate whether insulin can inhibit GP by a direct effect in this model under conditions of euglycemia. Depancreatized dogs were made euglycemic (approximately 6 mmol/l), rather than moderately hyperglycemic (approximately 10 mmol/l) as in our previous studies, by basal portal insulin infusion. After approximately 100 min of euglycemia, a hyperinsulinemic euglycemic clamp was performed by giving an additional infusion of insulin either portally (POR) or peripherally at about one-half the rate (1/2 PER) to match the peripheral venous insulin concentrations. The greater hepatic insulin load in POR resulted in greater suppression of GP (from 16.5 +/- 1.8 to 12.2 +/- 1.6 micromol. kg(-1). min(-1)) than 1/2 PER (from 17.8 +/- 1.9 to 15.6 +/- 2.0 micromol. kg(-1). min( 1), P < 0.001 vs. POR), consistent with insulin having a direct hepatic effect in suppressing GP. We conclude that the direct effect of insulin to inhibit GP is present in diabetic depancreatized dogs under conditions of acutely induced euglycemia. These results suggest that, in diabetes, the prevailing glycemic level is a determinant of the balance between insulin's direct and indirect effects on GP. PMID- 12376329 TI - Growth hormone secretion pattern is an independent regulator of growth hormone actions in humans. AB - The importance of gender-specific growth hormone (GH) secretion pattern in the regulation of growth and metabolism has been demonstrated clearly in rodents. We recently showed that GH secretion in humans is also sexually dimorphic. Whether GH secretion pattern regulates the metabolic effects of GH in humans is largely unknown. To address this question, we administered the same daily intravenous dose of GH (0.5 mg. m(-2). day(-1)) for 8 days in different patterns to nine GH deficient adults. Each subject was studied on four occasions: protocol 1 (no treatment), protocol 2 (80% daily dose at 0100 and 10% daily dose at 0900 and 1700), protocol 3 (8 equal boluses every 3 h), and protocol 4 (continuous GH infusion). The effects of GH pattern on serum IGF-I, IGF-binding protein (IGFBP) 3, osteocalcin, and urine deoxypyridinoline were measured. Hepatic CYP1A2 and CYP3A4 activities were assessed by the caffeine and erythromycin breath tests, respectively. Protocols 3 and 4 were the most effective in increasing serum IGF-I and IGFBP-3, whereas protocols administering pulsatile GH had the greatest effects on markers of bone formation and resorption. All GH treatments decreased CYP1A2 activity, and the effect was greatest for pulsatile GH. Pulsatile GH decreased, whereas continuous GH infusion increased, CYP3A4 activity. These data demonstrate that GH pulse pattern is an independent parameter of GH action in humans. Gender differences in drug metabolism and, potentially, gender differences in growth rate may be explained by sex-specific GH secretion patterns. PMID- 12376330 TI - Soy protein, casein, and zein regulate histidase gene expression by modulating serum glucagon. AB - Glucagon has been postulated as an important physiological regulator of histidase (Hal) gene expression; however, it has not been demonstrated whether serum glucagon concentration is associated with the type and amount of protein ingested. The purpose of the present work was to study the association between hepatic Hal activity and mRNA concentration in rats fed 18 or 50% casein, isolated soy protein, or zein diets in a restricted schedule of 6 h for 10 days, and plasma glucagon and insulin concentrations. On day 10, five rats of each group were killed at 0900 (fasting), and then five rats were killed after being given the experimental diet for 1 h (1000). Rats fed 50% casein or soy diets showed higher Hal activity than the other groups studied. Rats fed 50% zein diets had higher Hal activity than rats fed 18% casein, soy, or zein diets, but lower activity than rats fed 50% casein or soy diets. Hal mRNA concentration followed a similar pattern. Hal activity showed a significant association with serum concentrations of glucagon. Serum glucagon concentration was significantly correlated with protein intake. Thus the type and amount of protein consumed affect Hal activity and expression through changes in serum glucagon concentrations. PMID- 12376331 TI - Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat. AB - The importance of insulin for the in vivo effects of growth hormone (GH) on lipid and lipoprotein metabolism was investigated by examining the effects of GH treatment of hypophysectomized (Hx) female rats with and without concomitant insulin treatment. Hypophysectomy-induced changes of HDL, apolipoprotein (apo)E, LDL, and apoB levels were normalized by GH treatment but not affected by insulin treatment. The hepatic triglyceride secretion rate was lower in Hx rats than in normal rats and increased by GH treatment. This effect of GH was blunted by insulin treatment. The triglyceride content in the liver changed in parallel with the changes in triglyceride secretion rate, indicating that the effect of the hormones on triglyceride secretion was dependent on changed availability of triglycerides for VLDL assembly. GH and insulin independently increased editing of apoB mRNA, but the effects were not additive. The expression of fatty-acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and sterol regulatory element binding protein-1c (SREBP-1c) was increased by GH treatment. Insulin and GH had no additive effects on these genes; instead, insulin blunted the effect of GH on SREBP-1c mRNA. In contrast to the liver, adipose tissue expression of SREBP-1c, FAS, or SCD-1 mRNA was not influenced by GH. In conclusion, the increased hepatic expression of lipogenic enzymes after GH treatment may be explained by increased expression of SREBP-1c. Insulin does not mediate the effects of GH but inhibits the stimulatory effect of GH on hepatic SREBP-1c expression and triglyceride secretion rate. PMID- 12376332 TI - Phosphorylation of eukaryotic initiation factor eIF2Bepsilon in skeletal muscle during sepsis. AB - We reported that the inhibition of protein synthesis in skeletal muscle during sepsis correlated with reduced eukaryotic initiation factor eIF2B activity. The present studies define changes in eIF2Bepsilon phosphorylation in gastrocnemius of septic animals. eIF2B kinase activity was significantly elevated 175% by sepsis compared with sterile inflammation, whereas eIF2B phosphatase activity was unaffected. Phosphorylation of eIF2Bepsilon-Ser(535) was significantly augmented over 2-fold and 2.5-fold after 3 and 5 days and returned to control values after 10 days of sepsis. Phosphorylation of glycogen synthase kinase-3 (GSK-3), a potential upstream kinase responsible for the elevated phosphorylation of eIF2Bepsilon, was significantly reduced over 36 and 41% after 3 and 5 days and returned to control values after 10 days of sepsis. The phosphorylation of PKB, a kinase thought to directly phosphorylate and inactivate GSK-3, was significantly reduced approximately 50% on day 3, but not on days 5 or 10, postinfection compared with controls. Treatment of septic rats with TNF-binding protein prevented the sepsis-induced changes in eIF2Bepsilon and GSK-3 phosphorylation, implicating TNF in mediating the effects of sepsis. Thus increased phosphorylation of eIF2Bepsilon via activation of GSK-3 is an important mechanism to account for the inhibition of skeletal muscle protein synthesis during sepsis. Furthermore, the study presents the first demonstration of changes in eIF2Bepsilon phosphorylation in vivo. PMID- 12376333 TI - Intermittent increases in cytosolic Ca2+ stimulate mitochondrial biogenesis in muscle cells. AB - Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 microM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an approximately 40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+ intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+ stimulates mitochondrial biogenesis in muscle cells. PMID- 12376334 TI - Regulation of exercise carbohydrate metabolism by estrogen and progesterone in women. AB - To assess the roles of endogenous estrogen (E2) and progesterone (P4) in regulating exercise carbohydrate use, we used pharmacological suppression and replacement to create three distinct hormonal environments: baseline (B), with E2 and P4 low; estrogen only (E), with E2 high and P4 low; and estrogen/progesterone (E + P), with E2 and P4 high. Blood glucose uptake (R(d)), total carbohydrate oxidation (CHO(ox)), and estimated muscle glycogen utilization (EMGU) were assessed during 60 min of submaximal exercise by use of stable isotope dilution and indirect calorimetry in eight eumenorrheic women. Compared with B (1.26 +/- 0.04 g/min) and E + P (1.27 +/- 0.04 g/min), CHO(ox) was lower with E (1.05 +/- 0.02 g/min). Glucose R(d) tended to be lower with E and E + P relative to B. EMGU was 25% lower with E than with B or E + P. Plasma free fatty acids (FFA) were inversely related to EMGU (r(2) = 0.49). The data suggest that estrogen lowers CHO(ox) by reducing EMGU and glucose R(d). Progesterone increases EMGU but not glucose R(d). The opposing actions of E(2) and P(4) on EMGU may be mediated by their impact on FFA availability or vice versa. PMID- 12376335 TI - Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes. AB - We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation. PMID- 12376336 TI - Effect of treatment of diabetic rats with dehydroepiandrosterone on vascular and neural function. AB - Nutritional supplementation with dehydroepiandrosterone (DHEA) may be a candidate for treating diabetes-induced vascular and neural dysfunction. DHEA is a naturally occurring adrenal androgen that has antioxidant properties and is reportedly reduced in diabetes. Using a prevention protocol, we found that dietary supplementation of streptozotocin-induced diabetic rats with 0.1, 0.25, or 0.5% DHEA caused a concentration-dependent prevention in the development of motor nerve conduction velocity and endoneurial blood flow impairment, which are decreased in diabetes. At 0.25%, DHEA significantly prevented the diabetes induced increase in serum thiobarbituric acid-reactive substances and sciatic nerve conjugated diene levels. This treatment also reduced the production of superoxide by epineurial arterioles of the sciatic nerve. DHEA treatment (0.25%) significantly improved vascular relaxation mediated by acetylcholine in epineurial vessels of diabetic rats. Sciatic nerve Na+-K+-ATPase activity and myoinositol content was also improved by DHEA treatment, whereas sorbitol and fructose content remained elevated. These studies suggest that DHEA, by preventing oxidative stress and perhaps improving sciatic nerve Na+-K+-ATPase activity, may improve vascular and neural dysfunction in diabetes. PMID- 12376337 TI - AICAR and phlorizin reverse the hypoglycemia-specific defect in glucagon secretion in the diabetic BB rat. AB - Individuals with type 1 diabetes demonstrate a hypoglycemia-specific defect in glucagon secretion. To determine whether intraislet hyperinsulinemia plays a role in the genesis of this defect, glucagon-secretory responses to moderate hypoglycemia induced by either insulin or a novel combination of the noninsulin glucose-lowering agents 5-aminoimidazole-4-carboxamide (AICAR) and phlorizin were compared in diabetic BB rats (an animal model of type 1 diabetes) and nondiabetic BB rats. The phlorizin-AICAR combination was able to induce moderate and equivalent hypoglycemia in both diabetic and nondiabetic BB rats in the absence of marked hyperinsulinemia. Diabetic BB rats demonstrated impaired glucagon and epinephrine responses during insulin-induced hypoglycemia compared with nondiabetic rats. In contrast, both glucagon (9- to 10-fold increase) and epinephrine (5- to 6-fold increase) responses were markedly improved during phlorizin-AICAR hypoglycemia. Combining phlorizin, AICAR, and insulin attenuated the glucagon response to hypoglycemia by 70% in the diabetic BB rat. Phlorizin plus AICAR had no effect on counterregulatory hormones under euglycemic conditions. We conclude that alpha-cell glucagon secretion in response to hypoglycemia is not defective if intraislet hyperinsulinemia is prevented. This suggests that exogenous insulin plays a pivotal role in the etiology of this defect. PMID- 12376338 TI - A mathematical model of metabolic insulin signaling pathways. AB - We develop a mathematical model that explicitly represents many of the known signaling components mediating translocation of the insulin-responsive glucose transporter GLUT4 to gain insight into the complexities of metabolic insulin signaling pathways. A novel mechanistic model of postreceptor events including phosphorylation of insulin receptor substrate-1, activation of phosphatidylinositol 3-kinase, and subsequent activation of downstream kinases Akt and protein kinase C-zeta is coupled with previously validated subsystem models of insulin receptor binding, receptor recycling, and GLUT4 translocation. A system of differential equations is defined by the structure of the model. Rate constants and model parameters are constrained by published experimental data. Model simulations of insulin dose-response experiments agree with published experimental data and also generate expected qualitative behaviors such as sequential signal amplification and increased sensitivity of downstream components. We examined the consequences of incorporating feedback pathways as well as representing pathological conditions, such as increased levels of protein tyrosine phosphatases, to illustrate the utility of our model for exploring molecular mechanisms. We conclude that mathematical modeling of signal transduction pathways is a useful approach for gaining insight into the complexities of metabolic insulin signaling. PMID- 12376339 TI - Sharing signals: connecting lung epithelial cells with gap junction channels. AB - Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells. PMID- 12376340 TI - Focus on proteins, surfaces, et al. PMID- 12376341 TI - Content-dependent activity of lung surfactant protein B in mixtures with lipids. AB - The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approximately SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels < or =0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations. PMID- 12376342 TI - Plasticity of cholinergic and tachykinergic nerves: the convergence of the twain. PMID- 12376343 TI - Interleukin-1beta-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway. AB - Interleukin (IL)-1beta causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1beta. Ferrets were instilled intratracheally with IL-1beta (0.3 microg/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1beta. The IL-1beta induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1beta-induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1beta. These results show that IL-1beta-enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons. PMID- 12376344 TI - Hypoxia, anoxia, and O2 sensing: the search continues. PMID- 12376345 TI - Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species. AB - The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1alpha protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (rho(0)-cells) fail to generate reactive oxygen species or stabilize HIF-1alpha protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1alpha protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1alpha protein in response to hypoxia and anoxia. The rho(0)-A549 cells and rho(0)-HT1080 cells failed to accumulate HIF-1alpha protein in response to hypoxia. However, both rho(0)-A549 and rho(0)-HT1080 were able to stabilize HIF-1alpha protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1alpha protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1alpha protein. PMID- 12376346 TI - Iron increases expression of iron-export protein MTP1 in lung cells. AB - Accumulation of reactive iron in acute and chronic lung disease suggests that iron-driven free radical formation could contribute to tissue injury. Safe transport and sequestration of this metal is likely to be of importance in lung defense. We provide evidence for the expression and iron-induced upregulation of the metal transporter protein-1 (MTP1) genes in human and rodent lung cells at both the protein and mRNA levels. In human bronchial epithelial cells, a 3.8-fold increase in mRNA level and a 2.4-fold increase in protein level of MTP1 were observed after iron exposure. In freshly isolated human macrophages, as much as an 18-fold increase in the MTP1 protein level was detected after incubation with an iron compound. The elevation in expression of MTP1 gene was also demonstrated in iron-instilled rat lungs and in hypotransferrinemic mouse lungs. This is similar to our previous findings with divalent metal transporter-1 (DMT1), an iron transporter that is required for iron uptake and intracellular iron trafficking. These studies suggest the presence of iron mobilization and/or detoxification pathways in the lung that are crucial for iron homeostasis and lung defense. PMID- 12376347 TI - Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP. AB - Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life and prevention of infant respiratory distress syndrome. We have developed a new in vitro model to further investigate regulation of type II cell differentiation. Epithelial cells isolated from human fetal lung were cultured in serum-free medium on plastic. Cells treated with dexamethasone + cAMP analog and isobutylmethylxanthine for 4 days exhibited increased phosphatidylcholine synthesis and content of disaturated phosphatidylcholine species, manyfold increases in all surfactant proteins with processing to mature forms, and abundant lamellar bodies. DNA microarray analysis identified approximately 3,100 expressed genes, including subsets of genes induced 2- to >100-fold (approximately 2.5%) or repressed 2- to 18-fold (approximately 1.2%) by hormone treatment. Of the highly regulated genes, most were coregulated in an additive or synergistic manner by dexamethasone and cAMP agents. Approximately 90% of the regulated genes identified by this initial microarray analysis have not been previously recognized as hormone responsive. One newly identified hormone-induced gene is Nkx2.1 (thyroid transcription factor 1), which has a critical role in surfactant protein gene expression. Our findings indicate that glucocorticoid + cAMP is sufficient and necessary for precocious induction of functional type II cells in this in vitro system and that these hormones act primarily in combination to regulate expression of a subset of specific genes. PMID- 12376348 TI - Neutrophils play a critical role in development of LPS-induced airway disease. AB - We investigated the role of neutrophils in the development of endotoxin-induced airway disease via systemic neutrophil depletion of C3H/HeBFeJ mice and coincident inhalation challenge with lipopolysaccharide (LPS) over a 4-wk period. Mice were made neutropenic with intraperitoneal injections of neutrophil antiserum before and throughout the exposure period. Experimental conditions included LPS-exposed, antiserum-treated; LPS-exposed, control serum-treated; air exposed, antiserum-treated; and air-exposed, control serum-treated groups. Physiological, biological, and morphological assessments were performed after a 4 wk exposure and again after a 4-wk recovery period. After the 4-wk exposure, LPS induced inflammation of the lower airways was significantly attenuated in the neutropenic mice, although airway responsiveness (AR) to methacholine (MCh) remained unchanged. After the recovery period, LPS-exposed neutrophil-replete mice had increased AR to MCh when compared with the LPS-exposed neutropenic animals. Morphometric data indicate that the 4-wk exposure to LPS leads to a substantial expansion of the subepithelial area of the medium-sized airways (90 129 microm diameter) in nonneutropenic mice but not neutropenic mice, and this difference persisted even after the recovery period. Expression of bronchial epithelial and subepithelial transforming growth factor-beta1 (TGF-beta1) was diminished in the challenged neutropenic mice compared with the neutrophil sufficient mice. These studies demonstrate that neutrophils play a critical role in the development of chronic LPS-induced airway disease. PMID- 12376349 TI - Attenuation of antigen-induced airway hyperresponsiveness in CGRP-deficient mice. AB - Bronchial hyperresponsiveness and eosinophilia are major characteristics of asthma. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has various biological actions. In the present study, we questioned whether CGRP might have pathophysiological roles in airway hyperresponsiveness and eosinophilia in asthma. To determine the exact roles of endogenous CGRP in vivo, we chose to study antigen-induced airway responses using CGRP gene-disrupted mice. After ovalbumin sensitization and antigen challenge, we assessed airway responsiveness and measured proinflammatory mediators. In the sensitized CGRP gene-disrupted mice, antigen-induced bronchial hyperresponsiveness was significantly attenuated compared with the sensitized wild-type mice. Antigen challenge induced eosinophil infiltration in bronchoalveolar lavage fluid, whereas no differences were observed between the wild-type and CGRP-mutant mice. Antigen-induced increases in cysteinyl leukotriene production in the lung were significantly reduced in the CGRP-disrupted mice. These findings suggest that CGRP could be involved in the antigen-induced airway hyperresponsiveness, but not eosinophil infiltration, in mice. The CGRP-mutant mice may provide appropriate models to study molecular mechanisms underlying CGRP-related diseases. PMID- 12376350 TI - Retinoic acid attenuates O2-induced inhibition of lung septation. AB - Exposure of the newborn lung to hyperoxia is associated with impaired alveolar development. In newborn rats exposed to hyperoxia and studied at day 14 of life, retinoic acid (RA) treatment improved survival and increased lung collagen but did not improve alveolar development. To determine whether RA treatment during exposure to hyperoxia results in late improvement in alveolarization, we treated newborn rats with RA and hyperoxia from day 3 to day 14 and then weaned O2 to room air by day 20, and studied the animals on day 42. O2-exposed animals had larger mean lung volumes, larger alveoli, and decreased gas-exchange tissue relative to air-exposed animals, whereas RA-treated O2-exposed animals were not statistically different from air-exposed controls. Relative to control animals, elastin staining at day 14 was decreased in hyperoxia-exposed lung independent of RA treatment, and, at day 42, elastin staining was similar in all treatment groups. At day 14, elastin gene expression was similar in all treatment groups, whereas at day 42 lung previously exposed to hyperoxia showed increased elastin signal independent of RA treatment. These results indicate that RA treatment during hyperoxia exposure promotes septal formation without evidence of effects on elastin gene expression after 4 wk of recovery. PMID- 12376351 TI - Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth. AB - We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP) B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth. PMID- 12376352 TI - Increased epithelial cell proliferation in very premature baboons with chronic lung disease. AB - Coordinated proliferation of lung cells is required for normal lung growth and differentiation. Chronic injury to developing lung may disrupt normal patterns of cell proliferation. To examine patterns of cell proliferation in injured developing lungs, we investigated premature baboons delivered at 125 days gestation (approximately 67% of term) and treated with oxygen and ventilation for 6, 14, or 21 days (PRN). Each PRN treatment group contained 3 or 4 animals. During normal in utero lung development, the proportion of proliferating lung cells declined as measured by the cell-cycle marker Ki67. In the PRN group, the proportion of proliferating lung cells was 2.5-8.5-fold greater than in corresponding gestational controls. By 14 days of treatment, the proportion of cells that expressed pro-surfactant protein B (proSP-B) was ~2.5-fold greater than in gestational controls. In the PRN group, 41% of proliferating cells expressed proSP-B compared with 5.8% in the gestational controls. By 21 days of treatment, proliferation of proSP-B-expressing epithelial cells declined substantially, but the proportion of proliferating non-proSP-B-expressing cells increased approximately sevenfold. These data show that the development of chronic lung disease is associated with major alterations in normal patterns of lung-cell proliferation. PMID- 12376353 TI - Sequential targeted deficiency of SP-A and -D leads to progressive alveolar lipoproteinosis and emphysema. AB - Surfactant proteins-A and -D (SP-A and SP-D) are members of the collectin protein family. Mice singly deficient in SP-A and SP-D have distinct phenotypes. Both have altered inflammatory responses to microbial challenges. To further investigate the functions of SP-A and SP-D in vivo, we developed mice deficient in both proteins by sequentially targeting the closely linked genes in embryonic stem cells using graded resistance to G-418. There is a progressive increase in bronchoalveolar lavage phospholipid, protein, and macrophage content through 24 wk of age. The macrophages from doubly deficient mice express high levels of the matrix metalloproteinase MMP-12 and develop intense but patchy lung inflammation. Stereological analysis demonstrates significant air space enlargement and reduction in alveolar septal tissue per unit volume, consistent with emphysema. These changes qualitatively resemble the lung pathology seen in SP-D-deficient mice. These doubly deficient mice will be useful in dissecting the potential overlap in function between SP-A and SP-D in host defense. PMID- 12376354 TI - Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages. AB - Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles. PMID- 12376355 TI - Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice. AB - Plasminogen activator inhibitor-1 (PAI-1)-deficient transgenic mice have improved survival and less fibrosis after intratracheal bleomycin instillation. We hypothesize that PAI-1 deficiency limits scarring through unopposed plasminogen activation. If this is indeed true, then we would expect increased urokinase-type plasminogen activator (uPA) expression to result in a similar reduction in scarring and improvement in mortality. To test our hypothesis, using the tetracycline gene regulatory system, we have generated a transgenic mouse model with the features of inducible, lung-specific uPA production. After doxycycline administration, these transgenic animals expressed increased levels of uPA in their bronchoalveolar lavage (BAL) fluid that accelerated intrapulmonary fibrin clearance. Importantly, this increased plasminogen activator production led to a reduction in both lung collagen accumulation and mortality after bleomycin induced injury. These results suggest that PAI-1 deficiency does protect against the effects of bleomycin-induced lung injury through unopposed plasmin generation. By allowing the manipulation of plasminogen activation at different phases of the fibrotic process, this model will serve as a powerful tool in further investigations into the pathogenesis of pulmonary fibrosis. PMID- 12376356 TI - In vitro sensitization of human bronchus by beta2-adrenergic agonists. AB - Incubation of human distal bronchi from 48 patients for 15 h with 10(-7) M fenoterol induced sensitization characterized by an increase in maximal contraction to endothelin-1 (ET-1) and acetylcholine (ACh). Incubation of human bronchi with 10(-6), 3 x 10(-6), and 10(-5) M forskolin (an adenyl cyclase activator) reproduced sensitization to ET-1 and ACh. The sensitizing effect of fenoterol was inhibited by coincubation with gliotoxine (a nuclear factor-kappaB inhibitor), dexamethasone, indomethacin (a cyclooxygenase inhibitor), GR-32191 (a TP prostanoid receptor antagonist), MK-476 (a cysteinyl leukotriene type 1 receptor antagonist), SR-140333 + SR-48968 + SR-142801 (neurokinin types 1, 2, and 3 tachykinin receptor antagonists) with or without HOE-140 (a bradykinin B(2) receptor antagonist), SB-203580 (an inhibitor of the 38-kDa mitogen-activated protein kinase, p38(MAPK)), or calphostin C (a protein kinase C blocker). Our results suggest that chronic exposure to fenoterol induces proinflammatory effects mediated by nuclear factor-kappaB and pathways involving leukotrienes, prostanoids, bradykinin, tachykinins, protein kinase C, and p38(MAPK), leading to the regulation of smooth muscle contraction to ET-1 and ACh. PMID- 12376357 TI - Role of anti-L-selectin antibody in burn and smoke inhalation injury in sheep. AB - We hypothesized that the antibody neutralization of L-selectin would decrease the pulmonary abnormalities characteristic of burn and smoke inhalation injury. Three groups of sheep (n = 18) were prepared and randomized: the LAM-(1-3) group (n = 6) was injected intravenously with 1 mg/kg of leukocyte adhesion molecule (LAM) (1-3) (mouse monoclonal antibody against L-selectin) 1 h after the injury, the control group (n = 6) was not injured or treated, and the nontreatment group (n = 6) was injured but not treated. All animals were mechanically ventilated during the 48-h experimental period. The ratio of arterial PO2 to inspired O2 fraction decreased in the LAM-(1-3) and nontreatment groups. Lung lymph flow and pulmonary microvascular permeability were elevated after injury. This elevation was significantly reduced when LAM-(1-3) was administered 1 h after injury. Nitrate/nitrite (NO(x)) amounts in plasma and lung lymph increased significantly after the combined injury. These changes were attenuated by posttreatment with LAM-(1-3). These results suggest that the changes in pulmonary transvascular fluid flux result from injury of lung endothelium by polymorphonuclear leukocytes. In conclusion, posttreatment with the antibody for L-selectin improved lung lymph flow and permeability index. L-selectin appears to be principally involved in the increased pulmonary transvascular fluid flux observed with burn/smoke insult. L-selectin may be a useful target in the treatment of acute lung injury after burn and smoke inhalation. PMID- 12376358 TI - Role of PKC, tyrosine kinases, and Rho kinase in alpha-adrenoreceptor-mediated PASM contraction. AB - Our objectives were to identify the relative contributions of intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in the pulmonary artery smooth muscle (PASM) contractile response to the alpha-adrenoreceptor agonist phenylephrine (PE) and to assess the role of PKC, tyrosine kinases (TK), and Rho kinase (ROK) in that response. Our hypothesis was that multiple signaling pathways are involved in the regulation of [Ca2+]i, myofilament Ca2+ sensitization, and vasomotor tone in response to alpha-adrenoreceptor stimulation of PASM. Simultaneous measurement of [Ca2+]i and isometric tension was performed in isolated canine pulmonary arterial strips loaded with fura 2-AM. PE-induced tension development was due to sarcolemmal Ca2+ influx, Ca2+ release from inositol 1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2+ stores, and myofilament Ca2+ sensitization. Inhibition of either PKC or TK partially attenuated the sarcolemmal Ca2+ influx component and the myofilament Ca2+ sensitizing effect of PE. Combined inhibition of PKC and TK did not have an additive attenuating effect on PE-induced Ca2+ sensitization. ROK inhibition slightly decreased [Ca2+]i but completely inhibited myofilament Ca2+ sensitization. These results indicate that PKC and TK activation positively regulate sarcolemmal Ca2+ influx in response to alpha-adrenoreceptor stimulation in PASM but have relatively minor effects on myofilament Ca2+ sensitivity. ROK is the predominant pathway mediating PE-induced myofilament Ca2+ sensitization. PMID- 12376359 TI - Nitric oxide inhibits ADP-ribosyl cyclase through a cGMP-independent pathway in airway smooth muscle. AB - There is evidence for a role of cyclic ADP-ribose (cADPR) in intracellular Ca2+ regulation in smooth muscle. cADPR is synthesized and degraded by ADP-ribosyl cyclase and cADPR hydrolase, respectively, by a bifunctional protein, CD38. Nitric oxide (NO) inhibits intracellular Ca2+ mobilization in airway smooth muscle. The present study was designed to determine whether this inhibition is due to regulation of ADP-ribosyl cyclase and/or cADPR hydrolase activity. Sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine, NO donors, produced a concentration-dependent decrease in ADP-ribosyl cyclase, but not cADPR hydrolase, activity. The NO scavenger carboxy-PTIO prevented and reversed, and reduced glutathione prevented, the inhibition of ADP-ribosyl cyclase by SNP, suggesting S nitrosylation by NO as a mechanism. N-ethylmaleimide, which covalently modifies protein sulfhydryl groups, making them incapable of nitrosylation, produced a marked inhibition of ADP-ribosyl cyclase, but not cADPR hydrolase, activity. SNP and N-ethylmaleimide significantly inhibited the ADP-ribosyl cyclase activity in recombinant human CD38 without affecting the cADPR hydrolase activity. These results provide a novel mechanism for differential regulation of CD38 by NO through a cGMP-independent pathway involving S-nitrosylation of thiols. PMID- 12376360 TI - An endothelin receptor antagonist, SB-217242, inhibits airway hyperresponsiveness in allergic mice. AB - Within the airways, endothelin-1 (ET-1) can exert a range of prominent effects, including airway smooth muscle contraction, bronchial obstruction, airway wall edema, and airway remodeling. ET-1 also possesses proinflammatory properties and contributes to the late-phase response in allergic airways. However, there is no direct evidence for the contribution of endogenous ET-1 to airway hyperresponsiveness in allergic airways. Allergic inflammation induced in mice by sensitization and challenge with the house dust mite allergen Der P1 was associated with elevated levels of ET-1 within the lung, increased numbers of eosinophils within bronchoalveolar lavage fluid and tissue sections, and development of airway hyperresponsiveness to methacholine (P < 0.05, n = 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB 217242 (30 mg x kg(-1) x day(-1)), during allergen challenge markedly inhibited airway eosinophilia (bronchoalveolar lavage fluid and tissue) and development of airway hyperresponsiveness. These findings provide direct evidence for a mediator role for ET-1 in development of airway hyperresponsiveness and airway eosinophilia in Der P1-sensitized mice after antigen challenge. PMID- 12376361 TI - Altered fatty acid composition of lung surfactant phospholipids in interstitial lung disease. AB - Deterioration of pulmonary surfactant function has been reported in interstitial lung disease; however, the molecular basis is presently unclear. We analyzed fatty acid (FA) profiles of several surfactant phospholipid classes isolated from large-surfactant aggregates of patients with idiopathic pulmonary fibrosis (IPF; n = 12), hypersensitivity pneumonitis (n = 5), and sarcoidosis (n = 12). Eight healthy individuals served as controls. The relative content of palmitic acid in phosphatidylcholine was significantly reduced in IPF (66.8 +/- 2.5%; means +/- SE; P < 0.01) but not in hypersensitivity pneumonitis (78.5 +/- 1.8%) and sarcoidosis (78.2 +/- 3.1%; control 80.1 +/- 0.7%). In addition, the phosphatidylglycerol FA profile was significantly altered in the IPF patients, with a lower relative content of its major FA, oleic acid, at the expense of saturated FA. In the phosphatidylcholine class, a significant correlation between the impairment of biophysical surfactant function and decreased percentages of palmitic acid was noted. We conclude that significant alterations in the FA profile of pulmonary surfactant phospholipids occur predominantly in IPF and may contribute to the disturbances of alveolar surface activity in this disease. PMID- 12376362 TI - Gelatinase activities in the airways of premature infants and development of bronchopulmonary dysplasia. AB - Matrix-degrading metalloproteinases may play a role in the pathophysiology of bronchopulmonary dysplasia (BDP). We, therefore, evaluated correlations between gelatinase activities [metalloproteinase (MMP)-2 and MMP-9] or tissue inhibitor of metalloproteinase (TIMP)-1 levels present in the airways during the initial phase of hyaline membrane disease and the onset of BPD. Tracheal aspirates were obtained within 6 h of birth (day 0) from 64 intubated neonates with a gestational age < or =30 wk. Forty-five neonates were resampled on day 3 or 5. Total MMP-2 level measured by zymography fell with time, whereas total MMP-9 level and TIMP-1 levels, assayed by ELISA, increased; the MMP-9 increase correlated with the increase in airway inflammatory cell numbers. Among the parameters measured on day 0, 3, or 5, lower total MMP-2 level, lower birth weight, and higher fraction of inspired oxygen on day 0 were significantly and independently associated with the development of BPD. In conclusion, MMP-9 level and TIMP-1 levels increased after birth but are not linked to BPD outcome. In contrast, low MMP-2 level at birth is strongly associated with the development of BPD. PMID- 12376363 TI - TGF-beta1 stimulates HO-1 via the p38 mitogen-activated protein kinase in A549 pulmonary epithelial cells. AB - In lung injury and progressive lung diseases, the multifunctional cytokine transforming growth factor-beta1 (TGF-beta1) modulates inflammatory responses and wound repair. Heme oxygenase-1 (HO-1) is a stress-inducible protein that has been demonstrated to confer cytoprotection against oxidative injury and provide a vital function in maintaining tissue homeostasis. Here we report that TGF-beta1 is a potent inducer of HO-1 and examined the signaling pathway by which TGF-beta1 regulates HO-1 expression in human lung epithelial cells (A549). TGF-beta1 (1-5 ng/ml) treatment resulted in a marked time-dependent induction of HO-1 mRNA in A549 cells, followed by corresponding increases in HO-1 protein and HO enzymatic activity. Actinomycin D and cycloheximide inhibited TGF-beta1-responsive HO-1 mRNA expression, indicating a requirement for transcription and de novo protein synthesis. Furthermore, TGF-beta1 rapidly activated the p38 mitogen-activated protein kinase (p38 MAPK) pathway in A549 cells. A chemical inhibitor of p38 MAPK (SB-203580) abolished TGF-beta1-inducible HO-1 mRNA expression. Both SB-203580 and expression of a dominant-negative mutant of p38 MAPK inhibited TGF-beta1 induced ho-1 gene activation, as assayed by luciferase activity of an ho-1 enhancer/luciferase fusion construct (pMHO1luc-33+SX2). These studies demonstrate the critical intermediacy of the p38 MAPK pathway in the regulation of HO-1 expression by TGF-beta1. PMID- 12376364 TI - Contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine PPHN model. AB - Ca2+-sensitive K+ (K(Ca)) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung K(Ca) channel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age matched, sham-operated animals were used as controls. PASMC K+ currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 +/- 63.6% in control vs. 133.1 +/- 16.0% in hypertensive fetuses). The contribution of the K(Ca) channel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a -14.6 +/- 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotoxin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal perinatal pulmonary vasoreactivity associated with PPHN. PMID- 12376365 TI - Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice. AB - The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL 4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis. PMID- 12376367 TI - Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus. AB - The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (-log EC50 shift = -1.49 +/- 0.16 and E(max) inhibition for 100 microM ACh = 30%) more than the same levels of histamine contraction. The M2 receptor-selective antagonist methoctramine (1 microM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy-N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U 46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pK(B) (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism. PMID- 12376366 TI - Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells. AB - Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1 expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition. PMID- 12376368 TI - Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and in vivo. AB - We investigated regulation of vascular endothelial growth factor (VEGF) expression by hypoxia in cultured and freshly isolated rat alveolar epithelial cells (AEC). In vitro, hypoxia increased VEGF mRNA and protein levels, with maximal stimulation at 0% O2 for 18 h. A similar upregulation of VEGF expression was found in alveolar epithelial type II (ATII) cells freshly isolated from rats exposed to 8% O2 for 24 h. In vitro, hypoxia-induced upregulation of VEGF mRNA was due to an increase in transcription, rather than an increase in RNA stability, inasmuch as the half-life of VEGF mRNA was unchanged. Upregulation of VEGF mRNA by hypoxia was mimicked by CoCl2 and desferrioxamine in normoxic AEC and was not prevented by inhibitors of reactive oxygen species, suggesting that hypoxic VEGF regulation involved an O2-dependent protein that requires ferrous ions but is independent of reactive oxygen species generation. In polarized ATII cells, VEGF protein was secreted at the apical and basolateral sides. Similarly, in rats, VEGF was secreted in bronchoalveolar lavage fluid. Hypoxia induced a twofold increase in VEGF protein at the apical side of ATII cells in culture and in bronchoalveolar lavage fluid. These findings suggest that release of VEGF synthesized by AEC may target not only endothelial cells but also other alveolar cells, including macrophages and epithelial cells. PMID- 12376369 TI - Graded response of K+ current, membrane potential, and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle. AB - Many studies indicate that hypoxic inhibition of some K+ channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+ current (I(k)), resting membrane potential (E(m)), and intracellular Ca2+ concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing PO2 levels gradually inhibited steady-state I(k) of rat resistance PASMCs and depolarized the cell membrane. The block of I(k) by hypoxia was voltage dependent in that low O2 tensions (3 and 0% O2) inhibited I(k) more at 0 and -20 mV than at 50 mV. As expected, the hypoxia-sensitive I(k) was also 4 aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]i as PO2 levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in I(k) and E(m) and an increase of [Ca2+]i. PMID- 12376370 TI - Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle. AB - Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8 Isoprostaglandin E1 and 8-isoprostaglandin E2 (8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2 and PGE2 implicated isoprostane activity at the PGE2 receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2 did not reverse KCl tone, suggesting that voltage-dependent Ca2+ influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+ currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2 receptor directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+ channel activation or the direct inhibition of voltage-operated Ca2+ influx or myosin light chain kinase. PMID- 12376371 TI - An intelligent biological information management system. AB - MOTIVATION: As biomedical researchers are amassing a plethora of information in a variety of forms resulting from the advancements in biomedical research, there is a critical need for innovative information management and knowledge discovery tools to sift through these vast volumes of heterogeneous data and analysis tools. In this paper we present a general model for an information management system that is adaptable and scalable, followed by a detailed design and implementation of one component of the model. The prototype, called BioSifter, was applied to problems in the bioinformatics area. RESULTS: BioSifter was tested using 500 documents obtained from PubMed database on two biological problems related to genetic polymorphism and extracorporal shockwave lithotripsy. The results indicate that BioSifter is a powerful tool for biological researchers to automatically retrieve relevant text documents from biological literature based on their interest profile. The results also indicate that the first stage of information management process, i.e. data to information transformation, significantly reduces the size of the information space. The filtered data obtained through BioSifter is relevant as well as much smaller in dimension compared to all the retrieved data. This would in turn significantly reduce the complexity associated with the next level transformation, i.e. information to knowledge. PMID- 12376372 TI - Condition specific transcription factor binding site characterization in Saccharomyces cerevisiae. AB - MOTIVATION: We demonstrate a computational process by which transcription factor binding sites can be elucidated using genome-wide expression and binding profiles. The profiles direct us to the intergenic locations likely to contain the promoter regions for a given factor. These sequences are multiply and locally aligned to give an anchor motif from which further characterization can take place. RESULTS: We present bases for and assumptions about the variability within these motifs which give rise to potentially more accurate motifs, capture complex binding sites built upon the basis motif, and eliminate the constraints of the currently employed promoter searching protocols. We also present a measure of motif quality based on the occurrence of the putative motifs in regions observed to contain the binding sites. The assumptions, motif generation, quality assessment and comparison allow the user as much control as their a priori knowledge allows. AVAILABILITY: IGRDB and the datasets mentioned herein are available at http://chipdb.wi.mit.edu/ PMID- 12376373 TI - Optimal algorithms for local vertex quartet cleaning. AB - MOTIVATION: Reconstructing evolutionary trees is an important problem in biology. A response to the computational intractability of most of the traditional criteria for inferring evolutionary trees has been a focus on new criteria, particularly quartet-based methods that seek to merge trees derived on subsets of four species from a given species-set into a tree for that entire set. Unfortunately, most of these methods are very sensitive to errors in the reconstruction of the trees for individual quartets of species. A recently developed technique called quartet cleaning can alleviate this difficulty in certain cases by using redundant information in the complete set of quartet topologies for a given species-set to correct such errors. RESULTS: In this paper, we describe two new local vertex quartet cleaning algorithms which have optimal time complexity and error-correction bound, respectively. These are the first known local vertex quartet cleaning algorithms that are optimal with respect to either of these attributes. PMID- 12376374 TI - Short inversions and conserved gene cluster. AB - MOTIVATION: Two independent sets of recent observations on newly sequenced microbial genomes pertain to the prevalence of short inversion as a gene order rearrangement process and to the lack of conservation of gene order within conserved gene clusters. We propose a model of inversion where the key parameter is the length of the inverted fragment. RESULTS: We show that there is a qualitative difference in the pattern of evolution when the inversion length is small with respect to the cluster size and when it is large. This suggests an explanation of the lack of parallel gene order in conserved clusters and raises questions about the statistical validity of putative functionally selected gene clusters if these have only been tested against inappropriate null hypotheses. PMID- 12376375 TI - Comparative ab initio prediction of gene structures using pair HMMs. AB - We present a novel comparative method for the ab initio prediction of protein coding genes in eukaryotic genomes. The method simultaneously predicts the gene structures of two un-annotated input DNA sequences which are homologous to each other and retrieves the subsequences which are conserved between the two DNA sequences. It is capable of predicting partial, complete and multiple genes and can align pairs of genes which differ by events of exon-fusion or exon-splitting. The method employs a probabilistic pair hidden Markov model. We generate annotations using our model with two different algorithms: the Viterbi algorithm in its linear memory implementation and a new heuristic algorithm, called the stepping stone, for which both memory and time requirements scale linearly with the sequence length. We have implemented the model in a computer program called DOUBLESCAN. In this article, we introduce the method and confirm the validity of the approach on a test set of 80 pairs of orthologous DNA sequences from mouse and human. More information can be found at: http://www.sanger.ac.uk/Software/analysis/doublescan/ PMID- 12376376 TI - Gene perturbation and intervention in probabilistic Boolean networks. AB - MOTIVATION: A major objective of gene regulatory network modeling, in addition to gaining a deeper understanding of genetic regulation and control, is the development of computational tools for the identification and discovery of potential targets for therapeutic intervention in diseases such as cancer. We consider the general question of the potential effect of individual genes on the global dynamical network behavior, both from the view of random gene perturbation as well as intervention in order to elicit desired network behavior. RESULTS: Using a recently introduced class of models, called Probabilistic Boolean Networks (PBNs), this paper develops a model for random gene perturbations and derives an explicit formula for the transition probabilities in the new PBN model. This result provides a building block for performing simulations and deriving other results concerning network dynamics. An example is provided to show how the gene perturbation model can be used to compute long-term influences of genes on other genes. Following this, the problem of intervention is addressed via the development of several computational tools based on first-passage times in Markov chains. The consequence is a methodology for finding the best gene with which to intervene in order to most likely achieve desirable network behavior. The ideas are illustrated with several examples in which the goal is to induce the network to transition into a desired state, or set of states. The corresponding issue of avoiding undesirable states is also addressed. Finally, the paper turns to the important problem of assessing the effect of gene perturbations on long-run network behavior. A bound on the steady-state probabilities is derived in terms of the perturbation probability. The result demonstrates that states of the network that are more 'easily reachable' from other states are more stable in the presence of gene perturbations. Consequently, these are hypothesized to correspond to cellular functional states. AVAILABILITY: A library of functions written in MATLAB for simulating PBNs, constructing state transition matrices, computing steady-state distributions, computing influences, modeling random gene perturbations, and finding optimal intervention targets, as described in this paper, is available on request from is@ieee.org. PMID- 12376377 TI - Bayesian automatic relevance determination algorithms for classifying gene expression data. AB - MOTIVATION: We investigate two new Bayesian classification algorithms incorporating feature selection. These algorithms are applied to the classification of gene expression data derived from cDNA microarrays. RESULTS: We demonstrate the effectiveness of the algorithms on three gene expression datasets for cancer, showing they compare well with alternative kernel-based techniques. By automatically incorporating feature selection, accurate classifiers can be constructed utilizing very few features and with minimal hand-tuning. We argue that the feature selection is meaningful and some of the highlighted genes appear to be medically important. PMID- 12376378 TI - Selecting signature oligonucleotides to identify organisms using DNA arrays. AB - MOTIVATION: DNA arrays are a very useful tool to quickly identify biological agents present in some given sample, e.g. to identify viruses causing disease, for quality control in the food industry, or to determine bacteria contaminating drinking water. The selection of specific oligos to attach to the array surface is a relevant problem in the experiment design process. Given a set S of genomic sequences (the target sequences), the task is to find at least one oligonucleotide, called probe, for each sequence in S. This probe will be attached to the array surface, and must be chosen in a way that it will not hybridize to any other sequence but the intended target. Furthermore, all probes on the array must hybridize to their intended targets under the same reaction conditions, most importantly at the temperature T at which the experiment is conducted. RESULTS: We present an efficient algorithm for the probe design problem. Melting temperatures are calculated for all possible probe-target interactions using an extended nearest-neighbor model, allowing for both non Watson-Crick base-pairing and unpaired bases within a duplex. To compute temperatures efficiently, a combination of suffix trees and dynamic programming based alignment algorithms is introduced. Additional filtering steps during preprocessing increase the speed of the computation. The practicability of the algorithms is demonstrated by two case studies: The identification of HIV-1 subtypes, and of 28S rDNA sequences from >or=400 organisms. PMID- 12376379 TI - Threading using neural nEtwork (TUNE): the measure of protein sequence-structure compatibility. AB - MOTIVATION: Fold recognition programs align a probe protein sequence onto protein three-dimensional (3D) structure templates. The alignment between the probe sequence and the most suitable template can be used to predict the 3D structure and often biological function of the probe. Here we present a new threading scoring function of protein sequence-structure compatibility. An artificial neural network model is trained to predict compatibility of amino acid side chains with structural environments. Log-odds scores of predicted probabilities from this model can then be used to construct protein sequence-structure alignments. RESULTS: Our model is tested on discrimination of native and decoy protein 3D structures. With a residue level structural description, its performance is comparable to those of pseudo-energy functions with atom level structural descriptions, better than the two functions with residue level structural descriptions. AVAILABILITY: The C++ source code of our neural network model is available at http://mathbio.nimr.mrc.ac.uk/~kxlin. PMID- 12376380 TI - Data mining of sequences and 3D structures of allergenic proteins. AB - MOTIVATION: Many sequences, and in some cases structures, of proteins that induce an allergic response in atopic individuals have been determined in recent years. This data indicates that allergens, regardless of source, fall into discreet protein families. Similarities in the sequence may explain clinically observed cross-reactivities between different biological triggers. However, previously available allergy databases group allergens according to their biological sources, or observed clinical cross-reactivities, without providing data about the proteins. A computer-aided data mining system is needed to compare the sequential and structural details of known allergens. This information will aid in predicting allergenic cross-responses and eventually in determining possible common characteristics of IgE recognition. RESULTS: The new web-based Structural Database of Allergenic Proteins (SDAP) permits the user to quickly compare the sequence and structure of allergenic proteins. Data from literature sources and previously existing lists of allergens are combined in a MySQL interactive database with a wide selection of bioinformatics applications. SDAP can be used to rapidly determine the relationship between allergens and to screen novel proteins for the presence of IgE or T-cell epitopes they may share with known allergens. Further, our novel similarity search method, based on five dimensional descriptors of amino acid properties, can be used to scan the SDAP entries with a peptide sequence. For example, when a known IgE binding epitope from shrimp tropomyosin was used as a query, the method rapidly identified a similar sequence in known shellfish and insect allergens. This prediction of cross-reactivity between allergens is consistent with clinical observations. AVAILABILITY: SDAP is available on the web at http://fermi.utmb.edu/SDAP/index.html PMID- 12376381 TI - Quantification of protein surfaces, volumes and atom-atom contacts using a constrained Voronoi procedure. AB - MOTIVATION: Geometric representations of proteins and ligands, including atom volumes, atom-atom contacts and solvent accessible surfaces, can be used to characterize interactions between and within proteins, ligands and solvent. Voronoi algorithms permit quantification of these properties by dividing structures into cells with a one-to-one correspondence with constituent atoms. As there is no generally accepted measure of atom-atom contacts, a continuous analytical representation of inter-atomic contacts will be useful. Improved geometric algorithms will also be helpful in increasing the speed and accuracy of iterative modeling algorithms. RESULTS: We present computational methods based on the Voronoi procedure that provide rapid and exact solutions to solvent accessible surfaces, volumes, and atom contacts within macromolecules. Furthermore, we define a measure of atom-atom contact that is consistent with the calculation of solvent accessible surfaces, allowing the integration of solvent accessibility and inter-atomic contacts into a continuous measure. The speed and accuracy of the algorithm is compared to existing methods for calculating solvent accessible surfaces and volumes. The presented algorithm has a reduced execution time and greater accuracy compared to numerical and approximate analytical surface calculation algorithms, and a reduced execution time and similar accuracy to existing Voronoi procedures for calculating atomic surfaces and volumes. PMID- 12376382 TI - Finding motifs in the twilight zone. AB - MOTIVATION: Gene activity is often affected by binding transcription factors to short fragments in DNA sequences called motifs. Identification of subtle regulatory motifs in a DNA sequence is a difficult pattern recognition problem. In this paper we design a new motif finding algorithm that can detect very subtle motifs. RESULTS: We introduce the notion of a multiprofile and use it for finding subtle motifs in DNA sequences. Multiprofiles generalize the notion of a profile and allow one to detect subtle patterns that escape detection by the standard profiles. Our MULTIPROFILER algorithm outperforms other leading motif finding algorithms in a number of synthetic models. Moreover, it can be shown that in some previously studied motif models, MULTIPROFILER is capable of pushing the performance envelope to its theoretical limits. AVAILABILITY: http://www cse.ucsd.edu/groups/bioinformatics/software.html PMID- 12376383 TI - Subtle motifs: defining the limits of motif finding algorithms. AB - MOTIVATION: What constitutes a subtle motif? Intuitively, it is a motif that is almost indistinguishable, in the statistical sense, from random motifs. This question has important practical consequences: consider, for example, a biologist that is generating a sample of upstream regulatory sequences with the goal of finding a regulatory pattern that is shared by these sequences. If the sequences are too short then one risks losing some of the regulatory patterns that are located further upstream. Conversely, if the sequences are too long, the motif becomes too subtle and one is then likely to encounter random motifs which are at least as significant statistically as the regulatory pattern itself. In practical terms one would like to recognize the sequence length threshold, or the twilight zone, beyond which the motifs are in some sense too subtle. RESULTS: The paper defines the motif twilight zone where every motif finding algorithm would be exposed to random motifs which are as significant as the one which is sought. We also propose an objective tool for evaluating the performance of subtle motif finding algorithms. Finally we apply these tools to evaluate the success of our MULTIPROFILER algorithm to detect subtle motifs. PMID- 12376384 TI - Fold-recognition detects an error in the Protein Data Bank. PMID- 12376385 TI - GENOTRACE: cDNA-based local GENOme assembly from TRACE archives. AB - GENOTRACE identifies the genomic organization for a cDNA using raw data from genome sequencing projects in progress (trace archives). Local genomic contigs are generated, allowing for example the design of PCR primers in intronic sequences to amplify coding regions of a gene, needed for example for mutation or SNP detection. AVAILABILITY: The package and examples of output files can be downloaded from http://rat.niob.knaw.nl/GENOTRACE PMID- 12376386 TI - Genquire: genome annotation browser/editor. AB - We present a software package, Genquire, that allows visualization, querying, hand editing, and de novo markup of complete or partially annotated genomes. The system is written in Perl/Tk and uses, where possible, existing BioPerl data models and methods for representation and manipulation of the sequence and annotation objects. An adaptor API is provided to allow Genquire to display a wide range of databases and flat files, and a plugins API provides an interface to other sequence analysis software. AVAILABILITY: Genquire v3.03 is open-source software. The code is available for download and/or contribution at http://www.bioinformatics.org/Genquire PMID- 12376387 TI - Genexp--a genetic network simulation environment. AB - An environment for simulation of dynamics of genetic regulatory networks is presented. The model is based on the recurrent neural network principle and allows to interactively simulate various genetic regulatory interactions under different features of the system. The results are displayed graphically. AVAILABILITY: http://proteom.biomed.cas.cz/genexp PMID- 12376388 TI - CINEMA-MX: a modular multiple alignment editor. AB - Analyzing and visualizing multiple sequence alignments is a common task in many areas of molecular biology and bioinformatics. Many tools exist for this purpose, but are not easily customizable for specific in-house uses. Here we report the development of an editor, CINEMA-MX, that addresses these issues. CINEMA-MX is highly modular and configurable, and we present examples to illustrate its extensibility. AVAILABILITY: The program and full source code, which are available from http://www.bioinf.man.ac.uk/dbbrowser/cinema-mx, are being released under a combination of the LGPL and GPL, for Unix or Windows platforms. PMID- 12376389 TI - GENIE: estimating demographic history from molecular phylogenies. AB - GENIE implements a statistical framework for inferring the demographic history of a population from phylogenies that have been reconstructed from sampled DNA sequences. The methods are based on population genetic models known collectively as coalescent theory. AVAILABILITY: GENIE is available from http://evolve.zoo.ox.ac.uk. All popular operating systems are supported. PMID- 12376390 TI - "Drink at least eight glasses of water a day." Really? Is there scientific evidence for "8 x 8"? AB - Despite the seemingly ubiquitous admonition to "drink at least eight 8-oz glasses of water a day" (with an accompanying reminder that beverages containing caffeine and alcohol do not count), rigorous proof for this counsel appears to be lacking. This review sought to find the origin of this advice (called "8 x 8" for short) and to examine the scientific evidence, if any, that might support it. The search included not only electronic modes but also a cursory examination of the older literature that is not covered in electronic databases and, most importantly and fruitfully, extensive consultation with several nutritionists who specialize in the field of thirst and drinking fluids. No scientific studies were found in support of 8 x 8. Rather, surveys of food and fluid intake on thousands of adults of both genders, analyses of which have been published in peer-reviewed journals, strongly suggest that such large amounts are not needed because the surveyed persons were presumably healthy and certainly not overtly ill. This conclusion is supported by published studies showing that caffeinated drinks (and, to a lesser extent, mild alcoholic beverages like beer in moderation) may indeed be counted toward the daily total, as well as by the large body of published experiments that attest to the precision and effectiveness of the osmoregulatory system for maintaining water balance. It is to be emphasized that the conclusion is limited to healthy adults in a temperate climate leading a largely sedentary existence, precisely the population and conditions that the "at least" in 8 x 8 refers to. Equally to be emphasized, lest the message of this review be misconstrued, is the fact (based on published evidence) that large intakes of fluid, equal to and greater than 8 x 8, are advisable for the treatment or prevention of some diseases and certainly are called for under special circumstances, such as vigorous work and exercise, especially in hot climates. Since it is difficult or impossible to prove a negative-in this instance, the absence of scientific literature supporting the 8 x 8 recommendation-the author invites communications from readers who are aware of pertinent publications. PMID- 12376391 TI - Nitric oxide in the kidney. PMID- 12376392 TI - In vivo electrophysiological responses of pedunculopontine neurons to static muscle contraction. AB - The pedunculopontine nucleus (PPN) has previously been implicated in central command regulation of the cardiorespiratory adjustments that accompany exercise. The current study was executed to begin to address the potential role of the PPN in the regulation of cardiorespiratory adjustments evoked by muscle contraction. Extracellular single-unit recording was employed to document the responses of PPN neurons during static muscle contraction. Sixty-four percent (20/31) of neurons sampled from the PPN responded to static muscle contraction with increases in firing rate. Furthermore, muscle contraction-responsive neurons in the PPN were unresponsive to brief periods of hypotension but were markedly activated during chemical disinhibition of the caudal hypothalamus. A separate sample of PPN neurons was found to be moderately activated during systemic hypoxia. Chemical disinhibition of the PPN was found to markedly increase respiratory drive. These findings suggest that the PPN may be involved in modulating respiratory adjustments that accompany muscle contraction and that PPN neurons may have the capacity to synthesize muscle reflex and central command influences. PMID- 12376393 TI - Hypothalamic NPY, AGRP, and POMC mRNA responses to leptin and refeeding in mice. AB - Food deprivation (FD) increases hypothalamic neuropeptide Y (NPY) and agouti related protein (AGRP) mRNA levels and decreases proopiomelanocortin (POMC) mRNA levels; refeeding restores these levels. We determined the time course of changes in hypothalamic NPY, AGRP, and POMC mRNA levels on refeeding after 24 h FD in C57BL mice by in situ hybridization. After 24 h deprivation, mice were refed with either chow or a palatable mash containing no calories or were injected with murine leptin (100 microg) without food. Mice were perfused 2 or 6 h after treatment. Food deprivation increased hypothalamic NPY mRNA (108 +/- 6%) and AGRP mRNA (78 +/- 7%) and decreased hypothalamic POMC mRNA (-15 +/- 1%). Refeeding for 6 h, but not 2 h, was sufficient to reduce (but not restore) NPY mRNA, did not affect AGRP mRNA, and restored POMC mRNA levels to ad libitum control levels. Intake of the noncaloric mash had no effect on mRNA levels, and leptin administration after deprivation (at a dose sufficient to reduce refeeding in FD mice) was not sufficient to affect mRNA levels. These results suggest that gradual postabsorptive events subsequent to refeeding are required for the restoration of peptide mRNA to baseline levels after food deprivation in mice. PMID- 12376394 TI - Vestibulosympathetic reflex during orthostatic challenge in aging humans. AB - Aging attenuates the increase in muscle sympathetic nerve activity (MSNA) and elicits hypotension during otolith organ engagement in humans. The purpose of the present study was to determine the neural and cardiovascular responses to otolithic engagement during orthostatic stress in older adults. We hypothesized that age-related impairments in the vestibulosympathetic reflex would persist during orthostatic challenge in older subjects and might compromise arterial blood pressure regulation. MSNA, arterial blood pressure, and heart rate responses to head-down rotation (HDR) performed with and without lower body negative pressure (LBNP) in prone subjects were measured. Ten young (27 +/- 1 yr) and 11 older subjects (64 +/- 1 yr) were studied prospectively. HDR performed alone elicited an attenuated increase in MSNA in older subjects (Delta106 +/- 28 vs. Delta20 +/- 7% for young and older subjects). HDR performed during simultaneous orthostatic stress increased total MSNA further in young (Delta53 +/ 15%; P < 0.05) but not older subjects (Delta-5 +/- 4%). Older subjects demonstrated consistent significant hypotension during HDR performed both alone (Delta-6 +/- 2 mmHg) and during LBNP (Delta-7 +/- 2 mmHg). These data provide experimental support for the concept that age-related impairments in the vestibulosympathetic reflex persist during orthostatic challenge in older adults. Furthermore, these findings are consistent with the concept that age-related alterations in vestibular function might contribute to altered orthostatic blood pressure regulation with age in humans. PMID- 12376395 TI - Analysis of afferent, central, and efferent components of the baroreceptor reflex in mice. AB - Studies of genetically modified mice provide a powerful approach to investigate consequences of altered gene expression in physiological and pathological states. The goal of the present study was to characterize afferent, central, and efferent components of the baroreceptor reflex in anesthetized Webster 4 mice. Baroreflex and baroreceptor afferent functions were characterized by measuring changes in renal sympathetic nerve activity (RSNA) and aortic depressor nerve activity (ADNA) in response to nitroprusside- and phenylephrine-induced changes in arterial pressure. The data were fit to a sigmoidal logistic function curve. Baroreflex diastolic pressure threshold (P(th)), the pressure at 50% inhibition of RSNA (P(mid)), and baroreflex gain (maximum slope) averaged 74 +/- 5 mmHg, 101 +/- 3 mmHg, and 2.30 +/- 0.54%/mmHg, respectively (n = 6). The P(th), P(mid), and gain for the diastolic pressure-ADNA relation (baroreceptor afferents) were similar to that observed for the overall reflex averaging 79 +/- 9 mmHg, 101 +/- 4 mmHg, and 2.92 +/- 0.53%/mmHg, respectively (n = 5). The central nervous system mediation of the baroreflex and the chronotropic responsiveness of the heart to vagal efferent activity were independently assessed by recording responses to electrical stimulation of the left ADN and the peripheral end of the right vagus nerve, respectively. Both ADN and vagal efferent stimulation induced frequency dependent decreases in heart rate and arterial pressure. The heart rate response to ADN stimulation was nearly abolished in mice anesthetized with pentobarbital sodium (n = 4) compared with mice anesthetized with ketamine-acepromazine (n = 4), whereas the response to vagal efferent stimulation was equivalent under both types of anesthesia. Application of these techniques to studies of genetically manipulated mice can be used to identify molecular mechanisms of baroreflex function and to localize altered function to afferent, central, or efferent sites. PMID- 12376396 TI - Differential evolution of blood pressure and renal lesions after RAS blockade in Lyon hypertensive rats. AB - The present work aimed to assess, in Lyon hypertensive (LH) rats, whether an early and prolonged inhibition of the renin-angiotensin system (RAS) could result in a blood pressure (BP) lowering and nephroprotection that persist after its withdrawal. Male LH rats received orally from 3 to 12 wk of age either an angiotensin-converting enzyme inhibitor perindopril at the doses of 0.4 and 3 mg x kg(-1) x day(-1) or an AT(1) receptor antagonist losartan at the dose of 10 mg x kg(-1) x day(-1). BP, histological changes in the kidney, and urinary protein excretion were examined during and 10 wk after cessation of the treatments. Both perindopril and losartan decreased BP, prevented renal lesions, and limited urinary protein excretion. After cessation of the treatment, BP returned to the level of never-treated LH rats in rats having received 3 mg x kg(-1) x day(-1) of perindopril while it remained slightly lower in those treated with 0.4 mg x kg( 1) x day(-1) of perindopril or with losartan. This lack of marked persistent antihypertensive effect contrasted with a durable decrease in urinary protein excretion and improvement of the renal histological lesions. In conclusion, it is possible to separate the BP-lowering effects of RAS blockade from those on glomerulosclerosis and urinary protein excretion. PMID- 12376397 TI - Tumor necrosis factor-alpha inhibits renin gene expression. AB - Renin, produced in renal juxtaglomerular (JG) cells, is a fundamental regulator of blood pressure. Accumulating evidence suggests that cytokines may directly influence renin production in the JG cells. TNF-alpha, which is one of the key mediators in immunity and inflammation, is known to participate in the control of vascular proliferation and contraction and hence in the pathogenesis of cardiovascular diseases. Thus TNF-alpha may exert its effects on the cardiovascular system through modulation of renal renin synthesis. Therefore we have tested the effect of TNF-alpha on renin transcription in As4.1 cells, which represent transformed mouse JG cells, and in native mouse JG cells in culture. Renin gene expression was also determined in mice lacking the gene for TNF-alpha (TNF-alpha knockout mice). TNF-alpha inhibited renin gene expression via an inhibition of the transcriptional activity, targeting the proximal 4.1 kb of the renin promoter in As4.1 cells. TNF-alpha also attenuated forskolin-stimulated renin gene expression in primary cultures of mouse JG cells. Mice lacking the TNF alpha gene had almost threefold higher basal renal renin mRNA abundance relative to the control strain. The general physiological regulation of renin expression by salt was not disturbed in TNF-alpha knockout mice. Our data suggest that TNF alpha inhibits renin gene transcription at the cellular level and thus may act as a modulator of renin synthesis in (physio)pathological situations. PMID- 12376398 TI - Novel mechanism for high-altitude adaptation in hemoglobin of the Andean frog Telmatobius peruvianus. AB - In contrast to birds and mammals, no information appears to be available on the molecular adaptations for O(2) transport in high-altitude ectothermic vertebrates. We investigated Hb of the aquatic Andean frog Telmatobius peruvianus from 3,800-m altitude as regards isoform differentiation, sensitivity to allosteric cofactors, and primary structures of the alpha- and beta-chains, and we carried out comparative O(2)-binding measurements on Hb of lowland Xenopus laevis. The three T. peruvianus isoHbs show similar functional properties. The high O(2) affinity of the major component results from an almost complete obliteration of chloride sensitivity, which correlates with two alpha-chain modifications: blockage of the NH(2)-terminal residues and replacement by nonpolar Ala of polar residues Ser and Thr found at position alpha131(H14) in human and X. leavis Hbs, respectively. The data indicate adaptive significance of alpha-chain chloride-binding sites in amphibians, in contrast to human Hb where chloride appears mainly to bind in the cavity between the beta-chains. The findings are discussed in relation to other strategies for high-altitude adaptations in amphibians. PMID- 12376399 TI - Reduced feeding during water deprivation depends on hydration of the gut. AB - Removal of drinking water at the start of the dark period reduced food intake in freely feeding rats within 45 min. Both first and later meals were smaller during 7.5 h of water deprivation, but meal frequency did not change. Ingestion of a normal-sized meal (3 g) rapidly increased plasma tonicity when drinking water was withheld, but intravenous infusions of hypertonic NaCl causing similar increases in plasma tonicity did not reduce feeding. Feeding during 6 h of water deprivation was restored by slowly infusing the volume of water normally drunk into the stomach, jejunum, or cecum, but not in the vena cava or hepatic portal vein. The infusions did not alter water or electrolyte excretion or affect food intake in rats allowed to drink. We conclude that the inhibition of feeding seen during water deprivation is mediated by a sensor that is located in the gastrointestinal tract or perhaps in the mesenteric veins draining the gut, but not the hepatic portal vein or the liver. In the absence of drinking water, signals from this sensor provoke the early termination of a meal. PMID- 12376400 TI - Synergy between angiotensin and aldosterone in evoking sodium appetite in baboons. AB - The synergy between ANG II and aldosterone (Aldo) in the induction of salt appetite, extensively studied in rats, has been tested in baboons. ANG II was infused intracerebroventricularly at 0.5 or 1.0 microg/h; Aldo was infused subcutaneously at 20 microg/h. Separate infusions over 7 days had no significant effect on the daily intake of 300 mM NaCl. Concurrent infusions, however, increased daily NaCl intake approximately 10-fold and daily water intake approximately 2.5-fold. In addition, the combined infusions caused 1) a reduction in daily food intake, 2) changes in blood composition indicative of increased vasopressin release, and 3) changes of urinary excretion rates of cortisol and Aldo indicative of increased ACTH release. Arterial blood pressure, measured in two baboons, rose during concurrent ANG II and Aldo treatment. These results indicate a potent synergy between central ANG II and peripheral Aldo in stimulating salt appetite in baboons. At the same time, other ANG II-specific brain mechanisms concerned with water intake, food intake, vasopressin release, ACTH release, and blood pressure regulation appear to have been activated by the same type of synergy. These central enhancement processes have never been previously demonstrated in primates. PMID- 12376401 TI - Hypocretin release in normal and narcoleptic dogs after food and sleep deprivation, eating, and movement. AB - Hypocretins (orexins) are recently discovered hypothalamic neuropeptides that have been implicated in the etiology of narcolepsy. The normal behavioral functions of these peptides are unclear, although a role in feeding has been suggested. We measured hypocretin-1 (Hcrt-1) in the cerebrospinal fluid of dogs during a variety of behaviors. We found that 48 h without food (24 h beyond normal 24-h fasting period) produced no significant change in Hcrt-1 levels nor did feeding after the deprivation. In contrast, 24 h of sleep deprivation produced on average a 70% increase in Hcrt-1 level compared with baseline levels. The amount of increase was correlated with the level of motor activity during the sleep-deprivation procedure. A 2-h period of exercise in the same dogs produced a 57% increase in Hcrt-1 levels relative to quiet waking levels, with the magnitude of the increase being highly correlated with the level of motor activity. The strong correlation between motor activity and Hcrt-1 release may explain some of the previously reported behavioral, physiological, and pathological phenomena ascribed to the Hcrt system. PMID- 12376402 TI - Maternal obesity alters adiposity and monoamine function in genetically predisposed offspring. AB - The impact of maternal obesity on brain monoamine function in adult offspring of dams selectively bred to express diet-induced obesity (DIO) or diet resistance (DR) was assessed by making dams obese or lean during gestation and lactation. After 12 wk on chow and 4 wk on a 31% fat diet, offspring hypothalamic nucleus size and [(3)H]nisoxetine binding to norepinephrine transporters (NET) and [(3)H]paroxetine binding to serotonin transporters (SET) were measured. Offspring of obese DIO dams became more obese than all other groups, but maternal obesity did not alter weight gain in DR offspring (25). Maternal obesity was associated with 10-17% enlargement of ventromedial nuclei (VMN) and dorsomedial nuclei in both DIO and DR offspring. Offspring of obese DIO dams had 25-88% lower NET binding in the paraventricular nuclei (PVN), arcuate nuclei, VMN, and the central amygdalar nuclei, while offspring of obese DR dams had 43-67% higher PVN and 90% lower VMN NET binding and a generalized increase in SET binding across all hypothalamic areas compared with other groups. Thus maternal obesity was associated with alterations in offspring brain monoamine metabolism, which varied as a function of genotype and the development of offspring obesity. PMID- 12376403 TI - Compensation for partial lipectomy in mice with genetic alterations of leptin and its receptor subtypes. AB - One hypothesis for the regulation of total body fat suggests that leptin is a lipostatic feedback signal that acts at brain sites involved in regulation of energy balance. The importance of leptin in recovery from partial surgical lipectomy was tested by performing bilateral epididymal lipectomy or sham surgery on wild-type and leptin-deficient ob/ob mice. Eight weeks later, nonexcised pads of lipectomized mice were increased but total carcass fat was lower than in sham operated ob/ob mice. In experiment 2, ob/ob mice, wild-type mice, and two db/db mutants, C57BL/6J db(Lepr)/db(Lepr) (BL/6J) mice possessing short-form and circulating leptin receptors and C57BL/6J db(3J)/db(3J) (BL/3J) mice expressing only circulating receptors, were lipectomized or sham operated. Sixteen weeks later, body mass and carcass lipid were not different between sham and lipectomized ob/ob mice, wild-type mice, or BL/6J db/db mice, whereas there was incomplete (decreased carcass fat) but suggestive recovery (increased retroperitoneal fat mass and cell number) in lipectomized BL/3J db/db mice. These data indicate that leptin is not required for the regulation of total body fat. PMID- 12376404 TI - Prostaglandin E(2)-synthesizing enzymes in fever: differential transcriptional regulation. AB - The febrile response to lipopolysaccharide (LPS) consists of three phases (phases I-III), all requiring de novo synthesis of prostaglandin (PG) E(2). The major mechanism for activation of PGE(2)-synthesizing enzymes is transcriptional upregulation. The triphasic febrile response of Wistar-Kyoto rats to intravenous LPS (50 microg/kg) was studied. Using real-time RT-PCR, the expression of seven PGE(2)-synthesizing enzymes in the LPS-processing organs (liver and lungs) and the brain "febrigenic center" (hypothalamus) was quantified. Phase I involved transcriptional upregulation of the functionally coupled cyclooxygenase (COX)-2 and microsomal (m) PGE synthase (PGES) in the liver and lungs. Phase II entailed robust upregulation of all enzymes of the major inflammatory pathway, i.e., secretory (s) phospholipase (PL) A(2)-IIA --> COX-2 --> mPGES, in both the periphery and brain. Phase III was accompanied by the induction of cytosolic (c) PLA(2)-alpha in the hypothalamus, further upregulation of sPLA(2)-IIA and mPGES in the hypothalamus and liver, and a decrease in the expression of COX-1 and COX 2 in all tissues studied. Neither sPLA(2)-V nor cPGES was induced by LPS. The high magnitude of upregulation of mPGES and sPLA(2)-IIA (1,257-fold and 133-fold, respectively) makes these enzymes attractive targets for anti-inflammatory therapy. PMID- 12376405 TI - "Sausage-string" appearance of arteries and arterioles can be caused by an instability of the blood vessel wall. AB - Vascular damage induced by acute hypertension is preceded by a peculiar pattern where blood vessels show alternating regions of constrictions and dilations ("sausages on a string"). The pattern occurs in the smaller blood vessels, and it plays a central role in causing the vascular damage. A related vascular pattern has been observed in larger vessels from several organs during angiography. In the larger vessels the occurrence of the pattern does not appear to be related to acute hypertension. A unifying feature between the phenomenon in large and small vessels seems to be an increase in vascular wall tension. Despite much research, the mechanisms underlying the sausage pattern have remained unknown. Here we present an anisotropic model of the vessel wall and show that the sausage pattern can arise because of an instability of the vessel wall. The model reproduces many of the key features observed experimentally. Most importantly, it suggests that the "sausaging" phenomenon is neither caused by a mechanical failure of the vessel wall due to a high blood pressure nor is it due to standing pressure waves caused by the beating of the heart. Rather, it is the expression of a general instability phenomenon. Experimental data suggest that the structural changes induced by the instability may cause secondary damage to the wall of small arteries and arterioles in the form of endothelial hyperpermeability followed by local fibrinoid necrosis of the vascular wall. PMID- 12376406 TI - Effect of mild carboxy-hemoglobin on exercising skeletal muscle: intravascular and intracellular evidence. AB - We studied muscle blood flow, muscle oxygen uptake (VO(2)), net muscle CO uptake, Mb saturation, and intracellular bioenergetics during incremental single leg knee extensor exercise in five healthy young subjects in conditions of normoxia, hypoxia (H; 11% O(2)), normoxia + CO (CO(norm)), and 100% O(2) + CO (CO(hyper)). Maximum work rates and maximal oxygen uptake (VO(2 max)) were equally reduced by approximately 14% in H, CO(norm), and CO(hyper). The reduction in arterial oxygen content (Ca(O(2))) (approximately 20%) resulted in an elevated blood flow (Q) in the CO and H trials. Net muscle CO uptake was attenuated in the CO trials. Suprasystolic cuff measurements of the deoxy-Mb signal were not different in terms of the rate of signal rise or maximum signal attained with and without CO. At maximal exercise, calculated mean capillary PO(2) was most reduced in H and resulted in the lowest Mb-associated PO(2). Reductions in ATP, PCr, and pH during H, CO(norm), and CO(hyper) occurred earlier during progressive exercise than in normoxia. Thus the effects of reduced Ca(O(2)) due to mild CO poisoning are similar to H. PMID- 12376407 TI - Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells. AB - Mucosal and enterocyte IL-6 production is increased during sepsis and endotoxemia. Recent studies suggest that cAMP potentiates IL-6 production in endotoxin- or IL-1beta-stimulated enterocytes, but the molecular mechanisms are not known. We examined the role of the transcription factors NF-kappaB, activator protein (AP)-1, CCAAT/enhancer binding protein (C/EBP), and cAMP response element binding protein (CREB) in cAMP-induced IL-6 production in cultured Caco-2 cells, a human intestinal epithelial cell line. In addition, the role of the protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein (MAP) kinase signaling pathways was examined. Treatment of the cells with IL-1beta increased IL-6 production and activated the IL-6 promoter in cells transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter. These effects of IL-1beta were significantly potentiated by cAMP. When the binding sites for the individual transcription factors in the IL-6 promoter were mutated, results indicated that all four transcription factors may be involved in the cAMP induced activation of the IL-6 gene. Treatment of the Caco-2 cells with cAMP increased the DNA binding activity of CREB, C/EBP, and AP-1, but not NF-kappaB. By using specific blockers, evidence was found that both PKA and p38 MAP kinase (but not PKC or p42/44 MAP kinase) may be involved in the cAMP-induced potentiation of IL-6 production. The present results suggest that cAMP activates multiple transcription factors involved in the regulation of the IL-6 gene and that the activation of these transcription factors may at least in part explain why cAMP potentiates IL-6 production in stimulated enterocytes. PMID- 12376408 TI - Naltrexone infusion inhibits the development of preference for a high-sucrose diet. AB - We hypothesized that the opioid antagonist naltrexone would inhibit the redevelopment of a preference for a high-sucrose diet after an abstention period from this diet. Rats that chose between a starch or sucrose diet for 10 days preferred the sucrose diet. Rats were then given access to the starch diet alone for another 10-day period. A miniosmotic pump containing saline or naltrexone was then implanted (70 microg/h; 1.7 mg/day) for approximately 10 days. During the saline infusion, 77% of the total energy came from the sucrose diet, whereas during the naltrexone infusion, 33% of the total energy came from the sucrose diet. We repeated this study in another group of rats but did not restrict the sucrose diet. In this case naltrexone failed to decrease preference for the sucrose diet. Thus naltrexone infusion inhibited redevelopment of a preference for a sucrose diet after a period of restriction to a starch diet for 10 days but had no effect on preference if both diets were present throughout the study. PMID- 12376409 TI - Glucocorticoids, thyroid hormones, and iodothyronine deiodinases in embryonic saltwater crocodiles. AB - We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T(3)), and a combination of these two hormones (Dex + T(3)). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T(4)) ORD activities in liver and kidney microsomes, and a decrease in the high-K(m) rT(3) ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T(4) ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T(3) IRD activity in liver microsomes. However, this decrease did not change plasma-free T(3) concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development. PMID- 12376410 TI - Control of the development of the pulmonary surfactant system in the saltwater crocodile, Crocodylus porosus. AB - Pulmonary surfactant is a mixture of lipids and proteins that controls the surface tension of the fluid lining the inner lung. Its composition is conserved among the vertebrates. Here we hypothesize that the in ovo administration of glucocorticoids and thyroid hormones during late incubation will accelerate surfactant development in the saltwater crocodile, Crocodylus porosus. We also hypothesize that the increased maturation of the type II cells in response to hormone pretreatment will result in enhanced responsiveness of the cells to surfactant secretagogues. We sampled embryos at days 60, 68, and 75 of incubation and after hatching. We administered dexamethasone (Dex), 3,5,3'-triiodothyronine (T(3)), or a combination of both hormones (Dex + T(3)), 48 and 24 h before each prehatching time point. Lavage analysis indicated that the maturation of the phospholipids (PL) in the lungs of embryonic crocodiles occurs rapidly. Only T(3) and Dex + T(3) increased total PL in lavage at embryonic day 60, but Dex, T(3), and Dex + T(3) increased PL at day 75. The saturation of the PLs was increased by T(3) and Dex + T(3) at day 68. Swimming exercise did not increase the amount or alter the saturation of the surfactant PLs. Pretreatment of embryos with Dex, T(3), or Dex + T(3) changed the secretion profiles of the isolated type II cells. Dex + T(3) increased the response of the cells to agonists at days 60 and 68. Therefore, glucocorticoids and thyroid hormones regulate surfactant maturation in the crocodile. PMID- 12376411 TI - Regional responsiveness of renal perfusion to activation of the renal nerves. AB - We tested for regional differences in perfusion responses, within the renal medulla and cortex, to renal nerve stimulation in pentobarbital sodium anesthetized rabbits. Laser-Doppler flux (LDF) was monitored at various depths below the cortical surface (1-15 mm). Basal cortical LDF (1-3 mm, approximately 200-450 U) was greater than medullary LDF (5-15 mm, approximately 70-160 U), but there were no statistically significant differences in basal LDF within these regions. The background LDF signal during aortic occlusion was similar in the cortex (2 mm, 31 U) and outer medulla (7 mm, 31 U), but slightly greater in the inner medulla (12 mm, 44 U). During electrical stimulation of the renal nerves (0.5-8 Hz), cortical LDF and total renal blood flow were similarly progressively reduced with increasing stimulus frequency. Medullary LDF (measured between 5 and 15 mm) was overall less responsive than cortical LDF. For example, 4-Hz stimulation reduced inner medullary LDF (9 mm) by 19 +/- 6% but reduced cortical LDF (1 mm) by 54 +/- 11%. However, medullary LDF responses to nerve stimulation were similar at all depths measured. Our results indicate that while the vascular elements controlling medullary perfusion are less sensitive to the effects of electrical stimulation of the renal nerves than are those controlling cortical perfusion, sensitivity within these vascular territories appears to be relatively homogeneous. PMID- 12376412 TI - Hypotensive effect of ANG II and ANG-(1-7) at the caudal ventrolateral medulla involves different mechanisms. AB - The objective of the present study was to determine the contribution of the autonomic nervous system and nitric oxide to the depressor effect produced by unilateral microinjection of ANG-(1-7) and ANG II into the caudal ventrolateral medulla (CVLM). Unilateral microinjection of ANG-(1-7), ANG II (40 pmol), or saline (100 nl) was made into the CVLM of male Wistar rats anesthetized with urethane before and after intravenous injection of 1) methyl-atropine, 2.5 mg/kg; 2) prazosin, 25 microg/kg; 3) the nitric oxide synthase (NOS) inhibitor, N(G) nitro-L-arginine methyl ester (L-NAME), 5 mg/kg; or 4) the specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), 45 mg/kg. Arterial pressure and heart rate (HR) were continuously monitored. Microinjection of ANG-(1-7) or ANG II into the CVLM produced a significant decrease in mean arterial pressure (MAP; -11 +/- 1 mmHg, n = 12 and -10 +/- 1 mmHg, n = 10, respectively) that was not accompanied by consistent changes in HR or in cardiac output. The effect of ANG-(1-7) was abolished after treatment with methyl-atropine (-3 +/- 0.6 mmHg, n = 9) or L-NAME (-2.3 +/- 0.5 mmHg, n = 8) or 7-NI (-2.8 +/- 0.6 mmHg, n = 5). In contrast, these treatments did not significantly interfere with the ANG II effect (-10 +/- 2.6 mmHg, n = 8; -8 +/- 1.5 mmHg, n = 8; and -12 +/- 3.6 mmHg, n = 6; respectively). Peripheral treatment with prazosin abolished the hypotensive effect of ANG-(1-7) and ANG II. Microinjection of saline did not produce any significant change in MAP or in HR. These results suggest that the hypotensive effect produced by ANG II at the CVLM depends on changes in adrenergic vascular tonus and, more importantly, the hypotensive effect produced by ANG-(1-7) also involves a nitric oxide-related mechanism. PMID- 12376413 TI - Rearing temperature and the sympathetic nervous system regulation of white and brown adipose tissue. PMID- 12376414 TI - Effects of rearing temperature on sympathoadrenal activity in young adult rats. AB - Animals reared at 18 degrees C exhibit enhanced innervation of brown adipose tissue (BAT) and greater cold tolerance as adults, yet gain more weight when fed an enriched diet compared with rats reared at 30 degrees C. To explore this paradox, sympathoadrenal activity was examined using techniques of [(3)H]norepinephrine ([(3)H]NE) turnover and urinary catecholamine excretion in male and female rats reared until 2 mo of age at 18 or 30 degrees C. Gene expression in BAT was also analyzed for several sympathetically related proteins. Although [(3)H]NE turnover in heart did not differ between groups, [(3)H]NE turnover in BAT was consistently elevated in the 18 degrees C-reared animals, even 2 mo after removal from the cool environment. Gene expression for uncoupling proteins 1 and 3, GLUT-4, leptin, and the alpha(1A)-adrenergic receptor was more abundant in BAT and the increase in epinephrine excretion with fasting suppressed in 18 degrees C-reared animals. These studies demonstrate that obesity consequent to exposure to 18 degrees C in early life occurs despite tonic elevation of sympathetic input to BAT. Diminished adrenal epinephrine responsiveness to fasting may play a contributory role. PMID- 12376415 TI - Heart rate control and mechanical cardiopulmonary coupling to assess central volume: a systems analysis. AB - Small negative changes of central volume reduce cardiac output without significant alterations of arterial blood pressure (ABP), suggesting an adequate regulatory response. Furthermore, evidence has arisen supporting a Bainbridge reflex (tachycardia with hypervolemia) in humans. To investigate these phenomena, multivariate autoregressive techniques were used to evaluate the beat-to-beat interactions between respiration, R-R interval, and ABP at six levels of decreased and increased central volume. With reductions of central volume below control, baroreflex and respiratory sinus arrhythmia gains were reduced, while with increases of volume above control, gains increased for the first two levels but decreased again at the highest volume level, suggesting the presence of a Bainbridge reflex in healthy human subjects. The mechanical influence of respiration on central venous pressure (CVP) had an unexpected shift in phase at the point of mild central hypervolemia, with the expected negative relation at lower volumes (inspiration lowers CVP) but a positive relation at higher volumes (inspiration raises CVP). We conclude that multivariate techniques can quantify the relations between a variety of respiratory and hemodynamic parameters, allowing for the in vivo assessment of complex cardiorespiratory interactions during manipulations of central volume. The results identify the presence of a Bainbridge reflex in humans and suggest that short-term cardiovascular control is optimized at mild hypervolemia. PMID- 12376416 TI - Phenylephrine-induced elevations in arterial blood pressure are attenuated in heat-stressed humans. AB - To test the hypothesis that phenylephrine-induced elevations in blood pressure are attenuated in heat-stressed humans, blood pressure was elevated via steady state infusion of three doses of phenylephrine HCl in 10 healthy subjects in both normothermic and heat stress conditions. Whole body heating significantly increased sublingual temperature by ~0.5 degrees C, muscle sympathetic nerve activity (MSNA), heart rate, and cardiac output and decreased total peripheral vascular resistance (TPR; all P < 0.005) but did not change mean arterial blood pressure (MAP; P > 0.05). At the highest dose of phenylephrine, the increase in MAP and TPR from predrug baselines was significantly attenuated during the heat stress [DeltaMAP 8.4 +/- 1.2 mmHg; DeltaTPR 0.96 +/- 0.85 peripheral resistance units (PRU)] compared with normothermia (DeltaMAP 15.4 +/- 1.4 mmHg, DeltaTPR 7.13 +/- 1.18 PRU; all P < 0.001). The sensitivity of baroreflex control of MSNA and heart rate, expressed as the slope of the relationship between MSNA and diastolic blood pressure, as well as the slope of the relationship between heart rate and systolic blood pressure, respectively, was similar between thermal conditions (each P > 0.05). These data suggest that phenylephrine-induced elevations in MAP are attenuated in heat-stressed humans without affecting baroreflex control of MSNA or heart rate. PMID- 12376417 TI - Role of hypoxemia for the cardiovascular responses to apnea during exercise. AB - We sought to define the role of hypoxemia in eliciting the cardiovascular responses to apnea during exercise. Eleven men performed repeated apneas during 100-W steady-state exercise, either with normoxic gas (air) or 95% oxygen (oxygen). Beat-by-beat arterial blood pressure, arterial oxygen saturation, and heart rate (HR) were determined, and stroke volume (SV) was estimated from impedance cardiography calibrated with soluble gas rebreathing. There were large interindividual variabilities of HR, mean arterial pressure (MAP), and total peripheral resistance (TPR) at end-apnea (ea). However, for each individual, HR(ea), MAP(ea), and TPR(ea) were highly correlated between air and oxygen (R = 0.94, 0.78, and 0.93). HR decreased and MAP increased faster during apnea with air than with oxygen (ANOVA, P < 0.05), but MAP(ea) was not different between conditions. Cardiac output was reduced by 33% with air and by 11% with oxygen (P < 0.001 for air vs. oxygen). We conclude that the hypoxemia component cannot account for the wide interindividual differences of HR and TPR responses to apnea. However, hypoxemia augments the HR and TPR responses and may limit the MAP response to apnea by preventing a bradycardia-associated increase of SV. PMID- 12376418 TI - Beta-adrenoceptor control of G protein function in the neonate: determinant of desensitization or sensitization. AB - Neonatal beta-adrenoceptors (beta-ARs) are resistant to agonist-induced desensitization. We examined the functioning of G(i) and G(s) after repeated administration of beta-AR agonists to newborn rats. Isoproterenol (beta(1)/beta(2) agonist) obtunded G(i) function in the heart but not the liver; in contrast, terbutaline, a beta(2)-selective agonist, enhanced G(i) function. Isoproterenol, but not terbutaline, increased membrane-associated G((s)alpha), which would enhance receptor function. In addition, isoproterenol increased and terbutaline maintained the proportion of the short-splice (S) variant of G((s)alpha) in the membrane fraction; G((s)alpha)S is functionally more active than the long-splice variant. Either isoproterenol or terbutaline treatment increased G((s)alpha) in the cytosolic fraction, a characteristic usually associated with desensitization in the adult. Decreased G(i) activity, coupled with increased membrane-associated G((s)alpha) concentrations and maintenance or increases in membrane G((s)alpha)S, provide strong evidence that unique effects on G protein function underlie the ability of the immature organism to sustain beta-AR cell signaling in the face of excessive or prolonged stimulation; these mechanisms also contribute to tissue selectivity of the effects of beta-agonists with divergent potencies toward different beta-AR subtypes. PMID- 12376419 TI - Increased lipogenesis in isolated hepatocytes from cold-acclimated ducklings. AB - Thermogenic endurance and development of metabolic cold adaptation in birds may critically depend on their ability to synthesize and use fatty acids (FA) as fuel substrates. Hepatic lipogenesis and the capacity to oxidize FA in thermogenic tissues were measured in cold-acclimated (CA) ducklings (Cairina moschata) showing original mechanisms of metabolic cold adaptation in the absence of brown adipose tissue, the specialized thermogenic tissue of rodents. The rate of FA synthesis from [U-(14)C]glucose and from [1-(14)C]acetate, measured in incubated hepatocytes isolated from 5-wk-old thermoneutral (TN; 25 degrees C) or CA (4 degrees C) fed ducklings, was higher than in other species. Hepatic de novo lipogenesis was further increased by cold acclimation with both glucose (+194%) and acetate (+111%) as precursor. Insulin slightly increased (+11-14%) hepatic lipogenesis from both precursors in CA ducklings, whereas glucagon was clearly inhibitory (-29 to -51%). Enhanced de novo lipogenesis was associated with higher (+171%) hepatocyte activity of glucose oxidation and larger capacity (+50 to +100%) of key lipogenic enzymes. The potential for FA oxidation was higher in liver (+61%) and skeletal muscle (+29 to +81%) homogenates from CA than from TN ducklings, suggesting that the higher hepatic lipogenesis may fuel oxidation in thermogenic tissues. Present data underline the high capacity to synthesize lipids from glucose in species like muscovy ducks susceptible to hepatic steatosis. Lipogenic capacity can be further increased in the cold and may represent an important step in the metabolic adaptation to cold of growing ducklings. PMID- 12376420 TI - Oxygen-limited thermal tolerance in Antarctic fish investigated by MRI and (31)P MRS. AB - The hypothesis of an oxygen-limited thermal tolerance was tested in the Antarctic teleost Pachycara brachycephalum. With the use of flow-through respirometry, in vivo (31)P-NMR spectroscopy, and MRI, we studied energy metabolism, intracellular pH (pH(i)), blood flow, and oxygenation between 0 and 13 degrees C under normoxia (PO(2): 20.3 to 21.3 kPa) and hyperoxia (PO(2): 45 kPa). Hyperoxia reduced the metabolic increment and the rise in arterial blood flow observed under normoxia. The normoxic increase of blood flow leveled off beyond 7 degrees C, indicating a cardiovascular capacity limitation. Ventilatory effort displayed an exponential rise in both groups. In the liver, blood oxygenation increased, whereas in white muscle it remained unaltered (normoxia) or declined (hyperoxia). In both groups, the slope of pH(i) changes followed the alpha-stat pattern below 6 degrees C, whereas it decreased above. In conclusion, aerobic scope declines around 6 degrees C under normoxia, marking the pejus temperature. By reducing circulatory costs, hyperoxia improves aerobic scope but is unable to shift the breakpoint in pH regulation or lethal limits. Hyperoxia appears beneficial at sublethal temperatures, but no longer beyond when cellular or molecular functions become disturbed. PMID- 12376421 TI - Effect of hemorrhagic shock on gut barrier function and expression of stress related genes in normal and gnotobiotic mice. AB - We sought to determine whether gut-derived microbial factors influence the hepatic or intestinal inflammatory response to hemorrhagic shock and resuscitation (HS/R). Conventional and gnotobiotic mice contaminated with a defined microbiota without gram-negative bacteria were subjected to either a sham procedure or HS/R. Tissue samples were obtained 4 h later for assessing ileal mucosal permeability to FITC dextran and hepatic and ileal mucosal steady-state IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and TNF mRNA levels. Whereas HS/R significantly increased ileal mucosal permeability in conventional mice, this effect was not apparent in gnotobiotic animals. HS/R markedly increased hepatic mRNA levels for several proinflammatory genes in both conventional and gnotobiotic mice. HS/R increased ileal mucosal IL-6 and COX-2 mRNA expression in conventional but not gnotobiotic mice. If gnotobiotic mice were contaminated with Escherichia coli C25, HS/R increased ileal mucosal permeability and upregulated expression of IL-6 and COX-2. These data support the view that the hepatic inflammatory response to HS/R is largely independent of the presence of potentially pathogenic gram-negative bacteria colonizing the gut, whereas the local mucosal response to HS/R is profoundly influenced by the microbial ecology within the lumen during and shortly after the period of hemorrhage. PMID- 12376422 TI - Sensitive periods for the effect of dietary sodium restriction on intact and denervated taste receptor cells. AB - Unilateral chorda tympani nerve (CT) section combined with dietary sodium restriction leads to striking alterations in sodium taste function. The regenerated rat CT exhibits deficits in sodium sensitivity, and surprisingly, there are also functional alterations in the intact, contralateral nerve. The studies presented here describe the functional "sensitive periods" for these aberrations and the number of taste buds present during corresponding stages. The regenerated CT is sensitive to dietary sodium restriction during the first 2 wk after denervation, whereas the intact CT is sensitive to dietary manipulation during the first week postsection. Therefore, distinct mechanisms are responsible for the effects of sodium restriction combined with denervation, because separate sensitive periods exist for the regenerated and intact CT nerves. Identification of mature taste buds with an antibody directed at anti-keratin 19 revealed that there is a loss of ~85% of taste buds on the denervated side of the tongue under control and low-sodium diets within the first week postsection. Thus, sodium restriction does not differentially affect the loss of taste buds following denervation. PMID- 12376423 TI - Surgery for primary hyperparathyroidism. PMID- 12376424 TI - Medical school applications--a critical situation. PMID- 12376425 TI - Volume of procedures and outcome of treatment. PMID- 12376426 TI - Urinary stress incontinence. PMID- 12376427 TI - Your career: planning for the unexpected. PMID- 12376428 TI - Human genome pioneers are awarded Nobel prize. PMID- 12376429 TI - Rules on gene therapy are tightened after leukaemia alert. PMID- 12376431 TI - Genomes of the malaria mosquito and parasite are sequenced. PMID- 12376437 TI - Prospective cohort study of routine use of risk assessment scales for prediction of pressure ulcers. AB - OBJECTIVE: To evaluate whether risk assessment scales can be used to identify patients who are likely to get pressure ulcers. DESIGN: Prospective cohort study. SETTING: Two large hospitals in the Netherlands. PARTICIPANTS: 1229 patients admitted to the surgical, internal, neurological, or geriatric wards between January 1999 and June 2000. MAIN OUTCOME MEASURE: Occurrence of a pressure ulcer of grade 2 or worse while in hospital. RESULTS: 135 patients developed pressure ulcers during four weeks after admission. The weekly incidence of patients with pressure ulcers was 6.2% (95% confidence interval 5.2% to 7.2%). The area under the receiver operating characteristic curve was 0.56 (0.51 to 0.61) for the Norton scale, 0.55 (0.49 to 0.60) for the Braden scale, and 0.61 (0.56 to 0.66) for the Waterlow scale; the areas for the subpopulation, excluding patients who received preventive measures without developing pressure ulcers and excluding surgical patients, were 0.71 (0.65 to 0.77), 0.71 (0.64 to 0.78), and 0.68 (0.61 to 0.74), respectively. In this subpopulation, using the recommended cut-off points, the positive predictive value was 7.0% for the Norton, 7.8% for the Braden, and 5.3% for the Waterlow scale. CONCLUSION: Although risk assessment scales predict the occurrence of pressure ulcers to some extent, routine use of these scales leads to inefficient use of preventive measures. An accurate risk assessment scale based on prospectively gathered data should be developed. PMID- 12376438 TI - Using vital signs to diagnose impaired consciousness: cross sectional observational study. AB - OBJECTIVES: To determine whether any vital signs can be used to quickly identify brain lesions in patients with impaired consciousness. DESIGN: Cross sectional observational study. SETTING: Emergency department of an urban hospital, Japan. PARTICIPANTS: 529 consecutive patients (mean age 65 years) presenting with impaired consciousness (score <15 on the Glasgow coma scale) during 2000. MAIN OUTCOME MEASURES: The receiver operating characteristic curve was used to quantify the relation between the vital signs on arrival and the final diagnosis of a brain lesion. Stratum specific likelihood ratios were calculated to define strata with optimal discriminating power. RESULTS: 312 (59%) had a brain lesion which accounted for the impaired consciousness. The area under the receiver operating curve for systolic blood pressure was 0.90 (SE 0.01), indicating significantly higher accuracy (P<0.01) in the identification of a brain lesion than using diastolic pressure 0.82 (0.02) or pulse rate 0.63 (0.03). Likelihood ratios for systolic blood pressure lower than 90 mm Hg were less than 0.04, and those for systolic pressure higher than 170 mm Hg were greater than 6.09. CONCLUSIONS: Systolic blood pressure is useful for diagnosing brain lesions in patients with impaired consciousness. PMID- 12376439 TI - Where does blood go? Prospective observational study of red cell transfusion in north England. AB - OBJECTIVE: To collect population based information on transfusion of red blood cells. DESIGN: Prospective observational study over 28 days. SETTING: Hospital blood banks in the north of England (population 2.9 million). MAIN OUTCOME MEASURES: Indications for transfusion, number of units given, and the age and sex of transfusion recipients. PARTICIPANTS: All patients who received a red cell transfusion during the study period. Data completed by hospital blood bank staff. RESULTS: The destination of 9848 units was recorded (97% of expected blood use). In total 9774 units were transfused: 5047 (51.6%) units were given to medical patients, 3982 (40.7%) to surgical patients, and 612 (6.3%) to obstetric and gynaecology patients. Nearly half (49.3%) of all blood is given to female recipients, and the mean age of recipients of individual units was 62.7 years. The most common surgical indications for transfusion were total hip replacement (4.6% of all blood transfused) and coronary artery bypass grafting (4.1%). Haematological disorders accounted for 15.5% of use. Overall use was 4274 units per 100 000 population per year. CONCLUSION: In the north east of England more than half of red cell units are transfused for medical indications. Demand for red cell transfusion increases with age. With anticipated changes in the age structure of the population the demand for blood will increase by 4.9% by 2008. PMID- 12376440 TI - Socioeconomic position in childhood and adulthood and insulin resistance: cross sectional survey using data from British women's heart and health study. AB - OBJECTIVE: To assess the associations between childhood and adulthood social class and insulin resistance. DESIGN: Cross sectional survey. SETTING: 23 towns across England, Scotland, and Wales. PARTICIPANTS: 4286 women aged 60-79 years. MAIN OUTCOME MEASURES: Insulin resistance and other cardiovascular disease risk factors. RESULTS: Belonging to manual social classes in childhood and in adulthood was independently associated with increased insulin resistance, dyslipidaemia, and general obesity. The association between childhood social class and insulin resistance was stronger than that for adult social class. The effect, on insulin resistance and other risk factors, of belonging to a manual social class at either stage in the life course was cumulative, with no evidence of an interaction between childhood and adult social class. Women who were in manual social classes in childhood remained at increased risk of insulin resistance, dyslipidaemia, and obesity--even if they moved into non-manual social classes in adulthood--compared with women who were in non-manual social classes at both stages. CONCLUSIONS: Adverse social circumstances in childhood, as well as adulthood, are strongly and independently associated with increased risk of insulin resistance and other metabolic risk factors. PMID- 12376441 TI - Risk of renal stone events in primary hyperparathyroidism before and after parathyroid surgery: controlled retrospective follow up study. AB - AIM: To study the risk of renal stone episodes and risk factors for renal stones in primary hyperparathyroidism before and after surgery. DESIGN: Register based, controlled retrospective follow up study. SETTING: Tertiary hospitals in Denmark. PARTICIPANTS: 674 consecutive patients with surgically verified primary hyperparathyroidism. Each patient was compared with three age- and sex-matched controls randomly drawn from the background population. Hospital admissions for renal stone disease were compared between patients and controls. Risk factors for renal stones among patients were assessed. MAIN OUTCOME MEASURES: Number of renal stone episodes; comparison of hospital admissions for renal stones in patients and controls; assessment of risk factors for renal stones. RESULTS: Relative risk of a stone episode was 40 (95% confidence interval 31 to 53) before surgery and 16 (12 to 23) after surgery. Risk was increased 10 years before surgery, and became normal more than 10 years after surgery. Stone-free survival 20 years after surgery was 90.4% in patients and 98.7% in controls (risk difference 8.3%, 4.8% to 11.7%). Patients with preoperative stones had 27 times the risk of postoperative stone incidents than controls. Before surgery, males had more stone episodes than females and younger patients had more stone episodes than older patients. Neither parathyroid pathology, weight of removed tissue, plasma calcium levels, nor skeletal pathology (fractures) influenced the risk of renal stones. After surgery, younger age, preoperative stones and ureteral strictures were significant risk factors for stones. CONCLUSIONS: The risk of renal stones is increased in primary hyperparathyroidism and decreases after surgery. The risk profile is normal 10 years after surgery. Preoperative stone events increase the risk of postoperative stones. Stone formers and non-stone formers had the same risk of skeletal complications. PMID- 12376442 TI - Insecticide impregnated curtains to control domestic transmission of cutaneous leishmaniasis in Venezuela: cluster randomised trial. AB - OBJECTIVE: To measure the impact on transmission of leishmaniasis of curtains impregnated with insecticide. DESIGN: Cluster randomised controlled trial: household interview survey, observational study of people's behaviour, entomological study with light trap captures of sandflies inside houses. SETTING: 14 urban sectors in Trujillo, Venezuela. PARTICIPANTS: 2913 inhabitants of 569 houses. INTERVENTION: Sectors were paired according to their 12 month cumulative incidence of cutaneous leishmaniasis, one sector in each pair was randomly allocated to receive polyester curtains impregnated with lambdacyhalothrin (intervention group) while the other sector received curtains without insecticide or no curtains (control groups). After 12 months a follow up household survey was conducted. MAIN OUTCOME MEASURES: Reduction in abundance of sandflies indoors and 12 month incidence of clinical cases of cutaneous leishmaniasis. RESULTS: Transmission of cutaneous leishmaniasis occurred mainly in the domestic setting, with the incidence over 12 months of 4%. The mean number of sandflies per trap per night was 16. After follow up the 12 month incidence of cutaneous leishmaniasis was 0% in the intervention group and 8% in the six pairs in the control group that received unimpregnated curtains (mean difference 8, 95% confidence interval 4.22 to 11.78; P=0.001). There were significantly fewer sandflies in the intervention group (2 v 15, mean difference 13 sandflies per trap; 9 to 17; P<0.001). CONCLUSION: Curtains impregnated with insecticide provide a high degree of protection against indoor transmission of cutaneous leishmaniasis. PMID- 12376443 TI - Teething symptoms: cross sectional survey of five groups of child health professionals. PMID- 12376444 TI - Predictors of normotension on withdrawal of antihypertensive drugs in elderly patients: prospective study in second Australian national blood pressure study cohort. AB - OBJECTIVES: To identify simple long term predictors of maintenance of normotension after withdrawal of antihypertensive drugs in elderly patients in general practice. DESIGN: Prospective cohort study. SETTING: 169 general practices in Victoria, Australia. PARTICIPANTS: 503 patients aged 65-84 with treated hypertension who were withdrawn from all antihypertensive drugs and remained drug free and normotensive for an initial two week period; all were followed for a further 12 months. MAIN OUTCOME MEASURES: Relative likelihood of maintaining normotension 12 months after drug withdrawal; relative likelihood of early return to hypertension after drug withdrawal. RESULTS: The likelihood of remaining normotensive at 12 months was greater among younger patients (65-74 years), patients with lower "on-treatment" systolic blood pressure, patients on single agent treatment, and patients with a greater waist:hip ratio. The likelihood of return to hypertension was greatest for patients with higher "on treatment" systolic blood pressure. CONCLUSIONS: Age, blood pressure control, and the number of antihypertensive drugs are important factors in the clinical decision to withdraw drug treatment. Because of consistent rates of return to antihypertensive treatment, all patients from whom such treatment is withdrawn should be monitored indefinitely to detect a recurrence of hypertension. PMID- 12376445 TI - Developing the role of patients as teachers: literature review. AB - OBJECTIVES: To identify the roles and settings in which patients participate as teachers in medical education and the benefits to learners, their educational institutions, and participating patients. DESIGN: Review of publications from 1970 to October 2001 providing descriptions, evaluations, or research of programmes involving patients as teachers in medical education. DATA SOURCES: 1848 references were identified from various electronic databases. Applying inclusion criteria to abstracts generated 100 articles, from which 23 were selected after independent scrutiny. RESULTS: 13 articles discussed the role of patients in teaching physical examination skills, mostly musculoskeletal examination. Patients also taught pelvic and male genitorectal examination skills. Teaching roles varied, and 19 articles referred to patients' involvement as assessors. 18 articles described patients' training, with some patients being assessed. Reports of learners' experiences were all positive, many valuing the insights and confidence gained from practising skills on patients in a teaching role. Some learners preferred being taught by trained patients rather than doctors. Patients who were consulted enjoyed their teaching role. Several articles commented on the high quality of patients' teaching. Remuneration varied from payment of expenses to an hourly rate. Motivation for recruiting patients included the desire to reduce costs and the value attributed to the consumers' perspective. CONCLUSION: Involving patients as teachers has important educational benefits for learners. Patients offer unique qualities that can enhance the acquisition of skills and change attitudes towards patients. PMID- 12376446 TI - Squamous cell carcinomas of the head and neck. PMID- 12376447 TI - Bleeding risks of antithrombotic therapy. PMID- 12376448 TI - Evaluating complementary medicine: methodological challenges of randomised controlled trials. PMID- 12376449 TI - Ethical market in organs. Market of organs is unethical under any circumstances. PMID- 12376450 TI - Diagnosing brain death. Honesty is best policy. PMID- 12376451 TI - Adrenal insufficiency after treatment with fluticasone. Second line controller treatment might have been tried. PMID- 12376452 TI - Education on preventing HIV is paramount. PMID- 12376453 TI - September 11 might be shock treatment for addressing global health inequalities. PMID- 12376454 TI - Results of study on walk-in centres are only to be expected. PMID- 12376455 TI - More on doctors and pilots. Monitoring by Big Brother may not be a Bad Thing. PMID- 12376456 TI - Gastroenterology. PMID- 12376457 TI - My beautiful career. PMID- 12376459 TI - The doctors' support line. PMID- 12376462 TI - cDNA microarray profiling of rat mammary gland carcinomas induced by 2-amino-1 methyl-6-phenylimidazo[4,5-b]pyridine and 7,12-dimethylbenz[a]anthracene. AB - cDNA microarray analysis was used to examine gene expression profiles in normal female Sprague-Dawley rat mammary gland and in carcinomas induced by the cooked meat-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and the potent experimental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Nine tubulopapillary carcinomas (five from PhIP-treated rats and four from DMBA treated rats) and normal mammary gland from virgin, pregnant and lactating rats were examined on a rat 6.9k cDNA microarray. Although histologically identical, PhIP- and DMBA-induced carcinomas could be distinguished by hierarchical clustering and multi-dimensional scaling analyses of cDNA expression. In addition, the expression of 21 clones was statistically different between PhIP- and DMBA-induced carcinomas (F-test, P < 0.05). The data indicate that distinct chemical carcinogens induce unique gene expression patterns in mammary gland carcinomas. The specific chemical carcinogen-associated cDNA array profiles found in carcinomas may ultimately be applicable to better understanding cancer etiology. PhIP- and DMBA-induced carcinomas also shared similarities in cDNA expression profiles. By comparing the expression in carcinomas (PhIP plus DMBA induced) with normal rat mammary gland (at any stage of differentiation), 172 clones were found to be differentially expressed. Genes showing increased expression in carcinomas by cDNA microarray analysis (and further validated by immunohistochemistry and western blot analysis) include cyclin D1, PDGF-A chain, retinol binding protein 1, prohibitin and the transcription factor STAT5A. The similarities in gene expression between PhIP- and DMBA-induced carcinomas raise the possibility that several molecular pathways for rat mammary gland transformation are maintained irrespective of the carcinogenic initiating agent. PMID- 12376463 TI - Tamoxifen inhibits the growth of head and neck cancer cells and sensitizes these cells to cisplatin induced-apoptosis: role of TGF-beta1. AB - A number of studies have shown that tamoxifen increases the sensitivity of several types of solid tumours to cisplatin without increasing the associated side effects. The cellular mechanisms responsible for this increased sensitivity are currently unknown. In this study we have investigated whether tamoxifen alone or in combination with cisplatin could induce apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines. We have shown that tamoxifen treatment resulted in G(1) arrest in two cell lines, HN5 and HN6. Tamoxifen induced growth suppression was independent of p53 status but resulted in up-regulation of cyclin dependent kinase inhibitors (CDKIs) p21/Waf-1, p27/Kip1 and p15/INK4a. Furthermore, tamoxifen treatment resulted in an increased level of hypophosphorylated active RB. Cisplatin induced p53 independent apoptosis in both head and neck cancer cell lines. There was a significant sensitizing effect of tamoxifen on cisplatin-induced apoptosis in HN5 and HN6 cells, with the combined treatment being more effective in inducing apoptosis. Addition of tamoxifen did not result in significant inhibition of PKC activity in HN5 and HN6 cells. However, tamoxifen treatment resulted in increased secretion of TGF-beta1 by HN5 and HN6 cells. An anti-TGF-beta blocking antibody prevented both the blockade of cellular proliferation and the increased expression of CDKIs associated with tamoxifen treatment of HN5 and HN6 cells. These results show that tamoxifen alone induces a transient G(1) arrest that greatly sensitizes the cells to apoptosis induced by cisplatin. We have shown that the mechanism for this p53-independent G(1) arrest and apoptosis is at least partly due to the activation of TGF-beta1 resulting in the induction of p15/INK4b, p27/Kip-1, p21/Waf-1 and RB hypophosphorylation. These in vitro results suggest that combination of tamoxifen and cisplatin might be a more effective treatment for head and neck cancers than single modality therapy. PMID- 12376464 TI - Differential mutation frequency in mitochondrial DNA from thyroid tumours. AB - Lack of a chromatin structure and histone protection makes mitochondrial DNA susceptible to oxidative damage. Suboptimal DNA repair leads to a higher frequency of mitochondrial mutations, which are associated with aging, carcinogenesis and environmental insult. The instability of the hypervariable region II of the mitochondrial genome was investigated in radiation-associated thyroid tumours, which were diagnosed in children from Belarus after the accident at the Chernobyl nuclear power plant, and from 40 sporadic thyroid tumours from Munich. Two mutations were identified in two out of 126 tumours from Belarus, and eight mutations were found in six out of 40 tumours from Munich. All mutations were deletions or insertions of C in a poly-cytidine (C7TC6) microsatellite. The mutation frequency correlated with the age of the patients at surgery. Mutations with the typical pattern of base substitutions following oxidative DNA damage were not identified. PMID- 12376465 TI - Dual roles of Nur77 in selective regulation of apoptosis and cell cycle by TPA and ATRA in gastric cancer cells. AB - Nur77 is an orphan receptor. Although Nur77 affects cell proliferation and apoptosis through its capability of binding to a variety of response elements and regulating their transactivation activities, the intrinsic function of Nur77 is not yet fully understood; in particular, its regulation of apoptosis and proliferation has been characterized as cell type-dependent and agent context dependent. In this study, Nur77 can be seen to regulate apoptosis via its expression and translocation, rather than its transactivation activity in gastric cancer cells. Nur77 was constitutively expressed in BGC-823 cells. The tetradecanoylphorbol-1,3-acetate (TPA) treatment not only resulted in up regulation of the Nur77 mRNA level, but also led to translocation of Nur77 protein from the nucleus to the mitochondria, and caused the release of cytochrome c. This TPA-induced translocation of Nur77 was in association with the initiation of apoptosis in gastric cancer cells. Although all-trans retinoic acid (ATRA) could not induce apoptosis in BGC-823 cells due to failure of stimulating Nur77 translocation, expression of Nur77 in the nucleus was required for cell growth inhibition by ATRA. Transfection of antisense Nur77 receptor into BGC-823 cells resulted in resistance of cell growth against ATRA inhibition, and the cells were still arrested in the S phase. Furthermore, the action of Nur77 in TPA induced apoptosis was mediated through a protein kinase C signaling pathway, while mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways were responsible for the regulation of Nur77 mRNA expression. Taken together, the data revealed the dual functioning mechanisms of Nur77 in gastric cancer cells in response to TPA and ATRA. PMID- 12376466 TI - Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on beta1 integrins and the cytoskeleton. AB - Previous studies have shown that several proteolytic enzymes are associated with membrane protrusions at the leading edge of migrating tumor cells. In this study we demonstrate that seprase and the urokinase plasminogen activator receptor (uPAR), co-localize in the plasma membrane of LOX malignant melanoma cells. Cells were labeled with fluorochrome-conjugated monoclonal antibodies (mAb) directed against seprase and uPAR. Proximity between these two molecules was detected with resonance energy transfer (RET) imaging, single-cell emission spectrophotometry, and single-cell excitation spectrophotometry. Significant RET signals were detected on LOX cells when adherent to uncoated and extracellular matrix (ECM) coated surfaces. This indicates that seprase and uPAR are within approximately 7 nm in the plasma membrane of LOX cells. When LOX cells adhered to a 3D extracellular-like matrix, seprase-uPAR complexes were found to be associated with invadopodia. Further microscopy experiments demonstrated gelatinolytic activity, a functional attribute of seprase, in association with seprase-uPAR membrane domains. Formation of seprase-uPAR membrane complexes is dependent upon both the cytoskeleton and integrins. Specifically, the involvement of beta(1) integrins was demonstrated by the inhibition of RET by an inhibitory anti-beta(1) integrin mAb. Based on these findings, we speculate that formation of heterogeneous lytic domains in the invading membranes of LOX cells increases the efficiency of directed pericellular proteolysis. PMID- 12376467 TI - Regulation of the Mdr1 isoforms in a p53-deficient mouse model. AB - Both p53 and multidrug transporters play important roles in chemoresistance. A transcriptional dependence of the Mdr1 gene promoter by p53 was first established a decade ago, and despite intense study, the p53-Mdr1 relationship still remains vague in vivo. The general model proposes that wild-type p53 down regulates, while mutant p53 up regulates, the Mdr1 promoter. Given that many studies have utilized cancer cell lines, minimal promoters and non-specific cDNA expression for in vitro experiments, we first sought to confirm the model using dermal fibroblasts isolated from the p53-knockout mice. We show that the gene products of the mouse Mdr1 homologue (Mdr1a and Mdr1b), namely P-glycoprotein (P-gp), appear upregulated at both the protein and mRNA levels in p53(-/-) mFbs compared with p53(+/+) cells. We demonstrate that transient transfection of a mouse p53(WT) expression plasmid into short-term primary p53(-/-) fibroblasts can revert P-gp overexpression. The difference in P-gp levels has functional significance in that p53(-/-) fibroblasts are more resistant to doxorubicin and vincristine treatment and this resistance can be attenuated in the presence of the P-gp inhibitor, verapamil. Furthermore, we demonstrate that in kidney, spleen and testis, P-gp expression is elevated in the absence of p53. In contrast, other organs such as heart, liver, lung, brain, thymus and skeletal muscle, show no difference in expression between p53(+/+) and p53(-/-) mice. Thus, our data shows a tissue-specific regulation of P-gp isoforms by p53 in the context of a p53-null mouse model. PMID- 12376468 TI - Reporter gene transactivation by human p53 is inhibited in thioredoxin reductase null yeast by a mechanism associated with thioredoxin oxidation and independent of changes in the redox state of glutathione. AB - Reporter gene transactivation by human p53 is compromised in S. cerevisiae lacking the TRR1 gene encoding thioredoxin reductase. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Deltatrr1 yeast. The Deltatrr1 mutation affected the redox state of both molecules. About 34% of thioredoxin was in the disulfide form in wild-type yeast and increased to 70% in Deltatrr1 yeast. About 18% of glutathione was in the GSSG form in wild-type yeast and increased to 32% in Deltatrr1 yeast. The Deltatrr1 mutation also resulted in a 2.9-fold increase in total glutathione per mg extract protein. Highcopy expression of the GLR1 gene encoding glutathione reductase in Deltatrr1 yeast restored the GSSG:GSH ratio to wild-type levels, but did not restore p53 activity. Also, p53 activity was shown to be unaffected by a Deltaglr1 mutation, even though the mutation was known to result in glutathione oxidation. In summary, the results show that, although glutathione becomes more oxidized in Deltatrr1 cells, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. The results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment. PMID- 12376469 TI - Deficiency in the repair of UV-induced DNA damage in human skin fibroblasts compromised for the ATM gene. AB - Ataxia-telangiectasia (A-T), is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. A-T cells exhibit a greatly decreased survival and a reduction in DNA synthesis inhibition as well as p53 induction in response to ionizing radiation. Occasionally, some strains of A-T cells have been reported to manifest a slightly enhanced sensitivity with no consistent observations of a deficiency in either cell cycle control or the repair of DNA damage after treatment with ultraviolet (UV) light. In the present study it is shown that skin fibroblasts from four A-T patients, compared with the control, display enhanced sensitivity to the killing effect of UV-light, moderate radioresistant DNA synthesis, and a reduction in viral recovery in the host cell reactivation (HCR) assay. PCR based analysis indicated that three of these UV-sensitive A-T cell strains bear a large deletion in the ATM gene, and no ATM polypeptide was detected in their cell free extracts. Moreover, it is shown that, in non-replicative conditions, these A-T cells are less efficient than normal cells in repairing the T4 endonuclease V sensitive sites. These results constitute the first clear evidence showing the deficiency of A-T cells in the repair of UV-induced DNA damage, and provide further information on the relationship between cell cycle control and DNA repair in human cells. PMID- 12376470 TI - The redox protein thioredoxin-1 regulates the constitutive and inducible expression of the estrogen metabolizing cytochromes P450 1B1 and 1A1 in MCF-7 human breast cancer cells. AB - The oxidative metabolites of estrogen have been proposed to play an important role in the development of some human cancers. The two major pathways of estrogen metabolism, to the carcinogenic 4-hydroxyestradiol (4-OHE2) and to the non carcinogenic 2-hydroxyestradiol (2-OHE2), are mediated by cytochromes P450 CYP1B1 and CYP1A1, respectively. The expression of CYP1A1 and CYP1B1 is regulated by the aromatic hydrocarbon receptor/Ah receptor nuclear translocator (AhR/ARNT) transcription factor complex. CYP1B1 expression is elevated in a wide range of human cancers but is not found in corresponding normal tissue. Thioredoxin-1 (Trx 1) is a small redox protein that is overexpressed in a number of human cancers. We report that the expression of CYP1B1 mRNA and protein is increased by Trx-1 transfection of MCF-7 human breast cancer cells and decreased by a redox inactive mutant Trx-1. The Trx-1 inhibitor PX-12 inhibits CYP1B1 gene expression. Trx-1 transfected MCF-7 cells show increased AhR/ARNT DNA binding activity that is not due to altered AhR or ARNT protein expression. 2,3,7,8-Tetrachlorodibenzo-p dioxin (TCDD, dioxin) induced expression of CYP1B1 in MCF-7 cells is increased by Trx-1. Trx-1 does not effect the basal expression of CYP1A1, but increases CYP1A1 mRNA in response to TCDD. The redox inactive mutant Trx-1 completely blocks the induction of both CYP1B1 and CYP1A1 by TCDD. Expression of CYP1A1 but not CYP1B1 has been linked to estrogen receptor (ERalpha) status. Trx-1 transfected MCF-7 cells have decreased ERalpha expression, which may account for the lack of CYP1A1 induction by Trx-1 in the absence of ligand. The results suggest that Trx-1 is involved in the constitutive expression of CYP1B1 and is required for the induction of CYP1B1 and CYP1A1 by TCDD in human MCF-7 breast cancer cells. PMID- 12376471 TI - Modulation of the DNA damage response in UV-exposed human lymphoblastoid cells through genetic-versus functional-inactivation of the p53 tumor suppressor. AB - The global cellular response to UV-induced DNA damage has been analyzed in the p53-proficient human lymphoblastoid strain TK6 versus two isogenic derivatives wherein p53 activity was abrogated by diverse experimental approaches: (i) NH32, carrying a homozygous genetic knockout of p53; and (ii) TK6-5E, expressing the human papillomavirus E6 oncoprotein which binds and functionally inactivates p53 protein. Although widely employed as such, the extent to which intracellular E6 expression faithfully models the p53 deficient state still remains uncertain. Following irradiation with UV (either monochromatic 254 nm UV or broad-spectrum simulated sunlight), relative to wild-type TK6, p53-null NH32 exhibited virtually identical clonogenic survival and kinetics of G1-S progression but was nonetheless profoundly resistant to apoptosis. In addition, there were significant qualitative and quantitative differences between NH32 and TK6 with respect to UV mutagenesis at the endogenous hypoxanthine phosphoribosyltransferase (hprt) locus. However, important disparities were observed between genetically p53-deficient NH32 and E6-expressing TK6-5E regarding the manner in which they responded to UV-induced genotoxic stress in relation to wild-type TK6. Indeed, although NH32 and TK6-5E behaved similarly with respect to UV mutagenesis at the hprt locus, there were significant differences between these strains in clonogenic survival, apoptosis, and G1-S progression. Using a well-defined isogenic system, our data clearly reveal the influence of p53 inactivation on the global response of human cells to UV-induced DNA damage, and highlight an important caveat in the field of p53 biology by directly demonstrating that this influence varies substantially depending upon whether p53 function is abrogated genetically, or through E6 oncoprotein expression. PMID- 12376472 TI - Associations between carcinogen-DNA damage, glutathione S-transferase genotypes, and risk of lung cancer in the prospective Physicians' Health Cohort Study. AB - DNA damage from polycyclic aromatic hydrocarbons (PAH) and other aromatic/hydrophobic compounds has been implicated in case-control studies as a risk factor for lung cancer, as have common polymorphisms in the glutathione S transferase (GST) genes involved in carcinogen detoxification. However, their joint effects have not been evaluated in prospective studies, leaving open questions about predictive value of these biomarkers. In this matched case control study nested within the prospective Physicians' Health Study, we evaluated whether biomarkers measured in white blood cells (WBC) significantly predicted risk, alone and in combination, after controlling for level of smoking. The biomarkers reported here are aromatic/hydrophobic-DNA adducts and polymorphisms in genes coding for the GSTM1 and GSTP1 enzymes. Our study population was composed of 89 cases of primary lung cancer and 173 controls, matched in a 1:2 ratio on smoking, age and duration of follow up. Adducts were measured in WBC DNA by the nuclease P1-enhanced (32)P-post-labeling method. Genotypes (GSTM1 null versus non-null and GSTP1 Val versus GSTP1 Ile) were determined by genomic amplification and restriction fragment length polymorphism analysis. Among current smokers, adducts were significant predictors of lung cancer risk (after adjusting for GST genotypes, OR = 3.10, 95% CI 1.07, 9.01). The combined GSTM1 null/GSTP1 Val genotype was associated with lung cancer overall and especially among former smokers, before and after adjusting for adducts (OR for former smokers = 4.21, CI 1.08, 16.41; adjusted OR = 4.68, CI 1.17, 18.71). Among cases only, adducts were significantly higher among current or former smokers with the GSTM1 non-null/GSTP1 Ile genotype. The two risk factors (adducts and genotypes) appear to be independent predictors of risk. The findings underscore the complex and important role of biological susceptibility as a determinant of risk from carcinogens found in tobacco smoke and other environmental compounds. PMID- 12376473 TI - Polymorphic CAG repeats in the androgen receptor gene, prostate-specific antigen polymorphism and prostate cancer risk. AB - As the development of prostate cancer is androgen-dependent, it has been hypothesized that variation in transcriptional activity by the androgen receptor (AR) related to polymorphic CAG repeats in exon 1, influences prostate cancer risk. The AR regulates gene transcription by binding to androgen-response elements (AREs) in target genes, such as the prostate-specific antigen (PSA). In the ARE-I sequence of the PSA gene an adenine to guanine polymorphism is described. It has been hypothesized that the AR binds the two PSA alleles (A and G) with differing affinities and may, thereby, differentially influence prostate cancer risk. To examine the role of the polymorphisms in the AR and PSA genes in prostate cancer susceptibility, we conducted a case-control study of Austrian Caucasians with 190 newly diagnosed prostate cancer patients and 190 age-matched control men with benign prostatic hyperplasia (BPH). The polymorphisms were determined by polymerase chain reaction (PCR)-based methods using DNA from peripheral white blood cells. Logistic regressions were performed to calculate odds ratios (OR) and confidence limits (CL) and to control for possible confounders. Our data provide no evidence for an association between prostate cancer and CAG repeat length. However, we found a significant influence of the ARE-I PSA polymorphism on prostate cancer risk, when calculating the combination of the A/G and G/G genotypes relative to subjects with the A/A genotype (OR = 0.63; 95% CL 0.39-0.99; P = 0.048), suggesting that the G allele has a protective effect. In a case analysis according to Gleason score, the PSA G/G genotype was significantly more frequent in patients with Gleason score >7 (35.1%) than in patients with Gleason score <7 (21.5%), providing evidence that the PSA G/G genotype is associated with more advanced disease at time of diagnosis. However, the ambivalent role of the PSA during prostate carcinogenesis needs further investigation. PMID- 12376474 TI - Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice. AB - Cyclooxygenase (COX) enzyme expression is elevated in human and rodent lung tumors, and non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin reduce lung tumor formation in mice. These observations, along with the well characterized protection that NSAID treatment engenders for colon cancer, have prompted clinical trials testing whether celecoxib, a COX-2-specific inhibitor, can prevent lung cancer development in populations at high risk. Protection by celecoxib in murine models of pulmonary inflammation and lung tumorigenesis has not yet been evaluated, however, and we now report such studies. Chronic administration of butylated hydroxytoluene (BHT) to mice stimulates pulmonary inflammation characterized by vascular leakage and macrophage infiltration into the air spaces, increased PGE2 production, and translocation of 5-lipoxygenase (5 LO) from the cytosol to the particulate fraction. Dietary celecoxib limited macrophage infiltration, abrogated PGE2 production and reduced particulate 5-LO content. Celecoxib and aspirin were ineffective at preventing lung tumorigenesis in a two-stage carcinogenesis protocol in which 3-methylcholanthrene administration is followed by chronic BHT. Celecoxib also did not reduce the multiplicity of lung tumors after induction by urethane; lung tumors in celecoxib treated mice were larger than those in mice that did not receive celecoxib. Tumors induced in celecoxib-fed mice contained 60% less PGE2 than tumors in mice fed control diets, so reducing lung PGE2 levels was insufficient to prevent lung tumor formation. As the production of eicosanoids in addition to PGE2 is also inhibited by celecoxib, and as celecoxib has COX-independent interactions, its effects on tumor formation may vary in different organ systems. PMID- 12376475 TI - Critical role of allyl groups and disulfide chain in induction of Pi class glutathione transferase in mouse tissues in vivo by diallyl disulfide, a naturally occurring chemopreventive agent in garlic. AB - We have shown previously that the chemoprotective activity of diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, against benzo[a]pyrene (BP)-induced forestomach carcinogenesis in mice correlates strongly with its inductive effects on the expression of Pi class glutathione (GSH) transferase mGSTP1-1. The present structure-activity relationship studies were designed to define the role of allyl groups and the disulfide chain in mGSTP1-inducing activity of DADS. Hepatic mGSTP1 mRNA levels rose rapidly upon treatment of mice with DADS, reached a maximum between 12 and 24 h (< or =5.7-fold induction) and fell to control levels by 48 h after DADS treatment. Induction of mGSTP1 mRNA in the forestomach was maximal between 6 and 12 h after DADS treatment (< or =4.7 fold induction). The mGSTP1 mRNA expression was either unaltered (liver) or moderately increased (forestomach) upon treatment of mice with dipropyl disulfide (DPDS), which is a naturally occurring saturated analog of DADS. These results indicated that the allyl groups are critical for the mGSTP1-inducing activity of DADS. A statistically significant increase in the expression of mGSTP1 mRNA was also observed in the liver and forestomach of mice treated with diallyl monosulfide (DAMS), albeit to a much lesser extent compared with DADS. These results indicated that the oligosulfide chain length in garlic organosulfides (OSCs) is equally important for their mGSTP1-inducing activity. The role of the disulfide chain in DADS-mediated induction of mGSTP1 was further investigated by testing a pair of alkadienes (1,7-octadiene and 1,8-nonadiene) having structural similarity to DADS. Both DADS and the alkadienes carry allyl groups at both ends of a linear molecule and the distance between the allylic carbon atoms is similar in both compounds, but the central disulfide chain of DADS is replaced with an alkyl chain in the alkadienes. The alkadienes were either ineffective or moderately active in increasing mGSTP1 expression. In conclusion, the results of the present study clearly indicate that the presence of terminal allyl groups as well as the central disulfide chain is required for maximum induction of mGSTP1 in vivo by garlic-derived OSCs. PMID- 12376476 TI - Oral administration of the citrus coumarin, isopimpinellin, blocks DNA adduct formation and skin tumor initiation by 7,12-dimethylbenz[a]anthracene in SENCAR mice. AB - The current study was designed to evaluate the effects of oral administration of the citrus coumarin, isopimpinellin, on skin tumor initiation by topically applied benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA). To evaluate the effects of orally administered isopimpinellin on skin tumor initiation by B[a]P and DMBA, its effects on DNA adduct formation were first evaluated. Female SENCAR mice were pre-treated twice with corn oil, or isopimpinellin (70 mg/kg body wt per os) at 24 h and 2 h prior to topical treatment with B[a]P or DMBA. Another citrus coumarin, imperatorin, was also included in these experiments for comparison. Orally administered isopimpinellin and imperatorin significantly inhibited B[a]P-DNA adduct formation by 37 and 26%, respectively. Imperatorin also blocked DMBA-DNA adduct formation by 43%. In a second dose-response study, orally administered isopimpinellin (35, 70 and 150 mg/kg) blocked DMBA-DNA adduct formation by 23, 56 and 69%, respectively. For the tumor study, mice were pretreated orally with corn oil or isopimpinellin at 24 and 2 h prior to initiation with DMBA, and 2 weeks later promotion began with 12 O-tetradecanoylphorbol-13-acetate (TPA). Isopimpinellin significantly reduced the mean number of papillomas per mouse by 49, 73 and 78% compared to corn oil controls at 30, 70 and 150 mg/kg body wt, respectively. Orally administered isopimpinellin also significantly reduced the percentage of mice with papillomas at the highest dose tested (150 mg/kg). The effectiveness of isopimpinellin given topically over a broad dose range against DMBA tumor initiation was also evaluated for comparison. As part of this study, several parameters of systemic toxicity were evaluated following oral dosing with isopimpinellin and imperatorin. Mice were treated orally with corn oil, isopimpinellin or imperatorin (35, 70 and 150 mg/kg body wt per os) once daily for four consecutive days, killed at 24 h after the last dose, and livers, lungs, and kidneys evaluated histologically. In addition, urinary parameters of nephrotoxicity, blood parameters of liver and kidney function, and thrombin clotting time were assayed. No significant changes in blood clotting, or renal or hepatic function were observed. There was, however, a significant increase in liver wt accompanied by cytoplasmic vacuolation of hepatocytes. There were no histopathological changes in lungs or kidneys. Overall, these data indicate that isopimpinellin (and imperatorin) have chemopreventive effects when administered orally on skin tumor initiation by DMBA. PMID- 12376477 TI - Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells. AB - Tangeretin (5,6,7,8,4'-pentamethoxyflavone) is concentrated in the peel of citrus fruits. DNA flow cytometric analysis indicated that tangeretin blocked cell cycle progression at G1 phase in colorectal carcinoma COLO 205 cells. Over a 24 h exposure to tangeretin, the degree of phosphorylation of Rb was decreased after 12 h and G1 arrest developed. The protein expression of cyclins A, D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that tangeretin inhibited the activities of cyclin-dependent kinases 2 (Cdk2) and 4 (Cdk4) in a dose-dependent manner in the cell-free system. As the cells were exposed to tangeretin (50 microM) over 48 h a gradual loss of both Cdk2 and 4 kinase activities occurred. Tangeretin also increased the content of the Cdk inhibitor p21 protein and this effect correlated with the elevation in p53 levels. In addition, tangeretin also increased the level of the Cdk inhibitor p27 protein within 18 h. These results suggest that tangeretin either exerts its growth-inhibitory effects through modulation of the activities of several key G1 regulatory proteins, such as Cdk2 and Cdk4, or mediates the increase of Cdk inhibitors p21 and p27. PMID- 12376478 TI - Ethanol interactions with a choline-deficient, ethionine-supplemented feeding regime potentiate pre-neoplastic cellular alterations in rat liver. AB - To examine the effect of ethanol on hepatocarcinogenesis induced by a choline deficient, ethionine-supplemented (CDE) diet, rats were fed either an ethanol supplemented diet or ethanol-free, isocaloric diet for 2 months, followed by a CDE diet or control diet for up to 8 months. Changes to cellular composition and pattern of gene expression in the liver were determined at 0 and 3 days, and 1, 2 and 3 weeks after commencing the CDE diet, using histological/immunochemical techniques and northern analysis. Oval cells in the liver were identified morphologically and by expression of pi-glutathione S-transferase (pi-GST), alpha fetoprotein (AFP) and the embryonic isoform of pyruvate kinase (M2-PK). Oval cell numbers and changes in the pattern of gene expression induced by the CDE diet were accelerated by pre-treatment with ethanol. At all stages, the proportion of oval cells in the test group exceeded that in controls. After 1 week, oval cells had spread sufficiently from the periportal region to be observed pericentrally in test animals and by 3 weeks, extensive formation of ductal structures was apparent, which were absent in controls. Additionally, M2-PK and AFP mRNA were detected earlier, and in greater abundance in animals pre-treated with ethanol. After 8 months of CDE treatment, one or two small hepatic foci (<10 hepatocytes), strongly positive for pi-GST, were detected in the liver of ethanol-pre-treated animals. These foci were absent in CDE-treated animals; however, animals pre treated with ethanol followed by chronic CDE treatment showed increased size (>40 hepatocytes) and numbers of foci, correlating with the extent of liver damage and varying from 5 to 50% of the liver section. Our data suggest that ethanol pre treatment potentiates the short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances the appearance of pre-malignant hepatic foci that are observed with ethanol pre-treatment alone. PMID- 12376479 TI - Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. AB - Cell cycle progression is monitored by checkpoint mechanisms to ensure the integrity of the genome and the fidelity of sister chromatid separation. Failure of such checkpoint functions results in genomic instability, a condition that predisposes cells to neoplastic transformation and tumor progression. Recently, Scolnick and Halazonetis defined a new mitotic checkpoint that acts at prophase and delays chromosome condensation in response to mitotic stress, and identified a gene, named checkpoint with FHA and ring finger (Chfr), that seems to be required for delaying prophase in human cells. In the present study, we examined human Chfr mRNA expression in 15 human esophageal cancer cell lines and 43 primary esophageal cancers to investigate the potential involvement of Chfr in the pathogenesis of esophageal cancers. We report here that a significant proportion of human esophageal cancer has loss of expression of Chfr gene. Furthermore, we found aberrant hypermethylation of the promoter region of this checkpoint gene in four of 15 (26.7%) esophageal cancer cell lines and in seven of 43 (16.3%) primary cancers. PMID- 12376480 TI - Interrelationships among angiogenesis, proliferation, and apoptosis in the tumor microenvironment during N-methyl-N-nitrosourea androgen-induced prostate carcinogenesis in rats. AB - Proliferation, apoptosis and angiogenesis are critical biologic processes altered during carcinogenesis. Surrogate biomarkers of these processes represent potential intermediate endpoints for short-term intervention studies with preventive and therapeutic agents. We examined the interrelationships among these processes during prostate carcinogenesis induced by N-methyl-N-nitrosourea (MNU) in male Wistar-Unilever rats. Immunohistochemical and digital image analysis techniques were used to evaluate the proliferation index, the apoptotic index and microvessel density (MVD) in tissue representing stages of prostate carcinogenesis. The proliferation index in the normal glandular epithelium of the prostate is lower than that observed in hyperplastic foci and atypical hyperplasia (P < 0.01) and is further increased in carcinoma (P < 0.01). Apoptosis in the normal prostate epithelium or hyperplastic lesions is lower than in adenocarcinoma (P < 0.01). In parallel to proliferation index, MVD increases as prostate cancer progresses. As tumors enlarge, we observed a predictable change in biomarker expression within the tumor microenvironment. We examined prostate tumors vertical line 1 cm in diameter and biomarker expression was quantified within the peripheral (outer 1-2 mm), central (perinecrotic) and intermediate (remaining) areas of each tumor. The proliferation index is higher (P < 0.01) in the intermediate area than either in the peripheral area or central area. Similarly, the vascular density in the intermediate area is higher (P < 0.01) than either in the peripheral or central area. The apoptotic index is higher (P < 0.05) in the central perinecrotic core than that in either the intermediate or the peripheral area. In conclusion, we observe that angiogenesis, proliferation and apoptosis are linked biological processes predictably altered temporally and spatially during prostate carcinogenesis in the MNU model. These biomarker changes are similar to those reported in human prostate carcinogenesis and represent potential biomarkers for the assessment of dietary, chemopreventive and therapeutic agents. PMID- 12376481 TI - Detection of multiple gene hypermethylation in the development of esophageal squamous cell carcinoma. AB - Abnormal hypermethylation of CpG islands associated with tumor suppressor genes can lead to repression of gene expression and contribute significantly to tumorigenesis. Esophageal squamous cell carcinoma (ESCC) is thought to be developed through a multi-stage process, which involves basal cell hyperplasia (BCH), dysplasia (DYS), carcinoma in situ (CIS) and carcinoma. In the present study, we studied the hypermethylation of 10 selected genes in biopsies from normal individuals and resected tissues from ESCC patients. Tumor and neighboring normal and precancerous tissues including BCH, DYS and CIS were microdissected from the resected tissues by laser capture microdissection. Hypermethylation of CpG islands was examined in these samples for 10 genes: p16(INK4a), p15(INK4b), p14(ARF), human leukocyte antigen (HLA)-A, -B, -C, hMLH1, E-cadherin (E-cad), fragile histidine triad and von Hippel-Lindau (VHL). Methylation of two Alu sequences, which neighbor E-cad and VHL, respectively, was used as control to verify the procedure of DNA extraction and chemical modification. In 48 biopsy samples with BCH or DYS, the most frequent hypermethylated genes were p16(INK4a) (18.8%) and p14(ARF) (14.6%). Seventeen out of these 48 samples (35.4%) contained hypermethylation of at least one gene. In the resected tissues, 52% of the BCH and 81% of the tumors showed hypermethylation of at least one gene. Genes hypermethylated in earlier stage lesions were always found hypermethylated at the later stage lesions in the same patient. All of the genes were methylated at some stages and they were clustered into four groups according to their frequencies. The first group of genes, which consisted of p16(INK4a) and p14(ARF), was most frequently hypermethylated in all stages, and the frequencies increased from normal epithelial (0%) to BCH, to displasia/carcinoma in situ and ESCC. Other genes were hypermethylated less frequently. Our results suggest that hypermethylation of key genes, such as p16(INK4a), p14(ARF) and hMLH1, may be used in combination with other molecular changes, such as p53 mutation, in the development of biomarkers for predicting the risk for ESCC. PMID- 12376482 TI - 4-aminobiphenyl is a major etiological agent of human bladder cancer: evidence from its DNA binding spectrum in human p53 gene. AB - 4-aminobiphenyl (4-ABP) is a major etiological agent of human bladder cancer, and its metabolites are able to form DNA adducts that may induce mutation and initiate bladder carcinogenesis. Thirty to sixty percent of human bladder cancer has a mutation in the p53 gene, and the mutational spectrum bears two characteristics: compared with other cancers, the pattern of mutations is more evenly distributed along the p53 gene, and the mutational hotspots occur at both CpG sites, such as codons 175, 248 and 273, and non-CpG sites, such as codons 280 and 285, the latter two being unique mutational hotspots for bladder and other urinary tract cancers. These findings raise the possibility that the special p53 mutational features in human bladder cancer are due to the unique binding spectrum of metabolically activated 4-ABP in bladder cells. To address this question, here we have mapped the 4-ABP-DNA adduct distribution in the p53 gene at the nucleotide sequence level in human bladder cells. We found that, unlike benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide-DNA adduction, which preferentially occurs at CpG sites, 4-ABP-DNA adduction is not biased for CpG sites, and the adducts are more evenly distributed along the p53 gene; nonetheless, the p53 mutational hotspots in bladder cancer at codons 175, 248, 280 and 285 are also the preferential sites for 4-ABP adduct formation. These results strongly suggest that the unique binding spectrum of 4-ABP contributes greatly to the unique mutational spectrum in the p53 gene of human bladder cancer, and provide further molecular evidence to directly link 4-ABP to bladder cancer. PMID- 12376483 TI - Different genetic alterations in rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens. AB - Human beings are exposed to a multitude of carcinogens in their environment, and most cancers are considered to be chemically induced. Here we examined differences in genetic alterations in rat forestomach tumors induced by repeated exposure to a genotoxic carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methylnitrosourethane (MNUR), and chronic treatment with a non-genotoxic carcinogen, butylated hydroxyanisole (BHA) or caffeic acid (CA). A total of 132, 6-week-old male F344 rats were employed. Forty rats were treated with MNNG by intragastric administration at a dose of 20 mg/kg body wt once a week for 32 weeks, and 20 rats received 20 p.p.m. MNUR in their drinking water for 48 weeks. Further groups of 20 animals were administered 2% BHA or 2% CA in the diet for 104 weeks. The remaining rats were maintained without any supplement as controls. Multiple forestomach tumors were observed in all rats of the MNNG-, MNUR-, BHA- and CA-treated groups. Histopathologically, MNUR- and CA-treated groups showed almost the same pattern. On polymerase chain reaction-single strand conformation polymorphism analysis, H-ras and p53 gene mutations were observed at high and relatively low frequencies, respectively, in forestomach tumors induced by MNNG and MNUR. Most H-ras gene mutations were G-->A transitions in codons 7 and 12 of exon 1. On the other hand, forestomach tumors due to the non-genotoxic carcinogens, BHA and CA, had almost no mutations of the H-ras and p53 genes. Moreover, relative overexpression of cyclin D1 and p53 was detected in forestomach tumors induced by the genotoxic carcinogens, while their non genotoxic counterparts had a tendency to show low expression of those molecules. Mutations of the beta-catenin gene were not detected in any group. The present study demonstrates that rat forestomach tumors induced by genotoxic and non genotoxic carcinogens have different underlying genetic alterations, even if their pathological features are similar. PMID- 12376484 TI - Map kinase activation correlates with K-ras mutation and loss of heterozygosity on chromosome 6 in alveolar bronchiolar carcinomas from B6C3F1 mice exposed to vanadium pentoxide for 2 years. AB - Previous work showed a correlation between K-ras mutation and loss of heterozygosity (LOH) on chromosome 6 in the region of K-ras in lung carcinomas from B6C3F1 mice. We hypothesized that mitogen-activated protein kinase (MAPK) would be activated only in those lung neoplasms with both K-ras mutation and LOH. As MAPK activity can be correlated directly with signal detection using antibodies to phosphorylated MAPK, we were able to analyze lung carcinomas from B6C3F1 mice for the presence or absence of MAPK activity by western analysis. Vanadium pentoxide-induced mouse lung carcinomas, which had been shown to have a high frequency of K-ras mutations and LOH on chromosome 6 and for which frozen tumor tissue was available, were used for this study. Total MAPK expression levels were similar between normal lung and lung carcinomas. Phospho-MAPK was elevated in five of six lung carcinoma samples examined in which K-ras mutations and chromosome 6 LOH were identified and in four of five carcinomas with K-ras mutations that lacked LOH. Phospho-MAPK was undetectable or weakly expressed in seven carcinomas examined without K-ras mutations and in normal lung. By immunohistochemistry three K-ras positive/LOH negative samples exhibited multifocal areas of nuclear and cytoplasmic staining for phospho-MAPK. Large amounts of non-staining fibroblasts, lymphocytes and macrophages were also observed in these tumors. Two of these lung carcinomas were microdissected and chromosome 6 LOH was detected in regions of phospho-MAPK positive cells. These results suggest that MAPK is activated during vanadium pentoxide-induced B6C3F1 mouse lung tumorigenesis following K-ras mutation and loss of the wild-type K-ras allele. PMID- 12376485 TI - Induction of different types of uterine adenocarcinomas in Donryu rats due to neonatal exposure to high-dose p-t-octylphenol for different periods. AB - Inappropriate exposure to estrogens in the fetal and/or newborn period can exert irreversible influence, including carcinogenesis on the reproductive system in mammals. The present study was conducted to investigate uterine carcinogenesis in Donryu rats treated neonatally with a high-dose estrogenic compound, p-t octylphenol (OP) for different exposure periods. Female Donryu rats were subcutaneously administered 100 mg/kg/day OP every other day for the first 5 postnatal days (PNDs 1-5) or the first 2 weeks (PNDs 1-15). They received a single injection of 20 mg/kg N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) into a uterine horn at 11 weeks of age and were examined until 15 months of age. PNDs 1 5 OP-treated rats showed normal development of the female reproductive system, including uterine gland genesis and normal estrous cycling after vaginal opening. The treatment, however, accelerated an earlier occurrence of persistent estrus and increased the number of well differentiated uterine adenocarcinomas as compared with controls. This indicated that PNDs 1-5 OP treatment acts as a delayed modulator of the hypothalamus-pituitary-ovarian hormonal control system and the modulation increased the serum estrogen:progesterone ratio, resulting in induction of uterine tumors. On the contrary, PNDs 1-15 OP treatment demonstrated immediate and irreversible influences on the control system, called 'androgenization', and induced abnormal uterine development manifested by prolonged persistent estrus immediately after vaginal opening and also suppression of uterine gland genesis. In addition, uterine tumor malignancy in morphological and biological property clearly increased in this group although the total number of adenocarcinomas was not increased. The present study provides evidence that neonatal exposure to a high-dose OP enhances uterine carcinogenesis in rats, and the type of uterine tumors is changed by the periods of neonatal exposure to OP, suggesting that the mechanism of uterine tumor development is dependent upon neonatal exposure periods. PMID- 12376486 TI - Mutations induced by alpha-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats. AB - The antiestrogen tamoxifen is widely used for the treatment of breast cancer and more recently for the prevention of breast cancer. A concern over the use of tamoxifen as a chemopreventive agent is its carcinogenicity in rat liver, through a genotoxic mechanism involving alpha-hydroxylation, esterification, and DNA adduct formation, primarily by reaction with dG. In a recent study [Gamboa da Costa et al., Cancer Lett., 176, 37-45 (2002)], we demonstrated a significant increase in the mutant frequency in the lacI gene of Big Blue rats treated with tamoxifen, and a further increase in rats administered alpha-hydroxytamoxifen. In the present study, we have assessed mutation induction by tamoxifen and alpha hydroxytamoxifen in the liver cII gene of Big Blue rats and have characterized the types of mutations induced by alpha-hydroxytamoxifen in the liver lacI and cII genes. The mutant frequencies in the liver cII gene were 80 +/- 13 x 10(-6) in the control, 112 +/- 13 x 10(-6) in the tamoxifen-treated group (P < 0.01 vs. control), and 942 +/- 114 x 10(-6) in the alpha-hydroxytamoxifen-treated animals (P < 0.001 vs. control; P < 0.001 vs. tamoxifen). Molecular analysis of the mutants indicated that the alpha-hydroxytamoxifen-induced mutational spectrum differed significantly from the control spectrum, but was very similar to the spectrum induced by tamoxifen for both the lacI and cII genes [Davies et al., ENVIRON: Mol. Mutagen., 28, 430-433 (1996); Davies et al., Carcinogenesis, 20, 1351-1356 (1999)]. G:C --> T:A transversion was the major type of mutation induced by alpha-hydroxytamoxifen and tamoxifen, while G:C --> A:T transition was the main type of mutation in the control. These results support the hypothesis that alpha-hydroxytamoxifen is a major proximate tamoxifen metabolite causing the initiation of tumors in the liver of rats treated with tamoxifen. PMID- 12376487 TI - Association between aldehyde dehydrogenase gene polymorphisms and the phenomenon of field cancerization in patients with head and neck cancer. AB - Patients with squamous-cell carcinoma in the head and neck (HNSCC) often develop second primary esophageal squamous-cell carcinomas (ESCC). In addition, widespread epithelial oncogenic alterations are also frequently observed in the esophagus and can be made visible as multiple Lugol-voiding lesions (multiple LVL) by Lugol chromoendoscopy. Multiple occurrences of neoplastic change in the upper aerodigestive tract have been explained by the concept of 'field cancerization', usually associated with repeated exposure to carcinogens such as alcohol and cigarette smoke. However, the etiology of second ESCC in HNSCC patients remains unclear and acetaldehyde, the first metabolite of ethanol, has been implicated as the ultimate carcinogen in alcohol-related carcinogenesis. We first investigated the relation between second ESCC and multiple LVL in 78 HNSCC patients. Multiple LVL and second ESCC were observed in 29 (37%) and 21 (27%) patients, respectively. All of the second ESCC were accompanied by multiple LVL. This may indicate that episodes of multiple LVL are precursors for second ESCC. We then examined the association of multiple LVL with the patients' characteristics, including genetic polymorphisms of the alcohol metabolizing enzymes, alcohol dehydrogenase type 3 (ADH3) and aldehyde dehydrogenase type 2 (ALDH2). We also investigated acetaldehyde concentrations in the breath of 52 of the 78 patients. All the patients with multiple LVL were both drinkers and smokers. Multivariable logistic analysis showed that the inactive ALDH2 allele (ALDH2-2) was the strongest contributing factor for the development of multiple LVL (odds ratio 17.6; 95% confidence intervals 4.7-65.3). After alcohol ingestion, acetaldehyde in the breath was elevated to a significantly higher level in all patients with the ALDH2-2 allele than in those without it. The high levels of breath acetaldehyde were significantly modified by the slow metabolizing ADH3-2 allele. These results reveal strong evidence for a gene environmental interaction between the ALDH2-2 allele and alcohol consumption, for the risk of developing multiple LVL, resulting in the development of second ESCC in patients with HNSCC. Ultimately, increased local acetaldehyde exposure thus appears to be a critical determinant of the phenomenon of 'field cancerization'. PMID- 12376488 TI - Development of a multi-organ rat model for evaluating chemopreventive agents: efficacy of indole-3-carbinol-. Certain health supplements may cause both carcinogenic and anticarcinogenic effects. PMID- 12376491 TI - Guidelines for the diagnosis and treatment of cholangiocarcinoma: consensus document. PMID- 12376492 TI - Inherited genotype and prostate cancer outcomes. AB - Prostate cancer is the most commonly diagnosed noncutaneous tumor in North American men and confers significant morbidity and mortality to the general population. The use of screening tools to detect prostate cancer at an early stage may have beneficial effects on an individual's prognosis. However, the intense use of these screening modalities also detects tumors that may have a relatively benign course and for which intensive treatment is not necessary. There is a large body of research that evaluated biochemical, physiological, or somatic genetic measures in relation to prostate cancer progression or prognosis. Environmental exposures may also affect these outcomes. In contrast, inherited markers of genetic susceptibility to prostate cancer have largely been used to predict occurrence of disease rather than disease outcome. The use of inherited genetic markers to evaluate prostate cancer outcome could enhance our ability to identify those men who are more likely to develop clinically significant prostate cancer and to intervene in these men to reduce morbidity and mortality resulting from prostate cancer. PMID- 12376493 TI - Cigarette smoking and the risk of breast cancer in women: a review of the literature. AB - Animal experiments and in vitro studies have shown that compounds found in tobacco smoke, such as polycyclic hydrocarbons, aromatic amines, and N nitrosamines, may induce mammary tumors. The findings of smoking-specific DNA adducts and p53 gene mutations in the breast tissue of smokers also support the biological plausibility of a positive association between cigarette smoking and breast cancer, as does the detection of carcinogenic activity in breast fluid. However, epidemiological studies conducted over the past few decades have variably shown positive, inverse, or null associations. To help reconcile the discrepant findings, epidemiologists have paid increasing attention to measures of exposure to tobacco smoke that might be of the greatest etiological importance, to aspects of the smoker that might modify the association between smoking and breast cancer risk, and to the potentially different associations that might exist with different types of breast tumors, such as those with and without estrogen or progesterone receptors. Overall, the results of these studies suggest that smoking probably does not decrease the risk and indeed suggest that there may be an increased breast cancer risk with smoking of long duration, smoking before a first full-term pregnancy, and passive smoking. These findings require confirmation in future studies, as do suggestions of increased risk among women with certain genotypes. PMID- 12376494 TI - Molecular targets for cancer prevention: a meeting review of the third American Cancer Society-Schilling Research Conference. AB - The American Cancer Society-Schilling Research Conference, held at Seascape, California, on October 26-29, 2000, convened over 25 experts in interdisciplinary fields to discuss the prospects for molecular targets of cancer prevention. Promising molecular targets fell into four main classes: (a) genes in which altered expression or activation drives induction of cancer and for which inhibitor drugs are commercially available; (b) genes in which altered expression or activation is shown to be causal in two or more models but for which inhibitor/modulator drugs are not commercially available; (c) molecular targets for which drugs are available but of which the causal significance is unknown; and (d) known and unknown molecular targets of preventive dietary modifications. Recent developments in genomics and proteomics have brought us to the threshold of an extraordinarily promising era in our battle to reduce the burden of cancer. Knowledge of genes that drive or prevent cancer progression and genes that specify cancer susceptibility should bring molecular-targeted interventions to the individuals who will benefit most. PMID- 12376495 TI - Assessing cervical cancer risk in Hispanics. AB - Population-based cancer registries rely on various methods to assign Hispanic ethnic identifiers to patients in the registry. The methods may result in misclassification of patient ethnic identities. Such misclassification may obscure the real incidence of cervical cancer among Hispanic women. This review summarizes previous literature on the accuracy of methods used to ascertain Hispanic ethnicity in numerator and denominator data for the calculation of cancer incidence. In addition, cancer registry ethnicity ascertainment methods were examined for six United States states (California, Florida, Illinois, New Mexico, New York, and Texas) that have a high proportion of Hispanics. The percentage of persons classified as Hispanic who self-identified as Hispanic (predictive value positive) in various reported studies ranged from 54 to 76% for women. The accuracy of ethnicity assignments based on either the United States census list or the Generally Useful Ethnic Search System (GUESS) program show slight differences in percentages of self-identified Hispanics who were classified as Hispanic (sensitivity among women: 62-80% for 1980 United States census list, 63-82% for GUESS program). Higher sensitivity and lower predictive value positive is achieved with a greater number of sources used. In conclusion, decisions about collecting racial and ethnicity information are influenced by demographic changes, immigration trends, changes in ethnic and racial identity, legislative needs, and public policies. The rapidly growing Hispanic population and the excess incidence of cervical cancer in this population requires improving the accuracy of ethnicity information. PMID- 12376496 TI - Is the androgen receptor CAG repeat length significant for prostate cancer? PMID- 12376497 TI - Dietary fat and risk of lung cancer in a pooled analysis of prospective studies. AB - Lung cancer rates are highest in countries with the greatest fat intakes. In several case-control studies, positive associations have been observed between lung cancer and intakes of total and saturated fat, particularly among nonsmokers. We analyzed the association between fat and cholesterol intakes and lung cancer risk in eight prospective cohort studies that met predefined criteria. Among the 280,419 female and 149,862 male participants who were followed for up to 6-16 years, 3,188 lung cancer cases were documented. Using the Cox proportional hazards model, we calculated study-specific relative risks that were adjusted for smoking history and other potential risk factors. Pooled relative risks were computed using a random effects model. Fat intake was not associated with lung cancer risk. For an increment of 5% of energy from fat, the pooled multivariate relative risks were 1.01 [95% confidence interval (CI), 0.98 1.05] for total, 1.03 (95% CI, 0.96-1.11) for saturated, 1.01 (95% CI, 0.93-1.10) for monounsaturated, and 0.99 (95% CI, 0.90-1.10) for polyunsaturated fat. No associations were observed between intakes of total or specific types of fat and lung cancer risk among never, past, or current smokers. Dietary cholesterol was not associated with lung cancer incidence [for a 100-mg/day increment, the pooled multivariate relative risk was 1.01 (95% CI, 0.97-1.05)]. There was no statistically significant heterogeneity among studies or by sex. These data do not support an important relation between fat or cholesterol intakes and lung cancer risk. The means to prevent this important disease remains avoidance of smoking. PMID- 12376498 TI - Polymorphisms of the DNA repair gene xeroderma pigmentosum group A and risk of primary lung cancer. AB - Polymorphisms in DNA repair genes may be associated with differences in the repair capacity of DNA damage and may influence an individual's susceptibility to smoking-related cancer. We investigated the association between two polymorphisms of the DNA repair gene XPA and risk of lung cancer in the Korean population. Two XPA polymorphisms (A23G and G709A) were typed in 265 lung cancer patients and 185 healthy controls who were frequency-matched on age and sex. The XPA G709A polymorphism was not detected in cases and controls. The XPA 23 GG genotype was associated with a significantly decreased risk for lung cancer [odds ratio (OR), 0.56; 95% confidence interval (CI), 0.35-0.90] when the combined AA and AG genotype was used as the reference. The reduction in risk for the XPA 23 GG genotype was significant in males (OR, 0.51; 95% CI, 0.30-0.86), younger individuals (OR, 0.39; 95% CI, 0.19-0.80), and current smokers (OR, 0.46; 95% CI, 0.25-0.83). These results suggest that the XPA A23G polymorphism contributes to genetic susceptibility for lung cancer. PMID- 12376499 TI - Exposure levels and cytochrome P450 1A2 activity, but not N-acetyltransferase, glutathione S-transferase (GST) M1 and T1, influence urinary mutagen excretion in smokers. AB - We investigated the polymorphic enzymes cytochrome P450 1A2 (CYP1A2), N acetyltransferase (NAT2), glutathione S-transferase (GST) M1 (GSTM1), and T1 (GSTT1) in relation to cigarette smoking-associated urinary mutagenicity detected on YG1024 Salmonella typhimurium strain with S9 mix in 97 smokers. In each subject, cigarette smoke intake was checked by analysis of urinary nicotine plus its metabolites. NAT2 and CYP1A2 phenotypes were determined by the molar ratio of urinary caffeine metabolites detected by high-performance liquid chromatography, and GSTT1 and GSTM1 genotypes were determined by PCR. An increase in urinary mutagenicity was significantly related to levels of exposure to cigarette smoke and CYP1A2 N-hydroxylation activity (linear multiple regression analysis t = 4.51 and P < 0.001 and t = 3.09 and P = 0.003; F = 6.31, P < 0.001). Urinary mutagenicity was significantly higher in CYP1A2 extensive metabolizer smokers (n = 49) than in CYP1A2 poor metabolizer ones (n = 48; 2176 +/- 1525 versus 1384 +/- 1206 revertants/mmol creatinine, Mann-Whitney U-test, z = 2.65, P < 0.001). The highest mutagenic activity was seen in subjects CYP1A2 extensive metabolizer/NAT2 slow acetylators (n = 29) with respect to the other phenotype combinations (n = 68; 2392 +/- 1660 versus 1525 +/- 1238 revertants/mmol creatinine, Mann-Whitney U test, z = 2.37, P = 0.017). NAT2 acetylation activity was slightly but inversely related to urinary mutagenicity, and the association was not significant. No effect of GSTM1 and GSTT1 genotypes in lowering (detoxifying) urinary mutagens was found. The significant enhancement of urinary mutagenicity associated with increased CYP1A2 activity, as already seen for diet-caused urinary mutagenicity, allows for many analogies between the process of mutagen formation derived from cooked meat and that from cigarette smoke condensate. In conclusion, the intensity of tobacco smoke exposure, modulated by CYP1A2 activity, is the major determinant of mutagenic urine among smokers, whereas GSTM1 and GSTT1 genotypes have no influence on this biomarker. This study suggests that CYP1A2 should definitely be determined in future studies involving urinary mutagenicity in cases in which smoking is a factor. PMID- 12376500 TI - XPD codon 751 polymorphism, metabolism genes, smoking, and bladder cancer risk. AB - Cigarette smoking is the main risk factor for bladder cancer, accounting for at least 50% of bladder cancer in men. Cigarette smoke is a rich source of arylamines, which are detoxified by the NAT2 enzyme and activated by the NAT1 enzyme to highly reactive species that can form bulky adducts on DNA. DNA damage from such adducts is mainly repaired by the nucleotide excision repair pathway, in which the XPD protein functions in opening the DNA helix. We hypothesized that an XPD codon 751 polymorphism (Lys-to-Gln amino acid change) could affect the repair of smoking-induced DNA damage and could be associated with bladder-cancer risk. We also hypothesized that allelic variants of the NAT1 and NAT2 genes might modify the effect of the XPD codon 751 polymorphism on smoking-associated bladder cancer risk. We determined the XPD codon 751 genotype for 228 bladder-cancer cases and 210 controls who were frequency-matched to cases by age, sex, and ethnicity, and we used our previously published data on the NAT1 and NAT2 genotypes for these same individuals (J. A. Taylor et al., Cancer Res., 58: 3603 3610, 1998). We found a slight decrease in risk for the XPD codon 751 Gln/Gln genotype (adjusted odds ratio: 0.8; 95% confidence interval: 0.4-1.3) compared with subjects with the Lys/Lys or Lys/Gln genotypes. The analysis with smoking showed that smokers with the Lys/Lys or Lys/Gln genotypes were twice as likely to have bladder cancer than smokers with the Gln/Gln genotype (test of interaction P = 0.03). The combined presence of the NAT1/NAT2 high-risk genotype and the XPD Lys/Lys or Lys/Gln genotypes ignoring smoking had an odds ratio that was only slightly higher than expected, assuming no genotype-genotype interaction (P = 0.52). We found little evidence for a gene-gene-exposure, three-way interaction among the XPD codon 751 genotype, smoking, and the NAT1/NAT2 genotype. PMID- 12376501 TI - Risk factors for hyperplastic and adenomatous polyps: evidence for malignant potential? AB - Recent studies have suggested that hyperplastic polyps may be benign precursor lesions for a distinct subset of colorectal tumors. We conducted a clinic-based case-control study to evaluate risk factors for hyperplastic polyps. Cases with hyperplastic polyps (n = 219), adenomas (n = 437), and both types of polyps (n = 138), along with colonoscopy-negative controls (n = 708), were identified at a gastroenterology practice in the Minneapolis area during 1991-1994. A self administered questionnaire was used to collect risk factor information. Risk factors for hyperplastic and adenomatous polyps were generally similar to those for colorectal cancer. Male sex, smoking, and alcohol consumption were associated with increased risk of all polyp groups; nonsteroidal anti-inflammatory drug use, hormone replacement therapy use, and calcium intake were associated with reduced risk. There was no apparent association between increasing age and hyperplastic polyp risk (P = 0.21) in this analysis, although it was a strong risk factor for adenoma (P < 0.001). The odds ratio (OR) for hyperplastic polyps associated with >25 pack-years of smoking was 4.1 [95% confidence interval (CI), 2.2-7.6], whereas the OR for adenoma alone was 1.3 (95% CI, 0.8-2.3). The OR estimate for individuals diagnosed with both polyp types was 4.2 (95% CI, 1.9-9.3). These results suggest, as one possibility, that the consistent association of adenoma and smoking observed in previous studies may be partially attributable to the inclusion of individuals with both adenomas and hyperplastic polyps in the adenoma case group. To the contrary, individuals with both polyp types may be expressing a phenotype distinct from those who have only adenomas and should be considered separately. Further studies are necessary to establish which polyp phenotypes are related to smoking. Overall, the similarity of the risk profiles of colorectal hyperplastic polyps, adenoma, and cancer provides additional support for the growing body of evidence that some hyperplastic polyps may have neoplastic potential. PMID- 12376502 TI - Red meat intake, CYP2E1 genetic polymorphisms, and colorectal cancer risk. AB - N-Nitroso compounds are suspected colorectal cancer (CRC) carcinogens to which individuals on a diet high in red meat (RM) may be particularly exposed. Many of these compounds undergo alpha-hydroxylation by CYP2E1 to form DNA adducts. The gene coding for this enzyme is polymorphic and thus may constitute a susceptibility factor for CRC. We conducted a population-based case-control study in Hawaii to test the association of two functional polymorphisms in CYP2E1 (the G1259C RsaI substitution and a 5' 96-bp insertion variant) with CRC, as well as their modifying effects on the association of RM and processed meat (PM) with this cancer. We obtained interviews and blood samples for 521 patients with CRC (165 with rectal cancer) and 639 controls of Japanese, Caucasian, or Hawaiian origin. Genotyping was performed by PCR. After adjustment for CRC risk factors, subjects with the 5' insert variant were found to be at a 60% increased risk (95% confidence interval, 1.1-2.5) for rectal cancer. Subjects who carry the insert and who were predicted to have been exposed to increased levels of nitrosamines, based on their high intake of RM or PM, were at a markedly greater increased risk (2- and 3-fold for RM and PM, respectively) for rectal cancer. No clear association was found for colon cancer. A similar increase in rectal cancer risk was found for CYP2E1 insert carriers who consumed salted/dried fish or Oriental pickled vegetables. These data provide additional support for the hypothesis that nitrosamines are carcinogenic to the rectum in humans and that RM and, in particular, PMs are significant sources of exposure for these compounds. PMID- 12376503 TI - Pharmacokinetics of tea catechins after ingestion of green tea and (-) epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. AB - Green tea and tea polyphenols have been studied extensively as cancer chemopreventive agents in recent years. The bioavailability and metabolic fate of tea polyphenols in humans, however, are not clearly understood. In this report, the pharmacokinetic parameters of (-)-epigallocatechin-3-gallate (EGCG), (-) epigallocatechin (EGC), and (-)-epicatechin (EC) were analyzed after administration of a single oral dose of green tea or decaffeinated green tea (20 mg tea solids/kg) or EGCG (2 mg/kg) to eight subjects. The plasma and urine levels of total EGCG, EGC, and EC (free plus conjugated forms) were quantified by HPLC coupled to an electrochemical detector. The plasma concentration time curves of the catechins were fitted in a one-compartment model. The maximum plasma concentrations of EGCG, EGC, and EC in the three repeated experiments with green tea were 77.9 +/- 22.2, 223.4 +/- 35.2, and 124.03 +/- 7.86 ng/ml, respectively, and the corresponding AUC values were 508.2 +/- 227, 945.4 +/- 438.4, and 529.5 +/- 244.4 ng x h x ml(-1), respectively. The time needed to reach the peak concentrations was in the range of 1.3-1.6 h. The elimination half-lives were 3.4 +/- 0.3, 1.7 +/- 0.4, and 2.0 +/- 0.4 h, respectively. Considerable interindividual differences and variations between repeated experiments in the pharmacokinetic parameters were noted. Significant differences in these pharmacokinetic parameters were not observed when EGCG was given in decaffeinated green tea or in pure form. In the plasma, EGCG was mostly present in the free form, whereas EGC and EC were mostly in the conjugated form. Over 90% of the total urinary EGC and EC, almost all in the conjugated forms, were excreted between 0 and 8 h. Substantial amounts of 4'-O-methyl EGC, at levels higher than EGC, were detected in the urine and plasma. The plasma level of 4'-O-methyl EGC peaked at 1.7 +/- 0.5 h with a half life of 4.4 +/- 1.1 h. Two ring-fission metabolites, (-)-5-(3',4',5'-trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5 (3',4'-dihydroxyphenyl)-valerolactone (M6), appeared in significant amounts after 3 h and peaked at 8-15 h in the urine as well as in the plasma. These results may be useful for designing the dose and dose frequency in intervention studies with tea and for development of biomarkers of tea consumption. PMID- 12376504 TI - Androgen receptor polymorphisms and the incidence of prostate cancer. AB - The human androgen receptor gene contains polymorphic CAG and GGC repeats in exon 1. We investigated whether the number of CAG and/or GGC repeats is related to prostate cancer risk in a case-control study nested within the beta Carotene and Retinol Efficacy Trial. Among 300 cases and 300 controls, we did not observe any increase in risk associated with fewer CAG or GGC repeats. We observed a nonsignificant decrease in risk associated with each unit of decrease in CAG length [odds ratio (OR), 0.98; 95% confidence interval (CI), 0.93-1.03). Men with CAG <22 had a relative risk of prostate cancer of 0.89 (95% CI, 0.65-1.23) compared with men with CAG > or =22. There was no appreciable difference in the mean number of GGC repeats between cases and controls; the estimated change in the risk of prostate cancer associated with one fewer GGC repeat was 0.97 (95% CI, 0.88-1.06). The risk in men at or below the mean number of GGC repeats (17) was 0.80 (95% CI, 0.57-1.12). In contrast to prior reports, men with both short CAG (<22) and short GGC (< or =17) repeats were not at increased risk of prostate cancer (OR, 0.56; 95% CI, 0.32-0.98), compared with men with > or =22 CAG repeats and >17 GGC repeats. Our results do not support the hypothesis that a small number of CAG or GGC repeats in the androgen receptor gene increases a man's risk of prostate cancer. PMID- 12376505 TI - Serum androgen concentrations in young men: a longitudinal analysis of associations with age, obesity, and race. The CARDIA male hormone study. AB - Serum testosterone concentration appears to be higher in black men than white men, particularly at younger ages. The higher incidence of prostate cancer in blacks has been attributed, at least in part, to this difference. Other factors associated with androgen levels in men include age and obesity. However, most of the studies of adult androgen levels are limited by their cross-sectional design. We conducted longitudinal analyses (Generalized Estimating Equation) of the associations of age, body mass index (BMI), and waist circumference with total and free testosterone and sex hormone-binding globulin (SHBG) concentrations during an 8-year period and compared these hormonal factors between black (n = 483) and white (n = 695) male participants of the Coronary Artery Risk Development in Young Adults (CARDIA) Study. For men ages 24 years and older at the time of the first hormone measurement, increasing age was associated with a statistically significant decrease in serum total and free testosterone and an increase in SHBG (P < 0.05). BMI and waist circumference were inversely associated with total testosterone and SHBG, but only BMI was inversely associated with free testosterone. After adjustment for age and BMI, total testosterone was higher in blacks (0.21 ng/ml; P = 0.028) than whites, an approximately 3% difference. However, after further adjustment for waist circumference, there was no black-white difference (0.05 ng/ml; P = 0.62). These results indicate that the age-associated decrease in circulating testosterone and increase in SHBG begin during the 3rd decade of life, and that increasing obesity, particularly central obesity, is associated with decreasing total testosterone and SHBG. Results also suggest that the previously observed difference in total testosterone between black and white men could be attributed, for the most part, to racial differences in abdominal obesity. PMID- 12376506 TI - A longitudinal study of the effects of menopause on mammographic features. AB - Menopause has an important influence on risk of breast cancer. We have examined longitudinally the effect of menopause on mammographic densities, a strong risk factor for the disease, in women in the Canadian National Breast Screening Study. Baseline mammograms from women in the National Breast Screening Study, who were premenopausal at entry and had undergone menopause after entry, were compared with the mammogram that most closely followed menopause, using a computer assisted method of measurement. The changes seen in the mammograms of these subjects were compared with those in an age-matched group of women who were also premenopausal at entry, had been followed for the same length of time, and had not experienced menopause. The results of this longitudinal study show that menopause has effects on characteristics of the mammogram that, over the same period of time, are greater than the effects of age. These effects are a reduction in the area of radiologically dense tissue, an increase in the area of nondense tissue, and a decrease in the percentage of density. However, these changes do not fully account for the effects of age on mammographic densities seen in cross-sectional data. Menopause is associated with distinct changes in the mammogram that account for some, but not all, of the observed association of increasing age with decreasing percentage of mammographic density. The observed changes in the morphology of the breast may explain some of the effect that menopause has on breast cancer risk. PMID- 12376507 TI - Identification of 127 amino acid substitution variants in screening 37 DNA repair genes in humans. AB - The repair of damaged DNA requires the function of multiple proteins in generally damage-specific, nonredundant pathways. The relationship of DNA repair to cancer susceptibility is obvious in "cancer families," in which low frequency, high penetrance, loss-of-function variant alleles of genes with roles in the repair of damaged DNA have been associated with a high risk of disease. More important for the cancer incidence in the general population, many individuals exhibit reduced (60-75% of normal) repair capacity phenotypes that have been associated with several-fold increases in individual cancer risk. In a program to identify the molecular basis for the variation in repair capacity and the elevated cancer susceptibility, we have identified 127 amino acid substitution variants in resequencing 37 DNA repair genes in 36-164 unrelated individuals. Over 50% of the substitutions are exchanges of amino acid residues with dissimilar physical or chemical properties, at sites at which the common residue is identical in the human and mouse proteins. Five additional sequence changes resulting in proteins with altered termination of translation and one amino acid insertion variant were detected. The variant allele frequencies average 0.047, with individual variant allele frequencies ranging from <0.01 to 0.43. Homozygous variant individuals and individuals with multiple amino acid substitutions in a gene were observed. Most individuals exhibited variation in multiple genes in a repair pathway. Ten variant alleles accounted for 52% of the genetic variation among individuals, but a striking 23% of the total variation is associated with 108 variants with allele frequencies of less than 5%. Screening generally healthy individuals generates a catalogue of common variants that is a resource for molecular epidemiology studies endeavoring to use a genotype to phenotype paradigm to estimate the role of genetic variation and individual susceptibility in disease risk from environmental and lifestyle exposures in the general population of the United States. PMID- 12376508 TI - Validity of free testosterone and free estradiol determinations in serum samples from postmenopausal women by theoretical calculations. AB - In this study, we validated measurements of free testosterone (fT) and free estradiol (fE(2)) concentrations calculated from total serum concentrations of testosterone (T), estradiol (E(2)), and sex hormone-binding globulin (SHBG), measured by direct, commercial radioimmunoassays, by comparison with reference measurements obtained by dialysis plus an in-house radioimmunoassay after extraction and chromatographic purification. The study was conducted in serum samples from 19 postmenopausal women who were part of an ongoing prospective cohort study. We also performed sensitivity analyses to examine the robustness of the theoretical calculations. Sensitivity analyses showed that in this population, competitive binding of dihydrotestosterone and total T could be ignored in the calculation of fE(2), and competitive binding by dihydrotestosterone does not need to be taken into account for calculation of fT. Furthermore, variations in albumin and SHBG concentrations had negligible effects on fT and fE(2) calculations. Values of fT and fE(2), calculated from total T and E(2) concentrations obtained by the same in-house radioimmunoassay used for the dialysis method, correlated highly with the measurements by dialysis (Pearson's coefficients of correlation above 0.97). When calculating fT and fE(2) using total T and total E(2) concentrations obtained by different direct radioimmunoassays, almost all kits gave good correlations with the reference method for fT (Pearson's r > 0.83), but only a few gave good correlations for fE(2) (Diagnostic System Laboratories and DiaSorin; r > 0.80). The direct radioimmunoassays giving the best correlation for fT and fE(2) with the dialysis method were those that best measured total concentrations of T and E(2). Furthermore, mean values of fT and fE(2) corresponded well to mean values by the reference method if SHBG measurements were also well calibrated. We conclude that in postmenopausal women, theoretical calculations are valid for the determination of fT and fE(2) concentrations and can give reliable estimation of cancer risk in epidemiological studies when the total concentrations of T, E(2), and SHBG are measured accurately. PMID- 12376509 TI - The level of 8-hydroxyguanine, a possible repair product of oxidative DNA damage, is higher in urine of cancer patients than in control subjects. AB - Using high-performance liquid chromatography prepurification/isotope dilution gas chromatography/mass spectrometry technique, we examined whether the amount of 8 hydroxyguanine and 8-hydroxy-2'-deoxyguanosine excreted into urine is higher in cancer patients with advanced-stage disease than in the control group. The control group consisted of 38 healthy subjects, and the patient group comprised 42 cancer patients suffering from metastasis of their primary tumors into the bones. We have found that the amount of the modified base (but not the nucleoside) excreted into urine is about 50% higher in cancer patients than in the control group. Because the presence of the modified base in urine may represent the primary repair product of oxidative DNA damage in vivo, our results suggest an important role of DNA glycosylases (most likely OGG1) in removal of the damage induced as a result of cancer development. PMID- 12376510 TI - Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine values measured by an ELISA correlated well with measurements by high-performance liquid chromatography with electrochemical detection. AB - Measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) has recently become more popular as a means of assessing oxidative stress in the human body. Although using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) is a reliable method of measuring 8-oxo-dG, easier and simpler alternative methods may be preferred, if they are quantitatively accurate. The ELISA method is the likeliest candidate for a useful alternative. Anti-8-oxo-dG monoclonal antibody N45.1 has been shown to have better specificity for 8-oxo-dG than other anti-8-oxo-dG antibodies, but the urinary 8-oxo-dG values measured by ELISA using N45.1 have been claimed to only weakly correlate with the values obtained by HPLC-ECD. Because the commercial ELISA kit has been improved, we compared the urinary 8-oxo-dG values measured by the ELISA with the values obtained by HPLC-ECD. We sampled the urine of 72 healthy Japanese individuals and measured their urinary 8-oxo-dG levels by the ELISA with appropriate controls and by HPLC-ECD. When X was defined as the values of 8-oxo-dG measured by HPLC-ECD, and Y was defined as the values of 8-oxo-dG measured by the ELISA, simple regression analysis showed the most likely relationship to be Y = 1.83X + 0.8. The correlation coefficient was 0.88, which indicated a good correlation between X and Y. These results show that the ELISA can be applied to studies comparing relative urinary 8-oxo-dG values among several groups, if the studies do not require determination of the exact concentration of 8-oxo-dG in urine. PMID- 12376511 TI - Influence of metabolic genotypes on biomarkers of exposure to 1,3-butadiene in humans. AB - Carcinogenicity of 1,3-butadiene (BD) has been linked to its metabolic activation of genotoxic epoxides. The inherited variations in the activity of BD metabolizing enzymes may be responsible for individual differences that modulate the effects of BD exposure. In this study, 40 Italian subjects (30 BD-exposed workers and 10 clerks) were investigated to evaluate the role of genetic polymorphism of cytochromes P450 2E1, microsomal epoxide hydrolase, glutathione transferases GSTM1, GSTP1, GSTT1, and alcohol dehydrogenase, on urinary N-acetyl S-(3,4-hydroxybutyl)-L-cysteine (MI) and hemoglobin N-(2,3,4-trihydroxybutyl) valine adducts (THBVal). Median urinary MI and THBVal levels were 1.71 mg/g creatinine and 37.0 pmol/g globin in BD-exposed workers (exposure range, 4-201 microg/m(3)) and 1.42 mg/g creatinine and 35.3 pmol/g globin in unexposed subjects. No difference between the two groups was observed. Among all subjects, MI and THBVal levels were significantly correlated (r = 0.333). Smoking positively influenced the formation of THBVal. Higher THBVal levels were found in subjects with GSTM1 null and GSTT1 null genotypes; borderline influences were also noticed for CYP2E1(G(-35)T). An additive effect of combined polymorphisms for CYP2E1, GSTM1, and GSTT1 genes on the THBVal levels was suggested. A multiple linear regression analysis, where each factor contributed significantly, correlated THBVal levels with smoking, CYP2E1(G(-35)T), GSTT1, and GSTM1 genotypes (r = 0.698). Our results indicate that the THBVal level is influenced by genotypes, and that the analysis of combined polymorphisms may be the key to a better understanding of the role played by polymorphism of BD-metabolizing enzymes. PMID- 12376512 TI - Serum antibodies to Helicobacter pylori and the CagA antigen do not explain differences in the prevalence of precancerous gastric lesions in two Chinese populations with contrasting gastric cancer rates. AB - Incidence and mortality rates for gastric cancer in rural People's Republic of China differ greatly over short distances. In Shandong Province, we studied asymptomatic adult subjects from Bei Duan village (n = 196) in Linqu County (a high-risk area for gastric cancer) and from Shi Huang village (n = 192) in Cangshan County (a low-risk area for gastric cancer). The prevalence of advanced precancerous gastric lesions (APGL) was assessed by microscopic examination of endoscopic stomach biopsies. ELISAs were used to detect serum IgG to Helicobacter pylori whole-cell antigen and to the CagA protein. A logistic regression model was used to quantify the role of the two H. pylori seromarkers in explaining the differences in prevalence of APGL between the two villages after adjusting for age and sex. The prevalence of APGL was much greater in Bei Duan than in Shi Huang. Although H. pylori seroprevalence by the whole-cell ELISA was similar in the two populations, seroprevalence of CagA was significantly greater in Bei Duan. Although age, sex, and both H. pylori seromarkers were associated with APGL in the logistic regression model, the effect of village of residence remained strong after adjustment for all four covariates. Only a relatively small proportion of the difference in prevalence of APGL between these two rural Chinese populations can be explained by differences in H. pylori or CagA seroprevalence. PMID- 12376513 TI - Helicobacter pylori seropositivity and colorectal cancer risk: a prospective study of male smokers. AB - Because Helicobacter pylori colonization can produce systemic as well as local effects, it may be associated with carcinogenesis in extra gastric target organs. The currently available data regarding a possible link between H. pylori seropositivity and colorectal cancer risk are limited and inconclusive. In this prospective case-control study nested within the Alpha-Tocopherol, Beta-Carotene Study cohort of Finnish male smokers aged 50-69 years, we examined the association between H. pylori seropositivity and incident colorectal adenocarcinoma. Separate risk estimates were derived by colorectal cancer anatomical subsite and by H. pylori CagA seropositivity status. Demographic, dietary, and lifestyle variables were accounted for in the data analyses using information obtained from a prerandomization questionnaire and physical examination. Baseline serum samples from 118 cases and 236 matched controls were assayed for both H. pylori whole cell and H. pylori CagA antibodies. In total, 258 (73%) and 212 (60%) subjects expressed whole cell and CagA antibodies, respectively. H. pylori seropositivity, defined as one or both antibody assays positive, was present in 273 (77%) subjects. None of the seropositivity results were statistically different between cases and controls. Multivariate odds ratio (95% confidence interval) estimates for whole cell, cagA, and H. pylori seropositivity were 1.05 (0.63-1.74), 1.17 (0.74-1.84), and 0.91 (0.53-1.55), respectively. Stratification by colorectal cancer subsite yielded similarly unremarkable results. On the basis of these data, H. pylori carriage does not appear to be an important risk factor for colorectal adenocarcinoma. PMID- 12376514 TI - Oral contraceptive use and cyclin D1 overexpression in breast cancer among young women. AB - Cyclin D1, an important cell cycle regulator, is overexpressed in several human cancers including breast. Both estrogens and progestins activate the transcription of the gene; antiestrogens have been shown to reduce cyclin D1 protein levels. Cyclin D1 protein overexpression has been strongly associated with well-differentiated, estrogen receptor-positive tumors. Little is known, however, as to whether epidemiological risk factors are related to this molecularly defined subset of tumors. Using a population-based study of young women <45 years in New Jersey, we analyzed whether oral contraceptives (OCs) and other risk factors were associated with the overexpression of cyclin D1 in breast cancer tissue. We measured cyclin D1 status in paraffin-embedded, archived tissue from 78.8% of the breast cancer cases using immunohistochemistry. Cyclin D1 was overexpressed in 33.7% of the cases (123 of 365). We used unordered polytomous logistic regression to estimate the odds ratios (ORs) for two case groups--(a) breast cancer with cyclin D1 overexpression (n = 123) and (b) breast cancer without overexpression (n = 242)--compared with 462 population-based controls. The multivariate-adjusted OR for ever use of OCs was 1.6 [95% confidence interval (CI), 1.0-2.5] for cases that overexpressed cyclin D1 and 1.0 (95% CI, 0.7-1.5) for those with no overexpression. Among women who started using OCs at least 20 years before the reference date, the OR was increased 2-fold for breast cancer with cyclin D1 overexpression (OR, 2.2; 95% CI, 1.2-4.0) but not for breast cancer without cyclin D1 overexpression (OR, 1.1; 95% CI, 0.7-1.8). If replicated, these findings suggest that early OC use may be associated with the subset of mammary tumors that overexpress cyclin D1. PMID- 12376515 TI - Interaction of dietary folate intake, alcohol, and risk of hormone receptor defined breast cancer in a prospective study of postmenopausal women. AB - Alcohol intake is an established risk factor for breast cancer, but the underlying mechanism remains unknown. Four recent studies have described interactions of alcohol and low folate intake. We examined this interaction on the risk of postmenopausal breast cancer stratified by tumor receptor status for estrogen (ER) and progesterone (PR). The Iowa Women's Health Study is a prospective cohort study of 34,393 at-risk women. Alcohol use and folate intake from diet and supplements were estimated at baseline in 1986 through a semiquantitative food frequency questionnaire. Through 1999, 1,875 cases of breast cancer were identified through linkage to the Iowa Surveillance, Epidemiology, and End Results registry. Compared with nondrinkers with folate intakes above the 50(th) percentile, women with low folate and high alcohol were at 1.43-fold greater risk (1.02-2.02). When stratified by tumor receptor status for ER or PR, the risks for low folate/high alcohol were 2.1 (1.18-3.85), 1.0 (0.76-1.42), 1.2 (0.88-1.70), and 1.2 (0.69-2.02) for ER-, ER+, PR+, and PR- tumors, respectively. Because the results were limited primarily to ER- tumors, one plausible interpretation of these data is that alcohol influences breast cancer through its metabolite, acetaldehyde, rather than through effects on ER levels and receptor-mediated pathways. PMID- 12376516 TI - Aspirin use in relation to risk of prostate cancer. AB - Experimental studies have shown inhibitory effects of nonsteroidal anti inflammatory drugs on prostate cancer cell proliferation and reduction of prostate cancer metastasis, suggesting their possible preventive role for prostate cancer. We examined the association between regular aspirin use and the risk of prostate cancer among participants in the Health Professionals Follow-up Study, a prospective cohort of 47,882 United States men who were 40-75 years of age and without a history of prostate cancer in 1986. Biennial self-administered questionnaires were used to assess regular aspirin use from 1986 to 1996. We confirmed and staged incident cases of prostate cancer according to medical records and pathology reports. During 518,072 person-years of follow-up, 2,479 new cases of prostate cancer were ascertained. Of these, 608 were diagnosed as advanced (extraprostatic) prostate cancer and 258 as metastatic prostate cancer. We found no association between aspirin use and total prostate cancer. After accounting for prostate-specific antigen examinations and other potentially confounding variables, the relative risk of total prostate cancer for aspirin users compared with nonusers was 1.05 (95% confidence interval, 0.96-1.14). For metastatic prostate cancer, we observed a suggestive decrease in risk among men reporting greater frequency of aspirin use. The multivariate relative risk of metastatic prostate cancer among men using aspirin 22 or more days/month was 0.73 (95% confidence interval, 0.39-1.38) compared with nonusers. We noted no evidence of a linear dose-response relationship (P for trend = 0.40). The results from this cohort indicate that regular aspirin use is not likely to prevent the incidence of total prostate cancer, but we cannot exclude a possible benefit of frequent aspirin use on risk of developing metastatic prostate cancer. PMID- 12376517 TI - Ovarian cancer, cholesterol, and eggs: a case-control analysis. PMID- 12376518 TI - Utilization of breast cancer screening in a clinically based sample of women after BRCA1/2 testing. AB - We conducted a prospective, observational study to determine breast cancer screening practices among self-referred high-risk women who pursued genetic testing for BRCA1 and BRCA2 mutations. Of the 107 unaffected women included in this study, 41 were BRCA1/2 carriers and 66 women tested negative for a mutation previously identified in their family. All of the women underwent comprehensive pre- and posttest genetic counseling, and completed baseline and 12-month follow up telephone interviews. The baseline (pretest) interview assessed potential predictors of mammography use, including demographics and psychosocial variables. During the year after the receipt of BRCA1/2 test results, 47% of the noncarriers and 59% of the carriers reported that they had had a mammogram [chi(2) (1, n = 107) = 1.35; P = 0.24]. Only 39% of carriers ages 25-39 reported having a mammogram, versus 74% of carriers age > or =40 [chi(2) (1, n = 41) = 5.10; P = 0.02]. Among noncarriers ages 50 and older, 83% had an annual mammogram. Factors independently associated with mammography use included age (<40, > or =40; odds ratio, 7.5; confidence interval, 2.6-21.5) and test result (odds ratio, 4.6; confidence interval, 1.1-18.7). The effects of perceived likelihood of having a BRCA1/2 gene alteration and the interaction between test result and perceived likelihood were not significant. As expected, most carriers (95%) and noncarriers (77%) obtained a clinical breast exam within the year after the receipt of test results. These data suggest that in carriers, overall, the use of breast cancer screening including mammography is good, although there was a relatively low uptake rate of mammography in younger carriers. Noncarriers had very good adherence to general population screening guidelines. PMID- 12376519 TI - Intake of alcohol and alcoholic beverages and the risk of basal cell carcinoma of the skin. AB - We prospectively examined the intake of alcoholic beverages in relation to the risk of basal cell carcinoma BCC in two large cohorts of men and women. Alcohol intake was assessed with food frequency questionnaires every 2-4 years, and BCC was ascertained by self-report. We used a pooled logistic regression to model the association between alcohol intake and BCC adjusting for various health, sun exposure, and sun-sensitivity factors. During 8 years of follow-up in women (1986 1994) we recorded 3060 cases of BCC, and during 10 years (1986-1996), we recorded 3028 cases in men. Significant positive associations were observed between total alcohol intake (P for trend <0.0001), alcohol from liquor (P for trend = 0.003), and white wine (P for trend = 0.01) intake and risk of BCC. Compared with those who abstained, those who drank 0.1-4.9 g, 5.0-14.9 g, 15.0-14.9 g, and 30 g or more alcohol a day had multivariate relative risks of 1.11 [95% confidence interval (CI), 1.03, 1.19], 1.26 (95% CI, 1.12, 1.41), 1.29 (95% CI, 1.18, 1.42), and 1.12 (95% CI, 1.01, 1.26), respectively. Alcohol from beer had no association with BCC in either cohort, and red wine appeared to have an inverse association in women (P for trend = 0.004) but not in men. These associations remained unchanged after adjustment for individual vitamins, multivitamin use, outdoor walking, and exclusion of follow-up time after last physical examination among those who never had BCC. Alcohol intake was associated with BCC, but the association appeared to be different for each type of alcoholic beverage. Other studies are needed to confirm these results. PMID- 12376520 TI - Validating a dipstick method for detecting recent smoking. AB - This report evaluates the validity of a new method for verifying self-reported smoking status in patients presenting for pulmonary medicine treatment. A prospective comparison was made between self-reports of smoking status and a new semiquantitative, enzyme-linked, immunosorbent assay-based method testing for the presence of a prime nicotine metabolite, cotinine. Results were validated by gas chromatography/mass spectrometry. Data were collected in an urban, academic, tertiary health care setting. The study included 76 consecutive new patients presenting to participating clinical practices at the Pulmonology or Thoracic Surgery Services. Before taking a smoking history, patients were informed that their urine would be tested onsite for the presence of nicotine using a new method, the NicoMeter, for determining tobacco product exposure, followed by more standard laboratory testing. The level of agreement between the biochemical measurement types was excellent, kappa = 0.777. The new biochemical measurement type used was easy to use. Self-reported smoking status corresponded closely to biochemical testing. However, there was a 5.3-9.5% misclassification of smoking status among the group studied, depending upon the measurement type used. Among 32 lung cancer patients, 15.6%, most likely misrepresented their current smoking status. The NicoMeter appears to be a valid and useful method for confirming self reported smoking status. Lung cancer patients had a higher rate of inaccurate nonsmoking compared with patients with nonmalignant pulmonary disease. The findings have implications for investigators who accept self-reported smoking status without biochemical verification. PMID- 12376521 TI - Sensitivity of electrospray ionization mass spectrometry detection of codon 249 mutations in the p53 gene compared with RFLP. AB - Hepatocellular carcinoma (HCC) has several major etiological risk factors, including infection with hepatitis viruses and exposure to aflatoxin B(1). A specific missense mutation resulting from a guanine to thymine transversion at the third position of codon 249 in the p53 tumor suppressor gene has been reported in 10-70% of HCCs from areas of high dietary exposure to aflatoxin B(1.) This mutation has not only been detected in tumor samples but has also been measured in DNA isolated from the blood of patients with HCC in two separate studies by two independent methods: RFLP and short oligonucleotide mass analysis (SOMA), an electrospray ionization mass spectrometry technique. To compare the relative sensitivities of these methodologies, a set of serially diluted samples was analyzed by both techniques. The detection limits of RFLP and SOMA were 6% and 2.4% mutant alleles in the presence of wild-type alleles, respectively. When the DNA samples were predigested with HaeIII before SOMA, the detection limit was improved to 0.4% mutant allele in the presence of wild-type alleles. We have therefore found that SOMA is about 2.5-15-fold more sensitive than RFLP for detection of specific p53 mutations. A set of 26 DNA samples from HCC and normal liver was analyzed by RFLP and SOMA, and 5 samples were positive for the p53 mutation. An additional 4 samples were found to be positive for the mutation when SOMA was repeated after HaeIII predigestion. PMID- 12376522 TI - Buccal cell DNA yield, quality, and collection costs: comparison of methods for large-scale studies. AB - There is considerable interest in noninvasive and cost-effective methods for obtaining DNA in large-scale studies. In this randomized crossover study of 22 participants, we compared the DNA yield, quality, and associated costs of buccal cell DNA collected using cytobrushes (three brushes per collection) and swish (i.e., mouthwash) in self-administered procedures. There was a nonstatistically significant higher yield from the mouthwash compared with cytobrush collections (15.8 microg versus 12.0 microg, respectively; P = 0.53). PCR reactions that required short (0.3 kb) or intermediate (1.1 kb) DNA fragments were 100% successful for DNA from brush and mouthwash, whereas PCRs for reactions that required long fragments (7.8 kb) failed for all of the participants from cytobrush DNA and were 81% successful for DNA from the mouthwash source. The brush collections provided sufficient DNA for an estimated 150-225 PCR reactions requiring short and intermediate DNA fragments. The estimated per person costs for buccal brush DNA collections in large studies were less then half (8.50 dollars) those for the mouthwash method (18 dollars). In addition, we tested whether cytobrush instructions to rub cheeks before collection or collect cells only in the morning increased DNA yield and whether repeat brushings of the same cheek reduced DNA yield. These variations resulted in no significant differences in DNA yields. We conclude that the collection of DNA with cytobrushes using simple instructions is cost effective in large-scale studies, and yields sufficient quantity and quality of DNA for genotyping. PMID- 12376523 TI - In utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns. AB - Transplacental exposure to carcinogenic air pollutants from the combustion of fossil fuels is a growing health concern, given evidence of the heightened susceptibility of the fetus. These mutagenic/carcinogenic pollutants include aromatic compounds such as polycyclic aromatic hydrocarbons that bind to DNA, forming chemical-DNA adducts. We have investigated the genotoxic effects of transplacental exposure in humans by analyzing aromatic-DNA adducts and the frequency of gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in umbilical cord and maternal blood samples. Here we show, in a cross-sectional study of 67 mothers and 64 newborns from the Krakow Region of Poland, that aromatic-DNA adducts measured by (32)P-postlabeling are positively associated with HPRT mutant frequency in the newborns (beta = 0.56, P = 0.03) after controlling for exposure to tobacco smoke, diet, and socioeconomic status. In contrast to the fetus, HPRT mutations and DNA adducts do not reflect similar exposure periods in the mother, and the maternal biomarkers were not correlated. Adducts were higher in the newborn than the mother, indicating differential susceptibility of the fetus to DNA damage; but HPRT mutation frequency was 4-fold lower, consistent with the long lifetime of the biomarker. These results provide the first demonstration of a molecular link between somatic mutation in the newborn and transplacental exposure to common air pollutants, a finding that is relevant to cancer risk assessment. PMID- 12376524 TI - No association between a stop codon polymorphism in RAD52 and breast cancer risk. PMID- 12376525 TI - The MnSOD A16V mitochondrial targeting sequence polymorphism is not associated with increased risk of distal colorectal adenomas: data from a sigmoidoscopy based case control study. PMID- 12376526 TI - DNA repair gene XRCC3 241Met variant is not associated with risk of cutaneous malignant melanoma. PMID- 12376527 TI - Nectin-1alpha, an immunoglobulin-like receptor involved in the formation of synapses, is a substrate for presenilin/gamma-secretase-like cleavage. AB - Nectin-1 is a member of the immunoglobulin superfamily and a Ca(2+)-independent adherens junction protein involved in synapse formation. Here we show that nectin 1alpha undergoes intramembrane proteolytic processing analogous to that of the Alzheimer's disease amyloid precursor protein, mediated by a presenilin (PS) dependent gamma-secretase-like activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of Chinese hamster ovary cells activated a first proteolytic event, resulting in ectodomain shedding of nectin-1alpha. Subsequent cleavage of the remaining 26-kDa membrane-anchored C-terminal fragment (CTF) was inhibited independently by three specific gamma-secretase inhibitors and by expression of the dominant negative form of PS1. The PS/gamma-secretase-like cleavage product was detected in vivo following proteasome inhibitor treatment of cells. An in vitro gamma-secretase assay confirmed the generation of a 24-kDa nectin-1alpha intracellular domain, peripherally associated with the membrane fraction. We also found nectin-1alpha to interact with the N-terminal fragment of PS1. Finally, gamma-secretase inhibition resulted in beta-catenin release from cell junctions, concomitantly with the accumulation of the 26-kDa nectin-1alpha CTF, suggesting that high levels of nectin-1alpha CTF interfere with TPA-induced remodeling of cell-cell junctions. Our results are consistent with a previously reported role for PS/gamma-secretase in adherens junction function involving cleavage of cadherins. Similar to nectin-1, other members of the immunoglobulin superfamily involved in synapse formation may also serve as substrates for PS/gamma-secretase like intramembrane proteolytic activity. PMID- 12376528 TI - Overexpression of full-length but not N-terminal truncated isoform of microtubule associated protein (MAP) 1B accelerates apoptosis of cultured cortical neurons. AB - beta-amyloid (Abeta) is presumed to play a pathogenic role in Alzheimer's disease (AD). However, there is an imperfect correlation between Abeta deposition and neuronal loss or dementia. To clarify neuronal responses to Abeta, Abeta-induced gene expression in cultured cortical neurons was analyzed by differential display followed by Northern blotting. Here we report that nonaggregated or aggregated Abeta induced microtubule-associated protein 1B (MAP1B) mRNA, especially the alternative transcript containing exon 3U, before disruption of the cell membrane by Abeta. An alternative transcript containing exon 3U is translated into an N terminal truncated shorter isoform of MAP1B. Transfection experiments reveal that overexpression of this isoform does not accelerate neurite outgrowth or apoptosis of cortical neurons. In contrast, overexpression of MAP1B fragments containing the N-terminal 126 amino acids promoted neurite outgrowth and neuronal apoptosis. These results suggest that Abeta does not induce deleterious full-length MAP1B directly, but overexpression of full-length MAP1B might act as an effector of cell death in neurodegenerative disorders related to cytoskeletal abnormalities. PMID- 12376529 TI - Synergistic activity of the ninth and tenth FIII domains of human fibronectin depends upon structural stability. AB - The ninth and tenth FIII domains (FIII9-10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9 10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu(1408) to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9-10. We show that the introduction of mutations into the synergistic sequence of FIII9-10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin alpha(5)beta(1). Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu(1408) to Pro substitution in mutant FIII9-10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to alpha(5)beta(1). In keeping with the enhancement of functional activity, the Leu(1408) to Pro substitution in the FIII9-10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9-10 mutants and suggest that a Leu(1408) to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed. PMID- 12376530 TI - Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. AB - Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence. PMID- 12376531 TI - Ablation of internalization signals in the carboxyl-terminal tail of the cystic fibrosis transmembrane conductance regulator enhances cell surface expression. AB - The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999) J. Biol. Chem. 274, 3602-3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr(1424)-X X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function. PMID- 12376532 TI - P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells. AB - We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca(2+)(i) mobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca(2+)(i) mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca(2+)](i) in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5' nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca(2+)](i) in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca(2+)](i), suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca(2+)](i). Furthermore, adenosine, ADP, UDP, or adenosine 5'-O-(3-thiotriphosphate) did not induce Ca(2+)(i) mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4'-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca(2+)(i) response, whereas pertussis toxin, a G(i/o)-protein inhibitor, inhibited fluid flow-induced increases in [Ca(2+)](i) in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca(2+)(i) response to fluid flow. Finally, a decreased percentage of MC3T3 E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca(2+)](i), and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization in osteoblastic cells. PMID- 12376533 TI - Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3beta. AB - Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation in vivo. Here we show that binding of Reelin to these receptors stimulates phosphatidylinositol 3-kinase, resulting in activation of protein kinase B and inhibition of glycogen synthase kinase 3beta. We present genetic evidence that this cascade is dependent on apolipoprotein E receptor 2, very low density lipoprotein receptor, and Dab1. Reelin-signaling components are enriched in axonal growth cones, where tyrosine phosphorylation of Dab1 is increased in response to Reelin. These findings suggest that Reelin mediated phosphatidylinositol 3-kinase signaling in neuronal growth cones contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3beta kinase activities. PMID- 12376534 TI - Involvement of proteasome in the dynamic assembly of the androgen receptor transcription complex. AB - We have used the chromatin immunoprecipitation technique to analyze the formation of the androgen receptor (AR) transcription complex onto prostate-specific antigen (PSA) and kallikrein 2 promoters in LNCaP cells. Our results show that loading of holo-AR and recruitment of RNA polymerase II to the promoters occur transiently. The cyclic nature of AR transcription complex assembly is also illustrated by transient association of coactivators GRIP1 and CREB-binding protein and acetylated histone H3 with the PSA promoter. Treatment of cells with the pure antiandrogen bicalutamide also elicits occupancy of the promoter by AR. In contrast to the agonist-liganded AR, bicalutamide-bound receptor is not capable of recruiting polymerase II, GRIP1, or CREB-binding protein, indicating that the conformation of AR bound to anti-androgen is not competent to assemble transcription complexes. Proteasome is involved in the regulation of AR-dependent transcription, as a proteasome inhibitor, MG-132, prevents the release of the receptor from the PSA promoter, and it also blocks the androgen-induced PSA mRNA accumulation. Furthermore, occupancy of the PSA promoter by the 19 S proteasome subcomplex parallels that by AR. Collectively, formation of the AR transcription complex, encompassing AR, polymerase II, and coactivators, on a regulated promoter is a cyclic process involving proteasome function. PMID- 12376535 TI - Dopamine biosynthesis is regulated by S-glutathionylation. Potential mechanism of tyrosine hydroxylast inhibition during oxidative stress. AB - Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inhibited by the sulfhydryl oxidant diamide in a concentration-dependent manner. The inhibitory effect of diamide on TH catalytic activity is enhanced significantly by GSH. Treatment of TH with diamide in the presence of [(35)S]GSH results in the incorporation of (35)S into the enzyme. The effect of diamide-GSH on TH activity is prevented by dithiothreitol (DTT), as is the binding of [(35)S]GSH, indicating the formation of a disulfide linkage between GSH and TH protein cysteinyls. Loss of TH catalytic activity caused by diamide-GSH is partially recovered by DTT and glutaredoxin, whereas the disulfide linkage of GSH with TH is completely reversed by both. Treatment of intact PC12 cells with diamide results in a concentration dependent inhibition of TH activity. Incubation of cells with [(35)S]cysteine, to label cellular GSH prior to diamide treatment, followed by immunoprecipitation of TH shows that the loss of TH catalytic activity is associated with a DTT reversible incorporation of [(35)S]GSH into the enzyme. A combination of matrix assisted laser desorption/ionization/mass spectrometry and liquid chromatography/tandem mass spectrometry was used to identify the sites of S glutathionylation in TH. Six cysteines (177, 249, 263, 329, 330, and 380) of the seven cysteine residues in TH were confirmed as substrates for modification. Only Cys-311 was not S-glutathionylated. These results establish that TH activity is influenced in a reversible manner by S-glutathionylation and suggest that cellular GSH may regulate dopamine biosynthesis under conditions of oxidative stress or drug-induced toxicity. PMID- 12376536 TI - Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. AB - Vitamin B(1) in its active form thiamin pyrophosphate is an essential coenzyme that is synthesized by coupling of pyrimidine (hydroxymethylpyrimidine; HMP) and thiazole (hydroxyethylthiazole) moieties in bacteria. Using comparative analysis of genes, operons, and regulatory elements, we describe the thiamin biosynthetic pathway in available bacterial genomes. The previously detected thiamin regulatory element, thi box (Miranda-Rios, J., Navarro, M., and Soberon, M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9736-9741), was extended, resulting in a new, highly conserved RNA secondary structure, the THI element, which is widely distributed in eubacteria and also occurs in some archaea. Search for THI elements and analysis of operon structures identified a large number of new candidate thiamin-regulated genes, mostly transporters, in various prokaryotic organisms. In particular, we assign the thiamin transporter function to yuaJ in the Bacillus/Clostridium group and the HMP transporter function to an ABC transporter thiXYZ in some proteobacteria and firmicutes. By analogy to the model of regulation of the riboflavin biosynthesis, we suggest thiamin-mediated regulation based on formation of alternative RNA structures involving the THI element. Either transcriptional or translational attenuation mechanism may operate in different taxonomic groups, dependent on the existence of putative hairpins that either act as transcriptional terminators or sequester translation initiation sites. Based on analysis of co-occurrence of the thiamin biosynthetic genes in complete genomes, we predict that eubacteria, archaea, and eukaryota have different pathways for the HMP and hydroxyethylthiazole biosynthesis. PMID- 12376537 TI - Arf1 dissociates from the clathrin adaptor GGA prior to being inactivated by Arf GTPase-activating proteins. AB - The effectors of monomeric GTP-binding proteins can influence interactions with GTPase-activating proteins (GAPs) in two ways. In one case, effector and GAP binding to the GTP-binding protein is mutually exclusive. In another case, the GTP-binding protein bound to an effector is the substrate for the GTPase activating protein. Here predictions for these two mechanisms were tested for the Arf1 effector GGA and ASAP family Arf GAPs. GGA inhibited Arf GAP activity of ASAP1, AGAP1, ARAP1, and Arf GAP1 and inhibited binding of Arf1.GTPgammaS to AGAP1 with K(i) values correlating with the K(d) for the GGA.Arf1 complex. ASAP1 blocked Arf1.GTPgammaS binding to GGA with a K(i) similar to the K(d) for the ASAP.Arf1.GTPgammaS complex. No interaction of GGA with ASAP1 was detected. Consistent with GGA sequestering Arf from GAPs, overexpression of GGA slowed the rate of Arf dissociation from the Golgi apparatus following treatment with brefeldin A. Mutational analysis revealed the amino-terminal alpha-helix and switch I of Arf1 contributed to interaction with both GGA and GAPs. These data exclude the mechanism previously documented for Arf GAP1/coatomer in which Arf1 is inactivated in a tripartite complex. Instead, termination of Arf1 signals mediated through GGA require that Arf1.GTP dissociates from GGA prior to interaction with GAP and consequent hydrolysis of GTP. PMID- 12376538 TI - A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity. AB - alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer. PMID- 12376539 TI - Regulated interactions of the alpha 2A adrenergic receptor with spinophilin, 14-3 3zeta, and arrestin 3. AB - The present studies demonstrate that no single stretch of sequence in the third intracellular (3i) loop of the alpha(2A) adrenergic receptor (alpha(2A)-AR) can fully account for its previously described interactions with spinophilin (Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003-15008), 14-3-3zeta (Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462-13469), and arrestin 3 (Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842), suggesting that a three dimensional surface, rather than a linear sequence, provides the basis for these interactions as proposed for 3i loop tethering of the alpha(2A)-AR to the basolateral surface of Madin-Darby canine kidney cells (Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331-16336). Sequences at the extreme N-terminal and C-terminal ends of the 3i loop are critical for interaction with spinophilin but not for interaction with 14-3-3zeta or arrestin 3, for which the C-terminal half of the loop is more important. Competition binding for (35)S labeled alpha(2A)-AR 3i loop binding to glutathione S-transferase (GST) spinophilin amino acids 151-444 revealed a relative affinity of spinophilin congruent with arrestin > 14-3-3zeta for the unphosphorylated alpha(2A)-AR 3i loop. Agonist occupancy of the alpha(2A)-AR increases receptor association with spinophilin, and arrestin 3 appears to compete for this enrichment. However, when the G protein-coupled receptor kinase 2 substrate sequence was deleted from the 3i loop, arrestin 3 could not compete for the agonist-enriched binding of spinophilin to the mutant alpha(2A)-AR. These data are consistent with a model where sequential or competitive interactions among spinophilin, arrestin, and/or 14-3-3zeta play a role in alpha(2A)-AR functions. PMID- 12376540 TI - LL5beta is a phosphatidylinositol (3,4,5)-trisphosphate sensor that can bind the cytoskeletal adaptor, gamma-filamin. AB - We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5beta). The protein bound PtdIns(3,4,5)P(3) selectively in vitro. Strikingly, a substantial proportion of LL5beta became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P(3) levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5beta to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5beta that could not bind PtdIns(3,4,5)P(3) were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P(3) levels, LL5beta was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns(3,4,5)P(3) levels, it became significantly plasma-membrane localized. The distribution of LL5beta is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P(3) indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P(3). In addition, LL5beta bound to the cytoskeletal adaptor, gamma filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed gamma-filamin with GFP-LL5beta in the unidentified vesicles. PMID- 12376541 TI - A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating. AB - We have used sulfhydryl-modifying reagents to investigate the regulation of G protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2 aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism. PMID- 12376542 TI - Subtype-specific expression of group III metabotropic glutamate receptors and Ca2+ channels in single nerve terminals. AB - The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca(2+) channel activity. By imaging Ca(2+) in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca(2+) channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca(2+) channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for l(+)-2-amino-4 phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca(2+) response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca(2+) channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca(2+) channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca(2+) channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity. PMID- 12376544 TI - Mammalian vestigial-like 2, a cofactor of TEF-1 and MEF2 transcription factors that promotes skeletal muscle differentiation. AB - Expression of many skeletal muscle-specific genes depends on TEF-1 (transcription enhancer factor-1) and MEF2 transcription factors. In Drosophila, the TEF-1 homolog Scalloped interacts with the cofactor Vestigial to drive differentiation of the wing and indirect flight muscles. Here, we identify three mammalian vestigial-like genes, Vgl-1, Vgl-2, and Vgl-3, that share homology in a TEF-1 interaction domain. Vgl-1 and Vgl-3 transcripts are enriched in the placenta, whereas Vgl-2 is expressed in the differentiating somites and branchial arches during embryogenesis and is skeletal muscle-specific in the adult. During muscle differentiation, Vgl-2 mRNA levels increase and Vgl-2 protein translocates from the cytoplasm to the nucleus. In situ hybridization revealed co-expression of Vgl 2 with myogenin in the differentiating muscle of embryonic myotomes but not in newly formed somites prior to muscle differentiation. Like Vgl-1, Vgl-2 interacts with TEF-1. In addition, we show that Vgl-2 interacts with MEF2 in a mammalian two-hybrid assay and that Vgl-2 selectively binds to MEF2 in vitro. Co-expression of Vgl-2 with MEF2 markedly co-activates an MEF2-dependent promoter through its MEF2 element. Overexpression of Vgl-2 in MyoD-transfected 10T(1/2) cells markedly increased myosin heavy chain expression, a marker of terminal muscle differentiation. These results identify Vgl-2 as an important new component of the myogenic program. PMID- 12376543 TI - The vesicle- and target-SNARE proteins that mediate Glut4 vesicle fusion are localized in detergent-insoluble lipid rafts present on distinct intracellular membranes. AB - Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 ( approximately 35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-beta-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic. PMID- 12376545 TI - Intra- and intermolecular interactions between intracellular domains of receptor protein-tyrosine phosphatases. AB - The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The generally inactive membrane-distal PTP domains (RPTP-D2s) bind and are proposed to regulate the membrane-proximal PTP domains (RPTP-D1s). We set out to characterize the interactions between RPTP-D1s and RPTP-D2s in vivo by co-immunoprecipitation of hemagglutinin-tagged fusion proteins encoding the transmembrane domain and RPTP D1 and myc-tagged RPTP-D2. Seven RPTPs from four different subfamilies were used: RPTPalpha, RPTPepsilon, LAR, RPTPvarsigma, RPTPdelta, CD45, and RPTP(mu). We found that RPTP-D2s bound to RPTPs with different affinities. The presence of intrinsic RPTP-D2 altered the binding specificity toward other RPTP-D2s positively or negatively, depending on the identity of the RPTPs. Furthermore, the C terminus of RPTP-D2s and the "wedge" in RPTP-D1s played a central role in binding specificity. Finally, full-length RPTPalpha and LAR heterodimerized in an oxidative stress-dependent manner. Like RPTPalpha-D2, the LAR-D2 conformation was affected by oxidative stress, suggesting a common regulatory mechanism for RPTP complex formation. Taken together, interactions between RPTP-D1s and RPTP-D2s are a common but specific mechanism that is likely to be regulated. The RPTP-D2s and the wedge structures are crucial determinants of binding specificity, thus regulating cross-talk between RPTPs. PMID- 12376546 TI - Formation of mutually exclusive Rab11 complexes with members of the family of Rab11-interacting proteins regulates Rab11 endocytic targeting and function. AB - Several Rabs, including Rab11, regulate the traffic and sorting of proteins in the endosomal pathway. Recently, six novel Rab11 family interacting proteins (FIPs) were identified. Although they share little overall sequence homology, all FIPs contain a conserved Rab11-binding domain. Here we investigate the role of FIPs as Rab11-targeting proteins and show that the Rab11-binding domain assumes an alpha-helical structure, with the conserved residues forming a hydrophobic Rab11-binding patch. This hydrophobic patch mediates the formation of mutually exclusive complexes between Rab11 and various members of FIP protein family. Furthermore, the formation of Rab11/FIP complexes regulates Rab11 localization by recruiting it to distinct endocytic compartments. Thus, we propose that Rab11/FIP complexes serve as targeting patches, regulating Rab11 localization and recruitment of additional cellular factors to different endocytic compartments. PMID- 12376547 TI - Modulation of induction properties of glucocorticoid receptor-agonist and antagonist complexes by coactivators involves binding to receptors but is independent of ability of coactivators to augment transactivation. AB - Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonist-bound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes. These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors. We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity. Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities. DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities. The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved. The ability of a TIF2.4 fragment (i.e. TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2. Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids. These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes. In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s). PMID- 12376548 TI - NRAGE, a p75 neurotrophin receptor-interacting protein, induces caspase activation and cell death through a JNK-dependent mitochondrial pathway. AB - The p75 neurotrophin receptor (p75NTR) mediates signaling events leading to activation of the JNK pathway and cell death in a variety of cell types. We recently identified NRAGE, a protein that directly interacts with the p75NTR cytosolic region and facilitates p75NTR-mediated cell death. For the present study, we developed an inducible recombinant NRAGE adenovirus to dissect the mechanism of NRAGE-mediated apoptosis. Induced NRAGE expression resulted in robust activation of the JNK pathway that was not inhibited by the pharmacological mixed lineage kinase (MLK) inhibitor CEP1347. NRAGE induced cytosolic accumulation of cytochrome c, activation of Caspases-3, -9 and -7, and caspase-dependent cell death. Blocking JNK and c-Jun action by overexpression of the JNK-binding domain of JIP1 or dominant-negative c-Jun ablated NRAGE-mediated caspase activation and NRAGE-induced cell death. These findings identify NRAGE as a p75NTR interactor capable of inducing caspase activation and cell death through a JNK-dependent mitochondrial apoptotic pathway. PMID- 12376549 TI - Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy. AB - Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death. PMID- 12376550 TI - NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance. AB - Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine. PMID- 12376551 TI - RhoG signals in parallel with Rac1 and Cdc42. AB - RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo. First, we determined that RhoG was regulated by guanine nucleotide exchange factors that also activate Rac and/or Cdc42. Vav2 (which activates RhoA, Rac1, and Cdc42) and to a lesser degree Dbs (which activates RhoA and Cdc42) activated RhoG in vitro. Thus, RhoG may be activated concurrently with Rac1 and Cdc42. Second, some effectors of Rac/Cdc42 (IQGAP2, MLK-3, PLD1), but not others (e.g. PAKs, POSH, WASP, Par-6, IRSp53), interacted with RhoG in a GTP-dependent manner. Third, consistent with this differential interaction with effectors, activated RhoG stimulated some (JNK and Akt) but not other (SRF and NF-kappaB) downstream signaling targets of activated Rac1 and Cdc42. Finally, transient transduction of a tat-tagged Rac1(17N) dominant-negative fusion protein inhibited the induction of lamellipodia by the Rac-specific activator, Tiam1, but not by activated RhoG. Together, these data argue that RhoG function is mediated by signals independent of Rac1 and Cdc42 activation and instead by direct utilization of a subset of common effectors. PMID- 12376552 TI - Growth hormone-induced diacylglycerol and ceramide formation via Galpha i3 and Gbeta gamma in GH4 pituitary cells. Potentiation by dopamine-D2 receptor activation. AB - Growth hormone (GH) secretion is regulated by indirect negative feedback mechanisms. To address whether GH has direct actions on pituitary cells, lipid signaling in GH(4)ZR(7) somatomammotroph cells was examined. GH (EC(50) = 5 nm) stimulated diacylglycerol (DAG) and ceramide formation in parallel by over 10 fold within 15 min and persisting for >3 h. GH-induced DAG/ceramide formation was blocked by pertussis toxin (PTX) implicating G(i)/G(o) proteins and was potentiated 1.5-fold by activation of G(i)/G(o)-coupled dopamine-D2S receptors, which had no effect alone. Following PTX pretreatment, only PTX-resistant Galpha(i)3, not Galpha(o) or Galpha(i)2, rescued GH-induced DAG/ceramide signaling. GH-induced DAG/ceramide formation was also blocked in cells expressing Gbetagamma blocker GRK-ct. In GH(4)ZR(7) cells, GH induced phosphorylation of JAK2 and STAT5, which was blocked by PTX and mimicked by ceramide analogue C2 ceramide or sphingomyelinase treatment to increase endogenous ceramide. We conclude that in GH(4) pituitary cells, GH induces formation of DAG/ceramide via a novel Galpha(i)3/Gbetagamma-dependent pathway. This novel pathway suggests a mechanism for autocrine feedback regulation by GH of pituitary function. PMID- 12376554 TI - The 'spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families. AB - Recent studies have characterised a family of giant cytoskeletal crosslinkers encoded by the short stop gene in Drosophila and the dystonin/BPAG1 and MACF1 genes in mammals. We refer to the products of these genes as spectraplakins to highlight the fact that they share features with both the spectrin and plakin superfamilies. These genes produce a variety of large proteins, up to almost 9000 residues long, which can potentially extend 0.4 micro m across a cell. Spectraplakins can interact with all three elements of the cytoskeleton: actin, microtubules and intermediate filaments. The analysis of mutant phenotypes in BPAG1 in mouse and short stop in Drosophila demonstrates that spectraplakins have diverse roles. These include linking the plasma membrane and the cytoskeleton, linking together different elements of the cytoskeleton and organising membrane domains. PMID- 12376553 TI - New functional roles for non-collagenous domains of basement membrane collagens. AB - Collagens IV, XV and XVIII are major components of various basement membranes. In addition to the collagen-specific triple helix, these collagens are characterized by the presence of several non-collagenous domains. It is clear now that these ubiquitous collagen molecules are involved in more subtle and sophisticated functions than just the molecular architecture of basement membranes, particularly in the context of extracellular matrix degradation. Degradation of the basement membrane collagens occurs during numerous physiological and pathological processes such as embryonic development or tumorigenesis and generates collagen fragments. These fragments are involved in the regulation of functions differing from those of their original intact molecules. The non collagenous C-terminal fragment NC1 of collagen IV, XV and XVIII have been recently highlighted in the literature because of their potential in reducing angiogenesis and tumorigenesis, but it is clear that their biological functions are not limited to these processes. Proteolytic release of soluble NC1 fragments stimulates migration, proliferation, apoptosis or survival of different cell types and suppresses various morphogenetic events. PMID- 12376555 TI - Transforming growth factor-beta and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes. AB - Enhancement of tumor cell growth and invasiveness by transforming growth factor beta (TGF-beta) requires constitutive activation of the ras/MAPK pathway. Here we have investigated how MEK activation by epidermal growth factor (EGF) influences the response of fully differentiated and growth-arrested pig thyroid epithelial cells in primary culture to TGF-beta1. The epithelial tightness was maintained after single stimulation with EGF or TGF-beta1 (both 10 ng/ml) for 48 hours. In contrast, co-stimulation abolished the transepithelial resistance and increased the paracellular flux of [(3)H]inulin within 24 hours. Reduced levels of the tight junction proteins claudin-1 and occludin accompanied the loss of barrier function. N-cadherin, expressed only in few cells of untreated or single stimulated cultures, was at the same time increased 30-fold and co-localised with E-cadherin at adherens junctions in all cells. After 48 hours of co-stimulation, both E- and N-cadherin were downregulated and the cells attained a fibroblast like morphology and formed multilayers. TGF-beta1 only partially inhibited EGF induced Erk phosphorylation. The MEK inhibitor U0126 prevented residual Erk phosphorylation and abrogated the synergistic responses to TGF-beta1 and EGF. The observations indicate that concomitant growth factor-induced MEK activation is necessary for TGF-beta1 to convert normal thyroid epithelial cells to a mesenchymal phenotype. PMID- 12376556 TI - Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development. AB - Dictyostelium lacking myosin II cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin II is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and colocalize with the ConA cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of myosin activity, nor regulation of myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium myosin II and there may be no specific sequence required for localization of myosin to the cleavage furrow. PMID- 12376557 TI - Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2. AB - The ErbB family of transmembrane receptor tyrosine kinases plays an important role in the pathogenesis of many cancers. The four members of the family, ErbB1 4, form various homo- and heterodimers during the course of signal transduction. A second hierarchical level of molecular associations involving 10(2)-10(3) molecules, termed large-scale clustering, has also been identified, but the regulatory factors and biological consequences of such structures have not been systematically evaluated. In this report, we describe the states of association of ErbB2 and their relationship to local ErbB3 density and lipid rafts based on quantitative fluorescence microscopy of SKBR-3 breast cancer cells. Clusters of ErbB2 colocalized with lipid rafts identified by the GM1-binding B subunit of cholera toxin. Pixel-by-pixel analysis of fluorescence resonance energy transfer between labeled antibodies indicated that the homoassociation (homodimerization) of ErbB2 was proportional to the local density of ErbB2 and inversely proportional to that of ErbB3 and of the raft-specific lipid GM1. Crosslinking lipid rafts with the B subunit of cholera toxin caused dissociation of the rafts and ErbB2 clusters, an effect that was independent of the cytoskeletal anchoring of ErbB2. Crosslinking also decreased ErbB2-ErbB3 heteroassociation and the EGF- and heregulin-induced tyrosine phosphorylation of Shc. When cells were treated with the anti-ErbB2 monoclonal antibody 4D5 (parent murine version of Trastuzumab used in the immunotherapy of breast cancer), internalization of the antibody was inhibited by crosslinking of lipid rafts, but the antiproliferative activity of 4D5 was retained and even enhanced. We conclude that local densities of ErbB2 and ErbB3, as well as the lipid environment profoundly influence the association properties and biological function of ErbB2. PMID- 12376558 TI - Ultrastructural characterization of endoplasmic reticulum--Golgi transport containers (EGTC). AB - Recent observations made in live cells expressing green fluorescent protein (GFP) tagged cargo markers have demonstrated the existence of large, mobile transport intermediates linking peripheral ER exit sites (ERES) to the perinuclear Golgi. Using a procedure of rapid ethane freezing, we examined ultrastructurally the intermediates involved in ER-Golgi transport of the vesicular stomatitis virus (VSV) G protein. When released at the permissive temperature of 32 degrees C, VSVG is first found to be concentrated in pleiomorphic, membrane-bound structures (of about 0.4 to 1 microm in diameter) with extensive budding profiles. These structures are devoid of COPII components and Golgi markers, but are enriched in COPI, the retrograde cargo ERGIC53, and the tethering protein p115. The structures appear to be able to undergo fusion with the Golgi stack and are tentatively referred to as ER-Golgi transport containers, or EGTCs. VSVG protein exiting the ERES at 15 degrees C is first found in clusters or strings of COPII containing small vesicles, and morphological analysis indicates that these clusters and strings of COPII vesicles may coalesce by homotypic fusion to form the EGTCs. Together with the large transport containers mediating transport from the trans-Golgi network to the plasma membrane, EGTCs represents an emerging class of large membranous structures mediating anterograde transport between the major stations of the exocytic pathway. PMID- 12376560 TI - The integrin beta tail is required and sufficient to regulate adhesion signaling to Rac1. AB - Rac1 is a small Rho family GTPase that regulates changes in cell morphology associated with cell spreading and migration. Integrin-mediated adhesion is known to activate Rac1 and to regulate the interaction of Rac1 with downstream effectors. Currently, it is not clear how integrins signal Rac1 activation following cell adhesion. Integrin beta cytoplasmic domains (beta-tails) are known to be required for integrin-mediated cell spreading, and isolated beta tails expressed as tac-beta tail chimeras can inhibit cell spreading indicating that protein interactions with beta tails can regulate this process. Our recent studies demonstrated that the expression of constitutively activated Rac1 can restore cell spreading inhibited by tac beta tail chimeras, suggesting a role for Rac1 in the regulation of cell spreading by beta tails. Hence, we examined the role of beta tails in integrin activation of Rac1. By using recombinant wild-type and mutant integrin heterodimers, we demonstrate that integrin beta tails are required for adhesion to increase Rac1-GTP loading. We demonstrate that clustering tac-beta tail chimeras, on the surface of cells in suspension, activates Rac1. Thus, beta tails are not only required, but also sufficient for integrin-triggered Rac1 activation. Our findings indicate that integrin beta tails are an important link between integrin engagement and Rac1 signaling, and that protein interactions initiated at beta tails are sufficient for integrins to regulate Rac1 activity. PMID- 12376559 TI - Conditional ablation of the Mat1 subunit of TFIIH in Schwann cells provides evidence that Mat1 is not required for general transcription. AB - The mammalian Mat1 protein has been implicated in cell cycle regulation as part of the Cdk activating kinase (CAK), and in regulation of transcription as a subunit of transcription factor TFIIH. To address the role of Mat1 in vivo, we have used a Cre/loxP system to conditionally ablate Mat1 in adult mitotic and post-mitotic lineages. We found that the mitotic cells of the germ lineage died rapidly upon disruption of Mat1 indicating an absolute requirement of Mat1 in these cells. By contrast, post-mitotic myelinating Schwann cells were able to attain a mature myelinated phenotype in the absence of Mat1. Moreover, mutant animals did not show morphological or physiological signs of Schwann cell dysfunction into early adulthood. Beyond 3 months of age, however, myelinated Schwann cells in the sciatic nerves acquired a severe hypomyelinating morphology with alterations ranging from cells undergoing degeneration to completely denuded axons. This phenotype was coupled to extensive proliferation and remyelination that our evidence suggests was undertaken by the non-myelinated Schwann cell pool. These results indicate that Mat1 is not essential for the transcriptional program underlying the myelination of peripheral axons by Schwann cells and suggest that the function of Mat1 in RNA polymerase II-mediated transcription in these cells is regulatory rather than essential. PMID- 12376561 TI - Intrinsic actions of IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelial cells: inhibition or accentuation of attachment and survival is dependent upon the presence of fibronectin. AB - The insulin-like growth factor binding proteins (IGFBPs) have IGF-independent differential effects on cell function. We investigated whether they can affect integrin-receptor-mediated cell attachment to different extracellular matrix (ECM) components in Hs578T cells. Cell attachment to a general ECM gel was unaffected by IGFBP-1 and -6 but was significantly increased by IGFBP-4 and -5 and decreased by IGFBP-2 and -3. Similar results were obtained for attachment to laminin or collagen type IV. Attachment to fibronectin, however, was increased by IGFBP-3 and decreased by IGFBP-5. The actions of IGFBP-3 and -5 on cell attachment to ECM were lost in the presence of a soluble Arg-Gly-Asp (RGD) containing fibronectin fragment. Thrombospondin reversed the actions of IGFBP-3 on cell attachment, but IGFBP-5 still increased cell attachment. On plastic, neither IGFBP-3 nor -5 alone affected cell viability; although ceramide-induced apoptosis was enhanced by IGFBP-3 but reduced by IGFBP-5. The presence of RGD reversed the action of IGFBP-5 on cell death but attenuated that of IGFBP-3. With cells grown on fibronectin, the action of IGFBP-3 was reversed, and it conferred cell survival, whereas the survival effect of IGFBP-5 was lost. In summary we have demonstrated that IGFBP-3 and -5 both have intrinsic effects on cell survival. In each case the presence of fibronectin or fibronectin fragments determines whether susceptibility to apoptosis is increased or decreased. These effects on cell survival are paralleled by acute effects on integrin receptor function; IGFBP-3 and -5 were able to either enhance or inhibit cell attachment in the presence of fibronectin. Cell survival is tightly controlled by cues from the ECM and from growth factors, particularly the IGFs. Our findings indicate that, in addition to being crucial modulators of IGF actions, the IGFBPs have direct actions on cell attachment and survival that are specific and dependent upon the matrix components present. PMID- 12376562 TI - PTP-PEST controls motility through regulation of Rac1. AB - The cytoplasmic protein tyrosine phosphatase, PTP-PEST, associates with the focal adhesion proteins p130cas and paxillin and has recently been implicated in cell migration. In this study, we investigated the mechanism by which PTP-PEST regulates this phenomenon. We find that PTP-PEST is activated in an adhesion dependent manner and localizes to the tips of membrane protrusions in spreading fibroblasts. We show that the catalytic activity of PTP-PEST is a key determinant for its effects on motility. Overexpression of PTP-PEST, but not a catalytically inactive form, impairs haptotaxis, cell spreading and formation of membrane protrusions in CHOK1 cells. In addition, overexpression of PTP-PEST in Rat1 fibroblasts perturbs membrane ruffling and motility in response to PDGF stimulation. The expression level of PTP-PEST modulates the activity of the small GTPase, Rac1. PTP-PEST overexpression suppresses activation of Rac1 in response to both integrin-mediated adhesion or growth factor stimulation. In contrast, fibroblasts that lack PTP-PEST expression show enhanced Rac1 activity. Co expression of constitutively active Rac1 with PTP-PEST overcomes the inhibition of cell spreading and migration indicating that PTP-PEST acts by antagonizing Rac1 activation. Our data suggest a model in which PTP-PEST is activated by integrins and localized to regions where it can control motile events at the leading edge through inhibition of the small GTPase Rac1. PMID- 12376563 TI - JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms. AB - Phosphorylation of the N-terminal domain of Jun by the Jun kinases (JNKs) modulates the transcriptional activity of AP-1, a dimeric transcription factor typically composed of c-Jun and c-Fos, the latter being essential for osteoclast differentiation. Using mice lacking JNK1 or JNK2, we demonstrate that JNK1, but not JNK2, is specifically activated by the osteoclast-differentiating factor RANKL. Activation of JNK1, but not JNK2, is required for efficient osteoclastogenesis from bone marrow monocytes (BMMs). JNK1 protects BMMs from RANKL-induced apoptosis during differentiation. In addition, BMMs from mice carrying a mutant of c-Jun phosphorylation sites (JunAA/JunAA), as well as cells lacking either c-Jun or JunD, which is another JNK substrate, revealed that c-Jun phosphorylation and c-Jun itself, but not JunD, are essential for efficient osteoclastogenesis. Moreover, JNK1-dependent c-Jun phosphorylation in response to RANKL is not involved in the anti-apoptotic function of JNK1. Thus, these data provide genetic evidence that JNK1 activation modulates osteoclastogenesis through both c-Jun-phosphorylation-dependent and -independent mechanisms. PMID- 12376564 TI - Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton. AB - Live cell, time-lapse microscopy was used to study trafficking of caveolin-1-GFP in stably expressing CHO cells. Multiple cytological and biochemical tests verified that caveolin-1-GFP was a reliable marker for endogenous caveolin-1. At steady state, most caveolin-1-GFP was either at the cell surface associated with invaginated caveolae or near the centrosome in caveosomes. Live cell fluorescence imaging indicated that while much of the caveolin-1-GFP in caveolae at the cell surface was relatively sessile, numerous, highly motile caveolin-1-GFP-positive vesicles were present within the cell interior. These vesicles moved at speeds ranging from 0.3-2 microm/second and movement was abolished when microtubules were depolymerized with nocodazole. In the absence of microtubules, cell surface invaginated caveolae increased more than twofold and they became organized into linear arrays. Complete depolymerization of the actin cytoskeleton with latrunculin A, by contrast, triggered rapid and massive movements of caveolin positive structures towards the centrosomal region of the cell. The caveolar membrane system of CHO cells therefore appears to be comprised of three caveolin 1-containing compartments. These include caveolae that are confined to the cell surface by cortical actin filaments, the peri-centrosomal caveosomes and caveolar vesicles, which we call 'cavicles', that move constitutively and bi-directionally along microtubules between the cell surface and caveosomes. The behavior of cavicles suggests that they function as transport intermediates between caveolae and caveosomes. PMID- 12376565 TI - Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock. AB - The intermediate filament cytoskeleton is thought to confer physical resilience on tissue cells, on the basis of extrapolations from the phenotype of cell fragility that results from mutations in skin keratins. There is a need for functional cell assays in which the impact of stress on intermediate filaments can be induced and analyzed. Using osmotic shock, we have induced cytoskeleton changes that suggest protective functions for actin and intermediate filament systems. Induction of the resulting stress response has been monitored in keratinocyte cells lines carrying K5 or K14 mutations, which are associated with varying severity of epidermolysis bullosa simplex. Cells with severe mutations were more sensitive to osmotic stress and took longer to recover from it. Their stress-activated response pathways were induced faster, as seen by early activation of JNK, ATF-2 and c-Jun. We demonstrate that the speed of a cell's response to hypotonic stress, by activation of the SAPK/JNK pathway, is correlated with the clinical severity of the mutation carried. The response to hypo-osmotic shock constitutes a discriminating stress assay to distinguish between the effects of different keratin mutations and is a potentially valuable tool in developing therapeutic strategies for keratin-based skin fragility disorders. PMID- 12376566 TI - Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level. AB - Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking alpha(v)beta(3) antibody, a function blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking alpha(3)beta(1) antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis. PMID- 12376567 TI - The role of CD34 expression and cellular fusion in the regeneration capacity of myogenic progenitor cells. AB - Characterization of myogenic subpopulations has traditionally been performed independently of their functional performance following transplantation. Using the preplate technique, which separates cells based on their variable adhesion characteristics, we investigated the use of cell surface proteins to potentially identify progenitors with enhanced regeneration capabilities. Based on previous studies, we used cell sorting to investigate stem cell antigen-1 (Sca-1) and CD34 expression on myogenic populations with late adhesion characteristics. We compared the regeneration efficiency of these sorted progenitors, as well as those displaying early adhesion characteristics, by quantifying their ability to regenerate skeletal muscle and restore dystrophin following transplantation into allogenic dystrophic host muscle. Identification and utilization of late adhering populations based on CD34 expression led to differential regeneration, with CD34 positive populations exhibiting significant improvements in dystrophin restoration compared with both their CD34-negative counterparts and early adhering cell populations. Regenerative capacity was found to correspond to the level of myogenic commitment, defined by myogenic regulatory factor expression, and the rate and degree of induced cell differentiation and fusion. These results demonstrate the ability to separate definable subpopulations of myogenic progenitors based on CD34 expression and reveal the potential implications of defining myogenic cell behavioral and phenotypic characteristics in relation to their regenerative capacity in vivo. PMID- 12376568 TI - An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin. AB - We identified a novel fission yeast gene, ned1(+), with pleiotropic mutations that have a high incidence of chromosome missegregation, aberrantly shaped nuclei, overdeveloped endoplasmic reticulum-like membranes, and increased sensitivity to a microtubule destabilizing agent. Ned1 protein, which was phosphorylated in a growth-related manner, interacted in a yeast two-hybrid system with Dis3 as well as with Pim1/RCC1 (nucleotide exchange factor for Ran). Ned1 also interacted with an essential nucleoporin, a probable homologue of mammalian Nup98/96. The ned1 gene displayed a variety of genetic interactions with factors involved in nuclear transport and chromosome segregation, including the crm1 (exportin), spi1 (small GTPase Ran), pim1, and dis genes. A substitution mutation that affected the two-hybrid interaction with Dis3 increased chromosome instability, suggesting the functional importance of the interaction. Overproduction of Ned1 protein induced formation of an abnormal microtubule bundle within the nucleus, apparently independently of the spindle pole body, but dependent on pim1(+) activity. The ned1(+) gene belongs to an evolutionarily conserved gene family, which includes the mouse Lpin genes, one of whose mutations is responsible for lipodystrophy. PMID- 12376570 TI - Proteomic analysis in the neurosciences. AB - Proteomics is a field of study directed toward providing a comprehensive view of the characteristics and activity of every cellular protein. Rapid innovations in the core technologies required to characterize proteins on a global scale are poised to bring about a comprehensive understanding of how dynamic changes in protein expression, post-translational modification, and function affect complex signaling and regulatory networks. These advances have significant implications for understanding the multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease. PMID- 12376571 TI - Roles for the two-hybrid system in exploration of the yeast protein interactome. AB - Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research. PMID- 12376572 TI - Mapping and structural dissection of human 20 S proteasome using proteomic approaches. AB - The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250). PMID- 12376573 TI - Dynamics of protein turnover, a missing dimension in proteomics. AB - Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species. PMID- 12376574 TI - Hydrogen/deuterium exchange and aggregation of a polyvaline and a polyleucine alpha-helix investigated by matrix-assisted laser desorption ionization mass spectrometry. AB - The membrane-associated pulmonary surfactant protein C (SP-C), containing a polyvaline alpha-helix, and a synthetic SP-C analogue with a polyleucine helix (SP-C(Leu)) were studied by hydrogen/deuterium exchange matrix-assisted laser desorption ionization (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), formed abundant amyloid fibrils under experimental conditions. In CD(3)OD/D(2)O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchanged 40% of their exchangeable hydrogens within 1 min. This corresponds to exchange of labile side-chain hydrogen atoms, hydrogens on the N- and C-terminal heteroatoms, and amide hydrogen atoms in the unstructured N-terminal regions. After approximately 300 h, four exchangeable hydrogen atoms in SP-C(Leu) and 10 in SP-C remained unexchanged. During this time period the ion current corresponding to singly charged SP-C decreased to <10% of the initial value due to the formation of insoluble aggregates that are not detected by MALDI mass spectrometry. In contrast, the ion current for SP-C(Leu) was maintained over this time period, although the peptides were incubated together. In combination, hydrogen/deuterium exchange and aggregation data indicate that the polyleucine peptide refolds into a helix after opening, while the unfolded polyvaline peptide forms insoluble beta sheet aggregates rather than refolding into a helix. The SP-C helix, but not the SP-C(Leu) helix, is thus in a metastable state, which may contribute to the recently observed tendency of SP-C and its precursor to misfold and aggregate in vivo. PMID- 12376575 TI - Hyperphosphorylated C-terminal repeat domain-associating proteins in the nuclear proteome link transcription to DNA/chromatin modification and RNA processing. AB - Using an interaction blot approach to search in the human nuclear proteome, we identified eight novel proteins that bind the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of RNA polymerase II. Unexpectedly, five of the new phosphoCTD-associating proteins (PCAPs) represent either enzymes that act on DNA and chromatin (topoisomerase I, DNA (cytosine-5) methyltransferase 1, poly(ADP ribose) polymerase-1) or proteins known to bind DNA (heterogeneous nuclear ribonucleoprotein (hnRNP) U/SAF-A, hnRNP D). The other three PCAPs represent factors involved in pre-mRNA metabolism as anticipated (CA150, NSAP1/hnRNP Q, hnRNP R) (note that hnRNP U/SAF-A and hnRNP D are also implicated in pre-mRNA metabolism). Identifying as PCAPs proteins involved in diverse DNA transactions suggests that the range of phosphoCTD functions extends far beyond just transcription and RNA processing. In view of the activities possessed by the DNA directed PCAPs, it is likely that the phosphoCTD plays important roles in genome integrity, epigenetic regulation, and potentially nuclear structure. We present a model in which the phosphoCTD association of the PCAPs poises them to act either on the nascent transcript or on the DNA/chromatin template. We propose that the phosphoCTD of elongating RNA polymerase II is a major organizer of nuclear functions. PMID- 12376576 TI - DXA: potential for creating a metabolic map of organ-tissue resting energy expenditure components. AB - OBJECTIVE: This study tested the hypothesis that tissue-organ components can be derived from DXA measurements, and in turn, resting energy expenditure (REE) can be calculated from the summed heat productions of DXA-estimated brain, skeletal muscle mass (SM), adipose tissue, bone, and residual mass (RM). RESEARCH METHODS AND PROCEDURES: Subjects were divided into five groups of adults <50 years of age. The specific metabolic rate of RM was developed in 13 Group I healthy subjects and a DXA-brain mass prediction formula in 52 Group II subjects. SM, adipose tissue, and bone models were developed based on earlier reports. The composite REE prediction model (REEp) was tested in 154 Group III subjects in whom REEp was compared with measured REE (REEm). Features of the developed model were determined in 94 normal-weight men and women (Group IV) and seven spinal cord injury patients and healthy matched controls (Group V). RESULTS: REEp and REEm in Group III were highly correlated (y = 0.85x + 233; r = 0.82, p < 0.001), and no bias was detected. Both REEm (mean +/- SD, 1,579 +/- 324 kcal/d) and REEp (1,585 +/- 316 kcal/d) were also highly correlated (r values = 0.85 to 0.98; p values < 0.001) and provided similar group values to REE estimated by the Harris Benedict equations (1,597 +/- 279 kcal/d) and Wang's composite fat-free mass based REE equation (1,547 +/- 248 kcal/d). New insights into the sources and distribution of REE were provided by analysis of the demonstration groups. DISCUSSION: This approach offers a new practical and educational opportunity to examine REE in subject groups using modeling strategies that reveal the magnitude and distribution of fundamental somatic heat-producing units. PMID- 12376578 TI - Dietary weight loss decreases serum angiotensin-converting enzyme activity in obese adults. AB - OBJECTIVE: To study the effect of dietary weight loss, postural change, and an oral glucose load on serum angiotensin-converting enzyme (ACE) activity in obese adults. RESEARCH METHODS AND PROCEDURES: Sixteen obese adult men and women with a mean body mass index of 35.7 +/- 4.3 kg/m(2) were studied after 1 week on a maintenance energy lead-in diet and after 5 weeks on an identical but 40% reduced energy diet provided by the General Clinical Research Center (GCRC). ACE activity was measured spectrophotometrically. Plasma renin activity and serum aldosterone were measured by radioimmunoassay. RESULTS: All subjects lost weight, with a mean decrease in body weight of 7.0 +/- 2.1 kg or 6 +/- 3% of initial body weight (p < 0.00001). Systolic and diastolic blood pressure, supine plasma renin activity, and serum aldosterone levels decreased with weight loss (p < 0.05). Supine ACE activity decreased 23 +/- 12% with weight loss (p < 0.00001). Standing ACE activity, which was significantly higher than supine ACE activity before and after weight loss (p < 0.05), also decreased 18 +/- 17% with weight loss (p = 0.0007). A 75-g oral glucose load had no effect on serum ACE activity over a 3 hour period. DISCUSSION: In obese adults, serum ACE activity declines with modest weight loss, increases with postural change, and is unaffected by an oral glucose load. PMID- 12376577 TI - Effect of changes in fat distribution on the rates of change of insulin response in children. AB - OBJECTIVE: To develop mixed models for examining longitudinal associations between rates of change in visceral, subcutaneous abdominal, and total body fat with rates of change in fasting insulin (FI) and insulin sensitivity (SI) over 3 years in children. RESEARCH METHODS AND PROCEDURES: Seventy-seven children (mean age, 8.3 years at baseline) from Birmingham, Alabama, with three or more annual measures of FI and SI were included. Abdominal fat was measured by computed tomography, and total body fat and lean tissue mass were measured by DXA. Mixed models examined the longitudinal associations between the baseline level/rate of change of different fat compartments and the rate of change in FI or SI. RESULTS: An annual increase of approximately 5% in FI was associated with 1 cm(2)/yr of visceral fat gain per year (p < 0.05), independent of subcutaneous abdominal fat. A 1-cm(2) difference in initial subcutaneous abdominal fat was associated with an approximately 0.2% increase per year in FI (p < 0.02), independent of visceral fat. None of the rates of change in any of the fat measures was associated with the rate of change of SI. DISCUSSION: The rate of change in visceral fat was positively associated with the rate of change in FI, independent of increasing subcutaneous abdominal fat; however, subcutaneous abdominal fat may be more predictive of the rate of change of FI than visceral or total fat. Therefore, growth-related increases in abdominal fat, particularly subcutaneous abdominal fat, may contribute to accelerating increases in FI, but have no effect on SI. PMID- 12376579 TI - Hypophysectomy prevents ghrelin-induced adiposity and increases gastric ghrelin secretion in rats. AB - OBJECTIVE: The novel gastric hormone ghrelin has recently been identified as an important modulator of energy homeostasis. Leptin-responsive hypothalamic neuropeptide Y/Agouti-related protein neurons are believed to mediate afferent ghrelin signals. Little is known, however, about ghrelin-induced efferent signals. We therefore investigated if hypothalamic-pituitary axes have a role in transferring ghrelin-induced changes of energy balance to the periphery. RESEARCH METHODS AND PROCEDURES: We subcutaneously injected hypophysectomized, as well as adrenalectomized, thyroidectomized, and sham-operated control rats with GH secretagogues [ghrelin, growth hormone (GH)-releasing peptide] for 1 week. Body weight, food intake, and body composition (chemical carcass analysis) were analyzed and compared with vehicle-treated controls. In addition, we quantified circulating levels of endogenous ghrelin in hypophysectomized and GH-treated normal rats. RESULTS: GH-secretagogue treatment of sham-operated control rats dose-proportionally increased food intake, body weight, and fat mass compared with vehicle-injected controls (p < 0.01). These effects, however, were not observed in ghrelin-treated hypophysectomized, thyroidectomized, or adrenalectomized rats, indicating an essential role for the pituitary axis in ghrelin-induced adiposity. Circulating levels of endogenous ghrelin were reduced by administration of GH in normal rats and were about 3-fold higher in hypophysectomized rats (n = 20, p = 0.001), suggesting a regulatory feedback loop involving the stomach and the pituitary to regulate gastric ghrelin secretion. DISCUSSION: According to these results, the endocrine pituitary is mediating ghrelin-induced changes toward a positive energy balance and is involved in the regulation of ghrelin secretion through a gastro-hypophyseal feedback loop. PMID- 12376580 TI - Impact of body mass index on changes in common carotid artery wall thickness. AB - OBJECTIVE: To examine associations between changes in body mass index (BMI) and changes in carotid artery intima-media thickness (IMT) in a community-based sample. RESEARCH METHODS AND PROCEDURES: Carotid artery IMT and BMI were assessed at baseline (between 1987 and 1990) and in three subsequent examinations at 3 year intervals in participants in the Atherosclerosis Risk in Communities cohort. The 9,316 African-American and white men and women in the analysis were 45 to 64 years of age at baseline. Cross-sectional associations between BMI and IMT were assessed using general linear models. Longitudinal associations were examined using mixed models analysis. RESULTS: Cross-sectional associations between BMI and IMT were confirmed. At baseline, a 1-kg/m(2) increase in BMI was associated with an increase in IMT that ranged from 2.5 to 7.5 micro m among the ethnic gender groups examined. Changes in BMI were not associated with changes in IMT in models that adjusted for aging and other covariates. Results were similar across ethnic-gender groups. DISCUSSION: Among free-living, 45- to 64-year-old adults, changes in common carotid artery IMT associated with changes in BMI are either very small or absent. PMID- 12376581 TI - Effect of rosiglitazone on insulin sensitivity and body composition in type 2 diabetic patients [corrected]. AB - OBJECTIVE: To investigate the effects of rosiglitazone (RSG) on insulin sensitivity and regional adiposity (including intrahepatic fat) in patients with type 2 diabetes. RESEARCH METHODS AND PROCEDURES: We examined the effect of RSG (8 mg/day, 2 divided doses) compared with placebo on insulin sensitivity and body composition in 33 type 2 diabetic patients. Measurements of insulin sensitivity (euglycemic hyperinsulinemic clamp), body fat (abdominal magnetic resonance imaging and DXA), and liver fat (magnetic resonance spectroscopy) were taken at baseline and repeated after 16 weeks of treatment. RESULTS: There was a significant improvement in glycemic control (glycosylated hemoglobin -0.7 +/- 0.7%, p < or = 0.05) and an 86% increase in insulin sensitivity in the RSG group (glucose-disposal rate change from baseline: 17.5 +/- 14.5 micro mol glucose/min/kg free fat mass, p < 0.05), but no significant change in the placebo group compared with baseline. Total body weight and fat mass increased (p < or = 0.05) with RSG (2.1 +/- 2.0 kg and 1.4 +/- 1.6 kg, respectively) with 95% of the increase in adiposity occurring in nonabdominal regions. In the abdominal region, RSG increased subcutaneous fat area by 8% (25.0 +/- 28.7 cm(2), p = 0.02), did not alter intra-abdominal fat area, and reduced intrahepatic fat levels by 45% ( 6.7 +/- 9.7%, concentration relative to water). DISCUSSION: Our data indicate that RSG greatly improves insulin sensitivity in patients with type 2 diabetes and is associated with an increase in adiposity in subcutaneous but not visceral body regions. PMID- 12376582 TI - Plasma adiponectin levels are not associated with fat oxidation in humans. AB - OBJECTIVE: To test the hypothesis that low adiponectin is associated with low fat oxidation in humans. RESEARCH METHODS AND PROCEDURES: We measured plasma adiponectin concentrations in 75 healthy, nondiabetic Pima Indians (age, 28 +/- 7 years; 55 men and 20 women; body fat, 29.7 +/- 7.5%) and 18 whites [(age, 33 +/- 8 years; 14 men and 4 women; body fat, 28.2 +/- 10.8% (means +/- SD)] whose body composition was measured by DXA and 24-hour energy expenditure (24-hour EE) by a respiratory chamber. Respiratory quotient (an estimate of whole-body carbohydrate/lipid oxidation rate) was calculated over 24 hours (24-hour RQ). RESULTS: Before correlational analyses, waist-to-thigh ratio (WTR) and percentage of body fat (PFAT) were adjusted for age, sex, and race; 24-hour EE was adjusted for fat mass and fat-free mass, and 24-hour RQ were adjusted for energy balance. Plasma adiponectin concentrations were negatively correlated with WTR (r = -0.42, p < 0.0001) and PFAT (r = -0.46, p < 0.0001). There was no correlation between plasma adiponectin concentrations and 24-hour RQ, (r = 0.09, p = 0.36) before or after adjustment for PFAT (r = 0.001, p = 0.99, respectively, partial correlation), and no correlation was found between plasma adiponectin concentrations and 24-hour EE (r = -0.12, p = 0.27). DISCUSSION: Our cross sectional data do not suggest physiological concentrations of fasting plasma adiponectin play a role in the regulation of whole-body fat oxidation or energy expenditure in resting conditions. Whether administration of adiponectin to individuals with low levels of this hormone will increase their fat oxidation rates/energy expenditure remains to be established. PMID- 12376583 TI - Binge size increases with body mass index in women with binge-eating disorder. AB - OBJECTIVE: To determine whether meal size is related to body mass index (BMI) in obese subjects with binge-eating disorder (BED). RESEARCH METHODS AND PROCEDURES: Five groups of subjects each consumed two laboratory-test meals on nonconsecutive days. Forty-two women, categorized by BMI and BED diagnosis, were instructed to "binge" during one meal and to eat "normally" during another. Eighteen women had BMI values >38 kg/m(2) (more-obese) and 17 had BMI values between 28 to 32 kg/m(2) (less-obese). Twelve of the more-obese and nine of the less-obese individuals met Diagnostic and Statistical Manual (DSM)-IV criteria for BED. Seven normal-weight women also participated as controls. RESULTS: Subjects with BED ate significantly more in both meals than subjects without BED. Binge meals were significantly larger than normal meals only among subjects with BED. The more-obese subjects with BED ate significantly more than the less-obese subjects with BED, but only when they were asked to binge. Intake of the binge meal was significantly, positively correlated with BMI among subjects with BED. Subjects with BED reported significantly higher satiety ratings after the binge than after the normal meal, but subjects without BED reported similar ratings after both meals. Regardless of instructions and diagnosis, obese subjects consumed a significantly higher percentage of energy from fat (38.5%) than did normal-weight subjects (30.8%). DISCUSSION: During binge meals, the energy intake of subjects with BED is greater than that of individuals of similar body weight without BED and is positively correlated with BMI. PMID- 12376584 TI - Physical activity and the metabolic syndrome in a tri-ethnic sample of women. AB - OBJECTIVE: To determine the association of moderate-intensity physical activity (PA), vigorous-intensity PA, and maximal treadmill duration with the metabolic syndrome among African-American (n = 49), Native-American (n = 46), and white (n = 51) women (ages, 40 to 83 years), enrolled in the Cross-Cultural Activity Participation Study. RESEARCH METHODS AND PROCEDURES: The metabolic syndrome was defined as three or more of the following risk factors: waist circumference >88 cm, blood pressure > or =130/85 mm Hg, fasting glucose > or =110 mg/dL, hypertriglyceridemia (> or =150 mg/dL), and high-density lipoprotein-cholesterol <50 mg/dL. PA was determined from detailed PA records that included all PA performed during two consecutive 4-day periods. Maximal treadmill duration was determined from a graded exercise test. Women were categorized into quartiles of moderate-intensity PA, vigorous-intensity PA, and maximal treadmill duration. Multiple logistic regression was used to estimate odds ratios of the metabolic syndrome as a function of the four PA categories, adjusted for age, ethnicity, study site, menopausal status, and use of hormone-replacement therapy. RESULTS: The adjusted odds ratio for the metabolic syndrome was 0.18 (95% confidence interval, 0.33 to 0.90) for women in the highest category of moderate-intensity PA compared with women in the lowest category (p = 0.01 for trend). Similar associations were observed for the metabolic syndrome with vigorous-intensity PA (p = 0.01 for trend) and maximal treadmill duration (p = 0.0004 for trend). DISCUSSION: Higher levels of moderate and vigorous-intensity PA and greater maximal treadmill duration were inversely associated with the metabolic syndrome among an ethnically diverse sample of women. PMID- 12376585 TI - Obesity in South Africa: the South African demographic and health survey. AB - OBJECTIVES: To ascertain the anthropometric profile and determinants of obesity in South Africans who participated in the Demographic and Health Survey in 1998. RESEARCH METHODS AND PROCEDURES: A sample of 13,089 men and women (age, > or =15 years) were randomly selected and then stratified by province and urban and nonurban areas. Height, weight, mid-upper arm circumference, and waist and hip circumference were measured. Body mass index (BMI) was used as an indicator of obesity, and the waist/hip ratio (WHR) was used as an indicator of abdominal obesity. Multivariate regression identified sociodemographic predictors of BMI and waist circumference in the data. RESULTS: Mean BMI values for men and women were 22.9 kg/m(2) and 27.1 kg/m(2), respectively. For men, 29.2% were overweight or obese (> or =25 kg/m(2)) and 9.2% had abdominal obesity (WHR > or =1.0), whereas 56.6% of women were overweight or obese and 42% had abdominal obesity (WHR >0.85). Underweight (BMI <18.5 kg/m(2)) was found in 12.2% of men and 5.6% of women. For men, 19% of the variation of BMI and 34% of the variation in waist circumference could be explained by age, level of education, population group, and area of residence. For women, these variables explained 16% of the variation of BMI and 24% of the variation in waist circumference. Obesity increased with age, and higher levels of obesity were found in urban African women. DISCUSSION: Overnutrition is prevalent among adult South Africans, particularly women. Determinants of overnutrition include age, level of education, ethnicity, and area of residence. PMID- 12376586 TI - Bupropion SR vs. placebo for weight loss in obese patients with depressive symptoms. AB - OBJECTIVE: This randomized, double-blind, placebo-controlled study evaluated the efficacy and tolerability of bupropion sustained-release (bupropion SR) in reducing weight and depressive symptoms in obese adults. RESEARCH METHODS AND PROCEDURES: Obese adults (body mass index, 30 to 44 kg/m(2)) not currently meeting criteria for major depression but with depressive symptoms (Beck Depression Inventory score 10-30) received bupropion SR 300 mg/d or placebo for 26 weeks with a 500 kcal/d-deficit diet. Patients who lost <5% of baseline weight at week 12 had bupropion SR dosage or placebo increased to 400 mg/d in a blinded fashion. RESULTS: The bupropion SR group (n = 193) lost an average of 4.4 kg (4.6% of baseline weight) vs. 1.7 kg (1.8% of baseline weight) on placebo (n = 191, p < 0.001, last-observation-carried-forward analysis). More patients in the bupropion SR group than in the placebo group (40% vs. 16% of intent-to-treat sample, 50% vs. 28% of completers, respectively) lost at least 5% of baseline weight (p < 0.05 at week 4, p < 0.001 at weeks 6 to 26). The percentage of patients reporting > or =50% decrease in depressive symptoms did not differ between groups, but depressive symptoms improved more with bupropion SR than with placebo among patients with a history of major depression (p < 0.05, weeks 4 to 26). In the sample as a whole, improvement in depressive symptoms was related to weight loss of > or =5% regardless of treatment (p < 0.0001). Bupropion SR was well-tolerated. DISCUSSION: Bupropion SR in combination with a 500 kcal/d-deficit diet facilitated weight loss. Weight loss of > or =5% may improve mood in obese patients with depressive symptoms. PMID- 12376587 TI - Relationship between obesity and health-related quality of life in men. AB - OBJECTIVE: Few studies examining the relationship between obesity and health related quality of life (HRQOL) have used a medical outpatient population or demonstrated a relationship in men. Furthermore, most studies have not adequately considered comorbid illness. The goal of this study was to examine the relationship between body mass index (BMI) and HRQOL in male outpatients while considering comorbid illness. RESEARCH METHODS AND PROCEDURES: This cross sectional study examined 1168 male outpatients from Durham Veterans' Affairs Medical Center. Multiple linear regression was used to examine the relationship of BMI with each subscale from the Medical Outcomes Study Short Form 36 while adjusting for age, race, comorbid illness, depression, and physical activity. RESULTS: Participants had a mean age of 54.7 +/- 5.6 years; 69% were white and 29% were African American. The distribution for BMI was as follows: 18.5 to <25 kg/m(2) (21%), 25 to <30 kg/m(2) (43%), 30 to <35 kg/m(2) (25%), 35 to <40 kg/m(2) (8%), and > or =40 kg/m(2) (3%). Mean Short Form 36 subscale scores were lower than U.S. norms by an average of 27%. Individuals with BMI > or =40 kg/m(2) had significantly lower scores compared with normal weight individuals on the Role-Physical and Vitality subscales. On the Physical Functioning and Physical Component subscales, lower scores were observed at BMI > or =35 kg/m(2). On the Bodily Pain subscale, lower scores were observed at BMI > or =25 kg/m(2). DISCUSSION: An inverse relationship between BMI and physical aspects of HRQOL exists in a population of male outpatients. Increased BMI was most prominently associated with bodily pain; this relationship should receive more attention in clinical care and research. PMID- 12376588 TI - Association of physical activity and visceral adipose tissue in older women and men. AB - OBJECTIVE: Physical inactivity, abdominal fat, and age are known risk factors for diabetes, cardiovascular disease, and certain cancers. Previous evidence supports an inverse relationship between physical activity (PA) and abdominal fat estimated by waist circumference. However, few investigations used computed tomography (CAT) scanning for precise measures of abdominal fat. RESEARCH METHODS AND PROCEDURES: Sixty-five female and 106 male (age, 64.5 +/- 5.2 years) participants in the Prostate, Lung, Colon and Ovarian Cancer Screening Trial underwent a cross-sectional L4-L5 CAT scan to differentiate visceral adipose tissue (VAT). Subjects were also interviewed by phone to determine PA and physical difficulties (PD). RESULTS: Women had lower VAT (170 +/- 84 vs. 205 +/- 95 cm(2), p = 0.014), lower VAT/total fat (29.9 +/- 7.2% vs. 42.6 +/- 10.2%, p < 0.001), and higher total fat (596 +/- 385 vs. 482 +/- 183 cm(2), p = 0.010) than men. PA was inversely correlated to VAT (r = -0.164, p = 0.034) and total fat (r = -0.231, p = 0.003) in men and women. Those who reported a PD had higher VAT (249 vs. 180 cm(2), p < 0.001) and total fat (652 vs. 500 cm(2), p = 0.008). Multiple regression analysis indicated total PA and PD were independently associated to VAT and total fat. DISCUSSION: This investigation suggests a beneficial effect of PA and a negative influence of PD on abdominal fat accumulation. Although the cross-sectional design limits cause-effect designations, these results are consistent with other studies showing PA/abdominal fat relation. PMID- 12376589 TI - Congenic BB.SHR (D4Mit6-Npy-Spr) rats: a new aid to dissect the genetics of obesity. AB - OBJECTIVE: The phenotypic characterization of congenic BB.LL rats recombining a segment of the SHR chromosome 4 (D4Mit6-Npy-Spr; 12 cM) into the BB/OK background indicated that these rats were not lymphopenic and did not develop diabetes, but they were significantly heavier (at 16 weeks of age) and showed higher serum triglycerides and total cholesterol concentration. RESEARCH METHODS AND PROCEDURES: BB.LL rats were longitudinally studied for facets of metabolic syndrome (body mass index, blood glucose, serum lipids, insulin, leptin, and systolic and diastolic blood pressure) from 2 to 12 months of age. RESULTS: In this study, it was shown that BB.LL are obese, hyperleptinemic, hyperinsulinemic, and dyslipidemic compared with their parental BB/OK rats. DISCUSSION: It can be concluded that there is a gene(s) in the introgressed segment causing incomplete metabolic syndrome, because they do not develop hypertension and diabetes. To identify the gene(s), the introgressed chromosomal segment must be systematically whittled down to generate recombinants and new subcongenic lines carrying a much smaller segment of the SHR/Mol rat to increase the chance of identification of the appropriate gene(s). PMID- 12376590 TI - The glucocorticoid receptor gene and its association to metabolic syndrome. AB - In recent decades, there has been an increasing interest in the role of endogenous glucocorticoids such as cortisol in the pathogenesis of metabolic syndrome. Studies in humans have suggested a positive association between obesity, hypertension, and insulin resistance, with alleles at the glucocorticoid receptor (GR) gene. For instance, the BclI polymorphism within the intron upstream of GR exon 2 has been associated with cardiovascular risk factors such as visceral obesity, hypertension, insulin resistance, and elevated cortisol concentrations. However, the location of the BclI polymorphism is not known, and the variant has so far not been compared with the wild-type receptor for its ability to be activated by glucocorticoids. Although several other mutations in the GR gene have been postulated as being relevant to the progression to type 2 diabetes and cardiovascular diseases, conflicting results makes it difficult to determine exactly what effect these GR variations have on metabolic syndrome incidence and progression. Further studies focusing on the most compelling GR mutations might offer a better understanding of metabolic syndrome pathogenesis and progression, aiding in the development of more effective treatments for this condition. PMID- 12376591 TI - Amylin, food intake, and obesity. AB - Amylin, also known as islet amyloid polypeptide, identified in 1987, is a naturally occurring hormone, released by the beta cells of the pancreas and consists of 37 amino acids. Amylin seems to decrease food intake through both central and peripheral mechanisms and indirectly by slowing gastric emptying. The mean basal amylin concentration is higher in obese than in lean human subjects. The amylin response to oral glucose is also greater in obese subjects, whether or not they have impaired glucose tolerance. The elevated amylin levels in obesity may lead to down-regulation of amylin receptors and lessen the impact of postprandial amylin secretion on satiety and gastric emptying. Amylin administration may overcome resistance at target tissues, delay gastric emptying, and have potential for inducing weight loss in obese individuals. PMID- 12376592 TI - Why don't smokers show an increased mortality risk with overweight? PMID- 12376593 TI - Spatial and temporal offsets between proxy records in a sediment drift. AB - Chronologies for Late Quaternary marine sediment records are usually based on radiocarbon ages of planktonic foraminifera. Signals carried by other sedimentary components measured in parallel can provide complementary paleoclimate information. A key premise is that microfossils and other indicators within a given sediment horizon are of equal age. We show here that haptophyte-derived alkenones isolated from Bermuda Rise drift sediments are up to 7000 years older than coexisting planktonic foraminifera. This temporal offset, which is apparently due to lateral transport of alkenones on fine-grained particles from the Nova Scotian margin, markedly influences molecular estimates of sea surface temperatures. More broadly, the observation raises questions about both the temporal and the geographic fidelity of paleoenvironmental records encoded by readily transported components of sediments. PMID- 12376594 TI - Projection of an immunological self shadow within the thymus by the aire protein. AB - Humans expressing a defective form of the transcription factor AIRE (autoimmune regulator) develop multiorgan autoimmune disease. We used aire- deficient mice to test the hypothesis that this transcription factor regulates autoimmunity by promoting the ectopic expression of peripheral tissue- restricted antigens in medullary epithelial cells of the thymus. This hypothesis proved correct. The mutant animals exhibited a defined profile of autoimmune diseases that depended on the absence of aire in stromal cells of the thymus. Aire-deficient thymic medullary epithelial cells showed a specific reduction in ectopic transcription of genes encoding peripheral antigens. These findings highlight the importance of thymically imposed "central" tolerance in controlling autoimmunity. PMID- 12376595 TI - Transition state stabilization by a catalytic RNA. AB - The hairpin ribozyme catalyzes sequence-specific cleavage of RNA through transesterification of the scissile phosphate. Vanadate has previously been used as a transition state mimic of protein enzymes that catalyze the same reaction. Comparison of the 2.2 angstrom resolution structure of a vanadate-hairpin ribozyme complex with structures of precursor and product complexes reveals a rigid active site that makes more hydrogen bonds to the transition state than to the precursor or product. Because of the paucity of RNA functional groups capable of general acid-base or electrostatic catalysis, transition state stabilization is likely to be an important catalytic strategy for ribozymes. PMID- 12376597 TI - Combined CT venography and pulmonary angiography: a comprehensive review. AB - The combination of computed tomographic (CT) venography and pulmonary angiography (CTVPA) was initially described in 1998 as a single comprehensive noninvasive imaging examination for suspected thromboembolic disease. It allowed the identification of pulmonary embolism as well as deep venous thrombosis (DVT) in the abdomen, pelvis, thighs, and calves. The venographic portion of CTVPA has now been studied by multiple researchers and has been shown to be an accurate imaging study for the thigh veins in comparison with lower extremity sonography. In contrast to sonography, however, CTVPA readily and rapidly permits evaluation of the inferior vena cava, the pelvic veins, the calf veins, and all of the superficial venous system. Complex venous anatomy can be surveyed, an additional sonographic study is not required, and only a few extra minutes and images are required over and above CT pulmonary angiography. A review of 957 recent cases of suspected pulmonary embolism examined with CTVPA revealed an overall 10.5% frequency of DVT, with a nearly equal distribution of thrombosis at the common femoral, superficial femoral, popliteal, and deep calf veins. Although a variety of protocols for CTVPA may be implemented, including a contiguous helical acquisition, obtaining 5- or 10-mm-thick images every 4 cm provides a high degree of accuracy and decreases overall radiation dose. PMID- 12376599 TI - Developmental lung anomalies in the adult: radiologic-pathologic correlation. AB - Encountering a developmental lung anomaly in the adult can be a challenge, as the abnormality may be mistaken for something more sinister. The common anomalies encountered are classified into three broad categories: bronchopulmonary (lung bud) anomalies, vascular anomalies, and combined lung and vascular anomalies. The imaging features of these developmental anomalies at conventional radiography, ventilation-perfusion lung nuclear scanning, angiography, computed tomography, and magnetic resonance imaging are useful in differential diagnosis of thoracic lesions. Lung bud anomalies include agenesis, congenital bronchial atresia, congenital lobar emphysema, congenital cystic adenomatoid malformation, pulmonary bronchogenic cysts, tracheal or pig bronchus, and accessory cardiac bronchus. Vascular anomalies include interruption or absence of a main pulmonary artery, anomalous origin of the left pulmonary artery from the right, anomalous pulmonary venous drainage (partial or complete), and pulmonary arteriovenous malformation. Combined lung and vascular anomalies include the hypogenetic lung (scimitar) syndrome and bronchopulmonary sequestration, both intralobar and extralobar. PMID- 12376600 TI - Multi-detector row and volume-rendered CT of the normal and accessory flow pathways of the thoracic systemic and pulmonary veins. AB - Multi-detector row computed tomography (CT) and volume rendering can be used as an interpretive aid to present the systemic and pulmonary venous anatomy of the thorax. Both of these venous systems are routinely imaged in clinical practice and are important in interpretation of diagnostic images in health and disease. Multi-detector row CT and three-dimensional volume rendering provide high-quality near-isotropic data (ie, the longitudinal resolution approximates the in-plane resolution). The data sets allow tailored postprocessing to produce images optimized for these vessels, which are often not fully appreciated at planar axial imaging alone. Venous structures of the thorax that can be demonstrated with multi-detector row CT and volume rendering include the jugular veins; the subclavian and brachiocephalic veins; the internal and lateral thoracic veins; the superior and inferior venae cavae; the coronary sinus, the cardiac and pericardiophrenic veins, and vein grafts; the azygos, hemiazygos, and accessory hemiazygos veins; the intercostal veins; the pulmonary veins; and other thoracic veins. PMID- 12376601 TI - Fat-containing lesions of the chest. AB - Although most lesions that occur in the chest have a nonspecific soft-tissue appearance, fat-containing lesions are occasionally encountered at cross sectional computed tomography (CT) or magnetic resonance imaging. The various fat containing lesions of the chest include parenchymal and endobronchial lesions such as hamartoma, lipoid pneumonia, and lipoma. Endobronchial hamartoma usually appears at CT as a lesion with a smooth edge, focal collections of fat, or fat collections that alternate with foci of calcification. Mediastinal fat-containing lesions include germ cell neoplasms, thymolipomas, lipomas, and liposarcomas. The most frequent CT manifestation of the germ cell neoplasm teratoma is a heterogeneous mass with soft-tissue, fluid, fat, and calcium attenuation. Cardiac lesions with fat content include lipomatous hypertrophy of the interatrial septum and arrhythmogenic right ventricular dysplasia. Diagnosis of the former is made with CT when a smooth, nonenhancing, well-marginated fat-containing lesion is identified in the interatrial septum. Finally, fat may herniate into the chest at several characteristic locations. When such a lesion is identified, the time required for differential diagnosis is significantly reduced, often allowing a definitive radiologic diagnosis. Sagittal and coronal reformatted images can add valuable information by showing diaphragmatic defects and hernia contents. PMID- 12376602 TI - Imaging of cystic masses of the mediastinum. AB - Cystic masses of the mediastinum are well-marginated round lesions that contain fluid and are lined with epithelium. Major cystic masses include congenital benign cysts (ie, bronchogenic, esophageal duplication, neurenteric, pericardial, and thymic cysts), meningocele, mature cystic teratoma, and lymphangioma. Many tumors (eg, thymomas, Hodgkin disease, germ cell tumors, mediastinal carcinomas, metastases to lymph nodes, nerve root tumors) can undergo cystic degeneration especially after radiation therapy or chemotherapy-and demonstrate mixed solid and cystic elements at computed tomography (CT) or magnetic resonance (MR) imaging. If degeneration is extensive, such tumors may be virtually indistinguishable from congenital cysts. A mediastinal abscess or pancreatic pseudocyst also appears as a fluid-containing mediastinal cystic mass. However, clinical history and manifestations, anatomic position, and certain details seen at CT or MR imaging allow correct diagnosis in many cases. Familiarity with the radiologic features of mediastinal cystic masses facilitates accurate diagnosis, differentiation from other cystlike lesions, and, thus, optimal patient treatment. PMID- 12376603 TI - Best cases from the AFIP: thymoma. PMID- 12376604 TI - Imaging of diaphragmatic injury: a diagnostic challenge? AB - Diaphragmatic injuries occur in 0.8%-8% of patients after blunt trauma. Although the diagnosis may be obvious at standard chest radiography or computed tomography (CT) in most situations, some more subtle signs require careful analysis of CT images and examination with magnetic resonance (MR) imaging in some specific situations. Each method of imaging evaluation has advantages and pitfalls according to the type of diaphragmatic rupture. MR imaging with breath-hold acquisition permits good visualization of diaphragmatic abnormalities, but this technique cannot be performed in emergency situations. Because of a dramatic reduction in motion and beam-hardening artifacts and significant improvement of spatial resolution, especially along the z axis, helical CT and multisection CT allow better demonstration of the most subtle signs, such as a focal indentation of the liver or a right-sided collar sign. In addition, helical CT and multisection CT are useful tools in the evaluation of patients with multiple traumatic injuries. PMID- 12376606 TI - Thoracic complications of illicit drug use: an organ system approach. AB - Illicit drug use constitutes a major health problem and may be associated with various thoracic complications. These complications vary depending on the specific drug used and the route of administration. Commonly abused drugs that may play a role in causing thoracic disease include cocaine, opiates, and methamphetamine derivatives. Intravenously abused oral medications may contain filler agents that may be responsible for disease. Thoracic complications may be categorized as pulmonary, pleural, mediastinal, cardiovascular, and chest wall complications. Pulmonary complications of drug abuse include pneumonia, cardiogenic edema, acute lung injury, pulmonary hemorrhage, and aspiration pneumonia. Filler agents such as talc may result in panacinar emphysema or high attenuation upper-lobe conglomerate masses. The primary pleural complication of illicit drug use is pneumothorax. Mediastinal and cardiovascular complications of illicit drug use include pneumomediastinum, cardiomyopathy, myocardial infarction, aortic dissection, and injection-related pseudoaneurysms. Chest wall complications include diskitis and vertebral osteomyelitis, epidural abscess, necrotizing fasciitis, costochondritis, and septic arthritis. Categorization of thoracic complications of illicit drug use may facilitate understanding of these disorders and allow accurate diagnosis. PMID- 12376607 TI - Viral pneumonias in adults: radiologic and pathologic findings. AB - Numerous viruses, including influenza virus, measles virus, Hantavirus, adenovirus, herpesviruses, varicella-zoster virus, cytomegalovirus, and Epstein Barr virus, can cause lower respiratory tract infection in adults. Viral pneumonia in adults can be classified into two clinical groups: so-called atypical pneumonia in otherwise healthy hosts and viral pneumonia in immunocompromised hosts. Influenza virus types A and B cause most cases of viral pneumonia in immunocompetent adults. Immunocompromised hosts are susceptible to pneumonias caused by cytomegalovirus, herpesviruses, measles virus, and adenovirus. The radiographic findings, which consist mainly of patchy or diffuse ground-glass opacity with or without consolidation and reticular areas of increased opacity, are variable and overlapping. Computed tomographic findings, which are also overlapping, consist of poorly defined centrilobular nodules, ground-glass attenuation with a lobular distribution, segmental consolidation, or diffuse ground-glass attenuation with thickened interlobular septa. The radiologic findings reflect the variable extents of the histopathologic features: diffuse alveolar damage (intraalveolar edema, fibrin, and variable cellular infiltrates with a hyaline membrane), intraalveolar hemorrhage, and interstitial (intrapulmonary or airway) inflammatory cell infiltration. Clinical information such as patient age, immune status, community outbreaks, symptom onset and duration, and presence of a rash remain important aids in diagnosis of viral causes. PMID- 12376608 TI - Interstitial lung diseases associated with collagen vascular diseases: radiologic and histopathologic findings. AB - Collagen vascular diseases that demonstrate features of interstitial lung disease include systemic lupus erythematosus, rheumatoid arthritis, progressive systemic sclerosis, dermatomyositis and polymyositis, ankylosing spondylitis, Sjogren syndrome, and mixed connective tissue disease. At histopathologic analysis, interstitial lung diseases associated with collagen vascular diseases are diverse and include nonspecific interstitial pneumonia, usual interstitial pneumonia, bronchiolitis obliterans organizing pneumonia (BOOP), apical fibrosis, diffuse alveolar damage, and lymphocytic interstitial pneumonia. Although proportions of interstitial pneumonias vary, nonspecific interstitial pneumonia accounts for a large proportion, especially in progressive systemic sclerosis, dermatomyositis and polymyositis, and mixed connective tissue disease. The more favorable prognosis in interstitial pneumonia associated with collagen vascular diseases than in idiopathic interstitial pneumonias may be explained by the larger proportion of nonspecific interstitial pneumonia than of usual interstitial pneumonia. High-resolution computed tomography seems to help characterize and determine the extent of interstitial lung disease in collagen vascular diseases. PMID- 12376609 TI - Asbestos: when the dust settles an imaging review of asbestos-related disease. AB - Asbestos-related neoplastic and nonneoplastic diseases of the lungs and pleura range from pleural effusion and pleural plaques to lung cancer and malignant mesothelioma. Pleural effusions are typically hemorrhagic exudates of mixed cellularity but do not typically contain asbestos bodies. The classic distribution of pleural plaques seen on chest radiographs is the posterolateral chest wall between the seventh and tenth ribs, lateral chest wall between the sixth and ninth ribs, the dome of the diaphragm, and the mediastinal pleura. Computed tomographic (CT) findings support this distribution but also show anterior and paravertebral plaques not well shown at chest radiography. Imaging features of diffuse pleural thickening include a continuous sheet, often involving the costophrenic angles and apices, that rarely calcifies. The typical CT features of round atelectasis are of a round or oval mass that abuts the pleura, a "comet tail" of bronchovascular structures going into the mass, and thickening of the adjacent pleura. Features of asbestosis on chest radiographs include ground-glass opacification, small nodular opacities, "shaggy" cardiac silhouette, and ill-defined diaphragmatic contours. CT, however, is more sensitive in their detection. Chest radiography in patients with malignant mesothelioma may show an effusion, pleural thickening, and as the tumor progresses, a more lobulated outline. CT can help identify the disease in its early stages. Asbestos-related cancers can occur anywhere in the lungs. Recognition of the clinical, radiologic, and pathologic features of these diseases will be important for some years to come. PMID- 12376610 TI - Lymphangioleiomyomatosis: pulmonary and abdominal findings with pathologic correlation. AB - Lymphangioleiomyomatosis (LAM) is a rare disease characterized by pulmonary cysts at computed tomography (CT) and proliferation of abnormal smooth muscle cells at lung biopsy. Almost all patients are female, and all have pulmonary cysts at high resolution CT. Although the presence of cysts may be suggested at conventional CT or chest radiography, high-resolution CT is superior for cyst detection and is essential for diagnosis. The cysts are typically round; in most cases, the cyst wall is barely seen at thin-section CT. They are typically diffusely distributed throughout the central and peripheral lung parenchyma. The lung bases are affected in all patients. Some patients also have increased lung attenuation or a reticular pattern. Expiratory CT shows no air trapping between the cysts, and most of the cysts decrease in size. Pneumothorax, pleural effusion, and chylothorax are complications of LAM. Certain abdominal findings may provide additional corroborative evidence of the diagnosis. Renal angiomyolipomas, the most frequent abdominal lesions, usually manifest as asymptomatic, small, bilateral tumors of fat attenuation in the renal cortex. Lymphangiomas are cystic retroperitoneal masses that occur in up to 20% of patients. Other CT findings are hypo- or hyperattenuating lymph nodes, a dilated thoracic duct, and ascites. PMID- 12376611 TI - Broncholithiasis: review of the causes with radiologic-pathologic correlation. AB - Broncholithiasis is defined as a condition in which calcified or ossified material is present within the bronchial lumen. Radiographic findings of broncholithiasis include airway obstruction such as atelectasis, mucoid impaction, bronchiectasis, and expiratory air trapping. Broncholithiasis is strongly suggested at computed tomography (CT) when an endobronchial or peribronchial calcified nodule is associated with findings of bronchial obstruction. Volume data acquisition by means of helical CT with sections less than 3 mm in thickness and multiplanar reformation along the bronchial tree are helpful in confirming the endobronchial location of the calcified material. The most common cause of broncholithiasis is erosion by and extrusion of a calcified adjacent lymph node into the bronchial lumen, a finding usually associated with tuberculosis or histoplasmosis. Other causes of broncholithiasis include (a) aspiration of bone tissue or in situ calcification of aspirated foreign material and (b) erosion by and extrusion of calcified or ossified bronchial cartilage plates. Primary endobronchial infections with dystrophic calcification, calcified endobronchial tumors, tracheobronchial diseases with mural calcification, and hypertrophied bronchial artery with intramural protrusion may mimic broncholithiasis. An awareness of the typical imaging findings of broncholithiasis, along with a knowledge of its various causes, help in establishing an accurate diagnosis to ensure proper case management. PMID- 12376612 TI - Nonneoplastic lesions of the tracheobronchial wall: radiologic findings with bronchoscopic correlation. AB - Nonneoplastic diseases of the central airways are uncommon but can be categorized as either focal or diffuse, although there is some overlap. Focal diseases include postintubation stenosis, postinfectious stenosis, posttransplantation stenosis, and various systemic diseases that may involve the airways and lead to focal stenosis (eg, Crohn disease, sarcoidosis, Behcet syndrome). Diffuse diseases of the central airways include Wegener granulomatosis, relapsing polychondritis, tracheobronchopathia osteochondroplastica, amyloidosis, papillomatosis, and rhinoscleroma. Conventional radiography is often the first step in the evaluation of suspected central airway disease and may be adequate in itself to identify the abnormality. However, computed tomography (CT) improves both the detection and characterization of central airway disease. Bronchoscopy remains the primary procedure for the diagnostic work-up of these disease entities. Nevertheless, a thorough radiologic evaluation with radiography and CT may demonstrate specific imaging findings (eg, calcification) that can help narrow the differential diagnosis and aid in the planning of bronchoscopy or therapeutic intervention. PMID- 12376613 TI - The retrotracheal space: normal anatomic and pathologic appearances. AB - A variety of diseases can arise from the normal contents of the retrotracheal space or from adjacent structures. Mediastinal diseases in the retrotracheal space typically manifest radiographically as a contour abnormality or an area of increased opacity, although computed tomography (CT) or magnetic resonance (MR) imaging is usually required for diagnosis. The most common aortic arch anomaly, a right subclavian artery that originates from an otherwise normal left-sided aortic arch, appears at posteroanterior chest radiography as an obliquely oriented soft-tissue area of increased opacity that extends superiorly to the right from the superior margin of the aortic arch. CT and MR imaging can reveal associated vascular or mediastinal abnormalities. Aortic aneurysms and pseudoaneurysms can manifest radiographically as fusiform or saccular masslike lesions that protrude into the retrotracheal space. Thoracic MR imaging and spiral CT angiography are the diagnostic procedures of choice for evaluating diverse pathologic conditions of the thoracic aorta. Esophageal diseases can manifest as an abnormality in the retrotracheal space, which may be the initial clue to the diagnosis. At CT, lymphatic malformations in the mediastinum manifest as lobular, multicystic tumors that surround and infiltrate adjacent mediastinal structures. Familiarity with the normal radiologic appearance of the retrotracheal space and with the clinical manifestations of diseases that affect the retrotracheal space and adjacent structures can facilitate detection, diagnosis, and management. PMID- 12376614 TI - Thoracic complications of esophageal disorders. AB - Abnormalities of the esophagus are common, and complications associated with these disorders and diseases can involve the mediastinum, tracheobronchial tree, and lungs. The most common complications include mediastinitis secondary to esophageal perforation or postoperative anastomotic leak, or both; empyema due to fistula formation; and aspiration pneumonia. The authors reviewed the radiologic appearances of those and other common thoracic complications associated with esophageal disorders to facilitate early detection, diagnosis, and management. Computed tomographic (CT) findings of acute mediastinitis secondary to esophageal perforation may include esophageal thickening, extraluminal gas, pleural effusion, single or multiple abscesses, and extraluminal contrast medium. The radiologic manifestations of pneumonia secondary to tracheoesophageal fistula are variable, depending on the spread and severity of the aspiration. The most common radiographic pattern is that of bronchopneumonia with scattered air-space opacities. CT has been regarded as the imaging modality of choice for the evaluation of suspected esophagopleural fistula, because the site of communication between the pleural space and the esophagus can often be seen. An awareness of the radiologic manifestations of these complications is thus required to facilitate early diagnosis. PMID- 12376615 TI - Clinical applications of radio-frequency tumor ablation in the thorax. AB - Minimally invasive alternatives to surgery for the treatment of malignancy are becoming more attractive owing to improvements in technology, reduced morbidity and mortality, and the ability to provide treatment in an outpatient setting. Radio-frequency (RF) ablation has become the imaging-guided ablative method of choice because of its relatively low cost, its capability of creating large regions of coagulative necrosis in a controlled fashion, and its relatively low toxicity. RF ablation in the thorax involves the use of computed tomography (CT) to localize the tumor and determine the optimal approach. The size of the tumor determines whether a cluster of electrodes or a single electrode of a particular length will be used to perform the ablation. CT fluoroscopy aids in guiding placement of the electrode. In patients with non-small cell lung malignancy who are not candidates for surgery owing to poor cardiorespiratory reserve, RF ablation alone or followed by conventional radiation therapy with or without chemotherapy may prove to be a treatment option. In patients with metastatic disease, RF ablation may be suitable for treatment of a small tumor burden or for palliation of larger tumors that cause symptoms such as cough, hemoptysis, or pain. Patients with chest wall or osseous metastatic tumors in whom other therapies have failed may benefit from RF ablation as an alternative to radiation therapy. PMID- 12376616 TI - Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. AB - Parkinson's disease (PD) is most commonly a sporadic illness, and is characterized by degeneration of substantia nigra dopamine (DA) neurons and abnormal cytoplasmic aggregates of alpha-synuclein. Rarely, PD may be caused by missense mutations in alpha-synuclein. MPTP, a neurotoxin that inhibits mitochondrial complex I, is a prototype for an environmental cause of PD because it produces a pattern of DA neurodegeneration that closely resembles the neuropathology of PD. Here we show that alpha-synuclein null mice display striking resistance to MPTP-induced degeneration of DA neurons and DA release, and this resistance appears to result from an inability of the toxin to inhibit complex I. Contrary to predictions from in vitro data, this resistance is not due to abnormalities of the DA transporter, which appears to function normally in alpha-synuclein null mice. Our results suggest that some genetic and environmental factors that increase susceptibility to PD may interact with a common molecular pathway, and represent the first demonstration that normal alpha synuclein function may be important to DA neuron viability. PMID- 12376617 TI - A nucleolar TAR decoy inhibitor of HIV-1 replication. AB - Tat is a critical regulatory factor in HIV-1 gene expression. It mediates the transactivation of transcription from the HIV-1 LTR by binding to the transactivation response (TAR) element in a complex with cyclin T1. Because of its critical and early role in HIV gene expression, Tat and its interaction with the TAR element constitute important therapeutic targets for the treatment of HIV 1 infection. Based on the known nucleolar localization properties of Tat, we constructed a chimeric small nucleolar RNA-TAR decoy that localizes to the nucleoli of human cells and colocalizes in the nucleolus with a Tat-enhanced GFP fusion protein. When the chimeric RNA was stably expressed in human T lymphoblastoid CEM cells it potently inhibited HIV-1 replication. These results demonstrate that the nucleolar trafficking of Tat is critical for HIV-1 replication and suggests a role for the nucleolus in HIV-1 viral replication. PMID- 12376618 TI - Biodirected epitaxial nanodeposition of polymers on oriented macromolecular templates. AB - Biodirected epitaxial nanodeposition of polymers was achieved on a template with an oriented molecular surface. Acetobacter xylinum synthesized a ribbon of cellulose I microfibrils onto a fixed, nematic ordered substrate of glucan chains with unique surface characteristics. The substrate directed the orientation of the motion due to the inverse force of the secretion during biosynthesis, and the microfibrils were aligned along the orientation of the molecular template. Using real-time video analysis, the patterns and rates of deposition were elucidated. Field emission scanning electron microscopy revealed that a strong molecular interaction allowed for the deposition of nascent biosynthesized 3.5-nm cellulose microfibrils with inter-microfibrillar spacings of 7-8 nm on the surface of the template. The cellulose was deposited parallel to the molecular orientation of the template. Directed cellulose synthesis and ordered movement of cells were observed only by using a nematic ordered substrate made from cellulose, and not from ordered crystalline cellulose substrates or ordered cellulose-related synthetic polymers such as polyvinyl alcohol. This unique relationship between directed biosynthesis and the ordered fabrication from the nano to the micro scales could lead to new methodologies for the design of functional materials with desired nanostructures. PMID- 12376619 TI - Protection of the intestinal mucosa by intraepithelial gamma delta T cells. AB - gammadelta intraepithelial T lymphocytes (IEL) represent a major T cell population within the intestine of unclear functional relevance. The role of intestinal gammadelta IEL was evaluated in the dextran sodium sulfate (DSS) induced mouse colitis model system. Large numbers of gammadelta T cells, but not alphabeta T cells, were localized at sites of DSS-induced epithelial cell damage. gammadelta IEL in DSS treated mice expressed keratinocyte growth factor (KGF), a potent intestinal epithelial cell mitogen. gammadelta cell-deficient mice (TCRdelta(-/-)) and KGF-deficient mice (KGF(-/-)), but not alphabeta cell deficient mice (TCRalpha(-/-)), were more prone than wild-type mice to DSS induced mucosal injury and demonstrated delayed tissue repair after termination of DSS treatment. Termination of DSS treatment resulted in vigorous epithelial cell proliferation in wild-type mice but not in TCRdelta(-/-) mice or KGF(-/-) mice. These results suggest that gammadelta IEL help preserve the integrity of damaged epithelial surfaces by providing the localized delivery of an epithelial cell growth factor. PMID- 12376620 TI - Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin. AB - Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1 interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing. PMID- 12376621 TI - The role of ipsilateral premotor cortex in hand movement after stroke. AB - Movement of an affected hand after stroke is associated with increased activation of ipsilateral motor cortical areas, suggesting that these motor areas in the undamaged hemisphere may adaptively compensate for damaged or disconnected regions. However, this adaptive compensation has not yet been demonstrated directly. Here we used transcranial magnetic stimulation (TMS) to interfere transiently with processing in the ipsilateral primary motor or dorsal premotor cortex (PMd) during finger movements. TMS had a greater effect on patients than controls in a manner that depended on the site, hemisphere, and time of stimulation. In patients with right hemiparesis (but not in healthy controls), TMS applied to PMd early (100 ms) after the cue to move slowed simple reaction time finger movements by 12% compared with controls. The relative slowing of movements with ipsilateral PMd stimulation in patients correlated with the degree of motor impairment, suggesting that functional recruitment of ipsilateral motor areas was greatest in the more impaired patients. We also used functional magnetic resonance imaging to monitor brain activity in these subjects as they performed the same movements. Slowing of reaction time after premotor cortex TMS in the patients correlated inversely with the relative hemispheric lateralization of functional magnetic resonance imaging activation in PMd. This inverse correlation suggests that the increased activation in ipsilateral cortical motor areas during movements of a paretic hand, shown in this and previous functional imaging studies, represents a functionally relevant, adaptive response to the associated brain injury. PMID- 12376623 TI - The COBRA family of putative GPI-anchored proteins in Arabidopsis. A new fellowship in expansion. AB - Identification of regulatory molecules that determine the extent and direction of expansion is necessary to understand how cell morphogenesis is controlled in plants. We recently identified COB (COBRA) as a key regulator of the orientation of cell expansion in the root. Analysis of the Arabidopsis genome sequence indicated that COB belongs to a multigene family consisting of 12 members, all predicted to encode glycosylphosphatidylinositol-anchored proteins. All but two of the COBL (COB-like) genes are expressed in most organs examined, suggesting possible redundancy. Sequence comparisons, phylogenetic analyses, and exon-intron positions revealed that the COB family is composed of two main subgroups sharing a common architecture, one subgroup being characterized by an additional N terminal domain. Identification of expressed sequence tags corresponding to potential orthologs in other plant species suggested that COB-related functions are required in all vascular plants. Together, these results indicate that COB family members are likely to be important new players at the plasma membrane-cell wall interface. PMID- 12376622 TI - Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula. AB - The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable. PMID- 12376624 TI - Hydrogen peroxide activates cell death and defense gene expression in birch. AB - The function of hydrogen peroxide (H(2)O(2)) as a signal molecule regulating gene expression and cell death induced by external stresses was studied in birch (Betula pendula). Ozone (O(3)), Pseudomonas syringae pv syringae (Pss), and wounding all induced cell death of various extents in birch leaves. This was temporally preceded and closely accompanied by H(2)O(2) accumulation at, and especially surrounding, the lesion sites. O(3) and Pss, along with an artificial H(2)O(2) producing system glucose (Glc)/Glc oxidase, elicited elevated mRNA levels corresponding to genes encoding reactive oxygen species detoxifying enzymes, Pal, Ypr10, and mitochondrial phosphate translocator 1. In addition to the regulation of gene expression, Glc/Glc oxidase also induced endogenous H(2)O(2) production in birch leaves, accompanied by cell death that resembled O(3) and Pss damage. Wound-induced gene expression differed from that induced by O(3) and Pss. Thus, it appears that at least two separate defense pathways can be activated in birch leaves by stress factors, even though the early H(2)O(2) accumulation response is common among them all. PMID- 12376625 TI - Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis. AB - Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO(4)) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation. PMID- 12376626 TI - Expression profiling of the whole Arabidopsis shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. AB - The recent publication of the complete sequence of the Arabidopsis genome allowed us to identify and characterize the last two members of the SHAGGY-like kinase (AtSK) gene family. As a result, the study of the overall spatio-temporal organization of the whole AtSK family in Arabidopsis has become an achievable and necessary aim to understand the role of each SHAGGY-like kinase during plant development. An analysis of the transcript level of the 10 members of the family has been performed using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction. Transcript levels in several organs, under different growth conditions, were analyzed. To calibrate the results obtained, a number of other genes, such as those coding for the two MAP3Kepsilons and the two MAP4Kalphas, as well as the stress response marker RD29A; the small subunit of the Rubisco photosynthetic enzyme Ats1A; the MEDEA chromatin remodeling factor; and the SCARECROW, ASYMMETRIC LEAVES 1, and SUPERMAN transcription factors all involved in key steps of plant development were used. The analysis of our data revealed that eight of the 10 genes of the AtSK family displayed a pseudo-constitutive expression pattern at the organ level. Conversely, AtSK13 responded to osmotic changes and saline treatment, whereas AtSK31 was flower specific and responded to osmotic changes and darkness. PMID- 12376627 TI - Soluble invertase expression is an early target of drought stress during the critical, abortion-sensitive phase of young ovary development in maize. AB - To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development. PMID- 12376628 TI - Down-regulation of TM29, a tomato SEPALLATA homolog, causes parthenocarpic fruit development and floral reversion. AB - We have characterized the tomato (Lycopersicon esculentum Mill.) MADS box gene TM29 that shared a high amino acid sequence homology to the Arabidopsis SEP1, 2, and 3 (SEPALLATA1, 2, and 3) genes. TM29 showed similar expression profiles to SEP1, with accumulation of mRNA in the primordia of all four whorls of floral organs. In addition, TM29 mRNA was detected in inflorescence and vegetative meristems. To understand TM29 function, we produced transgenic tomato plants in which TM29 expression was down-regulated by either cosuppression or antisense techniques. These transgenic plants produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Petals and stamens were green rather than yellow, suggesting a partial conversion to a sepalloid identity. Stamens and ovaries were infertile, with the later developing into parthenocarpic fruit. Ectopic shoots with partially developed leaves and secondary flowers emerged from the fruit. These shoots resembled the primary transgenic flowers and continued to produce parthenocarpic fruit and additional ectopic shoots. Based on the temporal and spatial expression pattern and transgenic phenotypes, we propose that TM29 functions in floral organ development, fruit development, and maintenance of floral meristem identity in tomato. PMID- 12376629 TI - Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. AB - A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants. PMID- 12376630 TI - Phase-specific circadian clock regulatory elements in Arabidopsis. AB - We have defined a minimal Arabidopsis CATALASE 3 (CAT3) promoter sufficient to drive evening-specific circadian transcription of a LUCIFERASE reporter gene. Deletion analysis and site-directed mutagenesis reveal a circadian response element, the evening element (EE: AAAATATCT), that is necessary for evening specific transcription. The EE differs only by a single base pair from the CIRCADIAN CLOCK ASSOCIATED 1-binding site (CBS: AAAAAATCT), which is important for morning-specific transcription. We tested the hypothesis that the EE and the CBS specify circadian phase by site-directed mutagenesis to convert the CAT3 EE into a CBS. Changing the CAT3 EE to a CBS changes the phase of peak transcription from the evening to the morning in continuous dark and in light-dark cycles, consistent with the specification of phase by the single base pair that distinguishes these elements. However, rhythmicity of the CBS-containing CAT3 promoter is dramatically compromised in continuous light. Thus, we conclude that additional information normally provided in the context of a morning-specific promoter is necessary for full circadian activity of the CBS. PMID- 12376631 TI - Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. AB - In plants, low temperature and dehydration activate a set of genes containing C repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plant's response to cold and drought evolved from a common CBF like transcription factor, first through gene duplication and then through promoter evolution. PMID- 12376632 TI - L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants. AB - L-Ascorbic acid (AsA) was found to be loaded into phloem of source leaves and transported to sink tissues. When L-[(14)C]AsA was applied to leaves of intact plants of three different species, autoradiographs and HPLC analysis demonstrated that AsA was accumulated into phloem and transported to root tips, shoots, and floral organs, but not to mature leaves. AsA was also directly detected in Arabidopsis sieve tube sap collected from an English green aphid (Sitobion avenae) stylet. Feeding a single leaf of intact Arabidopsis or Medicago sativa with 10 or 20 mM L-galactono-1,4-lactone (GAL-L), the immediate precursor of AsA, lead to a 7- to 8-fold increase in AsA in the treated leaf and a 2- to 3-fold increase of AsA in untreated sink tissues of the same plant. The amount of AsA produced in treated leaves and accumulated in sink tissues was proportional to the amount of GAL-L applied. Studies of the ability of organs to produce AsA from GAL-L showed mature leaves have a 3- to 10-fold higher biosynthetic capacity and much lower AsA turnover rate than sink tissues. The results indicate AsA transporters reside in the phloem, and that AsA translocation is likely required to meet AsA demands of rapidly growing non-photosynthetic tissues. This study also demonstrates that source leaf AsA biosynthesis is limited by substrate availability rather than biosynthetic capacity, and sink AsA levels may be limited to some extent by source production. Phloem translocation of AsA may be one factor regulating sink development because AsA is critical to cell division/growth. PMID- 12376633 TI - The KNAT2 homeodomain protein interacts with ethylene and cytokinin signaling. AB - Using a transgenic line that overexpresses a fusion of the KNAT2 (KNOTTED-like Arabidopsis) homeodomain protein and the hormone-binding domain of the glucocorticoid receptor (GR), we have investigated the possible relations between KNAT2 and various hormones. Upon activation of the KNAT2-GR fusion, we observed a delayed senescence of the leaves and a higher rate of shoot initiation, two processes that are also induced by cytokinins and inhibited by ethylene. Furthermore, the activation of the KNAT2-GR fusion induced lobing of the leaves. This feature was partially suppressed by treatment with the ethylene precursor 1 aminocyclopropane-1-carboxylic acid, or by the constitutive ethylene response ctr1 mutation. Conversely, some phenotypic traits of the ctr1 mutant were suppressed by the activation of the KNAT2-GR fusion. These data suggest that KNAT2 acts synergistically with cytokinins and antagonistically with ethylene. In the shoot apical meristem, the KNAT2 gene is expressed in the L3 layer and the rib zone. 1-Aminocyclopropane-1-carboxylic acid treatment restricted the KNAT2 expression domain in the shoot apical meristem and reduced the number of cells in the L3. The latter effect was suppressed by the activation of the KNAT2-GR construct. Conversely, the KNAT2 gene expression domain was enlarged in the ethylene-resistant etr1-1 mutant or in response to cytokinin treatment. These data suggest that ethylene and cytokinins act antagonistically in the meristem via KNAT2 to regulate the meristem activity. PMID- 12376634 TI - Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. AB - Each fiber of cotton (Gossypium hirsutum) is a single epidermal cell that rapidly elongates to 2.5 to 3.0 cm from the ovule surface within about 16 d after anthesis. A large number of genes are required for fiber differentiation and development, but so far, little is known about how these genes control and regulate the process of fiber development. To investigate gene expression patterns in fiber, a cDNA, GhTUB1, encoding beta-tubulin was isolated from a cotton fiber cDNA library. The analyses of RNA northern-blot hybridization and reverse transcriptase-polymerase chain reaction demonstrated that GhTUB1 transcripts preferentially accumulated at high levels in fiber, at low levels in ovules at the early stage of cotton boll development, and at very low levels in other tissues of cotton. The corresponding GhTUB1 gene including the promoter region was isolated by screening a cotton genomic DNA library. To demonstrate the specificity of the GhTUB1 promoter, the 5'-flanking region including the promoter and 5'-untranslated region was fused with the beta-glucuronidase reporter gene. The expression of the reporter chimera was examined in a large number of transgenic cotton plants. Histochemical assays demonstrated that GhTUB1::beta glucuronidase fusion genes were expressed preferentially at high levels in fiber and primary root tip of 1- to 3-d-old seedlings and at low levels in other tissues such as ovule, pollen, seedling cotyledon, and root basal portion. The results suggested that the GhTUB1 gene may play a distinct and required role in fiber development. In addition, the GhTUB1 promoter may have great potential for cotton improvement by genetic engineering. PMID- 12376635 TI - The calcium-binding activity of a vacuole-associated, dehydrin-like protein is regulated by phosphorylation. AB - A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 +/- 0.142 mM and 0.64 +/- 0.08 mM, and has an estimated 24.7 +/- 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions. PMID- 12376636 TI - Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities. AB - A small family of novel basic leucine zipper proteins that includes abscisic acid (ABA)-INSENSITIVE 5 (ABI5) binds to the promoter region of the lea class gene Dc3. The factors, referred to as AtDPBFs (Arabidopsis Dc3 promoter-binding factors), were isolated from an immature seed cDNA library. AtDPBFs bind to the embryo specification and ABA-responsive elements in the Dc3 promoter and are unique in that they can interact with cis-elements that do not contain the ACGT core sequence required for the binding of most other plant basic leucine zipper proteins. Analysis of full-length cDNAs showed that at least five different Dc3 promoter-binding factors are present in Arabidopsis seeds; one of these, AtDPBF 1, is identical to ABI5. As expected, AtDPBF-1/ABI5 mRNA is inducible by exogenous ABA in seedlings. Despite the near identity in their basic domains, AtDPBFs are distinct in their DNA-binding, dimerization, and transcriptional activity. PMID- 12376637 TI - Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells. AB - Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed. PMID- 12376638 TI - ACTCAT, a novel cis-acting element for proline- and hypoosmolarity-responsive expression of the ProDH gene encoding proline dehydrogenase in Arabidopsis. AB - Proline (Pro) is one of the most widely distributed osmolytes in water-stressed plants. We previously isolated from Arabidopsis a gene encoding Pro dehydrogenase (ProDH), a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The ProDH gene in Arabidopsis is up-regulated by rehydration after dehydration but is down-regulated by dehydration. ProDH is also induced by L-Pro and hypoosmolarity. The induction of ProDH expression under rehydration seems to be caused by both accumulated Pro and hypoosmolarity. We analyzed a DNA region that is located 5' to the transcription start site (a promoter region) of ProDH to identify cis-acting elements involved in L-Pro induced and hypoosmolarity-induced expression in transgenic tobacco (Nicotiana tabacum) and Arabidopsis plants. We found that a 9-bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to L-Pro and hypoosmolarity. Moreover, ACTCAT is a core cis-acting element, which we have called Pro- or hypoosmolarity-responsive element (PRE), that is necessary for L-Pro-responsive and hypoosmolarity-responsive expression of ProDH. Microarray and RNA gel-blot analyses showed that 21 L-Pro-inducible genes have the PRE sequences in their promoter regions. These results indicate that the PRE sequence play an important role in the L-Pro-responsive gene expression. PMID- 12376639 TI - Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays. AB - Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations. PMID- 12376640 TI - Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. AB - The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented. PMID- 12376641 TI - Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis. AB - Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A(4)B(4) configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom. PMID- 12376642 TI - Two distinct jacalin-related lectins with a different specificity and subcellular location are major vegetative storage proteins in the bark of the black mulberry tree. AB - Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research. PMID- 12376643 TI - Transcriptional and posttranscriptional regulation of Arabidopsis TCH4 expression by diverse stimuli. Roles of cis regions and brassinosteroids. AB - The Arabidopsis TCH4 gene is up-regulated in expression by diverse environmental and hormonal stimuli. Because TCH4 encodes a xyloglucan endotransglucosylase/hydrolase, this change in expression may reflect a recruitment of cell wall-modifying activity in response to environmental stress and growth. How diverse stimuli lead to the common response of TCH4 expression regulation is not known. Here, we show that induction of expression by the diverse stimuli of touch, darkness, cold, heat, and brassinosteroids (BRs) is conferred to reporter genes by the same 102-bp 5'-untranscribed TCH4 region; this result is consistent with the idea that shared regulatory elements are employed by diverse stimuli. Distal regions influence magnitude and kinetics of expression and likely harbor regulatory elements that are redundant with those located more proximal to the transcriptional start site. Substitution of the proximal regulatory region sequences in the context of distal elements does not disrupt inducible expression. TCH4 expression induction is transcriptional, at least in part because 5'-untranscribed sequences are sufficient to confer this regulation. However, 5'-untranslated sequences are necessary and sufficient to confer the marked transience of TCH4 expression, most likely through an effect on mRNA stability. Perception of BR is not necessary for TCH4::GUS induction by environmental stimuli because regulation is intact in the BR-insensitive mutant, bri1-2. The full response to auxin, however, requires the functioning of BRI1. Developmental expression of TCH4 is unlikely to be meditated by BR because TCH4::GUS is expressed in BR perception and biosynthetic mutants bri1-2 and det2 1, respectively. PMID- 12376644 TI - Inventory and functional characterization of the HAK potassium transporters of rice. AB - Plants take up large amounts of K(+) from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K(+) from the soil solution to its final destination is mediated by channels and transporters. To better understand K(+) movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1-17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K(+) influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K(+) influx in yeast and in roots of K(+)-starved rice seedlings demonstrated the involvement of HAK transporters in root K(+) uptake. We discuss that all HAK transporters may mediate K(+) transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast. PMID- 12376645 TI - Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen. AB - Lignins, along with condensed tannins (CTs) and salicylate-derived phenolic glycosides, constitute potentially large phenylpropanoid carbon sinks in tissues of quaking aspen (Populus tremuloides Michx.). Metabolic commitment to each of these sinks varies during development and adaptation, and depends on L phenylalanine ammonia-lyase (PAL), an enzyme catalyzing the deamination of L phenylalanine to initiate phenylpropanoid metabolism. In Populus spp., PAL is encoded by multiple genes whose expression has been associated with lignification in primary and secondary tissues. We now report cloning two differentially expressed PAL cDNAs that exhibit distinct spatial associations with CT and lignin biosynthesis in developing shoot and root tissues of aspen. PtPAL1 was expressed in certain CT-accumulating, non-lignifying cells of stems, leaves, and roots, and the pattern of PtPAL1 expression varied coordinately with that of CT accumulation along the primary to secondary growth transition in stems. PtPAL2 was expressed in heavily lignified structural cells of shoots, but was also expressed in non lignifying cells of root tips. Evidence of a role for Pt4CL2, encoding 4 coumarate:coenzyme A ligase, in determining CT sink strength was gained from cellular co-expression analysis with PAL1 and CTs, and from experiments in which leaf wounding increased PAL1 and 4CL2 expression as well as the relative allocation of carbon to CT with respect to phenolic glycoside, the dominant phenolic sink in aspen leaves. Leaf wounding also increased PAL2 and lignin pathway gene expression, but to a smaller extent. The absence of PAL2 in most CT accumulating cells provides in situ support for the idea that PAL isoforms function in specific metabolic milieus. PMID- 12376647 TI - The predicted candidates of Arabidopsis plastid inner envelope membrane proteins and their expression profiles. AB - Plastid envelope proteins from the Arabidopsis nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH(2)-terminal plastid-targeting peptides from all annotated open reading frames from Arabidopsis; second, to search for proteins with membrane-spanning domains among the predicted plastidial-targeted proteins; and third, to subtract known thylakoid membrane proteins. Five hundred forty-one proteins were selected as potential candidates of the Arabidopsis plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared with the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of "transport facilitation," indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue expression data from expressed sequence tags and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic-based efforts in the increased detection of integral membrane proteins, low-abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of expressed sequence tags suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analyses identified a number of AtPEM candidates with tissue-specific expression patterns. PMID- 12376646 TI - SHORT INTEGUMENTS1/SUSPENSOR1/CARPEL FACTORY, a Dicer homolog, is a maternal effect gene required for embryo development in Arabidopsis. AB - The importance of maternal cells in controlling early embryogenesis is well understood in animal development, yet in plants the precise role of maternal cells in embryogenesis is unclear. We demonstrated previously that maternal activity of the SIN1 (SHORT INTEGUMENTS1) gene of Arabidopsis is essential for embryo pattern formation and viability, and that its postembryonic activity is required for several processes in reproductive development, including flowering time control and ovule morphogenesis. Here, we report the cloning of SIN1, and demonstrate its identity to the CAF (CARPEL FACTORY) gene important for normal flower morphogenesis and to the SUS1 (SUSPENSOR1) gene essential for embryogenesis. SIN1/SUS1/CAF has sequence similarity to the Drosophila melanogaster gene Dicer, which encodes a multidomain ribonuclease specific for double-stranded RNA, first identified by its role in RNA silencing. The Dicer protein is essential for temporal control of development in animals, through the processing of small RNA hairpins that in turn inhibit the translation of target mRNAs. Structural modeling of the wild-type and sin1 mutant proteins indicates that the RNA helicase domain of SIN1/SUS1/CAF is important for function. The mRNA was detected in floral meristems, ovules, and early embryos, consistent with the mutant phenotypes. A 3.3-kb region 5' of the SIN1/SUS1/CAF gene shows asymmetric parent-of-origin activity in the embryo: It confers transcriptional activation of a reporter gene in early embryos only when transmitted through the maternal gamete. These results suggest that maternal SIN1/SUS1/CAF functions early in Arabidopsis development, presumably through posttranscriptional regulation of specific mRNA molecules. PMID- 12376648 TI - The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences. AB - The abscisic acid (ABA)-induced protein kinase PKABA1 is present in dormant seeds and is a component of the signal transduction pathway leading to ABA-suppressed gene expression in cereal grains. We have identified a member of the ABA response element-binding factor (ABF) family of basic leucine zipper transcription factors from wheat (Triticum aestivum) that is specifically bound by PKABA1. This protein (TaABF) has highest sequence similarity to the Arabidopsis ABA response protein ABI5. In two-hybrid assays TaABF bound only to PKABA1, but not to a mutant version of PKABA1 lacking the nucleotide binding domain, suggesting that binding of TaABF requires prior binding of ATP as would be expected for binding of a protein substrate by a protein kinase. TaABF mRNA accumulated together with PKABA1 mRNA during wheat grain maturation and dormancy acquisition and TaABF transcripts increased transiently during imbibition of dormant grains. In contrast to PKABA1 mRNA, TaABF mRNA is seed specific and did not accumulate in vegetative tissues in response to stress or ABA application. PKABA1 produced in transformed cell lines was able to phosphorylate synthetic peptides representing three specific regions of TaABF. These data suggest that TaABF may serve as a physiological substrate for PKABA1 in the ABA signal transduction pathway during grain maturation, dormancy expression, and ABA-suppressed gene expression. PMID- 12376649 TI - An essential role of s-adenosyl-L-methionine:L-methionine s-methyltransferase in selenium volatilization by plants. Methylation of selenomethionine to selenium methyl-L-selenium- methionine, the precursor of volatile selenium. AB - Selenium (Se) phytovolatilization, the process by which plants metabolize various inorganic or organic species of Se (e.g. selenate, selenite, and Se-methionine [Met]) into gaseous Se forms (e.g. dimethylselenide), is a potentially important means of removing Se from contaminated environments. Before attempting to genetically enhance the efficiency of Se phytovolatilization, it is essential to elucidate the enzymatic pathway involved and to identify its rate-limiting steps. The present research tested the hypothesis that S-adenosyl-L-Met:L-Met S methyltransferase (MMT) is the enzyme responsible for the methylation of Se-Met to Se-methyl Se-Met (SeMM). To this end, we identified and characterized an Arabidopsis T-DNA mutant knockout for MMT. The lack of MMT in the Arabidopsis T DNA mutant plant resulted in an almost complete loss in its capacity for Se volatilization. Using chemical complementation with SeMM, the presumed enzymatic product of MMT, we restored the capacity of the MMT mutant to produce volatile Se. Overexpressing MMT from Arabidopsis in Escherichia coli, which is not known to have MMT activity, produced up to 10 times more volatile Se than the untransformed strain when both were supplied with Se-Met. Thus, our results provide in vivo evidence that MMT is the key enzyme catalyzing the methylation of Se-Met to SeMM. PMID- 12376650 TI - Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis. AB - The Arabidopsis genome contains 14 genes encoding the serine protease DegP. Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane. We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically. Size exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers. Proteolytic activity was characterized using beta casein as a model substrate. DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures. DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen. The results of this work are discussed in the context of the recent elucidation of the structure of the E. coli homolog and the possible physiological role of the protease in the chloroplast lumen. PMID- 12376651 TI - Characterization of SP1, a stress-responsive, boiling-soluble, homo-oligomeric protein from aspen. AB - sp1 cDNA was isolated from aspen (Populus tremula) plants by immunoscreening an expression library using polyclonal antibodies against BspA protein. BspA, which is a boiling-stable protein, accumulates in aspen plants in response to water stress and abscisic acid application (Pelah et al., 1995). The sp1 cDNA was found to encode a 12.4-kD generally hydrophilic protein with a hydrophobic C terminus, which is different from the BspA protein and was termed SP1 (stable protein 1). Northern-blot analysis revealed that sp1 encodes a small mRNA (about 0.6 kb) that is expressed in aspen plants under non-stress conditions and is accumulated after salt, cold, heat, and desiccation stress, and during the recovery from stress. The SP1 detected in plants remained soluble upon boiling, migrated both as a 12.4 kD band and a much higher mass of 116 kD on a 17% (w/v) Tricine-sodium dodecyl sulfate-polyacrylamide gel. Comparative protease digestion patterns, amino acid analyses, and the N-terminal sequences of the 12.4- and 116-kD proteins revealed that SP1 is homo-oligomeric. Furthermore, gel filtration chromatography analysis indicated that SP1 exists in aspen plants as a complex, composed of 12 subunits of 12.4 kD. A large number of sequences deduced from expressed sequence tags and genomic sequences of other organisms with unknown function show high homology to SP1. Thus, SP1 may represent a new protein family. Here, we present the first report on this putative protein family: the cloning, isolation, and characterization of SP1, a stress-responsive, boiling-soluble, oligomeric protein. PMID- 12376652 TI - Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single cell, facultative C(4) system of Hydrilla verticillata. AB - The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C(4) plant. It typically exhibits C(3) photosynthetic characteristics, but exposure to low [CO(2)] induces a C(4) system in which the C(4) and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C(4) induction. This and enzyme kinetic data were consistent with it being the C(4) photosynthesis isoform. However, the C(4) signature serine of terrestrial plant C(4) isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C(3) sequences, was present. Western analyses of C(3) and C(4) leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C(3), non-graminaceous C(4), and Crassulacean acid metabolism PEPCs but not with the graminaceous C(4), and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C(4) angiosperms. PMID- 12376653 TI - An Arabidopsis mutant defective in jasmonate response is allelic to the auxin signaling mutant axr1. AB - A screen for Arabidopsis mutants that were insensitive to methyl jasmonate (MeJA) in an assay for seedling root growth yielded only alleles of previously isolated mutants jar1 and coi1, with one exception. Mapping of the locus and morphological characterization of the new mutant suggested it might be allelic to axr1, which had not previously been reported to show resistance to MeJA. The F(1) from a cross of the new mutant with axr1-3 did not show complementation, confirming that these are the same genes. The new allele is called axr1-24. In addition to MeJA and indole-3-acetic acid (IAA), axr1-24 had decreased sensitivity to 1 aminocyclopropane-1-carboxylic acid, 6-benzylamino-purine, epi-brassinolide, and abscisic acid. Both axr1-24 and the previously characterized axr1-3 allele were shown to be susceptible to the opportunistic pathogen Pythium irregulare, a trait found in other jasmonate response mutants, including jar1-1. The double mutant jar1-1/axr1-3 was more resistant to inhibition of root growth by MeJA and was more susceptible to P. irregulare infection than either single mutant, suggesting these genes might act in independent response pathways. In contrast, resistance to IAA in the double mutant was not different from axr1-3. Northern-blot analysis showed that IAA induced the jasmonate-responsive lipoxygenase 2, AOS, and AtVSP gene transcripts and induction was strongly impaired in axr1-3. However, transcript induction by MeJA was only minimally affected in axr1-3. This study demonstrates that in addition to auxin signaling, the AXR1 locus is involved in MeJA response, providing a mechanistic link between jasmonate and auxin-signaling pathways. PMID- 12376654 TI - A comparison of oligogalacturonide- and auxin-induced extracellular alkalinization and growth responses in roots of intact cucumber seedlings. AB - Oligogalacturonic acid (OGA) affects plant growth and development in an antagonistic manner to that of the auxin indole-3-acetic acid (IAA), the mechanism by which remains to be determined. This study describes the relationship between IAA and OGA activity in intact cucumber (Cucumis sativus) seedlings. Both OGA and IAA induced rapid and transient extracellular alkalinization; however, the characteristics of the OGA and IAA responses differed in their kinetics, magnitude, calcium dependence, and region of the root in which they induced their maximal response. IAA (1 microM) induced a saturating alkalinization response of approximately 0.2 pH unit and a rapid reduction (approximately 80%) in root growth that only partially recovered over 20 h. OGAs, specifically those with a degree of polymerization of 10 to 13, induced a maximal alkalinization response of 0.48 pH unit, but OGA treatment did not alter root growth. Saturating concentrations of OGA did not block IAA-induced alkalinization or the initial IAA-induced inhibition of root growth but allowed IAA-treated roots to recover their initial growth rate within 270 min. IAA-induced alkalinization occurs primarily in the growing apical region of the root, whereas OGA induced its maximal response in the basal region of the root. This study demonstrates that OGA and IAA act by distinct mechanisms and that OGA does not simply act by inhibition of IAA action. These results also suggest that IAA induced extracellular alkalinization is not sufficient to account for the mechanism by which IAA inhibits root growth. PMID- 12376655 TI - Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. AB - The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during compatible or incompatible interactions. A new member of the PR-5 family was identified that accumulated early in both types of interaction. Other pathogenesis-related proteins appeared in compatible interactions only, concomitantly with disease development. This study demonstrates the feasibility of using proteomics for the identification of known and novel proteins in xylem sap, and provides insights into plant-pathogen interactions in vascular wilt diseases. PMID- 12376656 TI - Splicing of the maize Sh1 first intron is essential for enhancement of gene expression, and a T-rich motif increases expression without affecting splicing. AB - Certain plant and animal introns increase expression of protein-coding sequences when placed in the 5' region of the transcription unit. The mechanisms of intron mediated enhancement have not been defined, but are generally accepted to be post or cotranscriptional in character. One of the most effective plant introns in stimulating gene expression is the 1,028-bp first intron of the Sh1 gene that encodes maize (Zea mays) sucrose synthase. To address the mechanisms of intron mediated enhancement, we used reporter gene fusions to identify features of the Sh1 first intron required for enhancement in cultured maize cells. A 145-bp derivative conferred approximately the same 20- to 50-fold stimulation typical for the full-length intron in this transient expression system. A 35-bp motif contained within the intron is required for maximum levels of enhancement but not for efficient transcript splicing. The important feature of this redundant 35-bp motif is T-richness rather than the specific sequence. When transcript splicing was abolished by mutations at the intron borders, enhancement was reduced to about 2-fold. The requirement of splicing for enhancement was not because of upstream translation initiation codons contained in unspliced transcripts. On the basis of our current findings, we conclude that splicing of the Sh1 intron is integral to enhancement, and we hypothesize that transcript modifications triggered by the T-rich motif and splicing may link the mRNA with the trafficking system of the cell. PMID- 12376657 TI - An early C-22 oxidation branch in the brassinosteroid biosynthetic pathway. AB - The natural occurrence of 22-hydroxylated steroids in cultured Catharanthus roseus cells and in Arabidopsis seedlings was investigated. Using full-scan gas chromatography-mass spectrometry analysis, (22S)-22-hydroxycampesterol (22-OHCR), (22S,24R)-22-hydroxyergost-4-en-3-one (22-OH-4-en-3-one), (22S,24R)-22-hydroxy 5alpha-ergostan-3-one (22-OH-3-one), 6-deoxocathasterone (6-deoxoCT), 3-epi-6 deoxoCT, 28-nor-22-OHCR, 28-nor-22-OH-4-en-3-one, 28-nor-22-OH-3-one, 28-nor-6 deoxoCT, and 3-epi-28-nor-6-deoxoCT were identified. Metabolic experiments with deuterium-labeled 22-OHCR were performed in cultured C. roseus cells and Arabidopsis seedlings (wild type and det2), and the metabolites were analyzed by gas chromatography-mass spectrometry. In both C. roseus cells and wild-type Arabidopsis seedlings, [(2)H(6)]22-OH-4-en-3-one, [(2)H(6)]22-OH-3-one, [(2)H(6)]6-deoxoCT, and [(2)H(6)]3-epi-6-deoxoCT were identified as metabolites of [(2)H(6)]22-OHCR, whereas the major metabolite in det2 seedlings was [(2)H(6)]22-OH-4-en-3-one. Analysis of endogenous levels of these brassinosteroids revealed that det2 accumulates 22-OH-4-en-3-one. The levels of downstream compounds were remarkably reduced compared with the wild type. Exogenously applied 22-OH-3-one and 6-deoxoCT were found to rescue det2 mutant phenotypes, whereas 22-OHCR and 22-OH-4-en-3-one did not. These results substantiate the existence of a new subpathway (22-OHCR --> 22-OH-4-en-3-one --> 22-OH-3-one --> 6-deoxoCT) and reveal that the det2 mutant is defective in the conversion of 22-OH-4-en-3-one to 22-OH-3-one, which leads to brassinolide biosynthesis. PMID- 12376658 TI - CO(2)-triggered chloride release from guard cells in intact fava bean leaves. Kinetics of the onset of stomatal closure. AB - The influence of CO(2) on Cl(-) release from guard cells was investigated within the intact leaf by monitoring the Cl(-) activity in the apoplastic fluid of guard cells with a Cl(-)-sensitive microelectrode. In illuminated leaves adapted to a CO(2) concentration within the cuvette of 350 microL L(-1), an increase of 250 microL L(-1) CO(2) triggered a transient rise in the apoplastic Cl(-) activity from 3 to 14 mM within 10 min. This Cl(-) response was similar to the Cl(-) efflux evoked by turning off the light, when the substomatal CO(2) was kept constant (CO(2) clamp). Without CO(2) clamp, substomatal CO(2) increased by 120 microL L(-1) upon "light off." The response to an increase in CO(2) within the cuvette from 250 to 500 microL L(-1) in dark-adapted leaves was equivalent to the response to an increase from 350 to 600 microL L(-1) in the light. No Cl(-) efflux was triggered by 2-min CO(2) pulses (150-800 microL L(-1)). After a switch from 350 microL L(-1) to CO(2)-free cuvette air, the guard cells were less sensitive to a rise in CO(2) and to light off, but the sensitivity to both stimuli partially recovered. Changes in CO(2) also caused changes of the guard cell apoplastic voltage, which were generally faster than the observed Cl(-) responses, and which also promptly occurred when CO(2) did not initiate Cl(-) efflux. The comparatively slow activation of Cl(-) efflux by CO(2) indicates that an intermediate effector derived from CO(2) has to accumulate to fully activate plasma membrane anion channels of guard cells. PMID- 12376659 TI - Genetic architecture of NaCl tolerance in Arabidopsis. AB - The little success of breeding approaches toward the improvement of salt tolerance in crop species is thought to be attributable to the quantitative nature of most, if not all the processes implicated. Hence, the identification of some of the quantitative trait loci (QTL) that contribute to natural variation in salt tolerance should be instrumental in eventually manipulating the perception of salinity and the corresponding responses. A good choice to reach this goal is the plant model system Arabidopsis, whose complete genome sequence is now available. Aiming to analyze natural variability in salt tolerance, we have compared the ability of 102 wild-type races (named ecotypes or accessions) of Arabidopsis to germinate on 250 mM NaCl, finding a wide range of variation among them. Accessions displaying extremely different responses to NaCl were intercrossed, and the phenotypes found in their F(2) progenies suggested that natural variation in NaCl tolerance during germination was under polygenic controls. Genetic distances calculated on the basis of variations in repeat number at 22 microsatellites, were analyzed in a group of either extremely salt tolerant or extremely salt-sensitive accessions. We found that most but not all accessions with similar responses to NaCl are phylogenetically related. NaCl tolerance was also studied in 100 recombinant inbred lines derived from a cross between the Columbia-4 and Landsberg erecta accessions. We detected 11 QTL harboring naturally occurring alleles that contribute to natural variation in NaCl tolerance in Arabidopsis, six at the germination and five at the vegetative growth stages, respectively. At least five of these QTL are likely to represent loci not yet described by their relationship with salt stress. PMID- 12376660 TI - Bundle sheath diffusive resistance to CO(2) and effectiveness of C(4) photosynthesis and refixation of photorespired CO(2) in a C(4) cycle mutant and wild-type Amaranthus edulis. AB - A mutant of the NAD-malic enzyme-type C(4) plant, Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO(2) response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C(4) cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO(2) to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (r(bs)) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl) propenoate, an inhibitor of PEPC. Values of r(bs) in A. edulis were 70 to 180 m(2) s(-1) mol(-1), increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO(2) to Rubisco in mesophyll cells of C(3) plants. The values of r(bs) in A. edulis are sufficient for C(4) photosynthesis to elevate CO(2) in BS cells and to minimize photorespiration. The calculated CO(2) concentration in BS cells, which is dependent on input of r(bs), was about 2,000 microbar under maximum rates of CO(2) fixation, which is about six times the ambient level of CO(2). High re-assimilation of photorespired CO(2) was demonstrated in both mutant and wild-type plants at limiting CO(2) concentrations, which can be explained by high r(bs). Increasing O(2) from near zero up to ambient levels under low CO(2), resulted in an increase in the gross rate of O(2) evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO(2) in BS cells. PMID- 12376661 TI - Endoplasmic microtubules configure the subapical cytoplasm and are required for fast growth of Medicago truncatula root hairs. AB - To investigate the configuration and function of microtubules (MTs) in tip growing Medicago truncatula root hairs, we used immunocytochemistry or in vivo decoration by a GFP linked to a MT-binding domain. The two approaches gave similar results and allowed the study of MTs during hair development. Cortical MTs (CMTs) are present in all developmental stages. During the transition from bulge to a tip-growing root hair, endoplasmic MTs (EMTs) appear at the tip of the young hair and remain there until growth arrest. EMTs are a specific feature of tip-growing hairs, forming a three-dimensional array throughout the subapical cytoplasmic dense region. During growth arrest, EMTs, together with the subapical cytoplasmic dense region, progressively disappear, whereas CMTs extend further toward the tip. In full-grown root hairs, CMTs, the only remaining population of MTs, converge at the tip and their density decreases over time. Upon treatment of growing hairs with 1 microM oryzalin, EMTs disappear, but CMTs remain present. The subapical cytoplasmic dense region becomes very short, the distance nucleus tip increases, growth slows down, and the nucleus still follows the advancing tip, though at a much larger distance. Taxol has no effect on the cytoarchitecture of growing hairs; the subapical cytoplasmic dense region remains intact, the nucleus keeps its distance from the tip, but growth rate drops to the same extent as in hairs treated with 1 microM oryzalin. The role of EMTs in growing root hairs is discussed. PMID- 12376662 TI - Photorespiratory NH(4)(+) production in leaves of wild-type and glutamine synthetase 2 antisense oilseed rape. AB - Exposure of oilseed rape (Brassica napus) plants to increasing leaf temperatures between 15 degrees C and 25 degrees C increased photorespiratory NH(4)(+) production from 0.7 to 3.5 micromol m(-2) s(-1). Despite the 5-fold increase in the rate of NH(4)(+) production, the NH(4)(+) concentration in root and leaf tissue water and xylem sap dropped significantly, whereas that in the leaf apoplastic fluid remained constant. The in vitro activity of glutamine synthetase (GS) in both leaves and roots also increased with temperature and in all cases substantially exceeded the observed rates of photorespiratory NH(4)(+) production. The surplus of GS in oilseed rape plants was confirmed using GS2 antisense plants with 50% to 75% lower in vitro leaf GS activity than in the wild type. Despite the substantial reduction in GS activity, there was no tendency for antisense plants to have higher tissue NH(4)(+) concentrations than wild-type plants and no overall correlation between GS activity and tissue NH(4)(+) concentration was observed. Antisense plants exposed to leaf temperatures increasing from 14 degrees C to 27 degrees C or to a trifold increase in the O(2) to CO(2) ratio did not show any change in steady-state leaf tissue NH(4)(+) concentration or in NH(3) emission to the atmosphere. The antisense plants also had similar leaf tissue concentrations of glutamine, glycine, and serine as the wild type, whereas glutamate increased by 38%. It is concluded that photorespiration does not control tissue or apoplastic levels of NH(4)(+) in oilseed rape leaves and, as a consequence, that photorespiration does not exert a direct control on leaf atmosphere NH(3) fluxes. PMID- 12376663 TI - Activation of phospholipases C and D is an early response to a cold exposure in Arabidopsis suspension cells. AB - The signaling events generated by a cold exposure are poorly known in plants. We were interested in checking the possible activation of enzymes of the phosphoinositide signaling pathway in response to a temperature drop. In Arabidopsis suspension cells labeled with (33)PO(4)(3-), a cold treatment induces a rapid increase of phosphatidic acid (PtdOH) content. This production was due to the simultaneous activation of phospholipase C (through diacylglycerol kinase activity) and phospholipase D, as monitored by the production of inositol triphosphate and of transphosphatidylation product, respectively. Moreover, inhibitors of the phosphoinositide pathway and of diacylglycerol kinase reduced PtdOH production. Enzyme activation occurred immediately after cells were transferred to low temperature. The respective contribution of both kind of phospholipases in cold-induced production of PtdOH could be estimated. We created conditions where phospholipids were labeled with (33)PO(4)(3-), but with ATP being nonradioactive. In such conditions, the apparition of radioactive PtdOH reflected PLD activity. Thus, we demonstrated that during a cold stress, phospholipase D activity accounted for 20% of PtdOH production. The analysis of composition in fatty acids of cold-produced PtdOH compared with that of different phospholipids confirmed that cold-induced PtdOH more likely derived mainly from phosphoinositides. The addition of chemical reagents modifying calcium availability inhibited the formation of PtdOH, showing that the cold-induced activation of phospholipase pathways is dependent on a calcium entry. PMID- 12376664 TI - (18)O spatial patterns of vein xylem water, leaf water, and dry matter in cotton leaves. AB - Three leaf water models (two-pool model, Peclet effect, and string-of-lakes) were assessed for their robustness in predicting leaf water enrichment and its spatial heterogeneity. This was achieved by studying the (18)O spatial patterns of vein xylem water, leaf water, and dry matter in cotton (Gossypium hirsutum) leaves grown at different humidities using new experimental approaches. Vein xylem water was collected from intact transpiring cotton leaves by pressurizing the roots in a pressure chamber, whereas the isotopic content of leaf water was determined without extracting it from fresh leaves with the aid of a purpose-designed leaf punch. Our results indicate that veins have a significant degree of lateral exchange with highly enriched leaf water. Vein xylem water is thus slightly, but progressively enriched in the direction of water flow. Leaf water enrichment is dependent on the relative distances from major veins, with water from the marginal and intercostal regions more enriched and that next to veins and near the leaf base more depleted than the Craig-Gordon modeled enrichment of water at the sites of evaporation. The spatial pattern of leaf water enrichment varies with humidity, as expected from the string-of-lakes model. This pattern is also reflected in leaf dry matter. All three models are realistic, but none could fully account for all of the facets of leaf water enrichment. Our findings acknowledge the presence of capacitance in the ground tissues of vein ribs and highlight the essential need to incorporate Peclet effects into the string-of lakes model when applying it to leaves. PMID- 12376665 TI - Chemical inactivation of the cinnamate 4-hydroxylase allows for the accumulation of salicylic acid in elicited cells. AB - The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid pathway, leading to the synthesis of lignin, pigments, and many defense molecules. Salicylic acid (SA) is an essential trigger of plant disease resistance. Some plant species can synthesize SA from CA by a mechanism not yet understood. A set of specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi irreversible inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA. Competitive inhibitors such as 2-hydroxy-1 naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow [BY]) cell suspension culture. This inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism. Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as 4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent mechanism-based inactivators of the C4H. PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the enzyme in BY cells. Inhibition was not reversed 46 h after cell treatment. Cotreatment of BY cells with the fungal elicitor beta-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation. PIP and 4PB do not trigger SA accumulation in nonelicited cells in which the SA biosynthetic pathway is not activated. Mechanism-based C4H inactivators, thus, are promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense. PMID- 12376666 TI - Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis. A Fourier transform infrared spectroscopy study. AB - A technique based on Fourier transform infrared (FT-IR) spectrometry was developed to detect the corresponding changes in chemical composition associated with the rapid changes in sodium and water content in 200 mM NaCl-stressed halophyte ice plants (Mesembryanthemum crystallinum). The changes in glycophyte Arabidopsis stressed with 50 mM NaCl were also examined for comparison. The obtained IR spectra were further processed by deconvolution and curve fitting to examine the chemical nature of the responding sources in the leaves. Using three stages of ice plant leaves, absorption bands corresponding to carbohydrates, cell wall pectin, and proteins were identified, with distinct IR spectra representing each developmental stage. Within 48 h of mild salt stress, the absorption band intensities in the fingerprint region increased continuously in both plants, suggesting that the carbon assimilation was not affected at the early stage of stress. The intensities of ester and amide I absorption bands decreased slightly in Arabidopsis but increased in ice plant, suggesting that the cell expansion and protein synthesis ceased in Arabidopsis but continued in ice plant. In both plants, the shift in amide I absorption band was observed hourly after salt stress, indicating a rapid conformational change of cellular proteins. Analyses of the ratio between major and minor amide I absorption band revealed that ice plant was able to maintain a higher-ordered form of proteins under stress. Furthermore, the changes in protein conformation showed a positive correlation to the leaf sodium contents in ice plant, but not in Arabidopsis. PMID- 12376667 TI - Coupling sap flow velocity and amino acid concentrations as an alternative method to (15)N labeling for quantifying nitrogen remobilization by walnut trees. AB - The temporal dynamics of N remobilization was studied in walnut (Juglans nigra x regia) trees growing in sand culture. Trees were fed with labeled N ((15)N) during 1999 and unlabeled N in 2000. Total N and (15)N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and (15)N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, gamma-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed. PMID- 12376668 TI - Distinct N-terminal regulatory domains of Ca(2+)/H(+) antiporters. AB - The regulation of intracellular Ca(2+) levels is achieved in part by high capacity vacuolar Ca(2+)/H(+) antiporters. An N-terminal regulatory region (NRR) on the Arabidopsis Ca(2+)/H(+) antiporter CAX1 (cation exchanger 1) has been shown previously to regulate Ca(2+) transport by a mechanism of N-terminal auto inhibition. Here, we examine the regulation of other CAX transporters, both within Arabidopsis and from another plant, mung bean (Vigna radiata), to ascertain if this mechanism is commonly used among Ca(2+)/H(+) antiporters. Biochemical analysis of mung bean VCAX1 expressed in yeast (Saccharomyces cerevisiae) showed that N-terminal truncated VCAX1 had approximately 70% greater antiport activity compared with full-length VCAX1. A synthetic peptide corresponding to the NRR of CAX1, which can strongly inhibit Ca(2+) transport by CAX1, could not dramatically inhibit Ca(2+) transport by truncated VCAX1. The N terminus of Arabidopsis CAX3 was also shown to contain an NRR. Additions of either the CAX3 or VCAX1 regulatory regions to the N terminus of an N-terminal truncated CAX1 failed to inhibit CAX1 activity. When fused to N-terminal truncated CAX1, both the CAX3 and VCAX1 regulatory regions could only auto inhibit CAX1 after mutagenesis of specific amino acids within this NRR region. These findings demonstrate that N-terminal regulation is present in other plant CAX transporters, and suggest distinct regulatory features among these transporters. PMID- 12376669 TI - The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins. AB - Analyses of the protein content and composition revealed dramatic changes in gene expression during in situ banana (Musa spp.) fruit formation/ripening. The total banana protein content rapidly increases during the first 60 to 70 d, but remains constant for the rest of fruit formation/ripening. During the phase of rapid protein accumulation, an inactive homolog of class III chitinases accounts for up to 40% (w/v) of the total protein. Concomitant with the arrest of net protein accumulation, the chitinase-related protein (CRP) progressively decreases and several novel proteins appear in the electropherograms. Hence, CRP behaves as a fruit-specific vegetative storage protein that accumulates during early fruit formation and serves as a source of amino acids for the synthesis of ripening associated proteins. Analyses of individual proteins revealed that a thaumatin like protein, a beta-1,3-glucanase, a class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins. Because during the ripening of prematurely harvested bananas, similar changes take place as in the in situ ripening bananas, CRP present in immature fruits is a sufficient source of amino acids for a quasi-normal synthesis of ripening-associated proteins. However, it is evident that the conversion of CRP in ripening-associated proteins takes place at an accelerated rate, especially when climacteric ripening is induced by ethylene. The present report also includes a discussion of the accumulation of the major banana allergens and the identification of suitable promoters for the production of vaccines in transgenic bananas. PMID- 12376670 TI - Effect of producing sons on maternal longevity in premodern populations. PMID- 12376671 TI - Getting older. PMID- 12376672 TI - Scientific evidence. Judge rejects cancer data in Maryland cell phone suit. PMID- 12376673 TI - Bacterial meningitis. Appeal to thwart deadly outbreak. PMID- 12376674 TI - Herpetology. 100 frogs a-leaping for biodiversity. PMID- 12376676 TI - Physics. Quantum experiment asks 'how big is big?'. PMID- 12376675 TI - Toxicology. Protecting liver from painkiller's lethal dose. PMID- 12376677 TI - Switzerland. Compromise allows transgenic trials. PMID- 12376679 TI - Astronomy. New results reawaken quasar distance dispute. PMID- 12376678 TI - Young investigators. European program to fund the best. PMID- 12376680 TI - Microbiology. The science of Pfiesteria: elusive, subtle, and toxic. PMID- 12376681 TI - Microbiology. Pfiesterian lifestyle: simple or complex? PMID- 12376682 TI - Solar system formation. The first rocks whisper of their origins. PMID- 12376683 TI - Microbiology. Domino effects from battles against microbes. PMID- 12376685 TI - Women in science. Can equality in sports be repeated in the lab? PMID- 12376684 TI - Profile: Denny More. Learning to speak the Amazon's languages. PMID- 12376686 TI - Ornithology. High-flying science seeks to reduce toll at towers. PMID- 12376687 TI - Environment. Biodiversity update--progress in taxonomy. AB - Taxonomy and systematics underpin our ability to conserve and benefit from biodiversity in sustainable ways as envisaged under the Convention on Biological Diversity (CBD). Despite progress in phylogenetics towards reconstructing the "Tree of Life" and in biodiversity informatics, the fundamental documentation of species necessary to complete the inventory of life has lagged behind. It is argued that this reflects a lack of appreciation of the role played by species level taxonomic information in underpinning conservation and sustainable use and under investment in the relevant institutions at the expense of supporting the centralised financial mechanism of the CBD. PMID- 12376688 TI - Single molecules. Molecular entanglements. PMID- 12376689 TI - RNA events. No end to nonsense. PMID- 12376690 TI - Evolution. Jaws of the fates. PMID- 12376691 TI - Optics. A new low for nonlinear optics. PMID- 12376692 TI - Biomineralization. At the cutting edge. PMID- 12376693 TI - Neuroscience. Reconstructing a 3D world. PMID- 12376694 TI - Sri Lanka: an amphibian hot spot. PMID- 12376696 TI - A classical nova, V2487 Oph 1998, seen in x-rays before and after its explosion. AB - Classical nova explosions are very energetic and frequent phenomena caused by explosive hydrogen burning on top of an accreting white dwarf. Observations of the recent nova V2487 Oph 1998 by the X-ray Multi-Mirror satellite (XMM-Newton) provide evidence that accretion (probably on a magnetic white dwarf) was reestablished as early as 2.7 years after the explosion. In addition, positional correlation with a source previously discovered by the Rontgen Satellite (ROSAT) in 1990 suggests that the site of a nova explosion had been seen in x-rays before the outburst. PMID- 12376695 TI - High abrasion resistance with sparse mineralization: copper biomineral in worm jaws. AB - Biominerals are widely exploited to harden or stiffen tissues in living organisms, with calcium-, silicon-, and iron-based minerals being most common. In notable contrast, the jaws of the marine bloodworm Glycera dibranchiata contain the copper-based biomineral atacamite [Cu2(OH)3Cl]. Polycrystalline fibers are oriented with the outer contour of the jaw. Using nanoindentation, we show that the mineral has a structural role and enhances hardness and stiffness. Despite the low degree of mineralization, bloodworm jaws exhibit an extraordinary resistance to abrasion, significantly exceeding that of vertebrate dentin and approaching that of tooth enamel. PMID- 12376697 TI - Magnetic superstructure in the two-dimensional quantum antiferromagnet SrCu2(BO3)2. AB - We report the observation of magnetic superstructure in a magnetization plateau state of SrCu2(BO3)2, a frustrated quasi-two-dimensional quantum spin system. The Cu and B nuclear magnetic resonance (NMR) spectra at 35 millikelvin indicate an apparently discontinuous phase transition from uniform magnetization to a modulated superstructure near 27 tesla, above which a magnetization plateau at 1/8 of the full saturation has been observed. Comparison of the Cu NMR spectrum and the theoretical analysis of a Heisenberg spin model demonstrates the crystallization of itinerant triplets in the plateau phase within a large rhomboid unit cell (16 spins per layer) showing oscillations of the spin polarization. Thus, we are now in possession of an interesting model system to study a localization transition of strongly interacting quantum particles. PMID- 12376698 TI - Stimulated Raman scattering in hydrogen-filled hollow-core photonic crystal fiber. AB - We report on stimulated Raman scattering in an approximately 1-meter-long hollow core photonic crystal fiber filled with hydrogen gas under pressure. Light was guided and confined in the 15-micrometer-diameter hollow core by a two dimensional photonic bandgap. Using a pulsed laser source (pulse duration, 6 nanoseconds; wavelength, 532 nanometers), the threshold for Stokes (longer wavelength) generation was observed at pulse energies as low as 800 +/- 200 nanojoules, followed by a coherent anti-Stokes (shorter wavelength) generation threshold at 3.4 +/- 0.7 microjoules. The pump-to-Stokes conversion efficiency was 30 +/- 3% at a pulse energy of only 4.5 microjoules. These energies are almost two orders of magnitude lower than any other reported energy, moving gas based nonlinear optics to previously inaccessible parameter regimes of high intensity and long interaction length. PMID- 12376699 TI - Rapid vapor deposition of highly conformal silica nanolaminates. AB - Highly uniform and conformal coatings can be made by the alternating exposures of a surface to vapors of two reactants, in a process commonly called atomic layer deposition (ALD). The application of ALD has, however, been limited because of slow deposition rates, with a theoretical maximum of one monolayer per cycle. We show that alternating exposure of a surface to vapors of trimethylaluminum and tris(tert-butoxy)silanol deposits highly conformal layers of amorphous silicon dioxide and aluminum oxide nanolaminates at rates of 12 nanometers (more than 32 monolayers) per cycle. This process allows for the uniform lining or filling of long, narrow holes. We propose that these ALD layers grow by a previously unknown catalytic mechanism that also operates during the rapid ALD of many other metal silicates. This process should allow improved production of many devices, such as trench insulation between transistors in microelectronics, planar waveguides, microelectromechanical structures, multilayer optical filters, and protective layers against diffusion, oxidation, or corrosion. PMID- 12376700 TI - Neural correlates for perception of 3D surface orientation from texture gradient. AB - A goal in visual neuroscience is to reveal how the visual system reconstructs the three-dimensional (3D) representation of the world from two-dimensional retinal images. Although the importance of texture gradient cues in the process of 3D vision has been pointed out, most studies concentrate on the neural process based on binocular disparity. We report the neural correlates of depth perception from texture gradient in the cortex. In the caudal part of the lateral bank of intraparietal sulcus, many neurons were selective to 3D surface orientation defined by texture gradient, and their response was invariant over different types of texture pattern. Most of these neurons were also sensitive to a disparity gradient, suggesting that they integrate texture and disparity gradient signals to construct a generalized representation of 3D surface orientation. PMID- 12376701 TI - Extracting 3D from motion: differences in human and monkey intraparietal cortex. AB - We compared three-dimensional structure-from-motion (3D-SFM) processing in awake monkeys and humans using functional magnetic resonance imaging. Occipital and midlevel extrastriate visual areas showed similar activation by 3D-SFM stimuli in both species. In contrast, intraparietal areas showed significant 3D-SFM activation in humans but not in monkeys. This suggests that human intraparietal cortex contains visuospatial processing areas that are not present in monkeys. PMID- 12376702 TI - Coordinate regulation of transcription and splicing by steroid receptor coregulators. AB - Recent observations indicating that promoter identity influences alternative RNA processing decisions have created interest in the regulatory interactions between RNA polymerase II transcription and precursor messenger RNA (pre-mRNA) processing. We examined the impact of steroid receptor-mediated transcription on RNA processing with reporter genes subject to alternative splicing driven by steroid-sensitive promoters. Steroid hormones affected the processing of pre-mRNA synthesized from steroid-sensitive promoters, but not from steroid-unresponsive promoters, in a steroid receptor-dependent and receptor-selective manner. Several nuclear receptor coregulators showed differential splicing effects, suggesting that steroid hormone receptors may simultaneously control gene transcription activity and exon content of the product mRNA by recruiting coregulators involved in both processes. PMID- 12376705 TI - Evidence-based consensus statements and clinical guidelines: do the means meet the ends? PMID- 12376703 TI - Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. AB - We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild type but not in CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents. PMID- 12376704 TI - Subthalamic GAD gene therapy in a Parkinson's disease rat model. AB - The motor abnormalities of Parkinson's disease (PD) are caused by alterations in basal ganglia network activity, including disinhibition of the subthalamic nucleus (STN), and excessive activity of the major output nuclei. Using adeno associated viral vector-mediated somatic cell gene transfer, we expressed glutamic acid decarboxylase (GAD), the enzyme that catalyzes synthesis of the neurotransmitter GABA, in excitatory glutamatergic neurons of the STN in rats. The transduced neurons, when driven by electrical stimulation, produced mixed inhibitory responses associated with GABA release. This phenotypic shift resulted in strong neuroprotection of nigral dopamine neurons and rescue of the parkinsonian behavioral phenotype. This strategy suggests that there is plasticity between excitatory and inhibitory neurotransmission in the mammalian brain that could be exploited for therapeutic benefit. PMID- 12376706 TI - Energy expenditure and physical activity of obese children: cross-sectional study. AB - OBJECTIVES: To investigate the total daily energy expenditure and physical activity pattern of a group of obese and non-obese Hong Kong children. DESIGN: Cross-sectional study. SETTINGS: University teaching hospital, Hong Kong. PARTICIPANTS: Eighteen obese children aged 6 to 17 years and 18 age- and sex matched non-obese children in the local Hong Kong community. MAIN OUTCOME MEASURES: Total daily energy expenditure and physical activity pattern were estimated for 3 days using heart rate monitoring. Body composition was measured by dual-energy X-ray absorptiometry. RESULTS: In obese children, both total fat mass and fat-free mass were greater than in non-obese children. Total daily energy expenditure and its sleep and sedentary components were higher in absolute terms (by 42%, 43%, and 126%, respectively) for obese children. When normalised for body weight, the basal metabolic rate was no different between obese and non obese children, while the total daily energy expenditure of the obese children was significantly lower (by 22%) than that of non-obese children. When normalised for fat-free mass, the basal metabolic rate and the sedentary component of total daily energy expenditure were significantly higher for obese children. Obese children spent 12% less time asleep, but 51% more time in sedentary activity and 30% less time physically active: a ratio of active-to-sedentary waking time of 0.6 for obese children and 1.9 for non-obese children. CONCLUSIONS: Although the basal metabolic rate may be influenced by body composition, the finding of a normal basal metabolic rate when normalised for body weight suggests that an intrinsic difference of metabolic rate is not a major contributory cause of obesity. The study pointed particularly to the potential benefit of increasing physical exercise time relative to sedentary activities to reduce the prevalence of childhood obesity. Obese and non-obese children had similar basal metabolic rates when adjusted by fat-free mass and fat mass. Obese children spent more time in sedentary activities. PMID- 12376707 TI - Prognosis of patients with ventricular fibrillation in out-of-hospital cardiac arrest in Hong Kong: prospective study. AB - OBJECTIVE: To determine the prognosis of patients with ventricular fibrillation in out-of-hospital cardiac arrest in Hong Kong and examine its relationship with the other links in the chain of survival. DESIGN: Prospective descriptive study. SETTING: Three accident and emergency departments, Hong Kong. PARTICIPANTS: Patients older than 18 years with non-traumatic out-of-hospital cardiac arrest who were transported to the hospitals by ambulance between 15 March 1999 and 15 October 1999. MAIN OUTCOME MEASURES: Demographic data, characteristics of the cardiac arrest and the response times of the emergency medical service according to the Utstein style, and survival to hospital discharge rate. RESULTS: Three hundred and twenty patients were included. The incidence of ventricular fibrillation in this group of patients was 14.1%. The chance of survival to hospital discharge was significantly higher for patients with ventricular fibrillation than those with other rhythms of cardiac arrest (4.4% versus 0.7%). Approximately 40.0% of all cardiac arrests were witnessed. The bystander cardiopulmonary resuscitation rate was low at 15.6%. The median intervals for recognition to activation of the emergency medical service, time to cardiopulmonary resuscitation, time to defibrillation, and time to advanced life support were 1, 8, 9, and 27 minutes, respectively. CONCLUSION: Patients with ventricular fibrillation in out-of-hospital cardiac arrest have a better chance of survival than those with other cardiac rhythms. Further improvement requires simultaneous strengthening of all four links in the chain of survival. PMID- 12376708 TI - Breast conservation treatment in Hong Kong-early results of 203 patients: retrospective study. AB - OBJECTIVE: To study the clinical outcomes of patients with invasive or non invasive breast cancer after breast conservation treatment. DESIGN: Retrospective study. SETTING: Clinical oncology department of a public hospital, Hong Kong. PATIENTS: Two hundred and three patients who received postlumpectomy radiotherapy at the Pamela Youde Nethersole Eastern Hospital between January 1994 and June 1999. INTERVENTIONS: Adjuvant radiotherapy with or without systemic adjuvant treatment. MAIN OUTCOME MEASURES: Actuarial local control rate, progression-free survival rate, disease-specific survival rate, and cosmetic score. RESULTS: The median follow-up was 3.5 years. Two of the 25 patients with carcinoma in situ only developed local recurrence; the 5-year actuarial local control rate was 91.3%. Among the 178 patients with invasive cancer, seven had a local recurrence and 12 developed distant metastases without local failure. The 5-year actuarial local control, progression-free survival, and disease-specific survival rates for patients with invasive cancer were 95.5%, 85.8%, and 95.2%, respectively. The risk of local recurrence was significantly increased for younger patients (age <40 years) and those with positive final margins. Cosmetic scores were rated good to excellent by 95.6% of patients. CONCLUSIONS: The early clinical outcomes of these patients are comparable to those in large overseas trials, which have demonstrated the equivalence of mastectomy and breast conservation treatment in terms of survival. In addition to mastectomy, with or without breast reconstruction, breast conservation treatment should be offered as an alternative to suitable Chinese women. To maximise local control, further excision or mastectomy is recommended for patients with positive final margins. PMID- 12376709 TI - Hepatic resection for colorectal liver metastases: prospective study. AB - OBJECTIVE: To assess the operative and long-term survival outcomes of hepatic resection for colorectal liver metastases during an 11-year period in a tertiary referral centre in Hong Kong. DESIGN: Prospective study. SETTING: University teaching hospital, Hong Kong. SUBJECTS AND METHODS: Between January 1989 and December 1999, 72 patients underwent hepatic resection for colorectal liver metastases. Clinical, pathological, and outcome data were prospectively collected and analysed. Factors affecting long-term survival were also evaluated. RESULTS: Twenty-five (34.7%) patients were found to have synchronous hepatic metastasis at the time of colorectal resection. Fifty-two (72.2%) patients underwent major hepatic resection. The operative morbidity and hospital mortality rates were 19% and 4%, respectively. The 5-year survival rate after hepatectomy was 31.9%. The median disease-free survival and median overall cumulative survival were 18.5 months and 30.8 months, respectively. On multivariate analysis, a high preoperative serum carcinoembryonic antigen level (>200 ng/mL) and tumour involvement of the resection margin at histology were the two independent risk factors that adversely affected survival outcome. CONCLUSION: Hepatic resection for colorectal liver metastases can be performed safely, with minimal operative mortality and acceptable morbidity, and results in satisfactory survival. High preoperative serum carcinoembryonic antigen level and histological involvement of resection margin by cancer adversely affect the survival outcome. PMID- 12376710 TI - Factors affecting uptake of cervical and breast cancer screening among perimenopausal women in Hong Kong. AB - OBJECTIVES: To identify factors affecting cervical and breast cancer screening attendance among women aged 44 to 55 years by comparing self-reported uptake of cervical smear and clinical breast examination between patients and a population sample. DESIGN AND SETTING: Telephone survey and audit of clinic records to confirm patients' self-report. PARTICIPANTS: Two thousand and sixty-seven women identified through random telephone dialling from the residence directory and 319 patients ever-registered at a family practice teaching clinic. MAIN OUTCOME MEASURES: Uptake of cervical smear and clinical breast examination. RESULTS: The proportion of women undergoing cervical smear tests and clinical breast examination in the previous 12 months were 35.4% and 22.6%, respectively, for randomly selected women, while the figures were 47.2% and 50.6%, respectively, for patients. Record audit confirmed high rates of screening for patients according to evidence-based protocols (85.1% had had a cervical smear within 3 years). For women in the random sample (mean age, 48.9 years; standard deviation, 3.3 years), those who were older, postmenopausal, not receiving hormone therapy, educated to primary level, and with no chronic diseases were least likely to have had screening. For clinic patients (mean age, 47.9 years; standard deviation, 2.8 years), lower education level was the only variable associated with no recent smears. CONCLUSIONS: Healthy perimenopausal and postmenopausal women in the community with lower educational level and not receiving hormone therapy were more likely to be underscreened. Attendance of 44- to 55-year-old women at a family medicine clinic that actively promotes preventive medicine was associated with high screening uptake. PMID- 12376711 TI - Phenotype and management of patients with familial adenomatous polyposis in Hong Kong: perspective of the Hereditary Gastrointestinal Cancer Registry. AB - OBJECTIVES: To report on the phenotypic spectrum and clinical management of Chinese patients suffering from the rare autosomal dominant colorectal cancer syndrome of familial adenomatous polyposis. DESIGN: Analysis of prospectively collected data from the database of a regional registry. SETTING: The Hereditary Gastrointestinal Cancer Registry, Hong Kong. PARTICIPANTS: One hundred and eight patients with proven familial adenomatous polyposis from 36 local Chinese families with the condition recruited to the Registry from 1995 to 2001. INTERVENTIONS: Screening programme for at-risk family members, prophylactic surgery at presymptomatic diagnosis, and surveillance programme for extracolonic lesions in affected individuals. MAIN OUTCOME MEASURES: Rate of colorectal cancer, type of surgical treatment, spectrum of extracolonic lesions, and management of the syndrome. RESULTS: Fifty patients suffered from colorectal cancer with a mortality rate of 78.0%. The strategy of presymptomatic diagnosis by screening and appropriate prophylactic surgery reduced the incidence of colorectal cancer. Affected individuals were prone to develop potentially serious extracolonic lesions including thyroid cancer (5.7%), desmoid tumour (15.7%), gastroduodenal adenomas (7.1%), duodenal microadenoma (17.1%), and pouch polyposis (17.4%). CONCLUSIONS: Screening and prophylactic surgery are effective ways to prevent colorectal cancer for patients with familial adenomatous polyposis. Lifelong regular surveillance is necessary to detect and manage extracolonic lesions. A dedicated registry is essential to coordinate clinical management and to compile data for furthering knowledge of this rare but complex syndrome. PMID- 12376712 TI - Acute care service utilisation and the possible impacts of a user-fee policy in Hong Kong. AB - OBJECTIVES: To examine the utilisation pattern of accident and emergency services and to study the possible impact of a user-fee policy on non-emergency attendances in Hong Kong. DESIGN: Retrospective study. METHODS: Four different scenarios are postulated to examine the impact on the number of accident and emergency attendances of a user-fee policy from 2000 to 2029. Patient volume data of accident and emergency attendances for 2000 were made available by the Hospital Authority of Hong Kong. RESULTS: Non-emergency use of the accident and emergency services is the main cause of over-utilisation and contributes to more than 70.0% of its use. Only 22.0% of patients attending accident and emergency departments were admitted to a ward for further treatment. By 2029, the number of accident and emergency attendances would increase by more than 47.0% if the present utilisation pattern prevails. However, if patients at triage levels 3, 4, and 5 were discouraged from using the accident and emergency services, the number of attendances would decrease by 76.4%. CONCLUSION: The proposed user-fee policy would act as a deterrent by preventing unnecessary use of accident and emergency services. However, the use of out-patient services may be increased as a result and attendance should be carefully monitored. Community health education and civic education relating to abuse of accident and emergency services would be effective in reducing over-utilisation of these services. PMID- 12376713 TI - Sudden unexpected death in epilepsy. AB - Sudden unexpected death in epilepsy is the most common category of seizure related death for patients who develop chronic epilepsy, accounting for up to 17% of epilepsy deaths. Sudden unexpected death in epilepsy is defined as a sudden, unexpected, non-accidental death in an individual with epilepsy with or without evidence of a seizure having occurred (excluding documented status epilepticus) and where autopsy does not reveal an anatomical or toxicological cause of death. Incidence rates range between 0.35 and 2.70 per 1000 person-years in the population-based studies and between 1.50 and 9.30 per 1000 person-years in selected cohorts. Seizure frequency appears to be an important factor in sudden unexpected death in epilepsy, although the exact pathogenetic mechanisms involved are unclear. PMID- 12376714 TI - Pyothorax-associated large B-cell lymphoma: case report with emphasis on the potential diagnostic challenge. AB - A rare case of pyothorax-associated large B-cell lymphoma occurring in Hong Kong is reported. The patient was a 64-year-old Chinese male who presented with shortness of breath and pleuritic pain. Radiological examination revealed left pleural thickening associated with bilateral pleural effusion. Open biopsy of the thickened parietal pleura revealed occasional large malignant lymphoid cells of B lineage admixed with fibrin and hyalinised fibrous tissue. These lymphoma cells were shown to harbour both Epstein-Barr virus and human herpesvirus type 8 by in situ hybridisation and immunohistochemical study, respectively. There was no associated lymphadenopathy and hepatosplenomegaly. The clinicoradiological presentation and pathological findings thus fulfilled the criteria of the so called pyothorax-associated large B-cell lymphoma. Awareness of this rare entity, together with diligent histological examination and proper application of ancillary investigative techniques, are essential for making a correct diagnosis. The co-infection with Epstein-Barr virus and human herpesvirus type 8 in this case also suggests a possible pathogenetic relationship between pyothorax associated large B-cell lymphoma and primary effusion lymphoma. PMID- 12376715 TI - Corrosive oesophageal injury following vinegar ingestion. AB - A 39-year-old woman drank one tablespoon of white vinegar in order to 'soften' crab shell stuck in her throat. Endoscopy revealed inflammation of the oropharynx and second-degree caustic injury of the oesophagus extending to the cardia. She had an uneventful recovery. This case report confirmed that vinegar could cause ulcerative injury to the oropharynx and oesophagus. The folklore application of vinegar 'dislodging' a foreign body in the throat should be strongly discouraged. PMID- 12376716 TI - Unusual muscle pain in two patients with diabetic renal failure. AB - We report on two patients with diabetic muscle infarct, a painful musculoskeletal disorder complicating longstanding diabetes with established microangiopathy. Both patients had renal failure that was treated by dialysis. The underlying pathophysiological process was considered to be an arterial vascular event mediated through ischaemia-reperfusion injury. Clinicians should be alert to this condition. T2-weighted magnetic resonance imaging was valuable in establishing the diagnosis. PMID- 12376717 TI - Patient empowerment--a patient-centred approach to improve care. PMID- 12376718 TI - The challenge of chronic conditions in Hong Kong. PMID- 12376719 TI - Nutcracker phenomenon presenting as left varicocele. PMID- 12376720 TI - Pott's puffy tumour. PMID- 12376721 TI - Viability of the health protection account in Hong Kong. PMID- 12376723 TI - Liver transplantation in Hong Kong. PMID- 12376724 TI - Schistosome glycoconjugates in host-parasite interplay. AB - Schistosomes are digenetic trematodes which cause schistosomiasis, also known as bilharzia, one of the main parasitic infections in man. In tropical and subtropical areas an estimated 200 million people are infected and suffer from the debilitating effects of this chronic disease. Schistosomes live in the blood vessels and strongly modulate the immune response of their host to be able to survive the hostile environment that they are exposed to. It has become increasingly clear that glycoconjugates of schistosome larvae, adult worms and eggs play an important role in the evasion mechanisms that schistosomes utilise to withstand the immunological measures of the host. Upon infection, the host mounts innate as well as adaptive immune responses to antigenic glycan elements, setting the immunological scene characteristic for schistosomiasis. In this review we summarise the structural data now available on schistosome glycans and provide data and ideas regarding the role that these glycans play in the various aspects of the glycobiology and immunology of schistosomiasis. PMID- 12376726 TI - Didecyl squarate--a practical amino-reactive cross-linking reagent for neoglycoconjugate synthesis. AB - The present paper describes the synthesis and use of the hydrophobic squaric decyl ester glycosides in neoglycoconjugate chemistry. The 2-aminoethyl glycosides of alpha-D-mannopyranose, lactose, globotriose, globotetraose, GM3, and sialyl Lewis(x), as well as the 2-(2-aminoethylthio)ethyl glycoside of alpha D-mannopyranose, beta-D-glucopyranose, and galabiose were reacted with squaric acid didecyl ester to afford the hydrophobic squaric decyl ester glycosides. These glycosides were efficient reagents for the conjugation to amino-functional microtiter plates, BSA and aminated Sepharose EAH 4B. The decyl ester moiety of the squaric decyl ester glycosides constitutes a traceless hydrophobic tag, which has the major advantage, as compared to the corresponding ethyl esters, that it enables easy purification of the glycosides with silica chromatography and that unreacted excesses glycosides from conjugation reaction mixtures can easily be recovered by means of C18 solid phase extraction. PMID- 12376727 TI - Intact Golgi synthesize complex branched O-linked chains on glycoside primers: evidence for the functional continuity of seven glycosyltransferases and three sugar nucleotide transporters. AB - We examined the functional co-localization and continuity of glycosyltransferases and sugar nucleotide transporters in the Golgi of two Chinese hamster ovary (CHO) cell lines that synthesize different types of O-linked oligosaccharides. CHO cells normally synthesize primarily Sia2,3Galbeta1,3GalNAc- on glycoproteins. CHO cells transfected with core-2 GlcNAc transferase (Core 2) can synthesize glycoproteins containing branched O-linked oligosaccharides with poly-N acetyllactosamines. CHO lines incubated with [(3)H]galactose and GalNAc-alpha phenyl (GAP) as a primer, synthesize labeled glycoside products that faithfully resemble those found on the endogenous acceptors: CHO cells make Sia2,3[(3)H]Gal(beta)1,3GAP, while CHO Core2 cells synthesize GAPs with complex branched chains including poly-N-acetyllactosamines. To determine if isolated Golgi preparations make similar products, we prepared Golgi by established homogenization methods, documented their intactness, and added tracer UDP [(3)H]Gal, unlabeled sugar nucleotides, and GAP. CHO Golgi preparations synthesized only Sia2,3[(3)H]Gal(beta)1,3GAP. CHO Core2, also made this product and a small amount of Core-2 GlcNAc transferase-dependent products. No endogenous glycoproteins were labeled. However, when either cell line was gently permeabilized with streptolysin-O or given hypo-osmotic shock, both GAP and endogenous acceptors were efficiently glycosylated within an intact functional Golgi lumen and remained there. Significantly, Golgi from CHO Core2 cells made mostly branched GAP products including some with poly-N-acetyllactosamines as complex as those made and secreted by living cells incubated with GAP. These results suggest that the lumen of the Golgi apparatus is functionally continuous or interconnected. Once glycosides diffuse into the Golgi lumen, they have access to all the sugar nucleotide transporters and glycosyltransferases used for complex GAP-based products without requiring metabolic energy or inter-vesicular transport. Glycosylation of artificial acceptors could be used to track the functional continuity or co-localization of multiple glycosyltransferases and transporters under conditions where Golgi morphology disintegrates and/or reappears. PMID- 12376728 TI - Comparison of oligosaccharides derived from salivary mucin of Japanese secretor and non-secretor individuals of blood group type-A. AB - Comparison of oligosaccharide components derived from salivary mucin was performed between secretor and non-secretor individuals. Salivary mucin was collected from four secretors and three non-secretors having blood group type-A. Compositional analysis showed that the contents of galactose and N acetylglucosamine in the non-secretor were higher than those in the secretor. The O-linked oligosaccharides obtained by treatment with alkaline borohydride were separated by gel filtration using Sephadex G-50. The results indicated that the size of the type-A active oligosaccharides from the secretor was similar to or smaller than that of the non-secretor. Ion-exchange chromatography showed that the secretors had strong type-A activities in both the neutral and acidic fractions but the non-secretors showed type-A activity mainly in the neutral fraction. These results suggest that compositional differences in blood group substances exist between secretors and non-secretors. PMID- 12376725 TI - Plant lectins: occurrence, biochemistry, functions and applications. AB - Growing insights into the many roles of glycoconjugates in biorecognition as ligands for lectins indicates a need to compare plant and animal lectins. Furthermore, the popularity of plant lectins as laboratory tools for glycan detection and characterization is an incentive to start this review with a brief introduction to landmarks in the history of lectinology. Based on carbohydrate recognition by lectins, initially described for concanavalin A in 1936, the chemical nature of the ABH-blood group system was unraveled, which was a key factor in introducing the term lectin in 1954. How these versatile probes are produced in plants and how they are swiftly and efficiently purified are outlined, and insights into the diversity of plant lectin structures are also given. The current status of understanding their functions calls for dividing them into external activities, such as harmful effects on aggressors, and internal roles, for example in the transport and assembly of appropriate ligands, or in the targeting of enzymatic activities. As stated above, attention is given to intriguing parallels in structural/functional aspects of plant and animal lectins as well as to explaining caveats and concerns regarding their application in crop protection or in tumor therapy by immunomodulation. Integrating the research from these two lectin superfamilies, the concepts are discussed on the role of information-bearing glycan epitopes and functional consequences of lectin binding as translation of the sugar code (functional glycomics). PMID- 12376729 TI - Localized prostate cancer. PMID- 12376738 TI - Catalog of 86 single-nucleotide polymorphisms (SNPs) in three uridine diphosphate glycosyltransferase genes: UGT2A1, UGT2B15, and UGT8. AB - We report here three high-density maps of variations found among 48 Japanese individuals in three uridine diphosphate glycosyltransferase (UGT) genes, UGT2A1, UGT2B15, and UGT8. A total of 86 single-nucleotide polymorphisms (SNPs) were identified through systematic screening of genomic regions containing these genes: 8 in 5' flanking regions, 7 in coding regions, 67 in introns, 3 in 3' untranslated regions, and 1 in a 3' flanking region. We also discovered 14 variations of other types. Of the 86 SNPs, 63 (73%) were considered to be novel on the basis of comparison of our data with the Database of SNPs (dbSNP) of the National Center for Biotechnology Information. Among the seven SNPs identified in exonic sequences, five were non-synonymous changes that would result in amino acid substitutions. The collection of SNPs derived from this study will serve as an additional resource for studies of complex genetic diseases and responsiveness to drug therapy. PMID- 12376740 TI - Structural analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency. AB - Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. More than 90% of CAH cases are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed "apparent gene conversion," from the neighboring CYP21Ppseudogene. A chimeric CYP21P/CYP21gene with its 5' end corresponding to CYP21P and 3' end corresponding to CYP21 has been identified. This type of gene is nonfunctional because it produces a truncated protein. We found two distinct chimeric genes in CAH patients. Both genes had a sequence with -300 nucleotides of the 5' head as the CYP21P gene. The coding region consisted of a fusion molecule with the CYP21P gene in two different regions. One of the junctions was located in the chi-like sequence of GCTGGGC in the third intron and the other was in the minisatellite consensus TGGCAGGAGG of exon 5 of the CYP21P gene. In addition, analysis of restriction fragment length polymorphism for these two 3.3-kb chimeric molecules showed that these sequences arose as a consequence of unequal crossover between the CYP21Pand CYP21 genes. It is plausible that both consensus sequences are responsible for the gene conversion of these two chimeric genes. PMID- 12376739 TI - Familial 14-Mb deletion at 21q11.2-q21.3 and variable phenotypic expression. AB - We report a familial case with a proximal interstitial deletion of chromosome 21q [del(21q)]. Although the mother in the family was phenotypically normal, her first child was affected with both sensorineural hearing loss and moderate mental retardation, and the second affected child had mild mental retardation but not sensorineural hearing loss. We determined breakpoints of the del(21q) in the mother and her two affected children by fluorescence in situ hybridization analysis using 45 DNA clones and the molecular analysis using 21 DNA markers. The proximal breakpoint of the del(21q) was located at a region between 0.33 Mb and 0.46 Mb distal to the centromere, and the distal breakpoint was at a region between 14.6 Mb and 14.9 Mb. The finding indicates that the three individuals had an approximate 14-Mb deletion within 21q11.2-q21.3. Molecular analysis showed that both affected children shared the same maternal haplotype of their del(21q), but a crossover was detected in the paternally inherited normal chromosome 21. These data suggest that unmasking of deleterious genes on the paternally derived chromosome 21 of the two children as a result of the deletion may affect the extent of their mental retardation and/or sensorineural hearing loss. Usher syndrome 1E may be a candidate disease locus related to the sensorineural hearing loss of the first child. PMID- 12376741 TI - Yfm1, a multicopy marker specific for the Y chromosome and beneficial for forensic, population, genetic, and spermatogenesis-related studies. AB - A recently developed microsatellite marker on the Y chromosome, Yfm1, which was originally cloned from a cosmid clone mapped near the DAZ (Deleted in AZoospermia) genes, was used to classify Y chromosomes using an automatic sequencer. Yfm1 could detect multicopies on Y chromosomes in a single polymerase chain reaction, showing four main classes, A, A*, B, and C, according to the number of copies and peak patterns. Compound haplotype analysis of the Y chromosome using the Yfm1 marker with three other biallelic markers on the Y chromosome, SRY, DXYS5Y, and YAP, resulted in nine different haplotypes among different populations, including Japanese. Haplotype II (defined by YAP insertion) observed in the Japanese population was consistently associated with Yfm1 class A or A*, which showed the lowest number of copies of Yfm1. Haplotypes III and IV were consistently associated with Yfm1 class B. On the other hand, haplotype I showed a variety of Yfm1 patterns that were dubbed class C when not appropriately classified as A, A*, or B. These relationships among Yfm1 microsatellite and Y-specific biallelic markers could supply useful population genetic information. Moreover, because we have already shown that men with haplotype II have significantly lower spermatogenic ability than those with other haplotypes, Yfm1 class A or A* with the least number of copies may be related to the haplotype II-specific structure of the Y chromosome, such as deletion of DAZ or DAZ repeats, reflecting the lower spermatogenic abilities of Japanese haplotype II men. Thus, Yfm1 represents a very useful marker for analysis of genetic structure in different populations and studies on Y chromosome lineage specific genotype-phenotype correlations. PMID- 12376742 TI - Ovarian cancer of endometrioid type as part of the MSH6gene mutation phenotype. AB - The MSH6 gene is one of the DNA mismatch repair genes involved in development of inherited cancers, predominantly of the colorectum and endometrium. Herein we describe the first Polish MSH6 family and the pathological and clinical data about the ovarian cancer diagnosed in the proband. Our results and reports by others indicate that, besides colorectal and endometrial cancer, the late-onset endometrioid type of ovarian cancer can be a feature of families with MSH6 germline mutations. PMID- 12376743 TI - Single-nucleotide polymorphisms in the class II region of the major histocompatibility complex in Japanese patients with immunoglobulin A nephropathy. AB - Immunoglobulin A nephropathy (IgAN) is a form of chronic glomerulonephritis of unknown etiology and pathogenesis. Immunogenetic studies have not conclusively indicated that human leukocyte antigen (HLA) is involved. As a first step in investigating a possible relationship between HLA class II genes and IgAN, we analyzed the extent of linkage disequilibrium (LD) in this region of chromosome 6p21.3 in a Japanese test population and found extended LD blocks within the class II locus. We designed a case-control association study of single-nucleotide polymorphisms (SNPs) in each of those LD blocks, and determined that SNPs located in the HLA- DRA gene were significantly associated with an increased risk of IgAN ( P = 0.000001, odds ratio = 1.91 [95% confidence interval 1.46-2.49]); SNPs in other LD blocks were not. Our data imply that some haplotype of the HLA- DRA locus has an important role in the development of IgAN in Japanese patients. PMID- 12376744 TI - No intraindividual variation of disomy rate in sperm samples. AB - We assessed possible inter- and intraindividual variations in the frequency of disomy in sperm cells from three men with abnormal sperm analysis parameters. Mono- and dual-color fluorescence in situ hybridization (FISH) was applied to sperm cells from different samples of the men. Four men with a normal sperm profile were used as controls. The samples were taken separately over a period of 6 months. FISH probes used for the disomy rate analysis were clones from the satellite region of chromosomes 8, 18, X, and Y. The study group showed a significantly higher disomy rate compared with the control group, whereas there was no significant difference in the disomy rate between three different samples from the same individuals. These results suggest that the sampling time has no importance in assessing the rate of nondisjunction in sperm cells. PMID- 12376745 TI - Two families with Wilson disease in which siblings showed different phenotypes. AB - We investigated two families with Wilson disease in which siblings showed different clinical phenotypes and different ages at onset. In Family 1, the second and fourth male children demonstrated onset of the neurological type of Wilson disease at 16 and 28 years of age, respectively, and the first female child developed the hepatic type at 38 years of age. In Family 2, the second male child showed neurological symptoms at 32 years of age and was diagnosed as having the hepatoneurological type of Wilson disease; then the 35-year-old first female child was found to have the hepatic type by familial screening. We performed mutation analysis of the ATP7B gene for these patients, and found that the mutation was a compound heterozygote in both families. Previous reports of siblings with Wilson disease have shown an identical clinical phenotype and similar ages at onset. In addition, hepatic-type cases generally occur at lower ages compared with the neurological type. In the present investigation, however, younger patients showed neurological symptoms earlier than their older siblings, and clinical phenotypes differed among siblings in both families. These cases appear to be rare. Individual differences in copper accumulation in hepatic cells and intolerance to copper toxicity might be the reason for this phenomenon. Furthermore, there might be a difference in the dominance of the allele expressing ATP7B protein among these cases, resulting in different clinical phenotypes, because all patients of both families were found to be compound heterozygotes. PMID- 12376746 TI - Molecular cloning and characterization of a novel human Rab ( Rab2B) gene. AB - Rab proteins are small-molecular-weight guanosine triphosphatases (GTPases) that control vesicular traffic in eukaryotic cells. The small GTPase Rab2 is a resident of pre-Golgi intermediates and is required for protein transport from the endoplasmic reticulum to the Golgi complex. We identified a novel human Rab (Rab2B) gene that was 2312 bp in length and encoded a protein of 216 amino acid residues. The protein shared high homology with mouse Rab2 (identity 83%, similarity 91%). The expression pattern of the human Rab2B gene showed that there is a transcript in kidney, prostate, lung, liver, thymus, colon, pancreas, and skeletal muscle, and low levels in placenta, whereas specific bands of the transcript could not be detected in heart, brain, spleen, testis, ovary, small intestine, and leukocyte. Overexpression has been observed in colon adenocarcinoma CX-1. The Rab2B gene consists of nine exons and eight introns and is mapped to chromosome 14q11.1-14q11.2 by bioinformatics analysis. PMID- 12376747 TI - Comparative study on deletions of the dystrophin gene in three Asian populations. AB - The frequency and distribution of deletions of 19 deletion-prone exons clustered in two hot spots in the proximal and central regions of the dystrophin gene were compared in three populations from Singaporean, Japan, and Vietnam. DNA samples obtained from 105 Singaporean, 86 Japanese, and 34 Vietnamese Duchenne muscular dystrophy patients were examined by polymerase chain reaction amplification. Deletions of the examined exons were found in 51.2% of Japanese patients but in 40.0% or less of the Singaporeans and Vietnamese. About two thirds of the deletions were localized in the central region and the remaining deletions were clustered at the proximal region. The most commonly deleted exons at the central deletion hot spot were exon 50 in the Singaporean, exons 49 and 50 in the Japanese, and exon 51 in the Vietnamese population. At the proximal deletion hot spot, the most commonly deleted exons were exons 6 and 8 in the Singaporeans, exons 12 and 17 in the Japanese, and exons 8 and 12 in the Vietnamese. Two cases each from Singapore and Japan had large-scale gross mutations spanning both deletion hot spots. Our results suggest that, although the presence and frequency of the two deletion hot spots may be similar in the three Asian populations analyzed, the distribution and frequency of deletions among the different exons can vary as a result of population-specific intronic sequences that predispose individuals to preferential deletion breakpoints. PMID- 12376748 TI - A girl with 1p36 deletion syndrome and congenital fiber type disproportion myopathy. AB - Chromosome 1p36 deletion syndrome is characterized by hypotonia, moderate to severe developmental and growth retardation, and characteristic craniofacial dysmorphism. Muscle hypotonia and delayed motor development are almost constant features of the syndrome. We report a 4-year-old Japanese girl with 1p36 deletion syndrome whose muscle pathology showed congenital fiber type disproportion (CFTD) myopathy. This is the first case report of 1p36 deletion associated with CFTD. This association may indicate that one of the CFTD loci is located at 1p36. Ski proto-oncogene -/- mice have phenotypes that resemble some of the features observed in patients with 1p36 deletion syndrome. Because fluorescent in situ hybridization analysis revealed that the human SKI gene is deleted in our patient, some genes in 1p36, including SKI proto-oncogene, may be involved in muscle hypotonia and delayed motor development in this syndrome. PMID- 12376749 TI - Potyviruses, novel and known, in cultivated and wild species of the family Apiaceae in Australia. AB - Three potyviruses were identified by gene sequencing and found to be widespread in species of Apiaceae in Australia. Only celery mosaic virus was found in celery crops and in one of 180 specimens of feral carrot ( Daucus carota). Another related but distinct novel potyvirus, carrot virus Y, was the only virus found in carrot crops and all except one feral carrot. A more distantly related novel potyvirus, apium virus Y, was found in plants of sea celery ( Apium prostratum), cultivated parsley ( Petroselinum crispum) and the immigrant weed species poison hemlock ( Conium maculatum). These three potyviruses, together with celery yellow mosaic virus of South America and the closely related carrot thin leaf virus and carrot virus B of North America, form a distinct subgenus of the Potyviridae most closely related to turnip mosaic virus and two potyviruses of yam; yam mosaic virus from the Ivory Coast and Japanese yam mosaic virus. Celery mosaic and carrot virus Y are probably recent migrants to Australia, but apium virus Y may have been endemic longer. In ELISA tests using polyclonal antibodies against virions of celery mosaic virus, some isolates of carrot virus Y were indistinguishable from celery mosaic virus, whereas others gave smaller absorbancy values, and those of apium virus Y did not react. This study shows the value of virus identification based on gene sequencing for planning control measures. PMID- 12376750 TI - Herpesviral, but no papovaviral sequences, are detected in cloacal papillomas of parrots. AB - Internal papillomatosis of parrots (IPP) is a tumour disease with unknown etiology, characterised by progressive development of papillomas in the oral and cloacal mucosa. Based on epidemiologic data, infectious agents, particularly DNA tumour viruses, are considered to be involved. In this study, cloacal papillomas were investigated by PCR for the presence of herpesvirus, papillomavirus and avian polyomavirus genomes, respectively. Using consensus and specific primers, 5 out of 12 papillomas were tested positive for herpesvirus; all papillomas were tested negative for papillomavirus and avian polyomavirus. The DNA sequence of one of the PCR products showed 86.5% homology to the corresponding region of the psittacine herpesvirus 1 DNA polymerase gene. Using a PCR with primers based on this sequence, additional 4 papillomas were tested positive. By in situ hybridisation, herpesviral sequences were detected in epithelial cells of the papilloma, but not in surrounding tissues. As 75% of the tumours proved to be positive, these data suggest an involvement of a herpesvirus in the etiology of IPP; the distinct role, however, needs to be investigated. PMID- 12376751 TI - Two-dimensional electrophoresis of proteins discriminates aphid clones of Sitobion avenae differing in BYDV-PAV transmission. AB - Aset of 39 F1 Sitobion avenae clones was obtained by selfing a poorly efficient BYDV-PAV vector clone. These clones were genetically typed by 11 microsatellite loci, and tested for BYDV-PAV4 transmission to barley. The 39 clones displayed a continuum in transmission percentages, from 0% to 88% with a significant clone effect. From this set, two highly efficient (HEV) and two poorly efficient (PEV) vectoring clones were more precisely characterized for transmission of two other PAV isolates. The molecular bases of the lower transmissibility of BYDV-PAV4 by PEV clones and of the aphid vectoring properties were investigated respectively by comparing the sequences corresponding to structural proteins (CP and RTD) of BYDV, and by using proteomic analysis of aphids in two dimensional electrophoresis (2-DE) with immobilized pH gradients (IPG) after an improved protein extraction. Four residues specific to BYDV-PAV4 located in the CP sequence (A(24) and L(130)) or in the RTD region (M(334) and S(456)) could be responsible for the lower transmissibility of this isolate by PEV clones. Among a total of 2150 well-resoluted spots scored on S. avenae proteinic pattern, only twelve proteins were qualitatively or quantitatively different between clones. Four out of them discriminated HEV and PEV groups. PMID- 12376752 TI - NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells. AB - Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation. PMID- 12376753 TI - Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration. AB - Hantaviruses cause two diseases of man, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Pathogenic and non-pathogenic hantaviruses use beta3 and beta1 integrins, respectively, to enter endothelial cells. Beta3 integrins were recently reported to bind receptors that regulate vascular permeability suggesting that hantavirus beta3 integrin interactions may regulate endothelial cell function and contribute to viral pathogenesis. In this study we investigated the ability of pathogenic and non-pathogenic hantaviruses to regulate beta3 and beta1 integrin directed endothelial cell functions. We found that pathogenic NY-1, SNV, HTN, SEO and PUU viruses blocked endothelial cell migration on beta3, but not beta1, integrin ligands. Migration is similarly inhibited by antibodies to beta3 integrins which selectively block vitronectin directed endothelial cell migration. As a result, the ability of endothelial cells to migrate on integrin ligands was selectively inhibited by only pathogenic hantaviruses. Infection by NY-1 virus inhibited endothelial cell migration as early as 24-48 h post-infection. In contrast, non-pathogenic PH and TUL viruses had no effect on the ability of endothelial cells to migrate on either beta3 or beta1 integrin ligands from 1 to 5 days post-infection. These findings indicate that only hantaviruses which use beta3 integrins, and are associated with HPS and HFRS diseases, functionally dysregulate endothelial cell migration. These findings further demonstrate that hantaviruses regulate only beta3 integrin directed endothelial cell functions and have no effect on beta1 integrin functions. Since beta3 integrins are linked to changes in vascular permeability and the maintenance of vascular integrity, these findings suggest a means by which hantavirus usage and regulation of beta3 integrins may contribute to hantavirus pathogenesis. PMID- 12376754 TI - Identification of two homologous antigenic peptides derived from L1 HPV-16 and 18 proteins specific for the HLA-B*3901 allele. AB - In this work we present evidence that the homologous peptides IHSMNSTIL and IHSMNSSIL derived from L1 HPV-16 and 18 proteins respectively, and with high specificity for the allele HLA-B*3901, according with an algorithm prediction program, induced T cell stimulation in patients with advanced cervical cancer positive for HPV-16 or 18 infection and for the HLA-B*3901 allele. Interestingly, T lymphocytes derived from a patient with HPV-18 infection and stimulated with the peptide IHSMNSTIL were capable to kill a cervical cancer cell line named Rova, derived from the tumor of the same patient. In addition, the cytotoxic activity was strongly increased when this cell line was previously treated with hrIFN-gamma. These results suggest that the CTL immune response to L1 HPV-16 and 18 protein derived epitopes is maintained in patients with advanced cervical cancer within specific alleles, and opens the possibility that homologous epitopes may be used in the generation of prophylactic vaccines for cervical tumors bearing different HPV-types. PMID- 12376755 TI - Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus. AB - The variability in coat protein gene sequences of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) that naturally infect orchids worldwide was investigated. Samples were collected from Korea, Singapore and Taiwan. The sequence data were compared with available published coat protein gene sequences of CymMV and ORSV, including those from Japan and Thailand. Among CymMV isolates, the homology was 89.1%-99.7% and 93.2%-100% at the nucleotide and amino acid levels, respectively. Among the ORSV isolates, the homology was 95.5%-100% and 93%-100% at the nucleotide and amino acid levels, respectively. No particular region of variability could be defined in either of the viruses. In deduced amino acid sequence, the N-terminal was more conserved than the C-terminal in both CymMV and ORSV. By comparing all sequences determined in this study and those that are published in the GenBank databases, we did not find clustering based on geographical distribution or sequence identity. Such high sequence conservation suggests that both CymMV and ORSV coat protein genes are suitable candidates to provide resistance to orchids cultivated in different geographical locations. PMID- 12376756 TI - Sequences in the hypervariable region 1 of hepatitis C virus show only minimal variability in the presence of antibodies against hypervariable region 1 during acute infection in chimpanzees. AB - We analyzed sequences of hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in six chimpanzees, experimentally infected with a single HCV inoculum, to clarify the correlation between HVR1 mutation and antibodies to HVR1. Two chimpanzees had been immunized with synthetic HVR1 peptides before HCV inoculation. All six animals became infected with HCV but cleared the infection within the acute phase. The major HVR1 sequences in longitudinal sera were unchanged in animals both with and without anti-HVR1 antibodies. Additionally, sequences of HVR1 variants in each chimpanzee converged after 11 to 19 weeks. The data show that anti-HVR1 antibodies are unlikely to drive variation in HVR1. PMID- 12376757 TI - Properties of a chimeric simian-human immunodeficiency virus expressing an hybrid HIV-1 Nef/SIVmac Nef protein. AB - The nef genes of human and simian immunodeficiency viruses code for a membrane associated protein critical for AIDS development. SIVmac Nef presents C-terminal a 27 amino acid extension absent of HIV-1 Nef. To estimate the influence of this C-terminal domain on virus properties, we constructed viruses derived from SIVmac239 by replacing SIV nef with HIV-1 Lai nef gene (SHIV NefLai4) or with a sequence encoding a Nef fusion protein: HIV-1 Lai Nef/SIV Nef-Cterm (SHIV-Cterm). The recombinant viruses replicated efficiently in vitro in CEMx174 cells and in activated macaque PBMCs. The addition of SIV Nef C-terminal domain to HIV-1 Nef in SHIVNefLai4 did not change the in vitro properties of the chimeric virus, both viruses being more infectious than a nef deleted virus. Although the half-life of Nef fusion protein was augmented, SHIV-Cterm remained slightly less infectious than SIVmac239. PMID- 12376758 TI - The complete genome sequence of gill-associated virus of Penaeus monodon prawns indicates a gene organisation unique among nidoviruses. AB - We report here a 5596 nt sequence comprising the 3'-end of the (+) ssRNA genome of gill-associated virus (GAV), an invertebrate nidovirus of Penaeus monodon prawns. The sequence extends from a subgenomic RNA start site 35 nt upstream of the 4923 nt ORF3 gene to a 3'-poly(A) tail of the 26235 nt genome of GAV. The putative 1640 amino acid (aa) ORF3 protein (MW = 182049 Da, pI = 6.62) contains 15 potential N-linked glycosylation sites, 15 potential O-linked glycosylation sites and six highly hydrophobic regions predicted to represent transmembrane (TM) domains. Three of the predicted TM domains occur in the amino-terminal 228 aa, two in the central portion, and one near the carboxy-terminus of ORF3. Only one short (83 aa) open reading frame (ORF4) was identified between ORF3 and the 3'-poly(A) tail. Completion of the genome sequence of GAV has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative ORF3 spike glycoprotein gene resides downstream of a gene (ORF2) encoding a structural protein associated with nucleocapsids. PMID- 12376759 TI - Characterisation of immunodominant protein encoded by the F1L gene of orf virus strains isolated in Italy. AB - We analysed the molecular properties of the immunodominant protein of different orf virus strains isolated in Italy. The F1L encoding genes and the deduced amino acid sequences of all strains were determined and compared, and they showed several mutations. Structural analysis was carried out in order to assess the influence of amino acid variations on protein structure demonstrating a conservation of the secondary structure. Western blot analysis and immunogold electron microscopy showed that all orf virus strains were antigenically identical. The results of our study confirmed the immunogenicity of the F1L protein; furthermore, our data suggest a possible involvement of the protein in the virus cycle. PMID- 12376760 TI - Taxonomic position of sugarcane streak mosaic virus in the family Potyviridae. AB - AcDNA library was generated from purified RNA of sugarcane streak mosaic virus- Andhra Pradesh (SCSMV-AP). Two overlapping clones covering 3160 nucleotides encoding partial CI, complete 6K2, VPg-NIa and NIb genes were sequenced. A comparison of this sequence along with the 3' terminal 1315 nucleotides of SCSMV AP determined earlier with the other members of the family Potyviridae indicated that it had only 30% identity at the amino acid level for the partial polyprotein open reading frame (ORF) with the members of Ipomovirus and Tritimovirus genera. Further, in the most conserved NIb region also there was only 40% identity with the type members of these genera. Based on this analysis, we suggest the taxonomic affiliation of SCSMV-AP into an undescribed new genus in the family Potyviridae. PMID- 12376761 TI - Complete sequence of the Pepino mosaic virus RNA genome. AB - We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical. PMID- 12376762 TI - Anti-idiotypic antibody specific for an infectious bursal disease virus neutralizing monoclonal antibody does not interfere with the virus infection. AB - Infectious bursal disease virus (IBDV) capsid protein, VP2, contains a hypervariable domain that is recognized by virus-neutralizing antibodies. The virus-neutralizing epitope is highly conformation-dependent and the domain is speculated to be involved in the virus-target cell interaction. In this study, a polyclonal anti-idiotypic antibody (anti-id) was generated by the sequential immunization of a rabbit with a virus-neutralizing monoclonal antibody GI-11 which recognizes the VP2 hypervariable domain. Although the anti-id, which mimics the conformational epitope in the VP2 hypervariable domain, was expected to inhibit the virus infection, the anti-id did not interfere with either the virus binding or the infection to the target cell. PMID- 12376763 TI - Rapid pathotyping of Newcastle disease virus using a single-chain Fv displayed on phage against the C-terminal end of the F2 polypeptide. AB - Filamentous bacteriophage display technology has been used to generate specific antibody fragments for differentiating virulent and avirulent Newcastle disease virus. A single-chain Fv fragment to the motif (112)RRQ(114), present at the F2 C terminal end of many virulent Newcastle disease virus isolates, was isolated from a phage display library derived from a rabbit immunized with a peptide conjugate. An ELISA evaluation was carried out to test its ability to differentiate between 11 avirulent and 34 virulent NDV isolates. The antibody fragment reacted with 25/28 virulent viruses with the putative motif (112)RRQ(114). The three exceptions were viruses with an arginine instead of glycine, at position 110 of the fusion protein, just preceding the cleavage site. Five of six virulent isolates, whose predicted motif was different from that usually found in virulent strains, also tested negative. However, the antibody did react with one isolate with the motif (112)KRQ(114). There was no apparent reactivity with any of the avirulent isolates tested. We conclude that this antibody may, in the future, be a useful aid for the pathotyping of NDV isolates. PMID- 12376765 TI - The family Closteroviridae revised. PMID- 12376766 TI - Traumatic brain injury and subarachnoid hemorrhage: in vivo occult pathology demonstrated by magnetic resonance spectroscopy may not be "ischaemic". A primary study and review of the literature. AB - OBJECTIVES: To look for evidence of early ischaemic neurochemical changes in patients suffering severe traumatic brain injury (TBI) and severe subarachnoid haemorrhage (SAH). Proton metabolite concentrations were measured in normal and abnormal areas of brain on T2 MR imaging, in regions considered particularly vulnerable to ischaemic injury. METHODS: Intensive care patients underwent T2 weighted imaging in a 1.5 Tesla MR scanner and proton magnetic resonance spectroscopy (single voxel or chemical shift imaging). Metabolite values in areas that appeared 'normal' and 'abnormal' on T2 MR imaging were compared with those obtained from normal controls. RESULTS: 18 TBI and 6 SAH patients were imaged at 1 to 26 days. N-acetyl aspartate (NAA) was lower in TBI and SAH patients compared to controls in both T2 normal and T2 abnormal areas (p<0.0005). SAH, but not TBI patients also had increased choline and creatine compared to controls in the T2 normal (p<0.02, p<0.02 respectively) and T2 abnormal (p=0.0003, p=0.003) areas. No lactate was found in TBI or SAH patients. CONCLUSIONS: Significant loss of normal functioning neurones was present in TBI and SAH, but no evidence of anaerobic metabolism using lactate as a surrogate marker, questioning the role of 'ischemia' as a major mechanism of damage. Increased choline and creatine were found in SAH patients suggestive of increased cell-wall turnover. Current theories of brain injury after TBI or SAH do not explain these observed neurochemical changes and further research is required. PMID- 12376767 TI - Intra-operative monitoring of brain tissue O2 (PtiO2) during aneurysm surgery. AB - BACKGROUND: Regional cerebral blood flow may be compromised during aneurysm surgery. This may occur during vessel occlusion by temporary cliping or result from the malposition of an aneurysm clip. In this report we monitored intra operatively the brain tissue oxygen concentration (PtiO2) to visualize regional ischaemic events. METHOD: During surgery of 10 intracranial aneurysms, monitoring of PtiO2 was performed using a polarographic microcatheter (Licox, GMS-Kiel Germany), which was placed in the vascular territory of the artery harboring the aneurysm. FINDINGS: No complications were observed after implantation of Licox electrodes. In 6 patients PtiO2 decreased during transient clipping. In two patients PtiO2 decreased below 2 mmHg without morphological or clinical signs cerebral ischemia. In four patients, without incidence during surgery, only minor oscillations were observed. CONCLUSION: Intra-operative monitoring of PtiO2 is a complimentary procedure to monitor cerebral perfusion and detect episodes of ischaemia. Given the rapid detection of these events, therapeutic intervention may be initiated before irreversible neuronal damage occurs. PMID- 12376768 TI - Radiosurgery of intracranial cavernous malformations. AB - BACKGROUND: The efficacy of radiosurgery in cases of surgically high risk symptomatic cavernous malformations (CMs) for reducing haemorrhagic risk and for seizure control has not been clearly documented and the radiation-induced complications of radiosurgery remain problematic. The authors present a retrospective clinical analysis of 22 cases of CMs treated by radiosurgery. METHODS: Twenty-two patients with symptomatic CMs were treated by linear accelerator (LINAC) radiosurgery or Gamma knife (GK) between 1995 and 1998. Medical records including radiological investigations were carefully reviewed to the last follow-up. The mean age of the patients was 34.1 years (12-56) and the male to female ratio was 12:10. Twenty patients reported at least one episode of bleeding and four had undergone microsurgery before radiosurgery. The remaining two patients presented with seizure without evidence of recent haemorrhage. In 16 cases, the CMs were deep-seated, and the others were located in the cerebral hemispheres; four were located at an eloquent area. LINAC radiosurgery using computed tomography scan was performed in 11 cases until May 1997, after which GK radiosurgery using magnetic resonance (MR) image was performed in 11 cases. The volume of the lesion ranged from 0.09 cc to 4.8 cc (mean 1.42 cc) and the mean marginal dose was 16.1 Gy (8-24). The median follow-up period after radiosurgery was 38.3 months (21-67). The rate of haemorrhage, seizure, and neurological deterioration following radiosurgery was analyzed, and the rate of haemorrhage was compared to that seen in natural course reports. FINDINGS: There was one case of haemorrhage during the follow-up period and the seizure was well controlled with anticonvulsants. In the group with prior haemorrhage, the bleeding rate of cavernous malformation after radiosurgery (1.55%/year) was lower than that of pre radiosurgical period (35.5%/year, t=1.296, P=0.04). Six patients showed neurological deterioration following radiosurgery, however, the neurological deficits persisted in only two of the patients with LINAC. The radiosurgical modality (LINAC vs. GK) showed a possible correlation to radiation induced neurological deficits (P=0.06). On the MR images at the last follow-up, the lesion was decreased in eleven patients, increased in one, and no change was found in 10 cases. The T2 weighted MR images revealed a perilesional high signal change in nine patients. This signal change was not statistically related to lesion size (P=0.236), location (P=0.658), nor radiation dose (P=0.363), but was dependent on the treatment modality (P=0.02). New-enhancing lesion and a new cyst were each found in one case, respectively, during the follow-up. INTERPRETATION: Radiosurgery may be a good alternative option for treatment of surgically high risk CMs. However, the optimal radiosurgical technique, dose adjustment, and proper delineation of the mass are prerequisites. Radiosurgery induced complications are still problematic and post-radiosurgery MR image changes need to be further elucidated. PMID- 12376769 TI - Effect of partial targeted N-butyl-cyano-acrylate embolization in brain AVM. AB - BACKGROUND: The management of cerebral arteriovenous malformations needs effective treatments. So far, no study has shown that partial targeted embolization treatment (PTET) reduces the risk of intracranial hemorrhage with respect to the natural history of the malformation. METHODS: The pre-treatment and post-treatment-initialization hemorrhage incidences of neuro-interventional patients were compared. Two hundred fifteen patient years from 519 patients were used to observe the short term course of the untreated disease. Five hundred patient years from 326 patients were used to observe hemorrhage after the start of treatment. The Kaplan-Meier estimator of hemorrhage free time under treatment was compared with results in the literature. Confounding influences resulting from selection processes or the disease parameters were studied. RESULTS: The yearly hemorrhage incidence rate of all untreated patients was observed as 0.089 (95% CI [0.053, 0.138]). This rate was 0.052 (95% CI [0.019, 0.114]) in the subgroup of patients who underwent PTET later. In the same group the observed annual rate after the start of PTET was 0.036 (95% CI [0.021, 0.057]). Crawford's results about intracranial hemorrhage during the natural course show the lowest risk values compared to other published studies [3]. There was a significant difference between the Crawford's reference data and the ICH incidence after the start of PTET in the neuro-interventional population (p=0.037). The morbidity risk in treated patients was 5.3% for a transitory and 2% for a persisting neurological deficit. Mortality results were compared with those of Crawford. CONCLUSION: The neuro-interventional patients under study show a lower hemorrhage risk than the population studied by Crawford. A significant superiority with respect to hemorrhage risk is established two years after the start of the PTET treatment. PMID- 12376770 TI - Functional MRI and 18F FDG-positron emission tomography for presurgical planning: comparison with electrical cortical stimulation. AB - BACKGROUND: In patients with mass lesions near "eloquent" cortical areas different preoperative mapping techniques can be used. Two of the most widely used approaches include positron emission tomography (PET) and functional MRI (fMRI). We employed both methods in the same patients undergoing presurgical evaluation and compared the results to those obtained by direct electrical cortical stimulation (DECS). METHOD: 22 patients with tumours of different aetiology near the central region were investigated. FMRI was performed using a T2(*)-weighted gradient-echo BOLD sequence at 1.5 T, PET was performed after injection of 122-301 MBq (18)F-Fluorodeoxyglucose (18-FDG) under rest and activation conditions. DECS was performed in all patients with recordings of muscles primarily involved in the investigated tasks. FINDINGS: In 19 patients all three modalities could be compared, 1 patient demonstrated discordance between fMRI and PET with DECS speaking in favour of fMRI, 6 patients had neighbouring results of PET and fMRI (between 1-2 cm distance), 12 patients had overlapping results. INTERPRETATION: The high incidence of neighbouring results is presumably related to fMRI specific artefacts. Advantages of fMRI are: Higher spatial and temporal resolution, more and different functional runs, shorter examination time, wider availability, longitudinal examinations, non-invasiveness and cost-effectiveness, easy registration to anatomical images. Advantages of PET are: higher signal-to-noise ratio, lesser susceptibility to artefacts (motion, draining veins), evaluation of tumour metabolism. It is our opinion that the neurosurgeon has to decide on a case-by-case basis which study suits his specific needs in the presurgical evaluation of his patient. PMID- 12376771 TI - New-onset psychogenic seizures after intracranial neurosurgery. AB - BACKGROUND: Patients with physical brain abnormalities have an increased risk of developing psychogenic nonepileptic seizures (PNES). Here we describe patients who developed PNES after intracranial neurosurgery for indications other than the control of refractory epileptic seizures and explore whether neurosurgical intervention is at risk factor for PNES. METHOD: We searched the database of 372 patients diagnosed with PNES at our department over the last 10 years and identified 17 patients (4.6%) in whom PNES first started after intracranial neurosurgery. Surgical procedures included the complete or partial resection of a meningioma, AV malformation, cavernoma, plexus papilloma, neurinoma, astrocytoma, oligodendroglioma, dysontogenetic cyst, the drainage of a brain abscess and removal of a subdural hematoma. PNES were documented by ictal video-EEG, ictal EEG, or ictal observation and examination in all cases. The diagnosis of additional epileptic seizures were confirmed by ictal EEG/video-EEG, or made on the basis of a clinical assessment by an experienced epileptologist. FINDINGS: Five patients had purely psychogenic postoperative seizure disorders, twelve had epileptic and psychogenic attacks. Median age at neurosurgery was 32 years (range 5-54), median latency between surgery and onset of PNES was 1 year (range 0-17 years). INTERPRETATION: PNES may develop after intracranial neurosurgery undertaken for other indications than the control of refractory epileptic seizures. Younger patients with a history of pre-operative psychiatric problems or epileptic seizures and surgical complications may be at higher risk. A diagnosis of PNES should be considered in patients who develop refractory seizures after neurosurgery. PMID- 12376772 TI - Surgical treatment of the thrombosed vein of galen aneurysm. AB - OBJECTIVE AND IMPORTANCE: Vein of Galen aneurysm is a rare congenital anomaly of cerebral circulation and occurs mainly in newborns and children. The spontaneously thrombosed vein of Galen aneurysm is especially rare and an uncommonly reported event. CLINICAL PRESENTATION: Two patients with a spontaneously thrombosed vein of Galen aneurysm were operated on at our institute. CT and MRI demonstrated space-occupying lesions of the pineal region and two other masses in the posterior fossa in the second case. The first described case should be referred to as the true type of aneurysm. The vascular malformation, revealed in the second case should be referred to as the false type. In this case the vein of Galen was enlarged to a gigantic size due to the blood drainage from the arteriovenous malformation supplying it from the inferior posterior cerebellar artery. TECHNIQUE: Thrombosed aneurysmal sacs were resected totally by subtentorial supracerebellar and median suboccipital approaches. CONCLUSION: The authors report two cases of successful surgical resection of a thrombosed vein of Galen aneurysm in children. Diagnostic features are considered and the informative value of magnetic resonance imaging is stressed. The principles of choosing the right approach and dissection techniques of thrombosed aneurysms of the vein of Galen are described. PMID- 12376773 TI - Reconstruction of suboccipital craniectomy with autologous bone chips. AB - BACKGROUND: In patients submitted to suboccipital craniectomy in whom the bone is not repositioned, there may be a significant aesthetic defect due to lack of bone tissue, sometimes accompanied by paresthaesia and painful symptoms. METHOD: In 15 patients submitted to suboccipital craniectomy, the bone chips were repositioned during wound closure. FINDINGS: At a mean follow up of 19 months (from 6 to 36 months), 2 patients (13%) complained of mild wound discomfort or occasional local pain. Twelve patients underwent control CT-scan. In three cases (25%) the bone fragments had been partly reabsorbed whereas in the other 9 (75%) they either formed a thin (4 patients) or consistent (5 patients) bony wall, with variable degree of adaptation to the contour of the contralateral occipital bone. The best cosmetic and functional results were obtained in young patients in whom the cerebellar parenchyma was well-preserved, as opposed to those in whom a CSF collection had replaced areas of cerebellar tissue. INTERPRETATION: In the majority of cases in whom an osteoplastic suboccipital craniotomy is not possible, repositioning of the bone chips from suboccipital craniectomy is able to restore a bone table, thus allowing morphological and functional recovery of the occipital region. PMID- 12376775 TI - Subarachnoid heamorrhage-induced chronic cerebral vasospasm in the rabbit: IV-DSA versus IA-DSA. AB - BACKGROUND: Chronic cerebral vasospasm is delayed-onset cerebral arterial narrowing in response to blood clots left in the subarachnoid space after aneurysmal subarachnoid haemorrhage (SAH). Rabbit models of vasospasm have been developed as in vivo experimental pathogenesis and the treatments of cerebral vasospasm using human vessels are not possible. The present study assessed the diagnostic accuracy of the intravenous digital subtraction angiography (IV-DSA) in chronic cerebral arterial spasm following induced SAH in the rabbit. METHOD: Ten rabbits' left leg veins catheterised by intravascular access needle and 3F catheters introduced to the right leg arteries probing the proximal of the vertebral arteries. Initially IV-DSA and intra-arterial digital subtraction angiography (IA-DSA) was performed. Three millilitres of fresh autologous arterial blood was injected into the cisterna magna of the ten rabbits' in order to produce in vivo model of chronic SAH. Angiograms were obtained 15 minutes and 72 hours after the SAH. FINDINGS: Diameters of the basilar arteries were similar to each other in both methods and reduced after the SAH. INTERPRETATION: The present study shows that IV-DSA is a relatively simple and effective method for demonstrating cerebral vessels, especially the basilar artery. PMID- 12376774 TI - Therapeutic effects of intracarotid infusion of spermine/nitric oxide complex on cerebral vasospasm. AB - BACKGROUND: Vasospasm is one of the underlying causes of morbidity and mortality in subarachnoid haemorrhage (SAH). The therapeutic effects of intracarotid infusion of spermine/nitric oxide complex (SPER/NO) on cerebral vasospasm in an experimental model of SAH were investigated. METHOD: Twenty-four adult male New Zealand white rabbits (2.6-3.4 kg in weight) were randomly divided into four groups (n=6), as follows: (I) control group (without SAH and drug), (II) SAH alone group (with SAH, without drug), (III) SAH placebo group (with SAH and saline), and (IV) SAH-SPER/NO group (with SAH and SPER/NO). The fresh autologous non-heparinized blood was injected into the cisterna magna to induce a SAH, after 24 hour SAH, the substance (saline or SPER/NO) was delivered to animals. All rabbits were scarified at 48-hours of induced SAH. The basilar artery with surrounding tissue was removed from the cranium and processed for paraffin embedding. Histopathological and stereological examinations of the basilar artery were done. FINDINGS: In the SPER/NO treated group of rabbits, the histopathological changes were less severe than in the SAH-alone and SAH-placebo groups. Regarding the intracarotid pressure, there was a statistically significant difference between SAH-alone and SAH-SPER/NO groups and also between SAH-SPER/NO and control groups (p<0.05). The mean cross sectional area of basilar arteries was 0.26 mm(2) in the control, whereas in SAH alone, placebo and SAH SPER/NO groups were 0.13, 0.15 and 0.20 mm(2), respectively. INTERPRETATION: It is well known that NO is a critical substance involved in cerebral vascular dynamics. Present results indicate that treatment of vasospasm with SPER/NO in SAH may be promising. However, further studies should be done on this substance to clarify its effect on vasospasm before using the drug in clinical situations. PMID- 12376776 TI - 'Mirror image' distal anterior cerebral artery aneurysms. A case report of two patients with review of literature. AB - We report two cases of patients with bilateral 'mirror image' or 'kissing' aneurysms at the distal anterior cerebral arteries who presented with subarachnoid haemorrhage and frontal intracerebral haematoma. PMID- 12376777 TI - Ruptured true posterior communicating artery aneurysm and cystic craniopharyngioma. PMID- 12376778 TI - Glioblastoma multiforme of the brain stem in a patient with acquired immunodeficiency syndrome. AB - Glioblastoma of the brain stem is rare and there is no description of such a lesion in patients suffering from acquired immunodeficiency syndrome. The majority of intracerebral mass lesions are due either to toxoplasmosis or primary central nervous system lymphomas so that it is usually not included in the differential diagnosis of enhancing lesions of the central nervous system in these patients. A 31-year-old human immunodeficiency virus (HIV) infected man presented with a four months history of slowly progressive deterioration of brainstem associated symptoms despite antitoxoplasmic therapy. Magnetic resonance imaging revealed a large ring enhancing lesion in the brainstem. Clinical and neuroradiological data could not establish a proper diagnosis and a stereotactic serial biopsy was undertaken. Histological examination of the specimen showed a glioblastoma multiforme (GBM) as the first reported case of GBM located in the brainstem in an acquired immunodeficiency syndrome (AIDS) patient. Patient management and effectiveness of stereotactic serial biopsy are discussed. PMID- 12376779 TI - Epidermoid cyst of the cisterna magna presenting with cervicomedullary compression after trauma. PMID- 12376780 TI - The role of fine-needle aspiration and intraoperative frozen section in the surgical management of solitary thyroid nodules. AB - PURPOSE: We evaluated the role of intraoperative frozen section (FS) in the surgical management of solitary thyroid nodules, as its true value is a subject of some controversy. METHODS: We reviewed the records of 206 consecutive patients operated on for solitary thyroid nodules. All patients had undergone both preoperative fine-needle aspiration (FNA) and intraoperative FS. The diagnostic findings of FNA cytology and FS histology were compared with the final histological results. RESULTS: There were 61 patients with cancer and 145 with various benign conditions. The sensitivity and specificity of FNA were 78.1% and 96.5%, respectively, demonstrating an overall accuracy of 91.3%. The sensitivity, specificity, and accuracy rates for FS were 83.3%, 95.2%, and 91.7%, respectively. FS altered the operative decision in 14 patients, but correctly so in only 8 patients. Correlated with FNA cytology, the yield of FS in assisting in the intraoperative decision making was 1.8%, 3.4%, and 5.2% for benign, malignant, and suspicious cytology, respectively. CONCLUSIONS: When the results of FNA and FS are interpreted as either benign or malignant, both are highly accurate predictors of the pathological nature of the nodule. However, the findings of the present study do not support the use of FS in the surgical management of solitary thyroid nodules, regardless of FNA cytology. PMID- 12376781 TI - Video-assisted endoscopic thyroid and parathyroid surgery using a gasless method of anterior neck skin lifting: a review of 130 cases. AB - PURPOSE: Endoscopic endocrine neck surgery is desirable from a cosmetic viewpoint. We compared the effectiveness of our new technique with that of conventional surgery in a clinical study. METHODS: We performed our original endoscopic method of video-assisted neck surgery (VANS) on 130 patients: 126 with thyroid tumors and 4 with parathyroid tumors. The percentage of patients who underwent VANS among all those who underwent neck surgery and the procedure involved were analyzed. Operating time and blood loss were compared between the first 40 patients and last 39, and all factors were statistically analyzed in the most recent 20 patients who underwent the VANS method and the most recent 20 who underwent conventional surgery. RESULTS: More than 60% of benign thyroid tumors and 5.3% of malignant thyroid tumors were operated on by the VANS method. Nearly total lobectomy was the most common procedure (57.7%), followed by total lobectomy (26.1%), for benign tumors. Malignancy was defined as papillary carcinoma less than 1 cm in diameter. Total lobectomy with lymph node clearance was performed for all malignant tumors. There was less bleeding when the VANS method (P < 0.001) was used than when conventional surgery was performed, and the operating time has been reduced with experience. CONCLUSION: The VANS method is feasible, practical, and safe, and has great cosmetic benefits. PMID- 12376782 TI - Evaluation of oxidative stress in laparoscopic cholecystectomy. AB - PURPOSE: We conducted a prospective study to evaluate the effect of CO2 pneumoperitoneum and increased intra-abdominal pressure on arterial blood gases, end-tidal CO2 (ETCO2), nitric oxide (NO), blood and tissue malondialdehyde (MDA), and total antioxidant (TAOx) levels during laparoscopic cholecystectomy. METHODS: Fifty selected patients with cholelithiasis were randomized to undergo either laparoscopic or open surgery. Blood samples were taken pre-, mid-, and postinsufflation, and 24 h postoperatively. To determine the tissue MDA level, tissue samples were taken from the gallbladder just after removal. RESULTS: The increased levels of ETCO2 and PCO2, caused by CO2 pneumoperitoneum resulted in a minimal decrease in blood pH during the laparoscopic surgery. Although low levels of blood MDA were seen 30 min after the start of laparoscopy, due to less oxidative stress response and tissue trauma, increased levels of tissue MDA levels indicated that the gallbladder was more traumatized during laparoscopic dissection and handling. NO levels were slightly lower in the laparoscopic cholecystectomy (LC) group, but there were no significant differences compared with the open cholecystectomy group (OC). TAOx levels were similar in both groups 30 min after the start the procedure, but were much lower in the LC group 24 h postoperatively. CONCLUSIONS: These findings suggest that the antioxidant defense system is stimulated less with less oxidative stress, providing further evidence to support the opinion that LC is a safe technique. PMID- 12376783 TI - Platelet scintigraphy in the diagnosis of arteriosclerosis obliterans. AB - PURPOSE: We evaluated the efficacy of platelet scintigraphy using autologous platelets labeled with 111In-oxine, to assess the degree of arteriosclerotic activity in arteriosclerosis obliterans (ASO). METHODS: Thirty-three patients with clinical signs of ASO, seen between January 1996 and August 1999, were enrolled in this study. Scintigraphic imaging results were compared with the findings of contrast angiography in 26 patients, 17 of whom were taking antiplatelet and/or anticoagulant drugs during the platelet imaging study. RESULTS: Angiography demonstrated atherosclerotic lesions at 38 sites from the abdominal aorta to the popliteal arteries in 23 patients. There was an accumulation of platelets at 17 of these sites (45%) and at 6 other sites without definite angiographic abnormalities. Lesions that resulted in less than 50% stenosis were detected slightly, but not significantly, less often than lesions with higher degrees of stenosis and occlusion (50% vs 30%, P = 0.73). The frequency of true-positive scintigraphic results was statistically higher in patients not taking antithrombotic agents than in those taking antithrombotic agents (70% vs 32%, P = 0.02). CONCLUSIONS: Our results suggest that imaging with 111In-oxine-labeled platelets may be useful for evaluating the pathophysiologic characteristics of atherosclerotic lesions in patients with ASO. PMID- 12376784 TI - The role of epidermal growth factor formulation on stress ulcer healing of the gastric mucosa. AB - PURPOSE: We investigated the role of epidermal growth factor (EGF) microemulsion (ME) formulation on stress ulcer healing and the levels of gastric mucosal malondialdehyde (MDA) and glutathione (GSH). METHODS: Forty-eight female Wistar rats were divided into five groups according to the treatments given. Ten rats were given intragastric (i.g.) serum physiologic (SP) for 7 days; ten rats were given i.g. ME for 7 days; ten rats were given i.g. EGF in SP 6 microg/kg per day for 7 days; ten rats were given i.g. ME + EGF (Sigma E-7755) 6 microg/kg per day for 7 days; and eight rats were exposed to cold and immobilization stress or no stress (NS), and not given any treatment, as controls. The mean ulcerated area, and the MDA and GSH levels of the gastric mucosa were measured. RESULTS: The mean ulcerated area of the ME+EGF treatment group was significantly less than that of the ME- and EGF-treated groups and the control groups. The gastric MDA levels were also found to be significantly lower in the ME+EGF treated group than in the ME-treated group or the control groups. However, there were no significant changes in the gastric GSH levels among all the groups. The gastric MDA levels were positively correlated with the ulcerated area. CONCLUSION: The ME formulation of EGF at the dose given in this study was more effective than EGF alone on the healing of stress ulceration of the gastric mucosa. PMID- 12376785 TI - Polyurethane-covered dacron mesh versus polytetrafluoroethylene DualMesh for intraperitoneal hernia repair in rats. AB - PURPOSE: Abdominal hernia repair using the intraperitoneal implantation of a prosthesis requires mesh with impervious properties, such as expanded polytetrafluoroethylene (ePTFE). A newly developed polyurethane-covered polyester mesh with impervious properties has recently been introduced as a less expensive alternative to PTFE, and we compare these materials herein. METHODS: The adhesion formation and stability of intraperitoneal abdominal hernia repairs with DualMesh (macroporous ePTFE mesh with a microporous component) and PolyesterComposite (the newly developed polyurethane-covered Dacron mesh) were compared in a rat model. Forty rats were randomly divided into two groups; ten animals from each group were killed after 14 days, and the other ten after 90 days. RESULTS: The number and intensity of adhesions were comparable in the PolyesterComposite and PTFE groups. Loose adhesions were seen in 13 animals and appeared only selectively at the fixation sutures. Both PolyesterComposite and PTFE induced the formation of a smooth neoperitoneum on the intraperitoneal surface and showed a complete ingrowing of the prosthesis in the surrounding tissue. There were no significant differences between the prostheses in terms of clinical herniation pressure and hydroxyproline concentration. CONCLUSIONS: PolyesterComposite and PTFE are both suitable prostheses for intrapertoneal implantation, but PolyesterComposite is less expensive, which is an important advantage for clinical use. PMID- 12376786 TI - Myxoid malignant fibrous histiocytoma of the breast: report of a case. AB - Malignant fibrous histiocytoma (MFH) is the most common type of soft tissue sarcoma, but it rarely develops as a primary tumor in the breast. Furthermore, no case of the myxoid variant of MFH in the breast has ever been documented. We report the case of a 52-year-old woman with a breast tumor that was immunohistochemically confirmed to be myxoid MFH. She underwent a radical mastectomy and is currently well with no evidence of local recurrence or metastatic spread after 3 years of follow-up. PMID- 12376787 TI - Recurrent squamous cell carcinoma of the breast with undifferentiated features: report of a case. AB - Squamous cell carcinoma of the breast is a rare type of cancer, the origin of which is still uncertain. We report a case of squamous cell carcinoma of the breast with a recurrent tumor that showed undifferentiated features. The patient was a 55-year-old woman who originally presented with a left breast mass in the upper outer quadrant. Echography showed a 46 x 29 x 23-mm mass with cavity formation, and aspiration cytology confirmed a diagnosis of squamous cell carcinoma. A modified radical mastectomy with level III lymph node dissection was performed. Pathologically, the tumor was composed of squamous cell carcinoma and noninvasive ductal carcinoma. A recurrent tumor showing undifferentiated features was detected in the left forechest 3 months after the operation, and tumorectomy with partial resection of the major and minor pectoralis muscles was performed. Despite intensive therapy including chemotherapy (CEF: cyclophosphamide, epirubicin, 5-fluorouracil) and irradiation (50 Gy), the patient died from pulmonary and skin metastases 20 months after her initial operation. The squamous cell carcinoma of the breast in this patient grew rapidly and her prognosis was poor. Immunohistochemical findings indicated the possibility that the squamous cell carcinoma developed from noninvasive ductal carcinoma of the comedo type, and that the undifferentiated cells from the site of recurrence developed from dedifferentiation of the squamous cell carcinoma. PMID- 12376788 TI - Colon carcinoma after thymectomy for myasthenia gravis: report of a case. AB - A 74-year-old Japanese man was admitted to our hospital with anemia, 4 years after a thymectomy for thymoma associated with myasthenia gravis. A diagnosis of sigmoid colon carcinoma was confirmed, followed by surgical resection. This case is presented to reinforce that physicians should bear in mind the possibility of extrathymic malignancies in patients with thymoma. PMID- 12376789 TI - Successful pulmonary resection of lung cancer in a patient with partial anomalous pulmonary venous connection: report of a case. AB - A rare case of the potentially grave combination of lung cancer and partial anomalous pulmonary venous connection (PAPVC) is described. PAPVC would cause many problems following major lung resection, even in a preoperatively asymptomatic patient, because of the inevitable development of right ventricular failure as a result of right ventricular volume overload caused by the left-to right physiologic shunt. On the other hand, if a patient has primary lung cancer, anatomical resection should be done to achieve curative treatment. We successfully performed a left lower lobectomy for lung cancer in a patient with abnormal venous drainage in the left upper lobe, with simultaneous correction of a PAPVC. PMID- 12376790 TI - Laparoscopic repair of a Morgagni-Larrey hernia: report of three cases. AB - Morgagni-Larrey hernia is a rare type of diaphragmatic hernia, the diagnosis of which is made incidentally by routine chest X-ray film. We describe a technique for the laparoscopic repair of Morgagni-Larrey hernia which was successfully performed in three adult patients; two women and one man. Two of the patients were asymptomatic and had herniation of only omentum into the right hemithorax; however, one was symptomatic and had herniation of the omentum and large bowel. Tension-free closure of the defects was done using Prolene mesh with a hernia stapler, helical fastener, and Endostitch. There were no early complications and the patients were discharged on the fourth postoperative day. The mean follow-up period was 41 months, and there has been no late morbidity or mortality related to this procedure. Using a laparoscopic approach to repair a Morgagni-Larrey hernia provides an excellent view of the surgical field and allows easy manipulation with minimal surgical trauma, followed by rapid recovery of the patient. PMID- 12376791 TI - Pseudoachalasia as a late complication of gastric wrap performed for morbid obesity: report of a case. AB - A 66-year-old woman presented with progressive dysphagia of 10 years' duration. She had undergone a Teflon gastric wrap operation for obesity 20 years earlier. Endoscopic and radiological examinations showed a dilated tortuous esophagus and a contracted stomach. The esophageal manometry findings were consistent with achalasia. She underwent an uneventful total gastrectomy, partial distal esophagectomy, and Roux-en-Y esophagojejunal anastomosis. When last seen, 2 months after her operation, she was not suffering from dysphagia. This case report serves to demonstrate that gastric reservoir wrapping is associated with significant morbidity. PMID- 12376792 TI - Giant Brunneroma as an unusual cause of upper gastrointestinal hemorrhage: report of a case. AB - Brunneroma, also known as Brunner's gland adenoma or harmartoma, is a rare, benign, proliferative lesion arising from the Brunner's glands of the duodenum, usually discovered incidentally at endoscopy. Occasionally, these lesions manifest as a rare cause of duodenal obstruction or upper gastrointestinal hemorrhage, and require excision. We report a case of an unusually large Brunneroma in a patient who presented with fresh melena and anemia. Although endoscopic polypectomy is the treatment of choice, we decided to perform surgical polypectomy through a laparotomy due to the huge dimensions of the lesion. The clinical and endoscopic features of this benign tumor and the therapeutic options are critically reviewed. PMID- 12376793 TI - Primary torsion of the greater omentum: report of a case. AB - Primary or idiopathic torsion of the greater omentum is an uncommon cause of acute abdominal pain, often mimicking other acute abdominal conditions. The diagnosis is usually made at laparotomy, with the presence of free serosanguinous fluid in the absence of any other intra-abdominal pathology being suggestive of this condition. Resection of the infarcted segment is the treatment of choice, offering rapid recovery and reducing the possibility of adhesion formation. We report a case of primary omental torsion and discuss the diagnostic and therapeutic implications of this entity. PMID- 12376794 TI - Multiple ileal diverticula causing an ileovesical fistula: report of a case. AB - We report a case of multiple ileal diverticula causing an ileovesical fistula in an 85-year-old man. The patient was admitted for investigation and treatment of intractable urethrocystitis, which he had suffered for 5 years. Cystography showed an ileovesical fistula, and contrast study of the small bowel revealed about 80 diverticula in the ileum. The segment involved by diverticula was resected and a pathological diagnosis of diverticulitis leading to ileovesical fistula was confirmed. His postoperative clinical course was uneventful. PMID- 12376795 TI - Appendiceal stump abscess as an early complication of laparoscopic appendectomy: report of a case. AB - We report the case of an appendiceal stump abscess that was treated by relaparoscopy 4 days after a laparoscopic appendectomy (LA). Surgeons should be aware of the possibility of appendiceal stump abscess occurring as an early complication of LA. When performing LA, the appendiceal stump should be as short as possible, and the ligation of the root of the appendix should be only moderately tight, so as not to cause ischemic change of the stump, indicated by discoloration or edema. The insertion of a drain for monitoring exudate, as well as sonography, and relaparoscopy are helpful for diagnosing and treating this complication. PMID- 12376796 TI - Large benign cystic teratoma of the mesosigmoid causing intestinal obstruction: report of a case. AB - We report the case of a large benign cystic teratoma of the mesosigmoid in a 2 year-old child. To the best of our knowledge, this type of lesion has never been described in the English literature, although there are a few reports of teratomas arising from the greater and lesser omentum. There was diagnostic confusion due to the rarity of a teratoma arising from the peritoneal folds and also because calcification was not detected radiologically. A brief review of the literature on the diagnosis and treatment of similar lesions is presented. PMID- 12376797 TI - Survival for 5 years after repeat liver resections and multimodality treatment for metastatic intestinal leiomyosarcoma: report of a case. AB - The liver is a common site of metastases from gastrointestinal or retroperitoneal leiomyosarcomas. Although repeat liver resections can prolong survival, to our knowledge, 5-year survival has never been achieved. We report the case of a woman who has survived for 5 years since her first liver resection, during which time five more resections have been performed, in combination with systemic chemotherapy and radiofrequency ablation. PMID- 12376798 TI - Surgical treatment of retroperitoneal leiomyosarcoma invading the inferior vena cava: report of three cases. AB - Retroperitoneal leiomyosarcoma is a rare neoplasm for which complete surgical removal provides the only effective treatment, as local recurrence adversely affects prognosis. However, invasion of major vessels may occur, making complete resection difficult. This report describes the cases of three patients who required concomitant resection of parts of the inferior vena cava because of direct tumor invasion. The major vessels should be isolated in preference to the tumor capsule during surgery to prevent sudden exsanguination or incomplete tumor resection. Resection of a recurrent sarcoma or a solitary metastasis can be effective in selected patients. PMID- 12376799 TI - Venous stasis ulcers due to primary, isolated deep venous insufficiency in a patient with systemic lupus erythematosus: report of a case. AB - Primary, isolated deep venous incompetence is rare, difficult to diagnose, and can lead to the development of venous stasis ulcers. We herein report a case demonstrating chronic venous stasis ulcers due to primary, isolated deep venous incompetence, which was misdiagnosed as vasculitis ulcers associated with systemic lupus erythematosus (SLE). Although primary, isolated deep venous incompetence is rare, it is important to bear this possibility in mind when a patient presents with leg ulcers. PMID- 12376800 TI - Atheroembolic signals detected by Doppler ultrasound scan monitoring in a patient with blue toe syndrome: report of a case. AB - It is generally accepted that clinical symptoms give the only clue to the presence of atheroemboli in patients with blue toe syndrome (BTS). We report a case of atheroemboli originating from the abdominal aortic aneurysm in which Doppler ultrasound successfully detected atheroembolic signals, which vanished immediately after surgery. To our knowledge, this is the first such case to be documented. When a 67-year-old man was given warfarin after aortocoronay bypass, digital cyanosis suddenly developed, which became worse and was very painful. Angiography and computed tomography scanning revealed an infrarenal aortic aneurysm with mural thrombus. Doppler ultrasound detected atheroemboli as high intensity transient signals in the bilateral tibioperoneal trunks. After aneurysmectomy and a bifurcated graft replacement, the cyanotic and painful toes improved immediately. Microscopically, cholesterin crystals were seen in the arterioles of the amputated digits. Thus, Doppler ultrasound could be a valuable test to determine the appropriate treatment for patients at risk of atheroembolic BTS. PMID- 12376801 TI - Pseudoaneurysm of the abdominal aorta caused by acupuncture therapy. AB - Acupuncture is a major treatment modality used in Oriental medicine to control chronic pain. However, several complications have been reported, including spinal cord injury, pneumothorax, and subcutaneous pseudoaneurysm, according to the puncture sites. We report the case of a pseudoaneurysm of the abdominal aorta caused by acupuncture. PMID- 12376802 TI - The use of bonewax to control massive presacral bleeding. AB - Massive presacral bleeding during retroperitoneal resection is unusual, and can be difficult to control. We describe a technique for managing this complication whereby bonewax is pushed through the presacral fascia and periosteum directly into the bleeding point in the sacrum, followed by abdominal packing. This maneuver proved successful for achieving hemostasis when we recently encountered this intraoperative complication. PMID- 12376803 TI - Cell biology of renal osteodystrophy. AB - Renal osteodystrophy, a well-recognized complication of chronic renal failure, encompasses a spectrum of skeletal disorders ranging from high-turnover lesions of secondary hyperparathyroidism, the most common histologic lesion in pediatric patients with end-stage renal disease, to low-turnover lesions of adynamic renal osteodystrophy, which has become a common skeletal lesion in adults with chronic renal failure. Several advances have been made in the understanding of the pathogenesis of secondary hyperparathyroidism, particularly the critical roles of calcium, phosphorus, and vitamin D in promoting excess parathyroid hormone (PTH) synthesis and secretion, and parathyroid gland hyperplasia in renal failure. These insights will guide the development of more effective strategies for the prevention and management of renal bone disease. PMID- 12376804 TI - Can bone marrow differentiate into renal cells? AB - A considerable plasticity of adult stem cells has been confirmed in a wide variety of tissues. In particular, the pluripotency of bone marrow-derived stem cells may influence the regeneration of injured tissues and may provide novel avenues in regenerative medicine. Bone marrow contains at least hematopoietic and mesenchymal stem cells, and both can differentiate into a wide range of differentiated cells. Side population (SP) cells, which are originally defined in bone marrow cells by high efflux of DNA-binding dye, seem to be a new class of multipotent stem cells. Irrespective of the approach used to obtain stem cells, the fates of marrow-derived cells following bone marrow transplantation can be traced by labeling donor cells with green fluorescence protein or by identifying donor Y chromosome in female recipients. So far, bone marrow-derived cells have been reported to differentiate into renal cells, including mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney, although controversy exists. Further studies are required to address this issue. Cell therapy will be promising when we learn to control stem cells such as bone marrow derived stem cells, embryonic stem cells, and resident stem cells in the kidney. Identification of factors that support stem cells or promote their differentiation should provide a relevant step towards cell therapy. PMID- 12376805 TI - Elevated bone turnover in rat polycystic kidney disease is not due to prostaglandin E2. AB - Hyperparathyroidism, secondary to renal disease, is thought to cause high bone turnover via prostaglandin E2 (PGE2). Diets high in n-3 fatty acids reduced PGE2. Thus the objective was to compare the effect of diets high in n-6 and n-3 fatty acids on hyperparathyroidism, bone turnover, and PGE2 in Han:SPRD- cy rats that develop polycystic kidney disease (PKD). Weanling male rats ( n=58) were randomized to diets made with either corn or flaxseed oil (5%) for 8 weeks, followed by measurement of plasma parathyroid hormone (PTH), osteocalcin, urinary N-telopeptide (NTX), and ex vivo release of PGE2from femur. Plasma PTH was elevated ( P<0.01) as a result of PKD. Mean values for plasma osteocalcin and urinary NTX were elevated ( P<0.01) by PKD but not altered by diet. In contrast, values for PGE2 were lowest in the PKD rats fed flaxseed oil compared with PKD rats fed corn oil and compared with non-affected rats fed either oil. Rats with PKD have high-turnover bone disease, likely due to hyperparathyroidism, that is unaffected by feeding corn or flaxseed oils. Since PGE2 release is lower in the presence of high bone turnover, the high bone turnover in evolving rat uremia is not likely to be mediated by PGE2. PMID- 12376806 TI - Cobalamin C deficiency complicated by an atypical glomerulopathy. AB - Cobalamin C (cbl C) deficiency, an inherited disorder of vitamin B12 metabolism, causes elevated levels of methylmalonic acid and homocysteine and decreased methionine in all body fluids. Renal complications of cbl C disease are thrombotic microangiopathy (TMA), chronic renal failure, tubulointerstitial nephritis and proximal renal tubular acidosis. There is, however, only one case report of primary glomerular pathology, focal segmental glomerulosclerosis, in a cbl C deficient patient. We report a case of an atypical glomerulopathy in a 16 year-old male patient with cbl C deficiency. The glomerulopathy manifested with proteinuria and progressive renal insufficiency. The renal histologic, immunofluorescent and ultrastructural findings were similar, but not identical, to idiopathic membranoproliferative glomerulonephritis (MPGN) but also overlapped with those of a TMA. The serum complement profile was normal; there were scanty glomerular deposits of C3, no deposits of IgG and ultrastructural findings that were similar to those seen in either MPGN type III or a TMA. On the basis of these findings we have designated the renal disease as an atypical glomerulopathy. PMID- 12376807 TI - A novel mutation of the epithelial Na+ channel causes type 1 pseudohypoaldosteronism. AB - Type I pseudohypoaldosteronism (PHA-1) is a rare salt wasting syndrome occurring soon after birth, characterized by apathy and severe dehydration accompanied by hyponatremia, hyperkalemia, and metabolic acidosis despite high plasma aldosterone concentrations. The molecular defect involved in the systemic autosomal recessive form of the syndrome has been identified. Mutations in all three genes encoding the epithelial sodium channel (ENaC) lead to a decrease in the channel function, resulting in the disease. We report here two new cases of the autosomal recessive form of PHA-1 in the same family. We found a new homozygous mutation of the gene encoding the alpha ENaC subunit (alphaR492stop). The function of the mutated ENaC channel was assessed in the Xenopus laevis oocyte expression system. The mutant ENaC activity measured with the two electrode voltage clamp method was drastically decreased compared with the wild type activity, in agreement with the salt-losing phenotype. PMID- 12376808 TI - Shiga toxin-associated hemolytic uremic syndrome: absence of recurrence after renal transplantation. AB - We evaluated the relationship between the acute phase and the development of end stage renal disease (ESRD) and the outcome of renal transplant in patients with Shiga toxin-associated hemolytic uremic syndrome (Stx-HUS). A 20-year retrospective study was performed of 66 renal transplants in 62 patients with Stx HUS compared with 189 renal allografts in 178 children with other diseases. Of 62 patients, 61 had >7 days of oliguria during the acute phase. Stx-HUS patient survival was not different from controls (92% vs. 83% 15 years after renal transplantation). In the cyclosporine (CsA) era, survival of grafts from living related (LRD) and cadaver (CD) donors in Stx-HUS and control patients was 83% versus 70% ( P<0.03) and 77% versus 49% ( P<0.05) at 10 years. Graft survival in Stx-HUS and dysplasia/obstructive uropathy patients was 79% versus 76% ( P=NS), but it was different from that of other diseases (79% vs. 58%, P<0.001). There was no clinical or histopathological evidence of Stx-HUS recurrence. In conclusion, in Stx-HUS patients the duration of the acute oliguric period was a good predictor for the progression to ESRD. Use of CsA and the absence of recurrence of the disease influenced the excellent prognosis in Stx-HUS patients after renal transplantation. The development of ESRD in Stx-HUS could be mediated by non-immunological factors. PMID- 12376809 TI - Short-term outcomes of Thymoglobulin induction in pediatric renal transplant recipients. AB - No data are currently available that describe the clinical outcomes associated with Thymoglobulin (rabbit polyclonal anti-thymocyte globulin) induction in pediatric renal transplant recipients. We report the outcomes of 17 pediatric renal transplant recipients (mean age 10.1+/-5.2 years) transplanted between 1 August 1999 and 31 July 2001. Eleven patients (65%) were Caucasian and 6 (35%) were African-American. Eleven (65%) recipients received cadaveric allografts. Two patients (12%) were second allograft recipients. One patient had primary allograft non-function secondary to vascular thrombosis. Two patients (12%) had delayed allograft function. Immunosuppression consisted of Thymoglobulin induction (mean number of doses 6+/-1.7) with tacrolimus (62%) or cyclosporine A (38%), mycophenolate mofetil, and prednisone. One year post transplant, patient and graft survival was 100% and 93%, respectively. No acute rejection episodes occurred during the first 6 months after transplantation in any of the recipients. Additionally, no rejection episode occurred among the 14 patients followed for 1 year after transplant. The incidences of asymptomatic cytomegalovirus (CMV) and Epstein-Barr virus (EBV) seroconversion at 1 year in seronegative recipients with a seropositive donor were 100% of 4 patients and 0% of 4 patients, respectively. No symptomatic CMV or EBV infections and no post transplant lymphoproliferative disease have occurred in any patient. These short term data suggest that Thymogobulin induction is safe and effective in combination with triple immunosuppressive therapy for preventing early rejection in pediatric renal transplant recipients. PMID- 12376810 TI - Citrate clearance in children receiving continuous venovenous renal replacement therapy. AB - Anticoagulation is usually indicated in patients receiving continuous renal replacement therapy (CRRT) to prevent clotting of the extra-corporeal circuit. While heparin is the most frequently used anticoagulant, regional citrate anticoagulation is becoming the preferred choice in those patients at high risk for bleeding. However, it has been widely claimed that to avoid citrate toxicity, CRRT with citrate anticoagulation should utilize diffusive clearance (e.g., continuous venovenous hemodialysis). We studied citrate clearance in five children who received citrate anticoagulation during CRRT with a COBE PRISMA machine and an M-60 (AN-69) filter. The blood flow rate ranged from 50 to 150 ml/min (2.1-8.0 ml/kg per min). Citrate was infused in the circuit circulation as an acid citrate dextrose (ACD) solution at a rate of 1.6-3.7% of the blood flow rate to maintain the circuit ionized calcium (iCa) <0.5 mmol/l. Calcium-free replacement fluid with reduced alkali (NaHCO3 20 mEq/l) was infused in pre-filter mode at a rate of 1,800-2,000 ml/h per 1.73 m(2). In a separate central line, CaCl2 (0.8%) was infused (rate 25-50% of ACD infusion) to maintain systemic iCa between 1.0 and 1.3 mmol/l. Citrate concentration was measured using an enzymatic assay. Total CRRT duration was 1,224 h. Twenty-four filters were changed due to clotting, with a mean filter life of 51 h. Mean (range) citrate levels (mmol/l) were (1) before initiating CRRT ( n=2): patient baseline 0.13 (0.1-0.15), (2) during CRRT ( n=7): circuit 4.54 (3.95-6.25), effluent 4.31 (3.95-5.46), and patient 0.69 (0.30-1.13). Sieving coefficients for urea and citrate were 0.88 0.97 and 0.88-1.0, respectively. Citrate clearance (31-38 ml/min per 1.73 m(2)) was similar to that of urea (31-38 ml/min per 1.73 m(2)), and when evaluated in two patients, remained unchanged after substituting half of the convective clearance [continuous venovenous hemofiltration (CVVH)] by diffusive clearance [continuous venovenous hemodiafiltration (CVVHDF)]. The post-filter citrate load (mean+/-SD) delivered to the five patients during CRRT was 1.06+/-0.62 mmol/kg per hour. With the exception of alkalosis in one patient, no other complications were observed. Renal function recovered in all patients. We conclude that citrate anticoagulation in children is feasible, effective, and safe. Sufficient citrate clearance to prevent its toxic accumulation is achieved by convective clearance (CVVH) alone and diffusive clearance (CVVHDF) does not appear to be mandatory when utilizing citrate anticoagulation during CRRT. PMID- 12376811 TI - Effective removal of methotrexate by high-flux hemodialysis. AB - The purpose of the present study was to examine the clearance of methotrexate (MTX) by high-flux hemodialysis (HD) in pediatric oncology patients. We present three patients who experienced nephrotoxicity and prolonged exposure to toxic MTX concentrations following high-dose infusions during treatment for osteogenic sarcomas. Each patient was successfully treated with high-flux HD, followed by carboxypeptidase G2 (CPDG2) in two cases. Minimal systemic toxicity occurred. We review the literature and discuss guidelines for early and aggressive treatment for this complication of high-dose MTX therapy. Clinically important removal of MTX depends upon prompt initiation of HD after detection of nephrotoxicity and delayed clearance of MTX. Therapy is indicated in cases where compassionate use of CPDG(2) may not be available, or while awaiting its delivery. PMID- 12376812 TI - Growth hormone therapy and lipid profile in children on chronic peritoneal dialysis. AB - The aim of the study was to assess the effect of recombinant human growth hormone (rhGH) on serum lipids in children treated with chronic peritoneal dialysis. We studied 26 patients aged 5-18 years, including 13 patients treated with rhGH at a dose of 1-1.1 IU/kg per week for 6 months and a control group of 13 patients. Serum total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), HDL-C, triglycerides (TG), apolipoproteins A-I and B-100 (apoA and apoB), lipoprotein (a) [Lp(a)], total protein, albumin, urea, and creatinine levels were measured in all children at baseline and at 1, 3, and 6 months of follow-up. We found a significant increase in the TG level after 1 month of administration of rhGH in the treatment group compared with both baseline (218.8+/-49.2 mg/dl vs. 175.9+/ 71.9 mg/dl, respectively, P<0.05) and the control group at 1 month of follow-up (146.5+/-44.3 mg/dl, P<0.001). We found no change in TC, LDL-C, HDL-C, apoA, and Lp(a) levels during treatment with rhGH. These data suggest that administration of rhGH to children treated with peritoneal dialysis results in only a transient increase of serum TG level and has no effect on TC, LDL-C, HDL-C, apoA, and Lp(a) levels. PMID- 12376813 TI - Cardiac rhythm disturbances in children on hemodialysis. AB - Cardiovascular complications are the leading causes of morbidity and mortality in adult dialysis patients. The aim of this study was to evaluate the cardiovascular system of children on hemodialysis (HD), with special focus on rhythm disturbances. Nine patients, aged 15.6+/-4.1 years, underwent electrocardiographic examination (ECG) including 12-lead ECG, Holter-ECG, QT dispersion, signal-averaged ECG, and exercise testing. Echocardiography and 24-h blood pressure measurement were also performed. Patients had been on HD for a median period of 7 months (range 1-29 months). Normal findings were obtained with 12-lead ECG, exercise testing, QT dispersion, and signal-averaged ECG. Holter-ECG showed short runs of slow monomorphic ventricular tachycardia in two patients. Echocardiography was normal except for ventricular hypertrophy in two patients. In conclusion, rhythm disturbances were rare, with slow monomorphic ventricular tachycardias being the only significant finding. The absence of late potentials and normal QT dispersion suggest that myocardial electrical excitability and recovery are preserved in children on HD. PMID- 12376814 TI - Secondary resistance to cyclosporin A in children with nephrotic syndrome. AB - We investigated the phenomenon of secondary resistance to cyclosporin (CsA) in children with steroid dependent (SD) or steroid resistant (SR) nephrotic syndrome. Secondary resistance was defined as an initial response to CsA with relapse on withdrawal of therapy and absent or diminished response on reinstitution of the drug. Thirty-two children with nephrotic syndrome who were treated with CsA were included in the study. Twenty-two of the children (15 of 15 SD and 7 of 17 SR) responded while ten demonstrated primary CsA resistance. Of these 22 responders, 20 relapsed when therapy was tapered or discontinued. Cyclosporin was reinstituted in 19. Ten responded, demonstrating CsA dependence, and nine exhibited secondary CsA resistance. Focal segmental glomerular sclerosis (FSGS) was present in one patient with CsA dependence on the initial biopsy and in two of six on a subsequent biopsy. In comparison, seven of nine patients with secondary CsA resistance and ten of ten with primary CsA resistance had FSGS on the initial or subsequent biopsy ( P=0.03). C4 and/or C1q were present on the initial biopsy in one patient with CsA dependence as compared to six of nine with secondary CsA resistance ( P=0.02). Four patients with secondary CsA resistance had an accelerated progression to end-stage renal disease (ESRD). We conclude that the presence of FSGS, or of C4 and/or C1q, appears to increase the risk of secondary CsA resistance and some of these children rapidly progress to ESRD. PMID- 12376815 TI - Correct evaluation of renal glomerular filtration rate requires clearance assays. AB - Iohexol clearance is an accepted, but time-consuming assay for the measurement of glomerular filtration rate (GFR). We investigated if simpler methods could predict GFR. Sixty-nine children with hematological-oncological disorders participated. A linear relationship was established by regression analysis between iohexol clearance ( n=734) and 1/s-creatinine ( r=0.45, n=727), s cystatin C ( r=0.41, n=518), and the Schwartz ( r=0.45, n=723), Counahan-Barratt ( r=0.48, n=723), and modified Counahan-Barratt formulae ( r=0.48, n=723). These correlations improved when one GFR measurement per individual was compared with each of the five parameters. We further investigated if iohexol clearance could accurately be replaced. The degree of variation in predicting GFR was estimated by the standard deviation of the residuals (S(res)). For 1/s-creatinine and s cystatin C, S(res) was 39 and 38 ml/min per 1.73 m(2). For the formulae of Schwartz, Counahan-Barratt, and modified Counahan-Barratt, the S(res) was 43, 40, and 40 ml/min per 1.73 m(2), respectively. The wide variations of the S(res) were not reduced when one GFR measurement per child was compared with the five parameters. Due to the large deviation in predicting GFR, we conclude that the five alternative methods studied cannot replace iohexol clearance for measurement of GFR. PMID- 12376816 TI - Role of non-polio enterovirus infection in pediatric hemolytic uremic syndrome. AB - Verocytotoxin-producing Escherichia coli(VTEC) infections cause most cases of hemolytic uremic syndrome (HUS); 10-30% of patients, however, are negative for VTEC infection. The etiology of HUS in VTEC-negative cases remains poorly understood. Before the association between VTEC infection and HUS was recognized, sporadic cases of HUS with enterovirus infection were reported in the literature. Since May 1988, most cases of HUS in Italy have been reported to the Italian surveillance system, and in 73% of these, evidence of VTEC infection was demonstrated. The aim of this study was to determine whether the frequency of enteroviral infections was different in the acute phase of VTEC-positive and VTEC negative HUS. Eighty-nine patients were investigated for enteroviral infection, of whom 58 were VTEC positive and 31 VTEC negative. Two serum samples from each patient were examined for seroconversion to enterovirus (coxsackie, echovirus, and picornavirus) by a complement fixation test. Serological evidence of acute infection with non-polio enterovirus was found in 33 patients (37%) [20/58 (34.5%) VTEC positive and 13/31 (41.9%) VTEC negative]. There was no statistically significant difference between the two groups. These results demonstrate that there are no significant differences for enteroviral infection in VTEC-positive and VTEC-negative patients and, therefore, enteroviral infections should not be considered a cause of HUS in VTEC-negative children. PMID- 12376817 TI - L-Arginine effects on blood pressure and renal function of intrauterine restricted rats. AB - We have previously demonstrated that 3-month-old rats submitted to 50% intrauterine food restriction showed a decreased number of nephrons with increased glomerular diameter, a fact that suggests compensatory hypertrophy. In the present study, we extended the investigation and performed serial blood pressure measurements and renal function evaluation in 8- and 12-week-old rats submitted to 50% intrauterine food restriction (groups R8 and R12) and in age matched control rats (groups C8 and C12). After weaning, six to eight animals from each group received oral supplements of 2% L-arginine ( L-arg) solution for 4 or 8 weeks (groups CA8, CA12, RA8, RA12). Our findings showed that mean blood pressure (MBP), which was significantly increased from 8 weeks on in R rats, markedly decreased after L-arg supplementation. In control animals, no alterations in MBP were observed with L-arg. Proteinuria was within normal limits in all groups studied but L-arg caused a significant decrease in this parameter in both the RA8 and RA12 groups. Glomerular filtration rate (GFR, ml/min per kg) was significantly decreased in the C8 control group (3.75+/-0.12) and in both restricted groups R8 and R12, (2.47+/-0.13 and 3.76+/-0.16, respectively) compared with the C12 group (6.09+/-0.31; P<0.05 for all comparisons). L-Arg caused an increase in GFR only in the younger groups, C8 and R8. In a separate set of experiments, acetylcholine (ACh)-induced relaxation was examined in mesenteric arteries. The R12 group showed a significant impairment of the response to ACh, which returned to normal values after L-arg supplementation. Urinary excretion of NO(x) (NO3- + NO2-) was significantly decreased in 8- and 12 week-old food-restricted rats relative to control rats. Our data indicate that, besides the known decrease in absolute nephron number, disturbances in the production/sensitivity to the L-arg-nitric oxide system may contribute to the early appearance of hypertension in the offspring of mothers submitted to significant food restriction. PMID- 12376818 TI - An adolescent with IgA nephropathy and Crohn disease: pathogenetic implications. AB - We describe a patient with IgA nephropathy associated with Crohn disease. IgA nephropathy first appeared at the age of 10 years. Combined therapy with prednisolone, cyclophosphamide, warfarin, and angiotensin-converting enzyme inhibitor resulted in clinical improvement over the following year, and remission was maintained. At the age of 13 years, the patient developed Crohn disease and IgA nephropathy recurred. Significant increases in serum IgA were associated with progression of Crohn disease. An elemental diet combined with oral prednisolone resulted in clinical improvement of Crohn disease and in remission of nephropathy and normalization of serum IgA concentration. The clinical course of the two diseases was linked, suggesting a common pathogenetic mechanism involving an IgA immune response to mucosal challenge in the intestine. PMID- 12376819 TI - Plasma therapy in von Willebrand factor protease deficiency. AB - We report a patient with relapsing hereditary hemolytic uremic syndrome (HUS) that began in the neonatal period with life-threatening jaundice and hemolytic anemia. He progressed to end-stage renal failure at 14 years of age and had a cerebrovascular accident while on dialysis. The cause of HUS was a constitutional deficiency in the von Willebrand factor cleaving protease. Hematological features of HUS significantly improved following bilateral nephrectomy. After renal transplantation, he had an early recurrence of HUS associated with two episodes of retinal and cerebral ischemia. Long-term treatment with fresh-frozen plasma exchanges prevented recurrence of HUS, cerebrovascular attacks, and early loss of the graft. PMID- 12376820 TI - Fibrinolysis in the hemolytic uremic syndrome. PMID- 12376822 TI - Severe acute abdominal pain in a patient with steroid-sensitive idiopathic nephrotic syndrome. PMID- 12376823 TI - Perlecan, the multidomain HS-proteoglycan of basement membranes, is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes. AB - The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation. PMID- 12376824 TI - Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis. AB - Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T cadherin expression and SMC phenotype. PMID- 12376825 TI - Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles. AB - We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles. PMID- 12376826 TI - Primary longitudinal adhesion structures: plectin-containing precursors of costameres in differentiating human skeletal muscle cells. AB - Plectin is a high molecular mass protein (ca 530 kDa) that binds actin, intermediate filaments, and microtubules. Mutations of the human plectin gene cause epidermolysis bullosa simplex with muscular dystrophy. In mature human skeletal muscle, plectin is localized between neighboring myofibrils and between myofibrils and the sarcolemma, both at the level of Z-discs. In the present study we have analyzed plectin expression patterns with emphasis on its sarcolemmal localization during human skeletal muscle differentiation in vitro. In myoblasts plectin showed a cytoplasmic intermediate filament-like distribution, whereas in myotubes plectin is also found at the level of the sarcolemma. In particular, in early myotubes a specific plectin isoform colocalizes with the costameric proteins vinculin and beta1D integrin in longitudinally orientated structures which increased in number and longitudinal extension upon further maturation. In mature myotubes processes perpendicular to the parallel system of longitudinal structures became apparent. Subsequent to the occurrence of spontaneous myofibrillar contractions, the number of longitudinal streaks decreased, and plectin and other costameric proteins were found in an orderly cross-striated sarcolemmal lattice overlying myofibrillar Z-discs. Our study demonstrates that plectin is preassembled together with vinculin and beta1D integrin into primary longitudinal adhesion structures. After the occurrence of spontaneous contractions, these structures reorient and mature costameres are assembled. PMID- 12376827 TI - Immunohistochemical studies on the expression pattern of molecular chaperones HSC70 and HSP25 and cell cycle-related proteins cyclin D1 and PCNA in rat liver after thioacetamide intoxication. AB - Intoxication of rats with thioacetamide (TAA) is a model system to investigate mechanisms involved in liver cell death and tissue reconstitution. Our study was undertaken to determine by immunohistochemistry the expression pattern of the cytoprotective chaperone proteins HSC70 and HSP25 and proliferation markers cyclin D1 and PCNA in livers of Wistar rats intraperitoneally injected with TAA at a single dose of 50 mg/kg. For each protein studied we observed distinct dynamic changes in appearance and localization in liver lobules. During 24-36 h after TAA injection the HSC70 cytoplasmic immunoreaction gradually disappeared from hepatocytes localized around central veins and a shift of immunostaining to cell nuclei took place. Then, 36-48 h after TAA injection the HSC70 cytoplasmic immunoreaction reappeared with the highest intensity in hepatocytes surrounding the areas of inflammatory cells. HSP25, undetectable in control hepatocytes began to appear at approximately 36 h after TAA injection and HSP25-immunopositive cells formed a characteristic ring around areas of inflammation. Of the proteins studied, the most rapid reaction to TAA was observed for cyclin D1. As early as 15 min after TAA administration cyclin D1-positive hepatocytes appeared in intermediate and periportal areas of liver lobules and a subsequent shift of staining to centrilobular hepatocytes took place at 36 and 48 h. There was no correlation of cyclin D1 localization either with PCNA-positive cells or mitotic cells. Our observations suggest that in TAA-treated livers HSP25 and HSC70 proteins can play an anti-inflammatory role, and the early and distinct cyclin D1 expression is not related to proliferation of hepatocytes. PMID- 12376828 TI - Analyses in transfected cells and in vitro of a putative peroxisomal targeting signal of rat liver serine:pyruvate aminotransferase. AB - Serine:pyruvate aminotransferase (SPT; EC 2.6.1.51) of rat liver is a unique enzyme in that it is located in both mitochondria and peroxisomes. To analyze a peroxisomal targeting signal (PTS) of SPT, we constructed in this study various peroxisomal SPT clones having mutations at the C-terminal 20-amino acid region in which a putative PTS is located, and we examined subcellular localization of mutated products expressed in transfected COS-1 cells. When the mutant SPTs were unstable in transfected COS-1 cells, their translocation into peroxisomes was examined using an in vitro peroxisomal import system. Deletion of the C-terminal tripeptide, NKL, and amino acid substitution of K2 (the second lysine from the C terminus), K4, or E15 abolished or impaired the peroxisomal import of the translated product, resulting in cytosolic accumulation in the cell. In the cases of mutation of R18G, D19A, or K2Q and the conversion to proline of L9, L13, V17, or A20, no products were detected in transfected cells. However, the results of an in vitro peroxisomal import experiment showed that the mutation of L9P, L13P, V17P, and A20P caused loss of the PTS function. When serine was introduced instead of N3 to generate a typical PTS1, the SKL motif, at the C-terminus, all of the proteins having mutations at P5, E11, R12, or E15 showed extensive localization in peroxisomes. These results suggest that the putative C-terminal PTS of SPT is not equivalent to the typical PTS1 shown in acyl-CoA oxidase and urate oxidase, because the PTS of SPT is not restricted to the C-terminal tripeptide. The results also suggest that the alpha-helical structure of the C terminal region of SPT is important for the stable conformation of the enzyme and the peroxisomal targeting function of its PTS. PMID- 12376829 TI - Peroxisomes of the nematode Caenorhabditis elegans: distribution and morphological characteristics. AB - We analyzed the distribution and morphological characteristics of peroxisomes in the nematode Caenorhabditis elegans by routine electron microscopy, immunoelectron microscopy, and morphometry. Peroxisomes were mainly contained in the epithelial cells of the digestive tract and pharyngeal gland, but some were observed in other cells. Their shape varied from round to twisted. The matrix of most peroxisomes was coarse and uneven, and contained electron-dense nucleoids and frequently tubular substructures. The diameter of peroxisomes in the gut (0.185 micro m) was smaller than that in pharyngeal gland (0.262 micro m). The volume density of peroxisomes per 100 micro m(2) of cytoplasm was 1.86 in the gut and 1.75 in the pharyngeal gland. After treatment with clofibrate, the diameter of peroxisomes increased approximately 1.11-fold in the gut and 1.2-fold in the pharyngeal gland. The volume density of peroxisomes also increased by 2.2-fold in the gut and 2.6-fold in the pharyngeal gland. The labeling density for catalase-2 was almost identical between gut and pharyngeal gland peroxisomes. The results show that in C. elegans peroxisomes mainly distribute in the epithelial cells of the gut and pharyngeal gland. Peroxisomes of the pharyngeal gland are larger than those of the gut, but peroxisomes of both tissues contain catalase-2 at similar concentrations. PMID- 12376830 TI - Rat heart GDNF: effect of chemical sympathectomy. AB - Developmental studies indicate a role for GDNF in survival of motor, autonomic, and sensory neurons. However, no study attempted to demonstrate its participation in autonomic nerve regeneration. In this work, chemical sympathectomy by 6 hydroxydopamine provided the model for assessing heart GDNF expression during denervation and axonal regrowth. A glyoxylic acid-based histochemical technique evaluated the noradrenergic innervation. ELISA determined GDNF levels after concentrating heart homogenates. Light and ultrastructural in situ hybridization and immunocytochemistry were used for identifying cells expressing GDNF mRNA and protein. In control rats, the GDNF cardiac levels were significantly higher in 37 day-old animals in comparison with those aging 60 days. In sympathectomized rats, GDNF cardiac levels were significantly higher 7 days after sympathectomy and dropped to control levels at day 30. GDNF mRNA was expressed in atrial and ventricular myocytes from normal and sympathectomized rats. GDNF immunoreactivity occurred on atrial granules and quantitative analysis in electron micrographs confirmed ELISA-obtained data. In ventricular myocytes gold particles occurred sparsely. These findings constitute the first evidence for GDNF synthesis by cardiomyocytes and postulate a role for this factor soon after cardiac sympathetic denervation, probably in nerve regeneration. In atrial myocytes, GDNF is probably secreted by regulated pathway. PMID- 12376831 TI - Glucose-6-phosphate dehydrogenase and mouse Kupffer cell activation: an ultrastructural dual staining enzyme-cytochemical study. AB - Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in Kupffer cell function, especially in phagocytosis activity. Although it was suggested that Kupffer G6PD may be upregulated in Kupffer phagocytosis/activation, direct morphological evidence has been lacking. Acid phosphatase (ACP), a representative lysosomal enzyme, can be used as a cytochemical marker for phagocyte activation. Using an ultrastructural enzyme-cytochemical dual staining method, I simultaneously localized G6PD and ACP activity in mouse Kupffer cells on a cell by-cell basis, and examined whether or not cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Glucose-6 phosphate dehydrogenase labelings were observed in the cytoplasm and on the cytosolic side of the endoplasmic reticulum, and ACP labelings were seen in the lysosomes. In phagocytosing Kupffer cells, in which ACP deposits were observed not only in the lysosomes but also on the phagosomal membranes and phagosomal contents, G6PD labelings were denser than dormant Kupffer cells. Enzyme cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Kupffer cell G6PD, activated in phagocytosing Kupffer cells, may play an important role not only in liver defense but also in liver disease pathogenesis/pathophysiology. PMID- 12376832 TI - The identification of war victims by reverse paternity is associated with significant risks of false inclusion. AB - Since February 2001 the process of DNA identification of war victims in Croatia relies on the database of over 3,000 9-locus (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) STR genotypes of relatives of missing persons. Instead of a targeted approach to DNA typing, the genotype of each skeletal remains analysed is compared to all genotypes in the database to identify potential parents and children. Although this approach has significantly increased the pace of identification by DNA typing, non-targeted matching in a database containing several thousand genotypes considerably decreases the significance of inclusion, especially when identification is based on reverse paternity analysis. To support this statistical prediction we present 3 cases of 10 STR loci matches and 1 case of 11 STR loci matches between a child, child's mother and skeletal remains that did not originate from a father of that child. PMID- 12376833 TI - Population genetic data, comparison of the repeat structure and mutation events of two short STRs. AB - The short tandem repeat (STR) systems D3S1545 and D7S1517, both small size STRs (fragment lengths <160 bp), were investigated in a population sample of German Caucasoids. New primer sequences more closely flanking the repetitive region were designed for D3S1545. For D3S1545, 7 alleles could be found (heterozygosity 0.68) while 11 alleles could be typed for D7S1517 (heterozygosity 0.83). Additionally, sequencing of selected alleles was carried out to establish the allele nomenclature and to clarify the structure of the tandem repeat arrays. D3S1545 showed a uniform GATA repeat structure but, in contrast, the repeat stretch of D7S1517 showed a compound structure characterised by different numbers of GAAA and CAAA repeats. Two isolated cases of a new mutation could be confirmed for D7S1517. The alleles of these two family constellations were characterised by sequencing and the probable mutational events were demonstrated. PMID- 12376834 TI - Skin tears away from the entrance wound in gunshots to the head. AB - The present investigation covers 116 contact shots to the head and shots into the mouth from the Freiburg forensic autopsy material including 20 gunshot wounds which showed stretchmark-like tears of the facial skin away from the entrance wound. In these cases the gunshot entrance wounds were localised either in the mouth, the forehead, or the submental region. The stretchmark-like tears were found in the region of the eyes and the nasolabial folds. Radial tears were seen on the lips and in the vicinity of the corners of the mouth, particularly in cases involving shots into the mouth. The stretchmark-like tears essentially followed the skin tension lines and the expression-related lines of the face. They were apparently caused by the subcutaneous or intraoral expansion of the muzzle gases and/or the radial forces of the bullet resulting in ballooning and overextension of the facial soft tissues. The weapons used were not only rifles and shotguns, but also revolvers and pistols. PMID- 12376835 TI - Ubiquitin expression in skin wounds and its application to forensic wound age determination. AB - The time-dependent expression of ubiquitin (Ub) was examined in murine skin wounds and 55 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-5 days, III: 7-14 days and IV: 17-21 days). In murine skin wound specimens, neutrophils, macrophages and fibroblasts showed intensive Ub-positive reactions in the nuclei. In the human wound specimens with wound ages between 4 h and 1 day, neutrophils with strong intranuclear positive reactions for Ub were observed. With increasing wound ages, the nuclei of macrophages and fibroblasts were more intensively stained with anti-Ub antibody. Morphometrically, the intranuclear Ub-positive ratios were very low in group I. The skin wound specimens in groups II and IV showed Ub-positive ratios of >10%, and all samples in group III had Ub-positive ratios of >20%. These results suggest that a ratio of >10% for Ub indicates a wound age of at least 1 day. In contrast, Ub-positive ratios of less than 10% indicate a wound age of <1 day. Moreover, there was a significant difference in the Ub-positive ratio between group III and the other three groups. Thus, Ub-positive ratios considerably exceeding 30%, possibly indicate a wound age of 7-14 days. From the viewpoint of a forensic pathology application, the present study showed that Ub is suitable as a marker of wound age determination. PMID- 12376836 TI - Autopsy features relevant for discrimination between suicidal and homicidal gunshot injuries. AB - A total of 624 consecutive gunshot autopsies from the Institutes of Legal Medicine in Munster and Hamburg was investigated retrospectively. In a subsample of 284 suicides and 293 homicides (n=577), a large variety of features such as firearm, ammunition, number and site of entrance wounds, shooting distance and direction of the internal bullet path were recorded and binary logistic regression analysis performed in the case of bullet paths. Females constituted 26.3% of the homicide victims and 10.6% of the suicides. Short-barrelled firearms outnumbered long arms in homicides by 6:1 and in suicides by 2:1. More than 1 gunshot injury was found in 5.6% of the suicides (maximum 5 gunshots) and in 53.9% of the homicides (maximum 23 gunshots). The suicidal gunshots were fired from contact or near contact range in 89% while this was the case in only 7.5% of the homicides. The typical entrance wound sites in suicides were the temple (36%), mouth (20%), forehead (11%) and left chest (15%) but uncommon entrance wound sites such as the eye, ear, and back of the neck and head were also encountered. In suicidal gunshots to the right temple (n=107), only 6% of the bullet paths were directed downwards and only 4% were directed from back-to front. In gunshots to the left chest (n=130), bullet paths running right-to-left or parallel occurred frequently in suicides (75%) and infrequently in homicide victims (19%). From 61 suicides who fired the gun inside their mouth, only 1 pointed the gun downwards. Consequently, some bullet path directions cannot be considered indicative of suicide: downwards and back-to-front in gunshots to the temple, left-to-right in gunshots to the left chest and downwards in mouth shots. The isolated autopsy findings can only be indicative of suicide or homicide but the combined analysis of several findings can be associated with a high probability. PMID- 12376837 TI - Unusual craniocerebral injury caused by a pneumatic nail gun. AB - A man was found unconscious near a ladder in a house. After resuscitation he was brought to a hospital and X-rays of the skull showed that two 12-cm long nails had completely penetrated the cranial cavity. The nails were operatively removed and after treatment for 5 weeks, the patient was transferred to a rehabilitation centre with a decreasing hemiparesis on the left side and general deterioration and then, after an attempted suicide to a psychiatric hospital. The perforating cranio-cerebral injury from a pneumatic nail gun known to reach only low muzzle velocities is a very unusual finding. PMID- 12376838 TI - Fatal isolated ruptures of bladder following minor blunt trauma. AB - Traumatic bladder ruptures are generally secondary to severe trauma and associated with pelvic fractures. Conversely, isolated bladder ruptures following minor blunt trauma are rare and seldom fatal. We describe six fatal cases (five males, one female, 39-82 years old) of isolated bladder rupture subsequent to minor blunt trauma. Three cases were out-of-hospital deaths and among the three hospital cases, only one was diagnosed as bladder rupture ante-mortem. All victims had a history of chronic alcohol abuse. The differentiation between spontaneous and traumatic (accidental or purposely inflicted) bladder ruptures is crucial but may be difficult to assess, especially in cases involving alcohol abuse and occurring in a domestic setting. PMID- 12376839 TI - Canine STR analyses in forensic practice. Observation of a possible mutation in a dog hair. AB - In a case of the death of a 7-year-old boy, the police investigations revealed a possible dog attack contrary to the witness testimonies. DNA investigations were carried out from hairs, saliva and bloodstains with 10 canine-specific STR loci by the use of fluorescently labelled multiplex PCR and the ABI PRISM 310 genetic analyzer. The analysis of one hair sample revealed one allele deviation from the profile of the putative Rottweiler perpetrator possibly caused by a mutation. The PCR fragments in question at the PEZ20 locus were sequenced and compared with the alleles detected in the Hungarian canine population and identified on a repeat number basis. The allele frequencies were determined based on typing of 242 genetically independent canine individuals from 72 breeds. The results suggested that two of the canine individuals could be the perpetrators. PMID- 12376840 TI - First Polish DNA "manhunt"--an application of Y-chromosome STRs. AB - This study presents the application of Y-chromosomal STR polymorphisms to male identification in the case of a serial rapist and woman murderer in Poland. Since August 1996 a rapist from Swinoujscie (northwest Poland) committed at least 14 rapes. In the year 2000 he brutally raped 8 young girls and murdered a 22-year old girl. DNA profiles obtained from semen stains left at the scenes of crime gave information that one and the same man had committed all the rapes. The Y chromosome haplotype (9 loci) obtained was used for the elimination process of 421 suspects. One man was found who had an identical DNA profile in all Y chromosome STR loci analysed and possessed common alleles in 9 out of 10 autosomal loci, strongly suggesting that the real rapist and the typed man were closely related males. Analysis of reference DNA obtained from the man's brother revealed an identical DNA STR profile to that identified at the crime scenes. To the best of our knowledge this is the first case in Poland and probably in Eastern Europe where DNA typing of a large population was used to identify the offender. PMID- 12376841 TI - Air rifle injury with an entrance through the nose: a case report and review of the literature. AB - A case of attempted homicide is reported where a 31-year-old woman was shot in the left nostril with a pellet from an air rifle. The projectile channel reconstruction showed penetration of the nasal septum, the maxillary and sphenoid cavities and the dura mater, with the pellet finally lodging in the anterior cranial fossa between the sinus cavernosus and the internal carotid artery. The patient was finally discharged from hospital in a good physical condition without any neurological symptoms. Although the muzzle velocity of the air rifle was within the legal limits, the present case demonstrates the potential lethality of air weapons considering the site of entrance of the pellet. PMID- 12376842 TI - Forensic approach of fatal dog attacks: a case report and literature review. AB - Over 1 million dog bites occur every year in the USA, however, fatal dog bites are rare and mostly affect children under 4 years of age and old people. Usually pet dogs are involved and only recently has public awareness of this health problem increased. As an example of a forensic approach we present the case of a 6-year-old girl who was killed by the three pet Rottweilers of her father. The present report includes the investigation of the death scene, the autopsy findings and the results of the examination of the dogs. Dog bite wounds in this case typically were limited to the head and neck regions and classic features of these wounds have been described in various studies. We emphasise the particulars of canine dental features, discuss the resulting bite wounds and, reviewing the literature, try to come up with a strategy for prevention. PMID- 12376843 TI - Kurdish population data for 11 STR loci (ACTBP2, CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D13S317 and D21S11). AB - In a Kurdish population sample composed of 950 unrelated individuals from Northern Iraq, 11 tetrameric short tandem repeat (STR) loci from 10 different chromosomes (i.e., ACTBP2, CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D13S317 and D21S11) were typed to establish a database for immigration cases. The combined power of discrimination (PD) and the combined power of exclusion (PE) of all 11 loci were 0.99999999999994 and 0.99996, respectively. PMID- 12376844 TI - Genetic analysis of human remains from a double inhumation in a frozen kurgan in Kazakhstan (Berel site, Early 3rd Century BC). AB - The discovery of a big barrow of the Saka period in eastern Kazakhstan between the Russian and the Chinese borders provided the opportunity to excavate a frozen burial site. In the burial chamber, there was a wooden sarcophagus with two human bodies. The skeletons of these two individuals, a man and a woman, were well preserved. A genetic study based on STRs and mitochondrial DNA analyses was undertaken in order to determine whether these human remains belonged to close relatives. Results were obtained for all the markers. Nevertheless, nuclear STRs did not allow a clear conclusion concerning the relationship, but analysis of mitochondrial DNA showed that these skeletons were not close relatives. PMID- 12376846 TI - The prosecutor's and defendant's Bayesian nomograms. AB - Two nomograms to calculate posterior odds and probabilities in forensic cases according to Bayes' theorem are presented. PMID- 12376845 TI - Genetic variation of the nine Profiler Plus loci in Russians. AB - This paper presents allele frequency distributions from a representative population sample of the Russian Federation for the nine loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820) which constitute the commercially available Profiler Plus PCR amplification kit. DNA samples of 402 Russian individuals from 57 regions of the Russian Federation were amplified in a multiplex reaction with subsequent genotyping using an ABI Prism 377 DNA sequencer. The population data obtained for genotype and allele frequencies conformed to Hardy-Weinberg expectations (HWE). PMID- 12376847 TI - Paternity diagnosis by using umbilical cords preserved for periods ranging from 9 months to 44 years. PMID- 12376848 TI - [Some aspects of the current controversy about the treatment of amblyopia]. AB - Occlusion has been used for centuries for treating amblyopia but in recent years an intensive debate about the treatment has arisen. The aim of this article is to summarize some of the new aspects of this discussion: on the one hand the effect of occlusion treatment during childhood is being questioned and on the other hand experimental approaches are being tested which are aimed at treating amblyopia in adults. Evidence from own experimental work is presented showing the influence of occlusion on the spatial localization of visual stimuli. A continuation of research on amblyopia and the effect of the therapy is urgently needed. PMID- 12376849 TI - [Drugs for supplementation in cataract surgery with a laryngeal mask]. AB - BACKGROUND: We compared intraocular pressure (IOP), vitreous pressure and several anaesthesiological parameters for patients who underwent cataract surgery with propofol anaesthesia, laryngeal mask and different supplementations with reference to the effect of S-ketamin in particular. PATIENTS AND METHODS: In 4 groups with 15 patients cataract surgery (phacoemulsification) was carried out using anaesthesia with propofol, laryngeal masks and spontaneous breathing if possible, supplementation with propofol (0.6 mg/kg, group 1), S-ketamin (0.3 mg/kg, group 2), ketamin (0.6 mg/kg, group 3) or fentanyl (0.5 microgram/kg, group 4); IOP measurement with tonopen XL and scoring vitreous pressure at different times during anaesthesia (score 0-3). RESULTS: For IOP and vitreous pressure, none of the different supplementations showed a significant difference. Insertion of the laryngeal mask did not cause a rise in intraocular pressure. The number of patients with spontaneous breathing during the operation in group 4 was significantly lower than in groups 1-3. No significant differences were observed between the different anaesthesiological parameters. CONCLUSION: S-Ketamin had no significant effect on IOP and vitreous pressure during phacoemulsification. It offers a safe "handling" of patients because of a high spontaneous breathing rate and lower concentration compared to Ketamin. PMID- 12376850 TI - [Intrastomal refractive surgery with ultra-short laser pulses. Results from initial in vitro experiments]. AB - PURPOSE: To investigate the possibilities and techniques for intrastromal ablation of the cornea by the use of ultra-short laser pulses. METHOD: A laser induced plasma with a diameter of 8 mu was generated 100-300 mu below Bowmans membrane by focussing of a Ti:Saphir laser (lambda=805 nm, pulse width: 115 fs, energy: 0.6-3.3 microJ). Scanning the laser beam enabled arbitrary transsections in the corneal stroma. All experiments were performed in porcine eyes 1-4 h post mortem. RESULTS: Two-dimensional sections were achieved applying a minimal laser energy of 0.6 microJ at a separation of the single laser effects of 10 micrometer. A 150 micrometer thick disc was excised 150 micrometer below Bowmans membrane and extracted through a keratotomy performed by the laser. Sharp-cut edges were observed by slit-lamp biomicroscopy. CONCLUSIONS: Ultra-short laser pulses are suitable for precise intrastromal ablation. PMID- 12376851 TI - [Cyclosporin A 2% eyedrops in therapy of atopic and vernal keratoconjunctivitis]. AB - BACKGROUND: The efficacy of Cyclosporin A 2%eyedrops (CsA2%) as an additive treatment of atopic (AKC) and vernal keratoconjunctivitis (VKC) was evaluated. PATIENTS AND METHODS: The symptoms and findings of 26 patients with AKC and 12 patients with VKC with no improvement under conventional therapy, were additionally treated with CsA2% eyedrops and compared over a minimum follow-up period of 3 months to more than 24 months. RESULTS: The therapy with CsA2% eyedrops was well tolerated and evaluated as effective by the patients. Subtarsal papillae were reduced in 69.2% of the AKC group and in 66.6% of the VKC patients. Trantas dots were reduced or disappeared in about 50% of the AKC group and in the VKC group they had disappeared in more than 50%. Subjective symptoms (e.g. itching) had also been reduced or eliminated. CONCLUSION: CsA2% eyedrops are effective in the treatment of AKC and VKC, reducing objective and subjective signs as well as the need of adding topical steroids in affected patients. PMID- 12376852 TI - [Quantitative and objective topometrical analysis of drusen of the optic nerve head with the Heidelberg retina tomograph (HRT)]. AB - BACKGROUND AND PURPOSE: Optic nerve head drusen (ONHD) are one of the most frequent causes for congenital swelling of the optic nerve head. Visual field and retinal nerve fiber layer defects are reported in cases of ONHD. The Heidelberg Retina Tomograph (HRT) allows a 3-dimensional topometric analysis of the optic nerve disc and measurement of the peripapillary mean retinal nerve fiber layer. PATIENTS AND METHODS: A total of 18 eyes from 9 patients with sonographically confirmed drusen were analyzed with the HRT. Data were compared to a control group of 18 eyes from 9 matched healthy individuals. Statistical analyses were performed by using ANOVA (univariate). All patients with ONHD underwent a computerised visual field test (30 degrees, Octopus 101). Due to a bad reliability factor of over 10 in the visual fields by 4 out of 18 eyes, only measurements from 14 eyes were included in the study. We correlated visual field and HRT parameters and calculated the Pearson's correlation coefficient (r). RESULTS: We found a significant difference in the measured parameter mean retinal nerve fiber layer (RNFL) thickness ( p<0.05) between the two groups. In the ONHD group a negative correlation coefficient was found between the peripapillary mean RNFL thickness and the loss variance (r=-0.50, p=0.03) as well as between the peripapillary RNFL cross-sectional area and the loss variance (r=-0.47, p=0.04). CONCLUSIONS: The HRT is able to detect a peripapillary RNFL thinning in cases with ONHD. The mean RNFL thickness correlated with the loss variance. The HRT should be used to perform a quantitative and objective topometric analysis in cases with ONHD. PMID- 12376853 TI - [Time-correlated measurement of autofluorescence. A method to detect metabolic changes in the fundus]. AB - The detection of metabolic changes opens the possibility for intervention of reversible pathological alterations. Measurements of oxygen saturation are limited to the blood vessel system. Detection of alterations in oxygen concentrations are up to 3 orders of magnitude more sensitive by autofluorescence of coenzymes than by measurement of oxygen saturation. Because of limited transmission of the ocular media no specific excitation of endogenous fluorophores can be realised. For this reason it was investigated if the fluorescence lifetime after pulse excitation can be detected at the human fundus. Applying a laser scanner ophthalmoscope and mode-locked Ar(+) laser as well as time-correlated single photon counting, lifetime images of the living fundus were obtained. In mono-exponential approximation, a mean lifetime of 5 ns was detected from the optic disc and large vessels whereas about 1.5 ns were detected in the parapapillary area. By evaluating the frequency of lifetimes, lipofuscin, free FAD, and collagen are probably detectable. Comparative measurements were performed in fundus specimens and on free FAD. PMID- 12376854 TI - [Rheophoresis. A systematic approach to therapy of age-related macular degeneration (AMD)?]. AB - BACKGROUND: Choroidal microcirculation is impaired in age-related macular degeneration (AMD), and leads to deposition of lipids and proteins in Bruch's membrane. Rheophoresis can improve choroidal microcirculation by eliminating high molecular weight, rheologically relevant plasma proteins. The objective of this post-certification study was to analyse the effect of rheophoresis in 10 AMD patients. PATIENTS AND METHODS: A total of 6 patients with early AMD and 4 with late AMD in one eye (initial visual acuity equivalent 0.2-0.8) received rheophoresis treatment 10 times over an 18-week period. Visual acuity and color vision were determined initially and after 3, 5 and 12 months and fluorescein angiography was performed. RESULTS: Patients with early AMD showed improvement of visual acuity (2 lines on ETDRS charts) in 2 out of 6 cases and a stable visual acuity in 4 out of 6 cases 1 year after rheophoresis, whereas patients with late AMD showed improvement of visual acuity (2 lines on ETDRS charts) in 1 out of 4 cases and a stable visual acuity in 3 out of 4 cases. In red-free fundus photography, a reduction in drusen size and number could be observed in 4 out of 10 cases. CONCLUSION: The results of this investigation seem to be in accordance with data from previously published controlled clinical trials. Recommendations for the indication of rheopheresis for AMD should be further defined and evaluated within the framework base of a multicentric cooperative study. PMID- 12376855 TI - [Sclerochoroidal calcification with subretinal membrane in a patient with high myopia]. AB - BACKGROUND: Sclerochoroidal calcifications are rare and benign lesions. The diagnosis is made upon their fundoscopic and echographic appearance. Because of their possible association with diseases involving disturbances of calcium and phosphate metabolism, these parameters should be investigated as well. CASE REPORT: We present a patient with the classical features of sclerochoroidal calcifications. In fluorescein and indocyanine-green angiographies a presumed subretinal neovascular membrane was observed. CONCLUSION: The diagnosis of sclerochoroidal calcifications should be complemented with angiographic investigations because of a possible association with a subretinal neovascular membrane. In two such cases reported previously in the literature, the neovascular membrane was treated by laser coagulation. In one case, as in ours, no treatment was undertaken. The good prognosis of sclerochoroidal calcifications remained unaltered in all cases. PMID- 12376856 TI - [Low vision--management of visually impaired patients by magnifying vision aids. I: Physiological and optical basic principles]. PMID- 12376857 TI - [Telecommunication and telepathology. New diagnostic routes for orthopaedics and orthopaedic pathology]. AB - In a pilot project of the Department of Bone Pathology of the University of Hamburg and the Orthopaedic Department of the University of Heidelberg, the cases of 121 patients with suspicion of a primary bone tumour have been discussed at weekly interdisciplinary conferences during the period from July 2001 to May 2002., The consequent differential diagnoses were made prior to the biopsy, the optimal location of the biopsy and the further strategy was determined according to the guidelines of the international bone tumour centres. The latter includes the decision if a conventional biopsy or a intraoperative pathology examination on frozen sections should be performed. In 27 cases an intraoperative pathology examination was performed and then assessed in Hamburg. In 24 cases this diagnosis was identical with the final diagnosis. In three cases no definitive diagnosis could be made from the frozen sections. Additionally the pathohistological diagnoses of the cases of the previous week have been discussed in the video-conferences. Through this a unusually close interdisciplinary cooperation over a large distance has evolved, that is highly appreciated especially by the young and less experienced colleagues at the department of bone pathology and the orthopaedic department in Heidelberg. The awareness of the potential and limitations of a medical subject leads to an improved safety in the diagnostic process for bone tumours. The interdisciplinary discussion of all aspects of the diseases may also optimise the therapy of bone tumours. PMID- 12376858 TI - [Histogenesis of giant cell tumors]. AB - The giant cell tumor of bone (GCT) is a local osteolytic tumor with variable degrees of aggressiveness. In rare cases pulmonary metastases can be observed. The lesion most frequently occurs in the epiphysis of long tubular bones of the knee region, predominantly affecting young adults after closure of the growth plate. The characteristic histological appearance of GCT displays a high number of osteoclast-like multinucleated giant cells, which resulted in the classification "osteoclastoma" or "giant cell tumor". Apart from the multinucleated giant cells, there are two mononuclear cell types in the GCT. The first one has a round morphology and resembles a monocyte. The second cell type is the spindle-shaped, fibroblast-like stromal cell. Cell culture experiments with GCT cells revealed the stromal cell to be the proliferating component of the GCT. The other two cell types, the monocyte and the multinucleated giant cell, were lost after a few cell culture passages. Furthermore, latest results from GCT reveal that the stromal cells secrete a variety of cytokines and differentiation factors, including MCP1, ODF and M-CSF. These molecules are monocyte chemoattractants and are essential for osteoclast differentiation, suggesting that the stromal cell stimulates blood monocyte immigration into tumor tissue and enhances their fusion into osteoclast-like, multinucleated giant cells. The multinucleated giant cell itself demonstrates properties of a normal osteoclast that is able to resorb bone leading to extended osteolysis. This new model of GCT genesis supports the hypothesis that the stromal cell is the neoplastic component whilst the monocytes and the multinucleated giant cells are just a reactive component of this tumor. Taking this into consideration, the nomenclature of the "giant cell tumor" needs to be reconsidered. PMID- 12376859 TI - [Comparative DNA cytometric investigations on aneurysmal bone cysts and giant cell tumors]. AB - Giant cell-rich bone lesions consist of tumor-like lesions and true neoplastic giant cell tumors. In this study it was investigated whether DNA cytometry may contribute to the differential diagnosis between aneurysmal bone cysts and giant cell tumors. Statistically significant differences in the frequency of tetraploid stemlines were detected. Nevertheless, the knowledge of age, localization and radiological signs in addition to morphological findings are essential to distinguish between these lesions. PMID- 12376860 TI - [Fibrous dysplasia]. AB - The observations in 222 cases of fibrous dysplasia of the Hamburg Bone Tumor Registry will be presented. This benign lesion is based on the appearance of postzygotic point mutations in a gene encoding the Gsa protein. It occurs as a mono- and a polyostotic variant and may affect every bone of the skeleton. Most often affected are the proximal femur, the skull and the ribs. Polyostotic lesions tend to occur on one side of the body. The monostotic form is 7.6 times more frequent, but both variants show no predilection for gender. Most cases are diagnosed during adulthood. On x-rays the lesion has a ground-glass appearance and is located intramedullary with a sharply defined edge. The cortical bone is arroded and the bone is expanded. Histologically typical signs are slender curved fibrous trabeculae with a C and Y shape embedded in a morphologically bland and moderately cellular fibrous stroma. Diagnostically important are collagen fibres emerging perpendicular from the surface of the trabeculae. Cartilage is present in 8% of the cases. Therapeutically, thorough clinical controls are indicated and operative procedures are rarely needed to prevent progressive deformities and fractures. The formerly applied radiotherapy is now obsolete because of the increased occurrence of malignant transformations. PMID- 12376861 TI - [Clinical pathological aspects of Mazabraud's syndrome]. AB - Mazabraud's syndrome is a rare, sporadic disorder characterised by the association of mainly polyostotic fibrous dysplasia and intramuscular myxoma. Fibrous dysplasia is mostly diagnosed at a younger age, while myxomas only occur during adulthood. We report a case of a 42-year-old female with Mazabraud's syndrome where a polyostotic fibrous dysplasia was already diagnosed and at presentation two newly formed intramuscular myxomas were found in the gluteal muscle. PMID- 12376862 TI - [Expression analysis of protein tyrosine kinases of the FAK (focal adhesion kinase) family in osteosarcoma]. AB - AIMS: Expression analysis of the protein tyrosine kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase2 (Pyk2) in high grade osteosarcomas. MATERIALS AND METHODS: Expression of the kinases was evaluated qualitatively by immunohistochemical staining and quantitatively by real-time PCR. RESULTS: Osteoblastic cells of high grade osteosarcomas show a distinct FAK expression but an overexpression at the transcriptional level could not be detected. The Pyk2 mRNA expression was decreased in osteosarcomas. CONCLUSION: An altered relationship of FAK and Pyk2 was observed for different tumors and could also be important for osteosarcoma development. PMID- 12376863 TI - [Scanning electron microscopic characterization of resorption lacunae and perforations in the cancellous bone of the human femoral head]. AB - STAND: Light microscopic investigation of histologic and grinding sections indicates structural differences by resorption lacunae and perforations QUESTION: Is it possible to characterize different types of resorption lacunae and perforations on the basis of ultrastructural differences within them? AIM. Morphological characterization of resorption lacunae and perforations in cancellous bone of the human femoral head METHODS: Samples from the femoral head of 28 patients without skeletal diseases were macerated, dried, gold sputtered and analysed by scanning electron microscopy. RESULTS. We were able to distinguish two types of resorptions lacunae as well as two types of perforation: The longitudinal extended resorption (LER), the reticulate patched resorption (RPR), the lacunar perforation (LP), the tunneling perforation (TP). Moreover we demarcate perforations from blood vessel canals. CONCLUSION: The differentiated types of resorption lacunae and perforations suggest the influence of local factors which regulates osteoclastic activity or indicate different subspecies of osteoclasts. PMID- 12376864 TI - [cDNA array approach to cytokine expression profile of aseptic loosened hip arthroplasty]. AB - FACTS: Aseptic loosening of hip arthroplasties are the main reason for revision operations. Basic research indicates a significant relevance of the interface membrane formed between the implant an the surrounding bone. Their cellular composition and the influence of various factors on the process of aseptic loosening has attracted scientific interest. Cytokines are essential for intracellular communication. QUESTION: Is it possible to reveal differences in the expression profile of cytokines between well-fixed and failed hip arthroplasties using the cDNA array approach? AIM. Generation of a cytokine expression profile characteristic for failed hip arthroplasty. METHODS AND RESULTS: Radioactively labeled cDNA probes were synthesised from mRNA isolated from the interface membrane of six patients with aseptic loosened hip arthroplasty. Using a phosphorimager the analysis of the cDNA arrays revealed nine cytokines which were overexpressed compared with the reference tissue (Calgranulin A, Calgranulin B, IL-10, MCP-1, RANTES, TFDG1, TNFR2, RAI, THYB10). CONCLUSION: In this study four out of these nine cytokines were found to be connected with the process of aseptic loosening for the first time. PMID- 12376865 TI - [Paleopathology of ancient Egyptian mummies and skeletons. Investigations on the occurrence and frequency of specific diseases during various time periods in the necropolis of Thebes-West]. AB - The scientific investigation of mummies and skeletons provides considerable data for the reconstruction of the living conditions and diseases of past populations. We describe the data on four completely analyzed tomb complexes from the huge necropolis of Thebes-West in Upper Egypt dating to different time periods. A total of 211 individuals from the so-called "Middle Kingdom" (MK, c. 2050-1750 BC) were compared to 273 individuals from the "New Kingdom" (NK) to "Late Period" (LP, in total 1550-500 BC). The age at death and the sex ratio were comparable between both groups. There was a high rate of early death with a maximum between the 2nd and 3rd decade of life but infant/adolescent burials were comparably rare. This early death is assumed to be due to an elevated prevalence of various infectious diseases. Likewise, a high rate of tuberculosis infections was seen in those individuals regardless of which time period they came from. Metabolic disorders with osseous manifestations, such as scurvy, osteomalacia and chronic anemia (cribra orbitalia, porotic hyperostosis) were found with a high frequency in the MK populations but significantly less in the NK-LP populations. On the other hand signs of trauma were comparably high, and lesions due to degenerative joint and vertebral diseases were significantly higher in LP than in MK or NK individuals suggesting a higher mechanical load in the later populations. Cases of malignant (secondary) bone tumors and various soft tissue/organ diseases indicate that "civilization" disorders were present when the living conditions assured survival into advanced age. In summary, we provide circumstantial evidence that the systematic and concise analysis of mummy and skeletal remains can allow a reconstruction of major aspects of life and disease in historic populations, although a complete reconstruction is not possible. PMID- 12376866 TI - [Do polyvinylpyrrolidone (PVP) deposits still occur in internal organs at the turn of the millennium? Observations on three patients from the former USSR]. AB - PVP had been used as a plasma expander following the end of world war II up to relatively recently but after its intracellular storage became known, it was withdrawn from use. Nevertheless, it was used as a retarding agent for subcutaneous and intramuscular administration of drugs until the 1980s and as a consequence pseudotumors have been observed. Three patients from the former USSR are described with PVP storage in the gastric and duodenal mucosa as well as in lymph nodes. The reason for the administration in these patients and the substances applied remain obscure. It is known that PVP infusions are still performed in Taiwan and that it was also injected intraarticularly as an artificial joint lubricant in Russia in the early 1990s. Because cells with intracytoplasmic deposits of PVP can be misdiagnosed as tumor cells and for reasons of general health - "la maladie polyvinylique" [1] may develop - it is still necessary to retain knowledge of the histology of cellular PVP storage. PMID- 12376867 TI - [Growth factors and apoptosis rate in an unusual chorangioma]. AB - We describe an unusual type of cellular chorangioma with a high rate of proliferating cells and mitosis and high expression of the growth factor BFGF. The tumor was observed in the placenta of a 26-year-old gravida 1 with help syndrome. The pregnancy was terminated at 35+5 weeks by elective caesarean section. Chorangiomas are hamartoma malformations of the placenta which in some cases may be large enough to influence the course of pregnancy and associated complications are hydramnion, gestosis or hemorrhage. Complications for the fetus are due to hemodynamic alterations caused by the formation of an arteriovenous shunt. Because the histogenesis of the tumor is still unknown, we examined the expression of the growth factors VEGF and BFGF. The number of cells with an expression of VEGF in placental tissue and in the chorangioma was uniform, but the number of cells with an expression of BFGF was much higher in the chorangioma than in the placenta. We conclude that BFGF may have an influence on the growth of chorangiomas. There was no difference between the chorangioma and the placenta in the rate of apoptosis-inhibited cells. PMID- 12376869 TI - [Economic significance of work disability caused by musculoskeletal disorders]. AB - Musculoskeletal disorders are the most expensive form of work disability for companies. In Germany, they are responsible for almost 27% of all production downtimes caused by sick leave from work. The direct and indirect annual expenses of these disorders amount to approximately 24.5 billion Euro for the labor force and approximately 38 billion Euro for the total population. For analytical and practical reasons, measures for prevention of this socioeconomic damage should be implemented in companies. PMID- 12376870 TI - [Disk-related diseases of the lumbar spine as an example for the critical interaction between clinical diagnosis and occupational disease]. AB - First an overview of the significance of musculoskeletal diseases in terms of national economy and social politics is given, and then the historical development of the occupational disease "disk-related spinal disorders" is outlined. The most important court decisions and the actual state of jurisprudence on this matter are summarized, emphasizing the questions which still have to be answered in the course of medical evaluation of a spinal occupational disease. Based on a joint research project on the spinal effects of whole-body vibrations, an analysis of lumbar X-rays is presented which aimed at detecting specific patterns of response corresponding to the respective extent of strain. In spite of a statistically significant relationship between the clinical diagnosis of a lumbar syndrome and the severity of the degenerative radiological changes on the one hand and vibration exposure on the other hand, the evaluation of the lumbar X-rays did not show any clear radiological pattern related to the exposure. Furthermore, starting points for prevention are discussed. With regard to whole-body vibration, the technical possibilities of reducing the amount of vibration load are still not completely exhausted. However, during preventive measures of occupational health usually carried out as medical screening examinations, the occupational health physician again will face some of the same problems which have already been met with respect to the medical evaluation. Thus, a suggestion is made to modify the traditional concepts of the Professional Industrial Associations on occupational diseases in order to take into account the peculiarities of disk-related spinal disorders. PMID- 12376871 TI - [Development and trial use of multistep diagnosis of musculoskeletal disorders during a routine physical examination at the workplace]. AB - Musculoskeletal disorders represent the main cause for absenteeism. To determine the disorders exactly, a multistep inventory of examinations was developed. The method of diagnostics may be an indicator of the quality of occupational medical management. It may improve the availability of health data related to working conditions and may increase the chances of goal-oriented prevention in the field of working conditions as well as in the field of behavior. In addition, the standardized recording of data provides an opportunity for evaluation in epidemiological studies, which may be organized as longitudinal studies to look for the development of musculoskeletal disorders over time. PMID- 12376872 TI - [The essential tension of human-machine systems]. AB - The initially interchangeable terms ergonomics and human factors have taken on different meanings over the last 50 years. Especially in the area of occupational health hazards, the scope of ergonomics has been narrowed down to the analysis of musculoskeletal stress to determine critical values, which has led to the dose model. In contrast, human factors (sometimes replaced by engineering psychology) have focussed on the optimization of allocating tasks in human-machine systems. An example from the BMW plant in Regensburg shows that in a workplace where the musculoskeletal stress remains below the critical values humans are better than machines, but that situations leading to traumata can nevertheless arise. In conclusion, possibilities provided by virtual reality are discussed regarding the integration of optimal human-machine systems with principles of preventing occupational hazards. PMID- 12376873 TI - [Design Check. A screening method to assess physical stress]. AB - The "Arbeitsschutzgesetz" as the national German implementation of the EU Framework Directive on Health and Safety at Work (89/391/EEC) demands from the employer a documentation and evaluation of hazards to which employees are exposed. All measures have to be adapted to technical progress and to state-of the-art occupational medicine, hygiene, and ergonomics."Design Check" (DC) was developed for the ergonomic evaluation of industrial workplaces with special regard to assembly tasks. Designed as a screening tool, ergonomic bottlenecks of a work system are discovered quickly and easily with DC. DC aims to fix and evaluate unfavorable design conditions of workplaces and to reduce physical overload to the lowest level possible. Evaluation of the workplace covers anthropometric, physiological, and biomechanical aspects. Taking into consideration ranges in stature and force capacities instead of discrete values, DC allows a user group-oriented evaluation, independent from the individual worker.DC takes into account the ergonomic design principles from the machinery directive (98/37/EG). Thus, DC enables a prospective evaluation of individual workplaces with respect to their ergonomic design quality and to health-related hazards. PMID- 12376874 TI - [Orthopedics and occupational medicine. Building of ergonomic workplaces in the focus of industrial health management]. AB - Musculoskeletal disorders are a challenge for the occupational physician as they can occur in a work-related situation., They must, however, be separated from occupational diseases. At first glance, the change in a worker's capability can be taken to indicate lower efficiency. The ergonomic discussion in the Porsche Improvement Process has led to solutions which could usually be realized. The change in a worker's capability led to a work conception based on current ability. PMID- 12376876 TI - [Office workplace at a computer monitor from an occupational-orthopedic view]. AB - Typical back problems as well as complaints in the shoulder-arm-hand system are increasing as the volume of sedentary office work at the computer screen rises. Various legal rules precisely define preventive measures. However, basic orthopedic-biomechanical principles are not being taken into consideration. Orthopedists as well as occupational physicians are equally challenged to enforce those legal guidelines by beginning to raise awareness of the principles and thus contributing towards improvement of working conditions. PMID- 12376875 TI - [Repetitive strain injuries. Forearm pain caused by tissue responses to repetitive strain]. AB - According to the National Research Council, painful work-related upper limb disorders are caused by different pathophysiological mechanisms, one of which is repetitive strain injury (RSI). Forearm pain, tenderness, and paresthesias are thought to result from a continual risk of exceeding limits of "cumulative trauma load tolerance" (CTLT, cf. NRC 2001) in soft tissue by thousands of high frequency, repetitive movements. On the other hand, repetitive painful stimulations also produce neuroplastic changes in the spinal and supraspinal nociceptive systems. Thus, repetitive motor and nociceptive impulses become part of the same motor programs, which are also responsible for high-frequency movements and tissue damage. In this way RSI pain may be felt as a task-related response, even after all injuries are completely healed. Consequences of this neuroplastic CTLT model for RSI prevention and therapy are discussed. PMID- 12376878 TI - [Aseptic osteonecrosis in childhood: diagnosis and treatment]. PMID- 12376877 TI - [Cashier's workplace]. AB - Individuals working at the cashier's desk increasingly suffer from problems in the neck and shoulder region as well as elbow and wrist joints. Further complaints of this group include headache, eye irritation, insomnia, and nervousness. The law requires a structured setting for the cashier's desk itself as well as the working environment, i.e., lighting, climate, noise, etc., and the work organization, such as work routine, hours of work, and breaks. Various score models are available for evaluating the above-mentioned factors. Initial attempts to view the cashier's desk as a whole system have lead to promising cooperation between different manufacturers and government offices. PMID- 12376879 TI - [Paraneoplastic neurologic autoimmune diseases]. AB - All complications of tumor disease that do not result from the tumor itself, its metastases, or vascular, infectious, metabolic, or therapy-related causes are referred to as paraneoplastic neurologic syndromes. This earlier represented an excluding diagnosis and the association was established only statistically, but since the first description of the anti-Hu antibody in 1985, we know that around 2/3 of patients with paraneoplastic disorders display highly specific antibody reactions in the serum. These reactions not only prove a paraneoplastic etiology in almost 100% of cases, but, due to their highly specific associations with certain tumor types, also aid tumor detection in patients whose tumor types are not known. PMID- 12376880 TI - [Hereditary amyloidoses associated with transthyretin mutations]. AB - Hereditary amyloidoses form a clinically and genetically heterogeneous group of autosomal-dominantly inherited diseases characterized by the ubiquitous extracellular deposit of fibrillary aggregated proteins. Main components of these unsolvable deposits are physiologic proteins that became amyloidogenic through genetically determined conformation changes resulting in an increase in beta sheet structures. In the vast majority of cases, the offending protein is variant transthyretin (TTR), of which over 80 mutations are known. Among these, substitution of valine by methionine in position 30 (TTR-Met30) is the most commonly encountered. In typical cases, TTR amyloidoses present with polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, and gastrointestinal features, occasionally accompanied by vitreous opacities and renal insufficiency. Rarely, involvement of the leptomeningeal or meningovascular structures dominates the clinical picture. The clinical expression is highly variable, with many atypical manifestations. Asymptomatic mutations have recently been identified. The age of onset varies greatly between early adulthood and old age. Late-onset atypical manifestations and occurrence of asymptomatic carriers render identification of affected family members difficult despite autosomal dominant inheritance. Orthotopic liver transplantation (OLT) is the only effective therapy available today. This OLT stops progression of the disease, which is otherwise invariably fatal, by removal of the main production site of the amyloidogenic protein. However, cardiac involvement may progress after OLT for unknown reasons. The indication for OLT and its success depend on the grade of cardiovascular and autonomic dysfunction at the time of surgery, age, comorbidity, and type of mutation. Alternative treatment modalities with drugs stabilizing the native tetrameric conformation of TTR, inhibiting fibril formation or breaking beta-sheet structures, are currently being intensively studied. PMID- 12376881 TI - [Transplantation of myelinating cells as regenerative therapy for multiple sclerosis - experimental basis and present state of clinical studies]. AB - Currently available therapies for multiple sclerosis (MS) delay disease progression via immunomodulation or immunosuppression. A persisting neurological deficit is mostly irreversible. Thus, a reparative treatment is urgently warranted. After positive results in animal models, clinical trials to promote endogenous remyelination with intravenous immunoglobulins (IVIg) or the growth factor IGF-1 were performed, unfortunately without clinical improvement. Another possibility to achieve remyelination is the transplantation of myelinating cells into the central nervous system. Proof of principle and demonstration of the functionality were shown in numerous experiments, and a first clinical trial in patients with MS has started. Although there are still several open questions, many are specific to MS and can not be answered in an animal model. This first trial will show if cell transplantation is a feasible concept in MS and whether the transplanted cells will survive and form new myelin. Schwann cells are currently the most promising cells to be transplanted, due to the advantages of an autologous transplantation from the patient's sural nerve biopsy, possibility to expand the cells in culture, and the possibility that they may escape the ongoing inflammatory reaction in MS. Other cell types are available, including stem cells, which are in the centre of a lively discussion. The results of the ongoing trial must be awaited before other transplant studies are performed to tackle other yet unresolved problems. At the time, it seems unlikely that cell transplantation will become clinical practice in the near future. PMID- 12376882 TI - [Endemic spastic paraperesis (konzo)]. AB - Konzo is characterized by the abrupt onset of isolated and symmetric spastic paraparesis of the lower extremities which is permanent but nonprogressive. Epidemic outbreaks in subtropical and tropical regions are associated with drought-provoked agricultural and social crises rendering populations dependent on a diet of insufficiently processed bitter cassava. The shortcuts in processing allow large amounts of cyanogens to remain in the cassava consumed and hence there is a high dietary cyanide exposure. Mainly children and ill people, often malnourished (and with kwashiorkor), are not able to detoxify the cyanide sufficiently, consequently developing symptoms of upper motoneuron damage. Konzo generally seems not to be caused by ignorance concerning the correct processing of cassava, but predominantly directly and indirectly by poverty. PMID- 12376883 TI - [Visual evoked potentials in Creutzfeldt-Jakob disease]. AB - Neuropathological studies show frequent and extensive effects on the visual system in Creutzfeldt-Jakob disease (CJD), but deterioration of vision is not reported by all patients. We examined the function of the visual system by means of visual evoked potentials (VEP). We recorded monocular pattern-reversal VEP in six patients with sporadic CJD 1-13 months after first symptoms occurred. Three patients had normal vision, and in a further three, vision was impaired. All patients had pathological VEP with a delayed P100 component (six eyes) or loss of cortical response (five eyes). The patients with visual impairment vs those without were not different concerning VEP findings. The VEP are already pathological in initial CJD stages and point to an early effect on the visual system in CJD, irrespective of clinical visual deficits. PMID- 12376884 TI - [Peer review of routine clinical case reports - an instrument of quality management? Results of a pilot investigation]. AB - A scoring system for quality assessment of neurological routine case reports was developed in cooperation with the Quality Management Commission of the German Neurological Society (DGN). Five clinical departments of neurology submitted eight anonymized reports for each of seven tracer diagnoses (CNS hemorrhage, ischemic infarction in the anterior circulation, epilepsy, Parkinsonism, bacterial CNS infection, multiple sclerosis (initial diagnosis), polyneuropathy) which were reviewed by two neurologists from different departments. The reports reflect differences between departments concerning certain procedures, especially technical investigations and the use of standardized scores. Interrater reliability was low. However, there were significant and meaningful differences between departments, which can be used for quality improvement. The approach can be recommended for use in peer audits between hospitals. PMID- 12376885 TI - [Successful long-term treatment of multiple metastases from renal cell carcinoma: combination of stereotactically guided percutaneous single-dose convergent beam irradiation and surgery]. AB - The case of a patient suffering from renal cell carcinoma and recurrent brain metastases shows how the survival period can be significantly prolonged by a combination of stereotactically guided percutaneous single-dose convergent beam irradiation and surgery. This 58-year-old man's left kidney was completely excised because of a renal cell carcinoma. After 18 months and 25 months, respectively, a right frontal brain metastasis was operated on. In the next 7 years, biannual MRI checks were carried out which successively showed five different brain metastases, each of which was immediately subjected to single dose stereotactic irradiation (median dosage: 20 Gy prescribed to the 80% isodose). In the following 2 years, operations were carried out on two metastases which could not be treated by radiation because of their considerable size and partial compression of the ventricle. In the next 3 years, four more brain metastases were subjected to single-dose stereotactic irradiation. There were no metastases in the other organs. At present, the patient is in good clinical condition and mobile. A negative prognosis is usually delivered for patients suffering from renal cell carcinoma and brain metastasis. However, in individual cases, the survival period can be significantly prolonged by regular MRI examinations and a combination of neurosurgery and single-dose stereotactic irradiation. PMID- 12376886 TI - [LOVA hydrocephalus - a new entity of chronic hydrocephalus]. AB - The recently defined term "longstanding overt ventriculomegaly in adults" (LOVA) describes a unique entity of chronic occlusive hydrocephalus. The experiences so far using conventional DP valves were not encouraging because of a high percentage of overdrainage. The objective was to evaluate whether gravitational shunts could be used for this condition with an acceptable overdrainage risk. Twenty-three macrocephalic adults aged 17-72 years suffered from chronic progressive hydrocephalic conditions. They received two different types of gravitational shunt. Follow-up ranged from 6 months to 75 months. Only two patients presented small subdural effusions postoperatively, and only one required additional treatment for that. Eighty-two percent were shunt responders. Ventricular size was only marginally reduced in 22 of the 23 patients. There was no correlation between clinical benefit and the reduction in ventricular size. Gravitational shunts clearly have the potency for safe treatment of LOVA, significantly reducing the risk of overdrainage over conventional valves, and may be considered an equivalent alternative to third ventriculostomy. PMID- 12376887 TI - [Pesticide exposure and Parkinson's syndrome - the epidemiological and experimental evidence]. AB - Retrospective case-control studies among patients with idiopathic Parkinson's syndrome (IPS) show a positive association to the existence of a - mostly premorbid - exposure to pesticides. In acute pesticide intoxications, usually symptoms other than parkinsonism are found. Therefore, 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) continues to be the agent best documented both experimentally and clinically to cause a clinical syndrome comparable to IPS. It is debated whether still unknown effects between exogenous pesticide exposure and the xenobiotic enzyme system may lead to IPS in single genetically susceptible individuals. In practice, the present data on the problem of pesticide exposure in IPS are irrelevant for medicolegal considerations. PMID- 12376888 TI - [Meningioangiomatosis with associated meningioma in a 4-year-old girl presenting with a focal seizure]. AB - Meningioangiomatosis is regarded as a rare, benign, hamartomatous malformation. Histopathologically, the lesion is characterized by circumscribed transcortical and leptomeningeal meningovascular proliferation with focal calcifications. It may be classified into cases with predominant cellular or vascular features and may occur in association with neurofibromatosis, mostly of type 2, but sporadic cases are more frequently reported. Sporadic cases often present initially with seizures and can be treated surgically. However, a certain percentage of patients will need ongoing anticonvulsive therapy. The lesions are seldom associated with an overlying meningioma. These are usually benign lesions that must be strictly separated from an invasive anaplastic meningioma, which would warrant an adjuvant therapy. We report on a 4-year-old girl who presented with spontaneous, predominantly cellular meningioangiomatosis with associated fibrous meningioma. Focal immunopositivity of the meningioangiomatosis for CD34 was helpful in ruling out an invasive meningioma. PMID- 12376889 TI - [Paraneoplastic cerebellar degeneration associated with ovarian cancer: anti-Yo immunoreactivity in autoptic cerebellum and ovarian carcinoma]. AB - Paraneoplastic cerebellar degeneration is a rare disorder caused likely by autoimmune mechanisms in malignant oncologic diseases, and the most common tumors are ovarian, breast, lung cancer, and m. Hodgkin. An immune reaction is supposed to be directed against identical antigens of cerebellum and tumor, and paraneoplastic antibodies called anti-Yo, anti-Hu, anti-Ri, or anti-Tr are often detected in blood and cerebrospinal fluid. The course of paraneoplastic cerebellar degeneration as a complication of ovarian cancer is described. The relationship between the malignancy and pathologic changes in cerebellum was confirmed by positive immunohistochemical and immunofluorescence reaction between a patient's anti-Yo-positive serum and her own Purkinje's and ovarian cancer cells. PMID- 12376890 TI - [Diffusion-weighted MRI of spinal cord infarction. Description of two cases and review of the literature]. AB - Spinal cord infarctions occur rarely and are due to various aetiologies. In an emergency setting with acute spinal cord symptoms, magnetic resonance imaging (MRI) is used to exclude space-occupying lesions which require neurosurgical intervention. We report on two patients presenting with an anterior spinal artery syndrome caused by infarction of the thoracolumbar spinal cord including the conus medullaris. While T2-weighted images 4 h and 28 h after onset of clinical symptoms showed only slight unspecific signal changes, diffusion-weighted imaging revealed clear infarction and detected spinal cord ischaemia in an early stage, showing signal intensity conversion comparable to that in acute cerebral stroke. PMID- 12376891 TI - [Hauptmann-Thannhauser muscular dystrophy and differential diagnosis of myopathies associated with contractures]. AB - Hauptmann-Thannhauser muscular dystrophy is characterized by the clinical triad of early-onset contractures of elbow, Achilles tendons, and cervical spine, slowly progressive humeroperoneal muscle wasting and weakness, and life threatening cardiac involvement with conduction blocks manifesting in the third decade. Hauptmann-Thannhauser muscular dystrophy is due to mutations in the LMNA gene affecting the nuclear envelope proteins lamin A and C. We present a 16-year old German boy with typical muscular involvement and contractures and typical course of Hauptmann-Thannhauser muscular dystrophy due to the novel missense mutation R401C. The data of this family suggest a lower penetrance of muscular and especially cardiac symptoms than expected. Autosomal-dominant Hauptmann Thannhauser muscular dystrophy and X-chromosomal Emery-Dreifuss muscular dystrophy are not clearly distinguishable by phenotypic criteria. Other muscular diseases associated with contractures and congenital or childhood onset are reviewed. PMID- 12376892 TI - [The extraarticular proximal tibial fractures]. AB - Operative stabilization of proximal tibial fractures by use of conventional osteosynthesis is still problematic. The choice of the osteosynthetic treatment is strongly influenced by the situation of the surrounding soft tissue. Additional problems in this particular location may occur with malalignment in the fracture site after operation. Primary intraoperative malalignment may occur due to dislocating muscle forces or to the operative approach itself. Secondary dislocation is mainly due to the unstable fixation of the proximal fragment by the implant. Today many different implants with specific biomechanical properties are available. Each system requires a particular operative technique and can lead to individual implant-related problems. The new angle stable implant systems (e. g. LISS = "less invasive stabilization system"), offer significant advantages over conventional plate osteosyntheses and external fixation systems. Improvement of the geometry of standard intramedullary osteosyntheses and introduction of angle stability in the proximal interlocking screws (PTN = "proximal tibial nail") seemingly make this system the optimal solution, concerning biomechanics. On the background of our own clinical experiences and biomechanical investigations, the article discusses solutions for this particular problem. PMID- 12376893 TI - [Endoscopically assisted minimally invasive reconstruction of the anterior thoracolumbar spine in prone position]. AB - Irrespective of an anterior open or endoscopic approach, the combined postero anterior instrumentation of thoracolumbar fractures requires time consuming intraoperative maneuvers changing the patients position from prone to lateral.A standardised anterior endoscopically assisted approach for the segments Th4 to L4 is described, allowing the patient to remain in prone position, using a 4-5cm incision combined with a retractor system. The approach to the anterior spine in prone position is feasible by using a self holding retractor system for the region from Th4 to L4. Time of anaesthesia for the one stage combined procedure can be reduced by about 40 min, when changing the position of the patient is no longer necessary. The minimal incision in combination with the retractor system allows mainly the use of conventional instruments and implants, which provides reasonable lower costs. The advantages of the open and the endoscopical technique are combined. The main advantage of the prone position is the opportunity to access the anterior and posterior spine simultaneously, which is extremely helpful in reduction maneuvers. PMID- 12376894 TI - [Intra- and perioperative complications in the stabilization of per- and subtrochanteric femoral fractures by means of PFN]. AB - In the period from 2/98 up to 6/99, fractures of the proximal femur of 70 patients (phi 79.2 a), were treated with a proximal femur nail (PFN((R))). The aim of this retrospective analysis was to evaluate intra- and perioperative complications. The reports of the operation and the anaesthesia documentation sheets have been worked out, all X-rays have been measured, and evaluated along exactly defined parameters. Problems or complications were found in 18 cases (25,7%). In 7 cases (10,0%) these problems could be solved during operation. The "Z-effect" seen in 5 cases (7,1%) and the "cut-out" of the sliding-hip-screw in 6 cases (8,6%) were the most frequent complications. The main reason was a bad primary reposition in varus with a CCD-Angle less than 125 degrees. PMID- 12376895 TI - [Computer assisted pelvic and acetabular surgery. Clinical experiences and indications]. AB - CT based navigation has been used in spine surgery since 1994. Several clinical studies could show an increase in precision compared to the conventional technique and thus nowadays the navigated pedicle screw placement is a routine procedure in many hospitals. Based upon the experience in spine surgery the CT based navigation module was used for percutaneous screw fixations in minimally displaced pelvic ring and acetabular fractures. After preclinical experimental trials the C-arm navigation was used for 19 screw fixations. The postoperative control of the screw position was performed with postop. X-ray and CT. Overall 23 of the 24 screws were placed correctly. In one SI screw the postoperative CT could reveal a ventral cortex perforation of the sacrum without any clinical symptoms. Based upon this limited clinical experience we see the indication for CT based navigation in minimally displaced acetabular fractures or in SI screw fixations in case of sacral dysplasia. The C-arm based navigation with adequate image quality is our method of choice for SI screw fixation in traumatic or degenerative instabilities, especially if reduction maneuvers are necessary. PMID- 12376896 TI - [Acetabular fractures in the elderly. Results of a sophisticated treatment concept]. AB - Acetabular fractures in elderly patients are rare injuries, but their incidence is increasing. Poor bone quality due to osteoporosis and an increased operative risk due to concomitant disease are factors complicating surgical therapy. Literature does not provide generally accepted treatment protocols. In a 4-year period, 27 patients who were 65 years or older and who had an acute displaced fracture of the acetabulum were admitted to our department. Four minimally displaced and stable fractures were managed conservatively. Internal fixation was performed in 16 cases. According to the Merle d'Aubigne score, in 15 out of 18 surviving patients excellent or good results were found. Treatment strategy should be planned individually for each fracture, taking into account the patients biological age and general condition, fracture type, bone quality and associated injuries. Primary endoprosthetic replacement should only be considered when the acetabular bone stock allows stable cup fixation. Osteosynthesis in combination with early endoprosthetic replacement should be considered in acetabular fractures with associated femoral head or neck fractures or when significant articular steps and/or bone defects remain after open reduction and internal fixation. PMID- 12376897 TI - [Bilateral carotid dissection. A not to underestimate cause of neurological loss after road accident]. AB - The dissection of the internal carotid artery is a rare complication of acceleration traumas of the upper spine. 30% of these dissections are caused by road accidents and again less than 30% of these occur bilateral as shown here. The symptoms are fronto-temporal and periorbital starting headaches spreading out to the occiput and Horner's syndrome. Complete hemiplegia as in our case is an impressive exception but the doctor in attendance should think of the carotid dissection. The exclusion of this complication is obligatory because treatment and outcome depend on it.The dynamic effects of bilateral carotid dissections may, as shown here, lead to relapsing cerebral infarctions with persisting neurologic deficits up to manifest hemiparesis. But restitution can be accomplished if early diagnosed by DSA and/or MRI. Therapy of choice is early prevention of persisting neurologic deficits using effective dosed heparin and depending on the residual lumen of the vessel oral anticoagulants or platelet antagonists for one year. PMID- 12376898 TI - [The conservative treatment of femur fractures by Perkins traction. Management in adverse situations]. AB - In adverse situations and in hospitals of less prosperous countries, the operative treatment of fractures may for many reasons not be possible and conservative procedures remain the treatment of choice. This applies to the rule that under difficult conditions fracture treatment should be as conservative as possible and as operative as necessary, if at all feasible. This report goes on 109 Patients with femur fractures consecutively treated by Perkins traction in East African hospitals between Nov. 1991 and Sept. 1999. Out of them, 44 patients had wide open fractures, 41 of them caused by explosives and bullets. The Perkins procedure is a traction without a fixating splint. As soon as possible, the patient is forced to sit up in his bed, the removable parts of the springs are dropped and the patient starts with exercises flexing and stretching the knee. With the right traction weight and the bodys counterweight the fragments of the fracture find an alignment, and a malrotation of the distal fragment is prevented. Exercises as early as possible stimulate a rapid callus formation and prevent muscle atrophy and stiffness of the joints. We have seen 2 posttraumatic deep infections, 5 refractures in patients whose traction was removed too early or who fell down whilst walking with crutches. All patients had a good callus formation and in all patients, except two, the traction could be removed after 6 12 weeks, in the most cases even after 6-9 weeks. To the patients with refractures, the traction was reapplied and callus consolidation then occurred without major problems. At the time of removal of the traction all patients showed a flexion of the knee of at least 80 degrees -90 degrees and all were able to nearly fully stretch the knee joint.Compared with other methods of conservative treatment of femur fractures, Perkins traction has some advantages: simple management, immediate start of exercises, simple exercises, early callus formation, no stiffness of the joints and only few x-ray controls. Malalignment, non union, excessive shortening and rotation of the distal fragment are uncommon. PMID- 12376899 TI - [Septic arthritis of the shoulder following intra-articular injection therapy. Lethal course due to delayed initiation of therapy]. AB - Detection of a bacterial arthritis of the shoulder represents an absolute indication for intervention. Irrespective of the cause of the infection, the most decisive prognostic factors are early diagnosis and therapy. We report on two patients who suffered from generalized sepsis and resulting death after delayed treatment of iatrogenic joint infections of the shoulder caused by intra articular injection therapy. Both patients suffering from septic shock syndrome had been transferred to our hospital for surgical and intensive care treatment. They died in spite of maximal intensive care and aggressive surgical treatment. On the basis of the cases presented, it can be concluded that an acute infection of the shoulder joint must be excluded early when painfully limited range of motion in combination with clinical and laboratory signs of inflammation become apparent. Successful therapy of joint infection also requires early surgical treatment, including resection of infected tissue. If surgical joint revision is not performed or is performed too late, there is the risk of irreversible damage to the afflicted joint, even septic spread endangering the patient's life. PMID- 12376900 TI - [Steroid-induced osteonecrosis of femoral condyles and bilateral Freyberg's disease]. AB - We report on a patient with steroid-induced osteonecrosis of the femoral condyles after therapy of an acute lymphatic leukemia. Because of continuing bilateral knee pain, we performed osteochondral autografting of the right femoral condyle in two steps. During the follow-up period, the patient developed bilateral Freyberg's disease, which was also successfully treated by surgery. The MRIs which we performed as a follow-up 3 years later showed complete incorporation and vitality of the transplanted cylinders. No further clinical symptoms occurred. PMID- 12376901 TI - [Intraligamentous suture of a scapholunar ligament lesion in a 9-year-old child]. AB - Scapholunate dissociation is a well-known injury in adult patients. In pediatric patients, repair of this injury in the skeletally immature carpus has been previously reported. However, in none of these case studies is single ligamentous suture performed. We report a case of scapholunate dissociation in a 9-year-old boy after an initial Salter I injury of the distal radius. After 6 weeks of wrist immobilization, arthroscopy was performed due to persisting pain over the scapholunate gap, a positive Watson sign, and limited range of motion. This arthroscopy revealed intraligamentous rupture of the scapholunate ligament. Suture repair of the scapholunate ligament was performed. The suture was protected by a temporary K-wire arthrodesis for 8 weeks. One year after removal of the K-wire, the patient is completely free of symptoms and resumes all sport activities. PMID- 12376902 TI - [Waiting for the EuGH verdict to be put into practice or "Introduction of the four-day week on full pay"]. AB - The submitted model of working time transposes and interprets german industrial law. The result of this interpretation is a high level of acceptance of the employees, a fast education that is high qualified with costs that are still affordable. The advantage of this model compared with the shift-model that runs after the EuGH-decision is obvious if you look at the reality of our health care system. This is why it is important to have an efficient interpretation of the existing law. Of course it will be a necessity also in the future to create new models of working time and to adapt these models in a way that it fits into the structure of a hospital. It would be the wrong way to force a juridical and political decision, how it was done by the german government that gave a deadline to put the EuGH decision into operation, without the possibility of an interpretation that fulfils the demand of the hospital. PMID- 12376903 TI - [Extended kyphoplasty indications for stabilization of osteoporotic vertebral compression fractures]. AB - OBJECTIVES: Percutaneous vertebroplasty with polymethylmethacrylate allows minimally invasive stabilization of osteoporotic vertebral fractures. Fracture reduction is, however, not possible and the risk of uncontrolled epidural cement leakage with burst fractures is increased. Kyphoplasty, in contrast, allows a degree of fracture reduction and provides an extended spectrum of indications through open approaches, which enable spinal decompression and augmentation of incomplete burst fractures. METHODS. In kyphoplasty a contrast-filled balloon is inflated in the vertebra until a cavern is created. A degree of reposition may be achieved depending on fracture age. Augmentation is performed with high-viscosity polymethylmethacrylate under low pressure. In cases of neural compression, interlaminary spinal decompression and kyphoplasty through the posterior wall is performed. With anterior spinal procedures, kyphoplasty can be performed without extending the approach. RESULTS: Vertebral augmentation was performed by percutaneous, interlaminary, and anterior approaches for incomplete burst fractures. Four representative cases are presented from a collective of 120 augmentations. CONCLUSIONS: Percutaneous kyphoplasty, supplemented by open approaches, enables augmentation of osteoporotic incomplete burst fractures. PMID- 12376904 TI - [Methods and techniques of anesthesia for securing the airway in ENT medical interventions]. PMID- 12376905 TI - [CT and MRI of the petrous bone]. AB - Diseases of the petrous bone should now be diagnosed by means of high-resolution multislice spiral computed tomography (MSCT) and/or magnetic resonance imaging (MRI). The first step in the process of diagnosis, however, must be conventional X-ray photographs (according to Schuller, Mayer, Stenvers) for screening purposes, because of the high cost of the other procedures mentioned. Because of the excellent imaging of bone structures with MSCT, this technique is especially suitable for the diagnosis both of acquired pathologies and of congenital abnormalities of the external auditory meatus, the middle ear and the mastoid, of trauma-induced pathologies of the entire petrous bone, and of osteogenic diseases. MRI is the method of choice for examination of the labyrinthine system, the interior auditory meatus and the cerebellopontine angle because it gives much the best depiction of soft tissue. Sometimes when questions remain unsolved after computed tomography (CT) examination of the middle ear MRI can be applied to complement CT, and it can yield additional information. Lesions affecting the apex of the petrous pyramid should be examined by MRI. High-resolution CT through the bone window and thin-layer MRI are both components of the presurgical diagnosis before cochlear implant (CI) surgery. For postoperative monitoring a conventional transorbital X-ray of the petrous bone is sufficient; CT is indicated only in complicated cases, and MRI is absolutely contraindicated after CI. PMID- 12376906 TI - [Genotoxic effect of PCP and lindane on human epithelial tonsil cells]. AB - BACKGROUND AND OBJECTIVE: Experimental and epidemiological studies have provided evidence that pentachlorophenol (PCP) and gamma-hexachlorocyclohexane (lindane) may pose a potential carcinogenic risk for human epithelial cells of the upper aerodigestive tract. In the past, these two substances have been used for military and nonmilitary purposes, e.g., for impregnation of textiles and uniforms. In this study, we investigated the genotoxic effect of PCP and lindane on human mucosal tissue from the tonsils. METHODS: In epithelia obtained from the tonsillar mucosa removed during surgery, cell viability was evaluated by trypan blue staining. The specimens were incubated for 60 min with PCP (0.3, 0.75, and 1.2 mM) and lindane (0.5, 0.75, and 1.0 mM). The induction of DNA damage (single- and double-strand breaks) caused by PCP and lindane was measured using single cell microgel electrophoresis. Evaluation was performed with an image analyzer enhanced by fluorescence microscopy. RESULTS: After exposure to PCP and lindane, strong genotoxic effects are apparent. The DNA migration rose from 26 micrometer in the control solution up to nearly 90 micrometer after incubation with PCP and lindane ( p<0.0001). CONCLUSIONS: This study could demonstrate for the first time genotoxic effects of PCP and lindane on human tonsillar epithelium. It has to be considered that chronic exposure to both agents might increase the risk for cancer of the upper aerodigestive tract. PMID- 12376907 TI - [Intraoperative navigation in surgery of paranasal sinus and anterior skull base]. AB - BACKGROUND AND OBJECTIVE: Based on physical laws, stage of technical development and the user's individual skills a number of possible errors have to be considered for the application of CAS in paranasal sinus and anterior skull base surgery. PATIENTS/METHODS: Based on our experiences of 436 navigated cases hard- and software errors, errors of image acquisition and transfer, errors of patient registration, user related errors as well as strategic errors are analyzed. RESULTS: Any hindrance of the camera field leads to a limitation of functionality of optical systems in the same extent as electromagnetic systems can be affected by ferromagnetic materials. The mode of image acquisition is dependent from the CAS-system involved. The reconstruction algorithm requires particular attention. The patient registration based on the headset proved to be reliable for endonasal sinus surgery. CONCLUSIONS: In dealing with navigation devices in paranasal and anterior skull base surgery the user must pay critical attention to possible malfunction in order to guarantee a successful image guided surgical procedure. PMID- 12376908 TI - [Morphological and functional results of Palisade Cartilage Tympanoplasty]. AB - BACKGROUND AND OBJECTIVE: The application of cartilage in tympanoplasty has been generally accepted, because cartilage as a bradytrophic tissue allows stable and functionally reliable reconstruction of the eardrum even in difficult pathological conditions (such as subtotal defects, tympanosclerosis etc.). A special surgical technique using small cartilaginous chips for the reconstruction of the eardrum has been developed by J. Heermann, who introduced it as Palisade Cartilage Tympanoplasty (PCT). Although being increasingly applied in otosurgery, this technique has to date neither been evaluated regarding morphological and hearing results nor regarding its combination with titanium ossicular reconstruction prostheses. PATIENTS AND METHODS: Therefore we reviewed 84 of 94 ears (92 patients, 58 female, 34 male) 12 to 36 months after PCT. RESULTS: A recurrent defect was seen in 2 ears (1 adhesive otitis, 1 subtotal defect). There were no extrusions of prostheses. Preoperatively an ear-bone-gap of 0-10 dB was seen in 2 ears, 11-30 dB in 48 and 31-59 dB in 34 ears. Postoperatively the corresponding numbers were 25, 50 and 9 ears. CONCLUSIONS: The low rate of recurrent tympanic membrane defects (2.4%) shows that palisade cartilage tympanoplasty is particularly appropriate for the management of difficult indications in middle ear surgery. Further, it could be demonstrated that the PCT can be combined safely with titanium ossicular reconstruction prostheses. Regarding postoperative hearing results the negative preselection of pathological conditions has to be considered. PMID- 12376909 TI - [Endolaryngeal biopsies with local anesthesia]. PMID- 12376911 TI - [BAHA (bone-anchored hearing aid) in bilateral external ear dysplasia and congenital ear atresia]. PMID- 12376910 TI - [Otosialorrhoea - a rare case of a spontaneous salivary fistula of the external auditory canal]. AB - Spontaneous salivary fistulas are rare. Extremely rare are spontaneous salivary fistulas of the external auditory canal. We report a case of otorrhoea caused by such a fistula in connection with defects of the cartilagenous and the osseous external auditory canal (Fissures of Santorini, foramen of Huschke) and its successful surgical treatment. PMID- 12376912 TI - [Mind and matter - id and super-ego revisited?]. PMID- 12376913 TI - [Towards a neuropsychology of autonomy: re-presenting forgetting, subliminality and freedom]. AB - Recent neurobiological concepts about sensomotor processes in animals exhibit that voluntary motor behavior is not due primarily to external cues but to internal activation-processes ("initial activity"). Thus questions are raised as to the role of internal "limbic" valuation-processes in relation to contexts of the past. These are not limited to the actual memorial contents but also relate to subliminal aspects of the past. The inclusion of processes of "re-presenting forgetting" means a re-evaluation of autonomy within a neuropsychological theory of freedom. Consequently, subjective freedom may be realized by intrapsychic processes of re-presentation of the past. PMID- 12376914 TI - [Historical review and recent research trends of the antidepressant repetitive transcranial magnetic stimulation (rTMS)]. AB - The antidepressant effect of repetitive transcranial magnetic stimulation (rTMS) has been studied intensively over the last years. Numerous studies worldwide yielded variable results ranging from ineffectivity to an efficacy rate of more than 50 % for the reduction of depressive symptoms. A comparison of the effectiveness of TMS across studies is difficult due to the variety of chosen stimulation parameters, different inclusion criteria, and different designs. The discrepancies in results could be partially in account to these factors. From a pathophysiological point of view, there are no empirically justified parameters published carrying out an antidepressant TMS. Further studies are required to find optimal stimulation parameters using complementary methods like functional imaging techniques or regarding other factors that could interfere with its efficacy. An overview of studies published in the last ten years, a critical analysis of stimulation procedures and protocols, and possible future perspectives of this method are reviewed in this article. PMID- 12376915 TI - [Delusions of parasitosis: An up-to-date review]. AB - This paper gives an up-to-date review on delusional parasitosis (DP) and particularly meets clinical interests. Facing the lack of clear guidelines for clinical practice an efficient diagnostic staircase procedure is presented. The different approaches in the therapy of DP are reviewed and summed up in a checklist. The available information on the therapeutic gold standard, the neuroleptic agent pimozide, is discussed. Symptoms and clinical characteristics of DP are presented partly based on material from an internet website run by patients. Moreover, this paper delivers a comprehensive literature review on the pathogenesis of DP and its classification. To conclude, possible future therapies in DP, e. g. serotonin-dopamine-antagonists, as well as current methods in research on hallucinations are discussed. PMID- 12376916 TI - ["Stalking"--a popular conceptualisation of disturbing behaviour with limited practicability for forensic psychiatry]. AB - Aspects of "stalking" behaviour have been discussed predominantly in the Anglo American psychiatric literature. However following new legal regulations, "stalking" is expected to gain relevance for the German forensic psychiatry. "Stalkers" are a heterogeneous group presenting with a wide range of relational or even psychotic motives for their behaviour. Therefore careful diagnostics are required. Legal responsibility can be estimated by assessing the capacity of the offender to understand (cognitive functioning) and to control his behaviour (volitional functioning). The severity of possible restrictions can be assessed by comparing them to the known impairments in cases of erotomania. PMID- 12376917 TI - [Does caring for a schizophrenic family member increase the risk of becoming ill? Psychological and psychosomatic troubles in caregivers of Schizophrenia patients]. AB - OBJECTIVE: The aim of this study was to investigate to what extent caregivers of schizophrenia patients suffer from psychiatric and psychosomatic symptoms themselves; furthermore, whether there are differences between parents and spouses. METHOD: 51 parents and 52 spouses of people with schizophrenia were interviewed regarding psychiatric and psychosomatic troubles using standardized questionnaires and diagnostic methods. RESULTS: A considerably increased prevalence of depressive disorders was found compared to the level in the general population. As well as mothers and wives, caregivers of patients with severe impairments of psycho-social functioning were particularly affected. The severity of the patient's disease and the caregiver's mental problems are significant predictors of psychosomatic complaints in parents and spouses. In addition, caregivers visit physicians more frequently, in particular family doctors, psychiatrists and psychotherapists. DISCUSSION: The results support the hypothesis that the burden carried by caregivers of severely affected schizophrenia patients increases their risk of becoming ill, which, as a consequence, leads to a greater use of medical resources. Specific offers of health care and advice on preventative measures appear to be necessary in order to preclude health impairments to caregivers as early as possible. PMID- 12376918 TI - Demyelinating pseudotumor. AB - Demyelinating disease presenting as a solitary contrast-enhancing mass poses a diagnostic challenge for both radiologists and surgical pathologists. We report the cases of two female patients, aged 23 and 37 years, who exhibited the clinical and radiologic features of a space-occupying mass strongly suggestive of neoplasia. In both patients, magnetic resonance imaging showed a ring-enhancing parietal lesion. Intraoperative frozen sections in both patients displayed histologic features strongly suggestive of a glial neoplasm, including marked hypercellularity, a prominent astrocytic component, and easily identifiable mitotic figures. However, permanent sections showed additional and helpful histologic findings that included Creutzfeldt astrocytes and granular mitoses. Subsequent immunostaining showed that the hypercellularity was principally caused by macrophage infiltration (HAM-56 and CD68) and an associated reactive astrocytosis (glial fibrillary acidic protein). Additional confirmatory tests included special stains for myelin (Luxol-fast-blue), which demonstrated focal, sharply marginated loss of myelin, and for axons (silver stain for axons and neurofilament protein immunohistochemistry), which showed relative preservation of axons in areas of myelin loss. Together, the special stains confirmed the demyelinating nature of the lesions. The keys to avoiding misdiagnosing a demyelinating pseudotumor as a diffuse glioma include a general awareness of this potential pitfall, including the radiologic appearance of demyelinating pseudotumors as contrast-enhancing solitary masses that mimic tumor; knowledge of the characteristic histologic features, including Creutzfeldt astrocytes and granular mitoses; and a high index of suspicion for macrophage infiltration combined with a willingness to use appropriate confirmatory immunohistochemical studies in suspicious or uncertain cases. This approach will minimize the chance of misdiagnosis and subsequent use of inappropriate and deleterious therapies. PMID- 12376919 TI - Myxoinflammatory fibroblastic sarcoma: a tumor not restricted to acral sites. AB - We report on nine new cases of myxoinflammatory fibroblastic sarcoma; in six of them the location of the tumor was distal (acral), and proximal in three (forearm, arm, and thigh). Tumors varied in size from 1.5 to 18 cm, were well circumscribed, yellow-tan, and focally myxomatous. Histologically, they were similar in appearance and showed vaguely lobular architecture and oval, spindle, and epithelioid neoplastic cells with scattered, focally aggregated inflammatory cells. In all cases, in different numbers, bizarre giant cells with large, lobulated, or multiple nuclei were also admixed, some of them morphologically imitating Reed-Sternberg cells, lipoblasts, or ganglion cells; they showed distinct nucleoli or intranuclear inclusions. Myxoid areas were always present, to different extent. Immunohistochemically, tumor cells were uniformly positive for vimentin; some cells were also positive for CD68 and CD34. Ultrastructurally, tumor cells were nondescript, consistent with fibroblastic origin. On flow cytometry, two of the examined cases showed diploid pattern with low S-phase fraction. In none of the cases, metastases were observed, in one case the tumor recurred 5 years following surgery. We conclude that myxoinflammatory fibroblastic sarcoma is a distinct soft tissue tumor of low-grade malignancy and, until now, described only in extremities, although not confined to acral sites. PMID- 12376920 TI - Primitive neuroectodermal tumor of the cervix: a clinicopathologic and immunohistochemical study of two cases. AB - Two cases of primary primitive neuroectodermal tumors of the cervix are presented. The two female patients are 35 and 51 years of age who presented with abnormal uterine bleeding of several weeks' duration. On gynecologic examination, a mass in the cervical area was palpated and a biopsy was obtained. The initial biopsy was interpreted as possible small cell carcinoma in both women. A radical hysterectomy was performed in both patients. Grossly, in both cases, the uterine cervix showed an ill-defined tumor involving the ectocervix and endocervix, measuring 3.0 and 4.0 cm in greatest dimension, respectively, and showing areas of necrosis and hemorrhage. Histologic sections showed the presence of a malignant neoplasm arranged in cords and with a vague nesting pattern. Areas of hemorrhage and necrosis were also present. The neoplastic cells were characterized by having indistinct cell borders, small round to oval nuclei, and inconspicuous nucleoli. Mitotic figures were easily identified. In one patient, the tumor had metastasized to lymph nodes. Immunohistochemical studies revealed the neoplastic cells to be positive for antibodies for CD99 and focally for synaptophysin, while keratin, chromogranin, smooth muscle actin, desmin, and neurofilament protein were negative. Both patients received adjuvant chemotherapy and remain alive 5 and 18 months after initial diagnosis, respectively. The present cases highlight the importance of keeping primitive neuroectodermal tumors in the differential diagnosis of small cell neoplasms of the uterine cervix. PMID- 12376921 TI - Giant cell tumor of the skin: a morphologic and immunohistochemical study of five cases. AB - Giant cell tumor (GCT) of the skin is a rare entity that possesses similar gross and histologic features to GCT of bone. When located predominantly in the dermis GCT has been mistaken for benign fibrous histiocytoma and atypical fibroxanthoma. We report the clinical, morphologic, and immunohistochemical features of five cases of GCT of the skin. With one exception, all tumors are confined to the dermis. Patients' ages range from 6 to 78 years (median, 73 years) with a male to female ratio of 3:2. Gross and histologic features of the lesions are similar to those of GCT of bone (eg, brown fleshy tumor and a biphasic population of mononuclear cells admixed with osteoclast-like giant cells, respectively). The nuclei of the giant cells are similar to those of the mononuclear cells. A fascicular pattern with focal storiform arrangement of spindle neoplastic cells is noted in two cases. The osteoclast-like giant cells and some of the mononuclear cells are strongly positive for CD68, alpha-1-antitrypsin, and alpha 1-antichymotrypsin. Only the mononuclear cells express smooth muscle actin focally in one case. Both the osteoclast-like giant cells and the mononuclear cells are negative for cytokeratins (AE1/AE3 and CAM5.2) and S-100 protein in all cases. One patient developed lung metastases at presentation and local recurrence 4 months status post surgery. All patients are without evidence of disease 1 month to 12 years status post surgery. Cutaneous GCTs are low-grade sarcomas that can recur locally and infrequently metastasize. These tumors should be distinguished from a variety of cutaneous neoplasms that contain multinucleated giant cells. PMID- 12376922 TI - Diagnosis of gastrointestinal stromal tumor by endoscopic ultrasound-guided fine needle aspiration biopsy--a potential pitfall. AB - Endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNA) is considered to be a reliable and accurate method for the evaluation of submucosal lesions in the gastrointestinal tract. Herein, we report our experience with the diagnosis of 10 cases of gastrointestinal stromal tumor (GIST) using EUS-FNA. The materials obtained from the EUS-FNA were stained with the rapid Romanowsky or the Papanicolaou method for cytologic examination. The subsequent surgical resection specimens were submitted for histopathologic examination. Immunoperoxidase stains were performed on the cell blocks and/or representative histologic sections of the tumor using commercially available antibodies against c-kit (CD117), CD34, S 100, and smooth muscle actin. Of the 10 cases studied, there were five men and five women with an average age of 62 years (range, 38 to 87 years). Five tumors were located in the stomach, and five in the duodenum. Tumor size ranged from 3.5 to 16.2 cm. Immediate on-site evaluation and cytologic diagnoses were given in eight cases (80%) with an average of three passes. The diagnoses were confirmed by strong and diffuse tumor cell c-kit immunoreactivity in the cell blocks. However, the final diagnoses of two other cases (20%) were not established until surgical resections were obtained. Retrospectively, reviews of cytologic smears of both cases demonstrated rare cohesive sheets or clusters of spindle cells with cigar-shaped nuclei. These observations were initially misinterpreted as benign fibrous tissue and/or fragments of smooth muscle of the gastrointestinal wall such as one might encounter in a routine transgastric or transduodenal EUS-FNA. The current study showed that when combining cytologic and immunocytochemical studies, EUS-FNA is accurate and efficient in the diagnosis of GIST. It exemplified the importance of considering GIST in the differential diagnosis of gastrointestinal lesions and also demonstrated the potential pitfalls of EUS-FNA evaluation of submucosal lesions in the gastrointestinal tract. PMID- 12376923 TI - Metastatic epithelioid sarcoma to the brain: palisaded necrosis mimicking glioblastoma multiforme. AB - Epithelioid sarcomas are rare, morphologically distinct tumors that have a propensity to arise in the extremities. Brain metastasis from epithelioid sarcoma are a relatively rare occurrence. We report a case of brain metastasis in a 50 year-old man who was previously diagnosed with an epithelioid sarcoma arising in the elbow. Before the diagnosis of brain metastasis, he had developed an axillary lymph node metastasis. He presented with neurologic symptoms of progressively worsening headache and loss of vision on the right side. He underwent gross total resection of an occipital lobe mass. Histologically, the tumor was focally characterized by prominent perinecrotic pseudopalisading and demonstrated immunoreactivity with antibodies to cytokeratin AE1/3 and CAM5.2; the tumor did not stain with glial fibrillary acidic protein antibody. The literature is reviewed and the morphologic distinction between metastatic epithelioid sarcoma and other central nervous system neoplasms is discussed. PMID- 12376924 TI - Uterine leiomyoma after embolization by means of gelatin sponge particles alone: report of a case with histopathologic features. AB - We describe the histopathologic features of uterine leiomyoma after uterine artery embolization (UAE) in a 42-year-old woman. This patient, who was taking antiplatelet drugs for the treatment of cerebral disease, successfully underwent UAE using only gelatin sponge particles for a symptomatic uterine leiomyoma. Although menorrhagia improved moderately after the procedure, she underwent abdominal hysterectomy 11 months later because of recurrent uterine bleeding. Histopathology revealed that most of the area of the uterine leiomyoma was characterized by extensive coagulation necrosis, which support the positive result of the procedure. No significant abnormalities were noted in either the myometrium or endometrium, which also suggested that UAE using only gelatin sponge particles is an appropriate procedure to preserve the uterus. The histologic and radiologic features of this case are discussed. To the best of our knowledge, this is the first reported case of uterine leiomyoma after UAE using only gelatin sponge particles as a primary embolic agent. PMID- 12376925 TI - Endometrial stromal sarcoma of the retroperitoneum. AB - Endometrial stromal sarcoma (ESS) is an uncommon neoplasm whose occurrence outside the uterus is extremely rare in the absence of metastasis or extension of a primary uterine neoplasm. When ESS occurs in such locations it is often associated with the uterine adnexa or serosal surface of various organs. Although rare, ESS is usually considered in the differential diagnosis of spindle cell neoplasms in the female patient. We report a case of ESS arising in the retroperitoneum and discuss the morphologic and immunohistochemical features in the context of the differential diagnosis of a retroperitoneal low-grade spindle cell neoplasm occurring in the female patient. PMID- 12376926 TI - Bilateral ocular malformations in a newborn with normal karyotype: histologic findings. AB - Microphthalmos with cyst is a rare condition characterized by a small globe and an inferior uveoretinal coloboma. There is also a defect in the posterior aspect of the eye through which a cyst lined by neuroectodermically derived tissue protrudes into the orbit. A case of isolated bilateral colobomatous and cystic microphthalmos is reported in an otherwise healthy child, showing no evidence of chromosomal abnormalities. Microscopic findings in the enucleated eye consisted of iris and retinal dysgenesis, ectopia lentis, persistent anterior tunica vasculosa lentis and pupillary membrane, intrachoroidal smooth muscle, and optic nerve hypoplasia. In the orbital cyst, a thick membrane reminiscent of the retinal inner limiting membrane lay between the fibroadipose and vascularised outer wall and the inner neuroectodermal lining. PMID- 12376927 TI - Glomangiomatosis. AB - Neoplasms containing glomus cells are uncommon. Glomus cells within an angiomatosis, so called glomangiomatosis, is exceedingly rare with only three previously reported cases. We are describing the fourth case from a 17-year-old boy which involved his left hand, fingers, and distal forearm. We will review the previously reported cases. PMID- 12376928 TI - Wegener's granulomatosis is not a granulomatous disease. AB - The histologic diagnosis of Wegener's granulomatosis is usually challenging. It is made more so by the failure of most pathologists to realize that the disease is fundamentally neither a vasculitis nor a granulomatous reaction. This problem can contribute to delays in diagnosis and consequent adverse effects for patient prognosis. This article attempts to better describe the essence of Wegener's granulomatosis with the aim of enhancing diagnosis earlier in the course of the disease. PMID- 12376929 TI - V.R. Khanolkar: father of pathology and medical research in India. AB - Vasant Ramji Khanolkar was the first pathologist in India. He made major contributions to the epidemiology and understanding of cancer, blood groups, and leprosy. He was the first to show the existence of dhoti cancers, and was among the earliest to demonstrate the carcinogenicity of tobacco and the use of needle aspiration cytology for the diagnosis of neoplasms. He was an acclaimed teacher and was on the boards of numerous international organizations. He was a bibliophile and his writings are Oslerian in style. He serves as a role model to the few in India who are aware of him. He deserves to be called the "Father of pathology and medical research in India." PMID- 12376930 TI - Primary microcephaly: new approaches for an old disorder. PMID- 12376931 TI - Amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and 2-ketoglutaric aciduria. AB - A new metabolic disorder characterized by severe congenital microcephaly, death within the first year, and severe 2-ketoglutaric aciduria has been found among the Old-Order Amish of Lancaster County, Pennsylvania. Amish lethal microcephaly segregates as an autosomal recessive disorder and has an unusually high incidence of at least 1 in 500 births. When the infants are well, the urine organic acid profiles show isolated, extreme elevations of 2-ketoglutaric acid. However, during otherwise simple viral illnesses, the infants often develop a metabolic acidosis, which may follow a lethal course. Cranial magnetic resonance imaging of a single patient showed a smooth, immature brain similar to that of a 20-week fetus except for a moderate degree of cerebellar vermal hypoplasia. Assay of 2 ketoglutarate dehydrogenase in cultured lymphoblasts of one patient showed normal activity. Amish lethal microcephaly maps to 17q25 and may be caused by a defect in a mitochondrial inner membrane protein functioning as a 2-ketoglutarate transporter. PMID- 12376932 TI - Analysis of craniofacial development in children with hypohidrotic ectodermal dysplasia. AB - Ectodermal dysplasias (ED) are a heterogeneous group of inheritable disorders characterized by abnormal development of embryologic ectoderm derivatives. The purposes of this study were to: 1) create baseline cephalometric norms for male children with ED; 2) assess craniofacial growth and development in hypohidrotic ED male children with severe hypodontia, compared with non-ED children with class I dental relationships; 3) compare the craniofacial morphology of titanium dental implant-treated ED males with non-implant-treated ED males; and 4) correlate the severity of hypodontia to craniofacial dysmorphology. Cephalometric radiographs of class I individuals and implant-treated and nontreated ED groups were used to evaluate craniofacial morphology. Traditional cephalometric landmarks and measurements were used to compare groups using the generalized estimate equation analysis. Age, gender, and the number of permanent maxillary teeth present had a significant (P =.01) explanatory relationship with the craniofacial measures when comparing untreated ED children to norms. Mean craniofacial differences between ED and non-ED children still existed when the explanatory effects of these variables were controlled, indicating dysmorphology in several craniofacial structures (e.g., cranial base, mandibular length). The number of missing maxillary permanent teeth was significantly related with craniofacial dysmorphology in the ED population. Craniofacial morphology did not differ significantly between implant-treated and nontreated ED children, suggesting that treatment with intraosseous dental implants, as applied in this population, did not rescue normal craniofacial growth and development. PMID- 12376933 TI - Epidemiological evidence that maternal diabetes does not appear to increase the risk for Down syndrome. AB - In 1997, Narchi and Kulaylat, studying the incidence of Down syndrome in infants of gestational diabetic mothers, concluded that maternal diabetes increases the risk for Down syndrome, but failed to control the maternal age in their analysis. Using data from the Spanish Collaborative Study of Congenital Malformations (ECEMC), we analyzed the relationship between Down syndrome and maternal diabetes mellitus, and maternal gestational diabetes, controlling the maternal age through the pair-matching analysis, stratifying by maternal age and logistic regression analysis. The analyses show that maternal age is related either to Down syndrome as well as to both types of maternal diabetes. Thus, the overall analysis could be confounded by maternal age. Once we controlled the maternal age, the risk of maternal diabetes mellitus for Down syndrome is: odds ratio (OR) = 0.92 (0.41 2.07); P = 0.83. Controlling maternal age in gestational diabetes, the risk is OR = 1.18 (0.61-2.35); P > 0.70. Based on our results, we conclude that Down syndrome is related to maternal age, but does not seem to be related to any type of maternal diabetes. PMID- 12376934 TI - Kousseff syndrome caused by deletion of chromosome 22q11-13. AB - Kousseff syndrome was originally described by Boris Kousseff in 1984: Pediatrics 74:395-398 in three siblings whose main features were conotruncal heart defects, neural tube defects, and dysmorphic features. The proband is a white male who has spina bifida, shunted hydrocephalus, cleft palate, short stature, cognitive impairment, and the typical craniofacial features of velo-cardio-facial syndrome (VCFS), including low-set and dysplastic ears, broad base of the nose, narrow alae nasi, and retrognathia. The family history is significant for a brother who died at 2 weeks of age with myelomeningocele, hydrocephalus, transposition of the great vessels, and unilateral renal agenesis, and a sister who died at 11 days of age with myelomeningocele, truncus arteriosus, hypocalcemia, and autopsy findings of absent thymus and parathyroid glands, consistent with DiGeorge anomaly. Given the clinical findings, family history, and recent knowledge that open neural tube defects can occur in VCFS/DiGeorge anomaly, FISH analysis for 22q11-13 deletion was performed on the proband. A deletion was detected in him and subsequently confirmed in his father. Molecular analysis on autopsy material confirmed the deletion in the proband's deceased brother. We suggest that individuals with neural tube defects associated with other anomalies such as congenital heart defects or cleft palate be evaluated for 22q deletions. PMID- 12376935 TI - Male limited association of the dopamine receptor D2 gene TaqI a polymorphism and alcohol dependence. AB - Association studies of the TaqI A allele of the dopamine receptor D2 (DRD2) gene with alcohol dependence have produced conflicting findings. Although a wide series of clinical features have been considered in the different association studies performed, very few studies specifically analyzed the role of gender. We compared the TaqI A polymorphisms of the DRD2 gene in 120 French Caucasian alcohol-dependent inpatients (62 males and 58 females) and 107 healthy ethnically matched controls (66 males and 41 females). We observed that 55% of alcohol dependent males have at least one A1 allele, a prevalence that is significantly above that observed in the control males (38%). On the contrary, no differences were found in females between the alcohol-dependent inpatients and controls for the A1 allele prevalence. In our sample, this male-specific association was not explained by gender specificities of alcohol dependence, such as age at onset and severity measures (mean numbers of social, somatic, and withdrawal complications). On the other hand, alcohol-dependent women with the A1 allele reported more frequently a major depressive disorder (70% vs. 40%, P = 0.03). We thus replicated the allelic association of the A1 allele of the DRD2 gene with alcohol dependence, but showed a male-limited effect of this "vulnerability allele." Recent evidence for gender difference in dopamine D2-like receptor levels and affinity may explain this discrepancy. PMID- 12376936 TI - Late infantile neuronal ceroid lipofuscinosis: quantitative description of the clinical course in patients with CLN2 mutations. AB - We examined 26 individuals with clinical and electron microscopic signs of late infantile neuronal ceroid lipofuscinosis (LINCL). In 22 cases, we found both pathogenic alleles. Sixteen patients exclusively carried either one or a combination of the two common mutations R208X and IVS5-1G > C. In the remaining cases, four missense mutations could be detected, of which R127Q, N286S, and T353P represent novel, previously not described alleles. A clinical performance score was developed by rating motor, visual, and verbal functions and the incidence of cerebral seizures in 3-month intervals during the course of the disease. A Total Disability Score was derived by summing up the single scores for motor, visual, and verbal functions. The 16 individuals with the two common mutations were grouped together (referred to as standard patients), and the 5th, 50th, and 95th centiles were calculated and graphically depicted over time. The scores for motor function and language ability dropped earliest and progressed very similarly in the standard patients. The performance curves of two children with the N286S mutation slightly diverged from the 95th centile. However, the performance curves of one patient with atypical LINCL carrying the R127Q mutation fell far beyond the 95th centile. The presented performance rating clearly and quantitatively delineates the disease course of the LINCL patients and hence offers a useful tool for clinical evaluation of future therapeutic interventions. In addition, the described performance score system can be applied to other types of neuronal ceroid lipofuscinoses and could be adapted to various other neurodegenerative diseases of childhood. PMID- 12376937 TI - Genetic loci for pathological myopia are not associated with juvenile myopia. AB - The purpose of this study was to evaluate chromosomal regions previously linked to pathological myopia for linkage to juvenile myopia in a sample of myopic children and their families. Of 125 families with a myopic child participating in the Orinda longitudinal study of myopia, 53 submitted 221 buccal swab samples for genetic analysis. Myopia in proband children was defined as -0.75 D or more myopia in both meridians on cycloplegic autorefraction (1% tropicamide). Affected status in parents and siblings was obtained by survey. DNA was extracted from buccal mucosal cells, amplified by polymerase chain reaction (PCR), and then analyzed with seven markers for chromosome 12 and five markers for chromosome 18 in the regions previously associated with pathological myopia. LOD scores were not significant for any marker tested. The largest positive LOD score was 0.15 for GATA30F04. Model-free methods using a SimIBD approach suggested a possible linkage at one marker, GATA6H09 (P = 0.003), but these results were not supported by transmission disequilibrium test (TDT) analysis. The statistical power to detect LOD scores of > or =1.0, assuming homogeneity, was estimated at 93.2%. We found no confirmatory evidence of linkage between juvenile myopia and regions of chromosomes 12 and 18 previously associated with pathological myopia. PMID- 12376938 TI - Clinical study and haplotype analysis in two brothers with Partington syndrome. AB - Partington et al. [1988] described a three-generation family (MRXS1, MIM *309510, PRTS) with a syndromic form of X-linked mental retardation (XLMR). The clinical features in 10 affected males included mild to moderate MR, dystonic movements of the hands, and dysarthria. After refinement, the PRTS locus was mapped to marker DXS989 (with maximum LOD score of 3.1) with flanking markers DXS365 and DXS28. Since then, no other patients with a similar phenotype have been described. We present a detailed description of the neurological symptoms and the disease history of two brothers with the clinical features of PRTS. Psychomotor development was delayed in both, and neurological features included mild to moderate mental retardation, dysarthria, facial muscle weakness, severe dysdiadochokinesis, slow dystonic movements, and mild spasticity of the hands, without ataxia or spasticity of the legs. The symptoms were nonprogressive and extrapyramidal, and without cerebellar involvement. In general, behavior of the two brothers was friendly and quiet, although the elder brother had periods of depressed mood and outbursts of anger. Karyotypes and subsequent investigation of the subtelomeres as well as DNA analysis of the FMR1 gene, the androgen receptor gene, and the DM locus did not reveal a genetic abnormality. Haplotype analysis showed that the affected brothers share the PRTS region at Xp22.1. Mutation screening of the PDH-E1alpha gene did not reveal a pathogenic mutation. PMID- 12376939 TI - The decision to continue: the experiences and needs of parents who receive a prenatal diagnosis of holoprosencephaly. AB - Holoprosencephaly (HPE) is a condition characterized by a defect in the development of the midline embryonic forebrain. When detected prenatally, the diagnosis of HPE offers parents a poor but often uncertain prognosis. Since the majority of parents receiving a prenatal diagnosis of an abnormality terminate their pregnancies, few studies have examined parents' experiences and needs surrounding the decision to continue a pregnancy. We present a descriptive study of in-depth interviews with 24 parents who chose to continue their pregnancy after receiving a prenatal diagnosis of HPE. Parents were asked about their decision-making process to continue the pregnancy. Qualitative analysis was used to identify common themes that emerged from these parents' experiences. The results suggest that most parents did not make an active decision about continuing the pregnancy. Rather, they described a more subtle decision-making process that evolved over time and consisted of several factors. These factors included the parents' religious and personal beliefs, past experiences, and the uncertainty involved in the diagnosis of HPE. Throughout the decision-making process, they described informational, emotional, and supportive needs from family, friends, and health professionals. All of these factors contributed to the evolution of the parents' decision to continue the pregnancy and the acceptance of their decision. Results of this exploratory study suggest health care professionals need to work with parents as they make their decision to continue an affected pregnancy. The results also provide the groundwork for prospective investigation into parents' decision-making process as they receive and adjust to prenatal diagnoses of an abnormality. PMID- 12376940 TI - Clinical and genetic heterogeneity of Seckel syndrome. AB - Seckel syndrome is a rare autosomal recessive condition belonging to the group of osteodysplastic primordial "dwarfism" and characterized by the association of 1) severe pre- and postnatal growth retardation, 2) microcephaly with mental retardation, and 3) specific dysmorphic features. Recently, two disease loci have been mapped to chromosomes 3q22.1-q24 and 18p11.31-q11.2, respectively, by homozygosity mapping in consanguineous families. Here, we report on the exclusion of these loci in five consanguineous and one multiplex nonconsanguineous Seckel syndrome families and in two consanguineous families presenting type II osteodysplastic primordial dwarfism. These results support the view that Seckel syndrome is a clinically and genetically heterogeneous condition. PMID- 12376941 TI - Partial deletions of the long arm of chromosome 13 associated with holoprosencephaly and the Dandy-Walker malformation. AB - Two patients with partial deletions of the long arm of chromosome 13, del(13)(13q21-q34) and del(13)(13q22-q33), respectively, multiple congenital anomalies including holoprosencephaly (HPE) and the Dandy-Walker malformation (DWM) are described. The occurrence of HPE and the DWM in both of these patients suggests that, in addition to ZIC2, which is important for normal development of the forebrain, there is at least one other dosage-sensitive gene in 13q22-q33 that plays an important role in brain development. The DWM is anatomically and developmentally distinct from HPE. The presence of a DWM in each of these two patients with partial deletions of the long arm of chromosome 13 suggests that haploinsufficiency at a locus in 13q22-q33 may cause this anomaly. These findings suggest that microdeletions in 13q22-q33 may be found in a proportion of patients with an apparently isolated DWM. Therefore, careful high-resolution cytogenetic analysis (550 band level or greater) of 13q22-q33 may be considered in these patients. Furthermore, future molecular studies of this region may reveal candidate gene loci for the DWM. PMID- 12376942 TI - Two cases of the caudal duplication anomaly including a discordant monozygotic twin. AB - We present two unrelated patients with various duplications in the caudal region. One patient presented with a duplication of the distal spine from L4, left double ureter, duplication of the vagina and cervix, and duplication of the distal colon. The second patient was diagnosed with a duplication of the colon, bladder, vagina and uterus. The first patient had an unaffected monozygotic twin sister. Dominguez et al. [1993: Am J Dis Child 147:1048-1052] presented six similar cases, and introduced the name "caudal duplication syndrome." The pathogenesis of the caudal duplication anomaly is unclear. The possibility of a polytopic primary developmental field defect or a disruptive sequence are discussed. On the other hand, somatic or germline mutations in certain developmental genes could be involved, as illustrated by the mouse mutations disorganisation and fused. DNA analysis of the AXIN1 gene, the human homologue of the gene responsible for fused, performed in our first patient, did not show any apparent pathogenic mutation. PMID- 12376943 TI - Tibial/femoral hypoplasia with "hook" pelvis: a potentially unique dysostosis. AB - We report a 2-year-5-month-old girl with malformed lower limbs. The radiographic skeletal survey revealed agenesis of the ilio-pubic rami with pubic dehiscence, right hip dislocation, bilateral coxa vara, short femurs, femoro-tibial synchondrosis, bilateral hypoplastic tibiae more severe on the left side, and hypoplastic left calcaneus and talus. To the best of our knowledge, this combination of multiple congenital skeletal abnormalities has not been reported before. PMID- 12376944 TI - Trisomy 18 in a 20-year-old woman. AB - A 20-year-old female with trisomy 18 is described. Survival past infancy is rare in this disorder. Little is known concerning the factors that contribute to prolonged survival with this syndrome. This case provides an opportunity to review the management of older children and young adults with trisomy 18. PMID- 12376946 TI - Variable expression of mental retardation, autism, seizures, and dystonic hand movements in two families with an identical ARX gene mutation. AB - Two families, originally diagnosed as having nonsyndromic X-linked mental retardation (NSXLMR), were reviewed when it was shown that they had a 24-bp duplication (428-45 1dup(24bp)) in the ARX gene [Stromme et al., 2002: Nat Genet 30:441-445]. This same duplication had also been found in three other families: one with X-linked infantile spasms and hypsarrhythmia (X-linked West syndrome, MIM 308350) and two with XLMR and dystonic movements of the hands (Partington syndrome, MIM 309510). On review, manifestations of both West and Partington syndromes were found in some individuals from both families. In addition, it was found that one individual had autism and two had autistic behavior, one of whom had epilepsy. The degree of mental retardation ranged from mild to severe. A GCG trinucleotide expansion (GCG)10+7 and a deletion of 1,517 bp in the ARX gene have also been found in association with the West syndrome, and a missense mutation (1058C>T) in a family with a newly recognized form of myoclonic epilepsy, severe mental retardation, and spastic paraplegia [Scheffer et al., 2002: Neurology, in press]. Evidently all these disorders are expressions of mutations in the same gene. It remains to be seen what proportions of patients with infantile spasms, focal dystonia, autism, epilepsy, and nonsyndromic mental retardation are accounted for by mutations in the ARX gene. PMID- 12376945 TI - TM4SF2 gene involvement reconsidered in an XLMR family after neuropsychological assessment. AB - The TM4SF2 gene (localized at Xp11.4 between the loci DXS564 and DXS556) has been found to be mutated in one MRX family. In order to define the corresponding behavioral phenotype, global IQ and specific cognitive skills were assessed in seven males and three females of this family, independent of subject status. Mental retardation (MR) was mild in three patients and moderate in three others. Despite the broad variability of severity of MR, a cognitive profile specific to the MR in this family was documented. It was characterized by language disorder that was more marked in the articulatory component and spatial/verbal short-term memory dissociation with larger mnemonic span for spatial than for verbal cues. Linkage analysis was then performed on the basis of the cognitively determined status. Recombinations were observed with the loci DXS556 at Xp11.4 and DXS441 at Xq13.2 (maximum LOD score = 2.23 at theta = 0 for ALAS2). This localization region does not include the TM4SF2 gene that has been found mutated in both patients with MR and in one non-MR male subject of this family. The present results suggest two main hypotheses. First, TM4SF2 gene mutation could be involved in MR in this family, therefore representing accentuated intra familial phenotypic variability. Second, the structural particularity detected in the TM4SF2 gene might reflect a rare polymorphism rather than a pathogenic mutation, with the gene responsible for MR in this family being therefore more likely to be searched for in the pericentromeric region of the X chromosome. PMID- 12376947 TI - Lymphedema-lymphangiectasia-mental retardation (Hennekam) syndrome: a review. AB - The Hennekam syndrome is an infrequently reported heritable entity characterized by lymphedema, lymphangiectasia, and developmental delay. Here we add an additional 8 patients, and compare their findings to the 16 cases from the literature. The lymphedema is usually congenital, can be markedly asymmetrical, and, often, gradually progressive. Complications such as erysipelas are common. The lymphangiectasias are present in the intestines, but have also been found in the pleura, pericardium, thyroid gland, and kidney. Several patients have demonstrated congenital cardiac and blood vessel anomalies, pointing to a disturbance of angiogenesis in at least some of the patients. Facial features are variable, and are chiefly characterized, in a typical patient, by a flat face, flat and broad nasal bridge, and hypertelorism. Facial features are thought to mirror the extent of intrauterine facial lymphedema, or may be caused by lymphatic obstruction that affects the early migration of neural crest tissue. Other anomalies have included glaucoma, dental anomalies, hearing loss, and renal anomalies. The psychomotor development varies widely, even within a single family, from almost normal development to severe mental retardation. Convulsions are common. The existence of 10 familial cases, equal sex ratio, increased parental consanguinity rate (4/20 families), and absence of vertical transmission are consistent with an autosomal recessive pattern of inheritance. It seems likely that most (but not all) manifestations of the entity can be explained as sequences of impaired prenatal and postnatal lymphatic flow, suggesting that the causative gene(s) should have a major function in lymphangiogenesis. PMID- 12376948 TI - Difficulties in the diagnosis of neurofibomatosis-1 in children. PMID- 12376949 TI - Re-evaluation of MRX36 family after discovery of an ARX gene mutation reveals mild neurological features of Partington syndrome. PMID- 12376950 TI - Adjunct diagnostic test for Angelman syndrome: the tuning fork response. PMID- 12376951 TI - Documentary evidence for conjoined twins in the sixteenth century. PMID- 12376952 TI - Analysis of HIV-1 variation in blood and semen during treatment and treatment interruption. AB - The variability of populations of human immunodeficiency virus type 1 (HIV-1) in blood and semen, with respect to envelope and polymerase gene sequences, was examined longitudinally in a patient experiencing treatment failure, interruption of treatment, and successful reintroduction of therapy. During treatment failure, there was little evidence of compartmentalisation between blood and semen, with virus with identical resistance-associated mutations observed in both compartments and lack of clustering with respect to envelope gene sequences. After cessation of treatment, wild-type virus became the predominant population, displaying distinct envelope gene populations, indicating that wild-type virus had overgrown the resistant virus, rather than the resistant virus reverting to wild-type. Once successful therapy had been recommenced, it was possible to distinguish distinct populations of virus in the two compartments. These data support the hypothesis that the male genital tract represents a distinct HIV-1 reservoir. PMID- 12376953 TI - Persistence of earlier HIV-1 drug resistance mutations at new treatment failure. AB - The objective was to study the persistence of drug resistance mutations detected earlier at virological failure during second or third line antiretroviral therapy. Therefore, in HIV-1 infected patients, with a virological treatment failure, genotypic resistance testing was carried out before change of therapy and at the next treatment failure. The majority of primary and secondary resistance mutations persisted in both the reverse transcriptase (RT) and the protease genes. After changing from zidovudine- to stavudine-containing regimens, the thymidine analogue mutations (especially M41L and T215Y/F) were found at new treatment failure in almost all patients. The M184V mutation disappeared in most (64%) non-3TC treated patients, although it persisted in a few didanosine- and abacavir-treated subjects. The primary protease inhibitor (PI) mutations reverted back to wild type in most patients who did not receive a new PI. In contrast, after changing from indinavir to saquinavir or nelfinavir, the M46I/L and/or V82A/F/ST disappeared in only 9 of 21 occasions at the new treatment failure. Most secondary mutations persisted with the exception of N88D. In patients with multiple treatment failures, most NRTI mutations thus persist frequently at new failures with modified treatment. A similar pattern is seen for protease inhibitors. The data suggest that clinical cross-resistance may develop via common pathways within all categories of drugs in heavily treated patients. PMID- 12376954 TI - Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus. AB - The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV. PMID- 12376955 TI - Long-term immunogenicity of an inactivated virosome hepatitis A vaccine. AB - The aim of this study was to predict the long-term protection induced after immunisation with inactivated, aluminium-free virosome hepatitis A vaccine. The study population consisted of adult volunteers enrolled in four different clinical trials. Lower 95% confidence interval limits and seroconversion rate were calculated by using a linear mixed model to estimate the persistence of serum antibodies over time. To assess the robustness of the mathematical model, several sensitivity analyses were performed with more conservative protective threshold (20 mIU/ml vs. 10 mIU/ml), higher yearly decline rate, and exclusion of volunteers who had increasing titres over time. Based on 190 volunteers with at least two valid assessments of titres from year 3 onward, the median duration of protection was 55.5 years, with a lower limit of the 95% CI of 48.7 years. Duration below 25.3 years was predicted for only 5% of the subjects. Women tended to have higher titres to start with, but their rate of decline was higher, resulting in similar duration of protection overall. The use of a more conservative threshold, higher yearly decline rate, and exclusion of volunteers with increasing titres over time did not affect these results. According to this model, 95% of the volunteers should have anti-HAV titres above the minimum protective threshold for 20 years or more following immunisation with two doses of this aluminium-free vaccine. PMID- 12376956 TI - Real-time quantitation of hepatitis B virus (HBV) DNA in tumorous and surrounding tissue from patients with hepatocellular carcinoma. AB - Few data are available on the levels of HBV DNA in liver tissue of patients with hepatocellular carcinoma. In this study, HBV DNA was quantitated by a TaqMan real time PCR method and results were normalised to an endogenous reference gene. The assay could detect reproducibly viral sequences from over 10(7) to less than 50 copies/microg of liver DNA. The HBV DNA content in liver samples from 11 HBsAg positive patients (median: 10(5) copies/microg of DNA) was significantly higher (P < 0.001) compared to the viral DNA concentration detected in liver samples from 15 of 25 HBsAg-negative patients (median: 2.6 x 10(2) copies/microg). A liver DNA amount > or =1 HBV DNA copy per cell was detected in half of tissue samples from HBsAg-positive patients, and in none from HBsAg-negative ones. Liver tissue HBV DNA content was significantly higher in anti-HCV-negative than in anti HCV-positive cases (P < 0.001). These results show that the quantitation of liver HBV DNA by real-time PCR can be useful to understand HBV state in hepatocellular carcinoma and viral interplay in patients with multiple viral infections. PMID- 12376957 TI - Low hepatitis B prevalence among pre-school children in Denmark: saliva anti-HBc screening in day care centres. AB - Although Denmark has a low hepatitis B virus (HBV) prevalence, HBV transmission has been reported in Danish day-care centres. The aim of this study was to validate saliva anti-HBc testing as a method for HBV screening, the applicability of saliva sampling to pre-school children, and to determine the HBV prevalence in Danish day-care centres with a high proportion of immigrants. For validation, paired saliva and plasma samples were obtained from blood donors and injecting drug users. Employees and children in day-care centres with a high proportion of immigrant children were offered saliva screening followed by blood test if positive. The specificity and sensitivity of anti-HBc tests on saliva was 100% (102 blood donors and four injecting drug users) and 85.9% (61 of 71 anti-HBc positive injecting drug users), respectively. In all samples from HBsAg (n = 7) or anti-HBc IgM-positives (n = 9), anti-HBc was detected in saliva. Adequate saliva samples were obtained from 93% (588/634) of children and 100% (166/166) of employees participating in the day-care centre survey. Among children 55% were of non-Scandinavian origin and only one (0.2%, 95% CI [0.0; 1.0]) was HBV positive. Among employees the corresponding values were 22% and 7 (4.2%). The positive predictive value of the saliva test was 25% (1/4) among children and 88% (7/8) among adults. In conclusion, saliva testing is feasible for HBV screening among children in low prevalence populations, but any anti-HBc reactivity should be confirmed by plasma analysis. The HBV prevalence in pre-school children in Denmark is low even among immigrants from endemic areas. PMID- 12376958 TI - Evidence for a dynamic host-parasite relationship in e-negative hepatitis B carriers. AB - Anti-hepatitis Be (HBe) carriers are perceived as having low infectivity, with hepatitis B virus (HBV) DNA levels far below those seen in the HBeAg carrier. However, the temporal stability of HBV DNA in anti-HBe carriers remains poorly characterised. UK Department of Health guidelines use HBV DNA levels to define whether HBV-infected health care workers may perform exposure-prone procedures. Two samples separated by 1-23 years available from 147 carriers were analysed for precore variants and HBV DNA levels. Among 15 HBeAg carriers, HBV DNA was maintained at high levels. There was a 5 log(10) fold reduction in DNA in 11 individuals who developed anti-HBe during follow-up evaluation. Proportional changes in HBV DNA levels in anti-HBe carriers were similar to those in HBeAg carriers, although there was a trend for changes in viral DNA to be more marked in anti-HBe carriers followed up for longer periods. Closer sampling in 20 anti HBe carriers demonstrated large fluctuations of DNA levels over short periods. Serum transaminases and precore mutant status at the outset failed to predict those in whom HBV DNA levels fluctuated. HBV DNA was below the detection threshold (<400 copies/ml) in 36 anti-HBe carriers at first sampling and remained so in all but 5 of these carriers. Twelve individuals who were previously viraemic lost detectable HBV DNA during follow-up evaluation. While HBV DNA levels are found to fluctuate in carriers, our results indicate that once below the threshold of detectability, levels are unlikely to rise. This is an important factor when assessing health care workers for exposure-prone procedures. PMID- 12376959 TI - Characterization of integration patterns and flanking cellular sequences of hepatitis B virus in childhood hepatocellular carcinomas. AB - Hepatitis B virus (HBV) DNA integration into host chromosomes is detected in more than 80% of HBV-related hepatocellular carcinomas (HCC), yet its significance in tumor development remains obscure. In this study, we re-examined the integration pattern of HBV in childhood HCC tissues, which has less environmental confounding factors than adult HCC. The HBV junctions and flanking cellular sequences were amplified from five childhood HCC patients by the inverse polymerase chain reaction (IPCR) method using primers located near HBV direct repeats (DR) 1 and 2. The viral junctions in nine of the ten obtained IPCR clones were demonstrated to be located near HBV DR1, and their patterns were classified to type I integrants. Southern blot analyses demonstrate that the cellular junctions derived from two of the five HCC tissues were male specific and contained sequences homologous to human long interspersed DNA elements (LINE-1). HBV integrant of one HCC tissue (1217T) was integrated into a RNA binding motif Y chromosome (RBMY) gene. The expression of RBMY, which is normally found only in male germ cells, was detected in HCC tissue 1217T by RT-PCR but not in the corresponding non-tumor liver tissue. The prevalence of RBMY expression in liver tissues from the tumor and non-tumor parts of ten other HCC children and seven biliary atresia (BA) children was studied by RT-PCR. No RBMY transcripts were detected in the non-tumor parts of HCC patients or the cirrhotic livers of BA children, whereas 30% (three of ten) of HCC tissues specifically expressed RBMY. The results indicate that HBV integration and activation of RBMY gene expression in liver cells may be associated with the development of childhood HCC. PMID- 12376960 TI - Viral genotypes and response to interferon in patients with acute prolonged hepatitis B virus infection of adulthood in Japan. AB - Acute hepatitis B virus (HBV) infection was diagnosed in 57 adults admitted to Toranomon Hospital in Tokyo, Japan. Genotypes of HBV were determined by a serological method and compared to those in 1,077 patients with chronic hepatitis B. The distribution of genotypes were: genotype A (acute, 22.8% vs. chronic, 1.9%; P < 0.00001); B (14.0% vs. 9.4%); C (43.9% vs. 87.7%, P = 0.004); D (1.8% vs. 0.2%); F (1.8% vs. 0.2%); and unable to be typed (15.8% vs. 0.6%, P = 0.001). The infection persisted in seven (12%) of them. They included six (86%) of the seven patients who received prednisolone or glycyrrhizin during an acute phase of illness and one of the 41 (2%) who did not (P = 0.01). Interferon was given to the seven patients with acute prolonged HBV infection, and four of them responded by clearing hepatitis B e antigen (HBeAg) and surface antigen (HBsAg) from serum. Of the four responders, one was infected with HBV genotype B and three with genotype C. HBsAg persisted in the remaining three patients all of whom were infected with HBV genotype A, and HBeAg stayed positive in one of them. These results indicate that HBV genotype A prevails in Japanese patients with acute hepatitis B, and suggest a high efficacy of interferon in the adult patients with acute prolonged HBV infection, except in those infected with HBV genotype A. PMID- 12376961 TI - Hepatitis C virus replicates in sweat glands and is released into sweat in patients with chronic hepatitis C. AB - Hepatitis C virus (HCV) replicates in salivary glands of chronic hepatitis C patients and is released into the saliva, suggesting that HCV may replicate in other exocrine glands. The presence of positive and negative HCV RNA strands was demonstrated by in situ hybridization, and of HCV core protein by immunohistochemistry, in sweat glands and keratinocytes in healthy skin biopsies from 15 patients with chronic hepatitis C and 10 anti-HCV negative patients with chronic liver disease. Positive and negative HCV RNA strands were detected in 9.6 +/- 5.2% and 4.2 +/- 3.8%, respectively, of the epithelial cells of eccrine sweat glands. Core protein was detected in 6.0 +/- 3.93% of these cells. HCV RNA resistant to RNase digestion (encapsidated HCV RNA) was detected in 10/10 sweat samples from HCV-infected patients. Positive and negative HCV RNA strands were detected in 6.7 +/- 2.97% and 3.0 +/- 3.08% of the keratinocytes, respectively. HCV core protein was found in 4.5 +/- 2.76% of these cells. No HCV RNA or HCV core protein was detected in the skin biopsies from the 10 anti-HCV negative patients. In conclusion, HCV replicates in eccrine sweat glands cells and keratinocytes in healthy skin and is released into the sweat. PMID- 12376963 TI - Complete nucleotide sequence of a Coxsackievirus B-4 strain capable of establishing persistent infection in human pancreatic islet cells: effects on insulin release, proinsulin synthesis, and cell morphology. AB - The aim of the present investigation was to study the effect of infection of human pancreatic islet cells with a strain (VD2921) of Coxsackie B virus serotype 4 capable of establishing persistent infection in these cells, as well as to sequence the strain, to study the determinants of virulence and persistence. Groups of islets were infected and assessments of proinsulin, insulin content, and virus replication were made. Insulin release in response to high glucose was measured. Infected and control islets displayed a strong insulin response to high glucose 3-4 days as well as 7-8 days post-infection (dpi). At 11-17 dpi, the infected islets did not respond at all to high glucose, and the response of the control islets was at this late time point somewhat reduced. The insulin and proinsulin content of the infected islets did not differ significantly from that of the control islets. TCID(50) titrations showed that the VD2921 strain replicated in the islet cells during the whole study. Electron microscopic examination of infected islets did not reveal any virus-induced changes of cell morphology compared with the controls, although higher magnifications of the infected beta-cells showed virus-like particles in the cytoplasm. These results show that certain strains of Coxsackievirus B-4 in vitro can establish a persistent infection that might mimic, the more gradual loss of beta-cell function seen during the clinical course of autoimmune diabetes. The ability of this Coxsackievirus B-4 strain to establish a persistent infection might be due to substitution of 11 amino acids located at the surface of the structural protein VP1, adjacent to the predicted receptor binding canyon of the virus. PMID- 12376962 TI - Recombinant hepatitis C virus-like particles expressed by baculovirus: utility in cell-binding and antibody detection assays. AB - Hepatitis C virus (HCV) is difficult to study due to the lack of an efficient cell culture system or small animal model. As a result, HCV-cell interactions are not well-defined. In addition, several studies have identified a subset of patients in whom HCV RNA is present, but HCV antibody is not detected. We produced recombinant baculoviruses that expressed HCV structural proteins (core, E1 and E2, nt 342-2651) or control proteins. The HCV structural protein precursor was processed into immunoreactive proteins of appropriate size, and sucrose density sedimentation and electron microscopy of infected cell lysates demonstrated particle formation. To evaluate HCV antigenicity, particularly in patients who tested negative for HCV antibody in commercial HCV immunoassays but had persistent viremia, we evaluated the virus-like particles (VLPs) in solid phase immunoassays. VLPs reacted with sera from HCV antibody positive subjects in these solid phase immunoassays, but not with control sera. Plasma samples from 19% (5/26) of HCV antibody negative subjects who were persistently HCV RNA positive also reacted with the HCV VLPs. When incubated with MOLT-4 cells at 4 degrees C, HCV VLPs demonstrated cell binding, and behaved similar to plasma derived HCV preparations in a flow cytometry-based cell binding assay. These data suggest that recombinant HCV VLPs may allow identification of HCV antibody in patients, including some patients with persistent viremia and who are seronegative with current assays. In addition, HCV VLPs seem useful for evaluating HCV-cell interactions. PMID- 12376964 TI - Genetic diversity of echovirus 30 during a meningitis outbreak, demonstrated by direct molecular typing from cerebrospinal fluid. AB - Echovirus 30 is one of the enterovirus serotypes isolated most frequently in meningitis cases. The genetic diversity of echovirus 30 was investigated in patients hospitalised during an outbreak in 2000 in Clermont-Ferrand, France. A nested reverse transcription-PCR (RT-PCR) assay was developed for qualitative analysis of the echovirus 30 VP1 encoding sequence directly from cerebrospinal fluid. The viral sequences obtained for 22 patients were compared with those of virus isolates obtained from nine patients with echovirus 30 meningitis admitted to hospital in 1996-1997 and with echovirus 30 sequences from international databases. In 2000, meningitis cases were caused by two virus variants (C3 and C4) distinct genetically from the other two variants (C1 and C2) identified during the period 1996-1997. A detailed phylogenetic analysis established that the C1, C2, and C3 variants had close relatives among viruses previously identified in other geographical areas. The C4 variant had not been described earlier. The genomic differences observed between the four echovirus 30 variants arose at synonymous sites indicating that the viruses shared similar antigenic sites in the VP1 encoding sequence. Overall, these observations suggest wide circulation of different echovirus 30 variants and periodic importation of new viruses. The apparent displacement observed between virus variants did not result from a selective advantage caused by antigenic variation. PMID- 12376965 TI - Expression of viral and human dUTPase in Epstein-Barr virus-associated diseases. AB - Deoxyuridine triphosphatase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and pyrophosphate thus preventing the incorporation of uracil into replicating DNA. Previous studies of several virus models have suggested that viral dUTPases may be required for virus replication in resting cells whereas in proliferating cells cellular dUTPase may substitute for a mutant viral protein. Using monoclonal antibodies and immunohistochemistry, Epstein-Barr virus-associated non neoplastic and neoplastic diseases were studied for the expression of viral and human dUTPases. Oral hairy leukoplakia, an AIDS-associated lesion of the tongue, is known to support EBV replication in the upper epithelial cell layers. In agreement with this, strong focal expression of EBV dUTPase was detected in the upper epithelial cell layers of oral hairy leukoplakia whereas expression of human dUTPase was confined to the basal proliferative cell compartment. Furthermore, in infectious mononucleosis tonsils, rare scattered small lymphoid cells expressed EBV dUTPase, consistent with the expression pattern of other EBV lytic cycle antigens. These findings are in agreement with the notion that EBV replicates in resting cells. Three EBV-associated tumours, Hodgkin lymphoma, Burkitt lymphoma and nasopharyngeal carcinoma, lacked detectable expression of EBV dUTPase, in agreement with the notion that EBV infection is largely latent in these tumours. By contrast, expression of human dUTPase was observed regularly in these tumours. These results suggest that EBV dUTPase may be a suitable target for anti-viral therapy and that inhibitors of human dUTPase should prove useful for the treatment of human tumours, including EBV-associated cancers. PMID- 12376966 TI - Responses of IgM for enterovirus 71 infection. AB - A rapid serological assay was developed for detection of specific IgM to enterovirus 71, a human picornavirus that is usually associated with severe central nervous system complications. The sensitivity and specificity of this "in house" mu-capture enzyme linked-immunosorbent assay was assessed by testing 213 serum samples. With the conventional virus culture as a standard method, the sensitivity and specificity were 91.5 and 93.1%, respectively, for this newly developed immunoassay. This method allows for detection of the IgM responses from the patients either infected by genotype B or genotype C of enterovirus 71. IgM can be detected as early as the second day from the onset of disease. IgM responses exhibit 100% positive rate from enterovirus 71-infected patients with complications, including encephalitis, meningitis, polio-like syndrome, pulmonary edema, and fatal cases. These findings suggest that detection of specific IgM by the use of enzyme linked-immunosorbent assay is a rapid and valuable way for the diagnosis of enterovirus 71 infection. PMID- 12376967 TI - Rodent host specificity of European hantaviruses: evidence of Puumala virus interspecific spillover. AB - In order to investigate rodent host specificity of European hantaviruses, experimental infection of colonized and wild-trapped rodents was performed. In addition to the natural rodent reservoir, Clethrionomys glareolus, Puumala hantavirus (PUUV) could infect colonized Microtus agrestis and Lemmus sibiricus, but not Syrian hamsters or Balb/C mice. Neither C. glareolus, nor M. agrestis, could be readily infected by Tula hantavirus (TULV). Wild-trapped Apodemus flavicollis and A. agrarius, the natural reservoirs of Dobrava (DOBV) and Saaremaa (SAAV) hantaviruses, respectively, could both be infected by SAAV. NMRI mice could also be infected by SAAV, but with lower efficiency as compared to Apodemus mice. Balb/C and NMRI laboratory mice, but not C. glareolus, could be infected by DOBV. To our knowledge, this is the first time DOBV and SAAV have been shown to infect adult laboratory mice. Moreover, potential hantavirus spillover infections were investigated in wild-trapped rodents. In addition to the natural host C. glareolus, we also found M. arvalis and A. sylvaticus with a history of PUUV infection. We did not find any C. glareolus or A. sylvaticus infected with TULV, a hantavirus which is known to circulate in the same geographical regions of Belgium. PMID- 12376968 TI - Hantaviruses in Estonia. AB - Human serum samples collected from healthy individuals in 14 counties were screened by ELISA in order to investigate the presence of hantavirus infections in Estonia. Out of 1,234 serum samples, 124 were found positive for hantavirus specific IgG and were subsequently serotyped by a focus reduction neutralization test. A total of 112 samples neutralized at least one of the examined hantaviruses-Puumala (PUUV), Saaremaa (SAAV), Dobrava (DOBV), Hantaan, and Seoul viruses-and thereby, the focus reduction neutralization test confirmed the overall hantavirus seroprevalence rate in Estonia to be 9.1%. Most of the sera showed a specific reaction (at least 4-fold higher endpoint titer) of neutralizing antibodies to PUUV (5.1%), while 3.4% showed a SAAV- or SAAV/DOBV specific reaction. The fact that seven sera (0.6%) could not be serotyped may indicate the presence of an unknown hantavirus serotype. Hantavirus infections were confirmed in 13 of 14 investigated counties, with highly varying seroprevalence rates (1.0-28.4%). The sex ratio was 1.8:1.0 (M:F), and the antibody prevalence peaked in the age group 45-54 years. A total of 513 rodents of seven species trapped in seven counties were examined for the presence of hantavirus antigen, in order to study the distribution of hantavirus natural carriers. Two species, Clethrionomys glareolus and Apodemus agrarius, were found positive for hantaviral antigen in 13.7% and 4.5% of the investigated rodents, respectively. Analyses of viral sequences recovered from infected C. glareolus tissue samples showed that the infecting virus belonged to the PUUV genotype, confirming that PUUV circulates in mainland Estonia. The Estonian PUUV strains were placed in the closest proximity to Russian PUUV strains in phylogenetic trees, suggesting a common evolutionary history. Together with earlier data on SAAV in A. agrarius, the results revealed that two hantaviruses, PUUV and SAAV, are common in Estonia and that the incidence of human infection is high in both cases. PMID- 12376969 TI - A cell line that secretes inducibly a reporter protein for monitoring herpes simplex virus infection and drug susceptibility. AB - A cell line modified genetically (Vero-ICP10-SEAP) that responds to infection by herpes simplex virus (HSV) was established. The cell line was constructed by stable transfection of Vero cell with a plasmid encoding the secreted alkaline phosphatase (SEAP) driven by the promoter of the HSV-2 ICP10 gene. Following infection with HSV, the stable line secretes a high level of the SEAP in the supernatants as measured by a chemiluminescence-based assay. The detection system is sensitive to an HSV titer as low as a single plaque-forming unit (PFU), with a linear range up to the equivalent of 2.5 x 10(4) PFU inoculum after infection for 24 h. There was no detectable enhancement in SEAP activities following inoculations with several viruses other than HSV. The Vero-ICP10-SEAP cell line was also utilized to develop an assay for determination of antiviral susceptibility given that the induced SEAP activity appeared to reflect the numbers of plaque. Evaluations of the stable line with representative acyclovir (ACV)-sensitive and-resistant HSV isolates demonstrated that their drug susceptibilities were determined accurately. In summary, this novel SEAP reporter system is a sensitive means for rapid diagnosis, quantitation, and drug susceptibility testing for HSV, with potential to the development of an automated assay. PMID- 12376970 TI - Detection of human herpes virus type 6 DNA in precancerous lesions of the uterine cervix. AB - Human herpes virus type 6 (HHV-6) DNA has been suggested to be a cofactor to human papillomavirus (HPV) in cervical cancer. In a cross-sectional study, we investigated the association between HHV-6 DNA detected in cervical brushings and high-grade squamous intraepithelial lesions (HSIL), while controlling for genital infection with 27 genotypes of HPV. Of the 320 women recruited from an oncologic gynecology clinic, 50 had invasive cervical cancer, 65 had HSIL, 80 had low-grade squamous intraepithelial lesions (LSIL), and 125 were normal. Four of the seven HHV-6-positive women had HSIL. HHV-6 was associated with HSIL after adjusting for age and socioeconomic status (odds ratio [OR] of 10.9, 95% confidence interval [CI]: 1.1-107.1). This association was no longer significant after controlling for HPV (OR = 6.4, 95% CI = 0.3-128.5). HHV-6 was detected in cervical samples from women with precancerous and cancerous lesions of the cervix, but not significantly more frequently than in normal women. PMID- 12376971 TI - Characterization of measles sequences from Pune, India. AB - The goal of this study was to conduct the initial genetic characterization wild type measles viruses currently circulating in India. Reverse transcription polymerase chain reaction detected measles RNA in 11 of 14 throat swabs collected from sporadic and outbreak-associated cases in the city of Pune, during 1996 1998. Sequence analysis of the H and N genes showed that six sequences were genotype D4, three were genotype D8, and two were genotype A. Continued virologic surveillance in other areas of India as well as neighboring countries will indicate the extent of genetic diversity present among wild-type measles viruses circulating in India. PMID- 12376972 TI - Human antibody response to Toscana virus glycoproteins expressed by recombinant baculovirus. AB - The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. In this study, the viral envelope glycoproteins were expressed in soluble form in a baculovirus system. The recombinant sGN and sGC proteins were used as viral antigens in a Western blot assay to analyze the specific immune response in sera from patients with recognized virus-associated aseptic meningitis. The anti-glycoprotein and the anti-nucleoprotein N IgG responses were compared by an immunoassay based on the recombinant proteins. In this system, all the sera showed a high reactivity to the N protein, but they differed in the response to the glycoproteins. PMID- 12376973 TI - Outbreak of jaundice and hemorrhagic fever in the Southeast of Brazil in 2001: detection and molecular characterization of yellow fever virus. AB - Between January and March 2001, an outbreak of jaundice and hemorrhagic fever occurred in the state of Minas Gerais, Southeast region of Brazil, in which a mortality rate of 53% was reported. Seroconversion, virus isolation, histopathological and immunohistochemical findings, and reverse transcription polymerase chain reaction (RT-PCR) identified yellow fever virus (YFV) as the etiological agent responsible for the outbreak. Partial nucleotide sequence analysis from a fragment of the YFV genome spanning parts of nonstructural (NS) 5 gene and 3' noncoding region (3' UTR) showed that the YFV involved in this outbreak belongs to South American genotype I and differs from the Brazilian virus identified in 1996. PMID- 12376974 TI - Mutation analysis of the BCL10 gene in childhood solid malignancies. AB - BACKGROUND: BCL10, a gene involved in apoptosis signaling, has recently been identified at chromosome 1p22. This gene was found to be mutated in several types of lymphomas and other kinds of solid tumors. Especially in hepatocellular carcinoma, high mutation rates have been reported. These findings suggest that its inactivation may play an important pathogenetic role in tumorigenesis. Furthermore, abnormalities of chromosome 1 where the BCL10 gene is located are a common feature in pediatric solid tumors. Therefore, we analyzed 95 pediatric solid cancers for genomic BCL10 mutations. PROCEDURE: Three exons, which encode the whole coding region, were examined in 95 tumor tissues by PCR-SSCP method. Samples revealing aberrant band patterns were subjected to direct sequencing analysis. RESULTS: A total of six nucleotide changes were detected. Two were in intron 1 (IVS1 + 11C > G, IVS1 + 58G > C) and four were in exons 1 or 3 (Ala5Ser, Leu8Leu, Thr162Met and Gly213Glu). Of four exonic changes, three at codons 5, 162, and 213 resulted in amino acid substitution. In non-tumor tissues, however, similar mutation types were found, suggesting that all nucleotide changes detected were genetic polymorphisms. CONCLUSIONS: This study represents the first genetic analysis of the BCL10 gene in pediatric solid malignant tumors. Our results suggest that BCL10 mutation as a mechanism involved in tumorigenesis is unlikely to be associated with most childhood malignancies. PMID- 12376975 TI - Disialoganglioside GD2 and a novel tumor antigen: potential targets for immunotherapy of desmoplastic small round cell tumor. AB - BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is an aggressive and often misdiagnosed neoplasm of children and young adults. It is chemotherapy sensitive, yet patients often relapse off therapy because of residual microscopic disease at distant sites: peritoneum, liver, lymph node, and lung. Strategies directed at minimal residual disease (MRD) may be necessary for cure. Monoclonal antibodies selective for cell surface tumor-associated antigens may have utility for diagnosis and therapy of MRD, as recently demonstrated in advanced-stage neuroblastoma (JCO 16: 3053, 1998). We examined DSRCT samples for the expression of two tumor antigens that could serve as possible targets for antibody-based immunotherapeutic approaches. PROCEDURES: Using immunohistochemistry, we studied the expression of two antigens: (1) G(D2) using antibody 3F8 and (2) a novel antigen using antibody 8H9 in a panel of 46 freshly frozen DSRCT. G(D2) is a disialoganglioside, which is widely expressed among neuroectodermal tumors as well as adult sarcomas. 8H9 recognizes a 58 kDa surface antigen expressed among neuroectodermal, mesenchymal, and epithelial tumors with restricted expression on normal tissues. RESULTS: Thirty-two of 46 (70%) tumors were reactive with 3F8, and 44 of 46 (96%) with 8H9. Both G(D2) and the 58 kDa antigen were localized to tumor cell membrane and stroma. In general, immunoreactivity was stronger and more homogeneous with 8H9 than with 3F8. There was no correlation between expression of either antigen or clinical outcome. CONCLUSIONS: G(D2) and the novel tumor antigen recognized by 8H9 are potential targets for immunodiagnosis and antibody-based therapy of DSRCT. Med Pediatr Oncol 2002;39:547-551. PMID- 12376977 TI - SEER update of incidence and trends in pediatric malignancies: acute lymphoblastic leukemia. AB - BACKGROUND: Acute lymphoblastic leukemia (ALL) represents the most common malignancy of childhood. Its incidence peaks in children just before school entry age; i.e., in 2-3 year olds. It is known to be more common in white children in the USA; the incidence is also higher in boys than girls. PROCEDURE: We reviewed the 5,379 cases of ALL among persons under 20 years of age in the National Cancer Institute's Surveillance Epidemiology and End Results (SEER) database. RESULTS: The overall incidence of ALL was 26/10(6) person-years between 1973 and 1998, but increased from 19/10(6) person-years in 1973-77 to 28/10(6) person-years in 1993 98 (P < 0.0001). Rates were 44% higher among Whites compared to Blacks (27/10(6) person-years vs. 15/10(6) person-years, P < 0.0001). In 1992-1998, the incidence rate for Hispanics was 43/10(6) person-years, significantly higher than non Hispanics (28/10(6), P < 0.0001). White children with ALL had better 5-year survival rates than Black children with ALL (71% vs. 58%, P < 0.0001), and 5-year survival was poorest among black males. CONCLUSIONS: ALL incidence has increased over the examined 25-year period. The rate in US whites is higher than that of US Blacks, and the rates in the Hispanic subgroup are the highest of all. While the median survival period is now more than 10 years overall, the 5-year survival rate remains poor for Black males under 4 years of age. Socioeconomic factors do not account for this difference, which may relate to ALL subtype distribution. PMID- 12376978 TI - Mediastinal mass in childhood T-cell acute lymphoblastic leukemia: significance and therapy response. AB - BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-13% of childhood ALL cases. Patients with T-ALL frequently present with unfavorable features at diagnosis and thus are considered to have a higher risk to relapse. Within the last 10 years, the previously dismal prognosis of this ALL subtype has been improved by intensified chemotherapy. However, 30 40% of patients still relapse, so that additional prognostic factors such as the local response of the mediastinal mass to therapy might allow defining the patients at risk in a better manner. PROCEDURE: A retrospective analysis of 116 Austrian patients with T-ALL was performed to assess whether an initial mediastinal mass (70/116) and its response to chemotherapy as measured by thoracic X-rays (32/70) might predict outcome. RESULTS: Neither patients with a mediastinal tumor at the time of diagnosis nor patients with an incomplete response on day 35 or 70 of therapy had a worse prognosis, as compared with the group of patients with no initial tumor and complete regression on day 35 and 70. CONCLUSIONS: We failed to show that in children with T-ALL residual mediastinal tumors are of prognostic relevance. This might suggest that incomplete local response is not necessarily an indication for treatment intensification such as local irradiation, second-look operation, or high-dose chemotherapy with bone marrow rescue. However, due to the relatively small number of patients analyzed, our results have to be validated prospectively on a larger cohort of patients in future clinical trials. PMID- 12376979 TI - On the origin of EEG-slowing and encephalopathy during induction treatment of acute lymphoblastic leukemia. AB - BACKGROUND: Neurological complications and EEG slowing frequently occur in children undergoing induction treatment for acute lymphoblastic leukemia (ALL). Disease-related factors and treatment-related toxicity are believed to play causative roles. We wanted to elucidate the etiology further by serial EEG examinations and parallel CSF amino acid analyses. PROCEDURE: Twenty-nine children participated in the study. EEG examinations with quantitative computerized analysis were scheduled on day 1, 10, 29, and 59 of protocol I of BFM-ALL 90 and 95 Study Protocols. CSF analysis for amino acids was carried out on day 1, 15, 29, 45, and 59. RESULTS: A total of 21 of 25 available EEGs showed slight-to-moderate slowing already at diagnosis. The abundance of slow waves was significantly correlated to the white blood count and the CSF glutamine concentration. The EEGs significantly worsened during the first 10 days of treatment with prednisone, VCR, daunorubicin, and intrathecal methotrexate. The following treatment including asparaginase (ASP) gave rise to depletion of CSF from asparagine and a rise of aspartate; glutamine, and glutamate did not follow this pattern. The EEGs remained abnormal, but did not worsen further; the CSF amino acid changes were not related to the EEG. During the subsequent consolidation treatment, the EEGs normalized despite administration of cyclophosphamide, cytara bine, intrathecal methotrexate, and mercaptopurin. CONCLUSIONS: The greater part of EEG changes observed in the early treatment of ALL is due to disease-related factors. Treatment with prednisone, vincristine, and to a much lesser degree asparaginase aggravates the pre-existing encephalopathy. Depletion of CSF from asparagine does not give rise to additional changes. In the second month, the EEG normalizes despite ongoing treatment with different cytotoxic drugs. PMID- 12376980 TI - Platinum agents in the treatment of osteosarcoma: efficacy of cisplatin vs. carboplatin in human osteosarcoma cell lines. AB - BACKGROUND: Cisplatin (cDDP), when used either alone or, more often, in combination with other agents, especially adriamycin, achieves a high response rate in osteosarcoma. Its use, however, is limited by severe nephro- and neuro toxicity. Second generation platinum compounds, most notably carboplatin (CBDCA), have been developed in order to attempt to reduce these dose-limiting toxicities, and thus improve the therapeutic ratio. Studies evaluating the role of combination CT containing CBDCA vs. cDDP have demonstrated differing results depending on the tumor type tested and its role in the treatment of osteosarcoma has yet to be clarified. PROCEDURE: In this study, we compared the in vitro anti tumor activity of cDDP and CBDCA in a panel of three human osteosarcoma cell lines (HOS, MG63, and U2OS). RESULTS: cDDP and CBDCA (0-20 micromol) showed marked variation in cytotoxicity among the three cell lines. EC(50) values for CBDCA in HOS and MG63 cells were approximately two-fold higher than for cDDP and the ratio of AUC(CBDCA) to AUC(cDDP) varied from 1.8 in the HOS cell line to 2.3 in the MG63 cell line. Exposure of MG63 and HOS cells to either cDDP or CBDCA (1.67 and 13.5 micromol) caused a G2/M cell cycle arrest by 24 hr. Also evident was a sub G1 peak indicative of cell death by apoptosis. U2OS cells were relatively resistant to the cytotoxic effects of both drugs, although a cell cycle arrest in response to DNA damage was observed. This suggests that unlike MG63 and HOS cells, U2OS cells have either a more efficient repair pathway for platinum-induced DNA damage or are able to evade apoptosis. Examination of apoptotic events and cellular recovery demonstrated that both an 8-16-fold higher concentration and longer treatment period for CBDCA compared with cDDP was required to produce equivalent cell death and a loss of the ability of single cell clones to form colonies in both the HOS and MG63, but not the U2OS cell line. CONCLUSIONS: Our findings suggest that CBDCA at a two- to four-fold higher concentration than cDDP has potential therapeutic activity in platinum sensitive osteosarcomas, particularly when cDDP cytotoxicity compromises therapeutic efficacy. PMID- 12376981 TI - Response to initial treatment of multisystem Langerhans cell histiocytosis: an important prognostic indicator. AB - BACKGROUND: Reliable prediction of prognosis allowing risk-adapted therapy remains a major issue in the management of multisystem Langerhans cell histiocytosis (LCH). In a recent publication of the International LCH Study Group, response to initial therapy appears to be a reliable outcome predictor. The aim of this study is to test this observation in a cohort of patients treated with more intensive initial therapy. Furthermore, we compare the predictive value of response to initial therapy to some other well-established stratification systems. PROCEDURE: Response to initial combination chemotherapy (prednisolone, vinblastine, and etoposide) at 6 weeks and its prognostic value was evaluated retrospectively in 63 patients with multisystem LCH from the DAL-HX 83 and 90 Studies, and correlated to some established scoring systems from the literature. RESULTS: After 6 weeks of therapy, 50/63 (79%) patients qualified as responders, 4/63 (7%) patients showed intermediate response, and 9/63 (14%) patients did not respond. Probability of survival at 5 years was 0.94 +/- 0.03 for responders, 0.75 +/- 0.22 for patients with intermediate response, and only 0.11 +/- 0.10 for non-responders. CONCLUSIONS: Response to initial therapy appears to be a reliable prognostic predictor. Compared to the published international LCH-I Study, our results suggest that more intensive initial treatment allows a better discrimination between responders and non-responders. This allows to identify a subgroup of patients with extremely poor prognosis (mortality rate 90%) relatively early in the disease course. PMID- 12376982 TI - Localised Ewing sarcoma/PNET of bone--prognostic factors and international data comparison. AB - BACKGROUND: To determine if the distribution of prognostic factors accounted for the differences when the outcome for localised Ewing Sarcoma/PNET bone in Saudi Arabia was compared with results from countries with well developed health care systems. PROCEDURE: Retrospective analysis was undertaken of 163 consecutive patients of all ages, treated with radical intent at KFSHRC from 1975 to 1998. Standard chemotherapy was commenced in all patients. The local treatment modality was resection +/- radiation in 30% and radiation treatment alone in 67%. Size data were available for 51 patients treated from 1994 to 1998, inclusive. One third of these patients had tumors with volume >500 ml. RESULTS: Three year survival significantly increased with the year of diagnosis, 1975-1988 45%; 1989 1993 55%; and 1993-1998 63% (P = 0.006). Favorable prognostic factors were age < or =14 (P = 0.07); site, distal extremity, and skull (P = 0.08); and volume < or = 200 ml (P = 0.06). Secondary prognostic factors were response to induction chemotherapy, both histological, 100% necrosis, (P = 0.04) and clinical CR+PR, (P = 0.02). From 1994 to 1998, 3 year survival for tumors in the distal extremity and skull was 80% and for small tumors, < 200 ml, at any site was 82%. In comparison, the 3 year survival for patients with tumors at any other sites was 60%, and for tumors >200 ml, 55%. CONCLUSIONS: Overall survival progressively improved. From 1994 to 1998 the survival of patients with small tumors and/or favorable sites was similar to the best reported results. It was not possible to compare results by tumor size for large tumors, > 500 ml, due to the absence of data from elsewhere. A better staging system is required for the international comparison of results. PMID- 12376983 TI - Epithelial salivary glands neoplasms in children and adolescents: a forty-four year experience. AB - BACKGROUND: Epithelial neoplasms of salivary gland origin are relatively uncommon in children and adolescents. Over a 44-year period, there were 38 cases affecting children under 19 years of age in our Pediatric Hospital-Based Tumor Registry. PROCEDURE: Medical charts of 38 patients with epithelial neoplasms of salivary glands were reviewed. Data collected included demographic, clinical, and histological characteristics. Statistical analysis included descriptive statistics, Student t-test, and Kaplan-Meier method was used for survival analysis. RESULTS: The mean age was 11.8 years. There was a female preponderance of 1.9:1. The parotid gland was affected in most cases (65.8%). Twenty-seven patients had malignant tumors and eleven patients presented benign neoplasms. Pleomorphic adenoma was the most frequent benign tumor (7 out of 11) and mucoepidermoid carcinoma was the most frequent malignancy (17 out of 27). Five year overall survival rate was 81.6% for patients with malignant tumors. Grade of differentiation was the only significant prognostic factor for patients with mucoepidermoid carcinomas. CONCLUSIONS: Epithelial salivary gland tumors are very rare in children. Surgery is the best option to achieve high cure rates and radiotherapy must have precise indications because of their long-term side effects in young age. PMID- 12376985 TI - A comparative study of proteomics maps using graph theoretical biodescriptors. AB - This paper reports the development of new methods for mathematical characterization of effects of different toxic agents on the cellular proteome. We describe numerical characterization of proteomics maps based on mathematical invariants. A graph is first associated with a proteomics map by considering partial ordering of spots on 2-D gels by ordering proteins with respect to the mass and the charge, the two properties by which proteins are separated. The graph is then embedded over the map, and several graph theoretical invariants have been constructed. In particular we consider invariants that can be extracted from the Euclidean distance-adjacency matrix of the embedded graph, in which only Euclidean distances between adjacent vertices of a graph are considered. The approach is illustrated using proteomics patterns of normal liver cells of rats and those derived from liver cells of animals exposed to four peroxisome proliferators. In contrast to direct comparison of spot abundance our approach incorporates information on spots locations. The difference between the two approaches is that in the first case only changes in abundances are considered as a measure of perturbation of the proteome map, but in the second case not only the charge but also the mass of proteins are used for ordering protein spots. PMID- 12376986 TI - Orthogonalization of block variables by subspace-projection for quantitative structure property relationship (QSPR) research. AB - A subspace-projection method is developed to construct orthogonal block variable, which is originally from some kinds of series of topological indices or quantum chemical parameters. With the help of canonical correlation analysis, the orthogonal block variables were used to establish the structure-retention index correlation model. The regression of only few new orthogonal variables obtained by canonical correlation analysis against retention index shows significant improvement both in fitting and prediction ability of the correlation model. Moreover, the quantitative intercorrelation between the different block variables of topological indices can also be evaluated with the help of the subspace projection technique proposed in this work. PMID- 12376987 TI - Analysis of the relationship among the graphs isomorphic to multilayered cyclic fence graphs (MLCFG). AB - Multilayered cyclic fence graphs (MLCFG, E(m,n), F(m,n), D(m,n), G(m,n), X(m,n)) are proposed to be defined, all of which are composed of m 2n-membered cycles with periodic bridging. They are also cubic and bipartite. Hamiltonian wheel graph, H (n,[j(k)]), and parallelogram-shaped polyhex graph are also defined. All the members of MLCFGs are found to be isomorphic to the so-called "torus benzenoid graphs", while some members of MLCFGs are found to be related to the Hamilton wheel graphs. Through the construction of Hamilton wheel graph and the matrix representation by Kirby, a number of isomorphic relations among MLCFGs, Hamilton wheel graphs, and polyhex graphs were obtained. These relations among the MLCFG members were found also by the help of the characteristic quantities of MLCFGs. PMID- 12376988 TI - A chemistry field in search of applications statistical analysis of U.S. fullerene patents. AB - The paper is examining the U.S. patenting activity on the application of fullerenes and shows that despite some pessimistic manifestations in this respect in the current literature there are many promising approaches regarding the pragmatic aspects of this field of chemistry. PMID- 12376989 TI - Approaches to understanding the searching behavior of CrossFire users. AB - Due to the high costs of purchasing, supporting and training users of desktop chemical information systems, it is important to understand users' behavior in order that deficiencies in their search efficiency and effectiveness can be identified and addressed. CrossFire generates comprehensive log files that can be examined to determine the nature of search activity. At GSK (GlaxoSmithKline) a Log File Parser, CrossParse, has been developed in Visual Basic that enables analysis by individual user name, groups of users or the whole user population. Log files can be analyzed for occasions when specific structural features are built, specific types of search are done and how the results are manipulated. CrossParse produces output that can be saved and analyzed within Microsoft Excel. It also allows determination of numbers of active concurrent users on the CrossFire system. CrossParse has been used at GSK (ex-SmithKline Beecham sites) to examine the search behavior of medicinal and synthetic chemists. Additionally, it has been used by MIMAS (Manchester Information and Associated Services) to compare the search behavior of trained and untrained users in the higher education community and to identify any areas where improvements to training can be made. Use of CrossParse in both organizations has allowed identification of areas where users may have difficulties using CrossFire. This will provide valuable feedback to MDL Inc., the authors of the CrossFire application, and guide them in enhancing CrossFire. PMID- 12376990 TI - Algorithmically compressed data and the topological conjecture for the inner-core electrons. AB - Eight different properties of three classes of compounds, metal halides, halomethanes, and chlorofluorocarbons, have been modeled with the aim to check the validity of the odd complete graph conjecture suggested for encoding the contribution of the inner-core electrons to the molecular connectivity indices. Modeling using this conjecture is compared with modeling using connectivity indices derived by other well-known algorithms. The conjecture of odd complete graph for the inner-core electrons achieves to improve the modeling quality of the molecular connectivity indices and/or of the molecular connectivity terms. The importance of the recently introduced dual molecular connectivity indices and pseudoindices in further refining the modeling quality of the higher-order terms has also been stressed. PMID- 12376991 TI - The maximum common substructure as a molecular depiction in a supervised classification context: experiments in quantitative structure/biodegradability relationships. AB - The maximum common structure between two molecules (MCS) induces a similarity that enables one to group compounds sharing the same pattern. This text relates a study based on such a structural depiction in a context of quantitative structure/biodegradability relationships (QSBR). The similarity indices are based exclusively on the MCS. First, the results of statistical tests prove that these indices significantly group compounds of similar activity together. These first conclusions enable the elaboration of classification models using those structural similarities. In a second part, a population of classifiers relying on the maximum common structure and the k-nearest-neighbor algorithm is explored. Finally, a thorough examination of the best models is conducted. PMID- 12376992 TI - Prediction of glycine/NMDA receptor antagonist inhibition from molecular structure. AB - The design and blood brain barrier crossing of glycine/NMDA receptor antagonists are of significant interest in pharmaceutical research. The use of these antagonists in stroke or seizure reduction have been considered. Measuring the inhibitory concentrations, however, can be time-consuming and costly. The use of quantitative structure-activity relationships to estimate IC(50) values for these receptor antagonists is an attractive alternative compared to experimental measurement. A data set of 109 compounds with measured log(IC(50)) values ranging from -0.57 to 4.5 is used. Structural information is encoded with numerical descriptors for topological, electronic, geometric, and polar surface properties. A genetic algorithm with a computational neural network fitness evaluator is used to select the best descriptor subsets. Multiple linear regression and computational neural network models are developed. Additionally, a quantitative radial basis function neural network (QRBFNN) was developed with the intent of introducing nonlinearity at a faster speed. A genetic algorithm using the radial basis function network as a fitness evaluator was also developed to search descriptor space for optimum subsets. All models are tested using an external prediction set. The nonlinear computational neural network model has root-mean square errors of approximately half a log unit. PMID- 12376993 TI - Analysis of large screening data sets via adaptively grown phylogenetic-like trees. AB - As the use of high-throughput screening systems becomes more routine in the drug discovery process, there is an increasing need for fast and reliable analysis of the massive amounts of the resulting data. At the forefront of the methods used is data reduction, often assisted by cluster analysis. Activity thresholds reduce the data set under investigation to manageable sizes while clustering enables the detection of natural groups in that reduced subset, thereby revealing families of compounds that exhibit increased activity toward a specific biological target. The above process, designed to handle primarily data sets of sizes much smaller than the ones currently produced by high-throughput screening systems, has become one of the main bottlenecks of the modern drug discovery process. In addition to being fragmented and heavily dependent on human experts, it also ignores all screening information related to compounds with activity less than the threshold chosen and thus, in the best case, can only hope to discover a subset of the knowledge available in the screening data sets. To address the deficiencies of the current screening data analysis process the authors have developed a new method that analyzes thoroughly large screening data sets. In this report we describe in detail this new approach and present its main differences with the methods currently in use. Further, we analyze a well-known, publicly available data set using the proposed method. Our experimental results show that the proposed method can improve significantly both the ease of extraction and amount of knowledge discovered from screening data sets. PMID- 12376994 TI - Characteristic sequences for DNA primary sequence. AB - A DNA sequence can be identified with a word over an alphabet N = [A, C, G, T]. Characteristic sequences of a DNA sequence are given in term of classifications of bases of nucleic acids. Using the characteristic sequences, we construct a set of 2 x 2 matrices to represent DNA primary sequences, which are based on counting of the frequency of occurrence of all (0,1) triplets of characteristic sequences. Furthermore, the leading eigenvalues of these matrices are computed and considered as invariants for the DNA primary sequences. Similarity and dissimilarity analysis based on the characteristic sequences are given for eight exon-1 genes of beta-globin about eight species. PMID- 12376995 TI - A comparison of partial order technique with three methods of multi-criteria analysis for ranking of chemical substances. AB - An alternative to the often cumbersome and time-consuming risk assessments of chemical substances could be more reliable and advanced priority setting methods. An elaboration of the simple scoring methods is provided by Hasse Diagram Technique (HDT) and/or Multi-Criteria Analysis (MCA). The present study provides an in depth evaluation of HDT relative to three MCA techniques. The new and main methodological step in the comparison is the use of probability concepts based on mathematical tools such as linear extensions of partially ordered sets and Monte Carlo simulations. A data set consisting of 12 High Production Volume Chemicals (HPVCs) is used for illustration. It is a paradigm in this investigation to claim that the need of external input (often subjective weightings of criteria) should be minimized and that the transparency should be maximized in any multicriteria prioritisation. The study illustrates that the Hasse diagram technique (HDT) needs least external input, is most transparent and is least subjective. However, HDT has some weaknesses if there are criteria which exclude each other. Then weighting is needed. Multi-Criteria Analysis (i.e. Utility Function approach, PROMETHEE and concordance analysis) can deal with such mutual exclusions because their formalisms to quantify preferences allow participation e.g. weighting of criteria. Consequently MCA include more subjectivity and loose transparency. The recommendation which arises from this study is that the first step in decision making is to run HDT and as the second step possibly is to run one of the MCA algorithms. PMID- 12376996 TI - Global optimization of Lennard-Jones clusters by a parallel fast annealing evolutionary algorithm. AB - A parallel fast annealing evolutionary algorithm (PFAEA) was presented and applied to optimize Lennard-Jones (LJ) clusters. All the lowest known minima up to LJ(116) with both icosahedral and nonicosahedral structure, including the truncated octahedron of LJ(38), central fcc tetrahedron of LJ(98), the Marks' decahedron of LJ(75)(-)(77), and LJ(102)(-)(104), were located successfully by the unbiased algorithm. PFAEA is a parallel version of fast annealing evolutionary algorithm (FAEA) that combines the aspect of population in genetic algorithm and annealing algorithm with a very fast annealing schedule. A master slave paradigm is used to parallelize FAEA to improve the efficiency. The performance of PFAEA is studied, and the scaling of execution time with the cluster size is approximately cubic, which is important for larger scale energy minimization systems. PMID- 12376997 TI - Rule extraction from a mutagenicity data set using adaptively grown phylogenetic like trees. AB - A public bacterial mutagenicity database was classified into 2-D structural families using a set of specific algorithms and clustering techniques that find overlapping classes of compounds based upon chemical substructures. Structure activity relationships were learned from the biological activity of the compounds within each class and used to identify rules that define substructures potentially responsible for mutagenic activity. In addition, this method of analysis was used to compare the pharmacologically relevant substructure of test compounds with their potential toxic substructures making this a potentially valuable in silico profiling tool for lead selection and optimization. PMID- 12376998 TI - Fragmental approach in QSPR. AB - Methodological problems of using fragmental descriptors for construction of QSAR/QSPR equations are considered, and the main achievements in this field are summarized and discussed. If a structure-property data set is sufficiently large to allow building statistically significant models, then any topological index can be replaced with a set of fragmental descriptors. Several examples of using the fragmental approach for predicting retention indices and the normal boiling points of organic compounds are considered. Advantages of using fragmental descriptors, namely a "transparency" and interpretability of QSAR/QSPR models, are exemplified. PMID- 12377000 TI - A differential-operator approach to the permanental polynomial. AB - A recently published computational approach to the permanental polynomial scales very badly (approximately 2(n)) with problem size, relying as it does on examining the entire augmented adjacency matrix for nonzero products. The present study presents an entirely different algorithm that relies on symbolic computation of second partial derivatives. This approach has previously been applied to the matching polynomial but not the permanental polynomial. The differential-operator algorithm scales much better with problem size. For fullerene-type structures without perimeters, the two algorithms take about the same time to compute n = 32. On one n = 40 structure, the new algorithm was >45 times faster. Relative performance is even better for polycyclic aromatic hydrocarbon structures, which have perimeters. PMID- 12376999 TI - Comparative structural connectivity spectra analysis (CoSCoSA) models of steroid binding to the corticosteroid binding globulin. AB - A three-dimensional quantitative spectrometric data-activity relationship (3D QSDAR) model was developed that is built by combining NMR spectral information with structural information in a 3D-connectivity matrix. The 3D-connectivity matrix is built by displaying all possible carbon-to-carbon connections with their assigned carbon NMR chemical shifts and distances between the carbons. Selected 2D (13)C-(13)C COrrelation SpectroscopY (COSY) (through-bond nearest neighbors) and selected theoretical 2D (13)C-(13)C distance connectivity spectral slices from the 3D-connectivity matrix to produce a relationship among the spectral patterns for 30 steroids binding to corticosteroid binding globulin. We call this technique a comparative structural connectivity spectra analysis (CoSCoSA) modeling. A CoSCoSA principal component linear regression model based on the combination of (13)C-(13)C COSY and (13)C-(13)C distance spectra principal components (PCs) had an r(2) of 0.96 and a leave-one-out (LOO) cross-validation q(2) of 0.92. A CoSCoSA parallel distributed artificial neural network (PD-ANN) model based on the combination of (13)C-(13)C COSY and (13)C-(13)C distance spectra had an r(2) of 0.96, a leave-three-out q(3)(2) of 0.78, and a leave-ten out q(10)(2) of 0.73. CoSCoSA modeling attempts to uniquely combine the quantum mechanics information from the NMR chemical shifts with internal molecular atom to-atom distances into an accurate modeling technique. The CoSCoSA modeling technique has the flexibility and accuracy to outperform the cross-validated variance q(2) of previously published quantitative structure-activity relationship (QSAR), quantitative spectral data-activity relationship (QSDAR), self-organizing map (SOM), and electrotopological state (E-state) models. PMID- 12377001 TI - Application of associative neural networks for prediction of lipophilicity in ALOGPS 2.1 program. AB - This article provides a systematic study of several important parameters of the Associative Neural Network (ASNN), such as the number of networks in the ensemble, distance measures, neighbor functions, selection of smoothing parameters, and strategies for the user-training feature of the algorithm. The performance of the different methods is assessed with several training/test sets used to predict lipophilicity of chemical compounds. The Spearman rank-order correlation coefficient and Parzen-window regression methods provide the best performance of the algorithm. If additional user data is available, an improved prediction of lipophilicity of chemicals up to 2-5 times can be calculated when the appropriate smoothing parameters for the neural network are selected. The detected best combinations of parameters and strategies are implemented in the ALOGPS 2.1 program that is publicly available at http://www.vcclab.org/lab/alogps. PMID- 12377002 TI - Wavelength selection for multivariate calibration using a genetic algorithm: a novel initialization strategy. AB - Genetic algorithms and other procedures mimicking natural processes are being increasingly used for variable selection, to improve the predictive ability of partial least-squares multivariate calibration. Two issues are critical for the success of genetic algorithms: initialization (setting the first candidates for solving the problem at hand) and overfitting (the tendency to produce excellent results when training, but poor predictions toward fresh samples). A new procedure is presented for sensor selection problems, involving iterative reinitialization based on a statistical analysis of the included sensors. It is shown to give excellent results without the requirement of preparing independent test data sets. Monte Carlo simulations using a theoretical three-component example illustrate how partial least-squares regression greatly benefits from variable selection when the analyte of interest is diluted, and how the new initialization method compares with other strategies. The new genetic algorithm was applied to five experimental data sets. The target parameters were the concentrations of diluted analytes in four pharmaceutical mixtures studied by UV visible spectrophotometry and the octane number in gasolines analyzed by near infrared spectroscopy. PMID- 12377003 TI - Polarizabilities of solvents from the chemical composition. AB - From the experimental polarizability values, alpha, of a large set of solvents containing 426 compounds with very different chemical characteristics, an additive model for the estimation of the polarizability is proposed. The derived average atomic polarizability of 10 elements, C, H, O, N, S, P, F, Cl, Br, and I, allows the calculation of the molecular polarizability of solvents from their chemical composition alone, without any other structural consideration. The average errors are 2.31% and 1.93% for the working set of 340 solvents and the prediction set of 86 solvents, respectively. Semiempirical quantum methods, such as, AM1, PM3, and MNDO, gave errors of about 35%. The density functional theory (DFT) calculations give better results than the semiempirical ones but poorer results than those obtained by the additive approach. PMID- 12377004 TI - The nonpolar resonance effects and the non-Hammett behaviors. AB - In the study we tried to unify the observed non-Hammett behaviors in various fields by proposing the nonpolar resonance effect. This effect was shown to be important not only for carbon radicals but also for some closed-shell systems. Therefore, the odd electron or spin is not the essential cause of the effect. The real origin of the non-Hammett effect should be the HOMO(reaction-center) LUMO(acceptor) and LUMO(reaction-center)-HOMO(donor) interactions. This means that the nonpolar resonance effect is a universal effect. However, we found that the nonpolar resonance effect could not be well exhibited in many systems because of the serious competition from the polar Hammett effect. Finally, we showed that our proposal of the nonpolar resonance effect was valuable from a practical point of view, because using it we could perform much better correlation studies on some "tough" problems such as radical and multiple bond stabilities, UV and IR spectra, and molecular structures. PMID- 12377005 TI - Characterization of cyclo-polyphenacenes. AB - Cyclo-polyphenacenes belt-type compounds are considered here in terms of some simple chemico-graph-theoretic invariants. First the compounds of this group are neatly encoded in an unambiguous way. Then all the isomers in this system are categorized with respect to their "combinatorial curvature", and for the case of the 52 cyclo-hexaphenacenes it is found to correlate with steric stresses. A systematic effort is made to correlate the reactivity (via additive oxidation) of the various isomers with their Kekule-structure counts. PMID- 12377006 TI - A DNA algorithm for the graph coloring problem. AB - A DNA algorithm based on surfaces for the graph coloring problem is presented. First the whole combinatorial color assignments to the vertices of a graph are synthesized and immobilized on a surface; then a vertex is legally colored while those adjacent to it with illegal colors are deleted; and the cycle is repeated until finally the correct color assignments to the graph are reached. Compared with the other DNA algorithms, our algorithm is easy to implement and error resistant. PMID- 12377007 TI - Simple lattice simulation of chiral discrimination in monolayers. AB - A simulation method based on cellular automata on a hexagonal lattice is applied to model the behavior of chiral molecules on a surface, such as those in a monolayer of amphiphiles each having a single chiral center. The simulation method includes movement and orientation rules, with the objects ("molecules") on the lattice vertices possessing three different groups with one group pointing along each edge. Periodic boundary conditions are employed in the simulations. Interaction strengths are calculated between pairs of groups occupying the edges between vertices and summed for the entire system. The model successfully reproduces the formation of domains as a consequence of the movement rule. The movement rule can be adjusted to simulate homochiral discrimination or heterochiral discrimination for the case of racemic mixtures. The orientation rule results in a preference for orientations of the molecules that minimize the total interaction strength. PMID- 12377008 TI - Molecular quantum similarity-based QSARs for binding affinities of several steroid sets. AB - The application of Molecular Quantum Similarity Measures (MQSM) to correlate biological activities for three different sets of steroids is reported. A general protocol for the generation of descriptors is detailed, thus covering molecular superposition, electronic density fitting, and quantum similarity calculation issues. Satisfactory Quantitative Structure-Activity Relationship (QSAR) models (r(2) in [0.69,0.94] and q(2) in [0.59,0.73]), comparable to previous studies, are obtained in all cases, where steroid binding affinities to different enzymes are studied. In this work, MQSM, properly scaled using Carbo Index, are related to activity using a Partial Least Squares routine. PMID- 12377009 TI - In silico studies toward the discovery of new anti-HIV nucleoside compounds with the use of TOPS-MODE and 2D/3D connectivity indices. 1. Pyrimidyl derivatives. AB - Computational approaches are developed to design or rationally select, from structural databases, pyrimidyl nucleosides with anti-HIV activity. A data set of 141 nucleoside derivatives was selected from literature, and a discriminant function was derived with the use of TOPS-MODE descriptors. The model is able to classify correctly 83% of the compounds in a training set and 88.5% in a cross validation set. The use of an external prediction set selected from the most recent literature proved that the model has good predictive ability, with a good classification of 85% of the compounds in this set. This model permitted the structural interpretation of the anti-HIV activity of these nucleoside analogues. This interpretation is formulated as several rules concerning the influence of several structural features on the activity/inactivity of such compounds. A QSAR model for the most active compounds was developed with the combined use of 2D and 3D connectivity indices. This model explains 88% of the variance in the activity of these compounds in MT4 assay. The combination of both models will permit the selection of pyrimidyl nucleoside leads and their optimization to improve the potency of the selected ones. PMID- 12377010 TI - Use of catalyst pharmacophore models for screening of large combinatorial libraries. AB - Using a data set comprised of literature compounds and structure-activity data for cyclin dependent kinase 2, several pharmacophore hypotheses were generated using Catalyst and evaluated using several criteria. The two best were used in retrospective searches of 10 three-dimensional databases containing over 1,000,000 proprietary compounds. The results were then analyzed for the efficiency with which the hypotheses performed in the areas of compound prioritization, library prioritization, and library design. First as a test of their compound prioritization capabilities, the pharmacophore models were used to search combinatorial libraries that were known to contain CDK active compounds to see if the pharmacophore models could selectively choose the active compounds over the inactive compounds. Second as a test of their utility in library design again the pharmacophore models were used to search the active combinatorial libraries to see if the key synthons were over represented in the hits from the pharmacophore searches. Finally as a test of their ability to prioritize combinatorial libraries, several inactive libraries were searched in addition to the active libraries in order to see if the active libraries produced significantly more hits than the inactive libraries. For this study the pharmacophore models showed potential in all three areas. For compound prioritization, one of the models selected active compounds at a rate nearly 11 times that of random compound selection though in other cases models missed the active compounds entirely. For library design, most of the key fragments were over represented in the hits from at least one of the searches though again some key fragments were missed. Finally, for library prioritization, the two active libraries both produced a significant number of hits with both pharmacophore models, whereas none of the eight inactive libraries produced a significant number of hits for both models. PMID- 12377011 TI - A 3D QSAR pharmacophore model and quantum chemical structure--activity analysis of chloroquine(CQ)-resistance reversal. AB - Using CATALYST, a three-dimensional QSAR pharmacophore model for chloroquine(CQ) resistance reversal was developed from a training set of 17 compounds. These included imipramine (1), desipramine (2), and 15 of their analogues (3-17), some of which fully reversed CQ-resistance, while others were without effect. The generated pharmacophore model indicates that two aromatic hydrophobic interaction sites on the tricyclic ring and a hydrogen bond acceptor (lipid) site at the side chain, preferably on a nitrogen atom, are necessary for potent activity. Stereoelectronic properties calculated by using AM1 semiempirical calculations were consistent with the model, particularly the electrostatic potential profiles characterized by a localized negative potential region by the side chain nitrogen atom and a large region covering the aromatic ring. The calculated data further revealed that aminoalkyl substitution at the N5-position of the heterocycle and a secondary or tertiary aliphatic aminoalkyl nitrogen atom with a two or three carbon bridge to the heteroaromatic nitrogen (N5) are required for potent "resistance reversal activity". Lowest energy conformers for 1-17 were determined and optimized to afford stereoelectronic properties such as molecular orbital energies, electrostatic potentials, atomic charges, proton affinities, octanol water partition coefficients (log P), and structural parameters. For 1-17, fairly good correlation exists between resistance reversal activity and intrinsic basicity of the nitrogen atom at the tricyclic ring system, frontier orbital energies, and lipophilicity. Significantly, nine out of 11 of a group of structurally diverse CQ-resistance reversal agents mapped very well on the 3D QSAR pharmacophore model. PMID- 12377012 TI - A factorial design to optimize cell-based drug discovery analysis. AB - Drug discovery is dependent on finding a very small number of biologically active or potent compounds among millions of compounds stored in chemical collections. Quantitative structure-activity relationships suggest that potency of a compound is highly related to that compound's chemical makeup or structure. To improve the efficiency of cell-based analysis methods for high throughput screening, where information of a compound's structure is used to predict potency, we consider a number of potentially influential factors in the cell-based approach. A fractional factorial design is implemented to evaluate the effects of these factors, and lift chart results show that the design scheme is able to find conditions that enhance hit rates. PMID- 12377013 TI - A novel shape-feature based approach to virtual library screening. AB - The shape of and the chemical features of a ligand are both critical for biological activity. This paper presents a strategy that uses these descriptors to build a computational model for virtual screening of bioactive compounds. Molecules are represented in a binary shape-feature descriptor space as bit strings, and their relative activities are used to identify the subset of the bit string that is most relevant to bioactivity. This subset is used to score virtual libraries. We describe the computational details of the method and present an example validation experiment on thrombin inhibitors. PMID- 12377014 TI - Structure-activity relationships for the anti-HIV activity of flavonoids. AB - Quantitative structure-activity relationship (QSAR) models are useful in providing a biochemical understanding of the biological activity of natural and synthetic chemicals based solely on molecular structure. One- to three-parameter multiregression equations were generated for three different groups of flavonoids to model experimental flavonoid-induced cytotoxicity and inhibition of human immunodeficiency virus I replication in lymphocyte-infected cells, using quantum chemical and geometrical parameters derived from optimized molecular structures. Both biological properties were basically dependent on electronic parameters describing charge distribution on the two fused rings of the flavonoid molecule. Atomic charges in C3 and the carbonyl carbon as well as the dipolar moment were important electronic descriptors to define the studied biological properties of flavonoids. PMID- 12377015 TI - High-throughput, in silico prediction of aqueous solubility based on one- and two dimensional descriptors. AB - An aqueous solubility model has been developed. The model is based solely on one- and two-dimensional descriptors and an artificial neural network to ensure fast execution. 63 descriptors expressing physicochemical and topological properties were used. The final model consisted of a training set of 3042 molecules, a test set of 309 molecules and an independent validation set of 307 molecules. The squared correlation coefficients were 0.91 for the training set, 0.89 for the test set and 0.86 for the independent validation set. PMID- 12377016 TI - The importance of scaling in data mining for toxicity prediction. AB - While mining a data set of 554 chemicals in order to extract information on their toxicity value, we faced the problem of scaling all the data. There are numerous different approaches to this procedure, and in most cases the choice greatly influences the results. The aim of this paper is 2-fold. First, we propose a universal scaling procedure for acute toxicity in fish according to the Directive 92/32/EEC. Second, we look at how expert preprocessing of the data effects the performance of qualitative structure-activity relationship (QSAR) approach to toxicity prediction. PMID- 12377017 TI - Selecting screening candidates for kinase and G protein-coupled receptor targets using neural networks. AB - A series of neural networks has been trained, using consensus methods, to recognize compounds that act at biological targets belonging to specific gene families. The MDDR database was used to provide compounds targeted against gene families and sets of randomly selected molecules. BCUT parameters were employed as input descriptors that encode structural properties and information relevant to ligand-receptor interactions. In each case, the networks identified over 80% of the compounds targeting a gene family. The technique was applied to purchasing compounds from external suppliers, and results from screening against one gene family demonstrated impressive abilities to predict the activity of the majority of known hit compounds. PMID- 12377021 TI - Heterobinuclear complexes as building blocks in designing extended structures. AB - The heterobinuclear complex [CuPrL(NO(3))(3)] has been used as a building block for the construction of a two-dimensional coordination polymer with the formula [CuPrL(NO(3)(2)(IN)], 1 (L(2-) = the dianion of the compartmental Schiff-base ligand obtained from the 2:1 condensation of 3-methoxysalicylaldehyde with 1,3 propanediamine; IN(-) = the isonicotinate ion). The heterobinuclear units, [CuPrL(NO(3))(2)](+), are connected through the unsymmetrical exo-bidentate ligands, IN(-), leading to a unique extended structure. Crystal data for compound 1: FW, 790.94; monoclinic, space group P2(1)/n, a = 11.2837(3) A, b = 14.7785(4) A, c = 16.9745(4) A, beta = 100.427(1) degrees; V = 2783.86(12) A(3); Z = 4; R1 = 0.0210, wR2 = 0.0562 [I > 2sigma(I)]. PMID- 12377018 TI - Classification of biologically active compounds by median partitioning. AB - The median partitioning (MP) method was originally developed for the selection of diverse subsets from compound databases. Following this approach, property descriptors are used in subsequent steps to divide compounds into defined partitions from which representative molecules are selected. For descriptor analysis, MP was coupled to a genetic algorithm. MP subset selection does not depend on pairwise comparison of molecules and is therefore applicable to very large compound pools. Here the MP approach was evaluated for the classification of molecules according to biological activity. A total of 317 molecules belonging to 21 different activity classes were studied. MP compound classification calculations were carried out both in the presence and absence of 2000 randomly selected "background" molecules. The performance of MP was compared to cell-based partitioning and found to be at least comparable, with up to approximately 82% of active molecules occurring in "pure" partitions consisting only of molecules sharing the same activity. Different from cell-based methods, MP classification is based on "direct" and "sequential" contributions of molecular property descriptors. Our results suggest that MP in not only an effective method for the selection of diverse subsets but also for the classification of active compounds and searching for molecules with desired activity. PMID- 12377022 TI - New single source route to the molybdenum nitride Mo(2)N. AB - Dispersed Mo(2)N with lamellar morphology and high specific surface area was obtained from the decomposition of the (HMT)(2)(NH(4))(4)Mo(7)O(24) salt (HMT = hexamethylentertramine) in the temperature range 550-800 degrees C. During the thermal decomposition, reduction of Mo species by carbon from the HMT ligand occurs, with release of CO gas. PMID- 12377023 TI - Comparative preparations of homoleptic hydridic anions of iron and ruthenium using solution-based organometal hydrogenation techniques. AB - A study of the preparations of the complex hydridic anions [MH(6)](4)(-) (M = Fe and Ru) reveals a number of distinctive features. Here a soluble homoleptic ruthenium hydride has been prepared for the first time. For example, both FeX(2) and [Ru(eta(4)-1,5-COD)X(2)], X = Cl and Br, react with PhMgBr solutions under hydrogen to produce the title compounds. The benzene liberated in these reactions is more readily hydrogenated in the case of a homogeneous room temperature ruthenium hydride preparation to both cyclohexane and cyclohexene. The (1)H NMR spectroscopic data show that the two complex anions have hydride absorptions in the low-frequency region, delta -20.3 and -14.7, respectively. Further, (1)H spin lattice relaxation times (T(1)) for M-H are longer in the case of Ru vs Fe. PMID- 12377024 TI - Luminescence enhancement induced by aggregation of alkoxy-bridged rhenium(I) molecular rectangles. AB - Self-assembly of rhenium(I)-based molecular rectangles containing long alkyl chains has been achieved in one-pot synthesis by solvothermal methods. An enormous enhancement in the emission intensity, quantum yield, and lifetime of the rectangles has been observed when the solvent medium is changed from organic to aqueous. Addition of water favors the aggregation of Re(I) molecular rectangle resulting in the luminescence enhancement, and this phenomenon has been traced out using light scattering techniques. PMID- 12377025 TI - A tetranuclear heterobimetallic square formed from the cooperative ligand binding properties of square planar and tetrahedral metal centers. AB - Reaction of Rh(I) and Zn(II) metal centers with a ligand containing salicylaldiminato and thioether-phosphine moieties resulted in the formation of a tetranuclear heterobimetallic molecular square. The directionality required to form these structures is imparted by both the tetrahedral and square planar metal centers acting in concert with one another. PMID- 12377026 TI - Synthesis, structural characterization, and reactions of cyclic organohydroborate half-zirconocene compounds. AB - Cyclic organohydroborate complexes of zirconium monocyclopentadienyl CpZr[(mu H)(2)BC(5)H(10)](3), 1, and CpZr[(mu-H)(2)BC(8)H(14)](3), 2, were prepared from the reaction of CpZrCl(3) with 3 mol of K[H(2)BC(5)H(10)] and K[H(2)BC(8)H(10)], respectively, in diethyl ether. Compounds 1 and 2 react with the hydride ion abstracting agent B(C(6)F(5))(3) to form the same salt [CpZr(OEt)(OEt(2))(mu OEt)](2)[HB(C(6)F(5))(3)](2), 5. The complexes CpZr(Cl)[(mu-H)(2)BC(8)H(14)](2), 3, and CpZr(Cl)[(mu-H)(2)BC(8)H(14)](2) [where Cp = C(5)(CH(3))(5)], 4, were prepared from the reaction of CpZrCl(3) and CpZrCl(3) with K[H(2)BC(8)H(10)] in 1:2 molar ratios, respectively. An alpha-hydrogen of a BC(8)H(14) unit forms an agostic interaction with Zr in compound 3 but not in 4. All of the compounds were characterized by single-crystal X-ray diffraction analysis. PMID- 12377027 TI - Structural and photophysical properties of heterobimetallic 4f-zn iminophenolate cryptates. AB - Lanthanide complexes with the Schiff base axial macrobicyclic ligand L(1) react with Zn(II) nitrate in the presence of CaH(2) to yield Ln(III)-Zn(II) heterodinuclear cryptates with the formula [Ln(NO(3))(L(1) 3H)Zn](NO(3)).xH(2)O.yMeOH. The macrobicyclic receptor L(1) is an azacryptand N[(CH(2))(2)N=CH-R-CH=N-(CH(2))(2)](3)N (R = 1,3-(2-OH-5-Me-C(6)H(2))). The crystal structures of the Pr(III), Yb(III), and Lu(III) complexes, chemical formulas [Ln(NO(3))(L(1)-3H)Zn](NO(3)).xSolv (monoclinic, C2/c, Z = 8), as well as that of [Zn(2)(L(1)-3H)](NO(3)).H(2)O (15) (triclinic, P(-)1, Z = 2), have been determined by X-ray crystallography. The ligand is helically wrapped around the two metal ions, leading to pseudo-C(3) symmetries around the metals. The Ln(III)-Zn(II) distances lie in the range 3.3252(13) to 3.2699(14) A, while the Zn(II)-Zn(II) distance in 15 amounts to 3.1037(18) A. The three five-membered chelate rings of the ligand backbone coordinating the Ln(III) ion adopt a (lambdalambdadelta)(5) (or (deltadeltalambda)(5)) conformation while the three pseudochelate rings formed by the coordination of the ligand to the Zn(II) ion adopt a (lambda'lambda'lambda')(5) (or (delta'delta'delta')(5)) conformation. Thus in the solid state the conformation of the three cations is Lambda(deltadeltalambda)(5)(delta'delta'delta')(5) or its enantiomeric form Delta(lambdalambdadelta)(5)(lambda'lambda'lambda')(5). In solution, the helicates present a time-averaged C(3) symmetry, as shown by (1)H NMR, and the conformation of the cations is described as Lambda(deltadeltadelta)(5)(delta'delta'delta')(5) (or Delta(lambdalambdalambda)(5)(lambda'lambda'lambda')(5)). The photophysical properties of the cryptates depend on the nature of the Ln(III) ion, and (L 3H)(3)(-) is revealed to be a good sensitizer for Eu(III) and Tb(III) at low temperatures, but the emission at room temperature is limited by the low energy of the ligand (3)pipi state. While Eu(III) is most effectively sensitized by the ligand triplet state, the Tb(III) ((5)D(4)) sensitization occurs via the singlet state. The quantum yield of the metal-centered luminescence in the Eu-Zn cryptate amounts to 1.05% upon ligand excitation. The low energy of the ligand (3)pipi state allows efficient sensitization of the Nd(III) and Yb(III) cryptates, which emit in the near-infrared. PMID- 12377028 TI - Electronic structure of reduced symmetry peripheral fused-ring-substituted phthalocyanines. AB - Reduced symmetry phthalocyanines are finding use in an increasing number of industrial applications. A detailed understanding of the electronic structure of the pi-system will greatly facilitate the design of new complexes, which fit the specifications required in many of these emerging high technology fields. NMR, electronic absorption, magnetic circular dichroism (MCD), and fluorescence emission and excitation spectra have been recorded for five generic metal phthalocyanine (MPc) derivatives in which additional benzene rings are fused either radially or obliquely onto at least one of the four peripheral benzo groups. The spectroscopy of four radially substituted compounds, zinc mononaphthotribenzotetraazaporphyrine (Zn3B1N), zinc monobenzotrinaphthotetraazaporphyrine (Zn1B3N), and two cis and trans zinc dibenzodinaphthotetraazaporphyrine (Zn2B2N) isomers, is compared to that of the obliquely fused structural isomer of Zn3B1N (Zn3BoN) and the D(4)(h)() symmetry parent compounds, ZnPc and zinc naphthalocyanine (ZnNc). The selection of Zn(II) as the central metal eliminates the possibility of charge transfer between the metal and ring. None of the complexes studied contain any sigma-bonded peripheral substituents. (1)H NMR signals of the seven compounds are assigned on the basis of the coupling patterns, integrated proton numbers, and decoupling experiments. The SIMPFIT program was used to perform spectral band deconvolution analyses of absorption and MCD spectra. ZINDO molecular orbital calculations are described, and the optical spectra are assigned on the basis of the MO models that have been developed previously to account for the spectral properties of metal porphyrin (MP(-2)) and metal phthalocyanine (MPc(-2)) complexes. PMID- 12377029 TI - Trigonal bipyramidal geometry and tridentate coordination mode of the tripod in FeCl(2) complexes with tris(2-pyridylmethyl)amine derivatives bis-alpha substituted with bulky groups. structures and spectroscopic comparative studies. AB - A series of dichloroferrous complexes with ligands derived from the tris(2 pyridylmethyl)amine tripod has been prepared and characterized. The X-ray crystal structures of the complexes [bis(2-bromo-6-pyridylmethyl)(2 pyridylmethyl)amine]Fe(II)Cl(2) ((Br(2)TPA)Fe(II)Cl(2)) and [bis(2-phenyl-6 pyridylmethyl)(2-pyridylmethyl)amine]Fe(II)Cl(2), ((Ph(2)TPA)Fe(II)Cl(2)) are reported. In these complexes, the tripod coordinates in the tridentate mode, with a substituted pyridyl arm dangling away from the metal. Both complexes have a trigonal bipyramidal iron center with two equatorial chloride ions. Their crystal structures are compared with those of the [tris(2-pyridylmethyl)amine]Fe(II)Cl(2) and [(2-bromo-6-pyridylmethyl)bis(2-pyridylmethyl)amine]Fe(II)Cl(2) complexes ((TPA)Fe(II)Cl(2) and (BrTPA)Fe(II)Cl(2), respectively) in which the ligand coordinates in the tetradentate mode. For all complexes, the metal to ligand distances are systematically above the value of 2.0 A, and (1)H NMR displays paramagnetically shifted resonances with short relaxation times. This indicates that the iron is in a high-spin state. Electric conductivity measurements show that, for all complexes, the measured values lie within the same range, significantly below those expected for ionic complexes. Together with the analysis of the UV-visible and NMR data, this strongly suggests that the coordination mode of the tripod is retained in solution. PMID- 12377030 TI - New oxamato-bridged trinuclear Cu(II)-Cu(II)-Cu(II) complexes with hydrogen-bond supramolecular structures: synthesis and magneto-structural studies. AB - Three oxamato-bridged copper(II) complexes of formula [(Cu(H(2)O)(tmen)Cu(tmen))(mu-Cu(H(2)O)(Me(2)pba))](n)((PF(6))(2))(n).2nH(2)O (1), [(Cu(H(2)O)(tmen)Cu(NCS)(tmen))(mu Cu(H(2)O)(Me(2)pba))](2)(ClO(4))(2).4H(2)O (2), and [(Cu(H(2)O)(tmen)Cu(NCS)(tmen))(mu-Cu(H(2)O)(Me(2)pba))](2)(PF(6))(2).4H(2)O (3), where Me(2)pba = 2,2-dimethyl-1,3-propylenebis(oxamato) and tmen = N,N,N',N' tetramethylethylenediamine, have been synthesized and characterized. Their crystal structures were solved. Complex 1 crystallizes in the monoclinic system, space group P2(1), with a = 15.8364(3) A, b =8.4592(2) A, c = 15.952 A, beta = 101.9070(10) degrees, and Z = 2. Complex 2 crystallizes in the monoclinic system, space group P2(1)/c, with a = 6.69530(10) A, b = 18.2441(3) A, c = 31.6127(5) A, beta = 90.1230(10) degrees, and Z = 4. Complex 3 crystallizes in the monoclinic system, space group P2(1)/c, with a = 6.68970(10) A, b = 18.150 A, c = 32.1949(4) A, beta = 90.0820(10) degrees, and Z = 4. The three complexes have a central core in common: a trinuclear Cu(II) complex with the two terminal Cu(II) ions blocked by N,N,N',N'-tetramethylethylenediamine. The structure of complex 1 consists of trinuclear cationic entities connected by hydrogen bonds to produce a supramolecular one-dimensional array. The structure of complexes 2 and 3 consist of trinuclear cationic entities linked by pairs by hydrogen bonds between the water molecule of the central Cu(II) and one oxygen atom of the oxamato ligand of the neighboring entity, forming a hexanuclear complex. The magnetic properties of the three complexes were studied by susceptibility vs temperature measurement. For complexes 1-3 the fit was made by the irreducible tensor operator (ITO). The values obtained were J(1) = -386.48 cm(-1) and J(2) = 1.94 cm(-1) for 1, J(1) = 125.77 cm(-1) and J(2) = 0.85 cm(-1) for 2, and J(1) = -135.50 cm(-1) and J(2) = 0.94 cm(-1) for 3. In complex 1, the coordination polyhedron of the terminal Cu(II) atoms can be considered as square pyramidal; the apical positions are filled by the oxygen atom from a water molecule in the former and a F atom of the hexafluorophosphate anion in the latter showing a quasi-planar [Cu(CuMe(2)pba)Cu] network. For complexes 2 and 3, the square pyramidal environment of the terminal Cu(II) ions was strongly modified. To our knowledge, this is the first time that the longest distance (apical) in complexes with oxamato derivatives and bidentate amines as blocking ligands has been reported in one of the oxamato arms. The great difference in J(1) values between 1 and the other two complexes is interpreted as an orbital reversal of the magnetic orbitals of the terminal Cu(II) ions in 2 and 3. PMID- 12377031 TI - New dirhenium(III) compounds bridged by carboxylate ligands: N(C(4)H(9))(4)[Re(2)(OOCCF(3))Cl(6)] and Re(2)(OOCCCHCo(2)(CO)(6))(4)Cl(2). AB - The solvothermal reaction of (N(C(4)H(9))(4))(2)[Re(2)Cl(8)] with trifluoroacetic acid and acetic anhydride leads to the new rhenium trifluoroacetate dimer N(C(4)H(9))(4)[Re(2)(OOCCF(3))Cl(6)] (1) and to the rhenium carbonyl dimer Re(2)(mu(2)-Cl)(2)(CO)(8) as the rhenium-reduced byproduct. The reaction of the precursor complex, N(C(4)H(9))(4)[Re(2)(OOCCF(3))Cl(6)] (1), with the organometallic carboxylic acid (CO)(6)Co(2)HCCCOOH leads to the cluster of clusters compound Re(2)(OOCCCHCo(2)(CO)(6))(4)Cl(2) (2), which has the dimer structure of Re(2)(OOCR)(4)Cl(2). Cyclic voltammetric measurements show that Re(2)(OOCCCHCo(2)(CO)(6))(4)Cl(2) (2) has one reduction centered on the dirhenium core and a reduction centered on the cobalt atoms. DFT calculations have been used to rationalize the observed displacements of the voltammetric signals in Re(2)(OOCCCHCo(2)(CO)(6))(4)Cl(2) (2) compared to the parent ligand (CO)(6)Co(2)HCCCOOH and rhenium pivalate. PMID- 12377032 TI - Differing reactivities of (trimpsi)M(CO)(2)(NO) complexes [M = V, Nb, Ta; trimpsi = (t)BuSi(CH(2)PMe(2))(3)] with halogens and halogen sources. AB - Treatment of (trimpsi)V(CO)(2)(NO) (trimpsi = (t)BuSi(CH(2)PMe(2))(3)) with 1 equiv of PhICl(2) or C(2)Cl(6) or 2 equiv of AgCl affords (trimpsi)V(NO)Cl(2) (1) in moderate yields. Likewise, (trimpsi)V(NO)Br(2) (2) and (trimpsi)V(NO)I(2) (3) are formed by the reactions of (trimpsi)V(CO)(2)(NO) with Br(2) and I(2), respectively. The complexes (trimpsi)M(NO)I(2)(PMe(3)) (M = Nb, 4; Ta, 5) can be isolated in moderate to low yields when the (trimpsi)M(CO)(2)(NO) compounds are sequentially treated with 1 equiv of I(2) and excess PMe(3). The reaction of (trimpsi)V(CO)(2)(NO) with 2 equiv of ClNO forms 1 in low yield, but the reactions of (trimpsi)M(CO)(2)(NO) (M = Nb, Ta) with 1 equiv of ClNO generate (trimpsi)M(NO)(2)Cl (M = Nb, 6; Ta, 7). Complexes 6 and 7 are thermally unstable and decompose quickly at room temperature; consequently, they have been characterized solely by IR and (31)P[(1)H] NMR spectroscopies. All other new complexes have been fully characterized by standard methods, and the solid-state molecular structures of 1.3CH(2)Cl(2), 4.(3/4)CH(2)Cl(2), and 5.THF have been established by single-crystal X-ray diffraction analyses. A convenient method of generating Cl(15)NO has also been developed during the course of these investigations. PMID- 12377033 TI - Salen-type compounds of calcium and strontium. AB - Salen complexes of the heavy alkaline-earth metals, calcium and strontium, were prepared by the reaction of various salen(t-Bu)H(2) ligands with the metals in ethanol. Six new calcium and strontium compounds, [Ca(salen(t-Bu))(HOEt)(2)(thf)] (1), [Ca(salen(t-Bu))(HOEt)(2)] (2), [Ca(salpen(t-Bu))(HOEt)(3)] (3), [Ca(salophen(t-Bu))(HOEt)(thf)] (4), [Sr(salen(t-Bu))(HOEt)(3)] (5), and [Sr(salophen(t-Bu))(HOEt)(thf)(2)] (6), were formed in this way with the quatridentate Schiff-base ligands N,N'-bis(3,5-di-tert butylsalicylidene)ethylenediamine (salen(t-Bu)H(2)), N,N'-bis(3,5-di-tert butylsalicylidene)-1,3-propanediamine (salpen(t-Bu)H(2)), and N,N'-o phenylenebis(3,5-di-tert-butylsalicylideneimine (salophen(t-Bu)H(2)). Initially, ammonia solutions of the metals were combined with the salen(t-Bu)H(2) ligands, and in the reaction of strontium with salen(t-Bu)H(2), the unusual tetrametallic cluster [(OC(6)H(2)(t-Bu)(2)CHN(CH(2))(2)NH(2))Sr(mu(3)-salean(t-Bu)H(2))Sr(mu(3) OH)](2) (7) was produced (salean(t-Bu)H(4) = N,N'-bis(3,5-di-tert-butyl-2 hydroxybenzyl)ethylenediamine). In this compound, the imine bonds of the salen(t Bu)H(2) ligand were reduced to form the known ligands salean(t-Bu)H(4) and (HO)C(6)H(2)(t-Bu)(2)CHN(CH(2))(2)NH(2). Compounds 1, 5, 6, and 7 were structurally characterized by single-crystal X-ray diffraction. Crystal data for 1 (C(44)H(74)CaN(2)O(6)): triclinic space group P(-)1, a = 8.3730(10) A, b = 14.8010(10) A, c = 18.756(2) A, alpha = 72.551(10) degrees, beta = 81.795(10) degrees, gamma = 78.031(10) degrees, Z = 2. Crystal data for 5 (C(38)H(64)SrN(2)O(5)): monoclinic space group P2(1)/c, a = 23.634(3) A, b = 8.4660(10) A, c = 24.451(3) A, beta = 101.138(10) degrees, Z = 4. Crystal data for 6 (C(46)H(67)N(2)O(5)Sr): orthorhombic space group P2(1)2(1)2(1), a = 10.5590(2) A, b = 16.2070(3) A, c = 26.7620(6) A, Z = 4. Crystal data for 7 (C(98)H(156)N(8)O(8)Sr(4)): triclinic space group P(-)1, a = 14.667(1) A, b = 15.670(1) A, c = 18.594(2) A, alpha = 92.26(1) degrees, beta = 111.84(1) degrees, gamma = 117.12(1) degrees, Z = 4. PMID- 12377034 TI - Spontaneous reduction of a low-spin Fe(III) complex of a neutral pentadentate N(5) Schiff base ligand to the corresponding Fe(II) species in acetonitrile. AB - The iron complexes of a designed pentadentate Schiff base ligand N,N-bis(2 pyridylmethyl)amine-N-ethyl-2-pyridine-2-aldimine (SBPy(3)) have been synthesized. The low-spin mononuclear Fe(III) complex [(SBPy(3))Fe(DMF)](ClO(4))(3) (2), though stable in the solid state, is spontaneously reduced to the corresponding Fe(II) species [(SBPy(3))Fe(MeCN)](2+) in MeCN. Fe(II) complex [(SBPy(3))Fe(MeCN)](BF(4))(2) (3) has been isolated independently and characterized by crystallography. Electrochemical studies indicate that SBPy(3), like other pentadentate polypyridine ligands, stabilizes the Fe(II) center to a great extent (E(1/2) = 1.01 V vs SCE in MeCN). This fact is responsible for the ready reduction of 2. It is evident that such reactivity has brought complications in the syntheses of iron complexes of polypyridine ligands reported in previous accounts. Very low solubility of 2 in MeOH has allowed isolation of analytically pure 2 in the present work. Storage of dilute methanolic solution of 2 results in the formation of the mu-oxo Fe(III) dimer [(SBPy(3))FeOFe(SBPy(3))](ClO(4))(4) (5), the structure of which has also been determined. Fe(II) complex 3 reacts with CN(-) to afford cyanide adduct [(SBPy(3))Fe(CN)](BF(4)) (4) but does not exhibit any reactivity toward NO. The azomethine moiety (CH=N-py) of 2 is rapidly oxidized by H(2)O(2) to a pyridine-2 carboxamido (C(=O)-N-py) unit and affords [(PaPy(3))Fe(MeCN)](ClO(4))(2) (1), a complex previously reported by us. PMID- 12377035 TI - Nitric oxide photorelease from ruthenium salen complexes in aqueous and organic solutions. AB - The complexes [Ru(salen)(NO)Cl] and [Ru(salen)(NO)(H(2)O)](+) were shown to release the nitrosyl ligand as nitric oxide upon exposure to visible light in organic and aqueous solutions respectively, by means of UV-visible, EPR, and FTIR spectroscopies. The former was prepared by a new synthetic route and had its structure determined by single-crystal X-ray diffraction. A crystal of the dichloromethane solvate is orthorhombic, space group Fdd2 (No. 43) and formula C(16)H(14)ClN(3)O(3)Ru.CH(2)Cl(2), with Z = 16 and cell parameters a = 25.489(4), b = 33.435(4), and c = 9.3716(9) A. The electronic absorption spectra of the complexes were calculated using the INDO/S method. The water-soluble complex is a potential drug for antitumoral phototreatment. PMID- 12377036 TI - Kinetics and mechanism of the formation of nitroprusside from aquapentacyanoferrate(III) and NO: complex formation controlled by outer-sphere electron transfer. AB - The kinetics and mechanism of the reaction between nitric oxide and aquapentacyanoferrate(III) were studied in detail. Pentacyanonitrosylferrate (nitroprusside, NP) was produced quantitatively in a pseudo-first-order process. The complex-formation rate constant was found to be 0.252 +/- 0.004 M(-1) s(-1) at 25.5 degrees C, pH 3.0 (HClO(4)), and I = 0.1 M (NaClO(4)), for which the activation parameters are DeltaH++ = 52 +/- 1 kJ mol(-1), DeltaS++ = -82 +/- 4 J K(-1) mol(-1), and DeltaV++ = -13.9 + 0.5 cm(3) mol(-1). These data disagree with earlier studies on complex-formation reactions of aquapentacyanoferrate(III), for which a dissociative interchange (I(d)) mechanism was suggested. The aquapentacyanoferrate(II) ion was detected as a reactive intermediate in the reaction of aquapentacyanoferrate(III) with NO, by using pyrazine and thiocyanate as scavengers for this intermediate. In addition, the reactions of other [Fe(III)(CN)(5)L](n-) complexes (L = NCS(-), py, NO(2)(-), and CN(-)) with NO were studied. These experiments also pointed to the formation of Fe(II) species as intermediates. It is proposed that aquapentacyanoferrate(III) is reduced by NO to the corresponding Fe(II) complex through a rate-determining outer-sphere electron-transfer reaction controlling the overall processes. The Fe(II) complex rapidly reacts with nitrite producing [Fe(II)(CN)(5)NO(2)](4)(-), followed by the fast and irreversible conversion to NP. PMID- 12377037 TI - Synthesis, purification, and structural characterization of the dimethyldiselenoarsinate anion. AB - A novel arsenic-selenium solution species was synthesized by reacting equimolar sodium selenite and sodium dimethylarsinate with 10 mol equiv of glutathione (pH 7.5) in aqueous solution. The solution species showed a single (77)Se NMR resonance at 112.8 ppm. Size-exclusion chromatography (SEC) using an inductively coupled plasma atomic emission spectrometer (ICP-AES) as the simultaneous arsenic , selenium-, sulfur-, and carbon-specific detector revealed an arsenic-selenium moiety with an As:Se molar ratio of 1:2. Electrospray ionization mass spectrometry (ESI-MS) of the chromatographically purified compound showed a molecular mass peak at m/z 263 in the negative ion mode. Fragmentation of the parent ion (ESI-MS-MS) produced (CH(3))(2)As(-) and Se(2)(-) fragments. Arsenic and selenium extended X-ray absorption fine structure spectroscopy (EXAFS) of the purified species revealed two As-C interactions at 1.943 A and two As-Se interactions at 2.279 A. On the basis of these results this novel solution species is identified as the dimethyldiselenoarsinate anion. PMID- 12377038 TI - Iron(III) and iron(IV) corroles: synthesis, spectroscopy, structures, and no indications for corrole radicals. AB - A delicate control of reaction conditions allows the isolation of several distinctively different iron complexes of tris(pentafluorophenyl)- and tris(2,6 dichlorophenyl)corrole. As long as coordinating ligands are present, the iron(III) complexes are stable in solution. Otherwise they are aerobically oxidized to either mononuclear chloroiron(IV) or dinuclear (mu-oxo)iron(IV) complexes, in acidic and basic solutions, respectively (the latter holds only for tris(pentafluorophenyl)corrole). When treated with NaNO(2), the mononuclear chloroiron(IV) corroles are efficiently converted into diamagnetic iron nitrosyl complexes. The low- and intermediate-spin iron(III), iron nitrosyl, and chloroiron(IV) corroles were fully characterized by a combination of spectroscopic methods and X-ray crystallography. There was no indication for an open-shell corrole in any of the complexes. PMID- 12377039 TI - Copper-bispidine coordination chemistry: syntheses, structures, solution properties, and oxygenation reactivity. AB - Copper(I) and copper(II) complexes of two mononucleating and four dinucleating tetradentate ligands with a bispidine backbone (2,4-substituted (2-pyridyl or 4 methyl-2-pyridyl) 3,7-diazabicyclo[3.3.1]nonanone) have been prepared and analyzed structurally, spectroscopically, and electrochemically. The structures of the copper chromophores are square pyramidal, except for two copper(I) compounds which are four-coordinate with one noncoordinated pyridine. The other copper(I) structures have the two pyridine donors, the co-ligand (NCCH(3)), and one of the tertiary amines (N3) in-plane with the copper center and the other amine (N7) coordinated axially (Cu-N3 > Cu-N7, approximately 2.25 A vs 2.20 A). The copper(II) compounds with pyridine donors have a similar structure, but the axial amine has a weaker bond to the copper(II) center (Cu-N3 < Cu-N7, approximately 2.03 A vs 2.30 A). The structures with methylated pyridine donors are also square pyramidal with the co-ligands (Cl(-) or NCCH(3)) in-plane. With NCCH(3) the same structural type as for the other copper(II) complexes is observed, and with the bulkier Cl(-) the co-ligand is trans to N7, leading to a square pyramidal structure with the pyridine donors rotated out of the basal plane and only a small difference between axial and in-plane amines (2.15, 2.12 A). These structural differences, enforced by the rigid bispidine backbone, lead to large variations in spectroscopic and electrochemical properties and reactivities. Oxygenation of the copper(I) complexes with pyridine-substituted bispidine ligands leads to relatively stable mu-peroxo-dicopper(II) complexes; with a preorganization of the dicopper chromophores, by linking the two donor sets, these peroxo compounds are stable at room temperature for up to 1 h. The stabilization of the peroxo complexes is to a large extent attributed to the square pyramidal coordination geometry with the substrate bound in the basal plane, a structural motif enforced by the rigid bispidine backbone. The stabilities and structural properties are also seen to correlate with the spectroscopic (UV-vis and Raman) and electrochemical properties. PMID- 12377040 TI - Precursors to water-soluble dinitrogen carriers. Synthesis of water-soluble complexes of iron(II) containing water-soluble chelating phosphine ligands of the type 1,2-bis(bis(hydroxyalkyl)phosphino)ethane. AB - The reactions of the water-soluble chelating phosphines 1,2 bis(bis(hydroxyalkyl)phosphino)ethane (alkyl = n-propyl, DHPrPE; n-butyl, DHBuPE; n-pentyl, DHPePE) with FeCl(2).4H(2)O and FeSO(4).7H(2)O were studied as routes to water-soluble complexes that will bind small molecules, dinitrogen in particular. The products that form and their stereochemistry depend on the solvent, the counteranion, and the alkyl chain length on the phosphine. In alcoholic solvents, the reaction of FeCl(2).4H(2)O with 2 equiv of DHBuPE or DHPePE gave trans-Fe(L(2))(2)Cl(2). The analogous reactions in water with DHBuPE and DHPePE gave only cis products, and the reaction of FeSO(4).7H(2)O with any of the phosphines gave only cis-Fe(L(2))(2)SO(4). These results are interpreted as follows. The trans stereochemistry of the products from the reactions of FeCl(2).4H(2)O in alcohols is suggested to be the consequence of the trans geometry of the Fe(H(2)O)(4)Cl(2) complex, i.e., substitution of the water molecules by the phosphines retains the geometry of the starting material. The formation of cis-Fe(DHPrPE)(2)Cl(2) is an exception to this result because the coordination of two -OH groups forms two six-membered rings, as shown in the X ray structure of the molecule. DHBuPE and DHPePE reacted with FeSO(4).7H(2)O in water to initially yield cis-Fe(P(2))(2)SO(4) compounds, but subsequent substitution reactions occurred over several hours to give sequentially trans Fe(DHBuPE)(2)(H(2)O)(SO(4)) and then trans-[Fe(DHBuPE)(2)(H(2)O)(2)]SO(4). The rate constants and activation reactions for these aquation reactions were determined and are consistent with dissociatively activated mechanisms. The cis- and trans-Fe(L(2))(2)X (X = (Cl)(2) or SO(4)) complexes react with N(2), CO, and CH(3)CN to yield trans complexes with bound N(2), CO, or CH(3)CN. The crystal structures of the cis-Fe(DHPrPE)(2)SO(4), trans-Fe(DHPrPE)(2)(CO)SO(4), trans Fe(DHBuPE)(2)Cl(2), trans-[Fe(DHBuPE)(2)(CO)(Cl)][B(C(6)H(5))(4)], trans Fe(DMeOPrPE)(2)Cl(2), trans-Fe(DMeOPrPE)(2)Br(2), and trans [Fe(DHBuPE)(2)Cl(2)]Cl complexes are reported. As expected from using water soluble phosphines, the complexes reported herein are water soluble (generally greater than 0.5 M at 23 degrees C). PMID- 12377041 TI - Stabilization of molecular LiF and LiFHF inside metallamacrocyclic hosts. AB - Complexes of molecular LiF and LiFHF were synthesized using the metallamacrocyclic receptors [(cymene)Ru(C(5)H(3)NO(2))](3) (1), [CpRh(C(5)H(3)NO(2))](3) (2), and [CpIr(C(5)H(3)NO(2))](3) (3). LiBF(4) complexes of 1-3 were prepared and subsequently treated with F(-) or FHF(-) to give the desired products in an anion-exchange reaction. All complexes were characterized by multinuclear NMR spectroscopy ((1)H, (13)C, (19)F, (7)Li). Strong scalar coupling between (7)Li and (19)F is observed for the LiF and the LiFHF complexes ((1)J(LiF) = 91-103 Hz). The LiFHF adduct of 1 displays fluxional behavior with fast exchange of the two fluorine atoms. The structures of the complexes 1.LiBF(4), 2.LiBF(4), 1.LiF, 2.LiF, 1.LiFHF, and 3.LiFHF were determined by single-crystal X-ray analysis. Li-F bond lengths between 1.77 and 1.81 A were found. The LiFHF complexes show a hydrogen difluoride anion coordinated in a bent fashion via one fluorine atom to the lithium ion. PMID- 12377042 TI - Corynebactin and a serine trilactone based analogue: chirality and molecular modeling of ferric complexes. AB - Because the hydrolysis of ferric ion makes it very insoluble in aerobic, near neutral pH environments, most species of bacteria produce siderophores to acquire iron, an essential nutrient. The chirality of the ferric siderophore complex plays an important role in cell recognition, uptake, and utilization. Corynebactin, isolated from Gram-positive bacteria, is structurally similar to enterobactin, a well-known siderophore first isolated from Gram-negative bacteria, but contains L-threonine instead of L-serine in the trilactone backbone. Corynebactin also contains a glycine spacer unit in each of the chelating arms. A hybrid analogue (serine-corynebactin) has been prepared which has the trilactone ring of enterobactin and the glycine spacer of corynebactin. The chirality and relative conformational stability of the three ferric complexes of enterobactin, corynebactin, and the hybrid have been investigated by molecular modeling (including MM3 and pBP86/DN density functional theory calculations) and circular dichroism spectra. While enterobactin forms a Delta-ferric complex, corynebactin is Lambda. The hybrid serine-corynebactin forms a nearly racemic mixture, with the Lambda-conformer in slight excess. Each ferric complex has four possible isomers depending on the metal chirality and the conformation of the trilactone ring. For corynebactin, the energy difference between the two possible Lambda conformations is 2.3 kcal/mol. In contrast, only 1.5 kcal/mol separates the inverted Lambda- and normal Delta-configuration for serine-corynebactin. The small energy difference of the two lowest energy configurations is the likely cause for the racemic mixture found in the CD spectra. Both the addition of a glycine spacer and methylation of the trilactone ring (serine to threonine) favor the Lambda-conformation. These structural changes suffice to change the chirality from all Delta (enterobactin) to all Lambda (corynebactin). The single change (glycine spacer) of the hybrid ferric serine-corynebactin gives a mixture of Delta and Lambda, with the Lambda in slight excess. PMID- 12377043 TI - Gd(1.33)Pt(3)(Al,Si)(8) and Gd(0.67)Pt(2)(Al,Si)(5): two structures containing a disordered Gd/Al layer grown in liquid aluminum. AB - Gd(1.33)Pt(3)Al(8) was synthesized by the combination of Gd and Pt in excess liquid aluminum. Addition of silicon resulted in the incorporation of a small amount of this element into the material to form the isostructural Gd(1.33)Pt(3)Al(7)Si. Both compounds grow as rodlike crystals with hexagonal cross section. The structures were refined in the rhombohedral space group R( )3m, with cell parameters a = 4.3359(6) A and c = 38.702(8) A for the ternary and a = 4.3280(8) A and c = 38.62(1) A for the quaternary compound. The structure is comprised of stuffed arsenic-like PtAl(2) layers and disordered Gd/Al layers. Analysis of the hk0 zone reflections indicate the presence of an a = radical 3a supercell, but the structure is not ordered along c, as revealed by the highly diffuse reflections in the 0kl zone photos. Therefore, the compounds are disordered variants of the Gd(4)Pt(9)Al(24) type. Magnetic susceptibility studies reveal antiferromagnetic transitions at 15 K for the ternary and 7 K for the quaternary compound. Variation of the reactant ratio produces a different structure comprised of the same structural blocks, including the disordered Gd/Al layer. Gd(0.67)Pt(2)Al(5) and its quaternary analogue Gd(0.67)Pt(2)Al(4)Si form in the hexagonal system P6(3)/mmc with cell parameters a = 4.2907(3) A and c = 16.388(2) A for the ternary and a = 4.2485(6) A and c = 16.156(3) A for the quaternary compound. PMID- 12377044 TI - Substitution reactions of [Fe(4)S(4)Cl(4)](2-) with Bu(t)NC or Et(2)NCS(2)(-): trapping intermediates and detecting new pathways. AB - Kinetic studies on the substitution reaction between [Fe(4)S(4)Cl(4)](2-) and Bu(t)NC or Et(2)NCS(2)(-) are reported. The binding of small molecules and ions to Fe-S clusters is a fundamental step in substitution reactions but can be difficult to follow directly because these reactions are rapid and often associated with small spectroscopic changes. A novel kinetic method is reported which allows the time course of molecule and ion binding to Fe-S clusters to be followed by monitoring the lability of the cluster. Using a stopped-flow, sequential-mix apparatus, [Fe(4)S(4)Cl(4)](2-) and L (L = Et(2)NCS(2)(-) or Bu(t)NC) are rapidly mixed, and after a known time (delta) the resulting solution is mixed with a solution of PhS(-). The thiolate substitutes for the chloro ligands on the cluster, in a reaction which is easy to follow because of the large change in the visible absorption spectrum. The rate of this substitution is extremely sensitive to whether L is bound to the cluster or not. By correlation of delta with the rate of the reaction with PhS(-), the time course of the reaction between [Fe(4)S(4)Cl(4)](2-) and L can be mapped out. In studies where L = Bu(t)NC this technique has allowed the detection of an intermediate ([Fe(4)S(4)Cl(4)(CNBu(t))](2-)) which cannot be detected spectrophotometrically. In further studies, the substitution reactions of [Fe(4)S(4)Cl(4)](2-) with PhS( ), Et(2)NCS(2)(-), or Bu(t)NC are all perturbed by the addition of Cl(-). In all cases a common pathway for substitution is evident, but with Et(2)NCS(2)(-) an additional, slower pathway becomes apparent under conditions where the common pathway is completely inhibited by Cl(-). PMID- 12377045 TI - Expanding the remarkable structural diversity of uranyl tellurites: hydrothermal preparation and structures of K[UO(2)Te(2)O(5)(OH)], Tl(3)[(UO(2))(2)[Te(2)O(5)(OH)](Te(2)O(6))].2H(2)O, beta-Tl(2)[UO(2)(TeO(3))(2)], and Sr(3)[UO(2)(TeO(3))(2)](TeO(3))(2). AB - The reactions of UO(2)(C(2)H(3)O(2))(2).2H(2)O with K(2)TeO(3).H(2)O, Na(2)TeO(3) and TlCl, or Na(2)TeO(3) and Sr(OH)(2).8H(2)O under mild hydrothermal conditions yield K[UO(2)Te(2)O(5)(OH)] (1), Tl(3)[(UO(2))(2)[Te(2)O(5)(OH)](Te(2)O(6))].2H(2)O (2) and beta Tl(2)[UO(2)(TeO(3))(2)] (3), or Sr(3)[UO(2)(TeO(3))(2)](TeO(3))(2) (4), respectively. The structure of 1 consists of tetragonal bipyramidal U(VI) centers that are bound by terminal oxo groups and tellurite anions. These UO(6) units span between one-dimensional chains of corner-sharing, square pyramidal TeO(4) polyhedra to create two-dimensional layers. Alternating corner-shared oxygen atoms in the tellurium oxide chains are protonated to create short/long bonding patterns. The one-dimensional chains of corner-sharing TeO(4) units found in 1 are also present in 2. However, in 2 there are two distinct chains present, one where alternating corner-shared oxygen atoms are protonated, and one where the chains are unprotonated. The uranyl moieties in 2 are bound by five oxygen atoms from the tellurite chains to create seven-coordinate pentagonal bipyramidal U(VI). The structures of 3 and 4 both contain one-dimensional [UO(2)(TeO(3))(2)](2-) chains constructed from tetragonal bipyramidal U(VI) centers that are bridged by tellurite anions. The chains differ between 3 and 4 in that all of the pyramidal tellurite anions in 3 have the same orientation, whereas the tellurite anions in 4 have opposite orientations on each side of the chain. In 4, there are also additional isolated TeO(3)(2-) anions present. Crystallographic data: 1, orthorhombic, space group Cmcm, a = 7.9993(5) A, b = 8.7416(6) A, c = 11.4413(8) A, Z = 4; 2, orthorhombic, space group Pbam, a = 10.0623(8) A, b = 23.024(2) A, c = 7.9389(6) A, Z = 4; 3, monoclinic, space group P2(1)/n, a = 5.4766(4) A, b = 8.2348(6) A, c = 20.849(3) A, beta = 92.329(1) degrees, Z = 4; 4, monoclinic, space group C2/c, a = 20.546(1) A, b = 5.6571(3) A, c = 13.0979(8) A, beta = 94.416(1) degrees, Z = 4. PMID- 12377046 TI - HostDesigner: a program for the de novo structure-based design of molecular receptors with binding sites that complement metal ion guests. AB - This paper describes a novel approach to the discovery of host structures with binding sites that complement targeted metal ion guests. This approach uses a de novo structure-based design strategy that couples molecular building algorithms with scoring functions to prioritize candidate structures. The algorithms described herein have been implemented in a program called HostDesigner, the first structure-based design software specifically created for the discovery of metal ion receptors. HostDesigner generates and evaluates millions of candidate structures within minutes, rapidly identifying three-dimensional architectures that position binding sites to provide an optimal interaction with the metal ion. PMID- 12377047 TI - Structural and spectroscopic features of a cis (hydroxo)-Fe(III)-(carboxylato) configuration as an active site model for lipoxygenases. AB - In our preliminary communication (Ogo, S.; Wada, S.; Watanabe, Y.; Iwase, M.; Wada, A.; Harata, M.; Jitsukawa, K.; Masuda, H.; Einaga, H. Angew. Chem., Int. Ed. 1998, 37, 2102-2104), we reported the first example of X-ray analysis of a mononuclear six-coordinate (hydroxo)iron(III) non-heme complex, [Fe(III)(tnpa)(OH)(RCO(2))]ClO(4) [tnpa = tris(6-neopentylamino-2 pyridylmethyl)amine; for 1, R = C(6)H(5)], which has a characteristic cis (hydroxo)-Fe(III)-(carboxylato) configuration that models the cis (hydroxo) Fe(III)-(carboxylato) moiety of the proposed (hydroxo)iron(III) species of lipoxygenases. In this full account, we report structural and spectroscopic characterization of the cis (hydroxo)-Fe(III)-(carboxylato) configuration by extending the model complexes from 1 to [Fe(III)(tnpa)(OH)(RCO(2))]ClO(4) (2, R = CH(3); 3, R = H) whose cis (hydroxo)-Fe(III)-(carboxylato) moieties are isotopically labeled by (18)OH(-), (16)OD(-), (18)OD(-), (12)CH(3)(12)C(18)O(2)( ), (12)CH(3)(13)C(16)O(2)(-), (13)CH(3)(12)C(16)O(2)(-), (13)CH(3)(13)C(16)O(2)( ), and H(13)C(16)O(2)(-). Complexes 1-3 are characterized by X-ray analysis, IR, EPR, and UV-vis spectroscopy, and electrospray ionization mass spectrometry (ESI MS). PMID- 12377048 TI - Ba(SbF(6))(2).5XeF(2): first xenon(II) compound with barium. Synthesis, vibrational spectra, and crystal structure. AB - The reaction between Ba(SbF(6))(2) and excess XeF(2) in anhydrous HF at room temperature yields the white solid Ba(SbF(6))(2).5XeF(2) after the excess XeF(2) and the solvent have been removed under vacuum. Ba(SbF(6))(2).5XeF(2) crystallizes in the monoclinic space group C2/m, with a = 13.599(6) A, b = 12.086(4) A, c = 9.732(5) A, beta = 134.305(6) degrees, V = 1144.7 (8) A(3), and Z = 2. The coordination sphere of each barium atom consists of 12 fluorine atoms. The structure consists of alternating layers of Ba(SbF(6))(2).XeF(2) and 4 XeF(2) molecules. The Ba atoms in the Ba(SbF(6))(2).XeF(2) layer are in a nearly rhombic net array and are linked with trans F-bridging ligands of SbF(6)(-). A XeF(2) molecule is placed in the center of each rhombus of the Ba(2+) array so that its symmetry axis is perpendicular to the plane of the Ba(SbF(6))(2).XeF(2) layer. This layer is linked to its neighbors by a layer of centrosymmetric XeF(2) molecules. Raman spectra are in accord with all XeF(2) molecules being symmetrical. PMID- 12377049 TI - Reactions of thioethers with Mn(2)(CO)(7)(mu-S(2)) proceed with CO displacement and insertion of the sulfur atom into the Mn-Mn bond. AB - The reaction of Mn(2)(CO)(7)(mu-S(2)) (2) with SMe(2) yielded the new complexes Mn(2)(CO)(6)(mu-S(2))(mu-SMe(2)) (3) and Mn(4)(CO)(14)(SMe(2))(mu(3)-S(2))(mu(4) S(2)) (4) in 18 and 41% yields, respectively. The reaction of 2 with the cyclic thioether thietane SCH(2)CH(2)CH(2) yielded the new complexes Mn(2)(CO)(6)(mu S(2))(mu-SCH(2)CH(2)CH(2)) (5) and Mn(4)(CO)(14)(SCH(2)CH(2)CH(2))(mu(3) S(2))(mu(4)-S(2)) (6) in 12 and 52% yields, respectively, and the reaction of 2 with 1,4,9-trithiacyclododecane (12S3) yielded Mn(2)(CO)(6)(mu-12S3)(mu-S(2)) (7) and Mn(4)(CO)(14)(12S3)(mu(3)-S(2))(mu(4)-S(2)) (8) in 8 and 24% yields, respectively. Compounds 3 and 5-7 were characterized crystallographically. Compounds 3, 5, and 7 have similar structures in which the thioether ligand has replaced the bridging carbonyl ligand of 2 and its sulfur atom has been inserted into the manganese-manganese bond. The two manganese atoms are not mutually bonded, and two Mn(CO)(3) groups are held together through the bridging disulfido ligand and the bridging sulfur atom of the thioether ligand. Compound 6 contains a Mn(4)(mu(3)-S(2))(mu(4)-S(2)) moiety without metal-metal bonds. On the basis of spectroscopic data, compounds 4 and 8 are believed to have similar structures. PMID- 12377050 TI - Hydro/solvothermal synthesis and structures of new zinc phosphates of varying dimensionality. AB - Hydro/solvothermal reactions of ZnO, HCl, H(3)PO(4), 1,4-diazacycleheptane (homopiperazine), and H(2)O under a variety of conditions yielded three new organic-inorganic hybrid materials, [C(5)N(2)H(14)][Zn(HPO(4))(2)].xH(2)O (x = approximately 0.46), I, [C(5)N(2)H(14)][Zn(3)(H(2)O)(PO(4))(2)(HPO(4))], II, and [C(5)N(2)H(14)][Zn(2)(HPO(4))(3)].H(2)O, III. While I has a one-dimensional structure, II possesses a two-dimensional layered structure, and III has a three dimensional structure closely related to the ABW zeolitic architecture. All the compounds consist of vertex linking of ZnO(4), PO(4), and HPO(4) tetrahedral units. The fundamental building unit, single four-membered ring (S4R), is present in all the cases, and the observed differences in their structures result from variations in the connectivity between the S4R units. Thus I has a corner-shared S4R forming an infinite one-dimensional chain, II has two corner-shared chains fused through a 3-coordinated oxygen atom forming a strip and a layer with eight membered apertures, and III has S4R units connected via oxygen atoms to give rise to channels bound by eight T atoms (T = Zn, P) in all crystallographic directions. Crystal data: I, monoclinic, space group = P2(1)/n (No. 14), a = 8.6053(3) A, b = 13.7129(5) A, c = 10.8184(4) A, beta = 97.946(1) degrees, V = 1264.35(8) A(3), Z = 4; II, monoclinic, space group = P2(1)/c (No. 14), a = 11.1029(1) A, b = 17.5531(4) A, c = 8.2651(2) A, beta = 97.922(2) degrees, V = 1595.42(5) A(3), Z = 4; III, monoclinic, space group = P2(1) (No. 4), a = 8.0310(2) A, b = 10.2475(3) A, c = 10.570(3) A, beta = 109.651(1) degrees, V = 819.24(3) A(3), Z = 2. PMID- 12377051 TI - Reduction of octacyanomolybdate(V) by thioglycolic acid in aqueous media. AB - In aqueous media at 25 degrees C [Mo(CN)(8)](3-) is reduced by thioglycolic acid (HSCH(2)COOH, TGA), and the reaction is strongly accelerated by the presence of trace amounts of copper ions. Dipicolinic acid (dipic) is an effective inhibitor of the copper catalysis. Both with and without dipic the reaction has the stoichiometry 2[Mo(CN)(8)](3-) + 2TGA --> 2[Mo(CN)(8)](4-) + RSSR, where RSSR is the disulfide derived from formal oxidative dimerization of TGA. In the presence of dipic, PBN (N-tert-butyl-alpha-phenyl-nitrone), and with a large excess of TGA the rate law for consumption of [Mo(CN)(8)](3-) is first order in both [TGA] and [Mo(CN)(8)(3-)]. The complex pH dependence is consistent with (-)SCH(2)CO(2)(-) being highly reactive (k = 1.8 x 10(4) M(-1) s(-1)), the monoanion being less reactive, and HSCH(2)CO(2)H being unreactive. A mechanism is proposed in which the dianion undergoes electron transfer to [Mo(CN)(8)](3-), thus generating the thiyl radical. Analysis of the electron-transfer rate constant in terms of Marcus theory yields an effective self-exchange rate constant for the thiolate/thiyl redox couple that is in reasonable agreement with the value derived previously from the reaction of TGA with [IrCl(6)](2-). When copper catalysis is inhibited, the two reactions differ substantially in that the yield of (-)O(3)SCH(2)CO(2)(-) is significant for [IrCl(6)](2-) but undetectable for [Mo(CN)(8)](3-). PMID- 12377052 TI - Preparation of highly efficient manganese catalase mimics. AB - The series of compounds [Mn(bpia)(mu-OAc)](2)(ClO(4))(2) (1), [Mn(2)(bpia)(2)(muO)(mu-OAc)](ClO(4))(3).CH(3)CN (2), [Mn(bpia)(mu O)](2)(ClO(4))(2)(PF(6)).2CH(3)CN (3), [Mn(bpia)(Cl)(2)](ClO)(4) (4), and [(Mn(bpia)(Cl))(2)(mu-O)](ClO(4))(2).2CH(3)CN (5) (bpia = bis(picolyl)(N methylimidazol-2-yl)amine) represents a structural, spectroscopic, and functional model system for manganese catalases. Compounds 3 and 5 have been synthesized from 2 via bulk electrolysis and ligand exchange, respectively. All complexes have been structurally characterized by X-ray crystallography and by UV-vis and EPR spectroscopies. The different bridging ligands including the rare mono-mu-oxo and mono-mu-oxo-mono-mu-carboxylato motifs lead to a variation of the Mn-Mn separation across the four binuclear compounds of 1.50 A (Mn(2)(II,II) = 4.128 A, Mn(2)(III,III) = 3.5326 and 3.2533 A, Mn(2)(III,IV) = 2.624 A). Complexes 1, 2, and 3 are mimics for the Mn(2)(II,II), the Mn(2)(III,III), and the Mn(2)(III,IV) oxidation states of the native enzyme. UV-vis spectra of these compounds show similarities to those of the corresponding oxidation states of manganese catalase from Thermus thermophilus and Lactobacillus plantarum. Compound 2 exhibits a rare example of a Jahn-Teller compression. While complexes 1 and 3 are efficient catalysts for the disproportionation of hydrogen peroxide and contain an N(4)O(2) donor set, 4 and 5 show no catalase activity. These complexes have an N(4)Cl(2) and N(4)OCl donor set, respectively, and serve as mimics for halide inhibited manganese catalases. Cyclovoltammetric data show that the substitution of oxygen donor atoms with chloride causes a shift of redox potentials to more positive values. To our knowledge, complex 1 is the most efficient binuclear functional manganese catalase mimic exhibiting saturation kinetics to date. PMID- 12377053 TI - Oxovanadium(V) and cobalt(III) complexes of dithiocarbazate-based Schiff base ligands: formation of a thiadiazole ring by vanadium-induced cyclization of the coordinated ligand. AB - S-Methyl 3-((2-hydroxyphenyl)methyl)dithiocarbazate (H(2)L(1)) and its bromo derivative (H(2)L(2)), which are traditionally biprotic tridentate (ONS) ligands, behave in an unprecedented manner when allowed to react with [VO(acac)(2)] under an oxidative environment in acetonitrile-water medium containing a catalytic amount of alkali metal ion. The products obtained are oxovanadium(V) compounds [VOL(L(cyclic))] (L = L(1), 1a, and L(2), 1b) that contain one molecule of ligand which undergoes metal-induced cyclization to form a thiadiazole ring. Compound 1a crystallizes in the triclinic space group P(-)1 with a = 9.1830(9) A, b = 9.4165(12) A, c = 12.700(2) A, alpha = 100.988(8)(o), beta = 100.195(7)(o), gamma = 78.774(8)(o), V = 1046.3(2) A(3), and Z = 2. With cobalt(III), however, the products [CoL(HL)].H(2)O (L = L(1), 2a, and L(2), 2b) have hydrogen-bonded dimeric structures with each ligand virtually carrying 1.5 units of negative charge as confirmed by X-ray crystal structure analysis of 2a. It also crystallizes in triclinic space group P(-)1 with a = 12.0842(8) A, b = 13.5251(9) A, c = 14.1960(10) A, alpha = 78.122(6)(o), beta = 73.888(6)(o), gamma = 78.255(6)(o), V = 2154.7(3) A(3), and Z = 4. In solution, 2a is a symmetric molecule as indicated by (1)H NMR, involving a characteristic hydrogen-bonded O-H O broad feature in the downfield (at 14.5 ppm) connecting both monoprotonated (LH(-)) and deprotonated (L(2-)) forms of the ligand--a situation somewhat analogous to the classic H-F-H case as observed in bifluoride ion. PMID- 12377054 TI - A cis-IrL(CO) group responds to increasing steric bulk of L by M-L stretching, not M-C-O tilting or bending. AB - The crystal structures of [Ir(2-R-bq)(PPh(3))(2)(H)(CO)](+) (bq = benzoquinolinato; R = H, i-Pr, t-Bu) show that steric interference caused by contact between the bulky pendant R groups of the bq and the C of the cis-CO is relieved by Ir-N bond stretching in the Irbq system and bending of the trans Ph(3)P-Ir-PPh(3) groups, rather than by tilting or bending of the CO. Ir-CO is therefore more rigid than the Ir-N and Ir-P bonds. The Ir-N stretching is aided by the presence of a high trans effect H trans to N. PMID- 12377055 TI - Synthesis, structures, and magnetic and optical properties of a series of europium(III) and gadolinium(III) complexes with chelating nitronyl and imino nitroxide free radicals. AB - This paper reports the synthesis, structures, and magnetic and optical properties of a series of gadolinium(III) (1a-4a) and europium(III) (1b-4b) complexes with nitronyl or imino nitroxide radicals. The crystal structures of compounds 1a and 1b consist of [Ln(III)(radical)(2)(NO(3))(3)] entities in which the gadolinium(III) (1a) or europium(III) ion (1b) is 10-coordinated to two nitronyl nitroxide radicals and three nitrato ligands. The crystal structures of compounds 2a-4a and 2b-4b consist of [Ln(III)(hfac)(3)(radical)] entities in which the gadolinium(III) (2a-4a) or europium(III) ion (2b-4b) is 8-coordinated to one nitronyl (2a and 2b) or one imino (3a, 4a and 3b, 4b) nitroxide radical and three hexafluoroacetylacetonato ligands. The gadolinium(III) complexes (1a-4a) are isostructural with their europium(III) analogues (1b-4b). The magnetic properties of the gadolinium complexes were studied. Along the series 1a-4a only compound 2a exhibits a ferromagnetic Gd(III)-radical coupling (J(Gd-rad) = +1.7 cm(-1)), while for the others this coupling is antiferromagnetic (1a: J(Gd-rad1) = -4.05 cm(-1) and J(Gd-rad2) = -0.80 cm(-1); 3a: J(Gd-rad) = -2.6 cm(-1); 4a: J(Gd-rad) = -1.9 cm(-1)). The first full luminescence spectra of lanthanide complexes with free radical ligands are reported between 650 and 1200 nm. The rich vibronic structure in luminescence and absorption spectra indicates that several excited states define the absorption spectra between 400 and 800 nm. Qualitative trends can be established between magnetic ground state properties and the energies and fine structure of the title compounds. PMID- 12377056 TI - Effect of metal-ligand bond lengths on superexchange interactions in Jahn-Teller d(4) ion systems: spin dimer analysis of the magnetic structure of marokite CaMn(2)O(4). AB - In marokite CaMn(2)O(4), all six Mn-O bonds of each MnO(6) octahedron are different because of the Jahn-Teller distortion so that every Mn(3+) (d(4)) ion has six different superexchange interactions with its neighboring Mn(3+) ions. The spin exchange interactions of CaMn(2)O(4) were examined on the basis of spin dimer analysis to find what geometrical parameters of the Mn-O-Mn superexchange paths control the signs and strengths of their spin exchange interactions. Our work correctly describes the magnetic structure of CaMn(2)O(4) observed from neutron powder diffraction measurements and shows that the antiferromagnetic interactions of the Mn-O-Mn paths depend primarily on the asymmetry and the Mn-O bond length of the Mn-O-Mn bridge, but not on the 90 degree angle Mn-O-Mn bond angle. PMID- 12377057 TI - Paramagnetic cobalt(II) as a probe for kinetic and NMR relaxation studies of phosphate binding and the catalytic mechanism of Streptomyces dinuclear aminopeptidase. AB - Phosphate was proposed to be a bridging ligand in the structure 1xjo.pdb of Streptomyces dizinc aminopeptidase (sAP), which prompted further studies of phosphate binding to this enzyme. Phosphate inhibits sAP and its Co(2+) substituted derivatives in a noncompetitive manner from pH 6.0 to 9.0, with strongest inhibition observed at lower pHs (K(i) = 0.6, 8.2, and 9.1 mM for ZnZn , CoCo-, and CoZn-sAP, respectively, at pH 6.0), which indicates that phosphate does not compete with substrate binding to the dinuclear active site and that monobasic phosphate has a higher binding affinity. The inhibition K(i)-pH profiles for phosphate inhibition of both the native and the Co(2+)-substituted derivatives reveal a similar pK(a) around 7.0, reflecting that phosphate binding is not affected by the metal centers of different Lewis acidities. Modification of ZnZn- and CoCo-sAP with the arginine-specific reagent phenylglyoxal reveals a significant weakening in phosphate and substrate binding by showing approximately a 10-fold increase in the dissociation constant K(i) for phosphate binding and approximately 4-8-fold increase in K(m). The catalysis is also influenced by the modification as reflected by a significant decrease in k(cat) in both cases. Furthermore, phosphate and the transition-state inhibitor 1-aminobutyl phosphonate can protect arginine from the modification, strongly suggesting that Arg202 near the active site is involved in phosphate binding and in stabilizing the transition state. The effect on (31)P NMR relaxation of phosphate caused by the paramagnetic metal center in Co(2+)-substituted derivatives of sAP has been measured, which reveals that only one phosphate is bound to sAP with the Co(2+) (31)P distance in the range of 4.1-4.3 A. The (1)H NMR relaxation of the bulk water signal in the CoCo-sAP sample remains unchanged in the presence of phosphate, further indicating that phosphate may not bind to the active-site metals to displace any metal-bound water/hydroxide. These results strongly support that the phosphate binding site is Arg202 and that this residue plays an important role in the action of sAP. PMID- 12377058 TI - DFT investigation of the tri(amino)amine N(NH(2))(3)(2+) and the tri(azido)amine N(N(3))(3)(2+) dications and related mixed amino(azido)ammonium ions (N(3))(x)N(NH(2))(4-x)(+) (x = 0-4)(1). AB - Structures of the tri(amino)amine N(NH(2))(3)(2+) and the tri(azido)amine N(N(3))(3)(2+) dications were calculated at the density functional theory (DFT) B3LYP/6-311+G level. The tri(amino)amine dication (NH(2))(3)N(2+) (1) was found to be highly resonance stabilized with a high kinetic barrier for deprotonation. The structures of diamino(azido)amine dication (NH(2))(2)N(N(3))(2+) (2), amino(diazido)amine dication (NH(2))N(N(3))(2)(2+) (3), and tri(azido)amine dication (N(3))(3)N(2+) (4) were also found to be highly resonance stabilized. The structures and energetics of the related mixed amino(azido)ammonium ions (N(3))(x)N(NH(2))(4-x)(+) (x = 0-4) were also calculated. PMID- 12377059 TI - High nuclearity ruthenium-tin clusters from the reactions of triphenylstannane with pentaruthenium carbonyl carbido cluster complexes. AB - The reaction of Ru(5)(CO)(15)(mu(5)-C), 1, with Ph(3)SnH in the presence of UV irradiation has yielded the Ph(3)SnH adduct Ru(5)(CO)(15)(SnPh(3))(mu(5)-C)(mu H), 3, by SnH bond activation and cleavage of one Ru-Ru bond in the cluster of 1. The reaction of 1 with Ph(3)SnH at 127 degrees C yielded the high nuclearity cluster compound Ru(5)(CO)(10)(SnPh(3))(mu-SnPh(2))(4)(&mu(5)-C)(mu-H), 4, that contains five tin ligands. Four of these are SnPh(2) groups that bridge each edge of the base of the Ru(5) square pyramidal cluster. The reaction of Ph(3)SnH with the benzene-substituted cluster Ru(5)(CO)(12)(C(6)H(6))(mu(5)-C), 2, at 68 degrees C yielded two products: Ru(5)(CO)(11)(SnPh(3))(C(6)H(6))(mu(5)-C)(mu-H), 5, and Ru(5)(CO)(10)(SnPh(3))(2)(C(6)H(6))(mu(5)-C)(mu-H)(2), 6. Both contain square pyramidal Ru(5) clusters with one and two SnPh(3) groups, respectively. At 127 degrees C, the reaction of 2 with an excess of Ph(3)SnH has led to the formation of two new high-nuclearity cluster complexes: Ru(5)(CO)(8)(mu SnPh(2))(4)(C(6)H(6))(mu(5)-C), 7, and Ru(5)(CO)(7)(mu SnPh(2))(4)(SnPh(3))(C(6)H(6))(mu-H), 8. Both compounds contain square pyramidal Ru(5) clusters with SnPh(2) groups bridging each edge of the square base. Compound 8 contains a SnPh(3) group analogous to that of compound 4. When treated with CO, compound 8 is converted to 4. When heated to 68 degrees C, compound 5 was converted to the new compound Ru(5)(CO)(11)(C(6)H(6))(mu(4)-SnPh)(mu(3)-CPh), 9, by loss of benzene and the shift of a phenyl group from the tin ligand to the carbido carbon atom to form a triply bridging benzylidyne ligand and a novel quadruply bridging stannylyne ligand. PMID- 12377060 TI - A novel group of alkaline earth metal amides: syntheses and characterization of M[N(2,6-(i)Pr(2)C(6)H(3))(SiMe(3))](2)(THF)(2) (M = Mg, Ca, Sr, Ba) and the linear, two-coordinate Mg[N(2,6-(i)Pr(2)C(6)H(3))(SiMe(3))](2). AB - Novel alkaline earth metal aryl-substituted silylamides were prepared using alkane (Mg) and salt elimination reactions (Mg, Ca, Sr, and Ba). The salt elimination regime involved the treatment of the alkaline earth metal iodides with 2 equiv of the respective potassium amide KNDiip(SiMe(3)), (Diip = 2,6-i Pr(2)C(6)H(3)). The organomagnesium source for the alkane elimination was ((n)()Bu/(s)()Bu)(2)Mg. All compounds were characterized using (1)H, (13)C NMR, and IR spectroscopy, in addition to X-ray crystallography (except Mg[NDiip(SiMe(3))](2)THF(2)). Crystal data with Mo Kalpha (lambda = 0.710 73 A) are as follows: Mg[NDiip(SiMe(3))](2), 1, a = 9.4687(6) A, b = 9.6818(6) A, c = 17.9296(1) A, alpha = 96.487(1) degrees, beta = 94.537(1) degrees, gamma = 89.222(1) degrees, V = 1608.8(2) A(3), Z = 2 (two independent molecules), triclinic, space group P(-)1, R1 (all data) = 0.0508; (n)()BuMg[NDiip(SiMe(3))]THF(2), 2, a = 9.5413(1) A, b = 16.493(2) A, c = 9.8218(1) A, beta = 108.149(2) degrees, V = 1468.7(4) A(3), Z = 2, monoclinic, space group P2(1), R1(all data) = 0.1232; Ca[NDiip(SiMe(3))](2)THF(2), 4, a = 9.7074(1) A, b = 20.9466(4) A, c = 21.6242(3) A, alpha = 73.573(1) degrees, beta = 78.632(1) degrees, gamma = 89.621(1) degrees, V = 4129.1(1) A(3), Z = 4 (two independent molecules), triclinic, space group P(-)1, R1 (all data) = 0.0902; Sr[NDiip(SiMe(3))](2)THF(2), 5, a = 20.5874(5) A, b = 9.8785(2) A, c = 20.8522(5) A, beta = 102.035(2) degrees, V = 4147.6(2) A(3), Z = 4 (two independent molecules), monoclinic, space group P2/n, R1 (all data) = 0.0756; Ba[NDiip(SiMe(3))](2)THF(2), 6, a = 20.5476(2) A, b = 10.0353(2) A, c = 20.9020(4) A, beta = 101.657(1) degrees, V = 4221.0(1) A(3), Z = 4 (two independent molecules), monoclinic, space group P2/n, R1 (all data) = 0.0573. PMID- 12377061 TI - Carboxylate-bridged copper(II)-lanthanide(III) complexes [(Cu(3)Ln(2)(oda)(6)(H(2)O)(6)).12H(2)O](n)(Ln = Dy, Ho, Er, Y; oda = oxydiacetate). AB - The hydrothermal reaction of Ln(2)O(3) (Ln = Dy and Ho), Cu(OAc)(2).2H(2)O, and oxydiacetic acid in the approximate mole ratio of 1:3:8 resulted in the formation of two new members of the isostructural series of polymers formulated as [(Cu(3)Ln(2)(oda)(6)(H(2)O)(6)).12H(2)O](n), crystallizing in the hexagonal crystal system, space group P6/mcc (No. 192). Temperature-dependent magnetic susceptibilities and EPR spectra are reported for the heterometallic compounds Cu Dy 1, Cu-Ho 2, Cu-Er 3, and Cu-Y 4. The results are discussed in terms of the structure of the compounds, the electronic properties of the lanthanide ions, and the exchange interactions between the magnetic ions. PMID- 12377062 TI - Double silyl migration converting ORe[N(SiMe(2)CH(2)PCy(2))(2)] to NRe[O(SiMe(2)CH(2)PCy(2))(2)] substructures. AB - The reaction of (R(2)PCH(2)SiMe(2))(2)NM (PNP(R)M; R = Cy; M = Li, Na, MgHal, Ag) with L(2)ReOX(3) [L(2) = (Ph(3)P)(2) or (Ph(3)PO)(Me(2)S); X = Cl, Br] gives (PNP(Cy))ReOX(2) as two isomers, mer,trans and mer,cis. These compounds undergo a double Si migration from N to O at 90 degrees C to form (POP(Cy))ReNX(2) as a mixture of mer,trans and fac,cis isomers. Additional thermolysis effects migration of CH(3) from Si to Re, along with compensating migration of halide from Re to Si. DFT calculations on various structural isomers support the greater thermodynamic stability of the POP/ReN isomer vs PNP/ReO and highlight the influence of the template effect on the reactivities of these species. PMID- 12377063 TI - Rates and mechanism of fluoride and water exchange in UO(2)F(5)(3-) and [UO(2)F(4)(H(2)O)](2-) studied by NMR spectroscopy and wave function based methods. AB - The reaction mechanism for the exchange of fluoride in UO(2)F(5)(3-) and UO(2)F(4)(H(2)O)(2-) has been investigated experimentally using (19)F NMR spectroscopy at -5 degrees C, by studying the line broadening of the free fluoride, UO(2)F(4)(2-)(aq) and UO(2)F(5)(3-), and theoretically using quantum chemical methods to calculate the activation energy for different pathways. The new experimental data allowed us to make a more detailed study of chemical equilibria and exchange mechanisms than in previous studies. From the integrals of the different individual peaks in the new NMR spectra, we obtained the stepwise stability constant K(5) = 0.60 +/- 0.05 M(-1) for UO(2)F(5)(3-). The theoretical results indicate that the fluoride exchange pathway of lowest activation energy, 71 kJ/mol, in UO(2)F(5)(3-) is water assisted. The pure dissociative pathway has an activation energy of 75 kJ/mol, while the associative mechanism can be excluded as there is no stable UO(2)F(6)(4-) intermediate. The quantum chemical calculations have been made at the SCF/MP2 levels, using a conductor-like polarizable continuum model (CPCM) to describe the solvent. The effects of different model assumptions on the activation energy have been studied. The activation energy is not strongly dependent on the cavity size or on interactions between the complex and Na(+) counterions. However, the solvation of the complex and the leaving fluoride results in substantial changes in the activation energy. The mechanism for water exchange in UO(2)F(4)(H(2)O)(2-) has also been studied. We could eliminate the associative mechanism, the dissociative mechanism had the lowest activation energy, 39 kJ/mol, while the interchange mechanism has an activation energy that is approximately 50 kJ/mol higher. PMID- 12377066 TI - What will I do without you? PMID- 12377067 TI - Future eye implants focus on neurotransmitters. PMID- 12377068 TI - Two new projects to help Native Americans end substance abuse and domestic violence. PMID- 12377069 TI - From the Centers for Disease Control and Prevention. Acute flaccid paralysis syndrome associated with West Nile virus infection--Mississippi and Louisiana, July-August 2002. PMID- 12377070 TI - From the Centers for Disease Control and Prevention. Primary and secondary syphilis among men who have sex with men--New York City, 2001. PMID- 12377071 TI - From the Centers for Disease Control and Prevention. Trends in sexual risk behaviors among high school students--United States, 1991-2001. PMID- 12377074 TI - Beta-blocker therapy and depression. PMID- 12377075 TI - Beta-blocker therapy and depression. PMID- 12377076 TI - Medical residents' emotional well-being. PMID- 12377078 TI - Anesthesia and preeclampsia. PMID- 12377080 TI - Guidelines for treatment of anthrax. PMID- 12377082 TI - Guidelines for treatment of anthrax. PMID- 12377083 TI - Cost and cost-effectiveness of an early invasive vs conservative strategy for the treatment of unstable angina and non-ST-segment elevation myocardial infarction. AB - CONTEXT: In the Treat Angina with Aggrastat and Determine Cost of Therapy with an Invasive or Conservative Strategy (TACTICS)-Thrombolysis in Myocardial Infarction (TIMI) 18 trial, patients with either unstable angina or non-ST-segment elevation myocardial infarction (UA/NSTEMI) treated with the platelet glycoprotein (Gp IIb/IIIa) inhibitor tirofiban had a significantly reduced rate of major cardiac events at 6 months with an early invasive vs a conservative strategy. OBJECTIVE: To examine total 6-month costs and long-term cost-effectiveness of an invasive vs a conservative strategy. DESIGN: Randomized controlled trial including a priori economic end points. SETTING: Hospitalization for UA/NSTEMI with 6-month follow up period. PATIENTS: A total of 2220 patients with UA/NSTEMI; economic data from 1722 patients at US-non-VA hospitals. INTERVENTION: Early invasive strategy with routine catheterization and revascularization as appropriate vs a conservative strategy with catheterization performed only for recurrent ischemia or a positive stress test. MAIN OUTCOME MEASURE: Total 6-month costs and incremental cost effectiveness ratio. RESULTS: The average initial hospitalization costs among those in the invasive strategy group were $15714 vs $14047 among those in the conservative strategy group, a difference of $1667 (95% confidence interval [CI], $387-3091). The in-hospital costs were offset significantly at the 6-month follow up, with an average cost in the invasive group of $6098 vs $7180 in the conservative group, a difference of $1082 (95% CI, -$2051 to $76). The average total costs at 6 months, including productivity costs, for the invasive group was $21 813 vs $21 227 for the conservative group, a $586 difference (95% CI, -$1087 to $2486). The average 6-month costs excluding productivity costs in the invasive group was $19 780 vs $19 111 in the conservative group, a difference of $670, 95% CI; (-$1035 to $2321). Estimated cost per year of life gained for the invasive strategy, based on projected life expectancy, was $12739 for the base case, and ranged from $8371 to $25769, based on model assumptions. CONCLUSIONS: In patients with UA/NSTEMI treated with the Gp IIb/IIIa inhibitor tirofiban, the clinical benefit of an early invasive strategy was achieved with a small increase in cost, yielding favorable projected estimates of cost per year of life gained. These results support the broader use of an early invasive strategy in these patients. PMID- 12377084 TI - Mortality in Medicare beneficiaries following coronary artery bypass graft surgery in states with and without certificate of need regulation. AB - CONTEXT: Certificate of need regulation was designed to control health care costs by preventing health care facilities from expanding unnecessarily. While there have been several studies investigating whether these regulations have affected health care investment, few have evaluated the relationship between certificate of need regulation and quality of care. OBJECTIVE: To compare risk-adjusted mortality and hospital volumes for coronary artery bypass graft (CABG) surgery in states with and without certificate of need regulation. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study of 911 407 Medicare beneficiaries aged 65 years or older, who underwent CABG surgery between 1994 and 1999 in 1063 US hospitals. MAIN OUTCOME MEASURES: States (and the District of Columbia) with continuous (n = 27), none (n = 18), or intermittent (n = 6) certificate of need regulation; mortality (in-hospital or within 30 days of CABG surgery) rates; and mean annual hospital volumes for CABG surgery. RESULTS: Unadjusted mortality was 5.1% in states without certificate of need regulation compared with 4.4% in states with continuous regulation, and 4.3% in states with intermittent certificate of need regulation (P<.001 for each comparison). Adjusting for demographic and clinical factors, mortality remained higher in states without certificate of need regulation compared with states with continuous certificate of need regulation (odds ratio [OR], 1.22; 95% confidence interval [CI], 1.15 1.28; P<.001). Using the same groups for comparison, the mean annual hospital volume for CABG surgery was 84% lower in states without certificate of need regulation (104 vs 191; P<.001) and more patients underwent CABG surgery in low volume hospitals (<100 procedures annually) (30% vs 10% for states with continuous certificate of need programs; P<.001). Following the repeal of certificate of need regulation in states categorized as intermittent, the percentage of patients undergoing CABG surgery in low-volume hospitals tripled. CONCLUSIONS: Mortality rates for Medicare patients undergoing CABG surgery were higher in states without certificate of need regulation. Repeal of certificate of need regulations during the study period was associated with declines in hospital volume for CABG surgery. PMID- 12377085 TI - Plasma folate levels and risk of spontaneous abortion. AB - CONTEXT: Both folate deficiency and folic acid supplements have been reported to increase the risk of spontaneous abortion. The results are inconclusive, however, and measurements of folate have not been available in all studies. OBJECTIVE: To study the association between plasma folate levels and the risk of spontaneous abortion. DESIGN, SETTING, AND POPULATION: Population-based, matched, case control study of case women with spontaneous abortion and control women from January 1996 through December 1998 in Uppsala County, Sweden. Plasma folate measurements were available for 468 cases and 921 controls at 6 to 12 gestational weeks. MAIN OUTCOME MEASURE: Risk of spontaneous abortion vs maternal plasma folate level. RESULTS: Compared with women with plasma folate levels between 2.20 and 3.95 ng/mL (5.0 and 8.9 nmol/L), women with low (< or =2.19 ng/mL [< or =4.9 nmol/L]) folate levels were at increased risk of spontaneous abortion (adjusted odds ratio [OR], 1.47; 95% confidence interval [CI], 1.01-2.14), whereas women with higher folate levels (3.96-6.16 ng/mL [9.0-13.9 nmol/L] and > or =6.17 ng/mL [> or =14.0 nmol/L]) showed no increased risk of spontaneous abortion (OR, 0.84; 95% CI, 0.59-1.20; and OR, 0.74; 95% CI, 0.47-1.16, respectively). Low folate levels were associated with a significantly increased risk when the fetal karyotype was abnormal (OR, 1.95; 95% CI, 1.09-3.48) but not when the fetal karyotype was normal (OR, 1.11; 95% CI, 0.55-2.24) or unknown (OR, 1.45; 95% CI, 0.90-2.33). CONCLUSION: Low plasma folate levels were associated with an increased risk of early spontaneous abortion. PMID- 12377086 TI - Six-year follow-up of preventive interventions for children of divorce: a randomized controlled trial. AB - CONTEXT: Compared with their peers with nondivorced parents, adolescents with divorced parents are more likely to have mental health problems, drop out of school, and become pregnant. The long-term effects of intervention programs for this population are unknown. OBJECTIVE: To evaluate the long-term effectiveness of 2 programs designed to prevent mental health problems in children with divorced parents. DESIGN AND SETTING: Six-year follow-up of a randomized controlled trial of 2 intervention programs (mother program: 11 group and 2 individual sessions; mother plus child program: mother program and 11 group sessions for children) and a control condition (books on postdivorce adjustment), which was conducted in a large metropolitan US city from April 1998 through March 2000. PARTICIPANTS: A total of 218 families (91% of the original sample) with adolescents aged between 15 and 19 years were reinterviewed. MAIN OUTCOME MEASURES: Externalizing and internalizing problems, diagnosed mental disorders, drug and alcohol use, and number of sexual partners. RESULTS: Eleven percent of adolescents in the mother plus child program (95% confidence interval [CI], 3.8% 18.2%) had a 1-year prevalence of diagnosed mental disorder compared with 23.5% (95% CI, 13.8%-33.2%) of adolescents in the control program (P =.007). Adolescents in the mother plus child program had fewer sexual partners (mean [SE], 0.68 [0.16]) compared with adolescents in the control program (1.65 [0.37]; P =.01). Adolescents with higher initial mental health problems whose families were in the mother plus child program had lower externalizing problems (P =.007) and fewer symptoms of mental disorder (P =.02) compared with those in the control program. Compared with controls, adolescents whose mothers participated in the mother program and who had higher initial mental health problems had lower levels of externalizing problems (P<.001); fewer symptoms of mental disorder (P =.005); and less alcohol (P =.005), marijuana (P =.02), and other drug use (P =.01). CONCLUSIONS: In adolescents of divorced parents, the mother program and the mother plus child program reduced symptoms of mental disorder; rates of diagnoses of mental disorder; levels of externalizing problems; marijuana, alcohol, and other drug use; and number of sexual partners. PMID- 12377087 TI - Primary prevention of hypertension: clinical and public health advisory from The National High Blood Pressure Education Program. AB - The National High Blood Pressure Education Program Coordinating Committee published its first statement on the primary prevention of hypertension in 1993. This article updates the 1993 report, using new and further evidence from the scientific literature. Current recommendations for primary prevention of hypertension involve a population-based approach and an intensive targeted strategy focused on individuals at high risk for hypertension. These 2 strategies are complementary and emphasize 6 approaches with proven efficacy for prevention of hypertension: engage in moderate physical activity; maintain normal body weight; limit alcohol consumption; reduce sodium intake; maintain adequate intake of potassium; and consume a diet rich in fruits, vegetables, and low-fat dairy products and reduced in saturated and total fat. Applying these approaches to the general population as a component of public health and clinical practice can help prevent blood pressure from increasing and can help decrease elevated blood pressure levels for those with high normal blood pressure or hypertension. PMID- 12377088 TI - Clinical use of bone densitometry: scientific review. AB - CONTEXT: Osteoporosis causes substantial morbidity and costs $13.8 billion annually in the United States. Measurement of bone mass by densitometry is a primary part of diagnosing osteoporosis and deciding a preventive treatment course. Bone mineral densitometry has become more widely available and commonly used in practice. OBJECTIVE: To review evidence about the value of various clinical applications of bone densitometry. DATA SOURCES: A MEDLINE search was performed to update previous meta-analyses of the relationship between various measurements of bone density and risk of vertebral and hip fracture. We used data from the prospective Study of Osteoporotic Fractures to estimate risk of fracture from bone density and age in postmenopausal women. STUDY SELECTION AND DATA EXTRACTION: When available, meta-analyses and systematic reviews are emphasized in the review. DATA SYNTHESIS: Bone mineral density (BMD) predicts fracture and can be used in combination with age to estimate absolute risk of fractures in postmenopausal white women. Hip BMD predicts hip fracture more strongly than other measurements of BMD. There are insufficient data to translate BMD results into risk of fracture for men and nonwhite women. The benefits of treatments to prevent fractures depend on BMD: women with osteoporosis have a greater risk of fractures and greater benefit from treatments than women without osteoporosis. CONCLUSIONS: Guidelines based on systematic reviews and a cost-effectiveness analysis have suggested that it is worthwhile to measure BMD in white women older than 65 years and perhaps to use risk factors to select younger postmenopausal women for densitometry. Other potential clinical applications of BMD that have not yet been adequately studied include screening men or nonwhite women, monitoring BMD in patients receiving treatment, and using BMD to identify patients who should be evaluated for secondary causes of osteoporosis. PMID- 12377089 TI - Clinical use of bone densitometry: clinical applications. AB - Osteoporosis represents a difficult problem for physicians because, although many diagnostic tests are available, interpreting their results is not straightforward. As a result, many patients, even those with clear indications such as long-term steroid therapy or vertebral fractures on radiography, do not get screened or treated. Current evidence-based guidelines recommend screening for all white women older than 65 years and not already receiving an osteoporosis treatment and for many nonwhite women. For postmenopausal women who are younger than 65 years and have strong risk factors for osteoporosis, screening may also be beneficial. The optimal testing strategy depends on what is available locally. The best role for follow-up testing is still being defined, and interpretation of such testing is tricky. Reports of results can be hard to understand; a randomized controlled trial of clearer reports increased testing and decreased confusion about the meaning of test results. Densitometry might be more effectively used in practice if strategies such as having patients fill out a short questionnaire to assess for risk factors or creating a nurse-based system were used to identify patients. Clinicians need better approaches for identifying patients most likely to benefit from screening, systems that facilitate their application, and test results that are easy to interpret. PMID- 12377090 TI - Contact vaccinia--transmission of vaccinia from smallpox vaccination. PMID- 12377091 TI - Invasive vs conservative management of acute coronary syndromes: do the data support the guidelines? PMID- 12377092 TI - Improving primary care for patients with chronic illness: the chronic care model, Part 2. AB - This article reviews research evidence showing to what extent the chronic care model can improve the management of chronic conditions (using diabetes as an example) and reduce health care costs. Thirty-two of 39 studies found that interventions based on chronic care model components improved at least 1 process or outcome measure for diabetic patients. Regarding whether chronic care model interventions can reduce costs, 18 of 27 studies concerned with 3 examples of chronic conditions (congestive heart failure, asthma, and diabetes) demonstrated reduced health care costs or lower use of health care services. Even though the chronic care model has the potential to improve care and reduce costs, several obstacles hinder its widespread adoption. PMID- 12377095 TI - JAMA patient page. Miscarriage. PMID- 12377099 TI - Bone mineral density and the subsequent risk of cancer in the NHANES I follow-up cohort. AB - BACKGROUND: Bone mineral density (BMD) is a marker of long-term estrogen exposure. BMD measurement has been used in this context to investigate the association of estrogen with breast cancer risk in three cohorts. In order to assess further BMD as a predictor of estrogen related cancer risk, the association of BMD with colorectal and corpus uteri cancer was investigated in the NHANES I Epidemiologic Followup Study (NHEFS) cohort along with breast cancer and prostate cancer. METHODS: Participants were members of the NHEFS cohort who had BMD measurement in 1974-1975. Age, race, and BMI adjusted rate ratios and 95% confidence intervals were calculated for incidence of cancers of the corpus uterus, breast, colorectum, prostate, and of osteoporosis and hip fracture related to baseline BMD. RESULTS: Data were available for 6046 individuals. One hundred cases of breast cancer, 94 prostate cancers, 115 colorectal cancers, 29 uterine cancers, 110 cases of hip fracture and 103 cases of osteoporosis were reported between 1974 and 1993. Hip fracture and osteoporosis were both significantly inversely associated with BMD. Uterine cancer was positively associated (p = 0.005, test for linear trend) and colorectal cancer negatively associated (p = 0.03) with BMD. No association was found between elevated BMD and incidence of breast cancer (p = 0.74) or prostate cancer (p = 0.37) in the overall cohort, although a weak association was seen between BMD and subsequent breast cancer incidence when BMD was measured in post-menopausal women (p = 0.04). CONCLUSION: The findings related to cancers of the uterus and colorectum as well as the weak association of BMD with breast cancer strengthen the use of BMD as a marker of estrogen exposure and cancer risk. PMID- 12377100 TI - Patients affected with Fabry disease have an increased incidence of progressive hearing loss and sudden deafness: an investigation of twenty-two hemizygous male patients. AB - BACKGROUND: Fabry disease (FD, OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism due to the deficient activity of alpha-galactosidase A, a lysosomal enzyme. While the progressive systemic deposition of uncleaved glycosphingolipids throughout the body is known to have protean clinical manifestations, few data are available regarding the cochlear involvement. METHODS: We non-invasively investigated cochlear functions in 22 consecutive hemizygous males (age 19-64 years, mean 39) affected with classic FD. Conventional audiometry, tympanometry, ABR audiometry, otoacoustic emissions were performed in all patients, together with medical history record and physical examination as part of an exhaustive baseline evaluation prior to enzyme replacement therapy. RESULTS: A total of 12 patients (54.5%) with classic FD were found to have abnormal audition. Five patients had progressive hearing loss and seven patients (32%) experienced sudden deafness. In addition, a hearing loss on high-tone frequencies was found in 7 out of the 10 remaining patients without clinical impairment, despite their young age at time of examination. The incidence of hearing loss appeared significantly increased in FD patients with kidney failure (P < 0.01) or cerebrovascular lesions (P < 0.01), whereas there was no correlation with left ventricular hypertrophy. In addition, tinnitus aurium was also found in six patients (27%). CONCLUSION: This is the first evidence of a high incidence of both progressive hearing loss and sudden deafness in a cohort of male patients affected with classic Fabry disease. The exact pathophysiologic mechanism(s) of the cochlear involvement deserves further studies. PMID- 12377101 TI - Characterisation of silent and active genes for a variable large protein of Borrelia recurrentis. AB - BACKGROUND: We report the characterisation of the variable large protein (vlp) gene expressed by clinical isolate A1 of Borrelia recurrentis; the agent of the life-threatening disease louse-borne relapsing fever. METHODS: The major vlp protein of this isolate was characterised and a DNA probe created. Use of this together with standard molecular methods was used to determine the location of the vlp1B. recurrentis A1 gene in both this and other isolates. RESULTS: This isolate was found to carry silent and expressed copies of the vlp1B. recurrentis A1 gene on plasmids of 54 kbp and 24 kbp respectively, whereas a different isolate, A17, had only the silent vlp1B. recurrentis A17 on a 54 kbp plasmid. Silent and expressed vlp1 have identical mature protein coding regions but have different 5' regions, both containing different potential lipoprotein leader sequences. Only one form of vlp1 is transcribed in the A1 isolate of B. recurrentis, yet both 5' upstream sequences of this vlp1 gene possess features of bacterial promoters. CONCLUSION: Taken together these results suggest that antigenic variation in B. recurrentis may result from recombination of variable large and small protein genes at the junction between lipoprotein leader sequence and mature protein coding region. However, this hypothetical model needs to be validated by further identification of expressed and silent variant protein genes in other B. recurrentis isolates. PMID- 12377102 TI - The cost-effectiveness of the WINGS intervention: a program to prevent HIV and sexually transmitted diseases among high-risk urban women. AB - BACKGROUND: We evaluated the cost-effectiveness of the WINGS project, an intervention to prevent HIV and other sexually transmitted diseases among urban women at high risk for sexual acquisition of HIV. METHODS: We used standard methods of cost-effectiveness analysis. We conducted a retrospective analysis of the intervention's cost and we used a simplified model of HIV transmission to estimate the number of HIV infections averted by the intervention. We calculated cost-effectiveness ratios for the complete intervention and for the condom use skills component of the intervention. RESULTS: Under base case assumptions, the intervention prevented an estimated 0.2195 new cases of HIV at a cost of $215,690 per case of HIV averted. When indirect costs of HIV were excluded from the analysis, the intervention's cost-effectiveness ratios were $357,690 per case of HIV averted and $31,851 per quality-adjusted life year (QALY) saved. Under base case assumptions, the condom use skills component of the intervention prevented an estimated 0.1756 HIV infections and was cost-saving. When indirect HIV costs were excluded, the cost-effectiveness ratios for the condom use skills component of the intervention were $97,404 per case of HIV averted and $8,674 per QALY saved. CONCLUSIONS: The WINGS intervention, particularly the two sessions of the intervention which focussed on condom use skills, could be cost-effective in preventing HIV among women. PMID- 12377103 TI - Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor alpha by gallbladder epithelial cells: response to bacterial lipopolysaccharides. AB - BACKGROUND: In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor alpha (TNF-alpha) protein by these cells was also measured. RESULTS: Untreated mouse gallbladder cells expressed mRNA for TNF-alpha, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1beta (IL-1beta), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-alpha and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-alpha protein; however, they did synthesize and secrete TNF-alpha upon treatment with lipopolysaccharide. METHODS: Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFalpha, IL 1beta, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-alpha protein was measured by immunoassays. CONCLUSION: This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFalpha protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis. PMID- 12377104 TI - A reference database for tumor-related genes co-expressed with interleukin-8 using genome-scale in silico analysis. AB - BACKGROUND: The EST database provides a rich resource for gene discovery and in silico expression analysis. We report a novel computational approach to identify co-expressed genes using EST database, and its application to IL-8. RESULTS: IL-8 is represented in 53 dbEST cDNA libraries. We calculated the frequency of occurrence of all the genes represented in these cDNA libraries, and ranked the candidates based on a Z-score. Additional analysis suggests that most IL-8 related genes are differentially expressed between non-tumor and tumor tissues. To focus on IL-8's function in tumor tissues, we further analyzed and ranked the genes in 16 IL-8 related tumor libraries. CONCLUSIONS: This method generated a reference database for genes co-expressed with IL-8 and could facilitate further characterization of functional association among genes. PMID- 12377105 TI - The effect of massage on localized lumbar muscle fatigue. AB - BACKGROUND: There is not enough evidence to support the efficacy of massage for muscle fatigue despite wide utilization of the modality in various clinical settings. This study investigated the influence of massage application on localized back muscle fatigue. METHODS: Twenty-nine healthy subjects participated in two experimental sessions (massage and rest conditions). On each test day, subjects were asked to lie in the prone position on a treatment table and perform sustained back extension for 90 seconds. Subjects then either received massage on the lumbar region or rested for a 5 minute duration, then repeated the back extension movement. The median frequency (MDF), mean power frequency (MNF), and root mean square (RMS) amplitude of electromyographic signals during the 90 second sustained lumbar muscle contraction were analyzed. The subjective feeling of fatigue was then evaluated using the Visual Analogue Scale (VAS). RESULTS: MDF and MNF significantly declined with time under all conditions. There was no significant difference in MDF, MNF or RMS value change between before and after massage, or between rest and massage conditions. There was a significant increase in fatigue VAS at the end of the 2nd back extension with rest condition. There was a significant difference in fatigue VAS change between massage and rest condition. CONCLUSIONS: A significant difference was observed between massage and rest condition on VAS for muscle fatigue. On EMG analysis, there were no significant differences to conclude that massage stimulation influenced the myoelectrical muscle fatigue, which is associated with metabolic and electrical changes. PMID- 12377106 TI - Amyloid associated with elastin-staining laminar aggregates in the lungs of patients diagnosed with acute respiratory distress syndrome. AB - BACKGROUND: The heterogeneity of conditions underlying respiratory distress, whether classified clinically as acute lung injury (ALI) or the more severe acute respiratory distress syndrome (ARDS), has hampered efforts to identify and more successfully treat these patients. Examination of postmortem lungs among cases clinically diagnosed as ARDS identified a cohort that showed a consistent morphology at the light and electron microscope levels, and featured pathognomonic structures which we termed elastin-staining laminar structures (ELS). METHODS: Postmortem tissues were stained using the Verhoeff-Van Gieson procedure for elastic fibers, and with Congo red for examination under a polarizing microscope. Similar samples were examined by transmission EM. RESULTS: The pathognomonic ELS presented as ordered molecular aggregates when stained using the Verhoeff-van Gieson technique for elastic fibers. In several postmortem lungs, the ELS also displayed apple-green birefringence after staining with Congo red, suggesting the presence of amyloid. Remarkably, most of the postmortem lungs with ELS exhibited no significant acute inflammatory cellular response such as neutrophilic reaction, and little evidence of widespread edema except for focal intra-alveolar hemorrhage. CONCLUSIONS: Postmortem lungs that exhibit the ELS constitute a morphologically-identifiable subgroup of ARDS cases. The ordered nature of the ELS, as indicated by both elastin and amyloid stains, together with little morphological evidence of inflammation or edema, suggests that this cohort of ARDS may represent another form of conformational disease. If this hypothesis is confirmed, it will require a new approach in the diagnosis and treatment of patients who exhibit this form of acute lung injury. PMID- 12377108 TI - Characteristics of dermatophytoses in children treated at the Department of Dermatology and Venerology, Dr Josip Bencevic General Hospital, Slavonski Brod, Croatia, from February 1993 till February 2000. AB - Dermatophytoses are common infections in children. During the period from February 1993 till February 2000, 308 children with dermatophytoses were examined at the Department of Dermatology and Venerology, Dr.Josip Bencevic General Hospital, Slavonski Brod, Croatia. Out of these 308 pediatric patients, there were 157 (50.97%) male and 151 (49.03%) female children aged 3 months to 15 years. Microsporum spp. infection was detected in 251 (81.49%) and Trichophyton spp. infection in 57 (18.51%) cases. The epidemiology of the infection was analyzed according to age, sex, and seasonal progression PMID- 12377107 TI - Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences. AB - BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA. PMID- 12377109 TI - Medicaments as the possible cause of urticaria in children. AB - Medicaments are reported as the most common cause of urticaria. The objective of this study was to determine, by retrospective analysis of 132 pediatric patients treated at the Pulmonology and Allergology ward of the Department of Pediatrics, Banja Luka, over a 5-year period, the scope to which medicaments act as the possible cause of urticaria. Results of the study showed that the disease manifested mostly in male children (59.8), mainly of pre-school and school age rather than <1 year age group. Acute urticaria predominated, and it was recorded in 91.7% of cases. A medicament as the possible etiologic factor of acute urticaria was found in 29.8% of cases. Regarding chronic urticaria, in most cases the cause of disease remained unknown (63.6%), whereas a medicament and infection as the possible causal factors were found in 9.1% of cases. Before the occurrence of urticaria, 37 (28%) children took some medicament. Usually, these were antibiotics (45.9%), antipyretics (35.1%), or a combination of antibiotics and antipyretics (16.2%). Penicillin V, G or ampicillin were the most frequently used antibiotics (88.2%), whereas acetylsalicylic acid was the most frequently used antipyretic (53.8%). In 28% of the children suffering from acute urticaria, apart from taking some medicament, clinically manifested infection was also recorded, mostly of the respiratory system, so it could not be stated for sure whether the medicament or infection was the etiologic factor for the occurrence of disease. In only two cases it could be stated for sure that a medicament was the cause of urticaria, one acute and chronic urticaria each PMID- 12377110 TI - Lesion of a lower lip scar--an initial presentation of sarcoidosis. AB - The development of sarcoidosis at the site of previous scar is unusual and may be the first sign of systemic disease. Sarcoidosis on a lower lip scar persisting for 13 years, which pointed to examination for internal manifestations, is presented along with a review of the subject PMID- 12377111 TI - Cutaneous metastases--carcinoma en cuirasse. AB - Metastatic carcinoma of the skin develops per continuitatem, being disseminated by the lymphogenous and hematogenous pathways. From 3% to 5% of these metastases are described as cutaneous metastases. Metastases of the skin most frequently occur in breast cancer, and manifest as carcinoma en cuirasse characterized by thoracic wall lesions in the form of erythematous foci with induration as in scleroderma, or as exulcerating nodules scattered all over the skin surface. We studied patient with skin metastases according clinical and histological features. Carcinoma en cuirasse with sclerodermatomyositis like clinical appearances described in our female 49-year-old patient. She died one year after several excisions of skin metastases PMID- 12377112 TI - Search systems and medical data quoting from the internet. AB - The Internet as a new medium also has its use in medicine. Plenty of information which can be found on the world computer network, needs an efficient searching as well as an evaluation system and quoting of the information obtained. Twelve most popular search engines were chosen--9 general: Netscape, Altavista, HotBot, Goggle, Northern Light, Magellan, Infoseek, MSN; 2 specialized medical search engines: Medscape and Medline; and one Croatian search engine: Cross. The efficiency of searching was observed by analyzing the number of obtained pages, their contents and examples of the quoted references from the Internet. The searching was done using the mentioned search engines, and results varied from 2,189,793 network pages found on the Infoseek for the term 'skin cancer' to 0 pages with the search engine Cross for the term 'melanoma+therapy'. When comparing the results for a single term using various search engines, it is concluded that the number of pages varied because of different databases and specialized scooters that search and make indexes. It is possible to use logical operators and more advanced search systems with the majority of search engines. The quoting system is based on mentioning the names of the author and his work, the access date and url (uniform resource locator) addresses of the network pages. Permanent and reliable access to quotations could not be established with previous quotation systems. Dermatologic terms have been well dealt with concerning general population, whereas professionals are recommended to use Medline and reviewed web pages. There have been very few professional and professionally relevant and comprehensive pages on the Internet relative to the total of web pages. PMID- 12377113 TI - Complex cell signaling molecules from ancient molecular glue. AB - In this issue of Structure, Springer and colleagues argue that ancient extracellular domains of archaea involved in primitive cell adhesion have evolved into the extracellular domains of cell signaling molecules essential for the survival of animals and humans. PMID- 12377114 TI - The p47phox PX domain: two heads are better than one! AB - The recent X-ray structure of the PX domain of p47phox, a critical subunit of the NADPH oxidase, unexpectedly revealed the presence of two distinct lipid binding pockets within this single modular domain. This unusual feature allows the p47phox PX domain to integrate signal transduction events emerging from two different lipid signaling pathways. PMID- 12377115 TI - Recognition of acetylated proteins: lessons from an ancient family of enzymes. AB - The structure of a Sir2-like enzyme in complex with an acetylated peptide substrate has been solved, and provides the first glimpse into the mechanism of substrate recognition by this highly conserved family of enzymes. PMID- 12377116 TI - The ABCDs of periplasmic copper trafficking. AB - The structure of the CopC protein of Pseudomonas syringae pathovar tomato provides fascinating clues, not only to its role in the periplasmic space in copper resistance, but also to features important for copper trafficking and homeostasis that may be conserved in a variety of biological systems. PMID- 12377117 TI - The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot. AB - In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites. PMID- 12377118 TI - Microtubule structure at 8 A resolution. AB - We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 A, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through docking tubulin into lower-resolution maps. This work provides an improved picture of the interactions between adjacent protofilaments, which are responsible for microtubule stability, and also suggests that some structural features are different in microtubules from those in the zinc sheets with which the tubulin structure was determined. PMID- 12377119 TI - Sequence landmark patterns identify and characterize protein families. AB - The three-dimensional structures of homologous proteins are usually conserved during evolution, as are critical residues in a few short sequence motifs that often constitute the active site in enzymes. The precise spatial organization of such sites depends on the lengths and positions of the secondary structural elements connecting the motifs. We show how members of protein superfamilies, such as kinesins, myosins, and G(alpha) subunits of trimeric G proteins, are identified and classed by simply counting the number of amino acid residues between important sequence motifs in their nucleotide triphosphate-hydrolyzing domains. Subfamily-specific landmark patterns (motif to motif scores) are principally due to inserts and gaps in surface loops. Unusual protein sequences and possible sequence prediction errors are detected. PMID- 12377120 TI - Solution structure of CopC: a cupredoxin-like protein involved in copper homeostasis. AB - The structure of the metal-free form of CopC, a protein involved in copper homeostasis, has been obtained. The fold is a Greek key beta barrel similar to that of functionally unrelated blue copper proteins but with important structural variations. The protein binds one equivalent of copper (II) with relatively high affinity and contains a cluster of conserved residues (His1, Glu27, Asp89, and His91) which could form a water-accessible metal binding site. The structure also reveals a loop containing the M(X)(n)M motif which is present in a number of proteins also involved in copper homeostasis. The present structure represents a link between copper-trafficking proteins and cupredoxins. Within a structural and genomic analysis, the role of CopC in copper trafficking is discussed. PMID- 12377121 TI - Structure and interactions of PAS kinase N-terminal PAS domain: model for intramolecular kinase regulation. AB - PAS domains are sensory modules in signal-transducing proteins that control responses to various environmental stimuli. To examine how those domains can regulate a eukaryotic kinase, we have studied the structure and binding interactions of the N-terminal PAS domain of human PAS kinase using solution NMR methods. While this domain adopts a characteristic PAS fold, two regions are unusually flexible in solution. One of these serves as a portal that allows small organic compounds to enter into the core of the domain, while the other binds and inhibits the kinase domain within the same protein. Structural and functional analyses of point mutants demonstrate that the compound and ligand binding regions are linked, suggesting that the PAS domain serves as a ligand-regulated switch for this eukaryotic signaling system. PMID- 12377122 TI - Structure of the cathelicidin motif of protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein. AB - Cathelicidins are a family of antimicrobial proteins isolated from leucocytes and epithelia cells that contribute to the innate host defense mechanisms in mammalians. Located in the C-terminal part of the holoprotein, the cathelicidin derived antimicrobial peptide is liberated by a specific protease cleavage. Here, we report the X-ray structure of the cathelicidin motif of protegrin-3 solved by MAD phasing using the selenocysteine-labeled protein. Its overall structure represents a fold homologous to the cystatin family and adopts two native states, a monomer, and a domain-swapped dimer. This crystal structure is the first example of a structural characterization of the highly conserved cathelicidin motif and thus provides insights into the possible mechanism of activation of the antimicrobial protegrin peptide. PMID- 12377123 TI - Studies on the reaction mechanism of riboflavin synthase: X-ray crystal structure of a complex with 6-carboxyethyl-7-oxo-8-ribityllumazine. AB - Riboflavin synthase catalyzes the disproportionation of 6,7-dimethyl-8 ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione. We have determined the structure of riboflavin synthase from Schizosaccharomyces pombe in complex with the substrate analog, 6-carboxyethyl-7 oxo-8-ribityllumazine at 2.1 A resolution. In contrast to the homotrimeric solution state of native riboflavin synthase, we found the enzyme to be monomeric in the crystal structure. Structural comparison of the riboflavin synthases of S. pombe and Escherichia coli suggests oligomer contact sites and delineates the catalytic site for dimerization of the substrate and subsequent fragmentation of the pentacyclic intermediate. The pentacyclic substrate dimer was modeled into the proposed active site, and its stereochemical features were determined. The model suggests that the substrate molecule at the C-terminal domain donates a four-carbon unit to the substrate molecule bound at the N-terminal domain of an adjacent subunit in the oligomer. PMID- 12377124 TI - The structural basis for catalysis and specificity of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis. AB - The X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactococcus lactis is a dimeric enzyme catalyzing the removal of Xaa-Pro dipeptides from the N terminus of peptides. The structure of the enzyme was solved at 2.2 A resolution and provides a model for the peptidase family S15. Each monomer is composed of four domains. The larger one presents an alpha/beta hydrolase fold and comprises the active site serine. The specificity pocket is mainly built by residues from a small helical domain which is, together with the N-terminal domain, essential for dimerization. A C-terminal moiety probably plays a role in the tropism of X-PDAP toward the cellular membrane. These results give new insights for further exploration of the role of the enzymes of the SC clan. PMID- 12377125 TI - DNA recognition by the RUNX1 transcription factor is mediated by an allosteric transition in the RUNT domain and by DNA bending. AB - The Runt domain proteins are transcription regulators of major developmental pathways. Here we present the crystal structures of the Runt domain (RD) of the human protein RUNX1 and its DNA binding site in their free states and compare them with the published crystal structures of RD bound to DNA and to the partner protein CBFbeta. We demonstrate that (1) RD undergoes an allosteric transition upon DNA binding, which is further stabilized by CBFbeta, and that (2) the free DNA target adopts a bent-helical conformation compatible with that of the complex. These findings elucidate the mechanism by which CBFbeta enhances RD binding to DNA as well as the role of the intrinsic conformation of the DNA target in the recognition process. PMID- 12377126 TI - Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography--technical advance. AB - The combined use of a diamond anvil cell and ultrashort-wavelength undulator radiation has allowed the collection of high-resolution diffraction data from protein and virus crystals submitted to hydrostatic pressures beyond 2 kbar. Crystals of cubic cowpea mosaic virus (CPMV) can be compressed to at least 3.5 kbar. Diffraction from CPMV crystals displaying an unusual disorder at atmospheric pressure was considerably enhanced by application of pressure. These experiments suggest that pressure may be used in some cases to improve order in crystals. PMID- 12377127 TI - Hexameric ring structure of the ATPase domain of the membrane-integrated metalloprotease FtsH from Thermus thermophilus HB8. AB - FtsH is a cytoplasmic membrane-integrated, ATP-dependent metalloprotease, which processively degrades both cytoplasmic and membrane proteins in concert with unfolding. The FtsH protein is divided into the N-terminal transmembrane region and the larger C-terminal cytoplasmic region, which consists of an ATPase domain and a protease domain. We have determined the crystal structures of the Thermus thermophilus FtsH ATPase domain in the nucleotide-free and AMP-PNP- and ADP-bound states, in addition to the domain with the extra preceding segment. Combined with the mapping of the putative substrate binding region, these structures suggest that FtsH internally forms a hexameric ring structure, in which ATP binding could cause a conformational change to facilitate transport of substrates into the protease domain through the central pore. PMID- 12377128 TI - Structural and functional analysis of the kid toxin protein from E. coli plasmid R1. AB - We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action. PMID- 12377129 TI - Sequence and structural differences between enzyme and nonenzyme homologs. AB - To improve our understanding of the evolution of novel functions, we performed a sequence, structural, and functional analysis of homologous enzymes and nonenzymes of known three-dimensional structure. In most examples identified, the nonenzyme is derived from an ancestral catalytic precursor (as opposed to the reverse evolutionary scenario, nonenzyme to enzyme), and the active site pocket has been disrupted in some way, owing to the substitution of critical catalytic residues and/or steric interactions that impede substrate binding and catalysis. Pairwise sequence identity is typically insignificant, and almost one-half of the enzyme and nonenzyme pairs do not share any similarity in function. Heterooligomeric enzymes comprising homologous subunits in which one chain is catalytically inactive and enzyme polypeptides that contain internal catalytic and noncatalytic duplications of an ancient enzyme domain are also discussed. PMID- 12377130 TI - Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins. AB - The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M. mazei SLP. One domain displays a unique, highly symmetrical, seven-bladed beta propeller fold, and the other belongs to the polycystic kidney disease (PKD) superfamily fold. The third domain is predicted to adopt a beta helix fold. These domains have homologs in metazoan cell surface proteins, suggesting remarkable relationships between domains in archaeal SLPs and metazoan cell surface proteins. PMID- 12377131 TI - Appearance-based segmentation of medial temporal lobe structures. AB - A new paradigm for the characterization of structure appearance is proposed, based on a combination of gray-level MRI intensity data and a shape descriptor derived from a priori principal components analysis of 3D deformation vector fields. Generated without external intervention, it extends into 3D more classic, 2D manual landmark-based shape models. Application of this novel concept led to a method for the segmentation of medial temporal lobe structures from brain magnetic resonance images. The strategy employed for segmentation aims at synthesizing, using the appearance model, a deformation field that maps a new volume onto a reference target. Any information defined on the reference can then be propagated back on the new volume instance, thereby achieving segmentation. The proposed method was tested on a data set of 80 normal subjects and compared against manual segmentation as well as automated segmentation results from ANIMAL, a nonlinear registration and segmentation technique. Experimental results demonstrated the robustness and flexibility of the new method. Segmentation accuracy, measured by overlap statistics, is marginally lower (< 2%) than ANIMAL, while processing time is six times faster. Finally, the applicability of this concept toward shape deformation analysis is presented. PMID- 12377132 TI - Simulation of the effects of global normalization procedures in functional MRI. AB - We report on differences in sensitivity and false-positive rate across five methods of global normalization using resting-state fMRI data embedded with simulated activation. These methods were grand mean session scaling, proportional scaling, ANCOVA, a masking method, and an orthogonalization method. We found that global normalization by proportional scaling and ANCOVA decreased the sensitivity of the statistical analysis and induced artifactual deactivation even when the correlation between the global signal and the experimental paradigm was relatively low. The masking method and the orthogonalization method performed better from this perspective but are both restricted to certain experimental conditions. Based on the results of these simulations, we offer practical guidelines for the choice of global normalization method least likely to bias the experimental results. PMID- 12377133 TI - Functional magnetic resonance imaging of proactive interference during spoken cued recall. AB - Though lesions to frontal cortex can increase susceptibility to interference from previously established but irrelevant memories ("proactive interference"), the specific regions underlying this problem are difficult to determine because the lesions are typically large and heterogeneous. We used event-related functional magnetic resonance imaging to investigate proactive interference in healthy volunteers performing an "AB-AC" paired-associate cued-recall paradigm. At Study, participants intentionally encoded semantically related visual word pairs, which were changed three times (high interference), repeated three times (low interference), or presented only once. At Test, participants were presented with the first word of each pair and attempted to recall its most recent associate from the Study phase. To overcome the problem of image artifacts caused by speech related head motion, we cued speech during a gap between image acquisitions. Regions in left inferior frontal cortex and bilateral frontopolar cortex showed interference effects during both Study and Test. The pattern of responses in these regions differed, however. Left inferior frontal regions showed mainly reduced responses associated with low interference, whereas frontopolar regions showed mainly increased responses associated with high interference. When incorrect as well as correct trials were analyzed at Test, additional activation associated with high interference was observed in right dorsolateral prefrontal cortex. These data suggest that distinct regions within prefrontal cortex subserve different functions in the presence of proactive interference during cued recall. PMID- 12377134 TI - Human cortical electroencephalography (EEG) rhythms during the observation of simple aimless movements: a high-resolution EEG study. AB - In the present high-resolution electroencephalographic (EEG) study, we computed event-related desynchronization and synchronization (ERD/ERS) of alpha (about 10 Hz) and beta (about 20 Hz) rhythms in association with the execution (with visual feedback) and observation of brisk unilateral right and left aimless finger movements. A first scope was to test the topographical "functional equivalence" of cortical rhythmicity related to movement execution and observation, which would represent an ideal cortical observation/execution matching system. A second scope was to evaluate the hypothesis of a left or right hemisphere prevalence of the cortical rhythmicity related to the movement observation compared to the movement execution. EEG (128 electrodes) was recorded in 10 healthy right-handed volunteers. Surface Laplacian estimation spatially enhanced EEG data over a MRI constraint head model. Under both conditions, ERD peaked during the movement execution or observation and was replaced by a ERS "rebound" or "recovery," which peaked during the postevent period. Topographical results are in favor of a "functional equivalence" (i.e., similar ERD/ERS values in magnitude and timing) of alpha and beta rhythmicity in central scalp regions overlying premotor/primary sensorimotor cortex. On the contrary, the functional equivalence of alpha rhythmicity was negligible (i.e., different ERD/ERS values in magnitude and timing) in parietal-occipital scalp regions overlying posterior parietal and parieto-occipital cortex, which could be the neural substrate to distinguish among the own motor intensions and others' aimless movements (i.e., visuomotor transformation integrated with sensorimotor, postural, and kinematics representations). Finally, the pattern of hemispherical cortical rhythmicity did not support a "simple concentration" of movement observation functions in the left or right hemisphere. PMID- 12377135 TI - Effects of verbal working memory load on corticocortical connectivity modeled by path analysis of functional magnetic resonance imaging data. AB - We investigated the hypothesis that there are load-related changes in the integrated function of frontoparietal working memory networks. Functional magnetic resonance imaging time-series data from 10 healthy volunteers performing a graded n-back verbal working memory task were modeled using path analysis. Seven generically activated regions were included in the model: left/right middle frontal gyri (L/R MFG), left/right inferior frontal gyri (L/R IFG), left/right posterior parietal cortex (L/R PPC), and supplementary motor area (SMA). The model provided a good fit to the 1-back (chi(2) = 7.04, df = 8, P = 0.53) and 2 back conditions (chi(2) = 9.35, df = 8, P = 0.31) but not for the 3-back condition (chi(2) = 20.60, df = 8, P = 0.008). Model parameter estimates were compared overall among conditions: there was a significant difference overall between 1-back and 2-back conditions (chi(2)(diff) = 74.77, df = 20, P < 0.001) and also between 2-back and 3-back conditions (chi(2)(diff) = 96.28, df = 20, P < 0.001). Path coefficients between LIFG and LPPC were significantly different from zero in both 1-back and 2-back conditions; in the 2-back condition, additional paths from LIFG to LPPC via SMA and to RMFG from LMFG and LPPC were also nonzero. This study demonstrated a significant change in functional integration of a neurocognitive network for working memory as a correlate of increased load. Enhanced inferior frontoparietal and prefrontoprefrontal connectivity was observed as a correlate of increasing memory load, which may reflect greater demand for maintenance and executive processes, respectively. PMID- 12377136 TI - Cluster significance testing using the bootstrap. AB - Many of the statistical methods currently employed to analyze fMRI data depend on a response template. However, the true form of the hemodynamic response, and thereby the response template, is often unknown. Consequently, cluster analysis provides a complementary, template-free method for exploratory analysis of multidimensional fMRI data sets. Clustering algorithms currently being applied to fMRI data separate the data into a predefined number of clusters (k). A poor choice of k will result in erroneously partitioning well-defined clusters. Although several clustering algorithms have been successfully applied to fMRI data, techniques for statistically testing cluster separation are still lacking. To address this problem we suggest a method based on Fisher's linear discriminant and the bootstrap. Also introduced in this paper is a measure based on the projection of multidimensional data from two clusters onto the vector, maximizing the ratio of the between- to the within-cluster sums of squares. The resulting one-dimensional distribution may be readily visualized and used as a heuristic for estimating cluster homogeneity. These methods are demonstrated for the self organizing maps clustering algorithm when applied to event-related fMRI data. PMID- 12377137 TI - Spatial normalization and averaging of diffusion tensor MRI data sets. AB - Diffusion tensor magnetic resonance imaging (DT-MRI) is unique in providing information about both the structural integrity and the orientation of white matter fibers in vivo and, through "tractography", revealing the trajectories of white matter tracts. DT-MRI is therefore a promising technique for detecting differences in white matter architecture between different subject populations. However, while studies involving analyses of group averages of scalar quantities derived from DT-MRI data have been performed, as yet there have been no similar studies involving the whole tensor. Here we present the first step towards realizing such a study, i.e., the spatial normalization of whole tensor data sets. The approach is illustrated by spatial normalization of 10 DT-MRI data sets to a standard anatomical template. Both qualitative and quantitative approaches are described for assessing the results of spatial normalization. Techniques are then described for combining the spatially normalized data sets according to three definitions of average, i.e., the mean, median, and mode of a distribution of tensors. The current absence of, and hence need for, appropriate statistical tests for comparison of results derived from group-averaged DT-MRI data sets is then discussed. Finally, the feasibility of performing tractography on the group averaged DT-MRI data set is investigated and the possibility and implications of generating a generic map of brain connectivity from a group of subjects is considered. PMID- 12377138 TI - Patterns of cerebral atrophy in dementia with Lewy bodies using voxel-based morphometry. AB - Previous cross-sectional MRI studies based on region-of-interest analyses have shown that increased cerebral atrophy is a feature of both Dementia with Lewy bodies (DLB) and Alzheimer's disease (AD). Relative preservation of the hippocampus and temporal lobe structures in DLB compared to AD has been reported in region-of-interest-based studies. Recently, image processing techniques such as voxel-based morphometry (VBM) have been developed to provide an unbiased, visually informative, and comprehensive means of studying patterns of cerebral atrophy. We report the first study to use the voxel-based approach to assess patterns of cerebral atrophy in DLB compared to control subjects and AD. Regional gray matter volume loss was observed bilaterally in the temporal and frontal lobes and insular cortex of patients with DLB compared to control subjects. Comparison of dementia groups showed preservation of the medial temporal lobe, hippocampus, and amygdala in DLB relative to AD. Significant gray matter loss was also observed in the thalamus of AD patients compared to DLB. PMID- 12377139 TI - Manual and automated measurement of the whole thalamus and mediodorsal nucleus using magnetic resonance imaging. AB - The thalamus is an important relay structure in the brain that may be relevant to a variety of brain diseases. It is divided into multiple subnuclei with different cortical connections. The medial dorsal (MD) nucleus is particularly important because it forms key connections with the prefrontal cortex. The current study reports precise and efficient methods for measuring the whole thalamus and the MD with MRI that have a high degree of interrater reliability. A multispectral image acquisition and novel image processing technique were used to improve structure visibility. The tricolor image assigns a color to each of the T1, T2, and PD weighted images, represented by red, green, and blue, respectively. The manually defined regions were then used to train an artificial neural network (ANN) to automatically define both the whole thalamus and the MD. The ANN provides an efficient automated method, making studies using larger sample sizes more feasible. PMID- 12377140 TI - Attention-dependent changes of activation and connectivity in dichotic listening. AB - Functional studies of auditory spatial attention generally report enhanced neural responses in auditory cortical regions. However, activity in regions of the spatial attentional network as described in the visual modality is not consistently observed. Data analysis limitations due to oppositely lateralized activity depending on the side of attentional orientation and heterogeneity of paradigms makes it hard to untangle the possible causes of these various activation patterns. In the present article we present a PET study of auditory spatial attention in which we manipulated orientation of attention, attentional load, and difficulty of the task by means of the dichotic listening paradigm. Moreover, we designed a systematic, voxel-specific, method in order to deal with oppositely lateralized activity. The results show that when listeners are involved in auditory spatial attention tasks an interacting network of frontal, temporal, and parietal regions is activated. Selective orientation toward one side mostly yields activity and connectivity modulations in the hemisphere contralateral to the attended side while in divided attention activity is mostly bilateral. Taken together, our observations are consistent with the idea of a multimodal large-scale attentional network. PMID- 12377141 TI - Regional frontal cortical volumes decrease differentially in aging: an MRI study to compare volumetric approaches and voxel-based morphometry. AB - Recent neuroimaging studies suggest that the frontal lobes are the part of the brain most profoundly affected by the aging process. The present study investigated whether subregions within the frontal cortex show different patterns of brain aging. Magnetic resonance images of 57 healthy participants between 21 and 81 years old were used to measure regional frontal gray matter volumes in three ways: a manual tracing method, a semiautomatic "Talairach boxes" volumetric method, and voxel-based morphometry. Seven regions within each hemisphere were manually traced: precentral gyrus, inferior frontal gyrus, dorsolateral frontal cortex, ventral medial region, lateral orbital region, anterior cingulate, and frontal pole. With the semiautomatic approach, four regions were measured: lateral, orbital, and medial frontal regions and frontal pole. Advancing age was strongly associated with decreases in the volume of the whole frontal cortex. Differential age effects on the volumes of frontal subregions were dependent on the method applied. According to the manual approach, age-related volume decreases were strongest in the lateral and orbital frontal gray matter. The semiautomatic and voxel-based analyses found that age effects were most prominent within the lateral frontal and cingulate regions. Overall, it was concluded that although semiautomated and voxel-based methods can provide a reasonable estimate of regional brain volume, they cannot serve as a substitute for manual volumetry. PMID- 12377142 TI - The lateral asymmetry of the human brain studied by volumetric magnetic resonance imaging. AB - Improvements in in vivo imaging methods have boosted research on brain asymmetry aimed at further establishing putative anatomical substrates for brain functional lateralization and particularly to explain left-hemisphere specialization for language. We analyzed volume asymmetries for major anatomical divisions of the lateral (perisylvian) brain region and their relative white matter content. A total of 100 healthy right-handed subjects were examined with 3D magnetic resonance imaging (MRI). The insular plane was used to limit the lateral brain, and the sylvian fissure and central sulcus to define frontal, parietal, temporal, and temporo-parieto-occipital regions. Results revealed a frontal region showing similar volumes in both hemispheres, a parietal region and a temporal region both larger in the left hemisphere, and a temporo-parieto-occipital region with predominantly right-sided asymmetry. Volume measurements of the parietal, temporal, and temporo-parieto-occipital regions complemented each other and accounted for 58% of planum temporale area variations. All study regions showed significant asymmetry for relative white matter content (percentage of white matter relative to region volume). White matter asymmetry, however, was particularly relevant for the frontal and temporal regions showing a highly frequent left-sided pattern (frontal region, 90%; temporal region, 91% of subjects). Leftward asymmetry in these two regions occurred in both genders, although hemisphere differences were significantly larger in men. Results from this MRI volume analysis of structural asymmetries in the lateral brain region complement data obtained by other methods and suggest a high occurrence of leftward asymmetry for relative white matter content in language-related regions. PMID- 12377143 TI - Functional imaging of the mechanisms underlying the bilateral field advantage. AB - Visual stimulus comparisons across the vertical meridian are faster and more accurate than those restricted to a single hemifield (the bilateral field advantage). We set out to investigate the cerebral mechanisms underlying this effect using functional magnetic resonance imaging. Seven normal volunteers were presented pairs of shape stimuli bilaterally across the vertical meridian and unilaterally within a single hemifield. We found a network of additional areas activated in the unilateral condition over the bilateral condition which have been related to working memory in previous studies. The results suggest different processing strategies with different temporal characteristics in the bilateral and unilateral conditions, providing a novel explanation for the bilateral field advantage. PMID- 12377144 TI - The metabolic substrates of bradykinesia and tremor in uncomplicated Parkinson's disease. AB - The pathophysiological mechanisms of bradykinesia and resting tremor, i.e., two major features of Parkinson's disease (PD), remain incompletely understood despite extensive studies, including functional imaging investigations. Using high-resolution positron emission tomography (PET) and [(18)F]fluoro-2 deoxyglucose (FDG) in 17 nondemented patients with uncomplicated PD on a stable therapeutic regimen, we measured the resting-state cerebral metabolic rate of glucose (CMRGlc), a validated marker of synaptic density/activity. Following formulation of distinct a priori hypotheses about potentially involved brain regions based on previous experimental and clinical literature, correlations between CMRGlc and objective scores of bradykinesia and tremor were searched in a voxel-based fashion using SPM99. Bradykinesia scores were significantly positively correlated with bilateral putamen and globus pallidum CMRGlc, while tremor scores were negatively correlated with bilateral putamen and cerebellar vermis CMRGlc. There was a large overlap of putamenal voxels significantly but inversely correlated with both extrapyramidal features. For both bradykinesia and tremor, the observed patterns of subcortical correlations largely concurred with our a priori hypotheses, and point to the major role of disruption of the striatofrontal and corticocerebellar pathways in the genesis of these extrapyramidal features. The direction of these correlations was not entirely expected, however, which may be due to the patients' being studied on medication, contrary to most studies performed to date. The observation that overlapping portions of the putamen inversely correlated with bradykinesia and tremor was a novel and striking finding which points to the complexity of the underlying pathophysiology of PD. Because it allows greater control of the neurological status, studying patients on medication may partly explain our findings. Voxel based analysis of resting FDG-PET holds considerable potential for assessing the neural substrates of motor impairment in PD. PMID- 12377145 TI - Neural correlates for the acquisition of natural language syntax. AB - Some types of simple and logically possible syntactic rule never occur in human language grammars, leading to a distinction between grammatical and nongrammatical syntactic rules. Comparison of the neuroanatomical correlates underlying the acquisition of grammatical and nongrammatical rules can provide relevant evidence on the neural processes dedicated to language acquisition in a given developmental stage. Until present no direct evidence on the neural mechanisms subserving language acquisition at any developmental stage has been supplied. We used fMRI in investigating the acquisition of grammatical and nongrammatical rules in the specified sense in 14 healthy adults. Grammatical rules compared with nongrammatical rules specifically activated a left hemispheric network including Broca's area, as shown by direct comparisons between the two rule types. The selective role of Broca's area was further confirmed by time x condition interactions and by proficiency effects, in that higher proficiency in grammatical rule usage, but not in usage of nongrammatical rules, led to higher levels of activation in this area. These findings provide evidence for the neural mechanisms underlying language acquisition in adults. PMID- 12377146 TI - Amplitopicity of the human auditory cortex: an fMRI study. AB - Whereas specialized frequency-encoding patterns in the human auditory cortex are generally accepted, termed tonotopicity, a similar principle of intensity encoding--amplitopicity--is debated controversially. This functional magnetic resonance imaging study describes the relationship of the activation volume and the spatial distribution of activated clusters under different sound pressure levels (SPL) across the temporal plane including the transverse temporal gyrus (TTG). Nine healthy subjects with no hearing deficiencies were investigated using an echo-planar imaging technique at 1.5 T. A boxcar stimulation paradigm was applied with a 5-Hz pulsed sine tone of 1000 Hz frequency at three SPLs of 70, 82, and 90 dB. Linear cross-correlation analysis (correlation coefficient > 0.3 corresponding to P < 0.08) of the functional data set revealed bilateral BOLD response within the auditory cortex of the nine subjects with moderate increase of activation volume for higher sound pressure levels. With increasing sound pressure a two-dimensional drift of cortical activation was observed (a) from the ventral to the dorsal edge and (b) from lateral to medial parts of TTG. This latero-medial drift therefore mimics the well-accepted principle of tonotopy for frequency-encoding neurons. This study demonstrates the existence of an amplitopic pattern of intensity-encoding neuronal clusters that in part resembles the tonotopic distribution of frequency-encoding neurons. This finding has to be integrated into the understanding of the auditory organization for the interpretation of higher auditory functions such as sound perception or speech. PMID- 12377147 TI - A quantitative comparison of simultaneous BOLD fMRI and NIRS recordings during functional brain activation. AB - Near-infrared spectroscopy (NIRS) has been used to noninvasively monitor adult human brain function in a wide variety of tasks. While rough spatial correspondences with maps generated from functional magnetic resonance imaging (fMRI) have been found in such experiments, the amplitude correspondences between the two recording modalities have not been fully characterized. To do so, we simultaneously acquired NIRS and blood-oxygenation level-dependent (BOLD) fMRI data and compared Delta(1/BOLD) (approximately R(2)(*)) to changes in oxyhemoglobin, deoxyhemoglobin, and total hemoglobin concentrations derived from the NIRS data from subjects performing a simple motor task. We expected the correlation with deoxyhemoglobin to be strongest, due to the causal relation between changes in deoxyhemoglobin concentrations and BOLD signal. Instead we found highly variable correlations, suggesting the need to account for individual subject differences in our NIRS calculations. We argue that the variability resulted from systematic errors associated with each of the signals, including: (1) partial volume errors due to focal concentration changes, (2) wavelength dependence of this partial volume effect, (3) tissue model errors, and (4) possible spatial incongruence between oxy- and deoxyhemoglobin concentration changes. After such effects were accounted for, strong correlations were found between fMRI changes and all optical measures, with oxyhemoglobin providing the strongest correlation. Importantly, this finding held even when including scalp, skull, and inactive brain tissue in the average BOLD signal. This may reflect, at least in part, the superior contrast-to-noise ratio for oxyhemoglobin relative to deoxyhemoglobin (from optical measurements), rather than physiology related to BOLD signal interpretation. PMID- 12377148 TI - Cortical processing of noxious somatosensory stimuli in the persistent vegetative state. AB - The persistent vegetative state (PVS) is a devastating medical condition characterized by preserved wakefulness contrasting with absent voluntary interaction with the environment. We used positron emission tomography to assess the central processing of noxious somatosensory stimuli in the PVS. Changes in regional cerebral blood flow were measured during high-intensity electrical stimulation of the median nerve compared with rest in 15 nonsedated patients and in 15 healthy controls. Evoked potentials were recorded simultaneously. The stimuli were experienced as highly unpleasant to painful in controls. Brain glucose metabolism was also studied with [(18)F]fluorodeoxyglucose in resting conditions. In PVS patients, overall cerebral metabolism was 40% of normal values. Nevertheless, noxious somatosensory stimulation-activated midbrain, contralateral thalamus, and primary somatosensory cortex in each and every PVS patient, even in the absence of detectable cortical evoked potentials. Secondary somatosensory, bilateral insular, posterior parietal, and anterior cingulate cortices did not show activation in any patient. Moreover, in PVS patients, the activated primary somatosensory cortex was functionally disconnected from secondary somatosensory, bilateral posterior parietal, premotor, polysensory superior temporal, and prefrontal cortices. In conclusion, somatosensory stimulation of PVS patients, at intensities that elicited pain in controls, resulted in increased neuronal activity in primary somatosensory cortex, even if resting brain metabolism was severely impaired. However, this activation of primary cortex seems to be isolated and dissociated from higher-order associative cortices. PMID- 12377149 TI - Enhanced spatial localization of neuronal activation using simultaneous apparent diffusion-coefficient and blood-oxygenation functional magnetic resonance imaging. AB - Functional MRI (fMRI) can detect blood oxygenation level dependent (BOLD) hemodynamic responses secondary to local neuronal activity. The most commonly used method for detecting fMRI signals is the gradient-echo echo-planar imaging (EPI) technique because of its sensitivity and speed. However, it is known that much of the signal obtained with this approach arises from large veins, with additional contribution from the capillaries and venules. Early experiments using diffusion-weighted gradient-echo EPI have suggested that intravoxel incoherent motion (IVIM) weighting can selectively attenuate contributions from large vessels based on the differences in the mobility of the blood within them, thereby revealing the contributions from hemodynamic changes in capillaries, which are in close spatial proximity to the activated neural tissue. Using this differential sensitivity of the various neurovascular compartments to IVIM weighting, we present a new approach for deriving functional maps of neural activity. This method is based on task-induced changes of the apparent diffusion coefficients (ADC), a signal that we demonstrate is generated in vascular compartments that only partially overlap with those generating the BOLD signal. The approach allows both the ADC-based maps and the more commonly used BOLD-based maps to be acquired simultaneously. The spatial overlap between these maps can be used to create composite maps that permit improved localization of the underlying neuronal activity patterns by identifying signals generated in those vascular components that are in closest proximity to the active neuronal populations of interest. PMID- 12377150 TI - Dietary caffeine consumption modulates fMRI measures. AB - Caffeine is the most widely used stimulant in the world. The stimulant effects of caffeine are mediated through its antagonistic properties on neuronal adenosine receptors. In addition, caffeine blocks neurovascular adenosine receptors and decreases cerebral perfusion. Although the effects of caffeine on blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging measures are extremely important, there are few studies addressing this issue in the literature. Because chronic caffeine use causes an upregulation of adenosine receptors, the differential effects of caffeine in low and high users is of particular interest. The present study was designed to test the hypothesis that caffeine has differential effects on the BOLD signal in high and low caffeine users. We demonstrated that the BOLD signal change in visual cortex was significantly greater in high users than in low users in the presence of caffeine. In addition, the magnitude of the BOLD signal was significantly correlated with caffeine consumption. We propose that the outcome observed here was due to an upregulation of adenosine receptors in high users, resulting in differential contributions of the neural and vascular effects of adenosine in the two study populations. PMID- 12377151 TI - Neural substrates of human facial expression of pleasant emotion induced by comic films: a PET Study. AB - Laughter or smile is one of the emotional expressions of pleasantness with characteristic contraction of the facial muscles, of which the neural substrate remains to be explored. This currently described study is the first to investigate the generation of human facial expression of pleasant emotion using positron emission tomography and H(2)(15)O. Regional cerebral blood flow (rCBF) during laughter/smile induced by visual comics and the magnitude of laughter/smile indicated significant correlation in the bilateral supplementary motor area (SMA) and left putamen (P < 0.05, corrected), but no correlation in the primary motor area (M1). In the voluntary facial movement, significant correlation between rCBF and the magnitude of EMG was found in the face area of bilateral M1 and the SMA (P < 0.001, uncorrected). Laughter/smile, as opposed to voluntary movement, activated the visual association areas, left anterior temporal cortex, left uncus, and orbitofrontal and medial prefrontal cortices (P < 0.05, corrected), whereas voluntary facial movement generated by mimicking a laughing/smiling face activated the face area of the left M1 and bilateral SMA, compared with laughter/smile (P < 0.05, corrected). We demonstrated distinct neural substrates of emotional and volitional facial expression and defined cognitive and experiential processes of a pleasant emotion, laughter/smile. PMID- 12377152 TI - Unmasking motion-processing activity in human brain area V5/MT+ mediated by pathways that bypass primary visual cortex. AB - Most models of the human visual system argue that higher-order motion-processing cortical regions receive their inputs only via the primary visual cortex (striate cortex), rather than also via direct projections from the thalamus that bypass primary visual cortex. However, recent evidence in non-human primates, along with some evidence in humans with damaged primary visual cortex (e.g., "blindsight" for motion in the blind visual hemifield), have argued for the existence of a direct thalamic-to-extrastriate projection for motion processing. This evidence remains controversial. Here we tested the idea that direct thalamic input to extrastriate motion processing areas exists in humans but might be masked in scalp recordings by activity from early visual areas. To do this, we employed stimuli that induced strong refractory effects in primary visual cortex--thereby creating a brief "reversable lesion" in primary visual cortex--immediately before the presentation of a motion stimulus. Under these conditions, we then assessed whether motion areas of cortex were still able to process the motion stimuli by recording event-related potentials (ERPs) and event-related magnetic fields (ERFs/MEG). We found robust motion-related activity in extrastriate motion processing areas in the ERP and MEG signals even when primary visual cortex was heavily suppressed by our manipulation. This finding provides evidence for a direct thalamic functional pathway to extrastriate visual cortical motion processing areas in the human that bypasses primary visual cortex. PMID- 12377153 TI - The influence of explicit instructions and stimulus material on lateral frontal responses to an encoding task. AB - In this functional magnetic resonance imaging study, we explored the effects of both stimulus material and encoding task demands on activation in lateral prefrontal cortex (PFC). Two factors were manipulated: material type and task instructions. Subjects encoded words or abstract figures (factor 1: stimulus type) and were required to make either a meaning-based or a form-based (letter or shape) decision about each stimulus (factor 2: task instructions). Abstract figures engendered significantly higher levels of right PFC activity than did words. This effect was seen for meaning-based and form-based processing tasks and was significantly greater for the former. We did not observe a differential response of left lateral PFC to verbal and pictorial material. A double dissociation, however, was found within left PFC. A ventrolateral region (within left inferior frontal gyrus) showed the highest levels of activity when words were processed according to their meaning whereas activity in a more dorsolateral region (within left middle frontal gyrus) was greatest when words were processed according to their form (constituent letters). We have therefore observed a main effect of material type in producing lateralized activation of frontal lobes, although the strength of this effect is sensitive to the nature of the task that subjects are asked to perform. Left-side lateral PFC activity is also sensitive to task instructions but this effect was specific to verbal material. The complex patterns of frontal effect counsel against any simple dichotomy of frontal function at the level of either material or task type. PMID- 12377154 TI - Evidence for anterior cingulate cortex involvement in monitoring preparatory attentional set. AB - An important cognitive function underlying unified, voluntary behavior is attentional control. Two frontal regions, anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC), appear to be particularly involved in attentional control and monitoring. In this study, we investigated whether ACC is involved in monitoring the preparatory allocation of attention during task switching, or whether ACC is active only when subjects are processing target stimuli and selecting a response, via a cued-attention design. Event-related BOLD fMRI activity was examined using a cue-target paradigm in which subjects performed task switches that selectively required reallocation of attention when tasks changed. There were three cue conditions: informative switch, informative repeat, and neutral. There were four target conditions: informatively cued switch, informatively cued repeat, neutrally cued switch, and neutrally cued repeat. Significant ACC activity was observed following both informative switch and informative repeat cues, but not after neutral cues. No significant ACC activity was observed following any of the target conditions. Significant DLPFC activity was observed following all three cue conditions and following neutrally cued switch targets. Overall, our results suggest that ACC is involved in monitoring the preparatory allocation of attention for conflict at the level of activation of competing attentional sets. The results also support the role of DLPFC in holding cognitive goals in working memory and allocating attention to the appropriate processing systems to meet those goals. PMID- 12377155 TI - Tracking transplanted stem cell migration using bifunctional, contrast agent enhanced, magnetic resonance imaging. AB - The ability to track stem cell transplants in the brain by in vivo neuroimaging will undoubtedly aid our understanding of how these cells mediate functional recovery after neural transplantation. One major challenge for the development and refinement of stem cell transplantation is to map the spatial distribution and rate of migration in situ. Here we report a method for tracking transplanted stem cells in the ischemia-damaged rat hippocampus by magnetic resonance imaging (MRI). Before transplantation, stem cells were labeled in vitro either with a novel bifunctional contrast agent, gadolinium rhodamine dextran (GRID), identifiable by both MRI and fluorescence microscopy, or with PKH26, visible exclusively under fluorescence microscopy. At different time points following engraftment, the brains were evaluated by both histology and ex vivo MR imaging. Transplanted stem cells were identified by MRI only if prelabeled with GRID, whereas fluorescence microscopy detected transplanted cells using either label. The distribution of GRID-labeled stem cells identified by MRI corresponded to those detected using fluorescence microscopy. These results demonstrate that GRID enhanced MRI can reliably identify transplanted stem cells and their migration in the brain. PMID- 12377156 TI - Neural systems underlying spatial language in American Sign Language. AB - A [(15)O]water PET experiment was conducted to investigate the neural regions engaged in processing constructions unique to signed languages: classifier predicates in which the position of the hands in signing space schematically represents spatial relations among objects. Ten deaf native signers viewed line drawings depicting a spatial relation between two objects (e.g., a cup on a table) and were asked either to produce a classifier construction or an American Sign Language (ASL) preposition that described the spatial relation or to name the figure object (colored red). Compared to naming objects, describing spatial relationships with classifier constructions engaged the supramarginal gyrus (SMG) within both hemispheres. Compared to naming objects, naming spatial relations with ASL prepositions engaged only the right SMG. Previous research indicates that retrieval of English prepositions engages both right and left SMG, but more inferiorly than for ASL classifier constructions. Compared to ASL prepositions, naming spatial relations with classifier constructions engaged left inferior temporal (IT) cortex, a region activated when naming concrete objects in either ASL or English. Left IT may be engaged because the handshapes in classifier constructions encode information about object type (e.g., flat surface). Overall, the results suggest more right hemisphere involvement when expressing spatial relations in ASL, perhaps because signing space is used to encode the spatial relationship between objects. PMID- 12377157 TI - Improved optimization for the robust and accurate linear registration and motion correction of brain images. AB - Linear registration and motion correction are important components of structural and functional brain image analysis. Most modern methods optimize some intensity based cost function to determine the best registration. To date, little attention has been focused on the optimization method itself, even though the success of most registration methods hinges on the quality of this optimization. This paper examines the optimization process in detail and demonstrates that the commonly used multiresolution local optimization methods can, and do, get trapped in local minima. To address this problem, two approaches are taken: (1) to apodize the cost function and (2) to employ a novel hybrid global-local optimization method. This new optimization method is specifically designed for registering whole brain images. It substantially reduces the likelihood of producing misregistrations due to being trapped by local minima. The increased robustness of the method, compared to other commonly used methods, is demonstrated by a consistency test. In addition, the accuracy of the registration is demonstrated by a series of experiments with motion correction. These motion correction experiments also investigate how the results are affected by different cost functions and interpolation methods. PMID- 12377158 TI - Reading anomalous sentences: an event-related fMRI study of semantic processing. AB - We report a random-effects analysis of an event-related fMRI study (n = 28) of cerebral activity during the reading of sentences that ended with a word that was either congruent or incongruent with the previous sentence context. Event-related potential studies have shown that this task elicits a late negativity peaking around 400 ms poststimulus (N400) that is larger for incongruent than for congruent sentence endings. A direct comparison of the activation for incongruent words versus that for congruent words revealed significantly greater activation for incongruent words than congruent words in bilateral inferior frontal and inferio-medial temporal cortex, left lateral frontal cortex, left posterior fusiform gyrus, bilateral motor cortex, and supplementary motor area. These results are consistent with data from intracranial electrical recording studies of the N400 electrical potential. The results are discussed as they relate to the localization of the cerebral sites underlying semantic processing in general and the localization of the scalp recorded N400 event-related potential in particular. PMID- 12377159 TI - Volition to action--an event-related fMRI study. AB - Current concepts of the anterior cingulate cortex (ACC) increasingly emphasize its role as an interface between limbic and neocortical functions. It has been pointed out that ACC activation reflects the intentional amount of effort (volition) that a subject uses in a task. In previous electrophysiological source localization investigations during a choice reaction task, we described a strong early activation in the ACC region approximately 120-150 ms after stimulus presentation. The degree of midline ACC activation correlated negatively with reaction time. This observation together with the finding that ACC activation precedes information processing in cortical association areas provided preliminary support to the notion that the extent of ACC activation is related to a subject's task engagement. However, due to the inverse problem and the relatively low spatial resolution of the electrophysiological measurements, we were not able to make inferences about the validity and the exact localization of the observed midline activation maximum. We addressed this question and performed an event-related fMRI study in six healthy volunteers during a visual choice reaction task. Two checkerboard stimuli were presented either in the left or right visual hemifield in randomized order and with an interstimulus interval requiring an appropriate motor response (left-right button press). A bilateral BOLD maximum was observed in the region of the supplementary motor area confluent with the neighboring motor area of the dorsal ACC. The degree of ACC activation correlated significantly with reaction time. These results are in line with our previous electrophysiological findings and provide further evidence that early ACC activation during a choice reaction task reflects the intentional effort of a subject to carry out a task. PMID- 12377160 TI - Neuroimaging reveals automatic speech coding during perception of written word meaning. AB - The extent to which visual word perception engages speech codes (i.e., phonological recoding) remains a crucial question in understanding mechanisms of reading. In this study, we used functional magnetic resonance imaging techniques combined with behavioral response measures to examine neural responses to focused versus incidental phonological and semantic processing of written words. Three groups of subjects made simple button-pressing responses in either phonologically (rhyming-judgment) or semantically (category-judgment) focused tasks or both tasks with identical sets of visual stimuli. In the phonological tasks, subjects were given both words and pseudowords separated in different scan runs. The baseline task required feature search of scrambled letter strings created from the stimuli for the experimental conditions. The results showed that cortical regions associated with both semantic and phonological processes were strongly activated when the task required active processing of word meaning. However, when subjects were actively processing the speech sounds of the same set of written words, brain areas typically engaged in semantic processing became silent. In addition, subjects who performed both the rhyming and the semantic tasks showed diverse and significant bilateral activation in the prefrontal, temporal, and other brain regions. Taken together, the pattern of brain activity provides evidence of a neural basis supporting the theory that in normal word reading, phonological recoding is automatic and facilitates semantic processing of written words, while rapid comprehension of word meaning requires devoted attention. These results also raise questions about including multiple cognitive tasks in the same neuroimaging sessions. PMID- 12377161 TI - A functional magnetic resonance imaging study of overt letter verbal fluency using a clustered acquisition sequence: greater anterior cingulate activation with increased task demand. AB - Regional cerebral activation during a cognitive task can vary with task demand and task performance. In a functional magnetic resonance imaging study, we examined the effect of manipulating task demand on activation during verbal fluency by using "easy" and "hard" letters. A "clustered" image acquisition sequence allowed overt verbal responses to be made in the absence of scanner noise which facilitated "on-line" measurement of task performance. Eleven right handed, healthy male volunteers participated. Twice as many errors were produced with hard as with easy letters (20.8 +/- 13.6 and 10.1 +/- 10.7% errors, respectively). For both conditions, the distribution of regional activation was comparable to that reported in studies of covert verbal fluency, but with greater engagement of subcortical areas. The hard condition was associated with greater dorsal anterior cingulate activation than the easy condition. This may reflect the greater demands of the former, particularly in terms of arousal responses with increased task difficulty and the monitoring of potential response errors. PMID- 12377162 TI - Structural gray matter differences between first-episode schizophrenics and normal controls using voxel-based morphometry. AB - The aim of this study was to compare the gray matter segments from T1 structural MR images of the brain in first-episode schizophrenic subjects (n = 34) and normal control subjects (n = 36) using automated voxel-based morphometry (VBM). This study is novel in that few studies have examined subjects in their first episode of schizophrenia. The subjects were recruited for the Edinburgh High Risk project and regional brain volumes were previously measured using a semi automated volumetric region of interest (ROI) method of analysis. The primary interest was to compare the results from the compatible parts of the ROI study and the primary VBM approach. Our secondary interest was to compare the results of a study-specific template that was constructed from the control group to those using the generic T1 template (152 Montreal Neurological Institute brains) supplied with SPM99 (statistical parametric mapping). The images were processed and statistically analyzed using the SPM99 program. VBM analysis identified significant decreases in gray matter in the schizophrenics relative to the normal control group at the corrected voxel level (P < 0.05) in the right anterior cingulate, right medial frontal lobe, left middle temporal gyrus, left postcentral gyrus, and the left limbic lobe. There were no increases in gray matter in the schizophrenics relative to the control group. The construction of a customized template appeared to improve the detection of structural abnormalities. The analyses were subsequently restricted to voxels within the amygdala-hippocampal complex using the SPM small-volume correction. This identified gray matter decreases in the schizophrenics, at the corrected voxel level (P < 0.05), in the left and right uncus and parahippocampal gyri and the right amygdala. These results are compatible with and extend the relevant findings of the previous volumetric ROI analysis, when allowing for the differences between the methods and interpretation of their results. PMID- 12377163 TI - Activity in the fusiform gyrus predicts conscious perception of Rubin's vase-face illusion. AB - We localized regions in the fusiform gyrus and superior temporal sulcus that were more active when subjects viewed photographs of real faces than when they viewed complex inanimate objects and other areas in the parahippocampal gyrus and the lateral occipital lobe that showed more activity during the presentation of nonface objects. Event-related functional magnetic resonance imaging was then used to monitor activity in these extrastriate visual areas while subjects viewed Rubin's vase-face stimulus and indicated switches in perception. Since the spontaneous shifts in interpretation were too rapid for direct correlation with hemodynamic responses, each reported percept (faces or vase) was prolonged by suddenly adding subtle local contrast gradients (embossing) to one side or the other of the figure-ground boundary, stabilizing the percept. Under these conditions, only face-selective areas in the fusiform gyrus responded more strongly during the perception of faces. To control for effects of the physical change to Rubin's stimulus (i.e., addition of embossing), we compared activity when the face contours were embossed after the subject had just reported the onset of perception of either faces or vase. Activity in the fusiform face area responded more strongly under the first condition, despite the fact that the physical stimulus sequences were identical. Moreover, on a trial-to-trial basis, the activity was statistically predictive of the subjects' responses, suggesting that the conscious perception of faces could be made explicit in this extrastriate visual area. PMID- 12377164 TI - Functional magnetic resonance neuroimaging of drug dependence: naloxone precipitated morphine withdrawal. AB - This study investigated the potential utility of fMRI as a neuroimaging technique to examine drug dependence using a robust animal model of drug withdrawal. Two groups of rats chronically pretreated with incremental doses of morphine sulfate (2, 7, 15, 30, 40, 50, 50, and 50 mg/kg--subcutaneous injection) were subjected to opioid precipitated withdrawal (using the opioid antagonist, naloxone) and subsequently behaviorally assessed or gradient-echo imaged under urethane anesthesia. Whole brain, group statistical parametric maps revealed statistically significant changes in signal intensity following administration of 1 mg/kg naloxone (corrected for multiple comparisons: P < 0.05, T > 5.03). Control groups within the fully crossed designs did not exhibit any statistically significant changes in behavior or signal intensity changes. Regional patterns of modulated activity include the retrosplenial, piriform, insular, entorhinal, cingulate, visual and auditory cortices, posterior fields of the hippocampus, and in particular the dentate gyrus. Such areas are consistent with biochemical correlates of morphine withdrawal and time profiles derived from our behavioral observations (P < 0.02). A notable lack of signal intensity changes in a number of subcortical areas suggests a possible confound associated with fMRI under anesthesia. This paper reports the first whole brain fMRI examination of an animal model of drug withdrawal, we believe there is considerable scope for extrapolation of our methods to a multitude of pharmacological applications-most notably in conjunction with other techniques in the development of potential therapeutic agents for drug dependence. PMID- 12377165 TI - Reduced P300 amplitude in a visual recognition task in patients with schizophrenia. AB - We studied top-down visual processes in schizophrenia by analyzing visual event related potentials (ERPs) during a gestalt recognition task, after subjects (patients with schizophrenia, n = 10; controls, n = 14) were trained to perceive three different geometrical shapes. Recognition performance of patients was poorer under both the figure and the nonfigure conditions then that of normal controls. ERPs analysis indicated that P300 amplitudes of the patients were significantly smaller than those of controls during correct figure detection trials. Moreover, topographical analysis of the differences in ERPs during the figure vs the nonfigure condition showed an earlier, more positive and more widely distributed P300 in controls than in patients with schizophrenia. Our study supports the misconnection hypothesis of schizophrenia and highlights the difficulty of the patients to refer to previous experience in order to filter incoming information. In a visual recognition task, this misconnection syndrome might induce a failure to integrate information stored in the frontal and prefrontal sites with incoming stimuli. PMID- 12377166 TI - An optimized individual target brain in the Talairach coordinate system. AB - The goal of regional spatial normalization is to remove anatomical differences between individual three-dimensional brain images by warping them to match features of a single target brain. Current target brains are either an average, suitable for low-resolution brain mapping studies, or a single brain. While a single high-resolution target brain is desirable to match anatomical detail, it can lead to bias in anatomical studies. An optimization method to reduce the individual anatomical bias of the ICBM high-resolution brain template (HRBT), a high-resolution MRI target brain image used in many laboratories, is presented. The HRBT was warped to all images in a group of 27 normal subjects. Displacement fields were averaged to calculate the "minimal deformation target" (MDT) transformation for optimization. The greatest anatomical changes in the HRBT, following optimization, were observed in the superior precentral and postcentral gyri on the right, the right inferior occipital, the right posterior temporal lobes, and the lateral ventricles. Compared with the original HRBT, the optimized HRBT showed better anatomical matching to the group of 27 brains. This was quantified by the improvements in spatial cross-correlation and between the group of brains and the optimized HRBT (P < 0.05). An intended use of this processing is to create a digital volumetric atlas that represents anatomy of a normal adult brain by optimizing the HRBT to the group consisting of 100+ normal subjects. PMID- 12377167 TI - Evaluation of the reference tissue models for PET and SPECT benzodiazepine binding parameters. AB - Recently, reference tissue methods have been proposed to estimate binding potential from PET data. A reference region without specifically bound ligand is used as an indirect input function to enable the expression of the time concentration curve of a region of interest using a compartment model. However, PET dopaminergic and serotoninergic studies have shown differences between binding potential (BP) values obtained with reference tissue methods and those obtained with conventional kinetic modeling using an arterial input function. In this study, we measured the BP values for the benzodiazepine receptors in seven subjects using PET [(11)C]flumazenil and SPECT [(123)I]iomazenil radioligands. We compared the BP values obtained using the reference tissue methods with those obtained using the conventional kinetic method. These values were also compared with the absolute value of receptor density, B'(max). For the PET studies, a multi-injection approach employing labeled and unlabeled flumazenil was used to estimate the main binding parameters, BP and B'(max). For SPECT studies, a single injection protocol of [(123)I]iomazenil was used to estimate BP values. The BP values were estimated using one- and two-tissue compartment models for the target region. Similar BP values were obtained using either the one- or two-tissue compartment model. This is probably due to the rapid equilibrium between tissue compartments reached with these radioligands. For PET and SPECT, these BP values were highly correlated (r > 0.960) to the BP values obtained using the arterial input function. We also found high correlations between the BP values obtained using the simplified reference tissue method and the receptor density parameter B'(max) (r > 0.884). However, the reference tissue methods yielded lower BP values than those obtained using the conventional approach. Moreover, there was a bias on BP values that was not a simple scaling. It seems that the physiological values found in gray matter structures using these radioligands give acceptable BP values. We conclude that the reference tissue methods should be carefully evaluated for each radioligand. PMID- 12377168 TI - Intervoxel heterogeneity of event-related functional magnetic resonance imaging responses as a function of T(1) weighting. AB - Inflow effects on activation-related BOLD signal changes in event-related fMRI experiments were assessed by varying the repetition time (TR) and flip angle (FA) values for gradient-echo echo-planar imaging (GE-EPI). Surprisingly, both increases and decreases were detected in these signal changes with increased T(1) weighting (reduced TR, increased FA). The well-known "positive" effect is attributed to inflow of fresh spins in the slice, leading to an apparent reduction in T(1). The "negative" effect is attributed to voxels containing pure parenchyma, where large-vessel inflow effects are very small and the BOLD effect is dominated by microvascular blood volume and oxygenation changes. Because blood T(1) is greater than tissue T(1) at 1.5 T, the fractional BOLD effect decreases with increased T(1) weighting. To aid in the interpretation of these experimental results, numerical simulations were performed based on a physiological multicompartment model, including pure tissue, large vessels (arteries, veins), microvessels (arterioles, capillaries, venules), and cerebrospinal fluid. PMID- 12377169 TI - Bach speaks: a cortical "language-network" serves the processing of music. AB - The aim of the present study was the investigation of neural correlates of music processing with fMRI. Chord sequences were presented to the participants, infrequently containing unexpected musical events. These events activated the areas of Broca and Wernicke, the superior temporal sulcus, Heschl's gyrus, both planum polare and planum temporale, as well as the anterior superior insular cortices. Some of these brain structures have previously been shown to be involved in music processing, but the cortical network comprising all these structures has up to now been thought to be domain-specific for language processing. To what extent this network might also be activated by the processing of non-linguistic information has remained unknown. The present fMRI-data reveal that the human brain employs this neuronal network also for the processing of musical information, suggesting that the cortical network known to support language processing is less domain-specific than previously believed. PMID- 12377170 TI - Fast, fully automated global and local magnetic field optimization for fMRI of the human brain. AB - The aim of this novel technique is to allow researchers, particularly those operating at high static magnetic field strengths on fMRI applications, to tailor the static magnetic field within the brain. The optimum solution for their experimental needs is reached, utilizing the full potential of the active shims at their disposal. The method for shimming human brain, which incorporates automatic brain segmentation to remove nonbrain tissue from the optimization routine, is presented and validated. The technique is fast, robust, and accurate, achieving the global minimum to a static field homogeneity function of the in vivo brain. Both global and specified local regions of the brain can be selected on which to optimize the shims without requiring skilled intervention. The effectiveness of the automated local shim is demonstrated in an olfactory fMRI study where significant activations in the orbitofrontal cortex were very clear when the above method was employed. PMID- 12377171 TI - Electromyography as a recording system for eyeblink conditioning with functional magnetic resonance imaging. AB - This study was designed to develop a suitable method of recording eyeblink responses while conducting functional magnetic resonance imaging (fMRI). Given the complexity of this behavioral setup outside of the magnet, this study sought to adapt and further optimize an approach to eyeblink conditioning that would be suitable for conducting event-related fMRI experiments. This method involved the acquisition of electromyographic (EMG) signals from the orbicularis oculi of the right eye, which were subsequently amplified and converted into an optical signal outside of the head coil. This optical signal was converted back into an electrical signal once outside the magnet room. Electromyography (EMG)-detected eyeblinks were used to measure responses in a delay eyeblink conditioning paradigm. Our results indicate that: (1) electromyography is a sensitive method for the detection of eyeblinks during fMRI; (2) minimal interactions or artifacts of the EMG signal were created from the magnetic resonance pulse sequence; and (3) no electromyography-related artifacts were detected in the magnetic resonance images. Furthermore, an analysis of the functional data showed areas of activation that have previously been shown in positron emission tomography studies of human eyeblink conditioning. Our results support the strength of this behavioral setup as a suitable method to be used in association with fMRI. PMID- 12377172 TI - Attention to action: specific modulation of corticocortical interactions in humans. AB - The prefrontal cortex may exert cognitive control by a general mechanism of attentional selection of neuronal representations. We used functional magnetic resonance imaging to test this hypothesis in the motor system. Normal volunteers were scanned during performance of a simple motor task, with their attention either directed towards their actions, or diverted towards a visual search task, or neither. Attention to action increased activity in prefrontal, premotor and parietal cortex, compared with unattended performance of the same movements. Analysis of cortical activity by structural equation modelling of regional fMRI time series was used to measure effective connectivity among prefrontal, premotor and parietal cortices. Attention to action enhanced effective connectivity between dorsal prefrontal cortex and premotor cortex, whereas non-motor attention diminished it. These effects were not attributable to common inputs from parietal cortex to the prefrontal and premotor cortex. The results suggest a supra-modal role for the dorsal prefrontal cortex in attentional selection, operating within the motor system as well as sensory and mnemonic domains. PMID- 12377173 TI - A H(2)(15)O positron emission tomography study on mental imagery of movement sequences--the effect of modulating sequence length and direction. AB - Motor imagery is a state of mental rehearsal of single movements or movement patterns and has been shown to recruit motor networks overlapping with those activated during movement execution. We wished to examine whether the brain areas subserving control of sequential processes could be delineated by pure mental imagery, their activation levels reflecting the processing demands of a sequential task. We studied six right-handed volunteers (39.0 +/- 14 years) with H(2)(15)O positron emission tomography (PET) while they continuously mentally pursued with their right hand one of five sequences differing in complexity (i.e., increases in sequence length, single-finger repetitions, and reversals). Conditions were repeated twice, alternating with two rest scans. Each imagined single motor element was paced at a frequency of 1 Hz. Significant activation increases (P < 0.05, corrected) associated with imagination of right finger movement sequences (conditions I to V combined)--compared to the rest condition- were observed in left sensorimotor cortex (M1/S1) and the adjacent inferior parietal cortex. Further activation increases (P < 0.001, uncorrected) occurred in bilateral dorsal premotor (PMd) cortex, left caudal supplementary motor area, bilateral ventral premotor cortex, right M1, left superior parietal cortex, left putamen, and right cerebellum. Activation decreases occurred in bilateral prefrontal and right temporo-occipital cortex. Activation increases that correlated with sequence complexity were observed only in specific areas of the activated network, notably in left PMd, right superior parietal cortex, and right cerebellar vermis (P < 0.05, corrected). In conclusion, our study, by varying the sequence structure of imagined finger movements, identified task-related activity changes in parietopremotor-cerebellar structures, reflecting their role in mediating sequence control. PMID- 12377174 TI - Functional MRI of auditory cortex activated by multisite electrical stimulation of the cochlea. AB - Electrical stimulation of the ear of deaf patients via cochlear implants offers a unique occasion to study activity of central auditory pathways with fMRI, without bias due to scanner noise. Such measurements, however, require one to control the possible interference between fMRI acquisition and the implanted electrodes. A series of measurements on a customized phantom designed to characterize the level of induced currents during MRI acquisition is presented. These experiments demonstrate that the major artifactual contribution is due to radiofrequency interaction and that safe experimental conditions can be obtained with proper shielding of the stimulation cables. The induced currents could be reduced to low levels (<50 microA for a duration <2 ms), below the acoustic perceptual threshold of cochlear implant subjects. Subsequent fMRI experiments on a patient using an Ineraid cochlear implant were conducted. Results revealed bilateral localized activation of the primary auditory cortex. Stimulation of two different intracochlear electrodes elicited activity in two neighboring, but different, regions, in agreement with the known tonotopical organization of the auditory cortex. This work paves the way for fMRI studies of a broad selection of auditory paradigms without interference from unwanted noise. PMID- 12377175 TI - Temporal sensitivity of event-related fMRI. AB - The temporal resolution of event-related fMRI is limited by the low sampling rate of typical MR whole brain protocols and by the slow rate of the BOLD response. Within the assumptions of the General Linear Model, we explore the tolerance of regression analyses of fMRI data to errors in the timing of the model relative to the experimental data under a number of circumstances. Given the sensitivity of the analysis to temporal shifts of the model relative to the data, one can search for the time a neuronal event occurs with temporal resolution on the order of a few hundred milliseconds with 95% confidence. This confidence level is strongly dependent on the signal-to-noise ratio of the observed BOLD responses (approximately +/-200 ms in our example of visual stimulation data collected at 1.5 T). PMID- 12377176 TI - Distributional assumptions in voxel-based morphometry. AB - In this paper we address the assumptions about the distribution of errors made by voxel-based morphometry. Voxel-based morphometry (VBM) uses the general linear model to construct parametric statistical tests. In order for these statistics to be valid, a small number of assumptions must hold. A key assumption is that the model's error terms are normally distributed. This is usually ensured through the Central Limit Theorem by smoothing the data. However, there is increasing interest in using minimal smoothing (in order to sensitize the analysis to regional differences at a small spatial scale). The validity of such analyses is investigated. In brief, our results indicate that nonnormality in the error terms can be an issue in VBM. However, in balanced designs, provided the data are smoothed with a 4-mm FWHM kernel, nonnormality is sufficiently attenuated to render the tests valid. Unbalanced designs appear to be less robust to violations of normality: a significant number of false positives arise at a smoothing of 4 and 8 mm when comparing a single subject to a group. This is despite the fact that conventional group comparisons appear to be robust, remaining valid even with no smoothing. The implications of the results for researchers using voxel based morphometry are discussed. PMID- 12377177 TI - Separating relational from item load effects in paired recognition: temporoparietal and middle frontal gyral activity with increased associates, but not items during encoding and retention. AB - Working memory is affected by items stored and the relations between them. However, separating these factors has been difficult, because increased items usually accompany increased associations/relations. Hence, some have argued, relational effects are reducible to item effects. We overcome this problem by manipulating index length: the fewest number of item positions at which there is a unique item, or tuple of items (if length >1), for every instance in the relational (memory) set. Longer indexes imply greater similarity (number of shared items) between instances and higher load on encoding processes. Subjects were given lists of study pairs and asked to make a recognition judgement. The number of unique items and index length in the three list conditions were: (1) AB, CD: four/one; (2) AB, CD, EF: six/one; and (3) AB, AD, CB: four/two, respectively. Japanese letters were used in Experiments 1 (kanji-ideograms) and 2 (hiragana-phonograms); numbers in Experiment 3; and shapes generated from Fourier descriptors in Experiment 4. Across all materials, right dominant temporoparietal and middle frontal gyral activity was found with increased index length, but not items during study. In Experiment 5, a longer delay was used to isolate retention effects in the absence of visual stimuli. Increased left hemispheric activity was observed in the precuneus, middle frontal gyrus, and superior temporal gyrus with increased index length for the delay period. These results show that relational load is not reducible to item load. PMID- 12377178 TI - The influence of nitrous oxide and remifentanil on cerebral hemodynamics in conscious human volunteers. AB - Remifentanil is increasingly used in the context of anesthesia, e.g., in patients presenting for MRI examinations, not only as an analgesic but also to replace nitrous oxide. Therefore, a comparative analysis of the effects of commonly used doses of remifentanil and of nitrous oxide on cerebral hemodynamics is warranted. The present study used contrast-enhanced magnetic resonance (MR) perfusion measurement to compare the effects of nitrous oxide (N(2)O/O(2) = 50%; n = 9) and remifentanil (0.1 microg/kg/min; n = 10) on regional cerebral blood flow (rCBF), regional cerebral blood volume (rCBV), and regional mean transit time (rMTT) in spontaneously breathing human volunteers. Remifentanil increased rCBF above all in basal ganglia, whereas in supratentorial gray matter the increase in rCBF was equal or even more pronounced when using nitrous oxide. In contrast, nitrous oxide produced a greater increase in rCBV in gray-matter regions than did remifentanil. In summary, nitrous oxide increased rCBV in all gray-matter regions more than did remifentanil. However, the increase in rCBF, especially in basal ganglia, was typically less pronounced than during infusion of remifentanil. PMID- 12377179 TI - Modeling the link between functional imaging and neuronal activity: synaptic metabolic demand and spike rates. AB - Functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) measurements reflect changes in the hemodynamics which are thought to be related to local synaptic input to neuron populations. The local neuronal spiking activity, which is believed to form the basis of neuronal coding and communication, is not directly reflected in fMRI/PET measurements. We used a mean field neuronal model of recurrently coupled excitatory and inhibitory neuronal populations to characterize the relationship between the synaptic activity (reflected in the PET and fMRI measurements) and the neuronal spike rates, averaged over brain areas. We analyzed this relation for a number of cases. For a single brain area and in the absence of external input to its inhibitory neurons, the relation between average spike rates and synaptic activity is linear. However, departures from linearity are found when: (i) the local synaptic strengths vary, (ii) the external inputs vary, in the presence of external input to the inhibitory population, or (iii) the synchronization between oscillations of the average spike rates of two areas changes. We further show that an increase in the imaging signal can reflect a decrease in average spiking activity, in the presence of external input to the inhibitory population. Synaptic activity can also be associated with silent neuronal populations, when input to the excitatory population does not reach the activation threshold or for certain synchronizations between oscillations of two areas. In conclusion, caution should be used when interpreting neuroimaging results in terms of mean spike rates. PMID- 12377180 TI - Reflective self-awareness and conscious states: PET evidence for a common midline parietofrontal core. AB - A recent meta-analysis has shown precuneus, angular gyri, anterior cingulate gyri, and adjacent structures to be highly metabolically active in support of resting consciousness. We hypothesize that these regions constitute a functional network of reflective self-awareness thought to be a core function of consciousness. Seven normal volunteers were asked to think intensely on how they would describe the personality traits and physical appearance of themselves and a neutral reference person known to all the subjects (the Danish Queen). During each of the four conditions cerebral blood flow distribution was measured by the intravenous H(2)(15)O PET scanning technique. During scanning, no sensory or motor activity was intended. After each scan, the subjects reported the contents of their thoughts during the scan to ascertain that the instructions had been followed. The results confirmed our hypothesis: Statistical parametric mapping showed differential activity in precuneus and angular gyri during reflection on own personality traits and in anterior cingulate gyri during reflection on own physical traits. Connectivity analysis of synchrony showed these regions to be functionally connected during reflective self-awareness. The commonality between the neural networks of the resting conscious state and self-awareness reflects the phenomenological concept of a fundamental contribution of reflective self awareness to the contents and coherence of the conscious state. PMID- 12377181 TI - Politics and scientific publishing: noli me tangere. PMID- 12377182 TI - Safe passage: a plea for safety in hemophilia gene therapy. PMID- 12377183 TI - A new cellular system opposing HIV infection: implications for gene transfer? PMID- 12377184 TI - The adeno-associated virus crystal: impact inversely proportional to size. PMID- 12377185 TI - Five recombinant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat. AB - The purpose of our study was to evaluate lentiviral vector-mediated rat retinal transduction using simian immunodeficiency virus (SIV) pseudotyped with envelope proteins from vesicular stomatitis virus G glycoprotein (VSV-G), Mokola virus G protein (MK-G), amphotropic murine leukemia virus envelope (4070A-Env), influenza A virus hemagglutinin (HA), lymphocytic choriomeningitis virus G protein (LCMV G), and RD114 retrovirus envelope (RD114-Env). The six pseudotyped lentivirus vectors carried CMV-driven green fluorescent protein (GFP) or beta-galactosidase (beta-gal) reporter genes. Intravitreal and subretinal injections of each pseudotyped recombinant SIV were performed in cohorts of Wistar rats. Our results showed that no transgene expression was detected after intravitreal injection of each pseudotyped SIV vector. Also, no transduction could be detected following subretinal injection of RD114 pseudotyped SIV vectors. However, selective transduction of retinal pigment epithelium (RPE) cells was repeatedly obtained after subretinal delivery of VSV-G, MK-G, 4070A-Env, HA, and LCMV-G pseudotyped SIV. GFP expression was maximum as soon as 4 days postadministration for VSV-G, MK-G, 4070A-Env, and HA pseudotypes, with no evidence of pseudotransduction for VSV-G. Maximum transgene expression was observed 3 weeks postinjection for LCMV 6. Importantly, HA and VSV-G pseudotyped SIV lead to such a high level of transgene expression that GFP-related toxicity occurred. Therefore, when a high level of GFP synthesis is achieved, replacement of enhanced GFP (egfp, Aequorea victoria) by a low-toxicity GFP (Renilla reniformis) cDNA is necessary to allow long-term expression. PMID- 12377186 TI - HSV amplicon-mediated delivery of LIGHT enhances the antigen-presenting capacity of chronic lymphocytic leukemia. AB - Chronic lymphocytic leukemia (CLL) is a B lymphocyte malignancy that remains a largely incurable disease. CLL B cells possess the ability to process and present tumor antigens but lack expression of costimulatory molecules, rendering them inefficient effectors of T-cell activation. We previously demonstrated that helper virus-free preparations of herpes simplex virus (HSV) amplicon vectors encoding CD40L efficiently transduce CLL B cells and render them capable of eliciting specific anti-tumor T-cell responses. LIGHT (TNFSF14), a member of the tumor necrosis factor (TNF) superfamily, efficiently activates both T cells and antigen-presenting cells (APCs). We employed an HSV amplicon vector expressing human LIGHT (hf-HSV-LIGHT) to transduce CLL B cells and compared the immunomodulatory function and T-cell activation induced by hf-HSV-LIGHT transduction to that observed with a CD40L-expressing HSV amplicon (hf-HSV CD40L). hf-HSV-LIGHT transduction induced expression of endogenous B7.1, B7.2, and ICAM.1 on CLL cells, albeit to a lesser degree than that observed in response to transduction with hf-HSV-CD40L. hf-HSV-LIGHT enhanced the antigen-presenting capacity of CLL B cells, as measured by induction of T-cell proliferation in an allogeneic mixed lymphocyte tumor reaction. Finally, hf-HSV-LIGHT-transduced CLL B cells successfully stimulated the outgrowth of autologous cytotoxic T lymphocytes in vitro. In aggregate, these data suggest that hf-HSV-LIGHT transduction may be useful for induction of immune responses to CLL and other B cell lymphoid malignancies. PMID- 12377187 TI - Ectopic osteogenesis using adenoviral bone morphogenetic protein (BMP)-4 and BMP 6 gene transfer. AB - Bone morphogenetic proteins (BMPs) delivered on scaffolds can induce ectopic bone formation after subcutaneous injection. Adenoviral vectors (Ad) carrying BMP2, BMP7, and BMP9 cDNAs have been shown to produce bone through endochondral ossification. The present study was performed to elucidate the histological events leading to ectopic ossification for two novel first-generation adenoviral constructs encoding BMPs, AdBMP4 and AdBMP6. In vitro, the viral constructs produced and secreted the mature BMP4 and BMP6 proteins. In vivo, the calf muscles of athymic nude rats were injected with AdBMP4, AdBMP6, AdBMP2, or AdlacZ. Rats were sacrificed 3, 6, 9, 16, 21, 60, and 90 days postinjection. Whereas AdBMP4 produced ectopic bone through mechanisms similar to endochondral ossification, AdBMP6 seemed to induce bone by way of mechanisms similar to both intramembranous and endochondral ossification pathways. At the relatively low vector dose used in this study, AdBMP2 caused an initial recruitment of primitive mesenchymal cells, without further development to bone. From computed tomographic analysis, AdBMP6 produced the most rapid tissue calcification. The ultimate density of ectopic bone formed by AdBMP4 and AdBMP6 was comparable. The current study demonstrates that AdBMP4 and AdBMP6 are more potent than the prototypical osteogenic adenoviral vector AdBMP2 and seem to induce ectopic bone by different mechanisms. PMID- 12377188 TI - Development of a melanoma-specific adenovirus. AB - Concerns regarding the hepatotoxicity of adenovirus for cancer gene therapy have led to attempts to engineer viruses for tissue-specific gene expression and tissue-specific replication. The Tyrex2 (a tandem murine melanocyte-specific enhancer) system was used to express luciferase, purine nucleoside phosphorylase (PNP), and the essential adenoviral gene E1A. In nonmelanoma cell lines, the CMV promoter/enhancer (CMV p/e) was 969 times stronger than the Tyrex2 construct, whereas in melanoma cells it was only 2.6 times stronger. An adenovirus with Tyrex2 regulating PNP (Ad2Tyr2-PNP) was tested for cytotoxicity. In melanoma cells, treatment with Ad2Tyr2-PNP plus the prodrug 6-methylpurine deoxyriboside (6-MPDR) resulted in 90% cytotoxicity by day 4. In non-melanoma cell lines, only the CMV p/e resulted in significant cytotoxicity. We compared the intrinsic E1A promoter/enhancer (E1A p/e) system with the melanoma-specific constructs and found that the Tyrex2 system achieved higher levels of luciferase than the E1A p/e in all melanoma lines tested. In non-melanoma cell lines, the E1A p/e is 12.4 times stronger than the Tyrex2 construct. Tyrex2 was then used to regulate adenoviral E1A expression to construct a melanoma-specific replicating adenovirus. We were unable to achieve selective replication. As E1A is a known transactivator of the adenovirus major late promoter (MLP), we studied the ability of the MLP to express a transgene in the context of tissue-selective E1A expression. We were able to demonstrate high levels of luciferase activity with this construct; however, selectivity was lost. Melanoma-specific adenovirus expression was achieved with the Tyrex2 construct, and this led to melanoma specific cytotoxicity by the potent PNP suicide gene. Selective melanoma-specific replication was not successful. The MLP may be a useful promoter in the context of a tissue-specific replicating adenovirus. PMID- 12377189 TI - Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters. AB - We previously demonstrated that chimeric tRNA(Lys3)-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNA(Lys3) promoter variants and compared their expression levels from the internal tRNA(Lys3) promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNA(Lys3)-ribozyme containing a mutation in the tRNA B box. As detected by fluorescent in situ hybridization, transcripts from a construct with the tRNA promoter alone localized strictly to the cytoplasm, whereas transcripts from dual U6/tRNA promoter were present in both the cytoplasm and the nucleus. Inhibition of HIV-1 correlates well with expression levels of the chimeric constructs. The results presented demonstrate that U6 and tRNA promoters can be placed in tandem for high-level expression of small RNA therapeutic transcripts. PMID- 12377190 TI - Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents. AB - Neovascularization characterizes diabetic retinopathy and choroidal neovascularization associated with age-related macular degeneration, the most common causes of severe visual loss in the developed world. Gene transfer to the eye using adeno-associated viral (AAV) vectors is a promising new treatment for inherited and acquired ocular diseases. We used an AAV vector with rapid onset and high levels of gene expression in the retina to deliver three anti-angiogenic factors (pigment epithelium-derived factor, tissue inhibitor of metalloproteinase 3, and endostatin) to the eyes of mice in a mouse model of retinopathy of prematurity. All three vectors inhibited ischemia-induced neovascularization. PMID- 12377192 TI - Improved performance of a fully gutted adenovirus vector containing two full length dystrophin cDNAs regulated by a strong promoter. AB - Dystrophin gene transfer using gutted or helper-dependent adenoviruses (HDAd), which have most of their genes deleted, is a promising approach to treat Duchenne muscular dystrophy. In an attempt to boost the amount of dystrophin produced after gene transfer, we have constructed a fully deleted HDAd (HDCBDys2x) containing two human dystrophin cDNAs controlled by the powerful hybrid cytomegalovirus enhancer/beta-actin promoter. We demonstrated high dystrophin expression after infection of muscle cultures with HDCBDys2x. Similarly, high (mean=583) and moderate (mean=124) numbers of muscle fibers were transduced in anterior tibialis muscle after intramuscular injection of HDCBDys2x in neonate and adult dystrophindeficient (mdx) mice 10 days postinjection. In fact, in the neonatally injected mdx mice, the transferred dystrophin was five times more abundant than in normal human muscle. However, the high early transduction level was transient in both animal groups, and we observed a humoral response against the human dystrophin. In contrast, we demonstrated sustained dystrophin expression in immunodeficient mouse muscles. Dystrophin expression of HDCBDys2x could be further increased in the presence of an E1/E3-deleted (first-generation) adenovirus, thus demonstrating that the latter vector synthesizes trans-acting enhancing factors. We have achieved abundant dystrophin expression with our new, improved HDAd. It is anticipated that high longterm transgene expression will be possible by employing weaker immunogenic transgenes. PMID- 12377191 TI - Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity. AB - The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 microl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracyclineoff promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy. PMID- 12377193 TI - Production of recombinant adeno-associated type 5 (rAAV5) vectors using recombinant herpes simplex viruses containing rep and cap. AB - We developed a scaleable production system for adeno-associated virus type 5 (AAV5)-based vectors using a replication-defective recombinant herpes simplex type 1 virus (rHSV) containing the rep and cap genes of AAV5. Multiple rHSV isolates containing AAV5 rep and cap with normal or altered p5 promoter elements were constructed and tested in vector production. Compared with rAAV5 vector yields obtained by plasmid transfection, yields of rAAV5 using any of the rHSV isolates were low. Evidence suggests that the low vector yields are a consequence of the extensive and early cytopathology induced by the rHSV isolates. In addition, we found a correlation between the amount of Rep52 or Rep40 proteins and the amount of vector produced by each rHSV isolate, suggesting that packaging of vector DNA into virus particles is rate-limiting when using rHSV to generate rAAV5 vectors. PMID- 12377194 TI - Tumor treatment with complexes of cationic lipid and noncoding plasmid DNA results in the induction of cytotoxic T cells and systemic tumor elimination. AB - We have demonstrated recently that treatment of established peritoneal mesothelial tumors with complexes composed of cationic lipid and noncoding plasmid DNA (pNull) results in the inhibition of tumor growth accompanied by the induction of a tumor-specific cellular immune response. In this study, treatment of mice bearing intraperitoneal (i.p.) M3 melanoma tumors with i.p. injections of lipid/pNull complex was found to inhibit tumor growth and induce the development of a cytolytic response against several M3 melanoma-associated antigens. Depletion of CD8(+) T cells, as opposed to natural killer (NK) or CD4(+) T cells, essentially abrogated the therapeutic effect of lipid+pNull complex, thus supporting the involvement of cytotoxic CD8(+) T cells in the antitumor response. The antitumor effect of lipid/pNull complex was maximal following delivery into a tumor-bearing compartment. For example, i.p. delivery of complex was more effective than intravenous (i.v.) or subcutaneous (s.c.) treatment of i.p. M3 tumors. In addition, i.v. injection of complex displayed therapeutic activity against lung metastases caused by i.v. injection of tumor cells, and intratumoral injection of complex into solid s.c. tumors caused regression in most animals. Importantly, the immune response induced by local treatment of tumors with complex also offered systemic protection against tumor cells at distal sites, as illustrated by the eradication of both peritoneal tumors and lung metastases in mice treated with complex delivered i.p. Treatment with lipid/pNull complex, therefore, represents an attractive immune-based treatment modality that could potentially be applied to many tumor types. PMID- 12377195 TI - OX40 ligation enhances primary and memory cytotoxic T lymphocyte responses in an immunotherapy for hepatic colon metastases. AB - Previously, we showed that the eradication of murine hepatic metastatic colon carcinomas was achieved by using a combination therapy with adenovirus-mediated gene delivery of interleukin 12 (IL-12) and anti-4-1BB agonistic antibody. However, the therapeutic efficacy was compromised severely when mice with tumors larger than 8 x 8 mm(2) were treated. In this report, we studied the effect of OX40 costimulation through agonistic anti-OX40 in combination with the IL-12 + anti-4-1BB immunotherapy on primary and memory anti-tumor cytotoxic T lymphocyte responses in a large-tumor setting (8 x 8 to 12 x 12 mm(2)). Tumor-infiltrating leukocytes (TILs) isolated from mice treated with the combination therapy of IL 12, anti-4-1BB, and anti-OX40 showed a significantly higher ex vivo direct cytotoxic T lymphocyte (CTL) activity against parental tumors, as compared with those from mice treated with IL-12 and anti-4-1BB. In vivo depletion of CD4(+)T cells during combination therapy significantly decreased the number of tumor infiltrating CD8(+)T cells and their cytotoxic activity in mice treated with IL 12 + anti-4-1BB + anti-OX40 combination therapy but not in the group treated with IL-12 + anti-4-1BB, which indicated that in vivo OX40 engagement on CD4(+) T cells led to the higher CTL responses observed in animals treated with IL-12 + anti-4-BB + anti-OX40 combination therapy. Furthermore, the combination therapy of IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in treated mice, as compared with treatment with IL-12 and anti-4-1BB. More importantly, long-term surviving mice from the combination therapy with IL-12, anti-4-1BB, and anti-OX40 exhibited higher memory CTL responses against parental tumor cells. The results demonstrated that OX40 ligation of CD4(+) T cells facilitated the development of primary and memory CTL responses against tumorcells and that coordinated immune activation by IL-12, anti-4-1BB, and anti OX40 resulted in a significantly higher survival rate in mice with large tumor burdens. Thus, the combination therapy with the adenovirus encoding IL-12 (Adv.mIL-12) + anti-4-1BB + anti-OX40 antibodies may provide a better treatment modality for patients with advanced cancers, often associated with a state of immune suppression or tolerance. PMID- 12377196 TI - Regulated expression of diphtheria toxin in prostate cancer cells. AB - Despite their known potential for effectively killing cells, the therapeutic use of plant and bacterial toxins for the treatment of cancer has been slow to enter the clinical setting. This has been due in large part to the lack of gene regulatory elements that control expression of highly toxic genes in a sufficiently tight manner, such that the toxins are only expressed in specific target cells. "Leaky" promoters result in unwanted and harmful cell death. In this study, we tested a novel gene therapy strategy aimed at expressing diphtheria toxin (DT-A) in androgen-independent prostate cancer cells that express the protein BCL2. This strategy relies on both transcriptional regulation and inducibly regulated DNA recombination mediated by the site-directed Flp recombinase to control expression of the toxin. Adenoviruses are used to introduce the genetic elements required for this approach into cultured cells and xenografts. Administration of 4-hydroxytamoxifen, resulting in recombination and expression of the toxin, effectively kills the cancer cells. Our results suggest that following androgen ablation therapy for the treatment of prostate cancer, use of a regulated recombination system to target expression of DT-A to androgen independent cancer cells would be an effective way to arrest the development of recurrent tumors. PMID- 12377198 TI - Odontogenic tumours in children and adolescents: a study of 78 Nigerian cases. AB - BACKGROUND: There is paucity of literature on odontogenic tumours in children and adolescents. Available records are difficult to compare due to differences in study criteria. To contribute to the records, a 20-year study of odontogenic tumours on the basis of the WHO classification (Kramer et al., 1992) in Nigerian African children and adolescents < or =18 years of age was undertaken. MATERIAL: A retrospective survey of oral/jaw tumours and allied lesions in children and adolescents < or =18 years of age seen at the Maxillofacial Unit, Ahmadu Bello University Teaching Hospital, Kaduna, Nigeria between 1979 and 1998. Data collected were histopathologic type, age, clinical features, radiologic appearance, treatment and record of recurrence. METHOD: Odontogenic tumours selected using the WHO classification were used for further study. Data were collected from case notes, radiographs, histopathologic reports and follow-up records. Information retrieved was used to complete a questionnaire and subjected to analysis. RESULTS: Two hundred and fifty-two (252) subjects < or =18 years were recorded, from which 78 (31%) had odontogenic tumours. Among seven types of odontogenic tumours seen, ameloblastoma (54%), odontogenic myxoma (19%) and adenomatoid odontogenic tumour (9%) were predominant. All patients seen were from 6 to 18 years with more than half (53%) between 15 and 18 years of age. A patient with multiple, bilateral odontomas of the maxilla and mandible resembling Herrmann's syndrome was recorded. Seventy-three patients were treated using enucleation (37%), dentoalveolar resection with preservation of lower border (15%) and segmental resection (48%). Five patients absconded after tumour diagnosis. No tumour recurrence was recorded in 65 treated cases followed-up for between 2 months and 10 years. CONCLUSION: This report shows that while ameloblastoma was the predominant odontogenic tumour, its frequency in Nigerian African children was lower than in the adult population. A case resembling Herrmann's syndrome is also presented. PMID- 12377199 TI - Maxillary ameloblastomas: a review of the literature and of a 15-year database. AB - INTRODUCTION: Cases of maxillary ameloblastomas from 15-year database (1986-2000) collected in the Department of Cranio-Maxillofacial Surgery of the University Hospital of Zurich were evaluated. PATIENTS: Twenty-six patients suffering from ameloblastoma had been collected. Five of them, had a maxillary ameloblastoma, three females and two males. METHODS: A clinical retrospective study was performed. In addition a review of the literature was undertaken and the findings have been compared and contrasted. PATIENTS: The overall incidence of ameloblastoma within the mandible (21) was four times higher than in the maxilla (5). In 69 per cent of the cases (18) it occurred in men, in 31 percent (8) in women. The sex ratio differed with the maxillary ameloblastomas: 40 percent male (2) and 60 per cent female (3). Although slow growing and nearly painless, it can reach a considerable size within the mid-face involving such highly specialized structures as the orbit, skull-base and brain. Wide resections with a safety margin of healthy bone to prevent local recurrence were undertaken. Nevertheless, recurrence was frequent due to invasion of the adjacent bone. CONCLUSION: On the one hand, a recurrence was found after a simple curettage of a 'dental cyst'. On the other hand, extensive bone destruction, involvement of the nasal cavity, the ethmoidal and sphenoidal sinuses, infiltration of the skull-base and distant metastasis were observed. The current treatment of choice is partial maxillectomy with a 10-15 mm safety margin of healthy bone including the alveolar ridge, the hard palate, the mucosa of the maxillary sinus and the lateral nasal wall. For the removal of tumours close to or invading the retromaxillary space the temporal approach gives ample access. PMID- 12377200 TI - Vertical distraction osteogenesis of fibula transplants for mandibular reconstruction--a preliminary study. AB - INTRODUCTION: When reconstructing the mandible after tumour resection with a fibular graft, the mandible is often vertically deficient, making placement of dental implants impossible. PATIENTS: Segmental vertical distraction of the reconstructed mandible was performed in nine patients following tumour surgery between February 1998 and 2001. Their age was 14-65 years (average 46.3); all underwent radiotherapy with a dose of up to 55.6 Gy prior to tumour resection. Mandibular discontinuity was repaired with a microvascular fibular bone graft. All grafts had a vertical bone deficit ranging from 9 to 12 mm when compared with the non-resected part of the mandibles. METHODS: All patients underwent segmental vertical distraction of the transplants. The distraction devices were applied intraorally. Distraction of 1.0 mm/day was performed using a Martin distractor (TRACK 1.5) followed by 12 weeks retention time. RESULTS: The increase of vertical bone height was stable and enabled placement of dental implants without any complications. CONCLUSION: Vertical distraction osteogenesis may become a common procedure in the treatment of alveolar ridge deficiency resulting from transplanting fibulae for mandibular reconstruction following tumour surgery. PMID- 12377201 TI - Distraction osteogenesis of the mandible: evaluation of callus distraction by B scan ultrasonography. AB - INTRODUCTION: Distraction osteogenesis is a new and reliable technique for lengthening of hard and soft tissue in cranio-maxillofacial surgery. Since its first applications, X-rays were often the preferred diagnostic method to monitor this treatment. Apart from adding an additional radiation dose, analysis cannot detect and follow the osteogenic process in the distraction gap on the one hand, nor can it evaluate the soft tissue around the distracted area. In this paper, the authors report their experience with B-scan imaging to control mandibular distraction and to overcome the shortcomings of X-rays analysis. METHOD: Comparison of B-scanning with the traditional X-ray methods was performed in 12 patients. RESULTS: B-scan evaluation appeared to be a precise technique in monitoring the different phases of distraction. It is easy to repeat whenever necessary. Early and late complications of soft tissue healing, movements of the bone segment, as well as the osteogenesis were easily detectable. CONCLUSION: In the authors' opinion B-scan evaluation can play an important role in monitoring distraction osteogenesis. PMID- 12377202 TI - Modification of the Hunsuck sagittal split osteotomy using a nerve hook. Technical note. AB - A modification of the Hunsuck mandibular sagittal split osteotomy is described by which a nerve hook is used to facilitate the optimum placement of the medial bone cut. The simple technique assists with the location of the lingula, protects the inferior alveolar nerve as it enters the mandibular foramen, and enables a bone cut to be placed into the mandibular foramen itself. PMID- 12377203 TI - Neurosensory alterations of the inferior alveolar and mental nerve after genioplasty alone or associated with sagittal osteotomy of the mandibular ramus. AB - AIMS: The purpose of our protocol is to study neurosensory disturbances following genioplasty, sagittal split mandibular osteotomy, or both procedures in combination. Many authors assessed the incidence and degree of neurosensory disturbances of the inferior alveolar nerve following orthognathic surgery but often results are difficult to interpret and compare due to a lack of standardization of methods. PATIENTS: Fifty patients (24 males and 26 females) were tested with qualitative (touch sensation, sharp/blunt test, cold sensation and hot sensation) and quantitative methods (localization test, two point static and dynamic test) at least 1 year after orthognathic surgery. The patients were divided into the following groups: 10 patients in group 1 (controls); 12 patients in group 2 (genioplasty alone or in association with maxillary osteotomy or vertical mandibular ramus osteotomy); 10 patients in group 3 (sagittal split osteotomy alone); 18 patients in group 4 (sagittal split osteotomy with concomitant genioplasty). METHOD: On both sides four areas were tested: centre of chin and lip (cutaneous and mucosal sides), 2 cm lateral to the chin centre (cutaneous and mucosal sides), 3 cm lateral to the chin centre i.e. approximately at the mental foramen (cutaneous and mucosal sides) and vermilion. Tests were always performed by the same person. All patients were also asked to indicate whether the altered sensation was considered subjectively as being disabling. RESULTS: None of the patients showed persistent anaesthesia in the tested areas according to the qualitative tests. In group 2 the quantitative sensory tests revealed normal or slight hypoaesthesia (17%) in all areas tested; in 30% of the patients of group 3, minimal quantitative sensory disturbances were noted, while the incidence of objective sensory deficits increased in patients who had undergone a concomitant genioplasty (40% among group 4). Among the tested areas the vermilion and oral commissure were affected most often in all groups. Statistical analysis (using STATA 6.0) revealed that these differences were significant (p<0.05). There were also significant differences between group 1 and groups 3 and 4 for tactile sensitivity, location tests and sharp-blunt discrimination, while two point discrimination (quantitative test) showed statistically significant differences between group 1 and all other groups (2-4). No statistically significant differences among the four groups were found for thermal sensation (hot and cold). CONCLUSIONS: The combination of genioplasty and sagittal split osteotomy seems to be more detrimental for the lip sensibility than genioplasty or sagittal split alone. Thermal sensation is less affected than tactile sensation, location and two point discrimination tests (static and dynamic). Despite that, sensory deficit was never considered as disabling by the patients subjectively. PMID- 12377204 TI - Effects of streptomycin on the rat infraorbital nerve. AB - INTRODUCTION: It has been reported that streptomycin can have anti-neuralgic effects. However, the mode of action is unknown. This article was intended to investigate eventual neurolytic effects of streptomycin on peripheral nerves when applied topically. MATERIAL AND METHOD: The rat infraorbital nerve, used as the experimental model, was treated with 0.01 ml of streptomycin sulphate dissolved either in distilled water or in lidocaine via an infraorbital perineural injection. Saline was used in control animals. RESULTS: Cross-sections of the nerve, a week following treatment with streptomycin, revealed damaged axons with disintegration of both heavily and thinly myelinated fibres. Signs of regeneration were detected from the fourth postoperative week on. In control rats no damage was observed. CONCLUSION: Streptomycin can cause peripheral nerve damage when injected perineurally. These morphological changes may be responsible for anti-neuralgic effects of streptomycin. PMID- 12377205 TI - Function of the great auricular nerve following surgery for benign parotid disorders. AB - INTRODUCTION: The main concern in benign parotid surgery is complete removal of the lesion whilst avoiding any harm to the facial nerve. Some time ago, surgeons also began to spare other structures including the great auricular nerve. The purpose of this paper was to study these procedures prospectively and to evaluate the sensory recovery of this nerve. MATERIAL: Fourteen patients undergoing parotid surgery with preservation of the great auricular nerve (group A) have been studied and compared with 10 patients whose operations involved sacrificing the nerve (group B). METHODS: The function of the great auricular nerve has been tested (qualitatively and quantitatively) before the operation, and postoperatively during the first few days and at 3, 6, 9 and 12 months. RESULTS: Twelve months postoperatively, no area of anaesthesia was found in group A, whilst all patients in group B had some degree of sensory loss. In group A, the qualitative and quantitative tests documented complete recovery of various types of surface sensitivity in 80% of cases, with the remaining 20% showing only a moderate reduction in comparison with the unoperated side. CONCLUSION: From this study, it seems reasonable to spare the great auricular nerve during parotid surgery for benign disease because the procedure takes very little time, but guarantees a major improvement of postoperative sensitivity of the region innervated by the great auricular nerve. PMID- 12377206 TI - The great auricular nerve; does it penetrate the parotid gland? An anatomical and microscopical study. AB - Conflicting opinions exist in the literature regarding the exact anatomical course of the great auricular nerve. The aim of this work was to study the pathway of its anterior branch and endings in relation to the parotid gland. MATERIAL AND METHODS: Thirty-seven specimens were dissected from 19 fresh adult cadavers and studied including the parotid gland at the region of the termination of the anterior branch of the nerve. The causes of death were not due to pathology in the parotid region. All the specimens were fixed in formaldehyde, serially cut, stained by haematoxylin and eosin, and examined by light microscope. RESULTS: In most of the glands (21/37 = 57%), there was no evidence of well-organized nerve fibres of the great auricular nerve inside the parotid gland. In a few (5/37 = 13%), nerve fibers were seen to penetrate the interlobular septa and in 30% of the cases (11/37) nerve bundles were found deep in the gland along side small ducts and close to thin-walled blood vessels. CONCLUSION: These findings prove that there are anatomical variations of the endings of the anterior branch of the great auricular nerve. The significance of nerve bundles deep in the gland along ducts and near vessels remains to be explored. PMID- 12377207 TI - Vascular endothelial growth factor induces protein kinase C (PKC)-dependent Akt/PKB activation and phosphatidylinositol 3'-kinase-mediates PKC delta phosphorylation: role of PKC in angiogenesis. AB - Activation of the protein kinase Akt/PKB mediates VEGF-dependent endothelial cell survival and eNOS activation. Here we examined the role of PKC in mediating VEGF induced Akt activation. The PKC inhibitors GF109203X and calphostin C inhibited VEGF-induced Akt activation. Rottlerin and Go6976, inhibitors with specificities for PKC delta and alpha, respectively, also strongly inhibited VEGF-induced Akt activation. VEGF-induced Akt activation was prevented by down-regulation of PKC induced by prolonged pretreatment with the phorbol ester, PMA. VEGF induced phosphorylation of PKC delta at Thr 505 in the activation loop, and this phosphorylation was inhibited by LY294002, suggesting that modulation of PKC delta activation by VEGF occurs distal to phosphatidylinositol 3'-kinase. PKC and PI3K inhibitors both strongly reduced the stimulation of branching tubulogenesis by VEGF in vitro. The finding that PKC mediates VEGF-induced Akt activation identifies a novel signal transduction pathway through which Akt can be regulated by growth factors acting through receptor protein tyrosine kinases, and indicates that PKC-mediated Akt activity may play an essential role in VEGF-stimulated angiogenesis. PMID- 12377208 TI - Endocytosis of apoE-EGFP by primary human brain cultures. AB - Apolipoprotein E (apoE), a well characterized protein, forms lipoprotein complexes with cholesterol. Such complexes formed are endocytosed via the LDL receptor family by many cell types in particular within the human central nervous system (CNS). The apoE-endocytic pathway leads to apoE degradation. However, it has recently been indirectly shown that apoE can be retained intracellularly and then re-secreted. To investigate the fate of endocytosed apoE isoforms E2 and E3 within human CNS cells in real-time, we added the CNS form of these apoE isoforms, linked to a green fluorescent protein (EGFP), to cultured human foetal brain tissue. There was bi-directional trafficking of apoE-EGFP in neuron and astrocyte processes and 'stationary' perinuclear vesicles in type-I astrocytes. Thus, active apoE recycling in cells with defined processes suggests a role for apoE in mediating signalling through receptor-mediated endocytosis. PMID- 12377209 TI - Monocytes modulate the in vitro basal frequency of sister chromatid exchanges of plasma leukocyte cultures and mitotic activity of suppressor-cytotoxic T8 human lymphocytes. AB - The effect of monocytes (MNs) on baseline SCEs and kinetics of human lymphocytes in plasma leukocyte (PLCs) and whole blood cultures (WBCs) was studied. Baseline SCEs in PLCs were nearly two-fold over WBCs. No differences in SCEs were observed between PLCs and MN-depleted PLCs, indicating that SCEs from PLCs are independent of MNs. MNs titration into PLCs decreased proportionally SCEs. Reconstitution of depleted PLCs with concentration of MNs equivalent or higher than those of PLC decreased SCEs. No variations of lymphocyte kinetics in PLCs were observed in the absence/presence of MNs. The proportion of B and T-cell subsets among interphasic lymphocytes were similar in PLC in the absence/presence of MNs, but a significant increase in the proportion of mitotic T8 lymphocytes was observed. Accordingly, MNs modulate both the in vitro basal SCEs and the mitotic activity of T8, but not their cell-cycle kinetics. PMID- 12377210 TI - Molecular identification of PpHDAC1, the first histone deacetylase fron the slime mold Physarum polycephalum. AB - The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression itself. PMID- 12377211 TI - Centrosome-dependent anisotropic random walk of cytoplasmic vesicles. AB - We approach the problem of an apparently random movement of small cytoplasmic vesicles and its relationship to centrosome functioning. Motion of small vesicles in the cytoplasm of BSC-1 cells was quantified using computer-assisted microscopy. The vesicles move across the cytoplasm frequently changing their directions with negligible net displacement. The autocorrelation function for consecutive velocities of individual vesicles becomes indistinguishable from zero in 10s. Variance in the displacement is proportional to time. The motion of vesicles is anisotropic: It has diffusivity along the radii drawn from the centrosome several times higher than the tangential diffusivity. This anisotropy is abolished by ultraviolet microbeam irradiation of the centrosome when the microtubule array loses radial structure. We conclude that the motion of the vesicles in the cytoplasm can be described as diffusion-like random walk with centrosome-dependent anisotropy. The present analysis quantitatively corroborates the 'trial and error' model of vesicular transport. PMID- 12377212 TI - Cell renewal and apoptosis in macrostomum sp. [Lignano]. AB - In platyhelminths, all cell renewal is accomplished by totipotent stem cells (neoblasts). Tissue maintenance is achieved in a balance between cell proliferation and apoptosis. It is known that in Macrostomum sp. the epidermis undergoes extensive cell renewal. Here we show that parenchymal cells also exhibit a high rate of cell turnover. We demonstrate cell renewal using continuous 5'bromo-2-deoxyuridine (BrdU) exposure. About one-third of all cells are replaced after 14 days. The high level of replacement requires an equivalent removal of cells by apoptosis. Cell death is characterized using a combination of three methods: (1). terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), (2). specific binding of phosphatidyl-serine to fluorescent labelled annexin V and (3). identification of apoptotic stages by ultrastructure. The number of cells observed in apoptosis is insufficient to explain the homeostasis of tissues in Macrostomum. Apoptosis-independent mechanisms may play an additional role in tissue dynamics. PMID- 12377213 TI - Specific monoclonal antibodies against the surface of rat islet beta cells. AB - Type 1 diabetes arises from the autoimmune destruction of islet beta cells, with the participation of both arms of the immune system. To better characterize the beta cell membrane, we have raised monoclonal antibodies to the surface of the INS-1 insulinoma cell line. Twenty-two such antibodies were produced, 21 of the IgG class, all reactive to different cell membrane proteins from INS-1 and neonatal islet cells, yielding identical electrophoresis patterns, with molecular weights mainly between 45 and 60 kD. We have focused on three such antibodies that recognize different protein targets, and are specific for islet beta cells. The target protein of antibody AA4, also found on monkey islets, is expressed at significantly higher levels on beta cells (55.8 vs 30.6% of cells, plus 3-4 fold increase in average fluorescence intensity per cell) when neonatal rat islet cells are incubated with high (16 mM vs 3mM) glucose concentrations. Further identification of the target antigens is in progress and is expected to shed more light on the properties of beta cell membrane proteins, and their probable participation in various disease processes. PMID- 12377214 TI - Increased superoxide radicals generation from alveolar macrophages in immature guinea-pigs. AB - To study the effect of maturation on abilities of superoxide radicals (O-2) generation in the airways, we compared stimuli-induced O-2 generation by alveolar macrophages in immature (aged 10+/-2 days) and adult (aged 90+/-2 days) guinea pigs. The production of O-2 was assayed by chemiluminescence method, using a Cypridina luciferin analog as a highly sensitive and specific probe for O-2. Whereas no significant difference in cell components of bronchoalveolar lavage fluid was observed between immature and adult animals, O-2 generation induced by stimulation of alveolar macrophages was greater in immature than in adult animals, with significant differences observed after platelet-activating factor (100 nM) or phorbol myristate acetate (0.5 micro g/ml). The results suggest that alveolar macrophages from immature animals are far more potent O-2 generators than the same cells of adult animals. PMID- 12377215 TI - Different h2 receptor antihistamines dissimilarly retard the growth of xenografted human melanoma cells in immunodeficient mice. AB - Melanoma cells and tissues contain considerable amounts of histamine and express histamine receptors, suggesting the existence of autocrine and paracrine regulation by histamine. Our previous in vitro results suggested that histamine elevates melanoma cell growth through the H2 receptor. In this work we show that in vivo tumour proliferation in immunodeficient mice xenotransplanted with a human melanoma cell line is diminished by cimetidine, an H2 receptor antagonist, if combined with a tamoxifen derivate acting on cytochrome p450 molecules (DPPE). Ranitidine, another H2 receptor antagonist, has a weaker inhibitory effect, the kinetics and mechanism of which is probably dissimilar to that of the cimetidine/DPPE mixture. PMID- 12377217 TI - Activation of channel catfish (Ictalurus punctatus) T cells involves NFAT-like transcription factors. AB - Cyclosporin A (CsA) specifically inhibits mammalian T cells by preventing activation of transcription factors (termed nuclear factor of activated T cells (NFAT)) involved in cytokine gene expression. In this study, catfish peripheral blood lymphocytes (PBL) and antigen specific T cells were treated with CsA to gain insights into the intracellular processes involved in fish T cell activation. To this end, CsA was observed to inhibit the in vitro proliferation of Con A stimulated catfish PBL, and specific alloantigen stimulated T cells. However, the inhibitory effect of CsA on catfish T cells was obviated by treatment with Con A, antigen activation or culture supernatant from activated catfish T cells prior to the addition of CsA. The use of a phosphatase assay coupled with Western blot analysis employing a polyclonal antibody to mammalian NFAT indicated that CsA prevents the dephosphorylation and subsequent nuclear translocation of an NFAT-like molecule in catfish T cells. Finally, a nuclear protein selection protocol demonstrated that a catfish NFAT-like protein binds to a known murine IL-2 promoter sequence. These results suggest that cytokines are involved in the activation of teleost T cells, and argue that T cell activation processes are conserved over a wide phylogenetic distance. PMID- 12377218 TI - Light chain promoter regions in rainbow trout Oncorhynchus mykiss: function of a classical and an atypical Ig promoter. AB - Three rainbow trout (Oncorhynchus mykiss) light chain isotypes (L1, L2 and L3) have been identified at the cDNA level. Genomic clones have previously been obtained for L1 and L2, revealing different structures of the V/J/C clusters and divergent putative promoter regions. While the L1 putative promoter has a classical Ig promoter structure with TATA, E-box, and octamer, the L2 putative promoter lacks typical features of the Ig promoter. The L2 putative promoter region contains a kappa-Y motif, an E-box and two putative non-consensus TATA boxes. In this study the isolation of a genomic clone that encompassed a VJC cluster of the recently described L3 isotype is described. The structure of the L3 putative promoter region is very similar to that of the L1 promoter. The transcriptional activities of the promoters of the trout L1 and L2 isotypes were compared. The promoter region of the L1 isotype showed a strong and predictable B cell-specific activity. Despite the unusual structure of the L2 V gene promoter, the transcriptional activity of it was much stronger in B-cells as compared to non B-cells. Deletion analyses of L2 promoter constructs showed that the region containing the kappa-Y element is critical for the transcriptional activity of the L2 promoter. Both L1 and L2 promoters can cooperate with a B cell-specific enhancer from Atlantic cod (Gadus morhua). PMID- 12377219 TI - Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement. AB - The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 degrees C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF. At 35 degrees C there was no difference between the total (TC) activity and the activity of the AP, but at 10 degrees C the TC was notably higher than the AP. Total activity peaked at 30 degrees C and gradually grew smaller towards 0 degrees C. The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 degrees C. When compared to human serum complement, the peak human TC activity at 37 degrees C was four times higher than the TC of rainbow trout at 30 degrees C. Human TC activity was 10.1-fold lower at 25 degrees C when compared to the activity at 37 degrees C. At 37 degrees C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 degrees C there was no difference between human TC and AP. In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 degrees C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered. PMID- 12377220 TI - Effects of ovotransferrin on chicken macrophages and heterophil-granulocytes. AB - Ovotransferrin (OTF) is an acute phase protein in chickens, serum levels of which increase in inflammation and infections. To understand the significance of OTF in inflammation, we studied its in vitro effects on HD11 cells, a macrophage cell line, and heterophils isolated from blood using a panel of variables indicative of cellular activation. These included the production of interleukin-6 (IL-6), nitrite, matrix metalloproteinase (MMP), oxidation of dichlorofluorescein diacetate for respiratory burst and the degranulation of heterophils by the loss of fluorescein isothiocyanate positive cytoplasmic granules. The results show that ovotransferrin stimulates the production of IL-6, nitrite and MMP by HD11 cells and augments phorbol ester-induced respiratory burst. Ovotransferrin stimulated heterophils to produce IL-6, and MMP, but failed to produce nitrite, enhanced respiratory burst activity and degranulation. These results suggest that ovotransferrin can modulate macrophage and heterophil functions in chickens. PMID- 12377221 TI - Differential effects of age on chicken heterophil functional activation by recombinant chicken interleukin-2. AB - Interleukin-2 (IL-2) exercises an array of biological effects on many cells including the functional activation of cells of the innate immune response. Heterophils, the avian equivalent of the neutrophil, function as professional phagocytes to aid in regulation of innate host defenses. The objective of the present studies was to examine the effects of recombinant chicken IL-2 (rChIL-2) on functional activities of heterophils from chickens during the first 3 weeks after hatch. Peripheral blood heterophils were isolated and incubated with either COS cell-derived rChIL-2 or supernatants from mock-transfected COS cells. rChIL-2 had no effect on the functional activities of heterophils from day-of-hatch chickens, but significantly increased the phagocytosis and bactericidal activity of heterophils from 7- and 14-day-old chickens. rChIL-2 induced no direct stimulation of the respiratory burst by heterophils, but primed heterophils from 7- and 14-day-old birds for an enhanced respiratory burst in response to phorbol ester stimulation. Lastly, rChIL-2 had neither direct nor priming effects on heterophil degranulation. The enhancing effects on heterophil functional activity by rChIL-2 were abated by a neutralizing anti-chicken IL-2 mAb and were therefore specific for this cytokine. These results show that rChIL-2 can directly activate chicken heterophils to exert effector functions, and that heterophil activation by rChIL-2 is also an age-dependent event. PMID- 12377222 TI - Characterisation of echidna IgM provides insights into the time of divergence of extant mammals. AB - The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cmu)was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cmus, and approximately 30% amino acid identity with Cmu from birds and reptiles. Phylogenetic analysis of mammalian Cmu provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cmu sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130mya, and Australian and American marsupials separated approximately 65mya. PMID- 12377223 TI - The gut-associated lymphoid tissues of the northern brown bandicoot (Isoodon macrourus). AB - The gut associated lymphoid tissues (GALT) of a juvenile bandicoot has been examined using histological and immunohistochemical techniques. The mesenteric lymph nodes were hyperfollicular and had well defined paracortical and medullary areas. Lymphocytes were densely packed throughout the cortex and paracortex and the mantles of the follicles. The GALT contained two distinct areas of tissue organisation. One consisted of large areas of aggregated follicles, whilst the other consisted of more linearly distributed follicles. The distribution of T and B cells in the tissue beds was documented using antibodies to surface markers CD3, CD5 and CD79b. T-cells were present in high numbers in the cortical region of the lymph node, whilst B-cells were predominant in the mantle of the follicles. Dispersed CD3 positive T-cells were abundant in the villi lacteals and present in high numbers in follicular areas of gut. CD79b positive B-cells were not observed in the lacteals but were abundant in the mantles of follicles. This is similar to that observed in other metatherians. PMID- 12377225 TI - 5alpha, 8alpha-Epidioxysterol sulfate from a diatom Odontella aurita. AB - A 5alpha,8alpha-epidioxysterol sulfate was isolated from the cultured diatom Odontella aurita (NIES 589), and its structure was elucidated by spectroscopic methods. PMID- 12377224 TI - Maternal prolactin composition can permanently affect epidermal gammadeltaT cell function in the offspring. AB - There have been few studies aimed at determining the effects of maternal peptide hormones on the developing fetus and even fewer aimed at determining the long term consequences of abnormalities in maternal hormone exposure. In this study, we have examined the effect of maternal prolactin (PRL) on the production, seeding and long-term function of a T lymphocyte subset for which the precursors are only present during fetal life. Using this system, we can determine long-term consequences of maternal hormone exposure without concern for the subsequent influence of the offspring's endocrine milieu. Recombinant versions of the two major forms of the pituitary hormone, PRL, were administered to rats throughout pregnancy. Administration of a molecular mimic of phosphorylated PRL (PP-PRL) resulted in a marked increase in the level of apoptosis in the thymus of newborn pups, an effect that was not duplicated by administration of unmodified PRL. The increased thymic apoptosis in the animals exposed to PP-PRL resulted in decreased epidermal seeding of gammadeltaT cells and a markedly decreased gammadeltaT cell modulated epidermal response in the offspring. This decreased gammadeltaT cell modulated response persisted to adulthood. We conclude that maternal PRL composition during pregnancy can have a permanent effect on at least one component of the developing immune system. PMID- 12377226 TI - 6-Methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone from Salvia aegyptiaca. AB - From the whole plant of Salvia aegyptiaca, 6-methylcryptoacetalide, 6-methyl epicryptoacetalide and 6-methylcryptotanshinone have been isolated and characterized, mainly by spectroscopic means. In addition to these novel diterpenoids, the known compounds 3beta-hydroxy-olean-12-en-28-oic acid, 3beta hydroxy-oleana-11,13(18)-dien-28-oic acid, sitosterol-3beta-glucoside, sitosterol, stigmasterol, 5-hydroxy-7,3',4'-trimethoxyflavone and 5, 6-dihydroxy 7,3',4'-trimethoxyflavone were isolated. PMID- 12377227 TI - ent-Kaurane diterpenoid glycosides from a Malagasy endemic plant, Cussonia vantsilana. AB - Four ent-kaurane diterpenoid glycosides, cussovantosides A-D, were isolated from the dried leaves of Cussonia vantsilana Baker, together with six known compounds. The structures of the compounds were deduced on the basis of their physical and spectral data. PMID- 12377228 TI - Jatrophane and lathyrane diterpenoids from Euphorbia hyberna L. AB - A new diterpene tetraester, from the jatrophane family, and two new diterpene triesters, with a lathyrane skeleton, have been isolated from the chloroform extract of the roots of Euphorbia hyberna L. The structures of these compounds have been established by spectroscopic methods, including 2D NMR experiments. PMID- 12377229 TI - 27-Nor-triterpenoid glycosides from Mitragyna inermis. AB - From the bark of Mitragyna inermis, two 27-nor-triterpenoid glycosides, named inermiside I (1) and II (2), were isolated and their structures determined based on extensive 2D-NMR and MS spectral analysis as 6-deoxy-beta-D-glucopyranosyl-[3 O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-pyrocincholate and 6-deoxy beta-D-glucopyranosyl-pyrocincholate, respectively. In addition, the known quinovic acid (6), 3-O-[beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl] quinovoic acid (3),beta-D-glucopyranosyl-[3-O-(beta-D-glucopyranosyl)]-quinoviate (4) and cytotoxic 3-O-(beta-D-6-deoxy-glucopyranosyl)-quinovic acid (5) were also isolated. PMID- 12377230 TI - Polyoxypregnane glycosides from the flowers of Dregea volubilis. AB - Three novel polyoxypregnane glycosides, volubiloside A, B and C (1-3), were isolated from the flowers of Dregea volubilis Linn., and their structures were elucidated as drevogenin D-3-O-beta-D-glucopyranosyl (1-->4)-6-deoxy-3-O-methyl beta-D-allopyranosyl (1-->4)-beta-D-cymaropyranosyl (1-->4)-beta-D cymaropyranoside, drevogenin D-3-O-beta-D-glucopyranosyl (1-->4)-6-deoxy-3-O methyl-beta-D-allopyranosyl (1-->4)-beta-D-cymaropyranosyl (1-->4)-beta-D digitoxopyranoside and drevogenin P-3-O-beta-D-glucopyranosyl (1-->4)-6-deoxy-3-O methyl-beta-D-allopyranosyl (1-->4)-beta-D-cymaropyranosyl (1-->4)-beta-D cymaropyranoside, respectively, on the basis of extensive NMR experiments, MALDI TOF MS, and some chemical strategies. PMID- 12377231 TI - neo-Clerodane diterpenoids from Baccharis flabellata. AB - Three diterpenoid derivatives were isolated from the acetone extract of Baccharis flabellata. Their structures were elucidated as 2,19;15,16-diepoxy-neo-clerodan 3,13(16),14-trien-18-oic acid, 15,16-epoxy-5,10-seco-clerodan-1(10),2,4,13(16),14 pentaen-18,19-olide and 15,16-epoxy-neo-clerodan-1,3,13(16),14-tetraen-18,19 olide through spectroscopic analyses. PMID- 12377232 TI - Sesquiterpenes from Omphalotus illudens. AB - Three sesquiterpenes, illudosone hemiacetal (1a), isoomphadione (2) and illudiolone (3) were isolated from the liquid culture extract of Omphalotus illudens. Their structures were elucidated by spectroscopic techniques as well as by X-ray crystallographic analysis. PMID- 12377233 TI - Triterpenoids from the leaves of Psidium guajava. AB - Two triterpenoids, 20beta-acetoxy-2alpha,3beta-dihydroxyurs-12-en-28-oic acid (guavanoic acid, 3), and 2alpha,3beta-dihydroxy-24-p-z-coumaroyloxyurs-12-en-28 oic acid (guavacoumaric acid, 7), along with six known compounds 2alpha hydroxyursolic acid (1), jacoumaric acid (2), isoneriucoumaric acid (4), asiatic acid (5), ilelatifol D (6) and beta-sitosterol-3-O-beta-D-glucopyranoside (8), have been isolated from the leaves of Psidium guajava. Their structures were determined through spectroscopic methods. Compound 5 showed dose-dependent (10 500 microg/ml) spasmolytic activity in spontaneously contracting isolated rabbit jejunum preparations. PMID- 12377234 TI - Flavonoids from the aquatic plant Eriocaulon buergerianum. AB - Four flavonoids including (2S)-3',4'-methylenedioxy-5,7-dimethoxyflavan and hispidulin 7-(6-E-p-coumaroyl-beta-D-glucopyranoside), and one tocopherol were isolated from the capitulum of Eriocaulon buergerianum KOERN. Their structures were established by spectral and chemical evidence. PMID- 12377235 TI - Cimiracemates A-D, phenylpropanoid esters from the rhizomes of Cimicifuga racemosa. AB - Four phenylpropanoid esters, cimiracemates A-D (1-4), along with three known compounds, isoferulic acid, ferulic acid and methyl caffeate were isolated from the EtOAc fraction of the rhizome of Cimicifuga racemosa. The structures of the esters were elucidated by means of spectral data, including 2D NMR spectroscopy. PMID- 12377236 TI - Xanthones from Swertia punctata. AB - Isolation of 1-O-primeverosyl-3,8-dihydroxy-5-methoxyxanthone and 1-O gentiobiosyl-3,7-dimethoxy-8-hydroxyxanthone, along with five known xanthones, isobellidifolin, methylbellidifolin, isoswertianin, methylswertianin and norswertianin-1-O-beta-D-glucoside, from the roots of Swertia punctata is reported. In the aerial parts four xanthones, bellidifolin, methylbellidifolin, swertianolin and mangiferin, and flavone-C-glucoside, isoorientin were identified. The chemotaxonomic and pharmacological significance of these results is discussed. PMID- 12377237 TI - Naphthalene glucoside and other phenolics from the shoot and callus cultures of Drosophyllum lusitanicum. AB - The callus and, for the first time established, shoot cultures of Drosophyllum lusitanicum Link. (Droseraceae) yielded new naphthalene glucoside-5-hydroxy-4 methoxy-2-naphthalenecarboxylic acid 5-O-beta-glucoside (drosophylloside) and 5 hydroxy-4-methoxy-2-naphthalenecarboxylic acid methyl ester besides other phenolics like naphthalenes-5-hydroxy-4-methoxy-2-naphthalenecarboxylic acid (ancistronaphthoic acid B), hydroplumbagin 4-O-glucoside, naphthoquinones plumbagin and 3-chloroplumbagin, C-glycosylflavones- vitexin, isovitexin, orientin and isoorientin. The pattern of phenolics found supports affinity of Drosophyllum to the families-Droseraceae, Ancistrocladaceae and Dioncophyllaceae. PMID- 12377238 TI - Dibenzylbutane and aryltetralone lignans from seeds of Virola sebifera. AB - Two lignans rel-(8R, 8'R)-3,4:3',4'-bis-(methylenedioxy)-7.7'-dioxo-lignan and (7'R,8'S,8S)-2'-hydroxy-3,4:4',5'-bis-(methylenedioxy)-7-oxo-2,7'-cyclolignan were isolated from seeds of Virola sebifera. The cyclolignan showed two atropisomers as determined by 1H NMR spectroscopy at low temperature. PMID- 12377239 TI - Flavonoid glycosides from Centaurea pseudoscabiosa subsp. pseudoscabiosa from Turkey. AB - Three flavonoid glycosides were isolated and characterized, together with a further 13 substances belonging to various classes of compounds, in particular two phenolic acids, a coumarin, a sugar and nine flavonoids from the flowered aerial parts of Centaurea pseudoscabiosa subsp. pseudoscabiosa Boiss. et Buhse (Asteraceae). Some considerations about their evolutionary meaning are provided. PMID- 12377240 TI - Alkaloids from Menispermum dauricum. AB - The alkaloids, dechloroacutumidine and 1-epidechloroacutumine, together with three known alkaloids, acutumidine, acutumine, and dechloroacutumine, were isolated from the rhizomes of Menispermum dauricum and their structures established by spectral and chemical methods. The cytotoxicity of each compound against the growth of human cell lines was studied, and acutumine selectively inhibited T-cell growth. PMID- 12377241 TI - A dinitrogenous alkaloid from Cyrtanthus obliquus. AB - The ethanolic extract of bulbs of Cyrtanthus obliquus (L.f.) Ait yielded the new dinitrogenous alkaloid obliquine (1), 3S, 4aS, 11S, 10bS-3,4,4a,13,11,5,6 heptahydro-5[2-(4-hydroxyphenyl)ethyl]-3-methoxy-13-methyl-[1,3-dioxolo[4,5 g]indolo[3,3a-c]-isoquinolin-12-one, together with the five known structures 11alpha-hydroxygalanthamine, 3-epimacronine, narcissidine, tazettine and trisphaeridine. All structures were established using 1D and 2D NMR techniques and HREIMS. The alkaloids were tested for cytotoxicity against two mammalian cell lines and did not show activity at concentrations up to 100 microg/ml. PMID- 12377242 TI - Glycosides from Dicliptera riparia. AB - The dimeric monoterpenoid glycoside, dicliripariside A, and two flavonoid glycosides, dicliriparisides B and C, together with six known compounds, beta sitosterol, 2,5-dimethoxy-p-benzoquinone, vanillic acid, daucosterol, lugrandoside and poliumonside, were isolated from the aqueous ethanol extract of the whole plants of Dicliptera riparia Nees. Their structures were determined based on analyses of spectroscopic data. PMID- 12377244 TI - Iridoid and phenolic diglycosides from Canthium berberidifolium. AB - An iridoid diglycoside, 6-O-beta-D-apiofuranosyl-mussaenosidic acid, and four phenolic diglycosides, canthosides A-D, were isolated from the aerial part of Canthium berberidifolium, along with seven known compounds. Structural elucidations were based on analyses of spectroscopic data. PMID- 12377243 TI - Water-soluble constituents of caraway: aromatic compound, aromatic compound glucoside and glucides. AB - From the water-soluble portion of the methanolic extract of caraway (fruit of Carum carvi L.), an aromatic compound, an aromatic compound glucoside and a glucide were isolated together with 16 known compounds. Their structures were clarified as 2-methoxy-2-(4'-hydroxyphenyl)ethanol, junipediol A 2-O-beta-D glucopyranoside and L-fucitol, respectively. PMID- 12377245 TI - Screening for schistosomiasis with questionnaires. AB - New schistosomiasis control initiatives have been launched to reduce significantly the current global burden of this disease, mainly through regular administration of praziquantel to schoolchildren living in endemic areas. Because schistosomiasis is distributed focally, epidemiological tools for rapid and inexpensive identification of communities at highest risk of morbidity are required to target praziquantel most efficiently. This article outlines the development and validation of simple questionnaires for screening of Schistosoma haematobium and Schistosoma mansoni in sub-Saharan Africa. PMID- 12377246 TI - Ten years of NGDO action against river blindness. AB - For a decade, a dozen non-governmental development organizations (NGDOs) have organized themselves into a Geneva-based coordination group with the goal of global control of onchocerciasis through mass distribution of ivermectin (Mectizan(R)). Members of this group have worked with Ministries of Health and other partners to empower communities affected by the disease to take responsibility for their own treatment. The NGDO Group has played a key role in the governance of international onchocerciasis control effort, particularly as a partner within the African Programme for Onchocerciasis Control. Ten years on, it is now time to take stock of activities, review the lessons learned and confront future challenges. PMID- 12377247 TI - New policies for using anthelmintics in high risk groups. AB - The 'Informal Consultation on the Use of Praziquantel during Pregnancy/Lactation, and Albendazole/Mebendazole in Children under 24 Months' was held 8-9 April 2002, in Geneva, Switzerland. PMID- 12377248 TI - Transgenic mosquitoes in controlling malaria transmission. PMID- 12377249 TI - The prospects of light from DARC. PMID- 12377251 TI - Vaccination against helminths: influence on HIV/AIDS and TB. PMID- 12377252 TI - Chemokines, prolactin and parasite interactions. PMID- 12377253 TI - Getting a measure of bednets in Africa. PMID- 12377254 TI - Understanding parasite strategies: a state-dependent approach? AB - Understanding and predicting parasite strategies is of interest not only for parasitologists, but also for anyone interested in epidemiology, control strategies and evolutionary medicine. From an ecological and evolutionary perspective, parasites are an important feature of their hosts' selective environment, and may have diverse roles, ranging from the evolution of host sex to host-sexual selection behavior. Generally, it is the hosts and their biology that have been the focus of these evolutionary investigations, but we approach the subject from the parasites' perspective, illustrating the sophistication of parasite strategies in dealing with contrasting and unpredictable environments. PMID- 12377255 TI - Neospora caninum: a cause of immune-mediated failure of pregnancy? AB - Resistance to many intracellular protozoan parasites is dependent on T helper cell 1 cytokine responses. This has important repercussions for pregnant females because strong T helper cell 1 cytokine responses are incompatible with successful pregnancy. Thus, there are two possible consequences of infection with protozoans such as Leishmania major, Plasmodium falciparum and Toxoplasma gondii during pregnancy: (1) pregnancy is compromised; or (2) resistance to the parasite is compromised. The apicomplexan Neospora caninum is a parasite renowned for its association with abortion in cattle. Furthermore, a major route of transmission for this parasite is congenital. The evidence for the hypothesis that T helper cell 1 cytokines play a role in these events is reviewed here. PMID- 12377256 TI - How useful is PCR in the diagnosis of malaria? AB - Polymerase chain reaction (PCR) assays are the most sensitive and specific method to detect malaria parasites, and have acknowledged value in research settings. However, the time lag between sample collection, transportation and processing, and dissemination of results back to the physician limits the usefulness of PCR in routine clinical practice. Furthermore, in most areas with malaria transmission, factors such as limited financial resources, persistent subclinical parasitaemia, inadequate laboratory infrastructures in the poorer, remote rural areas preclude PCR as a diagnostic method. Even in affluent, non-endemic countries, PCR is not a suitable method for routine use. Nonetheless, PCR could be clinically useful in selected situations. PMID- 12377257 TI - Canine leishmaniasis: epidemiological risk and the experimental model. AB - Increasing risk factors are making zoonotic visceral leishmaniasis a growing public health concern in many countries. Domestic dogs constitute the main reservoir of Leishmania infantum and Leishmania chagasi, and play a key role in the transmission to humans. New reagents and tools allow the detailed investigation of canine leishmaniasis, permitting the monitoring of the immunological status of dogs in both natural and experimental infections. Such studies are essential to determine the basis of the canine protective immune response and to establish a laboratory model, a significant aspect for the development of vaccines against canine leishmaniasis. PMID- 12377258 TI - The clonal theory of parasitic protozoa: 12 years on. AB - The question of population structure in parasitic protozoa has recently gained a renewed topicality with significant contributions on medically important pathogens, such as Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. The proposals that initiated this debate are reviewed here and the subsequent developments of the clonal theory, in light of recent contributions, are examined. PMID- 12377259 TI - Selective pressures that decrease synonymous mutations in Plasmodium falciparum. AB - Rich and Ayala propose that the zero rate of non-amino-acid-changing (synonymous) mutations in some proteins of Plasmodium falciparum reflects a recent population bottleneck. Alternatively, Arnot and Saul propose sequence conservation in response to selective pressures other than the pressure to encode protein. Among these are fold pressure and purine-loading pressure. Genomes adapt to these by acquisition of introns and/or low-complexity (simple-sequence) segments in proteins. Adaptive explanations include facilitation of intragenic recombination (and hence diversification of the encoded protein) by DNA stem-loop secondary structures. PMID- 12377264 TI - Nicotine's oxidative and antioxidant properties in CNS. AB - Nicotine has been reported to be therapeutic in some patients with certain neurodegenerative diseases and to have neuroprotective effects in the central nervous system. However, nicotine administration may result in oxidative stress by inducing the generation of reactive oxygen species in the periphery and central nervous system. There is also evidence suggesting that nicotine may have antioxidant properties in the central nervous system. The antioxidant properties of nicotine may be intracellular through the activation of the nicotinic receptors or extracellular by acting as a radical scavenger in that it binds to iron. The possibility that nicotine might be used to treat some symptoms of certain neurodegenerative diseases underlies the necessity to determine whether nicotine has pro-oxidant, antioxidant or properties of both. This review discusses the studies that have addressed this issue, the behavioral effects of nicotine, and the possible mechanisms of action that result from nicotine administration or nicotinic receptor activation. PMID- 12377265 TI - Oxytocin secretion induced by osmotic stimulation in rats during the estrous cycle and after ovariectomy and hormone replacement therapy. AB - Hyperosmolality is a potent stimulus for the secretion of oxytocin. Oxytocinergic neurons are modulated by estrogen and oxytocin secretion in rats varies according to the phase of the estrous cycle, with higher activity during proestrus. We investigated the oxytocin secretion induced by an osmotic stimulus (0.5 M NaCl) in female rats. Plasma oxytocin and the oxytocin contents in the neurohypophysis and the paraventricular and supraoptic nuclei were determined during the morning (8-9 h) and afternoon (17-18 h) of the estrous cycle and after ovariectomy followed or not by hormone replacement. Plasma oxytocin peaked in control animals during proestrus. Oxytocin content decreased in the paraventricular and supraoptic nuclei during proestrus and estrus compared to diestrus and increased in the neurohypophysis during proestrus morning. No significant difference was observed in the oxytocin content of the neurohypophysis, nuclei or plasma between ovariectomized animals and ovariectomized animals treated with estrogen or estrogen plus progesterone. Therefore, any ovarian factor other than estrogen or progesterone seems to play a direct or indirect role in the increase in oxytocin secretion. The osmotic stimulus caused an increase in plasma oxytocin throughout the estrous cycle. A reduction in oxytocin content during diestrus and an increase during proestrus were observed in the paraventricular nuclei. In ovariectomized animals, the treatment with estrogen potentiated the response of oxytocin to the osmotic stimulus, with the response being even stronger in the case of estrogen plus progesterone. In conclusion, the ovarian steroids estrogen plus progesterone could modulate the osmoreceptor mechanisms related to oxytocin secretion. PMID- 12377266 TI - Nitric oxide synthase (NOS) coexists with activated neurons by skeletal muscle contraction in the brainstem of cats. AB - Contraction of skeletal muscle evokes increases in arterial blood pressure and heart rate. Some regions of the brainstem have been implicated for expression of the cardiovascular responses to muscle contraction. Previous studies have reported that static muscle contraction induced c-Fos protein in the nucleus of tractus solitarii (NTS), lateral reticular nucleus (LRN), lateral tegmental field (FTL), subretrofacial nucleus (SRF), A1 region and periaqueductal gray (PAG) of the brainstem. Furthermore, neuronal NADPH-diaphorase (NADPH-d), which is considered as a marker of neuronal nitric oxide synthase (nNOS), has been localized in those same regions. In this study, static muscle contraction was induced by electrical stimulation of the L7 and S1 ventral roots in anaesthetized cats. Distribution of c-Fos protein within neurons containing nNOS was evaluated by double labeling methods in order to determine if nNOS containing neurons in the brainstem were activated during muscle contraction. The results indicate that c-Fos protein colocalized with NADPH-d positive staining within the neurons of the SRF and PAG, but not within the NTS neurons. Distinct number of neurons with c-Fos protein was in close proximity to NADPH-d positive staining in the NTS, SRF, and PAG. Coexisting of c-Fos protein and NADPH-d positive staining was not observed in the LRN, FTL and A1 region. These findings demonstrate that nNOS containing neurons were activated by muscle contraction in the selective regions of the brainstem, and nNOS positive staining had close anatomic contacts with the neurons activated by contraction. This result provides neuroanatomic evidence suggesting that nitric oxide modulates the cardiovascular responses to muscle contraction within the NTS, SRF and PAG of the brainstem. PMID- 12377267 TI - Gastroprotective effect of Neem (Azadirachta indica) bark extract: possible involvement of H(+)-K(+)-ATPase inhibition and scavenging of hydroxyl radical. AB - The antisecretory and antiulcer effects of aqueous extract of Neem (Azadirachta indica) bark have been studied along with its mechanism of action, standardisation and safety evaluation. The extract can dose dependently inhibit pylorus-ligation and drug (mercaptomethylimidazole)-induced acid secretion with ED(50) value of 2.7 and 2 mg Kg(-1) b.w. respectively. It is highly potent in dose-dependently blocking gastric ulcer induced by restraint-cold stress and indomethacin with ED(50) value of 1.5 and 1.25 mg Kg(-1) b.w. respectively. When compared, bark extract is equipotent to ranitidine but more potent than omeprazole in inhibiting pylorus-ligation induced acid secretion. In a stress ulcer model, it is more effective than ranitidine but almost equipotent to omeprazole. Bark extract inhibits H(+)-K(+)-ATPase activity in vitro in a concentration dependent manner similar to omeprazole. It offers gastroprotection against stress ulcer by significantly preventing adhered mucus and endogenous glutathione depletion. It prevents oxidative damage of the gastric mucosa by significantly blocking lipid peroxidation and by scavenging the endogenous hydroxyl radical ((z.rad;)OH)-the major causative factor for ulcer. The (z.rad;)OH-mediated oxidative damage of human gastric mucosal DNA is also protected by the extract in vitro. Bark extract is more effective than melatonin, vitamin E, desferrioxamine and alpha-phenyl N-tert butylnitrone, the known antioxidants having antiulcer effect. Standardisation of the bioactive extract by high pressure liquid chromatography indicates that peak 1 of the chromatogram coincides with the major bioactive compound, a phenolic glycoside, isolated from the extract. The pharmacological effects of the bark extract are attributed to a phenolic glycoside which is apparently homogeneous by HPLC and which represents 10% of the raw bark extract. A single dose of 1g of raw extract per kg b.w. (mice) given in one day and application of 0.6g raw extract per kg b.w. per day by oral route over 15 days to a cumulative dose of 9g per kg was well tolerated and was below the LD(50). It is also well tolerated by rats with no significant adverse effect. It is concluded that Neem bark extract has therapeutic potential for the control of gastric hyperacidity and ulcer. PMID- 12377268 TI - Evidence for involvement of 5-hydroxytryptamine(1B) autoreceptors in the enhancement of serotonin turnover in the mouse brain following repeated treatment with fluoxetine. AB - The effect of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine on synthesis and turnover of 5-hydroxytryptamine (5-HT) was studied in the mouse brain in vivo. The concentration of 5-hydroxytryptophan (5-HTP), 5 hydroxyindoleacetic acid (5-HIAA) and 5-HT was measured in hypothalamus, hippocampus and frontal cortex after inhibition of the aromatic amino acid decarboxylase activity with m-hydroxybenzylhydrazine (NSD 1015). Fluoxetine 6.9 mg/kg s.c. was injected once daily for three weeks. Three days after the final daily injection of fluoxetine 5-HT synthesis (5-HTP accumulation) and turnover (5 HIAA/5-HT ratio) were significantly enhanced compared with saline-treated mice. The 5-HIAA/5-HT ratio was already significantly elevated after 3 days of fluoxetine treatment and continued to increase during treatment for 2-3 weeks. The increase in 5-HIAA/5-HT ratio was considerably larger (150-200% of controls) than the increase in 5-HTP accumulation (110-120%), which reached significance only after 3 weeks of treatment. The increase in 5-HT synthesis may be secondary to that of the turnover. The 5-HIAA/5-HT ratio returned to control values after a 14 days washout period. Simultaneous treatment with the long-acting 5-HT(1B) receptor antagonist, SB 224289 for 14 days counteracted the fluoxetine-induced increase in 5-HIAA/5-HT ratio that indicates involvement of 5-HT(1B) autoreceptors in the development of this increase. It is proposed that the fluoxetine-induced enhancement of 5-HT turnover was evoked by the long-lasting stimulation of 5-HT(1B) autoreceptors that resulted in an intraneuronal compensatory adaptation of the basal 5-HT release. PMID- 12377269 TI - Rat kidney antioxidant response to long-term exposure to flavonol rich red wine. AB - This study evaluated the antioxidant defense system of the rat kidney following chronic exposure to red wine rich in flavonols. Both ethanol and antioxidant non alcoholic wine components, mainly polyphenols, could contribute to the antioxidant status of kidney. Adult rats were given separately, water, ethanol (12.5%), red wine or alcohol-free red wine. After ten weeks of treatment, blood samples were obtained to determine plasma antioxidant capacity (FRAP, ferric reducing ability of plasma), uric acid and ethanol levels. Kidney tissues (cortex and papilla) were separated to perform measurements of reduced glutathione (GSH), glutathione disulfide (GSSG), lipid peroxidation (malondialdehyde, MDA) and the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The activity of (Na + K)-ATPase, a membrane-bound enzyme, was also assessed. Red wine in plasma, elevated the FRAP without changing the concentration of uric acid; in kidney, it diminished the MDA production and elevated the GSH/GSSG ratio and the activity of CAT and GSH-Px. The activity of SOD did not change. Despite the finding that renal (Na + K)-ATPase activity was upregulated by ethanol, it was not altered by either red wine or alcohol-free red wine. The effects on the antioxidant enzymes could be attributed to ethanol, but the increase in the FRAP and GSH/GSSG ratio is attributed to the non-alcoholic components of red wine. These data suggest that there is an enhancement of the antioxidant defense potential in kidney and plasma, after chronic red wine consumption. Both ethanol and the non-alcoholic antioxidant constituents of red wine could be responsible for these effects. PMID- 12377270 TI - Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes. AB - It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFalpha and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2', 7'-dichlorofluorescin (DCFH) to 2', 7'-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (p<0,001) while aqueous extract (50 microg/ml) by 43% (p<0,01). The ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production in monocytes by 76% (p<0,01). Effective concentrations (25-100 microg/ml) were well below the cytotoxic levels of the extracts which started at 1 mg/ml as assessed by LDH leakage and trypan blue exclusion. Penetration of some active substances into the cells was necessary for inhibition to take place as juged from the effect of preincubation time. These results demonstrate that artichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes. PMID- 12377272 TI - Dictyocaulus viviparus: re-emerging or never been away? AB - For over 40 years a highly effective vaccine against the bovine lungworm has been commercially available. The use of it successfully reduced the number of outbreaks in calves. However, the past decade has seen a dramatic increase in lungworm outbreaks in adult cows in the UK. This might indicate that Dictyocaulus viviparus is re-emerging as a significant parasite in the dairy cattle industry. Much is still unknown, and here the most important aspects requiring urgent attention are put into perspective. PMID- 12377271 TI - Biochemical and functional characterization of alpha-adrenergic receptors in the rabbit vagina. AB - Vascular and non-vascular smooth muscle within the vagina mediate important physiological changes during sexual arousal in women. In this study, we have characterized alpha-adrenergic receptors (AR) in rabbit vagina by assessment of radioligand binding, contractility of isolated tissue strips and genital hemodynamics. [3H]Prazosin and [3H]RX821002 (alpha-1 and alpha-2 AR selective antagonists) bound to rabbit vaginal membrane preparations with high affinity and limited capacity. Competition binding assays using both non-selective and subtype selective ligands for AR (phentolamine, prazosin, delequamine, rauwolscine and UK14304) further confirmed the presence of alpha-1 and alpha-2 AR in vaginal tissue. In organ bath preparations of vaginal tissue strips, norepinephrine induced contraction was attenuated by alpha-1 and alpha-2 AR antagonists (prazosin, tamsulosin, delequamine and phentolamine). In anesthetized rabbits, intravaginal injection of the alpha-1 AR selective antagonist REC 15/2615 (50 and 100 microg/kg) caused a 2 to 3-fold increase in genital tissue oxyhemoglobin (OHb) concentration. Similar increases in tissue OHb were observed with intravaginal injection of phentolamine (500 microg/kg) or a tri-mixture of vasodilators (PGE1, papaverine, phentolamine). REC 15/2615, phentolamine or the tri-mixture also enhanced the amplitude and/or duration of change in genital tissue OHb after pelvic nerve stimulation. Thus, vaginal tissue expresses functional alpha-1 and alpha-2 AR, which modulate vaginal smooth muscle contractility and genital engorgement. PMID- 12377273 TI - Interaction of Leishmania with the host macrophage. AB - The leishmaniases are a group of diseases with a spectrum of clinical manifestations ranging from self-healing cutaneous ulcers to severe visceral disease and even death. In mammals, the macrophage is the main host for the Leishmania amastigote. However, the macrophage is also the immune effector cell that, upon activation, is able to kill intracellular organisms. Therefore, understanding the parasite mechanisms which allow establishment of infection, and the host immune mechanisms that are responsible for parasite recognition and killing should lead to the development of new drugs and vaccines. PMID- 12377274 TI - New and re-emerging infectious diseases. PMID- 12377275 TI - Can we slow the development of anthelmintic resistance? An electronic debate. PMID- 12377276 TI - Real-time quantitative PCR in parasitology. AB - Standard techniques for counting parasites are often time-consuming, difficult and inaccurate, and occasionally unpleasant. Real-time quantitative polymerase chain reaction has recently been applied to parasitology, specifically Plasmodium, Toxoplasma, Leishmania and Neospora. These techniques are truly quantitative, give results over a range of 6-7 orders of magnitude, are quick to perform and require no manipulations post-amplification. They can be used to count genome numbers and to study levels of gene expression. The advantages and limitations of existing thermocyclers and applicable detection systems are discussed here, and promising new developments are highlighted. PMID- 12377277 TI - The worm turns. PMID- 12377278 TI - Unravelling the origins of Trypanosoma brucei rhodesiense. PMID- 12377279 TI - Turning a chloroquine-resistance reverser into an antimalarial. PMID- 12377281 TI - Oxidative permeabilization? PMID- 12377282 TI - Oxidative permeabilization? PMID- 12377283 TI - Extensive polymorphism and ancient origin of Plasmodium falciparum. AB - DNA sequence data reveal extensive polymorphism in the virulent, human malaria parasite Plasmodium falciparum. The extent of polymorphism at apparently neutral evolving loci points to a common ancestor for this species that is no more recent than approximately 150,000-200,000 years ago. In addition, there is evidence of balanced polymorphisms at certain antigen-encoding loci, some of which have been maintained for millions of years. Thus, we can reject the hypothesis that this species underwent a recent extreme bottleneck (i.e. one in which the population was reduced to a single haploid genotype). However, it is possible that less severe bottlenecks have occurred. PMID- 12377284 TI - Toxoplasma gondii and sex: essential or optional extra? AB - The evolutionary and biological significance of a female-biased sex ratio within apicomplexan parasites has been the subject of much discussion. It is proposed that the sex allocation theory, as applied to inbreeding populations, can explain the sex ratios observed for this diverse group of parasites. This is based on a mathematical model, which assumes that the majority of microgametes will succeed in fertilizing macrogametes. Is this a realistic assumption? It is possible, for different reasons, that the theory may not to be applicable to either malaria parasites or Toxoplasma gondii. PMID- 12377285 TI - In the blood--the remarkable ancestry of Plasmodium falciparum. AB - Discussions concerning the origin and spread of malaria are popular. Colourful and diverse players, such as early hominids, agriculturalists, conquistadors and various animals, tend to feature prominently in imagined horrible histories. So, what of recent studies on genomic diversity of Plasmodium falciparum that claim to give more precision to the subject? Have they restricted the freeform speculation or just enhanced it? Or, are they pointing to a more important understanding about the parasite that might affect its future? PMID- 12377286 TI - Breaking down the blood-brain barrier: signaling a path to cerebral malaria? AB - Cerebral malaria is a major killer in the developing world, but we still know very little about the causes of this disease. How does Plasmodium falciparum cause such a devastating neurological disease while it is in the brain vasculature? Why do some patients die, whereas others survive? What processes contribute to disease in the brain, and can we reverse them? Here, the latest evidence from post-mortem, in vitro and animal studies is reviewed to highlight the role of blood-brain barrier breakdown in cerebral malaria. Blood-brain barrier integrity is disturbed during severe malaria, causing leakage of cerebral vessels. Understanding how this happens and how it contributes to the pathogenesis of coma may provide new opportunities for the treatment of cerebral malaria. PMID- 12377288 TI - Battle of Leishmania on the Net. PMID- 12377287 TI - Protein kinases as drug targets in parasitic protozoa. AB - The importance of protein kinases in cell signaling and cell cycle control has led to detailed structural and functional studies in various eukaryotes, and hence to the synthesis of specific chemical inhibitors for managing disease. Here, the current progress in applying developments from the wider protein kinase field to parasitic protozoa is reviewed. The availability of genome sequence data for several parasites has led to the identification of many protein kinases. Reverse genetics studies, including gene knockout and 'chemical genetics', can help to define the roles of the protein kinases and validate them as drug targets. In addition, screening chemical libraries with active recombinant protein kinases can identify lead compounds for drug design. PMID- 12377289 TI - More, but not better. PMID- 12377290 TI - Depression and mental disorders in patients with diabetes, kidney disease, and obesity and eating disorders. PMID- 12377291 TI - Research on co-morbidity, contextual barriers, and stigma: an introduction to the special issue. PMID- 12377293 TI - Depression and public health: an overview. AB - Depressive disorders are a significant public health issue. They are prevalent, disabling, often chronic illnesses, which cause a high economic burden for society, related to both direct and indirect costs. Depressive disorders also influence significantly the outcome of comorbid medical illnesses such as cardiac diseases, diabetes, and cancer. In primary care, underrecognition and undertreatment of depressive disorders are common, despite their relatively high prevalence, which accounts typically for more than 10% of patients. Primary care physicians should be aware of the common risk factors for depressive disorders such as gender, neuroticism, life events and adverse childhood experiences, and they should be familiar with associated features such as a positive psychiatric family history and prior depressive episodes. In primary care settings, depressive disorders should be considered with patients with multiple medical problems, unexplained physical symptoms, chronic pain or use of medical services that is more frequent than expected. Management of depressive disorders in primary care should include treatment with the newer antidepressant agents (given the fact they are typically well tolerated and safe) and focus on concomitant unhealthy behaviors as well as treatment adherence, which may both affect patient outcome. Programs aimed at improving patient follow-up and the coordination of the primary care intervention with that of specialists have been found to improve patient outcomes and to be cost effective. PMID- 12377294 TI - Impact of major depression on chronic medical illness. PMID- 12377295 TI - Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress. AB - The stress system coordinates the adaptive responses of the organism to stressors of any kind.(1). The main components of the stress system are the corticotropin releasing hormone (CRH) and locus ceruleus-norepinephrine (LC/NE)-autonomic systems and their peripheral effectors, the pituitary-adrenal axis, and the limbs of the autonomic system. Activation of the stress system leads to behavioral and peripheral changes that improve the ability of the organism to adjust homeostasis and increase its chances for survival. The CRH and LC/NE systems stimulate arousal and attention, as well as the mesocorticolimbic dopaminergic system, which is involved in anticipatory and reward phenomena, and the hypothalamic beta endorphin system, which suppresses pain sensation and, hence, increases analgesia. CRH inhibits appetite and activates thermogenesis via the catecholaminergic system. Also, reciprocal interactions exist between the amygdala and the hippocampus and the stress system, which stimulates these elements and is regulated by them. CRH plays an important role in inhibiting GnRH secretion during stress, while, via somatostatin, it also inhibits GH, TRH and TSH secretion, suppressing, thus, the reproductive, growth and thyroid functions. Interestingly, all three of these functions receive and depend on positive catecholaminergic input. The end-hormones of the hypothalamic-pituitary-adrenal (HPA) axis, glucocorticoids, on the other hand, have multiple roles. They simultaneously inhibit the CRH, LC/NE and beta-endorphin systems and stimulate the mesocorticolimbic dopaminergic system and the CRH peptidergic central nucleus of the amygdala. In addition, they directly inhibit pituitary gonadotropin, GH and TSH secretion, render the target tissues of sex steroids and growth factors resistant to these substances and suppress the 5' deiodinase, which converts the relatively inactive tetraiodothyronine (T(4)) to triiodothyronine (T(3)), contributing further to the suppression of reproductive, growth and thyroid functions. They also have direct as well as insulin-mediated effects on adipose tissue, ultimately promoting visceral adiposity, insulin resistance, dyslipidemia and hypertension (metabolic syndrome X) and direct effects on the bone, causing "low turnover" osteoporosis. Central CRH, via glucocorticoids and catecholamines, inhibits the inflammatory reaction, while directly secreted by peripheral nerves CRH stimulates local inflammation (immune CRH). CRH antagonists may be useful in human pathologic states, such as melancholic depression and chronic anxiety, associated with chronic hyperactivity of the stress system, along with predictable behavioral, neuroendocrine, metabolic and immune changes, based on the interrelations outlined above. Conversely, potentiators of CRH secretion/action may be useful to treat atypical depression, postpartum depression and the fibromyalgia/chronic fatigue syndromes, all characterized by low HPA axis and LC/NE activity, fatigue, depressive symptomatology, hyperalgesia and increased immune/inflammatory responses to stimuli. PMID- 12377296 TI - Depression and immune function: central pathways to morbidity and mortality. AB - OBJECTIVE: The increased morbidity and mortality associated with depression is substantial. In this paper, we review evidence suggesting that depression contributes to disease and death through immune dysregulation. METHOD: This review focuses on recent human studies addressing the impact of depression on immune function, and the health consequences of those changes. RESULTS: There is growing evidence that depression can directly stimulate the production of proinflammatory cytokines that influence a spectrum of conditions associated with aging, including cardiovascular disease, osteoporosis, arthritis, type 2 diabetes, certain cancers, periodontal disease, frailty, and functional decline. Additionally, depression can down-regulate the cellular immune response; as a consequence, processes such as prolonged infection and delayed wound healing that fuel sustained proinflammatory cytokine production may be promoted by depression. CONCLUSIONS: These direct and indirect processes pose the greatest health risks for older adults who already show age-related increases in proinflammatory cytokine production. Thus, aging interacts with depression to enhance risks for morbidity and mortality. PMID- 12377297 TI - The role of adherence in mediating the relationship between depression and health outcomes. AB - Depression has been linked to poor health outcome in a number of studies; however, the mechanism underlying this relationship has received little attention. This paper explores the possibility that adherence mediates the relationship between depression and outcome. Principal findings regarding the relationship between depression, adherence, and outcome are reviewed. The data suggest that depression is related, at least moderately, to poorer adherence to a variety of treatment components. The relationship between adherence and outcome is more difficult to establish. In addition, current data, albeit limited, do not support the hypothesis that adherence mediates the relationship between depression and outcome. An alternative model in which adherence precedes and influences both mood state and health outcome is discussed. Finally, possible explanations for these relationships are explored and suggestions for future research provided. PMID- 12377298 TI - Studies of hormone action in the hippocampal formation: possible relevance to depression and diabetes. AB - OBJECTIVES: The goal is to review the plasticity and vulnerability of the hippocampus, a brain structure involved in episodic, declarative, contextual and spatial learning and memory, as well as its being a component in the control of autonomic and vegetative functions such as ACTH secretion. It discusses its possible role in the regulation of glucose homeostasis, and the need of hippocampal neurons for glucose because of their high metabolic activity. The hippocampus is also vulnerable to damage by stroke and head trauma and susceptible to damage during aging and repeated stress, and is sensitive to the effects of diabetes. METHODS: A summary of recent work in the author's laboratory and related work in the field using citations of literature based, in part, on Medline searches. CONCLUSIONS: In addition to its vulnerability, the hippocampus is also a plastic and adaptable brain region that is capable of considerable structural reorganization, including remodeling of dendrites and neurogenesis of dentate gyrus granule neurons in response to repeated stress. Animal models of Type 1 diabetes show accelerated remodeling of dendrites, and Type 2 diabetes remains to be studied in this regard. This is relevant to major depressive illness, in which a progressive atrophy of the hippocampal formation is reported and is accompanied by impairment of cognitive function in those subjects with hippocampal shrinkage. Therefore, hippocampal atrophy in depression, as well as in diabetes, may reflect either damage or plasticity involving structural reorganization that is potentially treatable. PMID- 12377299 TI - Epidemiologic evidence for the relation between socioeconomic status and depression, obesity, and diabetes. AB - Many of the leading causes of death and disability in the United States and other countries are associated with socioeconomic position. The least well-off suffer a disproportionate share of the burden of disease, including depression, obesity, and diabetes. Research suggests that the adverse effects of economic hardship on both mental and physical health and functioning are evident at young ages and persist across the lifecourse. Moreover, these associations are seen across cultures. Data from four large epidemiologic studies on the role of psychological characteristics, social factors, and behaviors in health and disease risk are presented that highlight the striking associations between socioeconomic factors and chronic diseases. Data from these studies demonstrate that the effects of economic disadvantage are cumulative, with the greatest risk of poor mental and physical health seen among those who experienced sustained hardship over time. PMID- 12377300 TI - Depression as a risk factor for cardiac mortality and morbidity: a review of potential mechanisms. AB - Depression increases the risk of cardiac mortality and morbidity in patients with coronary heart disease (CHD), but the mechanisms that underlie this association remain unclear. This review considers the evidence for several behavioral and physiological mechanisms that might explain how depression increases the risk for incident coronary disease and for subsequent cardiac morbidity and mortality. The candidate mechanisms include: (1). antidepressant cardiotoxicity; (2). association of depression with cardiac risk factors such as cigarette smoking, hypertension, diabetes, and reduced functional capacity; (3). association of depression with greater coronary disease severity; (4). nonadherence to cardiac prevention and treatment regimens; (5). lower heart rate variability (HRV) reflecting altered cardiac autonomic tone; (6). increased platelet aggregation; and (7). inflammatory processes. Despite recent advances in our understanding of these potential mechanisms, further research is needed to determine how depression increases risk for cardiac morbidity and mortality. PMID- 12377301 TI - Epidemiologic evidence on the comorbidity of depression and diabetes. AB - Data from population-based studies are important for the study of comorbidity. Cross-sectional research shows a consistent positive association of diabetes and depression. Prospective population-based studies are reviewed, showing that the temporal order may be from diabetes to depression, or from depression to diabetes, depending to some extent on the type of diabetes. The size of the effects is fairly consistent among the small number of studies and not trivial. Possibilities for future epidemiologic research on comorbidity are discussed. PMID- 12377302 TI - Depression in type 1 diabetes in children: natural history and correlates. AB - The combination of diabetes and depression in children and adolescents is largely unstudied. The purpose of this article is to review the literature on the natural history and correlates of comorbid diabetes and depression in children and adolescents. Children with diabetes have a two-fold greater prevalence of depression, and adolescents up to three-fold greater, than youth without diabetes. Correlates of depression and diabetes include gender, poorer metabolic control, and family behaviors. Very little is known about treatment in these youth, and more studies are indicated. PMID- 12377303 TI - Depression in diabetes and obesity: racial/ethnic/gender issues in older adults. AB - OBJECTIVES: This study focuses on the comorbidity between depression and diabetes, as well as depression and obesity, in a biracial community sample of older adults. METHODS: The data are drawn from a cross-sectional survey of five counties in North Carolina, USA, part of a longitudinal study of morbidity and mortality among elders in the urban and rural south. During the first wave of the survey, 4162 persons 65+ years of age participated in an interview at their homes. During this interview, data were collected to assess demographics, functional status, cognitive status, depression and self-report of diabetes. In addition, subjects were asked to estimate their height and weight for the interviewers, from which data body mass index (BMI) was estimated. RESULTS: In both uncontrolled and controlled analyses, female gender, lower education, functional impairment and cognitive impairment were associated with comorbid depression/diabetes and depression/high BMI. Age was not associated with comorbid depression/diabetes but younger age was associated with depression/cognitive impairment. African American race was strongly associated with depression/diabetes but not with depression/high BMI. CONCLUSIONS: More studies of comorbidity in the general population should be implemented to determine the relationship between these comorbid conditions and risk factors. Longitudinal studies are especially needed. PMID- 12377304 TI - Treatment of depression in diabetes: impact on mood and medical outcome. AB - Depression is prevalent as a co-morbid condition in diabetes. The efficacy of depression treatment with either pharmacological agents or psychotherapy has been demonstrated in the few available controlled trials. Depression has been associated with poor glycemic control and with accelerated rates of coronary heart disease in diabetic patients. Reported depression treatment trials demonstrate benefits of depression remission on glycemic control as well as mood and the potential for improvement in the course and outcome of diabetes. Because adverse effects of pharmacological agents on glycemic control have been observed, optimal therapies that improve both depression and measures of diabetes are still being sought. This review critically examines the efficacy of depression treatment in diabetes patients, the effects of depression treatment on the medical condition, and methodological issues important in the performance of treatment trials in the patient population. PMID- 12377305 TI - Abnormalities in glucose regulation associated with mental illness and treatment. PMID- 12377306 TI - Obesity-depression associations in the population. AB - This article summarizes data on the relationship between obesity and depression in the population. Both obesity and depression are increasingly prevalent and associated with numerous health complications including hypertension, coronary heart disease, and increased mortality. There does not appear to be a simple or single association between these disorders. Meta-analytic studies suggest no statistically significant relationship, although pooling all subjects may mask important variables that moderate or mediate potential covariations. Sociodemographic, psychosocial, and genetic factors may render certain obese individuals more prone to depression or vice versa. Physiological and behavioral variables that link obesity and depression have received limited study. There are likely multiple obesity-depression covariations in the population, rather than a single pattern of association. There is a need for longitudinal and mechanistic studies to understand casual pathways and greater collaboration between depression and obesity specialists. PMID- 12377307 TI - Eating disorders in young women with type 1 diabetes mellitus. AB - Research findings from the past decade regarding the association of type 1 diabetes mellitus and eating disorders are critically reviewed in this paper. Although there has been much debate regarding the specificity of this association, a recent large multisite case-controlled study demonstrated that the prevalence rates of both full syndrome and subthreshold eating disorders among adolescent and young adult women with diabetes are twice as high as in their nondiabetic peers. Further, a 4-year follow-up study showed that disordered eating behavior in young women with diabetes often persists and is associated with a threefold increase in the risk of diabetic retinopathy. These eating disturbances tend to be associated with impaired family functioning and with poor diabetes management. Health care professionals should maintain a high index of suspicion for the presence of an eating disturbance among young women with diabetes, particularly among those with persistently poor metabolic control and/or weight and shape concerns. Screening for such disturbances should begin during the prepubertal period among girls with diabetes. A brief psychoeducational intervention leads to a reduction in disturbed eating attitudes and behavior but is not sufficient to improve metabolic control. More intensive treatment approaches, which should include a family-based component, may be needed to improve metabolic control. The evaluation of these and other treatment approaches is indicated in view of the serious short- and long-term health risks associated with eating disorders in young women with diabetes. PMID- 12377308 TI - Depression in patients with chronic renal disease: what we know and what we need to know. AB - Depression is a common, but underdiagnosed and understudied problem in patients with renal disease. The overlap between symptoms of chronic medical illness and those of depression make for a particularly challenging diagnosis in this illness. The prevalence of depression varies with the diagnostic tool employed. The gold standard for the psychiatric diagnosis is the interview, using DSM-IV TR criteria. Researchers in the field of renal disease have often not distinguished between the diagnosis of major depression and high levels of depressive affect in studies. There are almost no data regarding the magnitude of depression in patients with chronic renal insufficiency, patients treated with peritoneal dialysis, and children with renal disease, compared with adults with end-stage renal disease treated with hemodialysis. The relationships between age, ethnicity, marital status and satisfaction, and perception of quality of life and level of depressive affect and diagnosis of depression, and medical outcomes have not been determined in patients with renal disease. The mediators which may underlie the deleterious effects of depression in patients with renal disease, and their relationship with stage of renal dysfunction have not been delineated. More emphasis must be placed on well-designed treatment studies and survival analyses in these populations, using longitudinal techniques. PMID- 12377309 TI - The treatment of depression in patients maintained on dialysis. PMID- 12377310 TI - Dose escalation in localized prostate cancer: make no assumptions. PMID- 12377311 TI - Age-related variations in the use of axillary dissection. PMID- 12377312 TI - Stereotactic radiosurgery for patients with ACTH-producing pituitary adenomas after prior adrenalectomy. PMID- 12377313 TI - Adverse effects: a Pandora's box for oncology. PMID- 12377314 TI - Estimating risks of radiotherapy complications as part of informed consent: the high degree of variability between radiation oncologists may be related to experience. AB - PURPOSE: Estimating the risks of radiotherapy (RT) toxicity is important for informed consent; however, the consistency in estimates has not been studied. This study aimed to explore the variability and factors affecting risk estimates (REs). METHODS AND MATERIALS: A survey was mailed to Australian radiation oncologists, who were asked to estimate risks of RT complications given 49 clinical scenarios. The REs were assessed for association with oncologist experience, subspecialization, and private practice. RESULTS: The REs were extremely variable, with a 50-fold median variability. The least variability (sevenfold) was for estimates of late, small intestinal perforation/obstruction after a one-third volume received 50 Gy with concurrent 5-fluorouracil (RE range 5-35%). The variation between the smallest and largest REs in 17 scenarios was >or=100-fold. The years of experience was significantly associated with REs of soft/connective-tissue toxicity (p = 0.01) but inversely associated with estimates of neurologic/central nervous system toxicity (p = 0.08). Ninety-six percent of respondents believed REs were important to RT practice; only 24% rated evidence to support their estimates as good. Sixty-seven percent believed national/international groups should pursue the issue further. CONCLUSION: Enormous variability exists in REs for normal tissue complications due to RT that is influenced by the years of experience. Risk estimation is perceived as an important issue without a good evidence base. Additional studies are strongly recommended. PMID- 12377315 TI - Cost-effectiveness of preoperative radiotherapy in rectal cancer: results from the Swedish Rectal Cancer Trial. AB - PURPOSE: The Swedish Rectal Cancer Trial (SRCT) demonstrated that a short-term regimen of high-dose fractionated preoperative radiotherapy (5 x 5 Gy) reduced the local recurrence rates and improved overall survival. This has had an impact on the primary treatment of rectal cancer. The current study investigated the cost-effectiveness of the new combined approach. METHODS AND MATERIALS: After an 8-year follow-up, in-hospital and outpatient costs related to the treatment of rectal cancer and its complications were analyzed for 98 randomly allocated patients who participated in the SRCT from a single Swedish health care region. The costs were then related to the clinical data from the SRCT regarding complications, local and distant recurrences, and survival. RESULTS: The total cost for a nonirradiated patient was US$30,080 compared with US$35,268 for an irradiated patient. The surgery-alone group had increased costs related to local recurrences, and the radiotherapy group had increased costs for irradiation and complications. With a survival benefit of 21 months (retrieved from the SRCT), the cost for a saved year was US$3654. Sensitivity analyses for different rates of local recurrences, the costs related to complications and less marked survival benefit showed that this figure could vary up to US$15,228. CONCLUSION: The cost for a life-year saved in these data was US$3654. This figure could reach US$15,228 in the most pessimistic setting of the sensitivity tests, a cost still comparable with other well-accepted medical interventions. PMID- 12377316 TI - Defining patient-based minimal clinically important effect sizes: a study in palliative radiotherapy for painful unresectable pelvic recurrences from rectal cancer. AB - PURPOSE: To measure patient-based minimal clinically important effect sizes (minimal incremental benefit that an individual would require to accept one treatment option over another) for pain relief between two contrasting palliative radiotherapy regimens for painful pelvic recurrences from rectal cancer. METHODS AND MATERIALS: Forty-three patients with a history of cancer pain without prior pelvic radiotherapy participated in decision aid-facilitated trade-off exercises. The clinical scenario and treatment options of a 5-day vs. a 20-day course of radiotherapy were described. The duration of pain relief for the 20-day regimen was increased until the respondents' preferences switched to the 20-day regimen. The exercises were repeated for different probabilities of benefit and pain intensity at the time of decision making. RESULTS: When the probability of pain relief was unchanged, the median switch point for the duration of pain relief was 6.7 and 7.2 months for severe and mild pain, respectively. The cumulative percentage frequency curve for the switch points approximated a sigmoid distribution. CONCLUSION: Determining the minimal clinically important effect sizes for symptom relief for palliative therapies is feasible. This type of information can be used to incorporate patient values into clinical trial designs. Modification of this method can be used to improve our understanding of shared (physician and patient) decision making. PMID- 12377317 TI - Phase I study of weekly gemcitabine as a radiation sensitizer for unresectable pancreatic cancer. AB - PURPOSE: To determine the maximal tolerated dose and dose-limiting toxicities (DLTs) of weekly gemcitabine with concurrent radiotherapy (RT) in patients with unresectable adenocarcinoma of the pancreas. METHODS AND MATERIALS: Patients who had locally advanced or recurrent unresectable pancreatic cancer were eligible. Gemcitabine was administered as a 30-min infusion once weekly for a total of five cycles during the course of RT. The starting dose of gemcitabine was 350 mg/m(2)/wk. Doses were escalated by increments of 25% in successive cohorts of 3 6 patients. RT was delivered at 180 cGy/d to a total dose of 5400-5580 cGy to the gross tumor volume. RESULTS: Nineteen patients were entered in this study through three dose levels (350-550 mg/m(2)/wk). The maximal tolerated dose was determined to be 440 mg/m(2)/wk. The DLTs were neutropenia, thrombocytopenia, and failure to receive all five cycles of gemcitabine. Other non-DLTs included 16 Grade III toxicities, which consisted of thrombosis, infection, nausea, vomiting, hypotension, constipation, diarrhea, and fatigue. One patient at each gemcitabine dose level experienced Grade IV vomiting, and the patient at the 550 mg/m(2) dose developed Grade IV anorexia. CONCLUSION: The maximal tolerated dose of gemcitabine when administered as a 30-min infusion once weekly during RT for unresectable pancreatic cancer was found to be 440 mg/m(2)/wk. The DLTs were neutropenia, thrombocytopenia, and failure to receive all five cycles of chemotherapy. Concurrent gemcitabine and RT is reasonably well tolerated and deserves additional evaluation against the current standard of care. PMID- 12377318 TI - Prostate biopsy status and PSA nadir level as early surrogates for treatment failure: analysis of a prostate cancer randomized radiation dose escalation trial. AB - PURPOSE: A positive biopsy after external beam radiotherapy in patients free of any evidence of treatment failure is not synonymous with eventual recurrence. Although biopsy positivity is a predictor of outcome, the utility of biopsy status as a surrogate end point, the effect of radiation dose on biopsy status, and the interrelationships of these associations to prostate-specific antigen (PSA) nadir level are not well-defined. These issues were investigated in a cohort of men with Stage T1-T3 prostate cancer who were randomized to receive between 70 Gy and 78 Gy and were prospectively biopsied at about 2 years after the completion of radiotherapy (RT). METHODS AND MATERIALS: Of the 301 assessable patients in the trial, 168 underwent planned sextant or greater prostate post-RT biopsies in the absence of biochemical or clinical failure; this group constituted the study cohort. Of the 168 patients, 87 were in the 70-Gy arm and 81 in the 78-Gy arm. Biopsies were classified into four groups: negative (no tumor), atypical/suspicious cells (not diagnostic of carcinoma), carcinoma with treatment effect (CaTxEffect), and carcinoma without treatment effect (CaNoTxEffect). Any diagnosis of carcinoma in the specimen was classified as biopsy positive. Freedom from failure (FFF) included biochemical failure and/or clinical failure. Kaplan-Meier curves were calculated from the completion of RT. For those alive in the study cohort, the median follow-up was 65 months. RESULTS: The rate of biopsy without tumor was 42%; with atypical cells, it was 28%, with CaTxEffect 21%, and with CaNoTxEffect 9%. The overall biopsy positivity rate (CaTxEffect + CaNoTxEffect) was 30%; 28% in the 70-Gy group and 32% in the 78-Gy group (p = 0.52). The distribution of PSA nadir levels was 73% 0.5 1.0, 5% >1.0-2.0, and 1% >2.0 ng/mL. Significantly more patients randomized to 78 Gy had a PSA nadir of 2.0 ng/mL. CONCLUSION: For patients free of treatment failure at the time of prostate biopsy 2 years after RT, the prognosis of no tumor cells was the same as that of atypical/suspicious cells and CaTxEffect was the same as CaNoTxEffect. The biopsy positivity rate was not altered by dose, suggesting that most of the outcome differences between the 70-Gy and 78-Gy groups were due to events occurring before prostate biopsy at 2 years and/or were not entirely dependent on biopsy status. Biopsy status is a strong prognostic factor, but, as an early end point, it may be misleading. PSA nadir appears to have little clinical value in patients treated to doses of >/=70 Gy who are failure free 2 years after RT. PMID- 12377319 TI - Treatment of prostate cancer with radiotherapy: should the entire seminal vesicles be included in the clinical target volume? AB - PURPOSE: When treating high-risk prostate cancer with radiation therapy, inclusion of the seminal vesicles (SVs) within the clinical target volume (CTV) can dramatically increase the volume of radiated normal tissues and hinder dose escalation. Because cancer may involve only the proximal portion of the frequently lengthy SVs, we performed a complete pathology review of prostatectomy specimens to determine the appropriate length of SV to include within the CTV when SV treatment is indicated. METHODS AND MATERIALS: A detailed pathologic analysis was performed for 344 radical prostatectomy specimens (1987-2000). All slides from each case were reviewed by a single pathologist (N.S.G.). Factors recorded for each case included length of each SV (cm), length of cancer involvement in each SV (cm) measured from the prostate-SV junction, and percentage of SV length involved. RESULTS: Fifty-one patients (15%) demonstrated SV involvement in 81 SVs (21 unilateral, 30 bilateral SV involvement). The median SV length was 3.5 cm (range: 0.7-8.5 cm). Factors associated with SV involvement included the pretreatment PSA level, biopsy Gleason score, and clinical T classification. The commonly used risk group stratification was very effective at predicting SV positivity. Only 1% of low-risk patients (PSA <10 ng/mL, Gleason or=10 ng/mL, biopsy Gleason >or=7, or clinical T stage >or=T2b). When treating the SV for prostate cancer, only the proximal 2.0-2.5 cm (approximately 60%) of the SV should be included within the CTV. PMID- 12377320 TI - Using the magnitude of PSA bounce after MRI-guided prostate brachytherapy to distinguish recurrence, benign precipitating factors, and idiopathic bounce. AB - PURPOSE: To identify events that precipitated a prostate-specific antigen (PSA) bounce and characterize the magnitude, duration, and time to PSA bounce after MRI guided prostate brachytherapy. METHODS AND MATERIALS: Between 1997 and 2001, 186 patients with low-risk prostate cancer underwent MRI-guided permanent 125I source implantation, with or without external beam radiotherapy. A PSA bounce was defined as a >or=15% elevation in PSA compared with the most recent value, followed by a decline to a level at or less than the prebounce value. At the time of PSA measurement, data were prospectively collected on whether the patient had recent ejaculation, ongoing radiation proctitis, or recent instrumentation. RESULTS: A total of 115 patients (61.8%) had a total of 156 PSA bounces. Of these, 36 patients had PSA bounces associated with ejaculation, proctitis, or instrumentation, and 79 experienced idiopathic PSA bounces (not associated with a precipitating event). The magnitude of the PSA bounce was significantly lower for the idiopathic PSA bounce (0.6 ng/mL) compared with that associated with ejaculation (p = 0.003), proctitis (p <0.0001), or instrumentation (p = 0.007). Patients with biopsy-proven local recurrence had a median PSA elevation of 1.2 ng/mL, significantly higher (p = 0.006) than the magnitude of the idiopathic PSA bounce, but not significantly different from the magnitude of the PSA bounce due to ejaculation, proctitis, or instrumentation. CONCLUSION: In patients treated with MRI-guided prostate brachytherapy, recent ejaculation, instrumentation, or ongoing radiation proctitis can cause a transient increase in PSA, the magnitude of which is significantly higher than that for idiopathic PSA bounce, but is similar to that in patients with recurrent disease. PMID- 12377321 TI - Comparison of MRI pulse sequences in defining prostate volume after permanent implantation. AB - PURPOSE: To determine the relative value of three MRI pulse sequences in defining the prostate volume after permanent implantation. METHODS AND MATERIALS: A total of 45 patients who received a permanent 125I implant were studied. Two weeks after implantation, an axial CT scan (2 mm thickness) and T1-weighted, T1 weighted fat saturation, and T2-weighted axial MRI (3-mm) studies were obtained. The prostate volumes were compared with the initial ultrasound planning volumes, and subsequently the CT, T1-weighted, and T1-weighted fat saturation MRI volumes were compared with the T2-weighted volumes. Discrepancies in volume were evaluated by visual inspection of the registered axial images and the registration of axial volumes on the sagittal T2-weighted volumes. In a limited set of patients, pre- and postimplant CT and T2-weighted MRI studies were available for comparison to determine whether prostate volume changes after implant were dependent on the imaging modality. RESULTS: T1-weighted and T1 weighted fat saturation MRI and CT prostate volumes were consistently larger than the T2-weighted MRI prostate volumes, with a volume on average 1.33 (SD 0.24) times the T2-weighted volume. This discrepancy was due to the superiority of T2 weighted MRI for prostate definition at the following critical interfaces: membranous urethra, apex, and anterior base-bladder and posterior base-seminal vesicle interfaces. The differences in prostate definition in the anterior base region suggest that the commonly reported underdose may be due to overestimation of the prostate in this region by CT. The consistent difference in volumes suggests that the degree of swelling observed after implantation is in part a function of the imaging modality. In patients with pre- and postimplant CT and T2 weighted MRI images, swelling on the T2-weighted images was 1.1 times baseline and on CT was 1.3 times baseline, confirming the imaging modality dependence of prostate swelling. CONCLUSION: Postimplant T2-weighted MRI images provided superior prostate definition in all critical regions of the prostate compared with CT and the other MRI sequences tested. In addition to defining an optimal technique, these findings call two prior observations into question. Under dosing at the anterior base region may be overestimated because of poor definition of the prostate-bladder muscle interface. The swelling observed after implantation was lower on T2-weighted images as well, suggesting that a fraction of postimplant swelling is a function of the imaging modality. These findings have implications for preimplant planning and postimplant evaluation. As implant planning techniques become more conformal, and registration methods become more efficient, T2-weighted MRI after implantation will improve the accuracy of postimplant dosimetry. PMID- 12377322 TI - Dawn of prostate brachytherapy: 1915-1930. AB - PURPOSE: To investigate the origins of prostate brachytherapy. METHODS AND MATERIALS: A review of contemporary journals and texts was conducted. RESULTS: Prostate brachytherapy was performed frequently by leading urologists before 1930. Both temporary and permanent implant techniques were developed using radium and radon through intracavitary and interstitial approaches. Transperineal implantation of permanent sources was first performed 80 years ago. CONCLUSION: Prostate brachytherapy has its origins in the early part of the last century. PMID- 12377323 TI - Randomized phase III trial of single versus fractionated thoracic radiation in the palliation of patients with lung cancer (NCIC CTG SC.15). AB - PURPOSE: This multi-institutional Phase III randomized study compared 10 Gy single-fraction radiotherapy (RT) with 20 Gy in five fractions in the palliation of thoracic symptoms from lung cancer. METHODS AND MATERIALS: The primary end point was palliation of thoracic symptoms at 1 month after RT, evaluated by a patient-completed daily diary card. Secondary end points included quality of life, toxicity, and survival. RESULTS: Most (69%) of 230 patients randomized had locally advanced disease unsuitable for curative treatment. The treatment arms were well balanced with respect to the known prognostic factors. At 1 month after RT, no difference was found in symptom control between the two arms, as judged by the daily diary scores. The changes in the scores on the Lung Cancer Symptom Scale indicated that the fractionated RT (five fractions) group had greater improvement in symptoms related to lung cancer (p = 0.009), pain (p = 0.0008), ability to carry out normal activities (p = 0.037), and better global quality of life (p = 0.039). The European Organization for Research and Treatment of Cancer QLQ-C30 scores showed that patients receiving five fractions had a greater improvement in scores with respect to pain (p = 0.04). No significant difference was found in treatment-related toxicity. Patients who received five fractions survived on average 2 months longer (p = 0.0305) than patients who received one fraction. CONCLUSION: Although the two treatment strategies provided a similar degree of palliation of thoracic symptoms, the difference in survival between the two study arms was of a clinically relevant magnitude. PMID- 12377324 TI - Phase I study of hyperfractionated accelerated radiotherapy and escalating doses of daily cisplatin for patients with locally advanced non-small-cell lung cancer. AB - PURPOSE: To determine the maximal tolerated dose of daily cisplatin (CDDP) administered during an aggressive program of hyperfractionated accelerated radiotherapy (t.i.d. RT) for locally advanced non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: Twenty patients with Stage III NSCLC were treated with RT administered three times daily and escalating doses of concurrent daily CDDP. The t.i.d. RT was delivered on 12 weekdays as follows: a total of 57.6 Gy was administered using 10-MV X-rays in 1.5-Gy fractions at 8 am and 4 pm and 1.8-Gy fractions at 12 pm. AP-PA fields were used in the morning and afternoon, and oblique fields, which included no spinal cord, were used at 12 pm. The first group of 5 patients was not given CDDP, the second group of 5 patients received 5 mg/m(2) of CDDP, and the last group of 10 patients received 7.5 mg/m(2). Toxicity was evaluated using the National Cancer Institute Common Toxicity Criteria. RESULTS: The first group of 5 patients received no CDDP. One of these patients developed both Grade 3 esophagitis and Grade 3 pneumonitis. The second group of 5 patients received 5 mg/m(2) of CDDP. One of these patients developed Grade 3 esophagitis. The last group of 10 patients received 7.5 mg/m(2) of CDDP. Four of these patients developed Grade 3 toxicity and one developed Grade 4 toxicity. None of the patients died as a result of toxicity. The median survival for the 20 patients was 19 months. The tumor response and patterns of failure were also evaluated. CONCLUSION: This study was performed to determine the maximal tolerated dose of CDDP when administered daily during an aggressive program of t.i.d. RT. A dose of 7.5 mg/m(2) daily was determined to be the maximal tolerated dose, because this dose caused Grade 3 or 4 toxicity in 50% of the patients who received it. PMID- 12377325 TI - Outcome and prognostic factors for patients with non-small-cell lung cancer and severe radiation pneumonitis. AB - PURPOSE: Radiation pneumonitis is a serious complication that develops after thoracic irradiation. The purpose of this study was to identify prognostic factors for severe radiation pneumonitis in patients with non-small-cell lung cancer. METHODS AND MATERIALS: The medical records of patients with non-small cell lung cancer and severe radiation pneumonitis were reviewed. Variables were analyzed by univariate and stepwise multivariate analysis using the Cox regression model. RESULTS: Among the 31 patients, the mortality rate approached 50% in the first 2 months after the onset of radiation pneumonitis. The variables significantly associated with survival in the univariate analysis were tumor histologic feature, grade and extent (out-of-field or in-field) of radiation pneumonitis, oxygenation index, and serum albumin (<35 g/L or >or=35 g/L), and uric acid levels at the onset of radiation pneumonitis. Only the extent of radiation pneumonitis and serum albumin level were independently associated with survival in the multivariate analysis. CONCLUSION: The mortality rate of non small-cell lung cancer patients with severe radiation pneumonitis is extremely high, and survival is much shorter in patients with out-of-field radiation pneumonitis or a low serum albumin level at the onset. Additional studies to investigate the factors precipitating out-of-field radiation pneumonitis should improve the management of irradiation complications. PMID- 12377326 TI - Quality of life in long-term survivors of oropharynx carcinoma. AB - PURPOSE: To collect data on the health-related quality of life (QOL) of long-term survivors and to determine to what extent QOL might be an appropriate end point in the comparison of treatment options in oropharyngeal carcinoma. METHODS AND MATERIALS: All patients treated between 1992 and 1998, in two French comprehensive cancer centers, by brachytherapy (BT) +/- external beam radiotherapy (EBRT) or surgery plus RT, or exclusive EBRT for T1-T3 (International Union Against Cancer staging system) oropharynx squamous cell carcinoma, were included. QOL was measured once in disease-free patients at least 2 years after treatment initiation. The European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire and the specific H&N35 module were self-administered by all participating patients. Sociodemographic data were collected using a questionnaire specifically designed for the study. The association between the QOL scores of the various treatment-, disease-, and patient-related variables was performed through bivariate analysis and then by multivariate analysis. The mean QOL scores of the EORTC QLQ-C30 questionnaire were compared with the mean scores in the general population. RESULTS: Of the 159 eligible patients, 113 agreed to participate (97 men and 16 women, median age 61 years, range 41-83). The initial treatment was EBRT plus BT in 49 patients, surgery plus RT in 27, and EBRT alone in 37. The median follow-up time was 62 months (range 24-110). Compared with the general population, the three scores indicating the most impaired QOL were emotional and social functioning and fatigue. The clinical significance of global QOL impairment was borderline. The physical functioning, role functioning, and pain scores did not significantly differ from those of the general population. In multivariate analysis, the initial treatment had no significant influence on any dimension of QOL, except global QOL and emotional functioning. Surprisingly, surgery plus RT, as the initial treatment, favorably influenced the emotional functioning score and EBRT plus BT negatively influenced the global QOL score. None of these treatment modalities influenced any symptom scales. Patient selection was, at least partially, responsible for these paradoxical results. CONCLUSION: The results of this study bring original and useful data about the QOL of long-term survivors of oropharynx carcinoma. In these patients, the QOL was significantly impaired, particularly in its psychosocial dimensions. The level of symptoms and functioning (except global QOL and emotional) was similar whatever the initial treatment. These results suggest the importance of coping processes. In a trial comparing treatment options from a long-term perspective, survival remains the most relevant end point, and a QOL evaluation should be a secondary end point. More prospective studies on QOL in head-and-neck cancer patients are needed to determine new strategies for rehabilitation management. PMID- 12377327 TI - Prognostic value of Chinese race in nasopharyngeal cancer. AB - PURPOSE: Nasopharyngeal carcinoma is rare in the United States and common in southern China. Evaluating American patients treated using a uniform technique and staged with CT scanning, we determined whether Chinese and non-Chinese patients differ in presentation and outcome. METHODS AND MATERIALS: Between 1979 and 1996, 172 patients treated at Massachusetts General Hospital received primary radiotherapy with curative intent for nasopharyngeal carcinoma. Forty-one patients (24%) were of Chinese descent, and 41% of cancers were classified as having lymphoepithelioma histologic features. Most patients received twice-daily radiotherapy and a brachytherapy boost, receiving a median dose of 72 Gy to the nasopharynx. RESULTS: At the initial presentation, the Chinese patients were significantly younger, less likely to smoke, more likely to have Stage IV disease, and more likely to have cancer with lymphoepithelioma histologic features. After controlling for stage, age, histologic type, and treatment variables, Chinese patients were significantly more likely to develop distant metastases (p <0.05). Although Chinese race does not predict for local control or overall survival, a younger age, continued tobacco use, total radiation dose, and lymphoepithelioma histologic features do. CONCLUSION: In a large retrospective analysis of nasopharyngeal carcinoma, Chinese and non-Chinese patients differed significantly in presentation-age, stage, and histologic features-and outcome. We suggest as an explanation differences in intrinsic tumor biology rather than differences in treatment techniques or staging systems. Additional trials in endemic countries are needed to confirm the optimal treatment of Chinese and Chinese-American patients. PMID- 12377328 TI - Prediction of radiotherapy outcome using dynamic contrast enhanced MRI of carcinoma of the cervix. AB - PURPOSE: To investigate whether analysis of MRI enhancement data using a pharmacokinetic model improved a previously found correlation between contrast enhancement and tumor oxygenation measured using PO2 histograph. To evaluate the prognostic value of gadolinium enhancement data for radiotherapy outcome, and to study the efficacy of combined enhancement and MRI volume data. METHODS AND MATERIALS: Fifty patients underwent dynamic gadolinium-enhanced MRI as part of their initial staging investigations before treatment. Gadolinium enhancement was analyzed using the Brix pharmacokinetic model to obtain the parameters amplitude and rate of contrast enhancement. Pretreatment tumor oxygen measurements (Eppendorf PO2 histograph) were available for 35 patients. RESULTS: Both standard and pharmacokinetic-derived enhancement data correlated with tumor oxygenation measurements, and poorly enhancing tumors had low tumor oxygen levels. However, only the pharmacokinetic-analyzed data correlated with patient outcome and patients with poorly (amplitude less than median) vs. well-enhancing tumors had significantly worse disease-specific survival (p = 0.024). For the 50 patients studied, no relationship was found between enhancement and volume data. Combining MRI volume and enhancement information highlighted large differences in outcome (p = 0.0054). At the time of analysis, only 55% of patients with large, poorly enhanced tumors were alive compared with 92% of patients with small, well enhanced tumors. CONCLUSION: These preliminary results suggest that pharmacokinetic modeling of dynamic contrast-enhanced MRI provides data that reflect tumor oxygenation and yields useful prognostic information in patients with locally advanced carcinoma of the cervix. Combining MRI-derived enhancement and volume data delineates large differences in radiotherapy outcome. PMID- 12377329 TI - Prognostic value of vascular endothelial growth factor in Stage IB carcinoma of the uterine cervix. AB - PURPOSE: To clarify the role of vascular endothelial growth factor (VEGF) expression as an independent prognostic factor in Stage IB cervical cancer. METHODS AND MATERIALS: A total of 117 patients with Stage IB cervical cancer who had undergone radical hysterectomy and pelvic lymph node dissection with complete histopathologic examination were included. Eighty-eight (75.2%) patients received postoperative radiotherapy and/or chemotherapy. VEGF expression was examined using immunohistochemistry. RESULTS: Of 117 patients, 35 (29.9%) showed high intensity VEGF expression and 69 (59%) had a high score for area of VEGF expression. Strong correlations were found between high VEGF intensity and both deep stromal invasion (p = 0.01) and positive pelvic lymph nodes (p = 0.03). The area of VEGF expression was significantly associated with tumor size (p = 0.02). In a multivariate analysis, high VEGF intensity (p = 0.009) and tumor size (p = 0.01) were significant prognostic factors for overall survival and disease-free survival (p = 0.001 and p = 0.003, respectively). However, the area of VEGF expression was not a prognostic factor for overall survival or disease-free survival. CONCLUSION: Our findings on the correlation between VEGF expression and prognosis were conflicting. Functional and quantitative tools to assess tumor angiogenesis in addition to the expression of VEGF need to be developed and would be helpful to support the finding that tumor angiogenesis correlates significantly with prognosis in early-stage cervical cancer. PMID- 12377330 TI - Operable Stages IB and II cervical carcinomas: a retrospective study comparing preoperative uterovaginal brachytherapy and postoperative radiotherapy. AB - PURPOSE: To evaluate our data concerning prognostic factors and treatment toxicity in a series of operable cervical carcinomas. METHODS AND MATERIALS: Between May 1972 and January 1994, 414 patients with cervical carcinoma, staged according to the 1995 FIGO staging system (286 Stage IB1, 38 Stage IB2, 56 Stage IIA, and 34 Stage IIB with 1/3 proximal parametrial involvement), underwent radical hysterectomy with (n = 380) or without (n = 34) bilateral pelvic lymph node dissection (N+: n = 68). Group I included 168 patients who received postoperative radiation therapy (RT): 64 patients had low-dose-rate vaginal brachytherapy with a median total dose (MTD) of 50 Gy; 93 patients had external beam pelvic RT (EBPRT) with an MTD of 45 Gy over 5 weeks, followed by low-dose rate vaginal brachytherapy (MTD: 20 Gy); and 11 patients had EBPRT alone (MTD: 50 Gy over 6 weeks). Group II included 246 patients treated with preoperative low dose-rate uterovaginal brachytherapy (MTD: 65 Gy); 32 of these 246 patients also received postoperative EBPRT (MTD: 45 Gy over 5 weeks) delivered to the parametria and pelvic nodes. Mean follow-up from the beginning of treatment was 106 months. RESULTS: First events included isolated locoregional recurrences (35 patients), isolated distant metastases (27 patients), and locoregional recurrences with synchronous metastases (13 patients). The 10-year disease-free survival (DFS) rate was 88% for Stage IB1, 44% for Stage IB2, 65% for Stage IIA, and 48% for Stage IIB. Multivariate analysis showed that independent factors influencing the probability of DFS were as follows: cervical site (exocervical or endocervical vs. both endo- and exocervical, relative risk [RR]: 1.77, p = 0.047), vascular space invasion (no vs. yes, RR: 1.95, p = 0.041), age (>51 years vs. 1 cm: 83% vs. 41%, respectively, p = 0.001). The overall postoperative complication rate was 10% in Group I and 9% in Group II (p = 0.7). The rate of postoperative ureteral complications requiring surgical intervention was lower in Group I than in Group II (0.6% vs. 2.3%, respectively, p = 0.03). The overall 10-year rate for Grade 3 and 4 late radiation complications was 10.4%. Postoperative EBPRT significantly increased the 10-year rate for Grade 3 and 4 late radiation complications (yes vs. no: 22% vs. 7%, respectively, p = 0.0002). CONCLUSION: The prognosis for patients with cervical carcinoma was not influenced by the sequence of adjuvant RT (preoperative uterovaginal brachytherapy vs. postoperative RT) for Stages IB, IIA, and IIB with 1/3 proximal parametrial involvement. However, postoperative EBPRT increased the risk of late radiation complications. PMID- 12377331 TI - Age-related variations in the use of axillary dissection: a survival analysis of 8038 women with T1-ST2 breast cancer. AB - PURPOSE: The use of axillary dissection (AD) in women with invasive breast cancer is increasingly questioned. This study analyzes the survival in women with T1-2 breast cancer according to age and AD use. METHODS AND MATERIALS: Data from the Breast Cancer Outcomes Unit Database were analyzed for 8038 women aged 50-89 years referred to the British Columbia Cancer Agency between 1989 and 1998 with invasive T1-2,M0 breast cancer. Tumor and treatment characteristics were compared between women treated with and without AD (AD+ vs. AD-) according to three age groups: 50-64, 65-74 and 75+ years. Regional relapse and actuarial 5-year overall and breast cancer-specific survival were compared between AD+ and AD- women. Multivariate analysis of age, tumor and treatment factors, and adjusted hazard ratios with AD omission were performed. RESULTS: AD was omitted more frequently with advancing age (4% vs. 8% vs. 22% in women aged 50-64, 65-74, and 75+ years, respectively, p <0.0001). Tumor characteristics were more favorable in AD- women, with fewer having Grade III disease, T2 tumors, or lymphovascular invasion (all p <0.0001). Women treated without AD were also less likely to undergo radiotherapy after lumpectomy or mastectomy (both p <0.0001). Systemic therapy use and regional relapse rates were comparable between AD- and AD+ women in each age specific cohort. Multivariate analysis identified age, tumor size, grade, lymphovascular invasion, estrogen receptor status, clinical nodal palpability, type of surgery, and radiotherapy use as independent variables affecting survival. Hazard ratios adjusted for these variables showed AD omission to be associated with lower overall survival in the entire cohort (hazard ratio 1.52, p <0.0001) and lower breast cancer-specific survival in women aged 65-74 years (hazard ratio 1.99, p = 0.02). CONCLUSION: AD was more frequently omitted with advancing age. The lack of differences in systemic therapy use, regional relapse, and breast cancer-specific survival among AD- compared with AD+ women aged 75+ years suggests that AD use may be selectively omitted in this elderly cohort. However, the lower survival associated with AD omission among women aged 65-74 years, and the lack of a survival advantage among AD- women aged 50-64 years despite more favorable tumor characteristics and comparable systemic therapy use support the hypothesis that definitive locoregional therapy has an impact on survival. PMID- 12377332 TI - Age as a predictor of outcome for women with DCIS treated with breast-conserving surgery and radiation: The University of Texas M. D. Anderson Cancer Center experience. AB - PURPOSE: To analyze the long-term outcome of breast conservation therapy in patients with ductal carcinoma in situ (DCIS) in a single institution and to analyze the prognostic importance, if any, of young patient age. METHODS AND MATERIALS: The hospital records of 150 patients with DCIS treated with surgical excision and radiotherapy at our institution between 1980 and 1997 were retrospectively reviewed. For most of the patients, intraoperative specimen radiographs or postoperative mammograms were available for use in assessing that an adequate surgical resection had been performed. The median patient age was 53 years (range 32-81), with 13% of patients or=40 years, p = 0.39). In all cases of local recurrence, patients underwent surgery with or without chemotherapy, and disease control was achieved. CONCLUSION: The results of this study demonstrate high rates of long-term overall survival, disease specific survival, and local control in patients with DCIS of the breast treated conservatively with segmental mastectomy and radiotherapy. On the basis of the excellent long-term local control and 100% disease-specific survival rates, we found that patient age does not affect the outcome if the margins are clear. Continued studies in young patients treated with breast conservative therapy for DCIS are needed. PMID- 12377333 TI - Results of whole brain radiotherapy in patients with brain metastases from breast cancer: a retrospective study. AB - PURPOSE: To analyze the factors that affect survival in patients with brain metastases (BM) from breast cancer who were treated with whole brain radiotherapy (WBRT). METHODS AND MATERIALS: We identified 116 women with breast cancer who were treated with WBRT alone between February 1984 and September 2000. All patients had treatment and follow-up data available in their medical charts, which we extracted for this retrospective study. We evaluated a number of potential predictors of survival after WBRT: age, primary tumor stage, control of primary tumor, presence of other systemic metastases, site of systemic metastases, Karnofsky performance status, Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis class, total dose of WBRT, and number of BM. Eighteen patients received a total dose >3000 cGy and 7 received a partial brain boost. RESULTS: For the entire cohort, the median survival from the start of WBRT was 4.2 months. The 1-year survival rate was 17%, and the 2-year survival rate was 2%. Using univariate analysis, only Karnofsky performance status (p = 0. 0084), recursive partitioning analysis class (p = 0. 0147), and total WBRT dose (p = 0.0001) were predictive of longer survival. In multivariate analysis, Karnofsky performance status was the only significant predictor. CONCLUSION: Overall survival in breast cancer patients with BM treated with WBRT is poor. We recommend breast cancer patients with BM be enrolled in prospective trials to improve results. PMID- 12377334 TI - Curative radiotherapy for primary orbital lymphoma. AB - PURPOSE: To review our institutional experience with primary orbital lymphoma and determine the prognostic factors for survival, local control, and distant metastases. In addition, we also analyzed the risk factors for complications in the radiotherapeutic management of this tumor. METHODS AND MATERIALS: Between 1973 and 1998, 47 patients (29 women [62%] and 18 men [38%], median age 69 years, range 32-89) with Stage IAE orbital lymphoma were treated with curative intent at one department. Five had bilateral orbital involvement. The tumor was located in the eyelid and extraocular muscles in 23 (44%), conjunctiva in 17 (33%), and lacrimal apparatus in 12 (23%). The histologic features according to the World Heath Organization classification of lymphoid neoplasms was follicular lymphoma in 25, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type in 8, diffuse large B-cell lymphoma in 12, mantle cell lymphoma in 6, and peripheral T-cell lymphoma in 1. For the purposes of comparison with the existing literature on orbital lymphomas, the grading system according to the Working Formulation was also recorded. The histologic grade was low in 33 (63%), intermediate in 18 (35%), and high in 1 (2%). All patients were treated with primary radiotherapy alone. The median dose for low-grade tumors was 3000 cGy (range 2000-4020); the median dose for intermediate and high-grade tumors was 4000 cGy (range 3000-5100). A lens-sparing approach was used in 19 patients (37%). Late complications for the lens and cornea were scored according to the subjective, objective, management, and analytic (SOMA) scale of the Late Effects of Normal Tissue (LENT) scoring system. The median follow-up was 55 months (range 6-232). RESULTS: The local control rate was 100% in the 52 orbits treated. The 5 year overall survival and relapse-free survival rate was 73.6% and 65.5%, respectively. Tumor grade and location did not predict for overall survival or relapse-free survival. Seven patients (15%) developed distant recurrence (brain 2, extremity 2, mediastinum 1, liver 1, and retroperitoneum 1). One patient (2%) developed cervical node metastasis. The 5- and 10-year cataract-free survival rate was 56.7% and 32.9%, respectively. Of the 12 lens complications, 8 were LENT Grade 1 and 4 were Grade 3 toxicity. Only male gender predicted for an increased risk of cataract formation. Radiotherapy dose and technique did not predict for cataract formation; however, none of the patients who underwent the lens-sparing technique developed Grade 3 lens toxicity or required surgical correction. Of the nine corneal events, two were Grade 1, four Grade 2, and three were Grade 3 toxicity. Ten dry eyes were recorded; all were mild, and no patient had severe dry eye syndrome. Neovascular glaucoma was seen in 1 patient. No injury to the retina or optic nerve was reported. CONCLUSION: Radiotherapy alone is a highly effective modality in the curative management of primary orbital lymphoma. Most complications were minimal and did not require medical or surgical intervention. Although the use of the lens-sparing technique did not influence the incidence of cataractogenesis, we continue to recommend this approach whenever possible, because our experience indicates a higher grade of toxicity occurs and a higher incidence of corrective surgery is needed in patients treated without lens protection. PMID- 12377335 TI - Potential reduction of the incidence of radiation-induced second cancers by using proton beams in the treatment of pediatric tumors. AB - PURPOSE: To assess the potential influence of improved dose distribution with proton beams compared to conventional or intensity-modulated (IM) X-ray beams on the incidence of treatment-induced secondary cancers in pediatric oncology. METHODS AND MATERIALS: Two children, one with a parameningeal rhabdomyosarcoma (RMS) and a second with a medulloblastoma, were used as models for the purpose of this study. After defining the target and critical structures, treatment plans were calculated and optimized, four for the RMS case (conventional X-ray, IM X rays, protons, and IM protons) and three for the irradiation of the spinal axis in medulloblastoma (conventional X-ray, IM X-rays, protons). Secondary cancer incidence was estimated using a model based on Publication No. 60 of the International Commission on Radiologic Protection. This model allowed estimation of absolute risks of secondary cancer for each treatment plan based on dose volume distributions for the nontarget organs. RESULTS: Proton beams reduced the expected incidence of radiation-induced secondary cancers for the RMS patient by a factor of >or=2 and for the medulloblastoma case by a factor of 8 to 15 when compared with either IM or conventional X-ray plans. CONCLUSIONS: The potential for a significant reduction in secondary cancers with pediatric cancers after using proton beams (forward planned or IM) in the treatment of RMS and MBD in children and adolescents represents an additional argument supporting the development of proton therapy for most radiotherapy indications in pediatric oncology. PMID- 12377336 TI - Hemithorax irradiation for Ewing tumors of the chest wall. AB - PURPOSE: In the Cooperative Ewing's Sarcoma Study 86 and the European Intergroup Cooperative Ewing's Sarcoma Study 92, hemithorax irradiation (RT) was performed in patients with Ewing tumors of the chest wall involving the pleura or contaminating the pleural cavity. In a retrospective analysis, the outcomes of these patients were evaluated and compared with those of patients with chest wall tumors who did not receive hemithorax RT. METHODS AND MATERIALS: Between 1985 and 1996, 138 patients presented with nonmetastatic Ewing tumors of the chest wall. They were treated in a multimodal treatment regimen that included polychemotherapy and local therapy depending on the tumor characteristics. Hemithorax RT was performed at a dose of 15 Gy for patients <14 years old and 20 Gy for patients >or=14 years old. Forty-two patients received hemithorax RT (Group 1) and 86 patients did not (Group 2). The data were insufficient for the other 10 patients. RESULTS: Comparing both groups, the initial pleural effusion, pleural infiltration, and intraoperative contamination of the pleural space were significantly more frequent in Group 1. The event-free survival rate after 7 years was 63% for patients in Group 1 and 46% for patients in Group 2 (not statistically significant). The 7-year local relapse rate (including combined local-systemic relapses) was 12% in Group 1 and 10% in Group 2; the corresponding systemic relapse rates were 22% and 39%. CONCLUSION: Patients with chest wall tumors who received hemithorax RT were negatively selected; yet the rate of event free survival was better for patients who received hemithorax RT than for those who did not (although the difference was not statistically significant). This result was due to a reduction of metastases, mainly lung metastases. Local control was equivalent between the two groups. These favorable results have caused us to continue using hemithorax RT to treat high-risk patients with Ewing tumors of the chest wall. PMID- 12377337 TI - Stereotactic radiosurgery for patients with ACTH-producing pituitary adenomas after prior adrenalectomy. AB - PURPOSE: To review the results of stereotactic radiosurgery for patients with adrenocorticotropic hormone (ACTH)-producing pituitary adenomas after bilateral adrenalectomy. METHODS AND MATERIALS: Eleven patients with ACTH-producing pituitary adenomas after bilateral adrenalectomy underwent radiosurgery between 1990 and 1999. Nine patients had documented tumor growth, hyperpigmentation, and elevated ACTH levels (median 920 ng/mL) at the time of radiosurgery. Five of these patients had tumor enlargement despite prior fractionated radiotherapy (median dose 50 Gy). Two patients were treated prophylactically within 1 month of their adrenalectomies to prevent future tumor growth. The median follow-up was 37 months (range 22-74). RESULTS: Tumor growth control was achieved in 9 patients (82%); 2 patients had had continued tumor growth after radiosurgery. The ACTH levels decreased a median of 66% (range -99% to +27%); 4 patients had normal ACTH levels. Three patients had radiation-related complications, including diplopia (n = 2), ipsilateral blindness (n = 1), testosterone/growth hormone deficiency (n = 1), and asymptomatic temporal lobe radiation necrosis (n = 1): all had received prior radiotherapy. One patient who had undergone three prior resections and radiotherapy died 59 months after radiosurgery despite two additional attempts at tumor resection. CONCLUSION: Although our experience is limited, it appears that radiosurgery provides tumor control for most patients with ACTH-producing pituitary adenomas who have undergone bilateral adrenalectomy. PMID- 12377338 TI - Implications of IMT-SPECT for postoperative radiotherapy planning in patients with gliomas. AB - PURPOSE: Using MRI, residual tumor cannot be differentiated from nonspecific postoperative changes in patients with brain gliomas after surgical resection. The goal of this study was to analyze the value of 123I-alpha-methyl-tyrosine single photon emission CT (IMT-SPECT) in radiotherapy planning of patients with brain gliomas after surgical resection. METHODS AND MATERIALS: In 66 patients with surgically resected brain gliomas (33 glioblastomas, 20 anaplastic astrocytomas, 7 anaplastic oligodendrogliomas, and 6 low-grade astrocytomas), IMT SPECT and MRI were performed for radiotherapy planning. On the MRI/IMT-SPECT fusion images, the volume with IMT uptake was compared with the volume of the hyperintensity areas of T(2)-weighted MRI and with the volume of contrast enhancement on T(1)-weighted MRI. The regions with IMT uptake and/or MRI changes (composite Vol-MRI/IMT), regions with overlay of IMT uptake and MRI changes (common Vol-MRI/IMT), area with IMT uptake without MRI changes (increase Vol MRI/IMT), and area with only MRI changes (Vol-MRI minus IMT) were analyzed separately. The planning target volume and boost volume defined using MRI information alone was compared with the planning target volume and boost volume defined by also using the SPECT information. RESULTS: Focally increased IMT uptake was observed in 25 (38%) of 66 patients, contrast enhancement on MRI was outlined in 59 (89%) of 66 patients, and hyperintensity areas on T(2)-weighted MRI were found in all 66 investigated patients. The mean composite Vol-T(2)/IMT was 73 cm(3). The relative increase Vol-T(2)/IMT, mean relative common Vol T(2)/IMT, and mean relative Vol-T(2) minus IMT was 4%, 6%, and 90% of the composite Vol-T(2)/IMT, respectively. The mean composite Vol-T(1)/IMT was 14 cm(3) and the mean relative increase Vol-T(1)/IMT, mean relative common Vol T(1)/IMT, and mean relative Vol-T(1) minus IMT was 21%, 4%, and 64% of the mean composite Vol-T(1)/IMT, respectively. In 19 (29%) of 66 patients, the focal IMT uptake was located outside the MRI changes. In this subgroup, the mean residual volume defined by focal IMT uptake in MRI/IMT-SPECT images, mean Vol-T(1), and mean Vol-T(2) was 19 cm(3), 10 cm(3), and 70 cm(3), respectively. The mean relative increase T(2)/IMT was 14% and T(1)/IMT was 61%. In this subgroup, the additional information of SPECT led to an increase in boost volume (mean relative increase BV-IMT) by 20%. CONCLUSION: In patients with surgically resected brain gliomas, the size and location of residual IMT uptake differs considerably from the abnormalities found on postoperative MRI. Because of the known high specificity of IMT uptake for tumor tissue, the findings on IMT-SPECT may significantly modify the target volumes for radiotherapy planning. This will help to focus the high irradiation dose on the tumor area and to spare normal brain tissue. PMID- 12377339 TI - Medulloblastoma in adults: treatment results and prognostic factors. AB - PURPOSE: To investigate the treatment outcome and prognostic factors of adult medulloblastoma patients who received postoperative craniospinal irradiation (RT). METHODS AND MATERIALS: Between 1983 and 2000, 30 adult patients (17 men and 13 women, age >or=16 years, median 27, range 16-45) underwent postoperative RT. The median duration of symptoms was 2 months (range 1-9). The tumor location was lateral in 16 (53%). A desmoplastic variant was seen in 12 (40%). Tumor resection was complete in 20 (67%) and incomplete in 10 (33%). All patients received craniospinal RT. The median dose to the whole brain was 40 Gy (range 36-51), to the posterior fossa 54 Gy (range 49-56), and to the spinal axis 36 Gy (range 24 40). The median interval between surgery and the start of RT was 31 days (range 12-69), and the median duration of RT was 45 days (range 34-89). Ten patients (33%) received adjuvant chemotherapy. The median follow-up was 51 months (range 5 215). RESULTS: The 5- and 8-year overall survival and disease-free survival rates were 65% and 51% and 63% and 50%, respectively. Twelve patients (40%) developed relapse, with a median follow-up of 51 months. The posterior fossa was the most common site of relapse (6 patients). The median time to relapse was 26 months (range 4-78). Fifty percent of the relapses occurred after 2 years, 17% after 5 years. In univariate analysis, M stage and the interval between surgery and the start of RT were significant prognostic factors for disease-free survival. At 5 years, 70% of M0 patients were estimated to be disease-free, but none of the 3 M3 patients reached 5 years without recurrence (p = 0.0002). The 5-year disease-free survival rate for the patients whose interval between surgery and the start of RT was <3 weeks, between 3 and 6 weeks, and >6 weeks was 0%, 85%, and 75%, respectively (p = 0.002). The 5-year posterior fossa control rate for patients who received >or=54 Gy or <54 Gy to the posterior fossa was 91% and 33%, respectively (p = 0.05). CONCLUSION: The survival results for medulloblastomas in adults compare favorably with those in children. However, late relapses, lateral tumor location, and desmoplastic histologic features are more frequent in adults. Spinal seeding at presentation is a poor prognostic factor for disease-free survival. A minimal dose of 54 Gy to the posterior fossa is essential for adequate tumor control. The interval between surgery and the start of RT, which was found to be a significant prognostic factor, is an interesting issue that requires further study. PMID- 12377340 TI - Toward a national consensus: teaching radiobiology to radiation oncology residents. AB - PURPOSE: The ASTRO Joint Working Group on Radiobiology Teaching, a committee composed of members having affiliations with several national radiation oncology and biology-related societies and organizations, commissioned a survey designed to address issues of manpower, curriculum standardization, and instructor feedback as they relate to resident training in radiation biology. METHODS AND MATERIALS: Radiation biology instructors at U.S. radiation oncology training programs were identified and asked to respond to a comprehensive electronic questionnaire dealing with instructor educational background, radiation biology course content, and sources of feedback with respect to curriculum planning and resident performance on standardized radiation biology examinations. RESULTS: Eighty-five radiation biology instructors were identified, representing 73 radiation oncology residency training programs. A total of 52 analyzable responses to the questionnaire were received, corresponding to a response rate of 61.2%. CONCLUSION: There is a decreasing supply of instructors qualified to teach classic, and to some extent, clinical, radiobiology to radiation oncology residents. Additionally, those instructors with classic training in radiobiology are less likely to be comfortable teaching cancer molecular biology or other topics in cancer biology. Thus, a gap exists in teaching the whole complement of cancer and radiobiology curricula, particularly in those programs in which the sole responsibility for teaching falls to one faculty member (50% of training programs are in this category). On average, the percentage of total teaching time devoted to classic radiobiology (50%), clinical radiobiology (30%), and molecular and cancer biology (20%) is appropriate, relative to the current makeup of the board examination. Nevertheless large variability exists between training programs with respect to the total number of contact hours per complete radiobiology course (ranging from approximately 10 to >50 h). A number of lecture topics, particularly in clinical radiobiology, are covered by fewer than 60% of training programs. A sizeable minority of radiation biology instructors are dissatisfied with the feedback they receive with respect to both course content and the performance of their residents on standardized radiobiology examinations administered by the American College of Radiology and/or the American Board of Radiology. PMID- 12377341 TI - Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model. AB - PURPOSE: The purpose of this study was to evaluate the efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in an adjuvant setting with external beam radiation therapy (EBRT) in an experimental prostate cancer model in preparation for a Phase I clinical study in humans. METHODS: For efficacy studies, i.m. DU145 and intraprostatic LNCaP C4-2 tumors were established in immune-deficient mice. Tumors were injected with the lytic, replication-competent Ad5-CD/TKrep adenovirus containing a cytosine deaminase (CD)/herpes simplex virus thymidine kinase (HSV-1 TK) fusion gene. Two days later, mice were administered 1 week of 5-fluorocytosine + ganciclovir (GCV) prodrug therapy and fractionated doses of EBRT (trimodal therapy). Tumor control rate of trimodal therapy was compared to that of EBRT alone. For toxicology studies, immune-competent male mice received a single intraprostatic injection (10(10) vp) of the replication-competent Ad5-CD/TKrep adenovirus. Two days later, mice were administered 4 weeks of 5-fluorocytosine + GCV prodrug therapy and 56 Gy EBRT to the pelvic region. The toxicity of trimodal therapy was assessed by histopathologic analysis of major organs and clinical chemistries. RESULTS: In both the i.m. DU145 and intraprostatic LNCaP C4-2 tumor models, trimodal therapy significantly improved primary tumor control beyond that of EBRT alone. In the DU145 model, trimodal therapy resulted in a tumor growth delay (70 days) that was more than twice that (32 days) of EBRT alone. Whereas EBRT failed to eradicate DU145 tumors, trimodal therapy resulted in 25% tumor cure. In the LNCaP C4-2 tumor model, EBRT slowed the growth of intraprostatic tumors, but resulted in no tumor cures, and 57% of the mice developed retroperitoneal lymph node metastases at 3 months. By contrast, trimodal therapy resulted in 44% tumor cure and reduced significantly the percentage (13%) of lymph node metastases relative to EBRT alone. Overall, trimodal therapy was associated with little toxicity. A comparison of the major histopathologic findings among the treatment groups indicated that most of the locoregional (prostate, seminal vesicles, urinary bladder) pathology was attributable to the combined effects of the Ad5-CD/TKrep vector and EBRT and that the prodrugs contributed little to this effect. Importantly, trimodal therapy did not exacerbate inflammation of the rectum and intestines beyond that of EBRT alone. CONCLUSION: Together, the results support the thesis that replication-competent adenovirus-mediated double suicide gene therapy may be a safe and effective adjuvant to EBRT and provide a sound scientific rationale for human trials. PMID- 12377342 TI - In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme: mechanistic considerations. AB - PURPOSE: Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor response to radiation in vivo, but the cellular mechanisms underlying the radiosensitizing effect are not understood. In the present study, we investigated several possible mechanisms using a murine sarcoma cell culture system. METHODS AND MATERIALS: Cells derived from a murine sarcoma, designated NFSA, were cultured in vitro and exposed to different (either single or split) doses of radiation with and without a pretreatment of SC-236 (4-[5-(4-chlorophenyl)-3 (trifluoromethyl)-1H-pyrazol-l-yl] benzene sulfonamide), a selective cyclooxygenase-2 (COX-2) inhibitor. The cells were assayed for clonogenic survival to determine the radiosensitizing effect of SC-236. In addition, MTT assay and TUNEL assay were performed to determine the effects of SC-236 and radiation on the cell survival and cell cycle distribution. RNase protection assay was performed on the total RNA extract using probes that encoded for selected cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases. To monitor the extent of COX-2 activity and its role in radiosensitization, the cellular content of prostaglandin E2, a major metabolite of COX-2 activity on arachidonic acid, was also determined. RESULTS: The cell clonogenic survival assay showed that SC-236 significantly enhanced tumor cell radiosensitivity: 50 microM SC-236 increased it by a factor of 1.51 at the 0.1 cell survival level. Treatment with SC-236 (50 microM, 3 days) removed the "shoulder" region on the radiation survival curve, suggesting that the drug inhibited repair of sublethal radiation damage. The inhibition was confirmed by split-dose experiments where two doses (3 Gy each) of radiation were given 4 h apart. The cells exposed to radiation only repaired the damage by a factor of 1.44, whereas those treated with SC-236 plus radiation repaired it by a factor of 1.1 only. Whereas SC-236 induced apoptosis in these NFSA cells, radiation did not. No further increase in apoptosis was observed when the cells were exposed to both SC-236 and radiation, suggesting that SC-236 did not render tumor cells more susceptible to radiation-induced apoptosis. The RNase protection assay showed that SC-236 (50 microM, 3 days) inhibited the expression of cyclins A and B, as well as cyclin-dependent kinase-1. Inhibition of these cell cycle regulatory elements by SC-236 was associated with the arrest of cells in the radiosensitive G2-M phase (67%), determined by flow cytometry. CONCLUSIONS: SC-236 significantly enhanced radiosensitivity of tumor cells; the magnitude of sensitivity was dependent on the drug's concentration. The likely mechanisms involve accumulation of cells in the radiosensitive G2-M phase of the cell cycle and inhibition of repair from sublethal radiation damage. PMID- 12377343 TI - Fractionated irradiation of H69 small-cell lung cancer cells causes stable radiation and drug resistance with increased MRP1, MRP2, and topoisomerase IIalpha expression. AB - PURPOSE: After standard treatment with chemotherapy and radiotherapy, small-cell lung cancer (SCLC) often develops resistance to both treatments. Our aims were to establish if fractionated radiation treatment alone would induce radiation and drug resistance in the H69 SCLC cell line, and to determine the mechanisms of resistance. METHODS AND MATERIALS: H69 SCLC cells were treated with fractionated X-rays to an accumulated dose of 37.5 Gy over 8 months to produce the H69/R38 subline. Drug and radiation resistance was determined using the MTT (3,-4,5 dimethylthiazol-2,5 diphenyltetrazolium bromide) cell viability assay. Protein expression was analyzed by Western blot. RESULTS: The H69/R38 subline was resistant to radiation (2.0 +/- 0.2-fold, p < 0.0001), cisplatin (14 +/- 7-fold, p < 0.001), daunorubicin (6 +/- 3-fold, p < 0.05), and navelbine (1.7 +/- 0.15 fold, p < 0.02). This was associated with increased expression of the multidrug resistance-associated proteins, MRP1 and MRP2, and topoisomerase IIalpha and decreased expression of glutathione-S-transferase pi (GSTpi) and bcl-2 and decreased cisplatin accumulation. Treatment with 4 Gy of X-rays produced a 66% decrease in MRP2 in the H69 cells with no change in the H69/R38 cells. This treatment also caused a 5-fold increase in topoisomerase IIalpha in the H69/R38 cells compared with a 1.5-fold increase in the H69 cells. CONCLUSIONS: Fractionated radiation alone can lead to the development of stable radiation and drug resistance and an altered response to radiation in SCLC cells. PMID- 12377344 TI - Correlations between in vivo tumor weight, oxygen pressure, 31P NMR spectroscopy, hypoxic microenvironment marking by beta-D-iodinated azomycin galactopyranoside (beta-D-IAZGP), and radiation sensitivity. AB - PURPOSE: The purpose of this study is to evaluate the amount of hypoxic fraction in a rodent tumor by means of polarographic oxygen electrode, phosphorus-31 magnetic resonance spectroscopy (31P-MRS), and a newly synthesized hypoxic marker, beta-D-iodinated azomycin galactopyranoside (beta-D-IAZGP). We also investigated the radiosensitivity for tumors of different weights. METHODS AND MATERIALS: Murine mammary carcinoma cells, FM3A, were subcutaneously implanted into the back of 5-week-old male C3H/He mice. beta-D-IAZGP radiolabeled with 123I or with 125I was injected intravenously into tumor-bearing mice, and marker distribution was measured by nuclear medicine procedures. Radiosensitivity of the tumor was measured by the in vivo/in vitro clonogenic assay. Tumor oxygenation status was also measured directly by polarographic oxygen electrodes and indirectly estimated from 31P-MR spectra. RESULTS: Higher accumulation of 123I beta-D-IAZGP was observed in the tumors than in normal tissues at 24 h after administration. As to biodistribution of 125I-beta-D-IAZGP, the tumor/blood ratio varied widely, but correlated significantly with tumor weight. Mean oxygen pressure (pO2) values and ratios of nucleoside triphosphate beta to inorganic phosphate (beta-ATP/Pi) and of phosphocreatine to inorganic phosphate (PCr/Pi) decreased significantly with the increase in tumor volume. As tumor volume increased, the surviving fraction of cells from tumors irradiated in vivo increased significantly. CONCLUSIONS: The increase in tumor volume was significantly correlated with a reduction in mean pO2, a decrease in the ratios of beta-ATP/Pi or PCr/Pi, an increase in uptake of beta-D-IAZGP, and an increase in radioresistance. Because the uptake of beta-D-IAZGP can be measured noninvasively by nuclear medicine techniques, it could be clinically useful for monitoring hypoxia in human tumors. PMID- 12377345 TI - Increased radiosensitivity with chronic hypoxia in four human tumor cell lines. AB - PURPOSE: It is well known that the radiosensitivity of tumor cells can be significantly reduced under hypoxic conditions. However, most of the reports in the literature refer to an experimental setup in which the supply of oxygen is kept low for a short period of time only. In tumors, chronic hypoxia would seem to be the more typical situation, because of an insufficient vascularization and the limited diffusion of oxygen into the tissue. Under such conditions, certain changes in the proliferation patterns of tumor cells, in which the cell cycle checkpoint protein p53 seems to play a role, have been shown to occur. We therefore decided to study radiosensitivity and cell cycle progression under conditions of chronic hypoxia in several human tumor cell lines differing in their p53 status. METHODS AND MATERIALS: Four human tumor cell lines (melanomas Be11 and MeWo and squamous carcinomas 4197 and 4451) were incubated for 3 h, 24 h, and 72 h under either oxic or hypoxic conditions and subsequently exposed to graded doses of X-rays. In some cases, cells were kept under hypoxia for the same periods of time, but then reoxygenated immediately before irradiation. Cell survival was assessed with the usual colony formation assay, and cell cycle distributions were determined by two-parameter flow cytometry after labeling with bromodeoxyuridine (BrdU). RESULTS: As expected, the oxygen enhancement ratio at 3 h was 2.0 or more in all cases. Differences, however, became evident with longer incubation times. At 24 h, the sensitivity of cells kept under hypoxic conditions both before and during irradiation was practically unchanged with cell lines Be11, 4197, and 4451, but clearly increased with MeWo. This resulted in an oxygen enhancement ratio of only 1.1 for the latter cell line when the sensitivity of aerated cells was used as reference. Cells kept under hypoxia for 24 h and reoxygenated shortly before irradiation, however, also showed an increase in sensitivity, so that the oxygen enhancement ratio based on differences in irradiation atmosphere alone was still around 2.0. At 72 h, the two p53 wild-type cell lines were not available for experiments, because they quickly degenerated under hypoxic conditions. Both mutant cell lines now showed similar results, the sensitivity being increased with irradiation under continued hypoxia as well as after reoxygenation. The oxygen enhancement ratios with reference to aerated cells were 1.3 and 1.5 for MeWo and 4451, respectively. Flow cytometric measurements after labeling with BrdU revealed that in all cell lines, the fraction of active S-phase cells during incubation tended to decrease under hypoxic conditions. Only in the p53 mutant cell lines, however, was this accompanied by an increase of the percentage of S-phase cells that were not actively incorporating BrdU. CONCLUSIONS: It is suggested that these quiescent cells in the S-phase compartment develop because of a general breakdown of cellular energy metabolism. In the p53 mutant cells, this may lead to a cessation of cell cycle progression in all phases alike, because checkpoint control has been lost; p53 wild-type cells, on the other hand, settle down preferentially in G(1) under the same conditions. Independently of the p53 status, however, energy depletion may be the cause of a decreased ability to cope with radiation damage and thus the cause of the observed increase in radiosensitivity. This would become more easily apparent in the p53 mutant cell lines, because they are less sensitive than the p53 wild types to hypoxia as such. PMID- 12377346 TI - Tumor control probability for selective boosting of hypoxic subvolumes, including the effect of reoxygenation. AB - PURPOSE: To study the effect on tumor control probability of selectively boosting the dose to hypoxic subvolumes. METHODS AND MATERIALS: A Monte Carlo model was developed that separates the tumor into two compartments, one of which receives a primary dose, and one of which receives a higher boost dose. During radiation delivery, each compartment consists of three clonogen subpopulations: those that are well oxygenated, those that are temporarily hypoxic (geometrically transient hypoxia), and those that are permanently hypoxic (geometrically stable hypoxia). The spatial location of temporary hypoxia within the tumor volume varies over time, whereas, the spatial location of permanent hypoxia does not. The effect of reoxygenation was included. Clonogen proliferation was not included in the model. RESULTS: A modest boost dose (120%-150% of the primary dose) increases tumor control probability to that found in the absence of permanent hypoxia. The entire hypoxic subvolume need not be included to obtain a significant benefit. However, only tumors with a geometrically stable hypoxic volume will have an improved control rate. CONCLUSIONS: Tumors with an identifiable geometrically stable hypoxic volume will have an improved control rate if the dose to the hypoxic volume is escalated. Further work is required to determine the spatiotemporal evolution of the hypoxic volumes before and during the course of radiotherapy. PMID- 12377347 TI - Preclinical biological assessment of proton and carbon ion beams at Hyogo Ion Beam Medical Center. AB - PURPOSE: To assess the biologic effects of proton and carbon ion beams before clinical use. METHODS AND MATERIALS: Cultured cells from human salivary gland cancer (HSG cells) were irradiated at 5 points along a 190 MeV per nucleon proton and a 320 MeV per nucleon carbon ion beam, with Bragg peaks modulated to 6 cm widths. A linac 4 MV X-ray was used as a reference. Relative biologic effectiveness (RBE) values at each point were calculated from survival curves. Cells were also irradiated in a cell-stack phantom to identify that localized cell deaths were observed at predefined depth. Total body irradiation of C3H/He mice was performed, and the number of regenerating crypts per jejunal section was compared to calculate intestinal RBE values. For carbon ion and referential 4 MV X-ray beams, mouse right legs were irradiated by four-fractional treatment and followed up for skin reaction scoring. RESULTS: RBE values calculated from cell survival curves at the dose that would reduce cell survival to 10% (D10) ranged from 1.01 to 1.05 for protons and from 1.23 to 2.56 for carbon ions. The cell stack phantom irradiation revealed localized cell deaths at predefined depth. The intestinal RBE values ranged from 1.01 to 1.08 for protons and from 1.15 to 1.88 for carbon ions. The skin RBE value was 2.16 at C320/6 cm spread-out Bragg peak (SOBP) center. CONCLUSION: The radiobiologic measurements of proton and carbon ion beams at Hyogo Ion Beam Medical Center are consistent with previous reports using proton beams in clinical settings and carbon ion beams with similar linear energy transfer (LET) values. PMID- 12377348 TI - Calculation of rotational setup error using the real-time tracking radiation therapy (RTRT) system and its application to the treatment of spinal schwannoma. AB - PURPOSE: The efficacy of a prototypic fluoroscopic real-time tracking radiation therapy (RTRT) system using three gold markers (2 mm in diameter) for estimating translational error, rotational setup error, and the dose to normal structures was tested in 5 patients with spinal schwannoma and a phantom. METHODS AND MATERIALS: Translational error was calculated by comparing the actual position of the marker closest to the tumor to its planned position, and the rotational setup error was calculated using the three markers around the target. Theoretically, the actual coordinates can be adjusted to the planning coordinates by sequential rotation of gamma degrees around the z axis, beta degrees around the y axis, and alpha degrees around the x axis, in this order. We measured the accuracy of the rotational calculation using a phantom. Five patients with spinal schwannoma located at a minimum of 1-5 mm from the spinal cord were treated with RTRT. Three markers were inserted percutaneously into the paravertebral deep muscle in 3 patients and surgically into two consecutive vertebral bones in two other patients. RESULTS: In the phantom study, the discrepancies between the actual and calculated rotational error were -0.1 +/- 0.5 degrees. The random error of rotation was 5.9, 4.6, and 3.1 degrees for alpha, beta, and gamma, respectively. The systematic error was 7.1, 6.6, and 3.0 degrees for alpha, beta, and gamma, respectively. The mean rotational setup error (0.2 +/- 2.2, -1.3 +/- 2.9, and 1.3 +/- 1.7 degrees for alpha, beta, and gamma, respectively) in 2 patients for whom surgical marker implantation was used was significantly smaller than that in 3 patients for whom percutaneous insertion was used (6.0 +/- 8.2, 2.7 +/- 5.9, and -2.1 +/- 4.6 degrees for alpha, beta, and gamma). Random translational setup error was significantly reduced by the RTRT setup (p < 0.0001). Systematic setup error was significantly reduced by the RTRT setup only in patients who received surgical implantation of the marker (p < 0.0001). The maximum dose to the spinal cord was estimated to be 40.6-50.3 Gy after consideration of the rotational setup error, vs. a planned maximum dose of 22.4-51.6 Gy. CONCLUSION: The RTRT system employing three internal fiducial markers is useful to reduce translational setup error and to estimate the dose to the normal structures in consideration of the rotational setup error. Surgical implantation of the marker to the vertebral bone was shown to be sufficiently rigid for the calculation of the rotational setup error. Fractionated radiotherapy for spinal schwannoma using the RTRT system may well be an alternative or supplement to surgical treatment. PMID- 12377349 TI - Clinical use of stereoscopic X-ray positioning of patients treated with conformal radiotherapy for prostate cancer. AB - PURPOSE: To evaluate accuracy and time requirements of a stereoscopic X-ray-based positioning system in patients receiving conformal radiotherapy to the prostate. METHODS AND MATERIALS: Setup errors of the isocenter with regard to the bony pelvis were measured by means of orthogonal verification films and compared to conventional positioning (using skin drawings and lasers) and infrared marker (IR) based positioning in each of 261 treatments. In each direction, the random error represents the standard deviation and the systematic error the absolute value of the mean position. Time measurements were done in 75 treatments. RESULTS: Random errors with the X-ray positioning system in the anteroposterior (AP), lateral, and longitudinal direction were (average +/- 1 standard deviation) 2 +/- 0.6 mm, 1.7 +/- 0.6 mm, and 2.4 +/- 0.7 mm. The corresponding values of conventional as well as IR positioning were significantly higher (p < 0.01). Systematic errors for X-ray positioning were 1.1 +/- 1.2 mm AP, 0.6 +/- 0.5 mm laterally, and 1.5 +/- 1.6 mm longitudinally. Conventional and IR marker-based positioning showed significantly larger systematic errors AP and laterally, but longitudinally, the difference was not significant. Depending on the axis looked at, errors of >or=5 mm occurred in 2%-14% of treatments after X-ray positioning, 13%-29% using IR markers, and 28%-53% with conventional positioning. Total linac time for one treatment session was 14 min 51 s +/- 4 min 18 s, half of which was used for the X-ray-assisted positioning procedure. CONCLUSION: X-ray-assisted patient positioning significantly improves setup accuracy, at the cost of an increased treatment time. PMID- 12377350 TI - Evaluation of concave dose distributions created using an inverse planning system. AB - PURPOSE: To evaluate and develop optimum inverse treatment planning strategies for the treatment of concave targets adjacent to normal tissue structures. METHODS AND MATERIALS: Optimized dose distributions were designed using an idealized geometry consisting of a cylindrical phantom with a concave kidney shaped target (PTV) and cylindrical normal tissues (NT) placed 5-13 mm from the target. Targets with radii of curvature from 1 to 2.75 cm were paired with normal tissues with radii between 0.5 and 2.25 cm. The target was constrained to a prescription dose of 100% and minimum and maximum doses of 95% and 105% with relative penalties of 25. Maximum dose constraint parameters for the NT varied from 10% to 70% with penalties from 10 to 1000. Plans were evaluated using the PTV uniformity index (PTV D(max)/PTV D(95)) and maximum normal tissue doses (NT D(max)/PTV D(95)). RESULTS: In nearly all situations, the achievable PTV uniformity index and the maximum NT dose exceeded the corresponding constraints. This was particularly true for small PTV-NT separations (5-8 mm) or strict NT dose constraints (10%-30%), where the achievable doses differed from the requested by 30% or more. The same constraint parameters applied to different PTV NT separations yielded different dose distributions. For most geometries, a range of constraints could be identified that would lead to acceptable plans. The optimization results were fairly independent of beam energy and radius of curvature, but improved as the number of beams increased, particularly for small PTV-NT separations or strict dose constraints. CONCLUSION: Optimized dose distributions are strongly affected by both the constraint parameters and target normal tissue geometry. Standard site-specific constraint templates can serve as a starting point for optimization, but the final constraints must be determined iteratively for individual patients. A strategy whereby NT constraints and penalties are modified until the highest acceptable PTV uniformity index is achieved is discussed. This strategy can be used, in simple patient geometries, to ensure the lowest possible normal tissue dose. Strategies for setting the optimum dose constraints and penalties may vary for different optimization algorithms and objective functions. Increasing the number of beams can significantly improve normal tissue dose and target uniformity in situations where the PTV-NT separation is small or the normal tissue dose limits are severe. Setting unrealistically severe constraints in such situations often results in dose distributions that are inferior to plans achieved with more lenient constraints. PMID- 12377351 TI - The impact of central lung distance, maximal heart distance, and radiation technique on the volumetric dose of the lung and heart for intact breast radiation. AB - PURPOSE: To investigate the impact of radiographic parameter and radiation technique on the volumetric dose of lung and heart for intact breast radiation. METHODS AND MATERIALS: Forty patients with both two-dimensional (2D) and computed tomographic (CT) simulations were enrolled in the study. Central lung distance (CLD), maximal heart distance (MHD), and maximal heart length (MHL) were measured under virtual simulation. Four plans were compared for each patient. Plan A used a traditional 2D tangential setup. Plan B used clinical target volume (CTV) based three-dimensional (3D) planning. Both plans C and D used a combination of a medial breast field with shallow tangents. Plan D is a further modification of plan C. RESULTS: Under the traditional tangential setup, the mean ipsilateral lung dose and volume at 20, 30, and 40 Gy correlated linearly with CLD (R = 0.85 approximately 0.91). The mean ipsilateral lung dose (Gy) approximated 4 times the CLD value (cm), whereas the percentage volume (%) of ipsilateral lung at 20, 30, and 40 Gy was about 10 times the CLD (cm). The mean heart dose and percentage volume at 20, 30, and 40 Gy correlated with MHD (R = 0.76 approximately 0.80) and MHL (R = 0.65 approximately 0.75). The mean heart dose (Gy) approximated 3 times the MHD value (cm), and the percentage volume (%) of the heart at 10, 20, 30, and 40 Gy was about 6 times MHD (cm). Radiation technique impacted lung and heart dose. The 3D tangential plan (plan B) failed to reduce the volumetric dose of lung and heart from that of the 2D plan (plan A). The medial breast techniques (plans C and D) significantly decreased the volume of lung and heart receiving high doses (30 and 40 Gy). Plan D further decreased the 20 Gy volumes. By use of the medial breast technique, the lung and heart dose were not impacted by original CLD and MHD/MHL. Therefore, the improvement from the tangential technique was more remarkable for patients with CLD >or= 3.0 cm (p < 0.001). CONCLUSIONS: The CLD and MHD impact the volumetric dose of lung and heart. The application of 3D planning for tangential breast irradiation does not decrease heart and lung dose. Adding a medial breast port significantly decreases percentage volume (PV) of lung and heart receiving high doses, especially when the CLD is excessive. PMID- 12377352 TI - High beta and electron dose from 192Ir: implications for "gamma" intravascular brachytherapy. AB - PURPOSE: Trains of multiple 192Ir seeds are used in many clinical trials for intravascular brachytherapy. 192Ir source is commonly considered as a gamma emitter, despite the understanding that this radionuclide also emits a wide range of electron and beta energies, with a similar range of energy. The high dose from betas and electrons in the submillimeter range due to unsealed ends of seed sources should be precisely quantified to fully understand the backdrop for complications associated with 192Ir coronary artery brachytherapy. METHODS AND MATERIALS: Monte Carlo simulations (MCNP4C code) were performed for a model 5 seed 192Ir train used in SCRIPPS, GAMMA, and the Washington Radiation for In Stent Restenosis (WRIST) randomized clinical trials. A stack of radiochromic films was also used to measure the dose distributions for an actual 6-seed train. RESULTS: In the submillimeter range very close to the source, Monte Carlo results show that betas and electrons deposit a higher dose than 192Ir photons (gamma and X-rays) over the interseed gap. A high luminal dose from the combined effects of betas, electrons, and photons emitted from 192Ir can be deposited, particularly between seeds. When prescribing 15 Gy at 2 mm, the combined dose can be as high as 160 Gy at 0.5 mm. Different peak doses near the interseed gaps were noted, which may be due to variability of seed-end surfaces and nonuniformity of seed activity within a real multiseed train. Dose-volume histograms (DVH) of lumen surfaces were evaluated for an eccentric seed train. The DVH parameters indicating the extent of hot spots in the lumen wall, DV(10), DV(5), DV(2), and DV(1) (dose received by 10, 5, 2, 1% respectively of the total lumen surface), can be as high as 55, 76, 81, and 155 Gy for a lumen with 3-mm diameter, and 75, 80, 110, and 158 Gy for a narrow 2-mm lumen. CONCLUSION: 192Ir multiple seed trains used in the SCRIPPS, GAMMA, and WRIST trials can deposit a very high dose to the luminal wall. A particularly high electron and beta dose can be delivered near the interseed gap if the source is not centered in the catheter and lumen. The dose from 192Ir betas and electrons may partially explain adverse outcomes reported from 192Ir multiseed clinical trials. Improvement of the encapsulation design to filter out the betas and electrons should be seriously considered. PMID- 12377353 TI - In regard to Padula et al., normalization of serum testosterone levels in patients treated with neoadjuvant hormonal therapy and three-dimensional conformal radiotherapy for prostate cancer. IJROBP 2002;52: 439-443. PMID- 12377355 TI - Repeated nicotine injections decrease operant ethanol self-administration. AB - Nicotine and alcohol are two of the most used drugs in the United States. However, it is not clear whether the co-use of these drugs is due to pharmacological or environmental reasons, or perhaps related to both. Although results from previous studies in animal models seem to indicate that nicotine has an effect on ethanol consumption, little has been done to determine how nicotine affects appetitive and consummatory phases of ethanol self-administration. In this study, we examined the effect of repeated treatment with nicotine (0, 0.35, and 0.7 mg/kg, s.c.), given 30 min before a daily operant session, on appetitive and consummatory phases of Long-Evans rats self-administering 10% (vol./vol.) ethanol in a sipper-tube model. Ethanol intake (consummatory phase) decreased at both doses of nicotine tested, and lever pressing (appetitive phase) decreased after injection of the high dose of nicotine. These results support the suggestion that nicotine affects ethanol self-administration. However, in this model, the findings demonstrate a reduction in drinking, rather than the enhancement that has been shown in findings obtained from other studies. PMID- 12377356 TI - Association between use of sedatives or hypnotics, alcohol consumption, or other risk factors and a single injurious fall or multiple injurious falls: a longitudinal general population study. AB - In this study, we investigated the association between risk factors, including use of sedatives or hypnotics or alcohol consumption, and injurious falls leading to hospitalization or death among 4023 subjects (1828 men and 2195 women) aged 20 89 years in Stockholm County, Sweden. Questionnaire data obtained from the 1984 1985 Stockholm Health of the Population Study (SHPS) were linked to official data registers on hospitalization and mortality. Of the 4023 subjects, 330 (121 men and 209 women) had been treated for or died of injurious falls during the 12-year follow-up period. High age was significantly associated with injurious falls among both men and women. Multivariate analyses showed that women who had used sedatives or hypnotics during the 2 weeks before an injurious fall were at increased risk [relative risk of 1.83 (95% confidence interval, 1.10-3.06)] for two or more injurious falls, but not for a single fall accident. High alcohol consumption and earlier self-reported injurious falls were significantly associated with injurious falls for women younger than 60 years of age and with earlier self-reported falls and living alone for men in the same age category. Among older women (>60 years of age), high alcohol consumption and use of sedatives or hypnotics were significantly associated with injurious falls, whereas living alone and earlier self-reported accidents were significant predictors for men in the same age category. These results support a cautious prescribing policy for sedatives and hypnotics, as well as an awareness of high alcohol consumption and its association with injurious falls. PMID- 12377357 TI - Roles of tyrosine kinase-, 1-phosphatidylinositol 3-kinase-, and mitogen activated protein kinase-signaling pathways in ethanol-induced contractions of rat aortic smooth muscle: possible relation to alcohol-induced hypertension. AB - Insights into the relations between and among ethanol-induced contractions in rat aorta, tyrosine kinases (including src family of cytoplasmic tyrosine kinases), 1 phosphatidylinositol 3-kinases (PI-3Ks), mitogen-activated protein kinases (MAPKs), and regulation of intracellular free Ca(2+) ([Ca(2+)](i)) were investigated in the present study. Ethanol-induced concentration-dependent contractions in isolated rat aortic rings were attenuated greatly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein), an src homology domain 2 (SH2) inhibitor peptide, a highly specific antagonist of p38 MAPK (SB-203580), a potent, selective antagonist of two specific MAPK kinases-MEK1/MEK2 (U0126)-and a selective antagonist of mitogen activated protein kinase kinase (MAPKK) (PD-98059), as well as by treatment with wortmannin or LY-294002 (both are selective antagonists of PI-3Ks). Inhibitory concentration 50 (IC(50)) levels obtained for these seven antagonists were consistent with reported inhibition constant (Ki) values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol-induced transient and sustained increases in [Ca(2+)](i) in primary single smooth muscle cells from rat aorta were markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, PD-98059, wortmannin, and LY-294002. A variety of specific antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate either the ethanol-induced contractions or the elevations of [Ca(2+)](i). Results of the present study support the suggestion that activation of tyrosine kinases (including the src family of cytoplasmic tyrosine kinases), PI-3Ks, and MAPK seems to play an important role in ethanol-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from rat aorta. These signaling pathways thus may be important in hypertension in human beings associated with chronic alcohol consumption. PMID- 12377358 TI - Neuropeptide Y administration into the amygdala does not affect ethanol consumption. AB - Evidence seems to indicate that the anxiolytic effects of centrally administered neuropeptide Y (NPY) are mediated by the central nucleus of the amygdala. Because findings seem to indicate that ethanol may be self-administered partially for its anxiolytic effects, it was hypothesized that NPY, microinjected into the central nucleus of the amygdala, would decrease ethanol intake. In this study, we examined the effects of NPY, administered into the central nucleus of the amygdala, on ethanol, sucrose, and food consumption, as well as the concomitant effects of NPY on cortical electroencephalographic activity. Wistar rats were implanted with cortical recording electrodes and cannulae above the central amygdaloid nuclei, after use of a sucrose-substitution procedure, to establish ethanol self-administration. Neuropeptide Y (0-250 pmol/0.5 micro l) was infused into the amygdala before drinking sessions, when 10% ethanol (10 E), 2% sucrose (2S), or food was available. Consumption, locomotor activity, and cortical electroencephalographic activity were then monitored concurrently. Neuropeptide Y had no effect on the intake of 10 E, 2S, or food, nor on the cortical electroencephalographic or locomotor activity. However, as reported previously, distinct changes in the electroencephalogram were associated with consumption of ethanol and sucrose. Cortical power in the 6-8 Hz frequency range was significantly increased during the beginning of the sucrose and ethanol sessions, with greater increases observed during the sucrose session. Overall, these findings support the suggestion that NPY administration into the central nucleus of the amygdala does not alter consumption of 10 E, 2S, or food, nor the cortical electroencephalographic or locomotor activity. PMID- 12377359 TI - Separate measures of ethanol seeking and drinking in the rat: effects of remoxipride. AB - Remoxipride, a dopamine D(2) antagonist, decreases responding that results in the presentation of small amounts (approximately 0.1 ml) of ethanol in limited-access paradigms. This type of operant response is a combined appetitive/consummatory response that is differentially affected by changing stimulus properties of consumed ethanol (i.e., taste, pharmacology) over the course of the session. In the present experimental design, ethanol-directed appetitive and consummatory responses were procedurally separated to investigate the specific effects of remoxipride on these distinct behaviors. Male Long-Evans rats were trained to make a series of lever-press responses once each day that resulted in access to a sipper tube spout containing 10% ethanol for 20 min. Three doses of remoxipride were tested: 5.0, 10.0, and 15.0 mg/kg (-30 min, i.p.). In Experiment 1, a response requirement of 20 was used, and both reinforced and nonreinforced sessions were examined. In nonreinforced sessions, subjects were permitted to lever press for 20 min, after which the session ended without sipper tube presentation. These sessions were conducted to remove the possibility that limiting responding might obscure a drug effect on the seeking response. In Experiment 2, a low response requirement (4) was used to investigate the effects of remoxipride on ethanol intake. Average baseline ethanol intake (Experiment 1) was 0.69 g/kg, with blood ethanol concentrations at the end of the session at 64 mg%. At all doses tested, remoxipride had no effect on the measures of ethanol consumption (e.g., total intake, lick latency, lick rate) in either experiment. However, remoxipride dose dependently decreased the number of appetitive responses made, while having no effect on response latency or rate, during both reinforced and nonreinforced sessions in Experiment 1. In these experiments, the systemic antagonism of the dopamine D(2) receptor decreased ethanol seeking without causing a general impairment of motor function. The procedural separation of seeking and intake responses revealed that appetitive responding was more sensitive than consummatory responding to remoxipride treatment. PMID- 12377360 TI - Dopamine D3 receptor antagonist effects on the motivational effects of ethanol. AB - Dopaminergic systems are thought to play important roles in the motivational effects of ethanol. In the present experiments, we examined the effects of U99194A, a putative dopamine D(3) receptor antagonist, on ethanol-induced conditioned place preference, locomotor stimulation, taste aversion, and self administration. In two separate studies with the use of a place conditioning procedure, adult male Swiss-Webster mice received six pairings of a tactile stimulus with ethanol (1 or 3 g/kg, i.p.), U99194A (20 mg/kg, i.p.), or ethanol + U99194A. For determination of ethanol-stimulated activity, subjects received U99194A at a dose of 0, 10, 20, or 30 mg/kg 15 min before ethanol at 0, 1, or 2 g/kg immediately before a 30-min locomotor activity test. In a taste conditioning procedure, subjects received five 1-h access periods to 0.2 M NaCl. After the first four access periods, subjects received ethanol at 0, 2, or 4 g/kg and U99194A at 0, 10, or 20 mg/kg. In an oral self-administration procedure, male C57BL/6J mice received U99194A at 0, 10, or 20 mg/kg, followed by 30-min access to 10% (wt./vol.) sucrose or 10% (vol./vol.) ethanol in 10% sucrose. The acquisition of ethanol-induced conditioned place preference was enhanced by U99194A. However, U99194A did not produce significant preference alone. U99194A did not alter locomotor stimulation produced by an injection of ethanol at 2 g/kg. U99194A also did not alter the acquisition of ethanol-induced conditioned taste aversion and did not change oral ethanol self-administration. These results support the suggestion that dopamine D(3) receptors have specific involvement in ethanol reward, as measured by place conditioning, but are not important for ethanol-stimulated activity, ethanol taste aversion, or ethanol intake. PMID- 12377361 TI - Short-term treatment for alcohol-related problems: four-session guided self change versus one session of advice--a randomized, controlled trial. AB - The aim of this study was to compare two short-term treatments for alcohol related problems. The study was performed at an outpatient clinic for substance misuse, and subjects (65 men and 28 women) were recruited through advertisements in the local newspaper. The subjects were randomized to either a four-session guided self-change group or a one-session advice group. Alcohol consumption, degree of alcohol dependence, negative consequences of drinking, and health related quality of life were measured or assessed, respectively, by using the timeline follow-back technique, the Short Alcohol Dependence Data (SADD) questionnaire, The Drinker Inventory of Consequences questionnaire, and the Nottingham Health Profile questionnaire. Biological markers for high alcohol consumption [carbohydrate-deficient transferrin (CDT) and gamma-glutamyl transferase (gamma-GT) levels] were analyzed. All assessments were made at baseline and at 9- and 23-month follow-up periods after treatment. Self-reported alcohol consumption was significantly reduced (P <.0001) in both treatment groups at the 23-month follow-up period, as were measures of alcohol dependence, negative consequences of drinking, and health-related quality of life, whereas no corresponding reduction was found in CDT or gamma-GT values. No statistically significant differences in self-reported alcohol consumption were found between the two groups. Patient satisfaction was significantly higher with the four session guided self-change treatment than with the one session of advice. This finding seems to indicate that individuals, although suffering from alcohol related problems of relatively low severity, appreciate more time with a therapist. PMID- 12377362 TI - Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-helix and beta-sheet conformations. AB - Because poly-L-lysine (PLL) can exist in the alpha-helix or beta-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and alpha-helix or beta-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the beta sheet conformation than for the alpha-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions. PMID- 12377363 TI - Differential effects of cholesterol on acyl chain order in erythrocyte membranes as a function of depth from the surface. An electron paramagnetic resonance (EPR) spin label study. AB - The purpose of this work is to analyze the effects of cholesterol modulation on acyl chain ordering in the membrane of human erythrocytes as a function of depth from the surface. Partial cholesterol depletion was achieved by incubation of erythrocytes with liposomes containing saturated phospholipids, or with methyl beta-cyclodextrin (MbetaCD). Cholesterol enrichment was achieved by incubation with liposomes formed by phospholipids/cholesterol, or with the complex MbetaCD/cholesterol. Acyl chain order was studied with electron paramagnetic resonance spectroscopy (EPR) using spin labels that sense the lipid bilayer at different depths. It is shown that the increase in cholesterol stiffens acyl chains but decreases the interaction among lipid headgroups, while cholesterol depletion causes the opposite behavior. It is likely that the observed cholesterol effects are related to those stabilizing the cholesterol-rich detergent-insoluble membrane domains (rafts), recently shown to exist in erythrocytes. PMID- 12377364 TI - Rheological study on lysozyme/tetramethylurea viscoelastic matrices. AB - Rheological properties of lysozyme viscoelastic matrices resulting from a sol-gel transition taking place in organic/aqueous media at room temperature were investigated. Gel-like structures, of transparent appearance, developed out of lysozyme (5.0 mmol/dm(3)) dispersed in tetramethylurea (TMU)/water binary mixtures, at TMU mass fraction (w) ranging from w(TMU) 0.6 to 0.9. The wide linear viscoelastic region (LVR) observed, up to strains of 10%, was invariant throughout the TMU concentration range investigated, indicating that the 3D structures of protein matrices, although fragile, are quite flexible and able to withstand great deformation before rupture. Storage (G') and loss (G") moduli continuously increased with increasing TMU concentration, the former at a greater rate, consequently leading systems to a decrease in the loss angle, tandelta. For gels developed out of binary systems at w(TMU)=0.9, creep curves revealed behaviour that very nearly approaches that of a perfect elastic solid. Although gelification under the experimental conditions employed is macroscopically accomplished in a time interval that does not exceed 24 h (for the gel developed out of the solvent mixture of lowest TMU concentration, w(TMU)=0.6), a slight decrease in loss angle can still be detected after that period. Such changes, however, have no effect on the LVR. Relaxation tests indicate that systems comprise at least two dynamically distinct contributions. PMID- 12377365 TI - Analysis of resonance energy transfer in model membranes: role of orientational effects. AB - The model of resonance energy transfer (RET) in membrane systems containing donors randomly distributed over two parallel planes separated by fixed distance and acceptors confined to a single plane is presented. Factors determining energy transfer rate are considered with special attention being given to the contribution from orientational heterogeneity of the donor emission and acceptor absorption transition dipoles. Analysis of simulated data suggests that RET in membranes, as compared to intramolecular energy transfer, is substantially less sensitive to the degree of reorientational freedom of chromophores due to averaging over multiple donor-acceptor pairs. The uncertainties in the distance estimation resulting from the unknown mutual orientation of the donor and acceptor are analyzed. PMID- 12377366 TI - Dynamic hydration numbers for biologically important ions. AB - The role of ionized groups in biological systems is determined by their affinity for water [Biophys. J. 72 (1997) 65-76]. The tightly bound water associated with biologically important ions increases their apparent size. We define the apparent dynamic hydration number of an ion here as the number of tightly bound water molecules that must be assigned to the ion to explain its apparent molecular weight on a Sephadex G-10 size exclusion column, and report the first accurate determination of tightly bound water for 23 ions of biological significance, including H(+) and HO(-). We also calculate the radius of the equivalent hydrated sphere (r(h)) for each ion. We find that the ratio of the hydrated volumes of two ions approximates the ratio of the square of the charges of the same two ions. Since the 'ionic strength' of the solution also depends upon the square of the charges on the ions, our results suggest that ionic strength effects may largely arise from local effects related to the hydrated volume of the ion--that is, from space filling, osmotic, water activity, surface tension and hydration shell overlap effects rather than from long-range electric field effects. PMID- 12377368 TI - EPR study of non-covalent spin labeled serum albumin and hemoglobin. AB - Electron Paramagnetic Resonance (EPR) was used to investigate the Tempyo spin label (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilized bovine serum albumin (BSA) and bovine hemoglobin (BH), as function of pH values in the range 2.5-11. The EPR spectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures. This behavior indicated a possible spin-spin interaction between the hemic iron and the nitroxide group. The changes in the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectral simulations and magnetic EPR parameters reveal the following: (i) one single paramagnetic species, with Gaussian line shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and (ii) a weighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation of correlation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changes of proteins in acidic and basic environment. PMID- 12377367 TI - Lysozyme viscoelastic matrices in tetramethylurea/water media: a small angle X ray scattering study. AB - Semi-solid viscoelastic matrices produced out of lysozyme in organic/aqueous media [tetramethylurea (TMU)/water] were characterized by small angle X-ray scattering (SAXS). The scattering curves were modeled in their form and interference factors. Radii of gyration of scattering particles were found to undergo a dramatic increase from 14 A in water to approximately 44 A in the matrices. Average correlation distances d=155 A were consistently verified for the scattering particles in the matrices, irrespective of solvent composition (in the 0.6